@ARTICLE{Andrews2022-ht,
  title    = "Challenges of Organoid Research",
  author   = "Andrews, Madeline G and Kriegstein, Arnold R",
  abstract = "Organoids are 3D cell culture systems derived from human
              pluripotent stem cells that contain tissue resident cell types
              and reflect features of early tissue organization. Neural
              organoids are a particularly innovative scientific advance given
              the lack of accessibility of developing human brain tissue and
              intractability of neurological diseases. Neural organoids have
              become an invaluable approach to model features of human brain
              development that are not well reflected in animal models.
              Organoids also hold promise for the study of atypical cellular,
              molecular, and genetic features that underscore neurological
              diseases. Additionally, organoids may provide a platform for
              testing therapeutics in human cells and are a potential source
              for cell replacement approaches to brain injury or disease.
              Despite the promising features of organoids, their broad utility
              is tempered by a variety of limitations yet to be overcome,
              including lack of high-fidelity cell types, limited maturation,
              atypical physiology, and lack of arealization, features that may
              limit their reliability for certain applications.",
  journal  = "Annu Rev Neurosci",
  volume   =  45,
  pages    = "23--39",
  month    =  jan,
  year     =  2022,
  address  = "United States",
  keywords = "human development; modeling human disease; neural development;
              neuroscience; organoids; stem cell models",
  language = "en"
}

Organoids are 3D cell culture systems derived from human pluripotent stem cells that contain tissue resident cell types and reflect features of early tissue organization. Neural organoids are a particularly innovative scientific advance given the lack of accessibility of developing human brain tissue and intractability of neurological diseases. Neural organoids have become an invaluable approach to model features of human brain development that are not well reflected in animal models. Organoids also hold promise for the study of atypical cellular, molecular, and genetic features that underscore neurological diseases. Additionally, organoids may provide a platform for testing therapeutics in human cells and are a potential source for cell replacement approaches to brain injury or disease. Despite the promising features of organoids, their broad utility is tempered by a variety of limitations yet to be overcome, including lack of high-fidelity cell types, limited maturation, atypical physiology, and lack of arealization, features that may limit their reliability for certain applications.

@ARTICLE{Paredes2022-ro,
  title    = "Nests of dividing neuroblasts sustain interneuron production for
              the developing human brain",
  author   = "Paredes, Mercedes F and Mora, Cristina and Flores-Ramirez,
              Quetzal and Cebrian-Silla, Arantxa and Del Dosso, Ashley and
              Larimer, Phil and Chen, Jiapei and Kang, Gugene and Gonzalez
              Granero, Susana and Garcia, Eric and Chu, Julia and Delgado, Ryan
              and Cotter, Jennifer A and Tang, Vivian and Spatazza, Julien and
              Obernier, Kirsten and Ferrer Lozano, Jaime and Vento, Maximo and
              Scott, Julia and Studholme, Colin and Nowakowski, Tomasz J and
              Kriegstein, Arnold R and Oldham, Michael C and Hasenstaub, Andrea
              and Garcia-Verdugo, Jose Manuel and Alvarez-Buylla, Arturo and
              Huang, Eric J",
  abstract = "The human cortex contains inhibitory interneurons derived from
              the medial ganglionic eminence (MGE), a germinal zone in the
              embryonic ventral forebrain. How this germinal zone generates
              sufficient interneurons for the human brain remains unclear. We
              found that the human MGE (hMGE) contains nests of proliferative
              neuroblasts with ultrastructural and transcriptomic features that
              distinguish them from other progenitors in the hMGE. When
              dissociated hMGE cells are transplanted into the neonatal mouse
              brain, they reform into nests containing proliferating
              neuroblasts that generate young neurons that migrate extensively
              into the mouse forebrain and mature into different subtypes of
              functional interneurons. Together, these results indicate that
              the nest organization and sustained proliferation of neuroblasts
              in the hMGE provide a mechanism for the extended production of
              interneurons for the human forebrain.",
  journal  = "Science",
  volume   =  375,
  number   =  6579,
  pages    = "eabk2346",
  month    =  jan,
  year     =  2022,
  language = "en"
}

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.

@ARTICLE{Pasca2022-zl,
  title    = "A nomenclature consensus for nervous system organoids and
              assembloids",
  author   = "Pașca, Sergiu P and Arlotta, Paola and Bateup, Helen S and Camp,
              J Gray and Cappello, Silvia and Gage, Fred H and Knoblich,
              J{\"u}rgen A and Kriegstein, Arnold R and Lancaster, Madeline A
              and Ming, Guo-Li and Muotri, Alysson R and Park, In-Hyun and
              Reiner, Orly and Song, Hongjun and Studer, Lorenz and Temple,
              Sally and Testa, Giuseppe and Treutlein, Barbara and Vaccarino,
              Flora M",
  abstract = "Self-organizing three-dimensional cellular models derived from
              human pluripotent stem cells or primary tissue have great
              potential to provide insights into how the human nervous system
              develops, what makes it unique and how disorders of the nervous
              system arise, progress and could be treated. Here, to facilitate
              progress and improve communication with the scientific community
              and the public, we clarify and provide a basic framework for the
              nomenclature of human multicellular models of nervous system
              development and disease, including organoids, assembloids and
              transplants.",
  journal  = "Nature",
  volume   =  609,
  number   =  7929,
  pages    = "907--910",
  month    =  sep,
  year     =  2022,
  address  = "England",
  language = "en"
}

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.

@ARTICLE{Andrews2022-ty,
  title    = "Tropism of {SARS-CoV-2} for human cortical astrocytes",
  author   = "Andrews, Madeline G and Mukhtar, Tanzila and Eze, Ugomma C and
              Simoneau, Camille R and Ross, Jayden and Parikshak, Neelroop and
              Wang, Shaohui and Zhou, Li and Koontz, Mark and Velmeshev, Dmitry
              and Siebert, Clara-Vita and Gemenes, Kaila M and Tabata, Takako
              and Perez, Yonatan and Wang, Li and Mostajo-Radji, Mohammed A and
              de Majo, Martina and Donohue, Kevin C and Shin, David and Salma,
              Jahan and Pollen, Alex A and Nowakowski, Tomasz J and Ullian,
              Erik and Kumar, G Renuka and Winkler, Ethan A and Crouch,
              Elizabeth E and Ott, Melanie and Kriegstein, Arnold R",
  abstract = "The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
              readily infects a variety of cell types impacting the function of
              vital organ systems, with particularly severe impact on
              respiratory function. Neurological symptoms, which range in
              severity, accompany as many as one-third of COVID-19 cases,
              indicating a potential vulnerability of neural cell types. To
              assess whether human cortical cells can be directly infected by
              SARS-CoV-2, we utilized stem-cell-derived cortical organoids as
              well as primary human cortical tissue, both from developmental
              and adult stages. We find significant and predominant infection
              in cortical astrocytes in both primary tissue and organoid
              cultures, with minimal infection of other cortical populations.
              Infected and bystander astrocytes have a corresponding increase
              in inflammatory gene expression, reactivity characteristics,
              increased cytokine and growth factor signaling, and cellular
              stress. Although human cortical cells, particularly astrocytes,
              have no observable ACE2 expression, we find high levels of
              coronavirus coreceptors in infected astrocytes, including CD147
              and DPP4. Decreasing coreceptor abundance and activity reduces
              overall infection rate, and increasing expression is sufficient
              to promote infection. Thus, we find tropism of SARS-CoV-2 for
              human astrocytes resulting in inflammatory gliosis-type injury
              that is dependent on coronavirus coreceptors.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  119,
  number   =  30,
  pages    = "e2122236119",
  month    =  jul,
  year     =  2022,
  keywords = "SARS-CoV-2 tropism; astrocyte reactivity; organoid models",
  language = "en"
}

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

@ARTICLE{Andrews2022-uu,
  title    = "How mechanisms of stem cell polarity shape the human cerebral
              cortex",
  author   = "Andrews, Madeline G and Subramanian, Lakshmi and Salma, Jahan and
              Kriegstein, Arnold R",
  abstract = "Apical-basal progenitor cell polarity establishes key features of
              the radial and laminar architecture of the developing human
              cortex. The unique diversity of cortical stem cell populations
              and an expansion of progenitor population size in the human
              cortex have been mirrored by an increase in the complexity of
              cellular processes that regulate stem cell morphology and
              behaviour, including their polarity. The study of human cells in
              primary tissue samples and human stem cell-derived model systems
              (such as cortical organoids) has provided insight into these
              processes, revealing that protein complexes regulate progenitor
              polarity by controlling cell membrane adherence within
              appropriate cortical niches and are themselves regulated by
              cytoskeletal proteins, signalling molecules and receptors, and
              cellular organelles. Studies exploring how cortical stem cell
              polarity is established and maintained are key for understanding
              the features of human brain development and have implications for
              neurological dysfunction.",
  journal  = "Nat Rev Neurosci",
  month    =  sep,
  year     =  2022,
  address  = "England",
  language = "en"
}

Apical-basal progenitor cell polarity establishes key features of the radial and laminar architecture of the developing human cortex. The unique diversity of cortical stem cell populations and an expansion of progenitor population size in the human cortex have been mirrored by an increase in the complexity of cellular processes that regulate stem cell morphology and behaviour, including their polarity. The study of human cells in primary tissue samples and human stem cell-derived model systems (such as cortical organoids) has provided insight into these processes, revealing that protein complexes regulate progenitor polarity by controlling cell membrane adherence within appropriate cortical niches and are themselves regulated by cytoskeletal proteins, signalling molecules and receptors, and cellular organelles. Studies exploring how cortical stem cell polarity is established and maintained are key for understanding the features of human brain development and have implications for neurological dysfunction.

@ARTICLE{Kodani2022-lj,
  title    = "Zika virus alters centrosome organization to suppress the innate
              immune response",
  author   = "Kodani, Andrew and Knopp, Kristeene A and Di Lullo, Elizabeth and
              Retallack, Hanna and Kriegstein, Arnold R and DeRisi, Joseph L
              and Reiter, Jeremy F",
  abstract = "Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and
              sex to cause congenital neurodevelopmental defects, including
              microcephaly. Inherited forms of microcephaly (MCPH) are
              associated with disrupted centrosome organization. Similarly, we
              found that ZIKV infection disrupted centrosome organization. ZIKV
              infection disrupted the organization of centrosomal proteins
              including CEP63, a MCPH-associated protein. The ZIKV
              nonstructural protein NS3 bound CEP63, and expression of NS3 was
              sufficient to alter centrosome architecture and CEP63
              localization. Loss of CEP63 suppressed ZIKV-induced centrosome
              disorganization, indicating that ZIKV requires CEP63 to disrupt
              centrosome organization. ZIKV infection or CEP63 loss decreased
              the centrosomal localization and stability of TANK-binding kinase
              1 (TBK1), a regulator of the innate immune response. ZIKV
              infection also increased the centrosomal accumulation of the
              CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1.
              Therefore, we propose that ZIKV disrupts CEP63 function to
              increase centrosomal DTX4 localization and destabilization of
              TBK1, thereby tempering the innate immune response.",
  journal  = "EMBO Rep",
  volume   =  23,
  number   =  9,
  pages    = "e52211",
  month    =  jul,
  year     =  2022,
  keywords = "Zika virus; centrosome; innate immunity; microcephaly",
  language = "en"
}

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

@ARTICLE{Crouch2022-fv,
  title    = "Ensembles of endothelial and mural cells promote angiogenesis in
              prenatal human brain",
  author   = "Crouch, Elizabeth E and Bhaduri, Aparna and Andrews, Madeline G
              and Cebrian-Silla, Arantxa and Diafos, Loukas N and Birrueta,
              Janeth Ochoa and Wedderburn-Pugh, Kaylee and Valenzuela, Edward J
              and Bennett, Neal K and Eze, Ugomma C and Sandoval-Espinosa,
              Carmen and Chen, Jiapei and Mora, Cristina and Ross, Jayden M and
              Howard, Clare E and Gonzalez-Granero, Susana and Lozano, Jaime
              Ferrer and Vento, Maximo and Haeussler, Maximilian and Paredes,
              Mercedes F and Nakamura, Ken and Garcia-Verdugo, Jose Manuel and
              Alvarez-Buylla, Arturo and Kriegstein, Arnold R and Huang, Eric J",
  abstract = "Interactions between angiogenesis and neurogenesis regulate
              embryonic brain development. However, a comprehensive
              understanding of the stages of vascular cell maturation is
              lacking, especially in the prenatal human brain. Using
              fluorescence-activated cell sorting, single-cell transcriptomics,
              and histological and ultrastructural analyses, we show that an
              ensemble of endothelial and mural cell subtypes tile the brain
              vasculature during the second trimester. These vascular cells
              follow distinct developmental trajectories and utilize diverse
              signaling mechanisms, including collagen, laminin, and midkine,
              to facilitate cell-cell communication and maturation.
              Interestingly, our results reveal that tip cells, a subtype of
              endothelial cells, are highly enriched near the ventricular zone,
              the site of active neurogenesis. Consistent with these
              observations, prenatal vascular cells transplanted into cortical
              organoids exhibit restricted lineage potential that favors tip
              cells, promotes neurogenesis, and reduces cellular stress.
              Together, our results uncover important mechanisms into vascular
              maturation during this critical period of human brain
              development.",
  journal  = "Cell",
  volume   =  185,
  number   =  20,
  pages    = "3753--3769.e18",
  month    =  sep,
  year     =  2022,
  address  = "United States",
  keywords = "angiogenesis; arterial endothelial cells; blood brain barrier;
              cortical organoids; endothelial cells; human prenatal brain
              development; mural cells; pericytes; smooth muscle cells; tip
              cells; venous and capillary endothelial cells; ventricular zone",
  language = "en"
}

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

@ARTICLE{Bhaduri2021-qf,
  title    = "An atlas of cortical arealization identifies dynamic molecular
              signatures",
  author   = "Bhaduri, Aparna and Sandoval-Espinosa, Carmen and Otero-Garcia,
              Marcos and Oh, Irene and Yin, Raymund and Eze, Ugomma C and
              Nowakowski, Tomasz J and Kriegstein, Arnold R",
  abstract = "The human brain is subdivided into distinct anatomical
              structures, including the neocortex, which in turn encompasses
              dozens of distinct specialized cortical areas. Early
              morphogenetic gradients are known to establish early brain
              regions and cortical areas, but how early patterns result in
              finer and more discrete spatial differences remains poorly
              understood(1). Here we use single-cell RNA sequencing to profile
              ten major brain structures and six neocortical areas during peak
              neurogenesis and early gliogenesis. Within the neocortex, we find
              that early in the second trimester, a large number of genes are
              differentially expressed across distinct cortical areas in all
              cell types, including radial glia, the neural progenitors of the
              cortex. However, the abundance of areal transcriptomic signatures
              increases as radial glia differentiate into intermediate
              progenitor cells and ultimately give rise to excitatory neurons.
              Using an automated, multiplexed single-molecule fluorescent in
              situ hybridization approach, we find that laminar gene-expression
              patterns are highly dynamic across cortical regions. Together,
              our data suggest that early cortical areal patterning is defined
              by strong, mutually exclusive frontal and occipital
              gene-expression signatures, with resulting gradients giving rise
              to the specification of areas between these two poles throughout
              successive developmental timepoints.",
  journal  = "Nature",
  volume   =  598,
  number   =  7879,
  pages    = "200--204",
  month    =  oct,
  year     =  2021,
  language = "en"
}

The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood(1). Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization approach, we find that laminar gene-expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene-expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.

@ARTICLE{Eze2021-aw,
  title    = "Single-cell atlas of early human brain development highlights
              heterogeneity of human neuroepithelial cells and early radial
              glia",
  author   = "Eze, Ugomma C and Bhaduri, Aparna and Haeussler, Maximilian and
              Nowakowski, Tomasz J and Kriegstein, Arnold R",
  abstract = "The human cortex comprises diverse cell types that emerge from an
              initially uniform neuroepithelium that gives rise to radial glia,
              the neural stem cells of the cortex. To characterize the earliest
              stages of human brain development, we performed single-cell
              RNA-sequencing across regions of the developing human brain,
              including the telencephalon, diencephalon, midbrain, hindbrain
              and cerebellum. We identify nine progenitor populations
              physically proximal to the telencephalon, suggesting more
              heterogeneity than previously described, including a highly
              prevalent mesenchymal-like population that disappears once
              neurogenesis begins. Comparison of human and mouse progenitor
              populations at corresponding stages identifies two progenitor
              clusters that are enriched in the early stages of human cortical
              development. We also find that organoid systems display low
              fidelity to neuroepithelial and early radial glia cell types, but
              improve as neurogenesis progresses. Overall, we provide a
              comprehensive molecular and spatial atlas of early stages of
              human brain and cortical development.",
  journal  = "Nat Neurosci",
  volume   =  24,
  number   =  4,
  pages    = "584--594",
  month    =  mar,
  year     =  2021,
  language = "en"
}

The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.

@ARTICLE{Pebworth2021-nn,
  title    = "Human intermediate progenitor diversity during cortical
              development",
  author   = "Pebworth, Mark-Phillip and Ross, Jayden and Andrews, Madeline and
              Bhaduri, Aparna and Kriegstein, Arnold R",
  abstract = "Studies of the spatiotemporal, transcriptomic, and morphological
              diversity of radial glia (RG) have spurred our current models of
              human corticogenesis. In the developing cortex, neural
              intermediate progenitor cells (nIPCs) are a neuron-producing
              transit-amplifying cell type born in the germinal zones of the
              cortex from RG. The potential diversity of the nIPC population,
              that produces a significant portion of excitatory cortical
              neurons, is understudied, particularly in the developing human
              brain. Here we explore the spatiotemporal, transcriptomic, and
              morphological variation that exists within the human nIPC
              population and provide a resource for future studies. We observe
              that the spatial distribution of nIPCs in the cortex changes
              abruptly around gestational week (GW) 19/20, marking a distinct
              shift in cellular distribution and organization during late
              neurogenesis. We also identify five transcriptomic subtypes, one
              of which appears at this spatiotemporal transition. Finally, we
              observe a diversity of nIPC morphologies that do not correlate
              with specific transcriptomic subtypes. These results provide an
              analysis of the spatiotemporal, transcriptional, and
              morphological diversity of nIPCs in developing brain tissue and
              provide an atlas of nIPC subtypes in the developing human cortex
              that can benchmark in vitro models of human development such as
              cerebral organoids and help inform future studies of how nIPCs
              contribute to cortical neurogenesis.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  118,
  number   =  26,
  month    =  jun,
  year     =  2021,
  keywords = "cortex; development; human; neuronal; progenitor",
  language = "en"
}

Studies of the spatiotemporal, transcriptomic, and morphological diversity of radial glia (RG) have spurred our current models of human corticogenesis. In the developing cortex, neural intermediate progenitor cells (nIPCs) are a neuron-producing transit-amplifying cell type born in the germinal zones of the cortex from RG. The potential diversity of the nIPC population, that produces a significant portion of excitatory cortical neurons, is understudied, particularly in the developing human brain. Here we explore the spatiotemporal, transcriptomic, and morphological variation that exists within the human nIPC population and provide a resource for future studies. We observe that the spatial distribution of nIPCs in the cortex changes abruptly around gestational week (GW) 19/20, marking a distinct shift in cellular distribution and organization during late neurogenesis. We also identify five transcriptomic subtypes, one of which appears at this spatiotemporal transition. Finally, we observe a diversity of nIPC morphologies that do not correlate with specific transcriptomic subtypes. These results provide an analysis of the spatiotemporal, transcriptional, and morphological diversity of nIPCs in developing brain tissue and provide an atlas of nIPC subtypes in the developing human cortex that can benchmark in vitro models of human development such as cerebral organoids and help inform future studies of how nIPCs contribute to cortical neurogenesis.

@ARTICLE{Ahanger2021-ke,
  title    = "Distinct nuclear compartment-associated genome architecture in
              the developing mammalian brain",
  author   = "Ahanger, Sajad Hamid and Delgado, Ryan N and Gil, Eugene and
              Cole, Mitchel A and Zhao, Jingjing and Hong, Sung Jun and
              Kriegstein, Arnold R and Nowakowski, Tomasz J and Pollen, Alex A
              and Lim, Daniel A",
  abstract = "Nuclear compartments are thought to play a role in
              three-dimensional genome organization and gene expression. In
              mammalian brain, the architecture and dynamics of nuclear
              compartment-associated genome organization is not known. In this
              study, we developed Genome Organization using CUT and RUN
              Technology (GO-CaRT) to map genomic interactions with two nuclear
              compartments-the nuclear lamina and nuclear speckles-from
              different regions of the developing mouse, macaque and human
              brain. Lamina-associated domain (LAD) architecture in cells in
              vivo is distinct from that of cultured cells, including major
              differences in LADs previously considered to be cell type
              invariant. In the mouse and human forebrain, dorsal and ventral
              neural precursor cells have differences in LAD architecture that
              correspond to their regional identity. LADs in the human and
              mouse cortex contain transcriptionally highly active sub-domains
              characterized by broad depletion of histone-3-lysine-9
              dimethylation. Evolutionarily conserved LADs in human, macaque
              and mouse brain are enriched for transcriptionally active neural
              genes associated with synapse function. By integrating GO-CaRT
              maps with genome-wide association study data, we found
              speckle-associated domains to be enriched for schizophrenia risk
              loci, indicating a physical relationship between these
              disease-associated genetic variants and a specific nuclear
              structure. Our work provides a framework for understanding the
              relationship between distinct nuclear compartments and genome
              function in brain development and disease.",
  journal  = "Nat Neurosci",
  volume   =  24,
  number   =  9,
  pages    = "1235--1242",
  month    =  jul,
  year     =  2021,
  language = "en"
}

Nuclear compartments are thought to play a role in three-dimensional genome organization and gene expression. In mammalian brain, the architecture and dynamics of nuclear compartment-associated genome organization is not known. In this study, we developed Genome Organization using CUT and RUN Technology (GO-CaRT) to map genomic interactions with two nuclear compartments-the nuclear lamina and nuclear speckles-from different regions of the developing mouse, macaque and human brain. Lamina-associated domain (LAD) architecture in cells in vivo is distinct from that of cultured cells, including major differences in LADs previously considered to be cell type invariant. In the mouse and human forebrain, dorsal and ventral neural precursor cells have differences in LAD architecture that correspond to their regional identity. LADs in the human and mouse cortex contain transcriptionally highly active sub-domains characterized by broad depletion of histone-3-lysine-9 dimethylation. Evolutionarily conserved LADs in human, macaque and mouse brain are enriched for transcriptionally active neural genes associated with synapse function. By integrating GO-CaRT maps with genome-wide association study data, we found speckle-associated domains to be enriched for schizophrenia risk loci, indicating a physical relationship between these disease-associated genetic variants and a specific nuclear structure. Our work provides a framework for understanding the relationship between distinct nuclear compartments and genome function in brain development and disease.

@ARTICLE{Andrews2021-xv,
  title    = "Tropism of {SARS-CoV-2} for Developing Human Cortical Astrocytes",
  author   = "Andrews, Madeline G and Mukhtar, Tanzila and Eze, Ugomma C and
              Simoneau, Camille R and Perez, Yonatan and Mostajo-Radji,
              Mohammed A and Wang, Shaohui and Velmeshev, Dmitry and Salma,
              Jahan and Kumar, G Renuka and Pollen, Alex A and Crouch,
              Elizabeth E and Ott, Melanie and Kriegstein, Arnold R",
  abstract = "The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
              readily infects a variety of cell types impacting the function of
              vital organ systems, with particularly severe impact on
              respiratory function. It proves fatal for one percent of those
              infected. Neurological symptoms, which range in severity,
              accompany a significant proportion of COVID-19 cases, indicating
              a potential vulnerability of neural cell types. To assess whether
              human cortical cells can be directly infected by SARS-CoV-2, we
              utilized primary human cortical tissue and stem cell-derived
              cortical organoids. We find significant and predominant infection
              in cortical astrocytes in both primary and organoid cultures,
              with minimal infection of other cortical populations. Infected
              astrocytes had a corresponding increase in reactivity
              characteristics, growth factor signaling, and cellular stress.
              Although human cortical cells, including astrocytes, have minimal
              ACE2 expression, we find high levels of alternative coronavirus
              receptors in infected astrocytes, including DPP4 and CD147.
              Inhibition of DPP4 reduced infection and decreased expression of
              the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for
              human astrocytes mediated by DPP4, resulting in reactive
              gliosis-type injury.",
  journal  = "bioRxiv",
  month    =  jan,
  year     =  2021,
  language = "en"
}

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

@ARTICLE{Bhaduri2021-ch,
  title    = "Identification of Lipid Heterogeneity and Diversity in the
              Developing Human Brain",
  author   = "Bhaduri, Aparna and Neumann, Elizabeth K and Kriegstein, Arnold R
              and Sweedler, Jonathan V",
  abstract = "The lipidome is currently understudied but fundamental to life.
              Within the brain, little is known about cell-type lipid
              heterogeneity, and even less is known about cell-to-cell lipid
              diversity because it is difficult to study the lipids within
              individual cells. Here, we used single-cell mass
              spectrometry-based protocols to profile the lipidomes of 154 910
              single cells across ten individuals consisting of five
              developmental ages and five brain regions, resulting in a unique
              lipid atlas available via a web browser of the developing human
              brain. From these data, we identify differentially expressed
              lipids across brain structures, cortical areas, and developmental
              ages. We inferred lipid profiles of several major cell types from
              this data set and additionally detected putative cell-type
              specific lipids. This data set will enable further interrogation
              of the developing human brain lipidome.",
  journal  = "JACS Au",
  volume   =  1,
  number   =  12,
  pages    = "2261--2270",
  month    =  nov,
  year     =  2021,
  language = "en"
}

The lipidome is currently understudied but fundamental to life. Within the brain, little is known about cell-type lipid heterogeneity, and even less is known about cell-to-cell lipid diversity because it is difficult to study the lipids within individual cells. Here, we used single-cell mass spectrometry-based protocols to profile the lipidomes of 154 910 single cells across ten individuals consisting of five developmental ages and five brain regions, resulting in a unique lipid atlas available via a web browser of the developing human brain. From these data, we identify differentially expressed lipids across brain structures, cortical areas, and developmental ages. We inferred lipid profiles of several major cell types from this data set and additionally detected putative cell-type specific lipids. This data set will enable further interrogation of the developing human brain lipidome.

@ARTICLE{Zhang2020-dj,
  title    = "Neurotoxic microglia promote {TDP-43} proteinopathy in
              progranulin deficiency",
  author   = "Zhang, Jiasheng and Velmeshev, Dmitry and Hashimoto, Kei and
              Huang, Yu-Hsin and Hofmann, Jeffrey W and Shi, Xiaoyu and Chen,
              Jiapei and Leidal, Andrew M and Dishart, Julian G and Cahill,
              Michelle K and Kelley, Kevin W and Liddelow, Shane A and Seeley,
              William W and Miller, Bruce L and Walther, Tobias C and Farese,
              Jr, Robert V and Taylor, J Paul and Ullian, Erik M and Huang, Bo
              and Debnath, Jayanta and Wittmann, Torsten and Kriegstein, Arnold
              R and Huang, Eric J",
  abstract = "Aberrant aggregation of the RNA-binding protein TDP-43 in neurons
              is a hallmark of frontotemporal lobar degeneration caused by
              haploinsufficiency in the gene encoding progranulin(1,2).
              However, the mechanism leading to TDP-43 proteinopathy remains
              unclear. Here we use single-nucleus RNA sequencing to show that
              progranulin deficiency promotes microglial transition from a
              homeostatic to a disease-specific state that causes endolysosomal
              dysfunction and neurodegeneration in mice. These defects persist
              even when Grn(-/-) microglia are cultured ex vivo. In addition,
              single-nucleus RNA sequencing reveals selective loss of
              excitatory neurons at disease end-stage, which is characterized
              by prominent nuclear and cytoplasmic TDP-43 granules and nuclear
              pore defects. Remarkably, conditioned media from Grn(-/-)
              microglia are sufficient to promote TDP-43 granule formation,
              nuclear pore defects and cell death in excitatory neurons via the
              complement activation pathway. Consistent with these results,
              deletion of the genes encoding C1qa and C3 mitigates microglial
              toxicity and rescues TDP-43 proteinopathy and neurodegeneration.
              These results uncover previously unappreciated contributions of
              chronic microglial toxicity to TDP-43 proteinopathy during
              neurodegeneration.",
  journal  = "Nature",
  volume   =  588,
  number   =  7838,
  pages    = "459--465",
  month    =  aug,
  year     =  2020,
  language = "en"
}

Aberrant aggregation of the RNA-binding protein TDP-43 in neurons is a hallmark of frontotemporal lobar degeneration caused by haploinsufficiency in the gene encoding progranulin(1,2). However, the mechanism leading to TDP-43 proteinopathy remains unclear. Here we use single-nucleus RNA sequencing to show that progranulin deficiency promotes microglial transition from a homeostatic to a disease-specific state that causes endolysosomal dysfunction and neurodegeneration in mice. These defects persist even when Grn(-/-) microglia are cultured ex vivo. In addition, single-nucleus RNA sequencing reveals selective loss of excitatory neurons at disease end-stage, which is characterized by prominent nuclear and cytoplasmic TDP-43 granules and nuclear pore defects. Remarkably, conditioned media from Grn(-/-) microglia are sufficient to promote TDP-43 granule formation, nuclear pore defects and cell death in excitatory neurons via the complement activation pathway. Consistent with these results, deletion of the genes encoding C1qa and C3 mitigates microglial toxicity and rescues TDP-43 proteinopathy and neurodegeneration. These results uncover previously unappreciated contributions of chronic microglial toxicity to TDP-43 proteinopathy during neurodegeneration.

@ARTICLE{Parker2020-tr,
  title    = "{Single-Cell} Analyses Identify Brain Mural Cells Expressing
              {CD19} as Potential {Off-Tumor} Targets for {CAR-T}
              Immunotherapies",
  author   = "Parker, Kevin R and Migliorini, Denis and Perkey, Eric and Yost,
              Kathryn E and Bhaduri, Aparna and Bagga, Puneet and Haris,
              Mohammad and Wilson, Neil E and Liu, Fang and Gabunia, Khatuna
              and Scholler, John and Montine, Thomas J and Bhoj, Vijay G and
              Reddy, Ravinder and Mohan, Suyash and Maillard, Ivan and
              Kriegstein, Arnold R and June, Carl H and Chang, Howard Y and
              Posey, Jr, Avery D and Satpathy, Ansuman T",
  abstract = "CD19-directed immunotherapies are clinically effective for
              treating B cell malignancies but also cause a high incidence of
              neurotoxicity. A subset of patients treated with chimeric antigen
              receptor (CAR) T cells or bispecific T cell engager (BiTE)
              antibodies display severe neurotoxicity, including fatal cerebral
              edema associated with T cell infiltration into the brain. Here,
              we report that mural cells, which surround the endothelium and
              are critical for blood-brain-barrier integrity, express CD19. We
              identify CD19 expression in brain mural cells using single-cell
              RNA sequencing data and confirm perivascular staining at the
              protein level. CD19 expression in the brain begins early in
              development alongside the emergence of mural cell lineages and
              persists throughout adulthood across brain regions. Mouse mural
              cells demonstrate lower levels of Cd19 expression, suggesting
              limitations in preclinical animal models of neurotoxicity. These
              data suggest an on-target mechanism for neurotoxicity in
              CD19-directed therapies and highlight the utility of human
              single-cell atlases for designing immunotherapies.",
  journal  = "Cell",
  volume   =  183,
  number   =  1,
  pages    = "126--142.e17",
  month    =  sep,
  year     =  2020,
  language = "en"
}

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

@ARTICLE{Huang2020-zf,
  title    = "Origins and Proliferative States of Human Oligodendrocyte
              Precursor Cells",
  author   = "Huang, Wei and Bhaduri, Aparna and Velmeshev, Dmitry and Wang,
              Shaohui and Wang, Li and Rottkamp, Catherine A and
              Alvarez-Buylla, Arturo and Rowitch, David H and Kriegstein,
              Arnold R",
  abstract = "Human cerebral cortex size and complexity has increased greatly
              during evolution. While increased progenitor diversity and
              enhanced proliferative potential play important roles in human
              neurogenesis and gray matter expansion, the mechanisms of human
              oligodendrogenesis and white matter expansion remain largely
              unknown. Here, we identify EGFR-expressing ``Pre-OPCs'' that
              originate from outer radial glial cells (oRGs) and undergo
              mitotic somal translocation (MST) during division. oRG-derived
              Pre-OPCs provide an additional source of human cortical
              oligodendrocyte precursor cells (OPCs) and define a lineage
              trajectory. We further show that human OPCs undergo consecutive
              symmetric divisions to exponentially increase the progenitor pool
              size. Additionally, we find that the OPC-enriched gene, PCDH15,
              mediates daughter cell repulsion and facilitates proliferation.
              These findings indicate properties of OPC derivation,
              proliferation, and dispersion important for human white matter
              expansion and myelination.",
  journal  = "Cell",
  volume   =  182,
  number   =  3,
  pages    = "594--608.e11",
  month    =  jul,
  year     =  2020,
  keywords = "EGFR; OPC; PCDH15; cortical expansion; oligodendrogenesis;
              self-repulsion",
  language = "en"
}

Human cerebral cortex size and complexity has increased greatly during evolution. While increased progenitor diversity and enhanced proliferative potential play important roles in human neurogenesis and gray matter expansion, the mechanisms of human oligodendrogenesis and white matter expansion remain largely unknown. Here, we identify EGFR-expressing ``Pre-OPCs'' that originate from outer radial glial cells (oRGs) and undergo mitotic somal translocation (MST) during division. oRG-derived Pre-OPCs provide an additional source of human cortical oligodendrocyte precursor cells (OPCs) and define a lineage trajectory. We further show that human OPCs undergo consecutive symmetric divisions to exponentially increase the progenitor pool size. Additionally, we find that the OPC-enriched gene, PCDH15, mediates daughter cell repulsion and facilitates proliferation. These findings indicate properties of OPC derivation, proliferation, and dispersion important for human white matter expansion and myelination.

@ARTICLE{Bhaduri2020-as,
  title    = "Cell stress in cortical organoids impairs molecular subtype
              specification",
  author   = "Bhaduri, Aparna and Andrews, Madeline G and Mancia Leon, Walter
              and Jung, Diane and Shin, David and Allen, Denise and Jung, Dana
              and Schmunk, Galina and Haeussler, Maximilian and Salma, Jahan
              and Pollen, Alex A and Nowakowski, Tomasz J and Kriegstein,
              Arnold R",
  abstract = "Cortical organoids are self-organizing three-dimensional cultures
              that model features of the developing human cerebral cortex(1,2).
              However, the fidelity of organoid models remains unclear(3-5).
              Here we analyse the transcriptomes of individual primary human
              cortical cells from different developmental periods and cortical
              areas. We find that cortical development is characterized by
              progenitor maturation trajectories, the emergence of diverse cell
              subtypes and areal specification of newborn neurons. By contrast,
              organoids contain broad cell classes, but do not recapitulate
              distinct cellular subtype identities and appropriate progenitor
              maturation. Although the molecular signatures of cortical areas
              emerge in organoid neurons, they are not spatially segregated.
              Organoids also ectopically activate cellular stress pathways,
              which impairs cell-type specification. However, organoid stress
              and subtype defects are alleviated by transplantation into the
              mouse cortex. Together, these datasets and analytical tools
              provide a framework for evaluating and improving the accuracy of
              cortical organoids as models of human brain development.",
  journal  = "Nature",
  volume   =  578,
  number   =  7793,
  pages    = "142--148",
  month    =  jan,
  year     =  2020,
  language = "en"
}

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex(1,2). However, the fidelity of organoid models remains unclear(3-5). Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development.

@ARTICLE{Song2020-fz,
  title    = "Cell-type-specific {3D} epigenomes in the developing human cortex",
  author   = "Song, Michael and Pebworth, Mark-Phillip and Yang, Xiaoyu and
              Abnousi, Armen and Fan, Changxu and Wen, Jia and Rosen, Jonathan
              D and Choudhary, Mayank N K and Cui, Xiekui and Jones, Ian R and
              Bergenholtz, Seth and Eze, Ugomma C and Juric, Ivan and Li,
              Bingkun and Maliskova, Lenka and Lee, Jerry and Liu, Weifang and
              Pollen, Alex A and Li, Yun and Wang, Ting and Hu, Ming and
              Kriegstein, Arnold R and Shen, Yin",
  abstract = "Lineage-specific epigenomic changes during human corticogenesis
              have been difficult to study owing to challenges with sample
              availability and tissue heterogeneity. For example, previous
              studies using single-cell RNA sequencing identified at least 9
              major cell types and up to 26 distinct subtypes in the dorsal
              cortex alone(1,2). Here we characterize cell-type-specific
              cis-regulatory chromatin interactions, open chromatin peaks, and
              transcriptomes for radial glia, intermediate progenitor cells,
              excitatory neurons, and interneurons isolated from
              mid-gestational samples of the human cortex. We show that
              chromatin interactions underlie several aspects of gene
              regulation, with transposable elements and disease-associated
              variants enriched at distal interacting regions in a
              cell-type-specific manner. In addition, promoters with increased
              levels of chromatin interactivity-termed super-interactive
              promoters-are enriched for lineage-specific genes, suggesting
              that interactions at these loci contribute to the fine-tuning of
              transcription. Finally, we develop CRISPRview, a technique that
              integrates immunostaining, CRISPR interference, RNAscope, and
              image analysis to validate cell-type-specific cis-regulatory
              elements in heterogeneous populations of primary cells. Our
              findings provide insights into cell-type-specific gene expression
              patterns in the developing human cortex and advance our
              understanding of gene regulation and lineage specification during
              this crucial developmental window.",
  journal  = "Nature",
  volume   =  587,
  number   =  7835,
  pages    = "644--649",
  month    =  oct,
  year     =  2020,
  language = "en"
}

Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone(1,2). Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.

@ARTICLE{Bhaduri2020-pa,
  title    = "Outer Radial Glia-like Cancer Stem Cells Contribute to
              Heterogeneity of Glioblastoma",
  author   = "Bhaduri, Aparna and Di Lullo, Elizabeth and Jung, Diane and
              M{\"u}ller, S{\"o}ren and Crouch, Elizabeth Erin and Espinosa,
              Carmen Sandoval and Ozawa, Tomoko and Alvarado, Beatriz and
              Spatazza, Julien and Cadwell, Cathryn Ren{\'e} and Wilkins, Grace
              and Velmeshev, Dmitry and Liu, Siyuan John and Malatesta, Martina
              and Andrews, Madeline Gail and Mostajo-Radji, Mohammed Andres and
              Huang, Eric Jinsheng and Nowakowski, Tomasz Jan and Lim, Daniel
              Amos and Diaz, Aaron and Raleigh, David Ronan and Kriegstein,
              Arnold Richard",
  abstract = "Glioblastoma is a devastating form of brain cancer. To identify
              aspects of tumor heterogeneity that may illuminate drivers of
              tumor invasion, we created a glioblastoma tumor cell atlas with
              single-cell transcriptomics of cancer cells mapped onto a
              reference framework of the developing and adult human brain. We
              find that multiple GSC subtypes exist within a single tumor.
              Within these GSCs, we identify an invasive cell population
              similar to outer radial glia (oRG), a fetal cell type that
              expands the stem cell niche in normal human cortex. Using live
              time-lapse imaging of primary resected tumors, we discover that
              tumor-derived oRG-like cells undergo characteristic mitotic somal
              translocation behavior previously only observed in human
              development, suggesting a reactivation of developmental programs.
              In addition, we show that PTPRZ1 mediates both mitotic somal
              translocation and glioblastoma tumor invasion. These data suggest
              that the presence of heterogeneous GSCs may underlie
              glioblastoma's rapid progression and invasion.",
  journal  = "Cell Stem Cell",
  volume   =  26,
  number   =  1,
  pages    = "48--63.e6",
  month    =  jan,
  year     =  2020,
  keywords = "cancer stem cell; glioblastoma; outer radial glia; single-cell
              sequencing; tumor heterogeneity",
  language = "en"
}

Glioblastoma is a devastating form of brain cancer. To identify aspects of tumor heterogeneity that may illuminate drivers of tumor invasion, we created a glioblastoma tumor cell atlas with single-cell transcriptomics of cancer cells mapped onto a reference framework of the developing and adult human brain. We find that multiple GSC subtypes exist within a single tumor. Within these GSCs, we identify an invasive cell population similar to outer radial glia (oRG), a fetal cell type that expands the stem cell niche in normal human cortex. Using live time-lapse imaging of primary resected tumors, we discover that tumor-derived oRG-like cells undergo characteristic mitotic somal translocation behavior previously only observed in human development, suggesting a reactivation of developmental programs. In addition, we show that PTPRZ1 mediates both mitotic somal translocation and glioblastoma tumor invasion. These data suggest that the presence of heterogeneous GSCs may underlie glioblastoma's rapid progression and invasion.

@ARTICLE{Markenscoff-Papadimitriou2020-bc,
  title    = "A Chromatin Accessibility Atlas of the Developing Human
              Telencephalon",
  author   = "Markenscoff-Papadimitriou, Eirene and Whalen, Sean and Przytycki,
              Pawel and Thomas, Reuben and Binyameen, Fadya and Nowakowski,
              Tomasz J and Kriegstein, Arnold R and Sanders, Stephan J and
              {State, Matthew W} and Pollard, Katherine S and Rubenstein, John
              L",
  abstract = "To discover regulatory elements driving the specificity of gene
              expression in different cell types and regions of the developing
              human brain, we generated an atlas of open chromatin from nine
              dissected regions of the mid-gestation human telencephalon, as
              well as microdissected upper and deep layers of the prefrontal
              cortex. We identified a subset of open chromatin regions (OCRs),
              termed predicted regulatory elements (pREs), that are likely to
              function as developmental brain enhancers. pREs showed temporal,
              regional, and laminar differences in chromatin accessibility and
              were correlated with gene expression differences across regions
              and gestational ages. We identified two functional de novo
              variants in a pRE for autism risk gene SLC6A1, and using CRISPRa,
              demonstrated that this pRE regulates SCL6A1. Additionally, mouse
              transgenic experiments validated enhancer activity for pREs
              proximal to FEZF2 and BCL11A. Thus, this atlas serves as a
              resource for decoding neurodevelopmental gene regulation in
              health and disease.",
  journal  = "Cell",
  volume   =  182,
  number   =  3,
  pages    = "754--769.e18",
  month    =  jun,
  year     =  2020,
  keywords = "ATAC-seq; autism; chromatin; enhancers; gene regulation; machine
              learning; neurodevelopment; neuropsychiatric disorders",
  language = "en"
}

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.

@ARTICLE{Bhaduri2020-fo,
  title    = "Are Organoids Ready for Prime Time?",
  author   = "Bhaduri, Aparna and Andrews, Madeline G and Kriegstein, Arnold R
              and Nowakowski, Tomasz J",
  abstract = "Innovations in organoid-based models of human tissues have made
              them an exciting experimental platform for studying development
              and disease. However, these models require systematic
              benchmarking against primary tissue to establish their value. We
              discuss key parameters that impact the utility of organoid
              models, primarily focusing on cerebral organoids as examples.",
  journal  = "Cell Stem Cell",
  volume   =  27,
  number   =  3,
  pages    = "361--365",
  month    =  sep,
  year     =  2020,
  language = "en"
}

Innovations in organoid-based models of human tissues have made them an exciting experimental platform for studying development and disease. However, these models require systematic benchmarking against primary tissue to establish their value. We discuss key parameters that impact the utility of organoid models, primarily focusing on cerebral organoids as examples.

@ARTICLE{Andrews2020-nm,
  title    = "{mTOR} signaling regulates the morphology and migration of outer
              radial glia in developing human cortex",
  author   = "Andrews, Madeline G and Subramanian, Lakshmi and Kriegstein,
              Arnold R",
  abstract = "Outer radial glial (oRG) cells are a population of neural stem
              cells prevalent in the developing human cortex that contribute to
              its cellular diversity and evolutionary expansion. The mammalian
              Target of Rapamycin (mTOR) signaling pathway is active in human
              oRG cells. Mutations in mTOR pathway genes are linked to a
              variety of neurodevelopmental disorders and malformations of
              cortical development. We find that dysregulation of mTOR
              signaling specifically affects oRG cells, but not other
              progenitor types, by changing the actin cytoskeleton through the
              activity of the Rho-GTPase, CDC42. These effects change oRG
              cellular morphology, migration, and mitotic behavior, but do not
              affect proliferation or cell fate. Thus, mTOR signaling can
              regulate the architecture of the developing human cortex by
              maintaining the cytoskeletal organization of oRG cells and the
              radial glia scaffold. Our study provides insight into how mTOR
              dysregulation may contribute to neurodevelopmental disease.",
  journal  = "Elife",
  volume   =  9,
  month    =  sep,
  year     =  2020,
  keywords = "human; human cortex; neuroscience; organoids; outer radial glia;
              regenerative medicine; stem cells",
  language = "en"
}

Outer radial glial (oRG) cells are a population of neural stem cells prevalent in the developing human cortex that contribute to its cellular diversity and evolutionary expansion. The mammalian Target of Rapamycin (mTOR) signaling pathway is active in human oRG cells. Mutations in mTOR pathway genes are linked to a variety of neurodevelopmental disorders and malformations of cortical development. We find that dysregulation of mTOR signaling specifically affects oRG cells, but not other progenitor types, by changing the actin cytoskeleton through the activity of the Rho-GTPase, CDC42. These effects change oRG cellular morphology, migration, and mitotic behavior, but do not affect proliferation or cell fate. Thus, mTOR signaling can regulate the architecture of the developing human cortex by maintaining the cytoskeletal organization of oRG cells and the radial glia scaffold. Our study provides insight into how mTOR dysregulation may contribute to neurodevelopmental disease.

@ARTICLE{Zhang2020-ie,
  title    = "Cortical Neural Stem Cell Lineage Progression Is Regulated by
              Extrinsic Signaling Molecule Sonic Hedgehog",
  author   = "Zhang, Yue and Liu, Guoping and Guo, Teng and Liang, Xiaoyi G and
              Du, Heng and Yang, Lin and Bhaduri, Aparna and Li, Xiaosu and Xu,
              Zhejun and Zhang, Zhuangzhi and Li, Zhenmeiyu and He, Miao and
              Tsyporin, Jeremiah and Kriegstein, Arnold R and Rubenstein, John
              L and Yang, Zhengang and Chen, Bin",
  abstract = "Neural stem cells (NSCs) in the prenatal neocortex progressively
              generate different subtypes of glutamatergic projection neurons.
              Following that, NSCs have a major switch in their progenitor
              properties and produce $\gamma$-aminobutyric acid (GABAergic)
              interneurons for the olfactory bulb (OB), cortical
              oligodendrocytes, and astrocytes. Herein, we provide evidence for
              the molecular mechanism that underlies this switch in the state
              of neocortical NSCs. We show that, at around E16.5, mouse
              neocortical NSCs start to generate GSX2-expressing (GSX2(+))
              intermediate progenitor cells (IPCs). In vivo lineage-tracing
              study revealed that GSX2(+) IPC population gives rise not only to
              OB interneurons but also to cortical oligodendrocytes and
              astrocytes, suggesting that they are a tri-potential population.
              We demonstrated that Sonic hedgehog signaling is both necessary
              and sufficient for the generation of GSX2(+) IPCs by reducing
              GLI3R protein levels. Using single-cell RNA sequencing, we
              identify the transcriptional profile of GSX2(+) IPCs and the
              process of the lineage switch of cortical NSCs.",
  journal  = "Cell Rep",
  volume   =  30,
  number   =  13,
  pages    = "4490--4504.e4",
  month    =  mar,
  year     =  2020,
  keywords = "Gli3; Gsx2; Shh; cerebral cortex; neural stem cells; olfactory
              bulb interneurons; oligodendrocytes",
  language = "en"
}

Neural stem cells (NSCs) in the prenatal neocortex progressively generate different subtypes of glutamatergic projection neurons. Following that, NSCs have a major switch in their progenitor properties and produce $γ$-aminobutyric acid (GABAergic) interneurons for the olfactory bulb (OB), cortical oligodendrocytes, and astrocytes. Herein, we provide evidence for the molecular mechanism that underlies this switch in the state of neocortical NSCs. We show that, at around E16.5, mouse neocortical NSCs start to generate GSX2-expressing (GSX2(+)) intermediate progenitor cells (IPCs). In vivo lineage-tracing study revealed that GSX2(+) IPC population gives rise not only to OB interneurons but also to cortical oligodendrocytes and astrocytes, suggesting that they are a tri-potential population. We demonstrated that Sonic hedgehog signaling is both necessary and sufficient for the generation of GSX2(+) IPCs by reducing GLI3R protein levels. Using single-cell RNA sequencing, we identify the transcriptional profile of GSX2(+) IPCs and the process of the lineage switch of cortical NSCs.

@ARTICLE{Yang2020-ft,
  title    = "{SMART-Q}: An Integrative Pipeline Quantifying Cell
              {Type-Specific} {RNA} Transcription",
  author   = "Yang, Xiaoyu and Bergenholtz, Seth and Maliskova, Lenka and
              Pebworth, Mark-Phillip and Kriegstein, Arnold R and Li, Yun and
              Shen, Yin",
  abstract = "Accurate RNA quantification at the single-cell level is critical
              for understanding the dynamics of gene expression and regulation
              across space and time. Single molecule FISH (smFISH), such as
              RNAscope, provides spatial and quantitative measurements of
              individual transcripts, therefore, can be used to explore
              differential gene expression among a heterogeneous cell
              population if combined with cell identify information. However,
              such analysis is not straightforward, and existing image analysis
              pipelines cannot integrate both RNA transcripts and cellular
              staining information to automatically output cell type-specific
              gene expression. We developed an efficient and customizable
              analysis method, Single-Molecule Automatic RNA Transcription
              Quantification (SMART-Q), to enable the analysis of gene
              transcripts in a cell type-specific manner. SMART-Q efficiently
              infers cell identity information from multiplexed immuno-staining
              and quantifies cell type-specific transcripts using a 3D Gaussian
              fitting algorithm. Furthermore, we have optimized SMART-Q for
              user experiences, such as flexible parameters specification,
              batch data outputs, and visualization of analysis results.
              SMART-Q meets the demands for efficient quantification of
              single-molecule RNA and can be widely used for cell type-specific
              RNA transcript analysis.",
  journal  = "PLoS One",
  volume   =  15,
  number   =  4,
  pages    = "e0228760",
  month    =  apr,
  year     =  2020,
  language = "en"
}

Accurate RNA quantification at the single-cell level is critical for understanding the dynamics of gene expression and regulation across space and time. Single molecule FISH (smFISH), such as RNAscope, provides spatial and quantitative measurements of individual transcripts, therefore, can be used to explore differential gene expression among a heterogeneous cell population if combined with cell identify information. However, such analysis is not straightforward, and existing image analysis pipelines cannot integrate both RNA transcripts and cellular staining information to automatically output cell type-specific gene expression. We developed an efficient and customizable analysis method, Single-Molecule Automatic RNA Transcription Quantification (SMART-Q), to enable the analysis of gene transcripts in a cell type-specific manner. SMART-Q efficiently infers cell identity information from multiplexed immuno-staining and quantifies cell type-specific transcripts using a 3D Gaussian fitting algorithm. Furthermore, we have optimized SMART-Q for user experiences, such as flexible parameters specification, batch data outputs, and visualization of analysis results. SMART-Q meets the demands for efficient quantification of single-molecule RNA and can be widely used for cell type-specific RNA transcript analysis.

@ARTICLE{Herrero2020-vo,
  title    = "Identification of amygdala-expressed genes associated with autism
              spectrum disorder",
  author   = "Herrero, Maria Jesus and Velmeshev, Dmitry and Hernandez-Pineda,
              David and Sethi, Saarthak and Sorrells, Shawn and Banerjee, Payal
              and Sullivan, Catherine and Gupta, Abha R and Kriegstein, Arnold
              R and Corbin, Joshua G",
  abstract = "BACKGROUND: Studies of individuals with autism spectrum disorder
              (ASD) have revealed a strong multigenic basis with the
              identification of hundreds of ASD susceptibility genes. ASD is
              characterized by social deficits and a range of other phenotypes,
              implicating complex genetics and involvement of a variety of
              brain regions. However, how mutations and mis-expression of
              select gene sets are associated with the behavioral components of
              ASD remains unknown. We reasoned that for genes to be associated
              with ASD core behaviors they must be: (1) expressed in brain
              regions relevant to ASD social behaviors and (2) expressed during
              the ASD susceptible window of brain development. METHODS:
              Focusing on the amygdala, a brain region whose dysfunction has
              been highly implicated in the social component of ASD, we mined
              publicly available gene expression databases to identify
              ASD-susceptibility genes expressed during human and mouse
              amygdala development. We found that a large cohort of known ASD
              susceptibility genes is expressed in the developing human and
              mouse amygdala. We further performed analysis of single-nucleus
              RNA-seq (snRNA-seq) data from microdissected amygdala tissue from
              five ASD and five control human postmortem brains ranging in age
              from 4 to 20 years to elucidate cell type specificity of
              amygdala-expressed genes and their dysregulation in ASD. RESULTS:
              Our analyses revealed that of the high-ranking ASD susceptibility
              genes, 80 are expressed in both human and mouse amygdala during
              fetal to early postnatal stages of development. Our human
              snRNA-seq analyses revealed cohorts of genes with altered
              expression in the ASD amygdala postnatally, especially within
              excitatory neurons, with dysregulated expression of seven genes
              predicted from our datamining pipeline. LIMITATIONS: We were
              limited by the ages for which we were able to obtain human
              tissue; therefore, the results from our datamining pipeline
              approach will require validation, to the extent possible, in
              human tissue from earlier developmental stages. CONCLUSIONS: Our
              pipeline narrows down the number of amygdala-expressed genes
              possibly involved in the social pathophysiology of ASD. Our human
              single-nucleus gene expression analyses revealed that ASD is
              characterized by changes in gene expression in specific cell
              types in the early postnatal amygdala.",
  journal  = "Mol Autism",
  volume   =  11,
  number   =  1,
  pages    = "39",
  month    =  may,
  year     =  2020,
  keywords = "ASD genes; Amygdala; Autism spectrum disorder; Brain development;
              Single nucleus RNA sequencing",
  language = "en"
}

BACKGROUND: Studies of individuals with autism spectrum disorder (ASD) have revealed a strong multigenic basis with the identification of hundreds of ASD susceptibility genes. ASD is characterized by social deficits and a range of other phenotypes, implicating complex genetics and involvement of a variety of brain regions. However, how mutations and mis-expression of select gene sets are associated with the behavioral components of ASD remains unknown. We reasoned that for genes to be associated with ASD core behaviors they must be: (1) expressed in brain regions relevant to ASD social behaviors and (2) expressed during the ASD susceptible window of brain development. METHODS: Focusing on the amygdala, a brain region whose dysfunction has been highly implicated in the social component of ASD, we mined publicly available gene expression databases to identify ASD-susceptibility genes expressed during human and mouse amygdala development. We found that a large cohort of known ASD susceptibility genes is expressed in the developing human and mouse amygdala. We further performed analysis of single-nucleus RNA-seq (snRNA-seq) data from microdissected amygdala tissue from five ASD and five control human postmortem brains ranging in age from 4 to 20 years to elucidate cell type specificity of amygdala-expressed genes and their dysregulation in ASD. RESULTS: Our analyses revealed that of the high-ranking ASD susceptibility genes, 80 are expressed in both human and mouse amygdala during fetal to early postnatal stages of development. Our human snRNA-seq analyses revealed cohorts of genes with altered expression in the ASD amygdala postnatally, especially within excitatory neurons, with dysregulated expression of seven genes predicted from our datamining pipeline. LIMITATIONS: We were limited by the ages for which we were able to obtain human tissue; therefore, the results from our datamining pipeline approach will require validation, to the extent possible, in human tissue from earlier developmental stages. CONCLUSIONS: Our pipeline narrows down the number of amygdala-expressed genes possibly involved in the social pathophysiology of ASD. Our human single-nucleus gene expression analyses revealed that ASD is characterized by changes in gene expression in specific cell types in the early postnatal amygdala.

@ARTICLE{Velmeshev2019-uk,
  title    = "Single-cell genomics identifies cell type-specific molecular
              changes in autism",
  author   = "Velmeshev, Dmitry and Schirmer, Lucas and Jung, Diane and
              Haeussler, Maximilian and Perez, Yonatan and Mayer, Simone and
              Bhaduri, Aparna and Goyal, Nitasha and Rowitch, David H and
              Kriegstein, Arnold R",
  abstract = "Despite the clinical and genetic heterogeneity of autism, bulk
              gene expression studies show that changes in the neocortex of
              autism patients converge on common genes and pathways. However,
              direct assessment of specific cell types in the brain affected by
              autism has not been feasible until recently. We used
              single-nucleus RNA sequencing of cortical tissue from patients
              with autism to identify autism-associated transcriptomic changes
              in specific cell types. We found that synaptic signaling of
              upper-layer excitatory neurons and the molecular state of
              microglia are preferentially affected in autism. Moreover, our
              results show that dysregulation of specific groups of genes in
              cortico-cortical projection neurons correlates with clinical
              severity of autism. These findings suggest that molecular changes
              in upper-layer cortical circuits are linked to behavioral
              manifestations of autism.",
  journal  = "Science",
  volume   =  364,
  number   =  6441,
  pages    = "685--689",
  month    =  may,
  year     =  2019,
  language = "en"
}

Despite the clinical and genetic heterogeneity of autism, bulk gene expression studies show that changes in the neocortex of autism patients converge on common genes and pathways. However, direct assessment of specific cell types in the brain affected by autism has not been feasible until recently. We used single-nucleus RNA sequencing of cortical tissue from patients with autism to identify autism-associated transcriptomic changes in specific cell types. We found that synaptic signaling of upper-layer excitatory neurons and the molecular state of microglia are preferentially affected in autism. Moreover, our results show that dysregulation of specific groups of genes in cortico-cortical projection neurons correlates with clinical severity of autism. These findings suggest that molecular changes in upper-layer cortical circuits are linked to behavioral manifestations of autism.

@ARTICLE{Pollen2019-vm,
  title    = "Establishing Cerebral Organoids as Models of {Human-Specific}
              Brain Evolution",
  author   = "Pollen, Alex A and Bhaduri, Aparna and Andrews, Madeline G and
              Nowakowski, Tomasz J and Meyerson, Olivia S and Mostajo-Radji,
              Mohammed A and Di Lullo, Elizabeth and Alvarado, Beatriz and
              Bedolli, Melanie and Dougherty, Max L and Fiddes, Ian T and
              Kronenberg, Zev N and Shuga, Joe and Leyrat, Anne A and West, Jay
              A and Bershteyn, Marina and Lowe, Craig B and Pavlovic, Bryan J
              and Salama, Sofie R and Haussler, David and Eichler, Evan E and
              Kriegstein, Arnold R",
  abstract = "Direct comparisons of human and non-human primate brains can
              reveal molecular pathways underlying remarkable specializations
              of the human brain. However, chimpanzee tissue is inaccessible
              during neocortical neurogenesis when differences in brain size
              first appear. To identify human-specific features of cortical
              development, we leveraged recent innovations that permit
              generating pluripotent stem cell-derived cerebral organoids from
              chimpanzee. Despite metabolic differences, organoid models
              preserve gene regulatory networks related to primary cell types
              and developmental processes. We further identified 261
              differentially expressed genes in human compared to both
              chimpanzee organoids and macaque cortex, enriched for recent gene
              duplications, and including multiple regulators of PI3K-AKT-mTOR
              signaling. We observed increased activation of this pathway in
              human radial glia, dependent on two receptors upregulated
              specifically in human: INSR and ITGB8. Our findings establish a
              platform for systematic analysis of molecular changes
              contributing to human brain development and evolution.",
  journal  = "Cell",
  volume   =  176,
  number   =  4,
  pages    = "743--756.e17",
  month    =  feb,
  year     =  2019,
  keywords = "cerebral organoids; chimpanzee; cortical development;
              human-specific evolution; mTOR; macaque; neural progenitor cells;
              radial glia; single-cell RNA sequencing",
  language = "en"
}

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

@ARTICLE{Schirmer2019-jc,
  title    = "Neuronal vulnerability and multilineage diversity in multiple
              sclerosis",
  author   = "Schirmer, Lucas and Velmeshev, Dmitry and Holmqvist, Staffan and
              Kaufmann, Max and Werneburg, Sebastian and Jung, Diane and
              Vistnes, Stephanie and Stockley, John H and Young, Adam and
              Steindel, Maike and Tung, Brian and Goyal, Nitasha and Bhaduri,
              Aparna and Mayer, Simone and Engler, Jan Broder and Bayraktar,
              Omer A and Franklin, Robin J M and Haeussler, Maximilian and
              Reynolds, Richard and Schafer, Dorothy P and Friese, Manuel A and
              Shiow, Lawrence R and Kriegstein, Arnold R and Rowitch, David H",
  abstract = "Multiple sclerosis (MS) is a neuroinflammatory disease with a
              relapsing-remitting disease course at early stages, distinct
              lesion characteristics in cortical grey versus subcortical white
              matter and neurodegeneration at chronic stages. Here we used
              single-nucleus RNA sequencing to assess changes in expression in
              multiple cell lineages in MS lesions and validated the results
              using multiplex in situ hybridization. We found selective
              vulnerability and loss of excitatory CUX2-expressing projection
              neurons in upper-cortical layers underlying meningeal
              inflammation; such MS neuron populations exhibited upregulation
              of stress pathway genes and long non-coding RNAs. Signatures of
              stressed oligodendrocytes, reactive astrocytes and activated
              microglia mapped most strongly to the rim of MS plaques. Notably,
              single-nucleus RNA sequencing identified phagocytosing microglia
              and/or macrophages by their ingestion and perinuclear import of
              myelin transcripts, confirmed by functional mouse and human
              culture assays. Our findings indicate lineage- and
              region-specific transcriptomic changes associated with selective
              cortical neuron damage and glial activation contributing to
              progression of MS lesions.",
  journal  = "Nature",
  volume   =  573,
  number   =  7772,
  pages    = "75--82",
  month    =  jul,
  year     =  2019,
  language = "en"
}

Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing-remitting disease course at early stages, distinct lesion characteristics in cortical grey versus subcortical white matter and neurodegeneration at chronic stages. Here we used single-nucleus RNA sequencing to assess changes in expression in multiple cell lineages in MS lesions and validated the results using multiplex in situ hybridization. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper-cortical layers underlying meningeal inflammation; such MS neuron populations exhibited upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated microglia mapped most strongly to the rim of MS plaques. Notably, single-nucleus RNA sequencing identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to progression of MS lesions.

@ARTICLE{Nobuta2019-ss,
  title    = "Oligodendrocyte Death in {Pelizaeus-Merzbacher} Disease Is
              Rescued by Iron Chelation",
  author   = "Nobuta, Hiroko and Yang, Nan and Ng, Yi Han and Marro, Samuele G
              and Sabeur, Khalida and Chavali, Manideep and Stockley, John H
              and Killilea, David W and Walter, Patrick B and Zhao, Chao and
              Huie, Jr, Philip and Goldman, Steven A and Kriegstein, Arnold R
              and Franklin, Robin J M and Rowitch, David H and Wernig, Marius",
  abstract = "Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy
              caused by mutations in Proteolipid Protein 1 (PLP1), encoding a
              major myelin protein, resulting in profound developmental delay
              and early lethality. Previous work showed involvement of unfolded
              protein response (UPR) and endoplasmic reticulum (ER) stress
              pathways, but poor PLP1 genotype-phenotype associations suggest
              additional pathogenetic mechanisms. Using induced pluripotent
              stem cell (iPSC) and gene-correction, we show that
              patient-derived oligodendrocytes can develop to the
              pre-myelinating stage, but subsequently undergo cell death.
              Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis
              including lipid peroxidation, abnormal iron metabolism, and
              hypersensitivity to free iron. Iron chelation rescued mutant
              oligodendrocyte apoptosis, survival, and differentiationin vitro,
              and post-transplantation in vivo. Finally, systemic treatment of
              Plp1 mutant Jimpy mice with deferiprone, a small molecule iron
              chelator, reduced oligodendrocyte apoptosis and enabled myelin
              formation. Thus, oligodendrocyte iron-induced cell death and
              myelination is rescued by iron chelation in PMD pre-clinical
              models.",
  journal  = "Cell Stem Cell",
  volume   =  25,
  number   =  4,
  pages    = "531--541.e6",
  month    =  oct,
  year     =  2019,
  keywords = "ferroptosis; gene correction; induced pluripotent stem cells;
              iron chelation; leukodystrophy; myelination; oligodendrocyte;
              patient models",
  language = "en"
}

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

@ARTICLE{Sorrells2019-ml,
  title    = "Immature excitatory neurons develop during adolescence in the
              human amygdala",
  author   = "Sorrells, Shawn F and Paredes, Mercedes F and Velmeshev, Dmitry
              and Herranz-P{\'e}rez, Vicente and Sandoval, Kadellyn and Mayer,
              Simone and Chang, Edward F and Insausti, Ricardo and Kriegstein,
              Arnold R and Rubenstein, John L and Manuel Garcia-Verdugo, Jose
              and Huang, Eric J and Alvarez-Buylla, Arturo",
  abstract = "The human amygdala grows during childhood, and its abnormal
              development is linked to mood disorders. The primate amygdala
              contains a large population of immature neurons in the
              paralaminar nuclei (PL), suggesting protracted development and
              possibly neurogenesis. Here we studied human PL development from
              embryonic stages to adulthood. The PL develops next to the caudal
              ganglionic eminence, which generates inhibitory interneurons, yet
              most PL neurons express excitatory markers. In children, most PL
              cells are immature (DCX+PSA-NCAM+), and during adolescence many
              transition into mature (TBR1+VGLUT2+) neurons. Immature PL
              neurons persist into old age, yet local progenitor proliferation
              sharply decreases in infants. Using single nuclei RNA sequencing,
              we identify the transcriptional profile of immature excitatory
              neurons in the human amygdala between 4-15 years. We conclude
              that the human PL contains excitatory neurons that remain
              immature for decades, a possible substrate for persistent
              plasticity at the interface of the hippocampus and amygdala.",
  journal  = "Nat Commun",
  volume   =  10,
  number   =  1,
  pages    = "2748",
  month    =  jun,
  year     =  2019,
  language = "en"
}

The human amygdala grows during childhood, and its abnormal development is linked to mood disorders. The primate amygdala contains a large population of immature neurons in the paralaminar nuclei (PL), suggesting protracted development and possibly neurogenesis. Here we studied human PL development from embryonic stages to adulthood. The PL develops next to the caudal ganglionic eminence, which generates inhibitory interneurons, yet most PL neurons express excitatory markers. In children, most PL cells are immature (DCX+PSA-NCAM+), and during adolescence many transition into mature (TBR1+VGLUT2+) neurons. Immature PL neurons persist into old age, yet local progenitor proliferation sharply decreases in infants. Using single nuclei RNA sequencing, we identify the transcriptional profile of immature excitatory neurons in the human amygdala between 4-15 years. We conclude that the human PL contains excitatory neurons that remain immature for decades, a possible substrate for persistent plasticity at the interface of the hippocampus and amygdala.

@ARTICLE{Mayer2019-tl,
  title    = "Multimodal {Single-Cell} Analysis Reveals Physiological
              Maturation in the Developing Human Neocortex",
  author   = "Mayer, Simone and Chen, Jiadong and Velmeshev, Dmitry and Mayer,
              Andreas and Eze, Ugomma C and Bhaduri, Aparna and Cunha, Carlos E
              and Jung, Diane and Arjun, Arpana and Li, Emmy and Alvarado,
              Beatriz and Wang, Shaohui and Lovegren, Nils and Gonzales,
              Michael L and Szpankowski, Lukasz and Leyrat, Anne and West, Jay
              A A and Panagiotakos, Georgia and Alvarez-Buylla, Arturo and
              Paredes, Mercedes F and Nowakowski, Tomasz J and Pollen, Alex A
              and Kriegstein, Arnold R",
  abstract = "In the developing human neocortex, progenitor cells generate
              diverse cell types prenatally. Progenitor cells and newborn
              neurons respond to signaling cues, including neurotransmitters.
              While single-cell RNA sequencing has revealed cellular diversity,
              physiological heterogeneity has yet to be mapped onto these
              developing and diverse cell types. By combining measurements of
              intracellular Ca(2+) elevations in response to neurotransmitter
              receptor agonists and RNA sequencing of the same single cells, we
              show that Ca(2+) responses are cell-type-specific and change
              dynamically with lineage progression. Physiological response
              properties predict molecular cell identity and additionally
              reveal diversity not captured by single-cell transcriptomics. We
              find that the serotonin receptor HTR2A selectively activates
              radial glia cells in the developing human, but not mouse,
              neocortex, and inhibiting HTR2A receptors in human radial glia
              disrupts the radial glial scaffold. We show highly specific
              neurotransmitter signaling during neurogenesis in the developing
              human neocortex and highlight evolutionarily divergent mechanisms
              of physiological signaling.",
  journal  = "Neuron",
  volume   =  102,
  number   =  1,
  pages    = "143--158.e7",
  month    =  feb,
  year     =  2019,
  keywords = "calcium imaging; differentiation; human neocortical development;
              intermediate progenitor cells; neurogenesis; neurotransmitter;
              radial glia; radial glia scaffold; serotonin; single-cell RNA
              sequencing",
  language = "en"
}

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca(2+) elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca(2+) responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling.

@ARTICLE{Sloan2019-bo,
  title    = "Neuroglial stem cell-derived inflammatory pseudotumor
              ({n-SCIPT)}: clinicopathologic characterization of a novel lesion
              of the lumbosacral spinal cord and nerve roots following
              intrathecal allogeneic stem cell intervention",
  author   = "Sloan, Emily A and Sampognaro, Paul J and Junn, Jacqueline C and
              Chin, Cynthia and Jacques, Line and Ramachandran, Prashanth S and
              DeRisi, Joseph L and Wilson, Michael R and Kriegstein, Arnold R
              and Bollen, Andrew W and Solomon, David A and Margeta, Marta and
              Engstrom, John W",
  journal  = "Acta Neuropathol",
  volume   =  138,
  number   =  6,
  pages    = "1103--1106",
  month    =  oct,
  year     =  2019,
  language = "en"
}

@ARTICLE{Adorjan2019-rx,
  title    = "Neuroserpin expression during human brain development and in
              adult brain revealed by immunohistochemistry and single cell
              {RNA} sequencing",
  author   = "Adorjan, Istvan and Tyler, Teadora and Bhaduri, Aparna and
              Demharter, Samuel and Finszter, Cintia Klaudia and Bako, Maria
              and Sebok, Oliver Marcell and Nowakowski, Tomasz J and
              Khodosevich, Konstantin and M{\o}llg{\aa}rd, Kjeld and
              Kriegstein, Arnold R and Shi, Lei and Hoerder-Suabedissen, Anna
              and Ansorge, Olaf and Moln{\'a}r, Zolt{\'a}n",
  abstract = "Neuroserpin is a serine-protease inhibitor mainly expressed in
              the CNS and involved in the inhibition of the proteolytic
              cascade. Animal models confirmed its neuroprotective role in
              perinatal hypoxia-ischaemia and adult stroke. Although
              neuroserpin may be a potential therapeutic target in the
              treatment of the aforementioned conditions, there is still no
              information in the literature on its distribution during human
              brain development. The present study provides a detailed
              description of the changing spatiotemporal patterns of
              neuroserpin focusing on physiological human brain development.
              Five stages were distinguished within our examined age range
              which spanned from the 7th gestational week until adulthood. In
              particular, subplate and deep cortical plate neurons were
              identified as the main sources of neuroserpin production between
              the 25th gestational week and the first postnatal month. Our
              immunohistochemical findings were substantiated by single cell
              RNA sequencing data showing specific neuronal and glial cell
              types expressing neuroserpin. The characterization of neuroserpin
              expression during physiological human brain development is
              essential for forthcoming studies which will explore its
              involvement in pathological conditions, such as perinatal
              hypoxia-ischaemia and adult stroke in human.",
  journal  = "J Anat",
  volume   =  235,
  number   =  3,
  pages    = "543--554",
  month    =  jan,
  year     =  2019,
  keywords = "human brain; neurodevelopment; neuroserpin; subplate",
  language = "en"
}

Neuroserpin is a serine-protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia-ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia-ischaemia and adult stroke in human.

@ARTICLE{Sorrells2018-dx,
  title    = "Human hippocampal neurogenesis drops sharply in children to
              undetectable levels in adults",
  author   = "Sorrells, Shawn F and Paredes, Mercedes F and Cebrian-Silla,
              Arantxa and Sandoval, Kadellyn and Qi, Dashi and Kelley, Kevin W
              and James, David and Mayer, Simone and Chang, Julia and Auguste,
              Kurtis I and Chang, Edward F and Gutierrez, Antonio J and
              Kriegstein, Arnold R and Mathern, Gary W and Oldham, Michael C
              and Huang, Eric J and Garcia-Verdugo, Jose Manuel and Yang,
              Zhengang and Alvarez-Buylla, Arturo",
  abstract = "New neurons continue to be generated in the subgranular zone of
              the dentate gyrus of the adult mammalian hippocampus. This
              process has been linked to learning and memory, stress and
              exercise, and is thought to be altered in neurological disease.
              In humans, some studies have suggested that hundreds of new
              neurons are added to the adult dentate gyrus every day, whereas
              other studies find many fewer putative new neurons. Despite these
              discrepancies, it is generally believed that the adult human
              hippocampus continues to generate new neurons. Here we show that
              a defined population of progenitor cells does not coalesce in the
              subgranular zone during human fetal or postnatal development. We
              also find that the number of proliferating progenitors and young
              neurons in the dentate gyrus declines sharply during the first
              year of life and only a few isolated young neurons are observed
              by 7 and 13 years of age. In adult patients with epilepsy and
              healthy adults (18-77 years; n = 17 post-mortem samples from
              controls; n = 12 surgical resection samples from patients with
              epilepsy), young neurons were not detected in the dentate gyrus.
              In the monkey (Macaca mulatta) hippocampus, proliferation of
              neurons in the subgranular zone was found in early postnatal
              life, but this diminished during juvenile development as
              neurogenesis decreased. We conclude that recruitment of young
              neurons to the primate hippocampus decreases rapidly during the
              first years of life, and that neurogenesis in the dentate gyrus
              does not continue, or is extremely rare, in adult humans. The
              early decline in hippocampal neurogenesis raises questions about
              how the function of the dentate gyrus differs between humans and
              other species in which adult hippocampal neurogenesis is
              preserved.",
  journal  = "Nature",
  volume   =  555,
  number   =  7696,
  pages    = "377--381",
  month    =  mar,
  year     =  2018,
  language = "en"
}

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

@ARTICLE{Paredes2018-uu,
  title    = "Does Adult Neurogenesis Persist in the Human Hippocampus?",
  author   = "Paredes, Mercedes F and Sorrells, Shawn F and Cebrian-Silla,
              Arantxa and Sandoval, Kadellyn and Qi, Dashi and Kelley, Kevin W
              and James, David and Mayer, Simone and Chang, Julia and Auguste,
              Kurtis I and Chang, Edward F and Gutierrez Martin, Antonio J and
              Kriegstein, Arnold R and Mathern, Gary W and Oldham, Michael C
              and Huang, Eric J and Garcia-Verdugo, Jose Manuel and Yang,
              Zhengang and Alvarez-Buylla, Arturo",
  journal  = "Cell Stem Cell",
  volume   =  23,
  number   =  6,
  pages    = "780--781",
  month    =  dec,
  year     =  2018,
  language = "en"
}

@ARTICLE{Nowakowski2018-ma,
  title    = "Regulation of cell-type-specific transcriptomes by {microRNA}
              networks during human brain development",
  author   = "Nowakowski, Tomasz J and Rani, Neha and Golkaram, Mahdi and Zhou,
              Hongjun R and Alvarado, Beatriz and Huch, Kylie and West, Jay A
              and Leyrat, Anne and Pollen, Alex A and Kriegstein, Arnold R and
              Petzold, Linda R and Kosik, Kenneth S",
  abstract = "MicroRNAs (miRNAs) regulate many cellular events during brain
              development by interacting with hundreds of mRNA transcripts.
              However, miRNAs operate nonuniformly upon the transcriptional
              profile with an as yet unknown logic. Shortcomings in defining
              miRNA-mRNA networks include limited knowledge of in vivo miRNA
              targets and their abundance in single cells. By combining
              multiple complementary approaches, high-throughput sequencing of
              RNA isolated by cross-linking immunoprecipitation with an
              antibody to AGO2 (AGO2-HITS-CLIP), single-cell profiling and
              computational analyses using bipartite and coexpression networks,
              we show that miRNA-mRNA interactions operate as functional
              modules that often correspond to cell-type identities and undergo
              dynamic transitions during brain development. These networks are
              highly dynamic during development and over the course of
              evolution. One such interaction is between radial-glia-enriched
              ORC4 and miR-2115, a great-ape-specific miRNA, which appears to
              control radial glia proliferation rates during human brain
              development.",
  journal  = "Nat Neurosci",
  volume   =  21,
  number   =  12,
  pages    = "1784--1792",
  month    =  nov,
  year     =  2018,
  language = "en"
}

MicroRNAs (miRNAs) regulate many cellular events during brain development by interacting with hundreds of mRNA transcripts. However, miRNAs operate nonuniformly upon the transcriptional profile with an as yet unknown logic. Shortcomings in defining miRNA-mRNA networks include limited knowledge of in vivo miRNA targets and their abundance in single cells. By combining multiple complementary approaches, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation with an antibody to AGO2 (AGO2-HITS-CLIP), single-cell profiling and computational analyses using bipartite and coexpression networks, we show that miRNA-mRNA interactions operate as functional modules that often correspond to cell-type identities and undergo dynamic transitions during brain development. These networks are highly dynamic during development and over the course of evolution. One such interaction is between radial-glia-enriched ORC4 and miR-2115, a great-ape-specific miRNA, which appears to control radial glia proliferation rates during human brain development.

@ARTICLE{Werling2018-yd,
  title    = "An analytical framework for whole-genome sequence association
              studies and its implications for autism spectrum disorder",
  author   = "Werling, Donna M and Brand, Harrison and An, Joon-Yong and Stone,
              Matthew R and Zhu, Lingxue and Glessner, Joseph T and Collins,
              Ryan L and Dong, Shan and Layer, Ryan M and
              Markenscoff-Papadimitriou, Eirene and Farrell, Andrew and
              Schwartz, Grace B and Wang, Harold Z and Currall, Benjamin B and
              Zhao, Xuefang and Dea, Jeanselle and Duhn, Clif and Erdman,
              Carolyn A and Gilson, Michael C and Yadav, Rachita and Handsaker,
              Robert E and Kashin, Seva and Klei, Lambertus and Mandell,
              Jeffrey D and Nowakowski, Tomasz J and Liu, Yuwen and Pochareddy,
              Sirisha and Smith, Louw and Walker, Michael F and Waterman,
              Matthew J and He, Xin and Kriegstein, Arnold R and Rubenstein,
              John L and Sestan, Nenad and McCarroll, Steven A and Neale,
              Benjamin M and Coon, Hilary and Willsey, A Jeremy and Buxbaum,
              Jose and Daly, Mark J and {State, Matthew W} and Quinlan, Aaron R
              and Marth, Gabor T and Roeder, Kathryn and Devlin, Bernie and
              Talkowski, Michael E and Sanders, Stephan J",
  abstract = "Genomic association studies of common or rare protein-coding
              variation have established robust statistical approaches to
              account for multiple testing. Here we present a comparable
              framework to evaluate rare and de novo noncoding
              single-nucleotide variants, insertion/deletions, and all classes
              of structural variation from whole-genome sequencing (WGS).
              Integrating genomic annotations at the level of nucleotides,
              genes, and regulatory regions, we define 51,801 annotation
              categories. Analyses of 519 autism spectrum disorder families did
              not identify association with any categories after correction for
              4,123 effective tests. Without appropriate correction,
              biologically plausible associations are observed in both cases
              and controls. Despite excluding previously identified
              gene-disrupting mutations, coding regions still exhibited the
              strongest associations. Thus, in autism, the contribution of de
              novo noncoding variation is probably modest in comparison to that
              of de novo coding variants. Robust results from future WGS
              studies will require large cohorts and comprehensive analytical
              strategies that consider the substantial multiple-testing burden.",
  journal  = "Nat Genet",
  volume   =  50,
  number   =  5,
  pages    = "727--736",
  month    =  apr,
  year     =  2018,
  language = "en"
}

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.

@ARTICLE{Bhaduri2018-ew,
  title    = "Identification of cell types in a mouse brain single-cell atlas
              using low sampling coverage",
  author   = "Bhaduri, Aparna and Nowakowski, Tomasz J and Pollen, Alex A and
              Kriegstein, Arnold R",
  abstract = "BACKGROUND: High throughput methods for profiling the
              transcriptomes of single cells have recently emerged as
              transformative approaches for large-scale population surveys of
              cellular diversity in heterogeneous primary tissues. However, the
              efficient generation of such atlases will depend on sufficient
              sampling of diverse cell types while remaining cost-effective to
              enable a comprehensive examination of organs, developmental
              stages, and individuals. RESULTS: To examine the relationship
              between sampled cell numbers and transcriptional heterogeneity in
              the context of unbiased cell type classification, we explored the
              population structure of a publicly available 1.3 million cell
              dataset from E18.5 mouse brain and validated our findings in
              published data from adult mice. We propose a computational
              framework for inferring the saturation point of cluster discovery
              in a single-cell mRNA-seq experiment, centered around cluster
              preservation in downsampled datasets. In addition, we introduce a
              ``complexity index,'' which characterizes the heterogeneity of
              cells in a given dataset. Using Cajal-Retzius cells as an example
              of a limited complexity dataset, we explored whether the detected
              biological distinctions relate to technical clustering.
              Surprisingly, we found that clustering distinctions carrying
              biologically interpretable meaning are achieved with far fewer
              cells than the originally sampled, though technical saturation of
              rare populations such as Cajal-Retzius cells is not achieved. We
              additionally validated these findings with a recently published
              atlas of cell types across mouse organs and again find using
              subsampling that a much smaller number of cells recapitulates the
              cluster distinctions of the complete dataset. CONCLUSIONS:
              Together, these findings suggest that most of the biologically
              interpretable cell types from the 1.3 million cell database can
              be recapitulated by analyzing 50,000 randomly selected cells,
              indicating that instead of profiling few individuals at high
              ``cellular coverage,'' cell atlas studies may instead benefit
              from profiling more individuals, or many time points at lower
              cellular coverage and then further enriching for populations of
              interest. This strategy is ideal for scenarios where cost and
              time are limited, though extremely rare populations of interest
              (< 1\%) may be identifiable only with much higher cell numbers.",
  journal  = "BMC Biol",
  volume   =  16,
  number   =  1,
  pages    = "113",
  month    =  oct,
  year     =  2018,
  keywords = "Bioinformatics; Cell atlas studies; Downsampling; Single-cell
              analysis",
  language = "en"
}

BACKGROUND: High throughput methods for profiling the transcriptomes of single cells have recently emerged as transformative approaches for large-scale population surveys of cellular diversity in heterogeneous primary tissues. However, the efficient generation of such atlases will depend on sufficient sampling of diverse cell types while remaining cost-effective to enable a comprehensive examination of organs, developmental stages, and individuals. RESULTS: To examine the relationship between sampled cell numbers and transcriptional heterogeneity in the context of unbiased cell type classification, we explored the population structure of a publicly available 1.3 million cell dataset from E18.5 mouse brain and validated our findings in published data from adult mice. We propose a computational framework for inferring the saturation point of cluster discovery in a single-cell mRNA-seq experiment, centered around cluster preservation in downsampled datasets. In addition, we introduce a ``complexity index,'' which characterizes the heterogeneity of cells in a given dataset. Using Cajal-Retzius cells as an example of a limited complexity dataset, we explored whether the detected biological distinctions relate to technical clustering. Surprisingly, we found that clustering distinctions carrying biologically interpretable meaning are achieved with far fewer cells than the originally sampled, though technical saturation of rare populations such as Cajal-Retzius cells is not achieved. We additionally validated these findings with a recently published atlas of cell types across mouse organs and again find using subsampling that a much smaller number of cells recapitulates the cluster distinctions of the complete dataset. CONCLUSIONS: Together, these findings suggest that most of the biologically interpretable cell types from the 1.3 million cell database can be recapitulated by analyzing 50,000 randomly selected cells, indicating that instead of profiling few individuals at high ``cellular coverage,'' cell atlas studies may instead benefit from profiling more individuals, or many time points at lower cellular coverage and then further enriching for populations of interest. This strategy is ideal for scenarios where cost and time are limited, though extremely rare populations of interest (< 1%) may be identifiable only with much higher cell numbers.

@ARTICLE{Raju2018-db,
  title    = "Secretagogin is Expressed by Developing Neocortical {GABAergic}
              Neurons in Humans but not Mice and Increases Neurite Arbor Size
              and Complexity",
  author   = "Raju, Chandrasekhar S and Spatazza, Julien and Stanco, Amelia and
              Larimer, Phillip and Sorrells, Shawn F and Kelley, Kevin W and
              Nicholas, Cory R and Paredes, Mercedes F and Lui, Jan H and
              Hasenstaub, Andrea R and Kriegstein, Arnold R and Alvarez-Buylla,
              Arturo and Rubenstein, John L and Oldham, Michael C",
  abstract = "The neocortex of primates, including humans, contains more
              abundant and diverse inhibitory neurons compared with rodents,
              but the molecular foundations of these observations are unknown.
              Through integrative gene coexpression analysis, we determined a
              consensus transcriptional profile of GABAergic neurons in
              mid-gestation human neocortex. By comparing this profile to genes
              expressed in GABAergic neurons purified from neonatal mouse
              neocortex, we identified conserved and distinct aspects of gene
              expression in these cells between the species. We show here that
              the calcium-binding protein secretagogin (SCGN) is robustly
              expressed by neocortical GABAergic neurons derived from caudal
              ganglionic eminences (CGE) and lateral ganglionic eminences
              during human but not mouse brain development. Through
              electrophysiological and morphometric analyses, we examined the
              effects of SCGN expression on GABAergic neuron function and form.
              Forced expression of SCGN in CGE-derived mouse GABAergic neurons
              significantly increased total neurite length and arbor complexity
              following transplantation into mouse neocortex, revealing a
              molecular pathway that contributes to morphological differences
              in these cells between rodents and primates.",
  journal  = "Cereb Cortex",
  volume   =  28,
  number   =  6,
  pages    = "1946--1958",
  month    =  jun,
  year     =  2018,
  language = "en"
}

The neocortex of primates, including humans, contains more abundant and diverse inhibitory neurons compared with rodents, but the molecular foundations of these observations are unknown. Through integrative gene coexpression analysis, we determined a consensus transcriptional profile of GABAergic neurons in mid-gestation human neocortex. By comparing this profile to genes expressed in GABAergic neurons purified from neonatal mouse neocortex, we identified conserved and distinct aspects of gene expression in these cells between the species. We show here that the calcium-binding protein secretagogin (SCGN) is robustly expressed by neocortical GABAergic neurons derived from caudal ganglionic eminences (CGE) and lateral ganglionic eminences during human but not mouse brain development. Through electrophysiological and morphometric analyses, we examined the effects of SCGN expression on GABAergic neuron function and form. Forced expression of SCGN in CGE-derived mouse GABAergic neurons significantly increased total neurite length and arbor complexity following transplantation into mouse neocortex, revealing a molecular pathway that contributes to morphological differences in these cells between rodents and primates.

@ARTICLE{Di_Lullo2017-mb,
  title    = "The use of brain organoids to investigate neural development and
              disease",
  author   = "Di Lullo, Elizabeth and Kriegstein, Arnold R",
  abstract = "Understanding the development and dysfunction of the human brain
              is a major goal of neurobiology. Much of our current
              understanding of human brain development has been derived from
              the examination of post-mortem and pathological specimens,
              bolstered by observations of developing non-human primates and
              experimental studies focused largely on mouse models. However,
              these tissue specimens and model systems cannot fully capture the
              unique and dynamic features of human brain development. Recent
              advances in stem cell technologies that enable the generation of
              human brain organoids from pluripotent stem cells (PSCs) promise
              to profoundly change our understanding of the development of the
              human brain and enable a detailed study of the pathogenesis of
              inherited and acquired brain diseases.",
  journal  = "Nat Rev Neurosci",
  volume   =  18,
  number   =  10,
  pages    = "573--584",
  month    =  sep,
  year     =  2017,
  language = "en"
}

Understanding the development and dysfunction of the human brain is a major goal of neurobiology. Much of our current understanding of human brain development has been derived from the examination of post-mortem and pathological specimens, bolstered by observations of developing non-human primates and experimental studies focused largely on mouse models. However, these tissue specimens and model systems cannot fully capture the unique and dynamic features of human brain development. Recent advances in stem cell technologies that enable the generation of human brain organoids from pluripotent stem cells (PSCs) promise to profoundly change our understanding of the development of the human brain and enable a detailed study of the pathogenesis of inherited and acquired brain diseases.

@ARTICLE{Nowakowski2017-et,
  title    = "Spatiotemporal gene expression trajectories reveal developmental
              hierarchies of the human cortex",
  author   = "Nowakowski, Tomasz J and Bhaduri, Aparna and Pollen, Alex A and
              Alvarado, Beatriz and Mostajo-Radji, Mohammed A and Di Lullo,
              Elizabeth and Haeussler, Maximilian and Sandoval-Espinosa, Carmen
              and Liu, Siyuan John and Velmeshev, Dmitry and Ounadjela, Johain
              Ryad and Shuga, Joe and Wang, Xiaohui and Lim, Daniel A and West,
              Jay A and Leyrat, Anne A and Kent, W James and Kriegstein, Arnold
              R",
  abstract = "Systematic analyses of spatiotemporal gene expression
              trajectories during organogenesis have been challenging because
              diverse cell types at different stages of maturation and
              differentiation coexist in the emerging tissues. We identified
              discrete cell types as well as temporally and spatially
              restricted trajectories of radial glia maturation and
              neurogenesis in developing human telencephalon. These
              lineage-specific trajectories reveal the expression of neurogenic
              transcription factors in early radial glia and enriched
              activation of mammalian target of rapamycin signaling in outer
              radial glia. Across cortical areas, modest transcriptional
              differences among radial glia cascade into robust typological
              distinctions among maturing neurons. Together, our results
              support a mixed model of topographical, typological, and temporal
              hierarchies governing cell-type diversity in the developing human
              telencephalon, including distinct excitatory lineages emerging in
              rostral and caudal cerebral cortex.",
  journal  = "Science",
  volume   =  358,
  number   =  6368,
  pages    = "1318--1323",
  month    =  dec,
  year     =  2017,
  language = "en"
}

Systematic analyses of spatiotemporal gene expression trajectories during organogenesis have been challenging because diverse cell types at different stages of maturation and differentiation coexist in the emerging tissues. We identified discrete cell types as well as temporally and spatially restricted trajectories of radial glia maturation and neurogenesis in developing human telencephalon. These lineage-specific trajectories reveal the expression of neurogenic transcription factors in early radial glia and enriched activation of mammalian target of rapamycin signaling in outer radial glia. Across cortical areas, modest transcriptional differences among radial glia cascade into robust typological distinctions among maturing neurons. Together, our results support a mixed model of topographical, typological, and temporal hierarchies governing cell-type diversity in the developing human telencephalon, including distinct excitatory lineages emerging in rostral and caudal cerebral cortex.

@ARTICLE{Bershteyn2017-ib,
  title    = "Human {iPSC-Derived} Cerebral Organoids Model Cellular Features
              of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial
              Glia",
  author   = "Bershteyn, Marina and Nowakowski, Tomasz J and Pollen, Alex A and
              Di Lullo, Elizabeth and Nene, Aishwarya and Wynshaw-Boris,
              Anthony and Kriegstein, Arnold R",
  abstract = "Classical lissencephaly is a genetic neurological disorder
              associated with mental retardation and intractable epilepsy, and
              Miller-Dieker syndrome (MDS) is the most severe form of the
              disease. In this study, to investigate the effects of MDS on
              human progenitor subtypes that control neuronal output and
              influence brain topology, we analyzed cerebral organoids derived
              from control and MDS-induced pluripotent stem cells (iPSCs) using
              time-lapse imaging, immunostaining, and single-cell RNA
              sequencing. We saw a cell migration defect that was rescued when
              we corrected the MDS causative chromosomal deletion and severe
              apoptosis of the founder neuroepithelial stem cells, accompanied
              by increased horizontal cell divisions. We also identified a
              mitotic defect in outer radial glia, a progenitor subtype that is
              largely absent from lissencephalic rodents but critical for human
              neocortical expansion. Our study, therefore, deepens our
              understanding of MDS cellular pathogenesis and highlights the
              broad utility of cerebral organoids for modeling human
              neurodevelopmental disorders.",
  journal  = "Cell Stem Cell",
  volume   =  20,
  number   =  4,
  pages    = "435--449.e4",
  month    =  jan,
  year     =  2017,
  keywords = "cerebral organoids; human lissencephaly; migration; outer radial
              glia; spindle orientation",
  language = "en"
}

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy, and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study, to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology, we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging, immunostaining, and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells, accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia, a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study, therefore, deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders.

@ARTICLE{Muller2017-zp,
  title    = "Single-cell profiling of human gliomas reveals macrophage
              ontogeny as a basis for regional differences in macrophage
              activation in the tumor microenvironment",
  author   = "M{\"u}ller, S{\"o}ren and Kohanbash, Gary and Liu, S John and
              Alvarado, Beatriz and Carrera, Diego and Bhaduri, Aparna and
              Watchmaker, Payal B and Yagnik, Garima and Di Lullo, Elizabeth
              and Malatesta, Martina and Amankulor, Nduka M and Kriegstein,
              Arnold R and Lim, Daniel A and Aghi, Manish and Okada, Hideho and
              Diaz, Aaron",
  abstract = "BACKGROUND: Tumor-associated macrophages (TAMs) are abundant in
              gliomas and immunosuppressive TAMs are a barrier to emerging
              immunotherapies. It is unknown to what extent macrophages derived
              from peripheral blood adopt the phenotype of brain-resident
              microglia in pre-treatment gliomas. The relative proportions of
              blood-derived macrophages and microglia have been poorly
              quantified in clinical samples due to a paucity of markers that
              distinguish these cell types in malignant tissue. RESULTS: We
              perform single-cell RNA-sequencing of human gliomas and identify
              phenotypic differences in TAMs of distinct lineages. We isolate
              TAMs from patient biopsies and compare them with macrophages from
              non-malignant human tissue, glioma atlases, and murine glioma
              models. We present a novel signature that distinguishes TAMs by
              ontogeny in human gliomas. Blood-derived TAMs upregulate
              immunosuppressive cytokines and show an altered metabolism
              compared to microglial TAMs. They are also enriched in
              perivascular and necrotic regions. The gene signature of
              blood-derived TAMs, but not microglial TAMs, correlates with
              significantly inferior survival in low-grade glioma.
              Surprisingly, TAMs frequently co-express canonical
              pro-inflammatory (M1) and alternatively activated (M2) genes in
              individual cells. CONCLUSIONS: We conclude that blood-derived
              TAMs significantly infiltrate pre-treatment gliomas, to a degree
              that varies by glioma subtype and tumor compartment.
              Blood-derived TAMs do not universally conform to the phenotype of
              microglia, but preferentially express immunosuppressive cytokines
              and show an altered metabolism. Our results argue against status
              quo therapeutic strategies that target TAMs indiscriminately and
              in favor of strategies that specifically target immunosuppressive
              blood-derived TAMs.",
  journal  = "Genome Biol",
  volume   =  18,
  number   =  1,
  pages    = "234",
  month    =  dec,
  year     =  2017,
  keywords = "Glioma; Immunotherapy; Macrophage; Single-cell sequencing",
  language = "en"
}

BACKGROUND: Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue. RESULTS: We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells. CONCLUSIONS: We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.

@ARTICLE{Ecker2017-pt,
  title    = "The {BRAIN} Initiative Cell Census Consortium: Lessons Learned
              toward Generating a Comprehensive Brain Cell Atlas",
  author   = "Ecker, Joseph R and Geschwind, Daniel H and Kriegstein, Arnold R
              and Ngai, John and Osten, Pavel and Polioudakis, Damon and Regev,
              Aviv and Sestan, Nenad and Wickersham, Ian R and Zeng, Hongkui",
  abstract = "A comprehensive characterization of neuronal cell types, their
              distributions, and patterns of connectivity is critical for
              understanding the properties of neural circuits and how they
              generate behaviors. Here we review the experiences of the BRAIN
              Initiative Cell Census Consortium, ten pilot projects funded by
              the U.S. BRAIN Initiative, in developing, validating, and scaling
              up emerging genomic and anatomical mapping technologies for
              creating a complete inventory of neuronal cell types and their
              connections in multiple species and during development. These
              projects lay the foundation for a larger and longer-term effort
              to generate whole-brain cell atlases in species including mice
              and humans.",
  journal  = "Neuron",
  volume   =  96,
  number   =  3,
  pages    = "542--557",
  month    =  nov,
  year     =  2017,
  keywords = "BRAIN initiative; anatomy; cell census; connectivity;
              electrophysiology; human brain; mouse brain; single-cell RNA-seq;
              single-cell epigenomics; single-cell transcriptomics",
  language = "en"
}

A comprehensive characterization of neuronal cell types, their distributions, and patterns of connectivity is critical for understanding the properties of neural circuits and how they generate behaviors. Here we review the experiences of the BRAIN Initiative Cell Census Consortium, ten pilot projects funded by the U.S. BRAIN Initiative, in developing, validating, and scaling up emerging genomic and anatomical mapping technologies for creating a complete inventory of neuronal cell types and their connections in multiple species and during development. These projects lay the foundation for a larger and longer-term effort to generate whole-brain cell atlases in species including mice and humans.

@ARTICLE{Subramanian2017-ff,
  title    = "Dynamic behaviour of human neuroepithelial cells in the
              developing forebrain",
  author   = "Subramanian, Lakshmi and Bershteyn, Marina and Paredes, Mercedes
              F and Kriegstein, Arnold R",
  abstract = "To understand how diverse progenitor cells contribute to human
              neocortex development, we examined forebrain progenitor behaviour
              using timelapse imaging. Here we find that cell cycle dynamics of
              human neuroepithelial (NE) cells differ from radial glial (RG)
              cells in both primary tissue and in stem cell-derived organoids.
              NE cells undergoing proliferative, symmetric divisions retract
              their basal processes, and both daughter cells regrow a new
              process following cytokinesis. The mitotic retraction of the
              basal process is recapitulated by NE cells in cerebral organoids
              generated from human-induced pluripotent stem cells. In contrast,
              RG cells undergoing vertical cleavage retain their basal fibres
              throughout mitosis, both in primary tissue and in older
              organoids. Our findings highlight developmentally regulated
              changes in mitotic behaviour that may relate to the role of RG
              cells to provide a stable scaffold for neuronal migration, and
              suggest that the transition in mitotic dynamics can be studied in
              organoid models.",
  journal  = "Nat Commun",
  volume   =  8,
  pages    = "14167",
  month    =  jan,
  year     =  2017,
  language = "en"
}

To understand how diverse progenitor cells contribute to human neocortex development, we examined forebrain progenitor behaviour using timelapse imaging. Here we find that cell cycle dynamics of human neuroepithelial (NE) cells differ from radial glial (RG) cells in both primary tissue and in stem cell-derived organoids. NE cells undergoing proliferative, symmetric divisions retract their basal processes, and both daughter cells regrow a new process following cytokinesis. The mitotic retraction of the basal process is recapitulated by NE cells in cerebral organoids generated from human-induced pluripotent stem cells. In contrast, RG cells undergoing vertical cleavage retain their basal fibres throughout mitosis, both in primary tissue and in older organoids. Our findings highlight developmentally regulated changes in mitotic behaviour that may relate to the role of RG cells to provide a stable scaffold for neuronal migration, and suggest that the transition in mitotic dynamics can be studied in organoid models.

@ARTICLE{Pollen2016-qj,
  title    = "Primate Neurons Flex Their Musclin",
  author   = "Pollen, Alex A and Kriegstein, Arnold R",
  abstract = "Sensory experience evokes long-lasting changes in neural circuits
              through activity-dependent gene expression. Ataman et al. (2016)
              report in Nature that primates evolved novel transcriptional
              responses to neuronal activity, including induction of
              musclin/osteocrin (OSTN), which may regulate specialized aspects
              of primate neural circuits.",
  journal  = "Neuron",
  volume   =  92,
  number   =  4,
  pages    = "681--683",
  month    =  nov,
  year     =  2016,
  address  = "United States",
  keywords = "MEF2C; NPR3; OSTN; activity-dependent gene expression;
              osteocrin/musclin; primate neocortex evolution",
  language = "en"
}

Sensory experience evokes long-lasting changes in neural circuits through activity-dependent gene expression. Ataman et al. (2016) report in Nature that primates evolved novel transcriptional responses to neuronal activity, including induction of musclin/osteocrin (OSTN), which may regulate specialized aspects of primate neural circuits.

@ARTICLE{Rani2016-zn,
  title    = "A Primate {lncRNA} Mediates Notch Signaling during Neuronal
              Development by Sequestering {miRNA}",
  author   = "Rani, Neha and Nowakowski, Tomasz J and Zhou, Hongjun and
              Godshalk, Sirie E and Lisi, V{\'e}ronique and Kriegstein, Arnold
              R and Kosik, Kenneth S",
  abstract = "Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved
              category of transcripts that have expanded greatly in primates,
              particularly in the brain. We identified an lncRNA, which has
              acquired 16 microRNA response elements for miR-143-3p in the
              Catarrhini branch of primates. This lncRNA, termed LncND
              (neurodevelopment), is expressed in neural progenitor cells and
              then declines in neurons. Binding and release of miR-143-3p by
              LncND control the expression of Notch receptors. LncND expression
              is enriched in radial glia cells (RGCs) in the ventricular and
              subventricular zones of developing human brain. Downregulation in
              neuroblastoma cells reduced cell proliferation and induced
              neuronal differentiation, an effect phenocopied by miR-143-3p
              overexpression. Gain of function of LncND in developing mouse
              cortex led to an expansion of PAX6+ RGCs. These findings support
              a role for LncND in miRNA-mediated regulation of Notch signaling
              within the neural progenitor pool in primates that may have
              contributed to the expansion of cerebral cortex.",
  journal  = "Neuron",
  volume   =  90,
  number   =  6,
  pages    = "1174--1188",
  month    =  jun,
  year     =  2016,
  language = "en"
}

Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved category of transcripts that have expanded greatly in primates, particularly in the brain. We identified an lncRNA, which has acquired 16 microRNA response elements for miR-143-3p in the Catarrhini branch of primates. This lncRNA, termed LncND (neurodevelopment), is expressed in neural progenitor cells and then declines in neurons. Binding and release of miR-143-3p by LncND control the expression of Notch receptors. LncND expression is enriched in radial glia cells (RGCs) in the ventricular and subventricular zones of developing human brain. Downregulation in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation, an effect phenocopied by miR-143-3p overexpression. Gain of function of LncND in developing mouse cortex led to an expansion of PAX6+ RGCs. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling within the neural progenitor pool in primates that may have contributed to the expansion of cerebral cortex.

@ARTICLE{Liu2016-tx,
  title    = "Single-cell analysis of long non-coding {RNAs} in the developing
              human neocortex",
  author   = "Liu, Siyuan John and Nowakowski, Tomasz J and Pollen, Alex A and
              Lui, Jan H and Horlbeck, Max A and Attenello, Frank J and He,
              Daniel and Weissman, Jonathan S and Kriegstein, Arnold R and
              Diaz, Aaron A and Lim, Daniel A",
  abstract = "BACKGROUND: Long non-coding RNAs (lncRNAs) comprise a diverse
              class of transcripts that can regulate molecular and cellular
              processes in brain development and disease. LncRNAs exhibit cell
              type- and tissue-specific expression, but little is known about
              the expression and function of lncRNAs in the developing human
              brain. Furthermore, it has been unclear whether lncRNAs are
              highly expressed in subsets of cells within tissues, despite
              appearing lowly expressed in bulk populations. RESULTS: We use
              strand-specific RNA-seq to deeply profile lncRNAs from
              polyadenylated and total RNA obtained from human neocortex at
              different stages of development, and we apply this reference to
              analyze the transcriptomes of single cells. While lncRNAs are
              generally detected at low levels in bulk tissues, single-cell
              transcriptomics of hundreds of neocortex cells reveal that many
              lncRNAs are abundantly expressed in individual cells and are cell
              type-specific. Notably, LOC646329 is a lncRNA enriched in single
              radial glia cells but is detected at low abundance in tissues.
              CRISPRi knockdown of LOC646329 indicates that this lncRNA
              regulates cell proliferation. CONCLUSION: The discrete and
              abundant expression of lncRNAs among individual cells has
              important implications for both their biological function and
              utility for distinguishing neural cell types.",
  journal  = "Genome Biol",
  volume   =  17,
  pages    = "67",
  month    =  apr,
  year     =  2016,
  keywords = "CRISPRi; Developing brain; Single-cell RNA-seq; lncRNA",
  language = "en"
}

BACKGROUND: Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that can regulate molecular and cellular processes in brain development and disease. LncRNAs exhibit cell type- and tissue-specific expression, but little is known about the expression and function of lncRNAs in the developing human brain. Furthermore, it has been unclear whether lncRNAs are highly expressed in subsets of cells within tissues, despite appearing lowly expressed in bulk populations. RESULTS: We use strand-specific RNA-seq to deeply profile lncRNAs from polyadenylated and total RNA obtained from human neocortex at different stages of development, and we apply this reference to analyze the transcriptomes of single cells. While lncRNAs are generally detected at low levels in bulk tissues, single-cell transcriptomics of hundreds of neocortex cells reveal that many lncRNAs are abundantly expressed in individual cells and are cell type-specific. Notably, LOC646329 is a lncRNA enriched in single radial glia cells but is detected at low abundance in tissues. CRISPRi knockdown of LOC646329 indicates that this lncRNA regulates cell proliferation. CONCLUSION: The discrete and abundant expression of lncRNAs among individual cells has important implications for both their biological function and utility for distinguishing neural cell types.

@ARTICLE{Nowakowski2016-cy,
  title    = "Transformation of the Radial Glia Scaffold Demarcates Two Stages
              of Human Cerebral Cortex Development",
  author   = "Nowakowski, Tomasz J and Pollen, Alex A and Sandoval-Espinosa,
              Carmen and Kriegstein, Arnold R",
  abstract = "The classic view of cortical development, embodied in the radial
              unit hypothesis, highlights the ventricular radial glia (vRG)
              scaffold as a key architectonic feature of the developing
              neocortex. The scaffold includes continuous fibers spanning the
              thickness of the developing cortex during neurogenesis across
              mammals. However, we find that in humans, the scaffold transforms
              into a physically discontinuous structure during the transition
              from infragranular to supragranular neuron production. As a
              consequence of this transformation, supragranular layer neurons
              arrive at their terminal positions in the cortical plate along
              outer radial glia (oRG) cell fibers. In parallel, the radial glia
              that contact the ventricle develop distinct gene expression
              profile and ``truncated'' morphology. We propose a supragranular
              layer expansion hypothesis that posits a deterministic role of
              oRG cells in the radial and tangential expansion of supragranular
              layers in primates, with implications for patterns of neuronal
              migration, area patterning, and cortical folding.",
  journal  = "Neuron",
  volume   =  91,
  number   =  6,
  pages    = "1219--1227",
  month    =  sep,
  year     =  2016,
  language = "en"
}

The classic view of cortical development, embodied in the radial unit hypothesis, highlights the ventricular radial glia (vRG) scaffold as a key architectonic feature of the developing neocortex. The scaffold includes continuous fibers spanning the thickness of the developing cortex during neurogenesis across mammals. However, we find that in humans, the scaffold transforms into a physically discontinuous structure during the transition from infragranular to supragranular neuron production. As a consequence of this transformation, supragranular layer neurons arrive at their terminal positions in the cortical plate along outer radial glia (oRG) cell fibers. In parallel, the radial glia that contact the ventricle develop distinct gene expression profile and ``truncated'' morphology. We propose a supragranular layer expansion hypothesis that posits a deterministic role of oRG cells in the radial and tangential expansion of supragranular layers in primates, with implications for patterns of neuronal migration, area patterning, and cortical folding.

@ARTICLE{Nowakowski2016-qh,
  title    = "Expression Analysis Highlights {AXL} as a Candidate Zika Virus
              Entry Receptor in Neural Stem Cells",
  author   = "Nowakowski, Tomasz J and Pollen, Alex A and Di Lullo, Elizabeth
              and Sandoval-Espinosa, Carmen and Bershteyn, Marina and
              Kriegstein, Arnold R",
  abstract = "The recent outbreak of Zika virus (ZIKV) in Brazil has been
              linked to substantial increases in fetal abnormalities and
              microcephaly. However, information about the underlying molecular
              and cellular mechanisms connecting viral infection to these
              defects remains limited. In this study we have examined the
              expression of receptors implicated in cell entry of several
              enveloped viruses including ZIKV across diverse cell types in the
              developing brain. Using single-cell RNA-seq and
              immunohistochemistry, we found that the candidate viral entry
              receptor AXL is highly expressed by human radial glial cells,
              astrocytes, endothelial cells, and microglia in developing human
              cortex and by progenitor cells in developing retina. We also show
              that AXL expression in radial glia is conserved in developing
              mouse and ferret cortex and in human stem cell-derived cerebral
              organoids, highlighting multiple experimental systems that could
              be applied to study mechanisms of ZIKV infectivity and effects on
              brain development.",
  journal  = "Cell Stem Cell",
  volume   =  18,
  number   =  5,
  pages    = "591--596",
  month    =  mar,
  year     =  2016,
  language = "en"
}

% The entry below contains non-ASCII chars that could not be converted
% to a LaTeX equivalent.

The recent outbreak of Zika virus (ZIKV) in Brazil has been linked to substantial increases in fetal abnormalities and microcephaly. However, information about the underlying molecular and cellular mechanisms connecting viral infection to these defects remains limited. In this study we have examined the expression of receptors implicated in cell entry of several enveloped viruses including ZIKV across diverse cell types in the developing brain. Using single-cell RNA-seq and immunohistochemistry, we found that the candidate viral entry receptor AXL is highly expressed by human radial glial cells, astrocytes, endothelial cells, and microglia in developing human cortex and by progenitor cells in developing retina. We also show that AXL expression in radial glia is conserved in developing mouse and ferret cortex and in human stem cell-derived cerebral organoids, highlighting multiple experimental systems that could be applied to study mechanisms of ZIKV infectivity and effects on brain development.

@ARTICLE{Muller2016-mp,
  title    = "Single-cell sequencing maps gene expression to mutational
              phylogenies in {PDGF-} and {EGF-driven} gliomas",
  author   = "M{\"u}ller, S{\"o}ren and Liu, Siyuan John and Di Lullo,
              Elizabeth and Malatesta, Martina and Pollen, Alex A and
              Nowakowski, Tomasz J and Kohanbash, Gary and Aghi, Manish and
              Kriegstein, Arnold R and Lim, Daniel A and Diaz, Aaron",
  abstract = "Glioblastoma multiforme (GBM) is the most common and aggressive
              type of primary brain tumor. Epidermal growth factor (EGF) and
              platelet-derived growth factor (PDGF) receptors are frequently
              amplified and/or possess gain-of-function mutations in GBM
              However, clinical trials of tyrosine-kinase inhibitors have shown
              disappointing efficacy, in part due to intra-tumor heterogeneity.
              To assess the effect of clonal heterogeneity on gene expression,
              we derived an approach to map single-cell expression profiles to
              sequentially acquired mutations identified from exome sequencing.
              Using 288 single cells, we constructed high-resolution
              phylogenies of EGF-driven and PDGF-driven GBMs, modeling
              transcriptional kinetics during tumor evolution. Descending the
              phylogenetic tree of a PDGF-driven tumor corresponded to a
              progressive induction of an oligodendrocyte progenitor-like cell
              type, expressing pro-angiogenic factors. In contrast,
              phylogenetic analysis of an EGFR-amplified tumor showed an
              up-regulation of pro-invasive genes. An in-frame deletion in a
              specific dimerization domain of PDGF receptor correlates with an
              up-regulation of growth pathways in a proneural GBM and enhances
              proliferation when ectopically expressed in glioma cell lines.
              In-frame deletions in this domain are frequent in public GBM
              data.",
  journal  = "Mol Syst Biol",
  volume   =  12,
  number   =  11,
  pages    = "889",
  month    =  nov,
  year     =  2016,
  keywords = "PDGFRA; copy‐number variation; glioblastoma; single‐cell
              RNA‐sequencing; tumor phylogeny",
  language = "en"
}

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM However, clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy, in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression, we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells, we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs, modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type, expressing pro-angiogenic factors. In contrast, phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data.

@ARTICLE{Retallack2016-pw,
  title    = "Zika virus cell tropism in the developing human brain and
              inhibition by azithromycin",
  author   = "Retallack, Hanna and Di Lullo, Elizabeth and Arias, Carolina and
              Knopp, Kristeene A and Laurie, Matthew T and Sandoval-Espinosa,
              Carmen and Mancia Leon, Walter R and Krencik, Robert and Ullian,
              Erik M and Spatazza, Julien and Pollen, Alex A and Mandel-Brehm,
              Caleigh and Nowakowski, Tomasz J and Kriegstein, Arnold R and
              DeRisi, Joseph L",
  abstract = "The rapid spread of Zika virus (ZIKV) and its association with
              abnormal brain development constitute a global health emergency.
              Congenital ZIKV infection produces a range of mild to severe
              pathologies, including microcephaly. To understand the
              pathophysiology of ZIKV infection, we used models of the
              developing brain that faithfully recapitulate the tissue
              architecture in early to midgestation. We identify the brain cell
              populations that are most susceptible to ZIKV infection in
              primary human tissue, provide evidence for a mechanism of viral
              entry, and show that a commonly used antibiotic protects cultured
              brain cells by reducing viral proliferation. In the brain, ZIKV
              preferentially infected neural stem cells, astrocytes,
              oligodendrocyte precursor cells, and microglia, whereas neurons
              were less susceptible to infection. These findings suggest
              mechanisms for microcephaly and other pathologic features of
              infants with congenital ZIKV infection that are not explained by
              neural stem cell infection alone, such as calcifications in the
              cortical plate. Furthermore, we find that blocking the
              glia-enriched putative viral entry receptor AXL reduced ZIKV
              infection of astrocytes in vitro, and genetic knockdown of AXL in
              a glial cell line nearly abolished infection. Finally, we
              evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We
              show that the macrolide antibiotic azithromycin reduced viral
              proliferation and virus-induced cytopathic effects in glial cell
              lines and human astrocytes. Our characterization of infection in
              the developing human brain clarifies the pathogenesis of
              congenital ZIKV infection and provides the basis for
              investigating possible therapeutic strategies to safely alleviate
              or prevent the most severe consequences of the epidemic.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  113,
  number   =  50,
  pages    = "14408--14413",
  month    =  nov,
  year     =  2016,
  keywords = "Zika virus; azithromycin; cortical development; microcephaly",
  language = "en"
}

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

@ARTICLE{Fandel2016-ay,
  title    = "Transplanted Human Stem {Cell-Derived} Interneuron Precursors
              Mitigate Mouse Bladder Dysfunction and Central Neuropathic Pain
              after Spinal Cord Injury",
  author   = "Fandel, Thomas M and Trivedi, Alpa and Nicholas, Cory R and
              Zhang, Haoqian and Chen, Jiadong and Martinez, Aida F and
              Noble-Haeusslein, Linda J and Kriegstein, Arnold R",
  abstract = "Neuropathic pain and bladder dysfunction represent significant
              quality-of-life issues for many spinal cord injury patients. Loss
              of GABAergic tone in the injured spinal cord may contribute to
              the emergence of these symptoms. Previous studies have shown that
              transplantation of rodent inhibitory interneuron precursors from
              the medial ganglionic eminence (MGE) enhances GABAergic signaling
              in the brain and spinal cord. Here we look at whether
              transplanted MGE-like cells derived from human embryonic stem
              cells (hESC-MGEs) can mitigate the pathological effects of spinal
              cord injury. We find that 6 months after transplantation into
              injured mouse spinal cords, hESC-MGEs differentiate into
              GABAergic neuron subtypes and receive synaptic inputs, suggesting
              functional integration into host spinal cord. Moreover, the
              transplanted animals show improved bladder function and
              mitigation of pain-related symptoms. Our results therefore
              suggest that this approach may be a valuable strategy for
              ameliorating the adverse effects of spinal cord injury.",
  journal  = "Cell Stem Cell",
  volume   =  19,
  number   =  4,
  pages    = "544--557",
  month    =  sep,
  year     =  2016,
  address  = "United States",
  keywords = "GABA; MGE; allodynia; bladder; conscious cystometry;
              electrophysiology; human pluripotent stem cells; hyperalgesia;
              interneuron; spinal cord injury",
  language = "en"
}

Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury.

@ARTICLE{Pollen2015-mc,
  title    = "Molecular identity of human outer radial glia during cortical
              development",
  author   = "Pollen, Alex A and Nowakowski, Tomasz J and Chen, Jiadong and
              Retallack, Hanna and Sandoval-Espinosa, Carmen and Nicholas, Cory
              R and Shuga, Joe and Liu, Siyuan John and Oldham, Michael C and
              Diaz, Aaron and Lim, Daniel A and Leyrat, Anne A and West, Jay A
              and Kriegstein, Arnold R",
  abstract = "Radial glia, the neural stem cells of the neocortex, are located
              in two niches: the ventricular zone and outer subventricular
              zone. Although outer subventricular zone radial glia may generate
              the majority of human cortical neurons, their molecular features
              remain elusive. By analyzing gene expression across single cells,
              we find that outer radial glia preferentially express genes
              related to extracellular matrix formation, migration, and
              stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using
              dynamic imaging, immunostaining, and clonal analysis, we relate
              these molecular features to distinctive behaviors of outer radial
              glia, demonstrate the necessity of STAT3 signaling for their cell
              cycle progression, and establish their extensive proliferative
              potential. These results suggest that outer radial glia directly
              support the subventricular niche through local production of
              growth factors, potentiation of growth factor signals by
              extracellular matrix proteins, and activation of self-renewal
              pathways, thereby enabling the developmental and evolutionary
              expansion of the human neocortex.",
  journal  = "Cell",
  volume   =  163,
  number   =  1,
  pages    = "55--67",
  month    =  sep,
  year     =  2015,
  language = "en"
}

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

@ARTICLE{Harwell2015-ng,
  title    = "Wide Dispersion and Diversity of Clonally Related Inhibitory
              Interneurons",
  author   = "Harwell, Corey C and Fuentealba, Luis C and Gonzalez-Cerrillo,
              Adrian and Parker, Phillip R L and Gertz, Caitlyn C and Mazzola,
              Emanuele and Garcia, Miguel Turrero and Alvarez-Buylla, Arturo
              and Cepko, Constance L and Kriegstein, Arnold R",
  abstract = "The mammalian neocortex is composed of two major neuronal cell
              types with distinct origins: excitatory pyramidal neurons and
              inhibitory interneurons, generated in dorsal and ventral
              progenitor zones of the embryonic telencephalon, respectively.
              Thus, inhibitory neurons migrate relatively long distances to
              reach their destination in the developing forebrain. The role of
              lineage in the organization and circuitry of interneurons is
              still not well understood. Utilizing a combination of genetics,
              retroviral fate mapping, and lineage-specific retroviral barcode
              labeling, we find that clonally related interneurons can be
              widely dispersed while unrelated interneurons can be closely
              clustered. These data suggest that migratory mechanisms related
              to the clustering of interneurons occur largely independent of
              their clonal origin.",
  journal  = "Neuron",
  volume   =  87,
  number   =  5,
  pages    = "999--1007",
  month    =  aug,
  year     =  2015,
  language = "en"
}

The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.

@ARTICLE{Gertz2015-pp,
  title    = "Neuronal Migration Dynamics in the Developing Ferret Cortex",
  author   = "Gertz, Caitlyn C and Kriegstein, Arnold R",
  abstract = "During mammalian neocortical development, newborn excitatory and
              inhibitory neurons must migrate over long distances to reach
              their final positions within the cortical plate. In the
              lissencephalic rodent brain, pyramidal neurons are born in the
              ventricular and subventricular zones of the pallium and migrate
              along radial glia fibers to reach the appropriate cortical layer.
              Although much less is known about neuronal migration in species
              with a gyrencephalic cortex, retroviral studies in the ferret and
              primate suggest that, unlike the rodent, pyramidal neurons do not
              follow strict radial pathways and instead can disperse
              horizontally. However, the means by which pyramidal neurons
              laterally disperse remain unknown. In this study, we identified a
              viral labeling technique for visualizing neuronal migration in
              the ferret, a gyrencephalic carnivore, and found that migration
              was predominantly radial at early postnatal ages. In contrast,
              neurons displayed more tortuous migration routes with a decreased
              frequency of cortical plate-directed migration at later stages of
              neurogenesis concomitant with the start of brain folding. This
              was accompanied by neurons migrating sequentially along several
              different radial glial fibers, suggesting a mode by which
              pyramidal neurons may laterally disperse in a folded cortex.
              These findings provide insight into the migratory behavior of
              neurons in gyrencephalic species and provide a framework for
              using nonrodent model systems for studying neuronal migration
              disorders. SIGNIFICANCE STATEMENT: Elucidating neuronal migration
              dynamics in the gyrencephalic, or folded, cortex is important for
              understanding neurodevelopmental disorders. Similar to the
              rodent, we found that neuronal migration was predominantly radial
              at early postnatal ages in the gyrencephalic ferret cortex.
              Interestingly, ferret neurons displayed more tortuous migration
              routes and a decreased frequency of radial migration at later
              ages coincident with the start of cortical folding. We found that
              ferret neurons use several different radial glial fibers as
              migratory guides, including those belonging to the recently
              described outer radial glia, suggesting a mechanism by which
              ferret neurons disperse laterally. It is likely that excitatory
              neurons horizontally disperse in other gyrencephalic mammals,
              including the primate, suggesting an important modification to
              the current model deduced primarily from the rodent.",
  journal  = "J Neurosci",
  volume   =  35,
  number   =  42,
  pages    = "14307--14315",
  month    =  oct,
  year     =  2015,
  keywords = "ferret; gyrencephaly; neuronal migration",
  language = "en"
}

During mammalian neocortical development, newborn excitatory and inhibitory neurons must migrate over long distances to reach their final positions within the cortical plate. In the lissencephalic rodent brain, pyramidal neurons are born in the ventricular and subventricular zones of the pallium and migrate along radial glia fibers to reach the appropriate cortical layer. Although much less is known about neuronal migration in species with a gyrencephalic cortex, retroviral studies in the ferret and primate suggest that, unlike the rodent, pyramidal neurons do not follow strict radial pathways and instead can disperse horizontally. However, the means by which pyramidal neurons laterally disperse remain unknown. In this study, we identified a viral labeling technique for visualizing neuronal migration in the ferret, a gyrencephalic carnivore, and found that migration was predominantly radial at early postnatal ages. In contrast, neurons displayed more tortuous migration routes with a decreased frequency of cortical plate-directed migration at later stages of neurogenesis concomitant with the start of brain folding. This was accompanied by neurons migrating sequentially along several different radial glial fibers, suggesting a mode by which pyramidal neurons may laterally disperse in a folded cortex. These findings provide insight into the migratory behavior of neurons in gyrencephalic species and provide a framework for using nonrodent model systems for studying neuronal migration disorders. SIGNIFICANCE STATEMENT: Elucidating neuronal migration dynamics in the gyrencephalic, or folded, cortex is important for understanding neurodevelopmental disorders. Similar to the rodent, we found that neuronal migration was predominantly radial at early postnatal ages in the gyrencephalic ferret cortex. Interestingly, ferret neurons displayed more tortuous migration routes and a decreased frequency of radial migration at later ages coincident with the start of cortical folding. We found that ferret neurons use several different radial glial fibers as migratory guides, including those belonging to the recently described outer radial glia, suggesting a mechanism by which ferret neurons disperse laterally. It is likely that excitatory neurons horizontally disperse in other gyrencephalic mammals, including the primate, suggesting an important modification to the current model deduced primarily from the rodent.

@ARTICLE{Chen2015-oa,
  title    = "A {GABAergic} projection from the zona incerta to cortex promotes
              cortical neuron development",
  author   = "Chen, Jiadong and Kriegstein, Arnold R",
  abstract = "$\gamma$-Aminobutyric acid (GABA) is the major inhibitory
              transmitter in the mature brain but is excitatory in the
              developing cortex. We found that mouse zona incerta (ZI)
              projection neurons form a GABAergic axon plexus in neonatal
              cortical layer 1, making synapses with neurons in both deep and
              superficial layers. A similar depolarizing GABAergic plexus
              exists in the developing human cortex. Selectively silencing
              mouse ZI GABAergic neurons at birth decreased synaptic activity
              and apical dendritic complexity of cortical neurons. The ZI
              GABAergic projection becomes inhibitory with maturation and can
              block epileptiform activity in the adult brain. These data reveal
              an early-developing GABAergic projection from the ZI to cortical
              layer 1 that is essential for proper development of cortical
              neurons and balances excitation with inhibition in the adult
              cortex.",
  journal  = "Science",
  volume   =  350,
  number   =  6260,
  pages    = "554--558",
  month    =  oct,
  year     =  2015,
  language = "en"
}

$γ$-Aminobutyric acid (GABA) is the major inhibitory transmitter in the mature brain but is excitatory in the developing cortex. We found that mouse zona incerta (ZI) projection neurons form a GABAergic axon plexus in neonatal cortical layer 1, making synapses with neurons in both deep and superficial layers. A similar depolarizing GABAergic plexus exists in the developing human cortex. Selectively silencing mouse ZI GABAergic neurons at birth decreased synaptic activity and apical dendritic complexity of cortical neurons. The ZI GABAergic projection becomes inhibitory with maturation and can block epileptiform activity in the adult brain. These data reveal an early-developing GABAergic projection from the ZI to cortical layer 1 that is essential for proper development of cortical neurons and balances excitation with inhibition in the adult cortex.

@ARTICLE{Ramos2015-sa,
  title    = "The long noncoding {RNA} Pnky regulates neuronal differentiation
              of embryonic and postnatal neural stem cells",
  author   = "Ramos, Alexander D and Andersen, Rebecca E and Liu, Siyuan John
              and Nowakowski, Tomasz Jan and Hong, Sung Jun and Gertz, Caitlyn
              and Salinas, Ryan D and Zarabi, Hosniya and Kriegstein, Arnold R
              and Lim, Daniel A",
  abstract = "While thousands of long noncoding RNAs (lncRNAs) have been
              identified, few lncRNAs that control neural stem cell (NSC)
              behavior are known. Here, we identify Pinky (Pnky) as a
              neural-specific lncRNA that regulates neurogenesis from NSCs in
              the embryonic and postnatal brain. In postnatal NSCs, Pnky
              knockdown potentiates neuronal lineage commitment and expands the
              transit-amplifying cell population, increasing neuron production
              several-fold. Pnky is evolutionarily conserved and expressed in
              NSCs of the developing human brain. In the embryonic mouse
              cortex, Pnky knockdown increases neuronal differentiation and
              depletes the NSC population. Pnky interacts with the splicing
              regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis.
              In NSCs, Pnky and PTBP1 regulate the expression and alternative
              splicing of a core set of transcripts that relates to the
              cellular phenotype. These data thus unveil Pnky as a conserved
              lncRNA that interacts with a key RNA processing factor and
              regulates neurogenesis from embryonic and postnatal NSC
              populations.",
  journal  = "Cell Stem Cell",
  volume   =  16,
  number   =  4,
  pages    = "439--447",
  month    =  mar,
  year     =  2015,
  language = "en"
}

While thousands of long noncoding RNAs (lncRNAs) have been identified, few lncRNAs that control neural stem cell (NSC) behavior are known. Here, we identify Pinky (Pnky) as a neural-specific lncRNA that regulates neurogenesis from NSCs in the embryonic and postnatal brain. In postnatal NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Pnky interacts with the splicing regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. These data thus unveil Pnky as a conserved lncRNA that interacts with a key RNA processing factor and regulates neurogenesis from embryonic and postnatal NSC populations.

@ARTICLE{Wu2014-hb,
  title    = "The dynamics of neuronal migration",
  author   = "Wu, Qian and Liu, Jing and Fang, Ai and Li, Rui and Bai, Ye and
              Kriegstein, Arnold R and Wang, Xiaoqun",
  abstract = "Proper lamination of the cerebral cortex is precisely
              orchestrated, especially when neurons migrate from their place of
              birth to their final destination. The consequences of failure or
              delay in neuronal migration cause a wide range of disorders, such
              as lissencephaly, schizophrenia, autism and mental retardation.
              Neuronal migration is a dynamic process, which requires dynamic
              remodeling of the cytoskeleton. In this context microtubules and
              microtubule-related proteins have been suggested to play
              important roles in the regulation of neuronal migration. Here, we
              will review the dynamic aspects of neuronal migration and brain
              development, describe the molecular and cellular mechanisms of
              neuronal migration and elaborate on neuronal migration diseases.",
  journal  = "Adv Exp Med Biol",
  volume   =  800,
  pages    = "25--36",
  year     =  2014,
  address  = "United States",
  language = "en"
}

Proper lamination of the cerebral cortex is precisely orchestrated, especially when neurons migrate from their place of birth to their final destination. The consequences of failure or delay in neuronal migration cause a wide range of disorders, such as lissencephaly, schizophrenia, autism and mental retardation. Neuronal migration is a dynamic process, which requires dynamic remodeling of the cytoskeleton. In this context microtubules and microtubule-related proteins have been suggested to play important roles in the regulation of neuronal migration. Here, we will review the dynamic aspects of neuronal migration and brain development, describe the molecular and cellular mechanisms of neuronal migration and elaborate on neuronal migration diseases.

@ARTICLE{Kriegstein2014-bc,
  title    = "Yoshiki Sasai (1962--2014)",
  author   = "Kriegstein, Arnold R",
  journal  = "Cell Stem Cell",
  volume   =  15,
  number   =  3,
  pages    = "265--266",
  month    =  sep,
  year     =  2014,
  address  = "United States",
  language = "en"
}

@ARTICLE{Kriegstein2014-vn,
  title    = "Yoshiki Sasai (1962--2014)",
  author   = "Kriegstein, Arnold R",
  journal  = "Neuron",
  volume   =  83,
  number   =  6,
  pages    = "1237--1238",
  month    =  sep,
  year     =  2014,
  address  = "United States",
  language = "en"
}

@ARTICLE{Southwell2014-fq,
  title    = "Interneurons from embryonic development to cell-based therapy",
  author   = "Southwell, Derek G and Nicholas, Cory R and Basbaum, Allan I and
              Stryker, Michael P and Kriegstein, Arnold R and Rubenstein, John
              L and Alvarez-Buylla, Arturo",
  abstract = "Many neurologic and psychiatric disorders are marked by
              imbalances between neural excitation and inhibition. In the
              cerebral cortex, inhibition is mediated largely by GABAergic
              ($\gamma$-aminobutyric acid-secreting) interneurons, a cell type
              that originates in the embryonic ventral telencephalon and
              populates the cortex through long-distance tangential migration.
              Remarkably, when transplanted from embryos or in vitro culture
              preparations, immature interneurons disperse and integrate into
              host brain circuits, both in the cerebral cortex and in other
              regions of the central nervous system. These features make
              interneuron transplantation a powerful tool for the study of
              neurodevelopmental processes such as cell specification, cell
              death, and cortical plasticity. Moreover, interneuron
              transplantation provides a novel strategy for modifying neural
              circuits in rodent models of epilepsy, Parkinson's disease, mood
              disorders, and chronic pain.",
  journal  = "Science",
  volume   =  344,
  number   =  6180,
  pages    = "1240622",
  month    =  apr,
  year     =  2014,
  language = "en"
}

Many neurologic and psychiatric disorders are marked by imbalances between neural excitation and inhibition. In the cerebral cortex, inhibition is mediated largely by GABAergic ($γ$-aminobutyric acid-secreting) interneurons, a cell type that originates in the embryonic ventral telencephalon and populates the cortex through long-distance tangential migration. Remarkably, when transplanted from embryos or in vitro culture preparations, immature interneurons disperse and integrate into host brain circuits, both in the cerebral cortex and in other regions of the central nervous system. These features make interneuron transplantation a powerful tool for the study of neurodevelopmental processes such as cell specification, cell death, and cortical plasticity. Moreover, interneuron transplantation provides a novel strategy for modifying neural circuits in rodent models of epilepsy, Parkinson's disease, mood disorders, and chronic pain.

@ARTICLE{Price2014-lp,
  title    = "The {Ink4a/Arf} locus is a barrier to direct neuronal
              transdifferentiation",
  author   = "Price, James D and Park, Ki-Youb and Chen, Jiadong and Salinas,
              Ryan D and Cho, Mathew J and Kriegstein, Arnold R and Lim, Daniel
              A",
  abstract = "Non-neurogenic cell types, such as cortical astroglia and
              fibroblasts, can be directly converted into neurons by the
              overexpression of defined transcription factors. Normally, the
              cellular phenotype of such differentiated cells is remarkably
              stable and resists direct cell transdifferentiation. Here we show
              that the Ink4a/Arf (also known as Cdkn2a) locus is a
              developmental barrier to direct neuronal transdifferentiation
              induced by transcription factor overexpression. With serial
              passage in vitro, wild-type postnatal cortical astroglia become
              progressively resistant to Dlx2-induced neuronal
              transdifferentiation. In contrast, the neurogenic competence of
              Ink4a/Arf-deficient astroglia is both greatly increased and does
              not diminish through serial cell culture passage.
              Electrophysiological analysis further demonstrates the neuronal
              identity of cells induced from Ink4a/Arf-null astroglia, and
              short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf
              p16(Ink4a) and p19(Arf) indicates that these gene products
              function postnatally as a barrier to cellular
              transdifferentiation. Finally, we found that mouse fibroblasts
              deficient for Ink4a/Arf also exhibit greatly enhanced
              transcription factor-induced neuronal induction. These data
              indicate that Ink4a/Arf is a potent barrier to direct neuronal
              transdifferentiation and further suggest that this locus
              functions normally in the progressive developmental restriction
              of postnatal astrocytes.",
  journal  = "J Neurosci",
  volume   =  34,
  number   =  37,
  pages    = "12560--12567",
  month    =  sep,
  year     =  2014,
  keywords = "Ink4a/Arf; astroglia; induced neuron; transcription factor;
              transdifferentiation",
  language = "en"
}

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that the Ink4a/Arf (also known as Cdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passage in vitro, wild-type postnatal cortical astroglia become progressively resistant to Dlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence of Ink4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced from Ink4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16(Ink4a) and p19(Arf) indicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient for Ink4a/Arf also exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate that Ink4a/Arf is a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

@ARTICLE{Ostrem2014-ds,
  title    = "Control of outer radial glial stem cell mitosis in the human
              brain",
  author   = "Ostrem, Bridget E L and Lui, Jan H and Gertz, Caitlyn C and
              Kriegstein, Arnold R",
  abstract = "Evolutionary expansion of the human neocortex is partially
              attributed to a relative abundance of neural stem cells in the
              fetal brain called outer radial glia (oRG). oRG cells display a
              characteristic division mode, mitotic somal translocation (MST),
              in which the soma rapidly translocates toward the cortical plate
              immediately prior to cytokinesis. MST may be essential for
              progenitor zone expansion, but the mechanism of MST is unknown,
              hindering exploration of its function in development and disease.
              Here, we show that MST requires activation of the Rho effector
              ROCK and nonmuscle myosin II, but not intact microtubules,
              centrosomal translocation into the leading process, or calcium
              influx. MST is independent of mitosis and distinct from
              interkinetic nuclear migration and saltatory migration. Our
              findings suggest that disrupted MST may underlie
              neurodevelopmental diseases affecting the Rho-ROCK-myosin pathway
              and provide a foundation for future exploration of the role of
              MST in neocortical development, evolution, and disease.",
  journal  = "Cell Rep",
  volume   =  8,
  number   =  3,
  pages    = "656--664",
  month    =  jul,
  year     =  2014,
  language = "en"
}

Evolutionary expansion of the human neocortex is partially attributed to a relative abundance of neural stem cells in the fetal brain called outer radial glia (oRG). oRG cells display a characteristic division mode, mitotic somal translocation (MST), in which the soma rapidly translocates toward the cortical plate immediately prior to cytokinesis. MST may be essential for progenitor zone expansion, but the mechanism of MST is unknown, hindering exploration of its function in development and disease. Here, we show that MST requires activation of the Rho effector ROCK and nonmuscle myosin II, but not intact microtubules, centrosomal translocation into the leading process, or calcium influx. MST is independent of mitosis and distinct from interkinetic nuclear migration and saltatory migration. Our findings suggest that disrupted MST may underlie neurodevelopmental diseases affecting the Rho-ROCK-myosin pathway and provide a foundation for future exploration of the role of MST in neocortical development, evolution, and disease.

@ARTICLE{Bershteyn2014-ih,
  title    = "Cell-autonomous correction of ring chromosomes in human induced
              pluripotent stem cells",
  author   = "Bershteyn, Marina and Hayashi, Yohei and Desachy, Guillaume and
              Hsiao, Edward C and Sami, Salma and Tsang, Kathryn M and Weiss,
              Lauren A and Kriegstein, Arnold R and Yamanaka, Shinya and
              Wynshaw-Boris, Anthony",
  abstract = "Ring chromosomes are structural aberrations commonly associated
              with birth defects, mental disabilities and growth retardation.
              Rings form after fusion of the long and short arms of a
              chromosome, and are sometimes associated with large terminal
              deletions. Owing to the severity of these large aberrations that
              can affect multiple contiguous genes, no possible therapeutic
              strategies for ring chromosome disorders have been proposed.
              During cell division, ring chromosomes can exhibit unstable
              behaviour leading to continuous production of aneuploid progeny
              with low viability and high cellular death rate. The overall
              consequences of this chromosomal instability have been largely
              unexplored in experimental model systems. Here we generated human
              induced pluripotent stem cells (iPSCs) from patient fibroblasts
              containing ring chromosomes with large deletions and found that
              reprogrammed cells lost the abnormal chromosome and duplicated
              the wild-type homologue through the compensatory uniparental
              disomy (UPD) mechanism. The karyotypically normal iPSCs with
              isodisomy for the corrected chromosome outgrew co-existing
              aneuploid populations, enabling rapid and efficient isolation of
              patient-derived iPSCs devoid of the original chromosomal
              aberration. Our results suggest a fundamentally different
              function for cellular reprogramming as a means of 'chromosome
              therapy' to reverse combined loss-of-function across many genes
              in cells with large-scale aberrations involving ring structures.
              In addition, our work provides an experimentally tractable human
              cellular system for studying mechanisms of chromosomal number
              control, which is of critical relevance to human development and
              disease.",
  journal  = "Nature",
  volume   =  507,
  number   =  7490,
  pages    = "99--103",
  month    =  jan,
  year     =  2014,
  language = "en"
}

Ring chromosomes are structural aberrations commonly associated with birth defects, mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome, and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division, ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations, enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of 'chromosome therapy' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition, our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control, which is of critical relevance to human development and disease.

@ARTICLE{Lui2014-qa,
  title    = "Radial glia require {PDGFD-PDGFR$\beta$} signalling in human but
              not mouse neocortex",
  author   = "Lui, Jan H and Nowakowski, Tomasz J and Pollen, Alex A and
              Javaherian, Ashkan and Kriegstein, Arnold R and Oldham, Michael C",
  abstract = "Evolutionary expansion of the human neocortex underlies many of
              our unique mental abilities. This expansion has been attributed
              to the increased proliferative potential of radial glia (RG;
              neural stem cells) and their subventricular dispersion from the
              periventricular niche during neocortical development. Such
              adaptations may have evolved through gene expression changes in
              RG. However, whether or how RG gene expression varies between
              humans and other species is unknown. Here we show that the
              transcriptional profiles of human and mouse neocortical RG are
              broadly conserved during neurogenesis, yet diverge for specific
              signalling pathways. By analysing differential gene co-expression
              relationships between the species, we demonstrate that the growth
              factor PDGFD is specifically expressed by RG in human, but not
              mouse, corticogenesis. We also show that the expression domain of
              PDGFR$\beta$, the cognate receptor for PDGFD, is evolutionarily
              divergent, with high expression in the germinal region of dorsal
              human neocortex but not in the mouse. Pharmacological inhibition
              of PDGFD-PDGFR$\beta$ signalling in slice culture prevents normal
              cell cycle progression of neocortical RG in human, but not mouse.
              Conversely, injection of recombinant PDGFD or ectopic expression
              of constitutively active PDGFR$\beta$ in developing mouse
              neocortex increases the proportion of RG and their subventricular
              dispersion. These findings highlight the requirement of
              PDGFD-PDGFR$\beta$ signalling for human neocortical development
              and suggest that local production of growth factors by RG
              supports the expanded germinal region and progenitor
              heterogeneity of species with large brains.",
  journal  = "Nature",
  volume   =  515,
  number   =  7526,
  pages    = "264--268",
  month    =  nov,
  year     =  2014,
  language = "en"
}

Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche during neocortical development. Such adaptations may have evolved through gene expression changes in RG. However, whether or how RG gene expression varies between humans and other species is unknown. Here we show that the transcriptional profiles of human and mouse neocortical RG are broadly conserved during neurogenesis, yet diverge for specific signalling pathways. By analysing differential gene co-expression relationships between the species, we demonstrate that the growth factor PDGFD is specifically expressed by RG in human, but not mouse, corticogenesis. We also show that the expression domain of PDGFR$β$, the cognate receptor for PDGFD, is evolutionarily divergent, with high expression in the germinal region of dorsal human neocortex but not in the mouse. Pharmacological inhibition of PDGFD-PDGFR$β$ signalling in slice culture prevents normal cell cycle progression of neocortical RG in human, but not mouse. Conversely, injection of recombinant PDGFD or ectopic expression of constitutively active PDGFR$β$ in developing mouse neocortex increases the proportion of RG and their subventricular dispersion. These findings highlight the requirement of PDGFD-PDGFR$β$ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains.

@ARTICLE{Gertz2014-cx,
  title    = "Diverse behaviors of outer radial glia in developing ferret and
              human cortex",
  author   = "Gertz, Caitlyn C and Lui, Jan H and LaMonica, Bridget E and Wang,
              Xiaoqun and Kriegstein, Arnold R",
  abstract = "The dramatic increase in neocortical size and folding during
              mammalian brain evolution has been attributed to the elaboration
              of the subventricular zone (SVZ) and the associated increase in
              neural progenitors. However, recent studies have shown that SVZ
              size and the abundance of resident progenitors do not directly
              predict cortical topography, suggesting that complex behaviors of
              the progenitors themselves may contribute to the overall size and
              shape of the adult cortex. Using time-lapse imaging, we examined
              the dynamic behaviors of SVZ progenitors in the ferret, a
              gyrencephalic carnivore, focusing our analysis on outer radial
              glial cells (oRGs). We identified a substantial population of
              oRGs by marker expression and their unique mode of division,
              termed mitotic somal translocation (MST). Ferret oRGs exhibited
              diverse behaviors in terms of division location, cleavage angle,
              and MST distance, as well as fiber orientation and dynamics. We
              then examined the human fetal cortex and found that a subset of
              human oRGs displayed similar characteristics, suggesting that
              diversity in oRG behavior may be a general feature. Similar to
              the human, ferret oRGs underwent multiple rounds of self-renewing
              divisions but were more likely to undergo symmetric divisions
              that expanded the oRG population, as opposed to producing
              intermediate progenitor cells (IPCs). Differences in oRG
              behaviors, including proliferative potential and daughter cell
              fates, may contribute to variations in cortical structure between
              mammalian species.",
  journal  = "J Neurosci",
  volume   =  34,
  number   =  7,
  pages    = "2559--2570",
  month    =  feb,
  year     =  2014,
  language = "en"
}

The dramatic increase in neocortical size and folding during mammalian brain evolution has been attributed to the elaboration of the subventricular zone (SVZ) and the associated increase in neural progenitors. However, recent studies have shown that SVZ size and the abundance of resident progenitors do not directly predict cortical topography, suggesting that complex behaviors of the progenitors themselves may contribute to the overall size and shape of the adult cortex. Using time-lapse imaging, we examined the dynamic behaviors of SVZ progenitors in the ferret, a gyrencephalic carnivore, focusing our analysis on outer radial glial cells (oRGs). We identified a substantial population of oRGs by marker expression and their unique mode of division, termed mitotic somal translocation (MST). Ferret oRGs exhibited diverse behaviors in terms of division location, cleavage angle, and MST distance, as well as fiber orientation and dynamics. We then examined the human fetal cortex and found that a subset of human oRGs displayed similar characteristics, suggesting that diversity in oRG behavior may be a general feature. Similar to the human, ferret oRGs underwent multiple rounds of self-renewing divisions but were more likely to undergo symmetric divisions that expanded the oRG population, as opposed to producing intermediate progenitor cells (IPCs). Differences in oRG behaviors, including proliferative potential and daughter cell fates, may contribute to variations in cortical structure between mammalian species.

@ARTICLE{Pollen2014-ow,
  title    = "Low-coverage single-cell {mRNA} sequencing reveals cellular
              heterogeneity and activated signaling pathways in developing
              cerebral cortex",
  author   = "Pollen, Alex A and Nowakowski, Tomasz J and Shuga, Joe and Wang,
              Xiaohui and Leyrat, Anne A and Lui, Jan H and Li, Nianzhen and
              Szpankowski, Lukasz and Fowler, Brian and Chen, Peilin and
              Ramalingam, Naveen and Sun, Gang and Thu, Myo and Norris, Michael
              and Lebofsky, Ronald and Toppani, Dominique and Kemp, 2nd,
              Darnell W and Wong, Michael and Clerkson, Barry and Jones,
              Brittnee N and Wu, Shiquan and Knutsson, Lawrence and Alvarado,
              Beatriz and Wang, Jing and Weaver, Lesley S and May, Andrew P and
              Jones, Robert C and Unger, Marc A and Kriegstein, Arnold R and
              West, Jay A A",
  abstract = "Large-scale surveys of single-cell gene expression have the
              potential to reveal rare cell populations and lineage
              relationships but require efficient methods for cell capture and
              mRNA sequencing. Although cellular barcoding strategies allow
              parallel sequencing of single cells at ultra-low depths, the
              limitations of shallow sequencing have not been investigated
              directly. By capturing 301 single cells from 11 populations using
              microfluidics and analyzing single-cell transcriptomes across
              downsampled sequencing depths, we demonstrate that shallow
              single-cell mRNA sequencing (~50,000 reads per cell) is
              sufficient for unbiased cell-type classification and biomarker
              identification. In the developing cortex, we identify diverse
              cell types, including multiple progenitor and neuronal subtypes,
              and we identify EGR1 and FOS as previously unreported candidate
              targets of Notch signaling in human but not mouse radial glia.
              Our strategy establishes an efficient method for unbiased
              analysis and comparison of cell populations from heterogeneous
              tissue by microfluidic single-cell capture and low-coverage
              sequencing of many cells.",
  journal  = "Nat Biotechnol",
  volume   =  32,
  number   =  10,
  pages    = "1053--1058",
  month    =  aug,
  year     =  2014,
  language = "en"
}

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing ( 50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

@ARTICLE{Clinton2014-bb,
  title    = "Radial glia in the proliferative ventricular zone of the
              embryonic and adult turtle, Trachemys scripta elegans",
  author   = "Clinton, Brian K and Cunningham, Christopher L and Kriegstein,
              Arnold R and Noctor, Stephen C and Mart{\'\i}nez-Cerde{\~n}o,
              Ver{\'o}nica",
  abstract = "To better understand the role of radial glial (RG) cells in the
              evolution of the mammalian cerebral cortex, we investigated the
              role of RG cells in the dorsal cortex and dorsal ventricular
              ridge of the turtle, Trachemys scripta elegans. Unlike mammals,
              the glial architecture of adult reptile consists mainly of
              ependymoradial glia, which share features with mammalian RG
              cells, and which may contribute to neurogenesis that continues
              throughout the lifespan of the turtle. To evaluate the morphology
              and proliferative capacity of ependymoradial glia (here referred
              to as RG cells) in the dorsal cortex of embryonic and adult
              turtle, we adapted the cortical electroporation technique,
              commonly used in rodents, to the turtle telencephalon. Here, we
              demonstrate the morphological and functional characteristics of
              RG cells in the developing turtle dorsal cortex. We show that
              cell division occurs both at the ventricle and away from the
              ventricle, that RG cells undergo division at the ventricle during
              neurogenic stages of development, and that mitotic Tbr2+
              precursor cells, a hallmark of the mammalian SVZ, are present in
              the turtle cortex. In the adult turtle, we show that RG cells
              encompass a morphologically heterogeneous population,
              particularly in the subpallium where proliferation is most
              prevalent. One RG subtype is similar to RG cells in the
              developing mammalian cortex, while 2 other RG subtypes appear to
              be distinct from those seen in mammal. We propose that the
              different subtypes of RG cells in the adult turtle perform
              distinct functions.",
  journal  = "Neurogenesis (Austin)",
  volume   =  1,
  number   =  1,
  pages    = "e970905",
  month    =  dec,
  year     =  2014,
  keywords = "adult; development; neurogenesis; radial glia; telencephalon;
              turtle; ventricular zone",
  language = "en"
}

To better understand the role of radial glial (RG) cells in the evolution of the mammalian cerebral cortex, we investigated the role of RG cells in the dorsal cortex and dorsal ventricular ridge of the turtle, Trachemys scripta elegans. Unlike mammals, the glial architecture of adult reptile consists mainly of ependymoradial glia, which share features with mammalian RG cells, and which may contribute to neurogenesis that continues throughout the lifespan of the turtle. To evaluate the morphology and proliferative capacity of ependymoradial glia (here referred to as RG cells) in the dorsal cortex of embryonic and adult turtle, we adapted the cortical electroporation technique, commonly used in rodents, to the turtle telencephalon. Here, we demonstrate the morphological and functional characteristics of RG cells in the developing turtle dorsal cortex. We show that cell division occurs both at the ventricle and away from the ventricle, that RG cells undergo division at the ventricle during neurogenic stages of development, and that mitotic Tbr2+ precursor cells, a hallmark of the mammalian SVZ, are present in the turtle cortex. In the adult turtle, we show that RG cells encompass a morphologically heterogeneous population, particularly in the subpallium where proliferation is most prevalent. One RG subtype is similar to RG cells in the developing mammalian cortex, while 2 other RG subtypes appear to be distinct from those seen in mammal. We propose that the different subtypes of RG cells in the adult turtle perform distinct functions.

@ARTICLE{Bershteyn2013-if,
  title    = "Cerebral organoids in a dish: progress and prospects",
  author   = "Bershteyn, Marina and Kriegstein, Arnold R",
  abstract = "A three-dimensional culture of cortical tissues derived from
              pluripotent stem cells offers an opportunity to model human brain
              development and disorders. In a recent issue of Nature, Lancaster
              et al. describe a new method for generating cerebral organoids in
              a dish and use it to model microcephaly.",
  journal  = "Cell",
  volume   =  155,
  number   =  1,
  pages    = "19--20",
  month    =  sep,
  year     =  2013,
  language = "en"
}

A three-dimensional culture of cortical tissues derived from pluripotent stem cells offers an opportunity to model human brain development and disorders. In a recent issue of Nature, Lancaster et al. describe a new method for generating cerebral organoids in a dish and use it to model microcephaly.

@ARTICLE{Visel2013-hi,
  title    = "A high-resolution enhancer atlas of the developing telencephalon",
  author   = "Visel, Axel and Taher, Leila and Girgis, Hani and May, Dalit and
              Golonzhka, Olga and Hoch, Renee V and McKinsey, Gabriel L and
              Pattabiraman, Kartik and Silberberg, Shanni N and Blow, Matthew J
              and Hansen, David V and Nord, Alex S and Akiyama, Jennifer A and
              Holt, Amy and Hosseini, Roya and Phouanenavong, Sengthavy and
              Plajzer-Frick, Ingrid and Shoukry, Malak and Afzal, Veena and
              Kaplan, Tommy and Kriegstein, Arnold R and Rubin, Edward M and
              Ovcharenko, Ivan and Pennacchio, Len A and Rubenstein, John L R",
  abstract = "The mammalian telencephalon plays critical roles in cognition,
              motor function, and emotion. Though many of the genes required
              for its development have been identified, the distant-acting
              regulatory sequences orchestrating their in vivo expression are
              mostly unknown. Here, we describe a digital atlas of in vivo
              enhancers active in subregions of the developing telencephalon.
              We identified more than 4,600 candidate embryonic forebrain
              enhancers and studied the in vivo activity of 329 of these
              sequences in transgenic mouse embryos. We generated serial sets
              of histological brain sections for 145 reproducible forebrain
              enhancers, resulting in a publicly accessible web-based data
              collection comprising more than 32,000 sections. We also used
              epigenomic analysis of human and mouse cortex tissue to directly
              compare the genome-wide enhancer architecture in these species.
              These data provide a primary resource for investigating gene
              regulatory mechanisms of telencephalon development and enable
              studies of the role of distant-acting enhancers in
              neurodevelopmental disorders.",
  journal  = "Cell",
  volume   =  152,
  number   =  4,
  pages    = "895--908",
  month    =  jan,
  year     =  2013,
  language = "en"
}

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

@ARTICLE{LaMonica2013-el,
  title    = "Mitotic spindle orientation predicts outer radial glial cell
              generation in human neocortex",
  author   = "LaMonica, Bridget E and Lui, Jan H and Hansen, David V and
              Kriegstein, Arnold R",
  abstract = "The human neocortex is increased in size and complexity as
              compared with most other species. Neocortical expansion has
              recently been attributed to protracted neurogenesis by outer
              radial glial cells in the outer subventricular zone, a region
              present in humans but not in rodents. The mechanisms of human
              outer radial glial cell generation are unknown, but are proposed
              to involve division of ventricular radial glial cells; neural
              stem cells present in all developing mammals. Here we show that
              human ventricular radial glial cells produce outer radial glial
              cells and seed formation of the outer subventricular zone via
              horizontal divisions, which occur more frequently in humans than
              in rodents. We further find that outer radial glial cell mitotic
              behaviour is cell intrinsic, and that the basal fibre, inherited
              by outer radial glial cells after ventricular radial glial
              division, determines cleavage angle. Our results suggest that
              altered regulation of mitotic spindle orientation increased outer
              radial glial cell number, and ultimately neuronal number, during
              human brain evolution.",
  journal  = "Nat Commun",
  volume   =  4,
  pages    = "1665",
  year     =  2013,
  language = "en"
}

The human neocortex is increased in size and complexity as compared with most other species. Neocortical expansion has recently been attributed to protracted neurogenesis by outer radial glial cells in the outer subventricular zone, a region present in humans but not in rodents. The mechanisms of human outer radial glial cell generation are unknown, but are proposed to involve division of ventricular radial glial cells; neural stem cells present in all developing mammals. Here we show that human ventricular radial glial cells produce outer radial glial cells and seed formation of the outer subventricular zone via horizontal divisions, which occur more frequently in humans than in rodents. We further find that outer radial glial cell mitotic behaviour is cell intrinsic, and that the basal fibre, inherited by outer radial glial cells after ventricular radial glial division, determines cleavage angle. Our results suggest that altered regulation of mitotic spindle orientation increased outer radial glial cell number, and ultimately neuronal number, during human brain evolution.

@ARTICLE{Nicholas2013-rz,
  title    = "Functional maturation of {hPSC-derived} forebrain interneurons
              requires an extended timeline and mimics human neural development",
  author   = "Nicholas, Cory R and Chen, Jiadong and Tang, Yunshuo and
              Southwell, Derek G and Chalmers, Nadine and Vogt, Daniel and
              Arnold, Christine M and Chen, Ying-Jiun J and Stanley, Edouard G
              and Elefanty, Andrew G and Sasai, Yoshiki and Alvarez-Buylla,
              Arturo and Rubenstein, John L R and Kriegstein, Arnold R",
  abstract = "Directed differentiation from human pluripotent stem cells
              (hPSCs) has seen significant progress in recent years. However,
              most differentiated populations exhibit immature properties of an
              early embryonic stage, raising concerns about their ability to
              model and treat disease. Here, we report the directed
              differentiation of hPSCs into medial ganglionic eminence
              (MGE)-like progenitors and their maturation into forebrain type
              interneurons. We find that early-stage progenitors progress via a
              radial glial-like stem cell enriched in the human fetal brain.
              Both in vitro and posttransplantation into the rodent cortex, the
              MGE-like cells develop into GABAergic interneuron subtypes with
              mature physiological properties along a prolonged intrinsic
              timeline of up to 7 months, mimicking endogenous human neural
              development. MGE-derived cortical interneuron deficiencies are
              implicated in a broad range of neurodevelopmental and
              degenerative disorders, highlighting the importance of these
              results for modeling human neural development and disease.",
  journal  = "Cell Stem Cell",
  volume   =  12,
  number   =  5,
  pages    = "573--586",
  month    =  may,
  year     =  2013,
  language = "en"
}

Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. However, most differentiated populations exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early-stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both in vitro and posttransplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to 7 months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease.

@ARTICLE{Hansen2013-zg,
  title    = "Non-epithelial stem cells and cortical interneuron production in
              the human ganglionic eminences",
  author   = "Hansen, David V and Lui, Jan H and Flandin, Pierre and Yoshikawa,
              Kazuaki and Rubenstein, John L and Alvarez-Buylla, Arturo and
              Kriegstein, Arnold R",
  abstract = "GABAergic cortical interneurons underlie the complexity of neural
              circuits and are particularly numerous and diverse in humans. In
              rodents, cortical interneurons originate in the subpallial
              ganglionic eminences, but their developmental origins in humans
              are controversial. We characterized the developing human
              ganglionic eminences and found that the subventricular zone (SVZ)
              expanded massively during the early second trimester, becoming
              densely populated with neural stem cells and intermediate
              progenitor cells. In contrast with the cortex, most stem cells in
              the ganglionic eminence SVZ did not maintain radial fibers or
              orientation. The medial ganglionic eminence exhibited unique
              patterns of progenitor cell organization and clustering, and
              markers revealed that the caudal ganglionic eminence generated a
              greater proportion of cortical interneurons in humans than in
              rodents. On the basis of labeling of newborn neurons in slice
              culture and mapping of proliferating interneuron progenitors, we
              conclude that the vast majority of human cortical interneurons
              are produced in the ganglionic eminences, including an enormous
              contribution from non-epithelial SVZ stem cells.",
  journal  = "Nat Neurosci",
  volume   =  16,
  number   =  11,
  pages    = "1576--1587",
  month    =  oct,
  year     =  2013,
  language = "en"
}

GABAergic cortical interneurons underlie the complexity of neural circuits and are particularly numerous and diverse in humans. In rodents, cortical interneurons originate in the subpallial ganglionic eminences, but their developmental origins in humans are controversial. We characterized the developing human ganglionic eminences and found that the subventricular zone (SVZ) expanded massively during the early second trimester, becoming densely populated with neural stem cells and intermediate progenitor cells. In contrast with the cortex, most stem cells in the ganglionic eminence SVZ did not maintain radial fibers or orientation. The medial ganglionic eminence exhibited unique patterns of progenitor cell organization and clustering, and markers revealed that the caudal ganglionic eminence generated a greater proportion of cortical interneurons in humans than in rodents. On the basis of labeling of newborn neurons in slice culture and mapping of proliferating interneuron progenitors, we conclude that the vast majority of human cortical interneurons are produced in the ganglionic eminences, including an enormous contribution from non-epithelial SVZ stem cells.

@ARTICLE{Chen2013-oe,
  title    = "Use of ``{MGE} enhancers'' for labeling and selection of
              embryonic stem cell-derived medial ganglionic eminence ({MGE})
              progenitors and neurons",
  author   = "Chen, Ying-Jiun J and Vogt, Daniel and Wang, Yanling and Visel,
              Axel and Silberberg, Shanni N and Nicholas, Cory R and Danjo,
              Teruko and Pollack, Joshua L and Pennacchio, Len A and Anderson,
              Stewart and Sasai, Yoshiki and Baraban, Scott C and Kriegstein,
              Arnold R and Alvarez-Buylla, Arturo and Rubenstein, John L R",
  abstract = "The medial ganglionic eminence (MGE) is an embryonic forebrain
              structure that generates the majority of cortical interneurons.
              MGE transplantation into specific regions of the postnatal
              central nervous system modifies circuit function and improves
              deficits in mouse models of epilepsy, Parkinson's disease, pain,
              and phencyclidine-induced cognitive deficits. Herein, we describe
              approaches to generate MGE-like progenitor cells from mouse
              embryonic stem (ES) cells. Using a modified embryoid body method,
              we provided gene expression evidence that mouse ES-derived
              Lhx6(+) cells closely resemble immature interneurons generated
              from authentic MGE-derived Lhx6(+) cells. We hypothesized that
              enhancers that are active in the mouse MGE would be useful tools
              in detecting when ES cells differentiate into MGE cells. Here we
              demonstrate the utility of enhancer elements [422 (DlxI12b),
              Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES
              cell differentiation experiments. We found that enhancers
              DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while
              enhancer 1056 is active in Olig2(+) cells. These data demonstrate
              unique techniques to follow and purify MGE-like derivatives from
              ES cells, including GABAergic cortical interneurons and
              oligodendrocytes, for use in stem cell-based therapeutic assays
              and treatments.",
  journal  = "PLoS One",
  volume   =  8,
  number   =  5,
  pages    = "e61956",
  month    =  may,
  year     =  2013,
  language = "en"
}

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

@ARTICLE{Woodworth2012-ch,
  title    = "{SnapShot}: cortical development",
  author   = "Woodworth, Mollie B and Greig, Luciano Custo and Kriegstein,
              Arnold R and Macklis, Jeffrey D",
  journal  = "Cell",
  volume   =  151,
  number   =  4,
  pages    = "918--918.e1",
  month    =  nov,
  year     =  2012,
  language = "en"
}

@ARTICLE{LaMonica2012-py,
  title    = "{OSVZ} progenitors in the human cortex: an updated perspective on
              neurodevelopmental disease",
  author   = "LaMonica, Bridget E and Lui, Jan H and Wang, Xiaoqun and
              Kriegstein, Arnold R",
  abstract = "Recent discoveries concerning the architecture and cellular
              dynamics of the developing human brain are revealing new
              differences between mouse and human cortical development. In
              mice, neurons are produced by ventricular radial glial (RG) cells
              and subventricular zone intermediate progenitor (IP) cells. In
              the human cortex, both ventricular RG and highly motile outer RG
              cells generate IP cells, which undergo multiple rounds of transit
              amplification in the outer subventricular zone before producing
              neurons. This creates a more complex environment for neurogenesis
              and neuronal migration, adding new arenas in which
              neurodevelopmental disease gene mutation could disrupt
              corticogenesis. A more complete understanding of disease
              mechanisms will involve use of emerging model systems with
              developmental programs more similar to that of the human
              neocortex.",
  journal  = "Curr Opin Neurobiol",
  volume   =  22,
  number   =  5,
  pages    = "747--753",
  month    =  apr,
  year     =  2012,
  language = "en"
}

Recent discoveries concerning the architecture and cellular dynamics of the developing human brain are revealing new differences between mouse and human cortical development. In mice, neurons are produced by ventricular radial glial (RG) cells and subventricular zone intermediate progenitor (IP) cells. In the human cortex, both ventricular RG and highly motile outer RG cells generate IP cells, which undergo multiple rounds of transit amplification in the outer subventricular zone before producing neurons. This creates a more complex environment for neurogenesis and neuronal migration, adding new arenas in which neurodevelopmental disease gene mutation could disrupt corticogenesis. A more complete understanding of disease mechanisms will involve use of emerging model systems with developmental programs more similar to that of the human neocortex.

@ARTICLE{Harwell2012-dq,
  title    = "Sonic hedgehog expression in corticofugal projection neurons
              directs cortical microcircuit formation",
  author   = "Harwell, Corey C and Parker, Philip R L and Gee, Steven M and
              Okada, Ami and McConnell, Susan K and Kreitzer, Anatol C and
              Kriegstein, Arnold R",
  abstract = "The precise connectivity of inputs and outputs is critical for
              cerebral cortex function; however, the cellular mechanisms that
              establish these connections are poorly understood. Here, we show
              that the secreted molecule Sonic Hedgehog (Shh) is involved in
              synapse formation of a specific cortical circuit. Shh is
              expressed in layer V corticofugal projection neurons and the Shh
              receptor, Brother of CDO (Boc), is expressed in local and
              callosal projection neurons of layer II/III that synapse onto the
              subcortical projection neurons. Layer V neurons of mice lacking
              functional Shh exhibit decreased synapses. Conversely, the loss
              of functional Boc leads to a reduction in the strength of
              synaptic connections onto layer Vb, but not layer II/III,
              pyramidal neurons. These results demonstrate that Shh is
              expressed in postsynaptic target cells while Boc is expressed in
              a complementary population of presynaptic input neurons, and they
              function to guide the formation of cortical microcircuitry.",
  journal  = "Neuron",
  volume   =  73,
  number   =  6,
  pages    = "1116--1126",
  month    =  mar,
  year     =  2012,
  language = "en"
}

The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry.

@ARTICLE{Chen2012-yi,
  title    = "Regulation of microtubule stability and organization by mammalian
              Par3 in specifying neuronal polarity",
  author   = "Chen, She and Chen, Jia and Shi, Hang and Wei, Michelle and
              Castaneda-Castellanos, David R and Bultje, Ronald S and Pei, Xin
              and Kriegstein, Arnold R and Zhang, Mingjie and Shi, Song-Hai",
  abstract = "Polarization of mammalian neurons with a specified axon requires
              precise regulation of microtubule and actin dynamics in the
              developing neurites. Here we show that mammalian partition
              defective 3 (mPar3), a key component of the Par polarity complex
              that regulates the polarization of many cell types including
              neurons, directly regulates microtubule stability and
              organization. The N-terminal portion of mPar3 exhibits strong
              microtubule binding, bundling, and stabilization activity, which
              can be suppressed by its C-terminal portion via an intramolecular
              interaction. Interestingly, the intermolecular oligomerization of
              mPar3 is able to relieve the intramolecular interaction and
              thereby promote microtubule bundling and stabilization.
              Furthermore, disruption of this microtubule regulatory activity
              of mPar3 impairs its function in axon specification. Together,
              these results demonstrate a role for mPar3 in directly regulating
              microtubule organization that is crucial for neuronal
              polarization.",
  journal  = "Dev Cell",
  volume   =  24,
  number   =  1,
  pages    = "26--40",
  month    =  dec,
  year     =  2012,
  language = "en"
}

Polarization of mammalian neurons with a specified axon requires precise regulation of microtubule and actin dynamics in the developing neurites. Here we show that mammalian partition defective 3 (mPar3), a key component of the Par polarity complex that regulates the polarization of many cell types including neurons, directly regulates microtubule stability and organization. The N-terminal portion of mPar3 exhibits strong microtubule binding, bundling, and stabilization activity, which can be suppressed by its C-terminal portion via an intramolecular interaction. Interestingly, the intermolecular oligomerization of mPar3 is able to relieve the intramolecular interaction and thereby promote microtubule bundling and stabilization. Furthermore, disruption of this microtubule regulatory activity of mPar3 impairs its function in axon specification. Together, these results demonstrate a role for mPar3 in directly regulating microtubule organization that is crucial for neuronal polarization.

@ARTICLE{Plath2012-in,
  title    = "Stem cells in the land of the rising sun: {ISSCR} 2012",
  author   = "Plath, Kathrin and Srivastava, Deepak and Alvarez-Buylla, Arturo
              and Tanaka, Elly M and Kriegstein, Arnold R",
  abstract = "The 2012 annual meeting of the International Society for Stem
              Cell Research (ISSCR) marked the Tenth Anniversary of the ISSCR.
              Held in Japan, the meeting showcased recent discoveries and
              surveyed the remarkable progress that has been made in a decade
              of stem cell research.",
  journal  = "Cell Stem Cell",
  volume   =  11,
  number   =  5,
  pages    = "607--614",
  month    =  nov,
  year     =  2012,
  address  = "United States",
  language = "en"
}

The 2012 annual meeting of the International Society for Stem Cell Research (ISSCR) marked the Tenth Anniversary of the ISSCR. Held in Japan, the meeting showcased recent discoveries and surveyed the remarkable progress that has been made in a decade of stem cell research.

@ARTICLE{Lui2011-tn,
  title    = "Development and evolution of the human neocortex",
  author   = "Lui, Jan H and Hansen, David V and Kriegstein, Arnold R",
  abstract = "The size and surface area of the mammalian brain are thought to
              be critical determinants of intellectual ability. Recent studies
              show that development of the gyrated human neocortex involves a
              lineage of neural stem and transit-amplifying cells that forms
              the outer subventricular zone (OSVZ), a proliferative region
              outside the ventricular epithelium. We discuss how proliferation
              of cells within the OSVZ expands the neocortex by increasing
              neuron number and modifying the trajectory of migrating neurons.
              Relating these features to other mammalian species and known
              molecular regulators of the mouse neocortex suggests how this
              developmental process could have emerged in evolution.",
  journal  = "Cell",
  volume   =  146,
  number   =  1,
  pages    = "18--36",
  month    =  jul,
  year     =  2011,
  language = "en"
}

The size and surface area of the mammalian brain are thought to be critical determinants of intellectual ability. Recent studies show that development of the gyrated human neocortex involves a lineage of neural stem and transit-amplifying cells that forms the outer subventricular zone (OSVZ), a proliferative region outside the ventricular epithelium. We discuss how proliferation of cells within the OSVZ expands the neocortex by increasing neuron number and modifying the trajectory of migrating neurons. Relating these features to other mammalian species and known molecular regulators of the mouse neocortex suggests how this developmental process could have emerged in evolution.

@ARTICLE{Hansen2011-ce,
  title    = "Deriving excitatory neurons of the neocortex from pluripotent
              stem cells",
  author   = "Hansen, David V and Rubenstein, John L R and Kriegstein, Arnold R",
  abstract = "The human cerebral cortex is an immensely complex structure that
              subserves critical functions that can be disrupted in
              developmental and degenerative disorders. Recent innovations in
              cellular reprogramming and differentiation techniques have
              provided new ways to study the cellular components of the
              cerebral cortex. Here, we discuss approaches to generate specific
              subtypes of excitatory cortical neurons from pluripotent stem
              cells. We review spatial and temporal aspects of cortical neuron
              specification that can guide efforts to produce excitatory neuron
              subtypes with increased resolution. Finally, we discuss
              distinguishing features of human cortical development and their
              translational ramifications for cortical stem cell technologies.",
  journal  = "Neuron",
  volume   =  70,
  number   =  4,
  pages    = "645--660",
  month    =  may,
  year     =  2011,
  language = "en"
}

The human cerebral cortex is an immensely complex structure that subserves critical functions that can be disrupted in developmental and degenerative disorders. Recent innovations in cellular reprogramming and differentiation techniques have provided new ways to study the cellular components of the cerebral cortex. Here, we discuss approaches to generate specific subtypes of excitatory cortical neurons from pluripotent stem cells. We review spatial and temporal aspects of cortical neuron specification that can guide efforts to produce excitatory neuron subtypes with increased resolution. Finally, we discuss distinguishing features of human cortical development and their translational ramifications for cortical stem cell technologies.

@ARTICLE{Wang2011-lb,
  title    = "Orienting fate: spatial regulation of neurogenic divisions",
  author   = "Wang, Xiaoqun and Lui, Jan H and Kriegstein, Arnold R",
  abstract = "Cleavage plane orientation has been thought to govern the fate of
              neural stem cell progeny, but supporting evidence in the
              neocortex has been sparse. A new study by Postiglione et al. in
              this issue of Neuron shows that mouse Inscuteable-mediated
              control of cleavage plane orientation regulates the output of
              neural progenitor cells.",
  journal  = "Neuron",
  volume   =  72,
  number   =  2,
  pages    = "191--193",
  month    =  oct,
  year     =  2011,
  language = "en"
}

Cleavage plane orientation has been thought to govern the fate of neural stem cell progeny, but supporting evidence in the neocortex has been sparse. A new study by Postiglione et al. in this issue of Neuron shows that mouse Inscuteable-mediated control of cleavage plane orientation regulates the output of neural progenitor cells.

@ARTICLE{Wang2011-tj,
  title    = "A new subtype of progenitor cell in the mouse embryonic neocortex",
  author   = "Wang, Xiaoqun and Tsai, Jin-Wu and LaMonica, Bridget and
              Kriegstein, Arnold R",
  abstract = "A hallmark of mammalian brain evolution is cortical expansion,
              which reflects an increase in the number of cortical neurons
              established by the progenitor cell subtypes present and the
              number of their neurogenic divisions. Recent studies have
              revealed a new class of radial glia-like (oRG) progenitor cells
              in the human brain, which reside in the outer subventricular
              zone. Expansion of the subventricular zone and appearance of oRG
              cells may have been essential evolutionary steps leading from
              lissencephalic to gyrencephalic neocortex. Here we show that
              oRG-like progenitor cells are present in the mouse embryonic
              neocortex. They arise from asymmetric divisions of radial glia
              and undergo self-renewing asymmetric divisions to generate
              neurons. Moreover, mouse oRG cells undergo mitotic somal
              translocation whereby centrosome movement into the basal process
              during interphase precedes nuclear translocation. Our finding of
              oRG cells in the developing rodent brain fills a gap in our
              understanding of neocortical expansion.",
  journal  = "Nat Neurosci",
  volume   =  14,
  number   =  5,
  pages    = "555--561",
  month    =  apr,
  year     =  2011,
  language = "en"
}

A hallmark of mammalian brain evolution is cortical expansion, which reflects an increase in the number of cortical neurons established by the progenitor cell subtypes present and the number of their neurogenic divisions. Recent studies have revealed a new class of radial glia-like (oRG) progenitor cells in the human brain, which reside in the outer subventricular zone. Expansion of the subventricular zone and appearance of oRG cells may have been essential evolutionary steps leading from lissencephalic to gyrencephalic neocortex. Here we show that oRG-like progenitor cells are present in the mouse embryonic neocortex. They arise from asymmetric divisions of radial glia and undergo self-renewing asymmetric divisions to generate neurons. Moreover, mouse oRG cells undergo mitotic somal translocation whereby centrosome movement into the basal process during interphase precedes nuclear translocation. Our finding of oRG cells in the developing rodent brain fills a gap in our understanding of neocortical expansion.

@ARTICLE{Ihrie2011-pd,
  title    = "Persistent sonic hedgehog signaling in adult brain determines
              neural stem cell positional identity",
  author   = "Ihrie, Rebecca A and Shah, Jugal K and Harwell, Corey C and
              Levine, Jacob H and Guinto, Cristina D and Lezameta, Melissa and
              Kriegstein, Arnold R and Alvarez-Buylla, Arturo",
  abstract = "Neural stem cells (NSCs) persist in the subventricular zone (SVZ)
              of the adult brain. Location within this germinal region
              determines the type of neuronal progeny NSCs generate, but the
              mechanism of adult NSC positional specification remains unknown.
              We show that sonic hedgehog (Shh) signaling, resulting in high
              gli1 levels, occurs in the ventral SVZ and is associated with the
              genesis of specific neuronal progeny. Shh is selectively produced
              by a small group of ventral forebrain neurons. Ablation of Shh
              decreases production of ventrally derived neuron types, while
              ectopic activation of this pathway in dorsal NSCs respecifies
              their progeny to deep granule interneurons and calbindin-positive
              periglomerular cells. These results show that Shh is necessary
              and sufficient for the specification of adult ventral NSCs.",
  journal  = "Neuron",
  volume   =  71,
  number   =  2,
  pages    = "250--262",
  month    =  jul,
  year     =  2011,
  language = "en"
}

Neural stem cells (NSCs) persist in the subventricular zone (SVZ) of the adult brain. Location within this germinal region determines the type of neuronal progeny NSCs generate, but the mechanism of adult NSC positional specification remains unknown. We show that sonic hedgehog (Shh) signaling, resulting in high gli1 levels, occurs in the ventral SVZ and is associated with the genesis of specific neuronal progeny. Shh is selectively produced by a small group of ventral forebrain neurons. Ablation of Shh decreases production of ventrally derived neuron types, while ectopic activation of this pathway in dorsal NSCs respecifies their progeny to deep granule interneurons and calbindin-positive periglomerular cells. These results show that Shh is necessary and sufficient for the specification of adult ventral NSCs.

@ARTICLE{Rowitch2010-rj,
  title    = "Developmental genetics of vertebrate glial-cell specification",
  author   = "Rowitch, David H and Kriegstein, Arnold R",
  abstract = "Oligodendrocytes and astrocytes are macroglial cells of the
              vertebrate central nervous system. These cells have diverse roles
              in the maintenance of neurological function. In the embryo, the
              genetic mechanisms that underlie the specification of macroglial
              precursors in vivo appear strikingly similar to those that
              regulate the development of the diverse neuron types. The switch
              from producing neuronal to glial subtype-specific precursors can
              be modelled as an interplay between region-restricted components
              and temporal regulators that determine neurogenic or gliogenic
              phases of development, contributing to glial diversity. Gaining
              insight into the developmental genetics of macroglia has great
              potential to improve our understanding of a variety of
              neurological disorders in humans.",
  journal  = "Nature",
  volume   =  468,
  number   =  7321,
  pages    = "214--222",
  month    =  nov,
  year     =  2010,
  address  = "England",
  language = "en"
}

Oligodendrocytes and astrocytes are macroglial cells of the vertebrate central nervous system. These cells have diverse roles in the maintenance of neurological function. In the embryo, the genetic mechanisms that underlie the specification of macroglial precursors in vivo appear strikingly similar to those that regulate the development of the diverse neuron types. The switch from producing neuronal to glial subtype-specific precursors can be modelled as an interplay between region-restricted components and temporal regulators that determine neurogenic or gliogenic phases of development, contributing to glial diversity. Gaining insight into the developmental genetics of macroglia has great potential to improve our understanding of a variety of neurological disorders in humans.

@ARTICLE{Nicholas2010-sk,
  title    = "Regenerative medicine: Cell reprogramming gets direct",
  author   = "Nicholas, Cory R and Kriegstein, Arnold R",
  journal  = "Nature",
  volume   =  463,
  number   =  7284,
  pages    = "1031--1032",
  month    =  feb,
  year     =  2010,
  address  = "England",
  language = "en"
}

@ARTICLE{Hansen2010-pi,
  title    = "Neurogenic radial glia in the outer subventricular zone of human
              neocortex",
  author   = "Hansen, David V and Lui, Jan H and Parker, Philip R L and
              Kriegstein, Arnold R",
  abstract = "Neurons in the developing rodent cortex are generated from radial
              glial cells that function as neural stem cells. These epithelial
              cells line the cerebral ventricles and generate intermediate
              progenitor cells that migrate into the subventricular zone (SVZ)
              and proliferate to increase neuronal number. The developing human
              SVZ has a massively expanded outer region (OSVZ) thought to
              contribute to cortical size and complexity. However, OSVZ
              progenitor cell types and their contribution to neurogenesis are
              not well understood. Here we show that large numbers of radial
              glia-like cells and intermediate progenitor cells populate the
              human OSVZ. We find that OSVZ radial glia-like cells have a long
              basal process but, surprisingly, are non-epithelial as they lack
              contact with the ventricular surface. Using real-time imaging and
              clonal analysis, we demonstrate that these cells can undergo
              proliferative divisions and self-renewing asymmetric divisions to
              generate neuronal progenitor cells that can proliferate further.
              We also show that inhibition of Notch signalling in OSVZ
              progenitor cells induces their neuronal differentiation. The
              establishment of non-ventricular radial glia-like cells may have
              been a critical evolutionary advance underlying increased
              cortical size and complexity in the human brain.",
  journal  = "Nature",
  volume   =  464,
  number   =  7288,
  pages    = "554--561",
  month    =  mar,
  year     =  2010,
  address  = "England",
  language = "en"
}

Neurons in the developing rodent cortex are generated from radial glial cells that function as neural stem cells. These epithelial cells line the cerebral ventricles and generate intermediate progenitor cells that migrate into the subventricular zone (SVZ) and proliferate to increase neuronal number. The developing human SVZ has a massively expanded outer region (OSVZ) thought to contribute to cortical size and complexity. However, OSVZ progenitor cell types and their contribution to neurogenesis are not well understood. Here we show that large numbers of radial glia-like cells and intermediate progenitor cells populate the human OSVZ. We find that OSVZ radial glia-like cells have a long basal process but, surprisingly, are non-epithelial as they lack contact with the ventricular surface. Using real-time imaging and clonal analysis, we demonstrate that these cells can undergo proliferative divisions and self-renewing asymmetric divisions to generate neuronal progenitor cells that can proliferate further. We also show that inhibition of Notch signalling in OSVZ progenitor cells induces their neuronal differentiation. The establishment of non-ventricular radial glia-like cells may have been a critical evolutionary advance underlying increased cortical size and complexity in the human brain.

@ARTICLE{Tsai2010-jd,
  title    = "Kinesin 3 and cytoplasmic dynein mediate interkinetic nuclear
              migration in neural stem cells",
  author   = "Tsai, Jin-Wu and Lian, Wei-Nan and Kemal, Shahrnaz and
              Kriegstein, Arnold R and Vallee, Richard B",
  abstract = "Radial glial progenitor cells exhibit bidirectional cell
              cycle-dependent nuclear oscillations. The purpose and underlying
              mechanism of this unusual 'interkinetic nuclear migration' are
              poorly understood. We investigated the basis for this behavior by
              live imaging of nuclei, centrosomes and microtubules in embryonic
              rat brain slices, coupled with the use of RNA interference (RNAi)
              and the myosin inhibitor blebbistatin. We found that nuclei
              migrated independent of centrosomes and unidirectionally away
              from or toward the ventricular surface along microtubules, which
              were uniformly oriented from the ventricular surface to the pial
              surface of the brain. RNAi directed against cytoplasmic dynein
              specifically inhibited nuclear movement toward the apical
              surface. An RNAi screen of kinesin genes identified Kif1a, a
              member of the kinesin-3 family, as the motor for basally directed
              nuclear movement. These observations provide direct evidence that
              kinesins are involved in nuclear migration and neurogenesis and
              suggest that a cell cycle-dependent switch between distinct
              microtubule motors drives interkinetic nuclear migration.",
  journal  = "Nat Neurosci",
  volume   =  13,
  number   =  12,
  pages    = "1463--1471",
  month    =  oct,
  year     =  2010,
  language = "en"
}

Radial glial progenitor cells exhibit bidirectional cell cycle-dependent nuclear oscillations. The purpose and underlying mechanism of this unusual 'interkinetic nuclear migration' are poorly understood. We investigated the basis for this behavior by live imaging of nuclei, centrosomes and microtubules in embryonic rat brain slices, coupled with the use of RNA interference (RNAi) and the myosin inhibitor blebbistatin. We found that nuclei migrated independent of centrosomes and unidirectionally away from or toward the ventricular surface along microtubules, which were uniformly oriented from the ventricular surface to the pial surface of the brain. RNAi directed against cytoplasmic dynein specifically inhibited nuclear movement toward the apical surface. An RNAi screen of kinesin genes identified Kif1a, a member of the kinesin-3 family, as the motor for basally directed nuclear movement. These observations provide direct evidence that kinesins are involved in nuclear migration and neurogenesis and suggest that a cell cycle-dependent switch between distinct microtubule motors drives interkinetic nuclear migration.

@ARTICLE{Elias2010-st,
  title    = "Connexin 43 mediates the tangential to radial migratory switch in
              ventrally derived cortical interneurons",
  author   = "Elias, Laura A B and Turmaine, Mark and Parnavelas, John G and
              Kriegstein, Arnold R",
  abstract = "The adult cerebral cortex is composed of excitatory and
              inhibitory neurons that arise from progenitor cells in disparate
              proliferative regions in the developing brain and follow
              different migratory paths. Excitatory pyramidal neurons originate
              near the ventricle and migrate radially to their position in the
              cortical plate along radial glial fibers. On the other hand,
              inhibitory interneurons arise in the ventral telencephalon and
              migrate tangentially to enter the developing cortex before
              migrating radially to reach their correct laminar position. Gap
              junction adhesion has been shown to play an important mechanistic
              role in the radial migration of excitatory neurons. We asked
              whether a similar mechanism governs the tangential or radial
              migration of inhibitory interneurons. Using short hairpin RNA
              knockdown of Connexin 43 (Cx43) and Cx26 together with rescue
              experiments, we found that gap junctions are dispensable for the
              tangential migration of interneurons, but that Cx43 plays a role
              in the switch from tangential to radial migration that allows
              interneurons to enter the cortical plate and find their correct
              laminar position. Moreover this action is dependent on the
              adhesive properties and the C terminus of Cx43 but not the Cx43
              channel. Thus, the radial phase of interneuron migration
              resembles that of excitatory neuron migration in terms of
              dependence on Cx43 adhesion. Furthermore, gap junctions between
              migrating interneurons and radial processes were observed by
              electron microscopy. These findings provide mechanistic and
              structural support for a gap junction-mediated interaction
              between migrating interneurons and radial glia during the switch
              from tangential to radial migration.",
  journal  = "J Neurosci",
  volume   =  30,
  number   =  20,
  pages    = "7072--7077",
  month    =  may,
  year     =  2010,
  language = "en"
}

The adult cerebral cortex is composed of excitatory and inhibitory neurons that arise from progenitor cells in disparate proliferative regions in the developing brain and follow different migratory paths. Excitatory pyramidal neurons originate near the ventricle and migrate radially to their position in the cortical plate along radial glial fibers. On the other hand, inhibitory interneurons arise in the ventral telencephalon and migrate tangentially to enter the developing cortex before migrating radially to reach their correct laminar position. Gap junction adhesion has been shown to play an important mechanistic role in the radial migration of excitatory neurons. We asked whether a similar mechanism governs the tangential or radial migration of inhibitory interneurons. Using short hairpin RNA knockdown of Connexin 43 (Cx43) and Cx26 together with rescue experiments, we found that gap junctions are dispensable for the tangential migration of interneurons, but that Cx43 plays a role in the switch from tangential to radial migration that allows interneurons to enter the cortical plate and find their correct laminar position. Moreover this action is dependent on the adhesive properties and the C terminus of Cx43 but not the Cx43 channel. Thus, the radial phase of interneuron migration resembles that of excitatory neuron migration in terms of dependence on Cx43 adhesion. Furthermore, gap junctions between migrating interneurons and radial processes were observed by electron microscopy. These findings provide mechanistic and structural support for a gap junction-mediated interaction between migrating interneurons and radial glia during the switch from tangential to radial migration.

@ARTICLE{Wang2010-ab,
  title    = "Blocking early {GABA} depolarization with bumetanide results in
              permanent alterations in cortical circuits and sensorimotor
              gating deficits",
  author   = "Wang, Doris D and Kriegstein, Arnold R",
  abstract = "A high incidence of seizures occurs during the neonatal period
              when immature networks are hyperexcitable and susceptible to
              hypersyncrhonous activity. During development,
              $\gamma$-aminobutyric acid (GABA), the primary inhibitory
              neurotransmitter in adults, typically excites neurons due to high
              expression of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). NKCC1
              facilitates seizures because it renders GABA activity excitatory
              through intracellular Cl(-) accumulation, while blocking NKCC1
              with bumetanide suppresses seizures. Bumetanide is currently
              being tested in clinical trials for treatment of neonatal
              seizures. By blocking NKCC1 with bumetanide during cortical
              development, we found a critical period for the development of
              $\alpha$-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
              synapses. Disruption of GABA signaling during this window
              resulted in permanent decreases in excitatory synaptic
              transmission and sensorimotor gating deficits, a common feature
              in schizophrenia. Our study identifies an essential role for
              GABA-mediated depolarization in regulating the balance between
              cortical excitation and inhibition during a critical period and
              suggests a cautionary approach for using bumetanide in treating
              neonatal seizures.",
  journal  = "Cereb Cortex",
  volume   =  21,
  number   =  3,
  pages    = "574--587",
  month    =  jul,
  year     =  2010,
  language = "en"
}

A high incidence of seizures occurs during the neonatal period when immature networks are hyperexcitable and susceptible to hypersyncrhonous activity. During development, $γ$-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in adults, typically excites neurons due to high expression of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). NKCC1 facilitates seizures because it renders GABA activity excitatory through intracellular Cl(-) accumulation, while blocking NKCC1 with bumetanide suppresses seizures. Bumetanide is currently being tested in clinical trials for treatment of neonatal seizures. By blocking NKCC1 with bumetanide during cortical development, we found a critical period for the development of $α$-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate synapses. Disruption of GABA signaling during this window resulted in permanent decreases in excitatory synaptic transmission and sensorimotor gating deficits, a common feature in schizophrenia. Our study identifies an essential role for GABA-mediated depolarization in regulating the balance between cortical excitation and inhibition during a critical period and suggests a cautionary approach for using bumetanide in treating neonatal seizures.

@ARTICLE{Martinez-Cerdeno2010-xt,
  title    = "Embryonic {MGE} precursor cells grafted into adult rat striatum
              integrate and ameliorate motor symptoms in {6-OHDA-lesioned} rats",
  author   = "Mart{\'\i}nez-Cerde{\~n}o, Ver{\'o}nica and Noctor, Stephen C and
              Espinosa, Ana and Ariza, Jeanelle and Parker, Philip and Orasji,
              Samantha and Daadi, Marcel M and Bankiewicz, Krystof and
              Alvarez-Buylla, Arturo and Kriegstein, Arnold R",
  abstract = "We investigated a strategy to ameliorate the motor symptoms of
              rats that received 6-hydroxydopamine (6-OHDA) lesions, a rodent
              model of Parkinson's disease, through transplantation of
              embryonic medial ganglionic eminence (MGE) cells into the
              striatum. During brain development, embryonic MGE cells migrate
              into the striatum and neocortex where they mature into GABAergic
              interneurons and play a key role in establishing the balance
              between excitation and inhibition. Unlike most other embryonic
              neurons, MGE cells retain the capacity for migration and
              integration when transplanted into the postnatal and adult brain.
              We performed MGE cell transplantation into the basal ganglia of
              control and 6-OHDA-lesioned rats. Transplanted MGE cells
              survived, differentiated into GABA(+) neurons, integrated into
              host circuitry, and modified motor behavior in both lesioned and
              control rats. Our data suggest that MGE cell transplantation into
              the striatum is a promising approach to investigate the potential
              benefits of remodeling basal ganglia circuitry in
              neurodegenerative diseases.",
  journal  = "Cell Stem Cell",
  volume   =  6,
  number   =  3,
  pages    = "238--250",
  month    =  mar,
  year     =  2010,
  language = "en"
}

We investigated a strategy to ameliorate the motor symptoms of rats that received 6-hydroxydopamine (6-OHDA) lesions, a rodent model of Parkinson's disease, through transplantation of embryonic medial ganglionic eminence (MGE) cells into the striatum. During brain development, embryonic MGE cells migrate into the striatum and neocortex where they mature into GABAergic interneurons and play a key role in establishing the balance between excitation and inhibition. Unlike most other embryonic neurons, MGE cells retain the capacity for migration and integration when transplanted into the postnatal and adult brain. We performed MGE cell transplantation into the basal ganglia of control and 6-OHDA-lesioned rats. Transplanted MGE cells survived, differentiated into GABA(+) neurons, integrated into host circuitry, and modified motor behavior in both lesioned and control rats. Our data suggest that MGE cell transplantation into the striatum is a promising approach to investigate the potential benefits of remodeling basal ganglia circuitry in neurodegenerative diseases.

@ARTICLE{Kriegstein2009-fa,
  title    = "Commentary: the prospect of cell-based therapy for epilepsy",
  author   = "Kriegstein, Arnold R and Pitk{\"a}nen, Asla",
  abstract = "About 30\% of patient with epilepsy do not respond to available
              antiepileptic drugs. In addition to seizure suppression, novel
              approaches are needed to prevent or alleviate epileptogenic
              process after various types of brain injuries. The use of cell
              transplants as factories to produce endogeneous anticonvulsants
              or as bricks to repair abnormal ictogenic and epileptogenic
              neuronal circuits has generated hope that cell-based therapies
              could become a novel therapeutic category in the treatment
              arsenal of epilepsy. Herein we summarize the current status and
              future perspectives of cell-based therapies in the treatment of
              epilepsy.",
  journal  = "Neurotherapeutics",
  volume   =  6,
  number   =  2,
  pages    = "295--299",
  month    =  apr,
  year     =  2009,
  language = "en"
}

About 30% of patient with epilepsy do not respond to available antiepileptic drugs. In addition to seizure suppression, novel approaches are needed to prevent or alleviate epileptogenic process after various types of brain injuries. The use of cell transplants as factories to produce endogeneous anticonvulsants or as bricks to repair abnormal ictogenic and epileptogenic neuronal circuits has generated hope that cell-based therapies could become a novel therapeutic category in the treatment arsenal of epilepsy. Herein we summarize the current status and future perspectives of cell-based therapies in the treatment of epilepsy.

@ARTICLE{Wang2009-yl,
  title    = "Defining the role of {GABA} in cortical development",
  author   = "Wang, Doris D and Kriegstein, Arnold R",
  abstract = "Of the many signals in the developing nervous system, GABA
              (gamma-aminobutyric acid) has been shown to be one of the
              earliest neurotransmitters present. Unlike in the adult, where
              this transmitter acts synaptically to inhibit neurons, during
              development, GABA can depolarize progenitor cells and their
              progeny due to their high intracellular chloride concentration.
              This early form of GABA signalling may provide the main
              excitatory drive for the immature cortical network and play a
              central role in regulating cortical development. Many features of
              GABA signalling are conserved in different species and are
              recapitulated during neurogenesis in the adult brain,
              demonstrating the importance of this versatile molecule in
              driving cortical formation. Here, we present recent evidence
              supporting the multiple functions of GABA during embryonic
              development and adult neurogenesis, from regulating progenitor
              proliferation to influencing the migration and maturation of
              newborn neurons.",
  journal  = "J Physiol",
  volume   =  587,
  number   = "Pt 9",
  pages    = "1873--1879",
  month    =  jan,
  year     =  2009,
  language = "en"
}

% The entry below contains non-ASCII chars that could not be converted
% to a LaTeX equivalent.

Of the many signals in the developing nervous system, GABA (gamma-aminobutyric acid) has been shown to be one of the earliest neurotransmitters present. Unlike in the adult, where this transmitter acts synaptically to inhibit neurons, during development, GABA can depolarize progenitor cells and their progeny due to their high intracellular chloride concentration. This early form of GABA signalling may provide the main excitatory drive for the immature cortical network and play a central role in regulating cortical development. Many features of GABA signalling are conserved in different species and are recapitulated during neurogenesis in the adult brain, demonstrating the importance of this versatile molecule in driving cortical formation. Here, we present recent evidence supporting the multiple functions of GABA during embryonic development and adult neurogenesis, from regulating progenitor proliferation to influencing the migration and maturation of newborn neurons.

@ARTICLE{Bultje2009-tx,
  title    = "Mammalian Par3 regulates progenitor cell asymmetric division via
              notch signaling in the developing neocortex",
  author   = "Bultje, Ronald S and Castaneda-Castellanos, David R and Jan, Lily
              Yeh and Jan, Yuh-Nung and Kriegstein, Arnold R and Shi, Song-Hai",
  abstract = "Asymmetric cell division of radial glial progenitors produces
              neurons while allowing self-renewal; however, little is known
              about the mechanism that generates asymmetry in daughter cell
              fate specification. Here, we found that mammalian partition
              defective protein 3 (mPar3), a key cell polarity determinant,
              exhibits dynamic distribution in radial glial progenitors. While
              it is enriched at the lateral membrane domain in the ventricular
              endfeet during interphase, mPar3 becomes dispersed and shows
              asymmetric localization as cell cycle progresses. Either removal
              or ectopic expression of mPar3 prevents radial glial progenitors
              from dividing asymmetrically yet generates different outcomes in
              daughter cell fate specification. Furthermore, the expression
              level of mPar3 affects Notch signaling, and manipulations of
              Notch signaling or Numb expression suppress mPar3 regulation of
              radial glial cell division and daughter cell fate specification.
              These results reveal a critical molecular pathway underlying
              asymmetric cell division of radial glial progenitors in the
              mammalian neocortex.",
  journal  = "Neuron",
  volume   =  63,
  number   =  2,
  pages    = "189--202",
  month    =  jul,
  year     =  2009,
  language = "en"
}

Asymmetric cell division of radial glial progenitors produces neurons while allowing self-renewal; however, little is known about the mechanism that generates asymmetry in daughter cell fate specification. Here, we found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or Numb expression suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex.

@ARTICLE{LoTurco2008-an,
  title    = "Manipulating midbrain stem cell self-renewal",
  author   = "LoTurco, Joseph J and Kriegstein, Arnold R",
  abstract = "In this issue of Cell Stem Cell, Falk and colleagues (Falk et
              al., 2008) demonstrate that differential responsiveness to
              TGF-beta signaling selectively modulates self-renewal of dorsal
              midbrain stem cells. This observation may lead to strategies for
              expanding specific neural stem cell subtypes.",
  journal  = "Cell Stem Cell",
  volume   =  2,
  number   =  5,
  pages    = "405--406",
  month    =  may,
  year     =  2008,
  language = "en"
}

In this issue of Cell Stem Cell, Falk and colleagues (Falk et al., 2008) demonstrate that differential responsiveness to TGF-beta signaling selectively modulates self-renewal of dorsal midbrain stem cells. This observation may lead to strategies for expanding specific neural stem cell subtypes.

@ARTICLE{Elias2008-av,
  title    = "Gap junctions: multifaceted regulators of embryonic cortical
              development",
  author   = "Elias, Laura A B and Kriegstein, Arnold R",
  abstract = "The morphological development of the cerebral cortex from a
              primitive neuroepithelium into a complex laminar structure
              underlying higher cognition must rely on a network of
              intercellular signaling. Gap junctions are widely expressed
              during embryonic development and provide a means of cell-cell
              contact and communication. We review the roles of gap junctions
              in regulating the proliferation of neural progenitors as well as
              the migration and differentiation of young neurons in the
              embryonic cerebral cortex. There is substantial evidence that
              although gap junctions act in the classical manner coupling
              neural progenitors, they also act as hemichannels mediating the
              spread of calcium waves across progenitor cell populations and as
              adhesive molecules aiding neuronal migration. Gap junctions are
              thus emerging as multifaceted regulators of cortical development
              playing diverse roles in intercellular communication.",
  journal  = "Trends Neurosci",
  volume   =  31,
  number   =  5,
  pages    = "243--250",
  month    =  apr,
  year     =  2008,
  language = "en"
}

The morphological development of the cerebral cortex from a primitive neuroepithelium into a complex laminar structure underlying higher cognition must rely on a network of intercellular signaling. Gap junctions are widely expressed during embryonic development and provide a means of cell-cell contact and communication. We review the roles of gap junctions in regulating the proliferation of neural progenitors as well as the migration and differentiation of young neurons in the embryonic cerebral cortex. There is substantial evidence that although gap junctions act in the classical manner coupling neural progenitors, they also act as hemichannels mediating the spread of calcium waves across progenitor cell populations and as adhesive molecules aiding neuronal migration. Gap junctions are thus emerging as multifaceted regulators of cortical development playing diverse roles in intercellular communication.

@ARTICLE{Elias2008-jl,
  title    = "A time and a place for nkx2-1 in interneuron specification and
              migration",
  author   = "Elias, Laura A B and Potter, Gregory B and Kriegstein, Arnold R",
  abstract = "The homeobox transcription factor, Nkx2-1, plays multiple roles
              during forebrain development. Using restricted genetic ablation
              of Nkx2-1, in this issue of Neuron, Butt et al. show that Nkx2-1
              in telencephalic progenitors regulates interneuron subtype
              specification, while N{\'o}brega-Pereira et al. demonstrate that
              postmitotic Nkx2-1 regulates migration and sorting of
              interneurons to the striatum or cortex by controlling the
              expression of the guidance receptor, Neuropilin-2.",
  journal  = "Neuron",
  volume   =  59,
  number   =  5,
  pages    = "679--682",
  month    =  sep,
  year     =  2008,
  language = "en"
}

The homeobox transcription factor, Nkx2-1, plays multiple roles during forebrain development. Using restricted genetic ablation of Nkx2-1, in this issue of Neuron, Butt et al. show that Nkx2-1 in telencephalic progenitors regulates interneuron subtype specification, while Nóbrega-Pereira et al. demonstrate that postmitotic Nkx2-1 regulates migration and sorting of interneurons to the striatum or cortex by controlling the expression of the guidance receptor, Neuropilin-2.

@ARTICLE{Wang2008-wf,
  title    = "{GABA} regulates stem cell proliferation before nervous system
              formation",
  author   = "Wang, Doris D and Kriegstein, Arnold R and Ben-Ari, Yehezkel",
  journal  = "Epilepsy Curr",
  volume   =  8,
  number   =  5,
  pages    = "137--139",
  month    =  sep,
  year     =  2008,
  language = "en"
}

@ARTICLE{Elias2008-op,
  title    = "Differential trafficking of {AMPA} and {NMDA} receptors by
              {SAP102} and {PSD-95} underlies synapse development",
  author   = "Elias, G M and Elias, L A B and Apostolides, P F and Kriegstein,
              A R and Nicoll, R A",
  abstract = "The development of glutamatergic synapses involves changes in the
              number and type of receptors present at the postsynaptic density.
              To elucidate molecular mechanisms underlying these changes, we
              combine in utero electroporation of constructs that alter the
              molecular composition of developing synapses with dual whole-cell
              electrophysiology to examine synaptic transmission during two
              distinct developmental stages. We find that SAP102 mediates
              synaptic trafficking of AMPA and NMDA receptors during
              synaptogenesis. Surprisingly, after synaptogenesis, PSD-95
              assumes the functions of SAP102 and is necessary for two aspects
              of synapse maturation: the developmental increase in AMPA
              receptor transmission and replacement of NR2B-NMDARs with
              NR2A-NMDARs. In PSD-95/PSD-93 double-KO mice, the maturational
              replacement of NR2B- with NR2A-NMDARs fails to occur, and PSD-95
              expression fully rescues this deficit. This study demonstrates
              that SAP102 and PSD-95 regulate the synaptic trafficking of
              distinct glutamate receptor subtypes at different developmental
              stages, thereby playing necessary roles in excitatory synapse
              development.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  105,
  number   =  52,
  pages    = "20953--20958",
  month    =  dec,
  year     =  2008,
  language = "en"
}

The development of glutamatergic synapses involves changes in the number and type of receptors present at the postsynaptic density. To elucidate molecular mechanisms underlying these changes, we combine in utero electroporation of constructs that alter the molecular composition of developing synapses with dual whole-cell electrophysiology to examine synaptic transmission during two distinct developmental stages. We find that SAP102 mediates synaptic trafficking of AMPA and NMDA receptors during synaptogenesis. Surprisingly, after synaptogenesis, PSD-95 assumes the functions of SAP102 and is necessary for two aspects of synapse maturation: the developmental increase in AMPA receptor transmission and replacement of NR2B-NMDARs with NR2A-NMDARs. In PSD-95/PSD-93 double-KO mice, the maturational replacement of NR2B- with NR2A-NMDARs fails to occur, and PSD-95 expression fully rescues this deficit. This study demonstrates that SAP102 and PSD-95 regulate the synaptic trafficking of distinct glutamate receptor subtypes at different developmental stages, thereby playing necessary roles in excitatory synapse development.

@ARTICLE{Milosevic2008-if,
  title    = "Progenitors from the postnatal forebrain subventricular zone
              differentiate into cerebellar-like interneurons and
              cerebellar-specific astrocytes upon transplantation",
  author   = "Milosevic, Ana and Noctor, Stephen C and Martinez-Cerdeno,
              Veronica and Kriegstein, Arnold R and Goldman, James E",
  abstract = "Forebrain subventricular zone (SVZ) progenitor cells give rise to
              glia and olfactory bulb interneurons during early postnatal life
              in rats. We investigated the potential of SVZ cells to alter
              their fate by transplanting them into a heterotypic neurogenic
              and gliogenic environment-the cerebellum. Transplanted cells were
              examined 1 to 7 weeks and 6 months post transplantation.
              Forebrain progenitors populated the cerebellum and differentiated
              into oligodendrocytes, cerebellar-specific Bergmann glia and
              velate astrocytes, and neurons. The transplanted cells that
              differentiated into neurons maintained an interneuronal fate:
              they were GABA-positive, expressed interneuronal markers, such as
              calretinin, and exhibited membrane properties that are
              characteristic of interneurons. However, the transplanted
              interneurons lost the expression of the olfactory bulb
              transcription factors Tbr2 and Dlx1, and acquired a
              cerebellar-like morphology. Forebrain SVZ progenitors thus have
              the potential to adapt to a new environment and integrate into
              diverse regions, and may be a useful tool in transplantation
              strategies.",
  journal  = "Mol Cell Neurosci",
  volume   =  39,
  number   =  3,
  pages    = "324--334",
  month    =  jul,
  year     =  2008,
  language = "en"
}

Forebrain subventricular zone (SVZ) progenitor cells give rise to glia and olfactory bulb interneurons during early postnatal life in rats. We investigated the potential of SVZ cells to alter their fate by transplanting them into a heterotypic neurogenic and gliogenic environment-the cerebellum. Transplanted cells were examined 1 to 7 weeks and 6 months post transplantation. Forebrain progenitors populated the cerebellum and differentiated into oligodendrocytes, cerebellar-specific Bergmann glia and velate astrocytes, and neurons. The transplanted cells that differentiated into neurons maintained an interneuronal fate: they were GABA-positive, expressed interneuronal markers, such as calretinin, and exhibited membrane properties that are characteristic of interneurons. However, the transplanted interneurons lost the expression of the olfactory bulb transcription factors Tbr2 and Dlx1, and acquired a cerebellar-like morphology. Forebrain SVZ progenitors thus have the potential to adapt to a new environment and integrate into diverse regions, and may be a useful tool in transplantation strategies.

@ARTICLE{Wang2008-cn,
  title    = "{GABA} regulates excitatory synapse formation in the neocortex
              via {NMDA} receptor activation",
  author   = "Wang, Doris D and Kriegstein, Arnold R",
  abstract = "The development of a balance between excitatory and inhibitory
              synapses is a critical process in the generation and maturation
              of functional circuits. Accumulating evidence suggests that
              neuronal activity plays an important role in achieving such a
              balance in the developing cortex, but the mechanism that
              regulates this process is unknown. During development, GABA, the
              primary inhibitory neurotransmitter in adults, excites neurons as
              a result of high expression of the Na(+)-K(+)-2Cl(-)
              cotransporter (NKCC1). Using NKCC1 RNA interference knockdown in
              vivo, we show that GABA-induced depolarization is necessary for
              proper excitatory synapse formation and dendritic development of
              newborn cortical neurons. Blocking NKCC1 with the diuretic
              bumetanide during development leads to similar persistent changes
              in cortical circuitry in the adult. Interestingly, expression of
              a voltage-independent NMDA receptor rescues the failure of NKCC1
              knockdown neurons to develop excitatory AMPA transmission,
              indicating that GABA depolarization cooperates with NMDA receptor
              activation to regulate excitatory synapse formation. Our study
              identifies an essential role for GABA in the synaptic integration
              of newborn cortical neurons and suggests an activity-dependent
              mechanism for achieving the balance between excitation and
              inhibition in the developing cortex.",
  journal  = "J Neurosci",
  volume   =  28,
  number   =  21,
  pages    = "5547--5558",
  month    =  may,
  year     =  2008,
  language = "en"
}

The development of a balance between excitatory and inhibitory synapses is a critical process in the generation and maturation of functional circuits. Accumulating evidence suggests that neuronal activity plays an important role in achieving such a balance in the developing cortex, but the mechanism that regulates this process is unknown. During development, GABA, the primary inhibitory neurotransmitter in adults, excites neurons as a result of high expression of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Using NKCC1 RNA interference knockdown in vivo, we show that GABA-induced depolarization is necessary for proper excitatory synapse formation and dendritic development of newborn cortical neurons. Blocking NKCC1 with the diuretic bumetanide during development leads to similar persistent changes in cortical circuitry in the adult. Interestingly, expression of a voltage-independent NMDA receptor rescues the failure of NKCC1 knockdown neurons to develop excitatory AMPA transmission, indicating that GABA depolarization cooperates with NMDA receptor activation to regulate excitatory synapse formation. Our study identifies an essential role for GABA in the synaptic integration of newborn cortical neurons and suggests an activity-dependent mechanism for achieving the balance between excitation and inhibition in the developing cortex.

@ARTICLE{Noctor2008-ot,
  title    = "Distinct behaviors of neural stem and progenitor cells underlie
              cortical neurogenesis",
  author   = "Noctor, Stephen C and Mart{\'\i}nez-Cerde{\~n}o, Ver{\'o}nica and
              Kriegstein, Arnold R",
  abstract = "Neocortical precursor cells undergo symmetric and asymmetric
              divisions while producing large numbers of diverse cortical cell
              types. In Drosophila, cleavage plane orientation dictates the
              inheritance of fate-determinants and the symmetry of newborn
              daughter cells during neuroblast cell divisions. One model for
              predicting daughter cell fate in the mammalian neocortex is also
              based on cleavage plane orientation. Precursor cell divisions
              with a cleavage plane orientation that is perpendicular with
              respect to the ventricular surface (vertical) are predicted to be
              symmetric, while divisions with a cleavage plane orientation that
              is parallel to the surface (horizontal) are predicted to be
              asymmetric neurogenic divisions. However, analysis of cleavage
              plane orientation at the ventricle suggests that the number of
              predicted neurogenic divisions might be insufficient to produce
              large amounts of cortical neurons. To understand factors that
              correlate with the symmetry of cell divisions, we examined rat
              neocortical precursor cells in situ through real-time imaging,
              marker analysis, and electrophysiological recordings. We find
              that cleavage plane orientation is more closely associated with
              precursor cell type than with daughter cell fate, as commonly
              thought. Radial glia cells in the VZ primarily divide with a
              vertical orientation throughout cortical development and undergo
              symmetric or asymmetric self-renewing divisions depending on the
              stage of development. In contrast, most intermediate progenitor
              cells divide in the subventricular zone with a horizontal
              orientation and produce symmetric daughter cells. We propose a
              model for predicting daughter cell fate that considers precursor
              cell type, stage of development, and the planar segregation of
              fate determinants.",
  journal  = "J Comp Neurol",
  volume   =  508,
  number   =  1,
  pages    = "28--44",
  month    =  may,
  year     =  2008,
  language = "en"
}

Neocortical precursor cells undergo symmetric and asymmetric divisions while producing large numbers of diverse cortical cell types. In Drosophila, cleavage plane orientation dictates the inheritance of fate-determinants and the symmetry of newborn daughter cells during neuroblast cell divisions. One model for predicting daughter cell fate in the mammalian neocortex is also based on cleavage plane orientation. Precursor cell divisions with a cleavage plane orientation that is perpendicular with respect to the ventricular surface (vertical) are predicted to be symmetric, while divisions with a cleavage plane orientation that is parallel to the surface (horizontal) are predicted to be asymmetric neurogenic divisions. However, analysis of cleavage plane orientation at the ventricle suggests that the number of predicted neurogenic divisions might be insufficient to produce large amounts of cortical neurons. To understand factors that correlate with the symmetry of cell divisions, we examined rat neocortical precursor cells in situ through real-time imaging, marker analysis, and electrophysiological recordings. We find that cleavage plane orientation is more closely associated with precursor cell type than with daughter cell fate, as commonly thought. Radial glia cells in the VZ primarily divide with a vertical orientation throughout cortical development and undergo symmetric or asymmetric self-renewing divisions depending on the stage of development. In contrast, most intermediate progenitor cells divide in the subventricular zone with a horizontal orientation and produce symmetric daughter cells. We propose a model for predicting daughter cell fate that considers precursor cell type, stage of development, and the planar segregation of fate determinants.

@ARTICLE{Noctor2007-wh,
  title    = "Contribution of intermediate progenitor cells to cortical
              histogenesis",
  author   = "Noctor, Stephen C and Mart{\'\i}nez-Cerde{\~n}o, Ver{\'o}nica and
              Kriegstein, Arnold R",
  abstract = "The mammalian cerebral cortex is the most cellularly complex
              structure in the animal kingdom. Almost all cortical neurons are
              produced during a limited embryonic period by cortical progenitor
              cells in a proliferative region that surrounds the ventricular
              system of the developing brain. The proliferative region
              comprises 2 distinct zones, the ventricular zone, which is a
              neuroepithelial layer directly adjacent to the ventricular lumen,
              and the subventricular zone, which is positioned superficial to
              the ventricular zone. Recent advances in molecular and cell
              biology have made possible the study of specific cell
              populations, and 2 cortical progenitor cell types, radial glial
              cells in the ventricular zone and intermediate progenitor cells
              in the subventricular zone, have been shown to generate neurons
              in the embryonic cerebral cortex. These findings have refined our
              understanding of cortical neurogenesis, with implications for
              understanding the causes of neurodevelopmental disorders and for
              their potential treatment.",
  journal  = "Arch Neurol",
  volume   =  64,
  number   =  5,
  pages    = "639--642",
  month    =  may,
  year     =  2007,
  address  = "United States",
  language = "en"
}

The mammalian cerebral cortex is the most cellularly complex structure in the animal kingdom. Almost all cortical neurons are produced during a limited embryonic period by cortical progenitor cells in a proliferative region that surrounds the ventricular system of the developing brain. The proliferative region comprises 2 distinct zones, the ventricular zone, which is a neuroepithelial layer directly adjacent to the ventricular lumen, and the subventricular zone, which is positioned superficial to the ventricular zone. Recent advances in molecular and cell biology have made possible the study of specific cell populations, and 2 cortical progenitor cell types, radial glial cells in the ventricular zone and intermediate progenitor cells in the subventricular zone, have been shown to generate neurons in the embryonic cerebral cortex. These findings have refined our understanding of cortical neurogenesis, with implications for understanding the causes of neurodevelopmental disorders and for their potential treatment.

@ARTICLE{Noctor2007-ty,
  title    = "Neural stem and progenitor cells in cortical development",
  author   = "Noctor, Stephen C and Martinez-Cerde{\~n}o, Veronica and
              Kriegstein, Arnold R",
  abstract = "Recent work has begun to identify neural stem and progenitor
              cells in the embryonic and adult brain, and is unravelling the
              mechanisms whereby new nerve cells are created and delivered to
              their correct locations. Radial glial (RG) cells, which are
              present in the developing mammalian brain, have been proposed to
              be neural stem cells because they produce multiple cell types.
              Furthermore, time-lapse imaging demonstrates that RG cells
              undergo asymmetric self-renewing divisions to produce immature
              neurons that migrate along their parent radial fibre to reach the
              developing cerebral cortex. RG cells also produce intermediate
              progenitor (IP) cells that undergo symmetric division in the
              subventricular zone of the embryonic cortex to produce pairs of
              neurons. The symmetric IP divisions increase cell number within
              the same cortical layer. This two-step process of neurogenesis
              suggests new mechanisms for the generation of cell diversity and
              cell number in the developing cortex and supports a model similar
              to that proposed for the development of the fruit fly CNS. In
              this model, a temporal sequence of gene expression changes in
              asymmetrically dividing self-renewed RG cells could lead to the
              differential inheritance of cell identity genes in cortical cells
              generated at different cell cycles.",
  journal  = "Novartis Found Symp",
  volume   =  288,
  pages    = "59--73; discussion 73--8, 96--8",
  year     =  2007,
  address  = "England",
  language = "en"
}

Recent work has begun to identify neural stem and progenitor cells in the embryonic and adult brain, and is unravelling the mechanisms whereby new nerve cells are created and delivered to their correct locations. Radial glial (RG) cells, which are present in the developing mammalian brain, have been proposed to be neural stem cells because they produce multiple cell types. Furthermore, time-lapse imaging demonstrates that RG cells undergo asymmetric self-renewing divisions to produce immature neurons that migrate along their parent radial fibre to reach the developing cerebral cortex. RG cells also produce intermediate progenitor (IP) cells that undergo symmetric division in the subventricular zone of the embryonic cortex to produce pairs of neurons. The symmetric IP divisions increase cell number within the same cortical layer. This two-step process of neurogenesis suggests new mechanisms for the generation of cell diversity and cell number in the developing cortex and supports a model similar to that proposed for the development of the fruit fly CNS. In this model, a temporal sequence of gene expression changes in asymmetrically dividing self-renewed RG cells could lead to the differential inheritance of cell identity genes in cortical cells generated at different cell cycles.

@ARTICLE{Kriegstein2007-sz,
  title    = "Cortical neurogenesis: transitioning from advances in the
              laboratory to cell-based therapies",
  author   = "Kriegstein, Arnold R",
  journal  = "J Vis Exp",
  number   =  6,
  pages    = "241",
  month    =  jul,
  year     =  2007,
  language = "en"
}

@ARTICLE{Elias2007-ms,
  title    = "Gap junction adhesion is necessary for radial migration in the
              neocortex",
  author   = "Elias, Laura A B and Wang, Doris D and Kriegstein, Arnold R",
  abstract = "Radial glia, the neuronal stem cells of the embryonic cerebral
              cortex, reside deep within the developing brain and extend radial
              fibres to the pial surface, along which embryonic neurons migrate
              to reach the cortical plate. Here we show that the gap junction
              subunits connexin 26 (Cx26) and connexin 43 (Cx43) are expressed
              at the contact points between radial fibres and migrating
              neurons, and acute downregulation of Cx26 or Cx43 impairs the
              migration of neurons to the cortical plate. Unexpectedly, gap
              junctions do not mediate neuronal migration by acting in the
              classical manner to provide an aqueous channel for cell-cell
              communication. Instead, gap junctions provide dynamic adhesive
              contacts that interact with the internal cytoskeleton to enable
              leading process stabilization along radial fibres as well as the
              subsequent translocation of the nucleus. These results indicate
              that gap junction adhesions are necessary for glial-guided
              neuronal migration, raising the possibility that the adhesive
              properties of gap junctions may have an important role in other
              physiological processes and diseases associated with gap junction
              function.",
  journal  = "Nature",
  volume   =  448,
  number   =  7156,
  pages    = "901--907",
  month    =  aug,
  year     =  2007,
  address  = "England",
  language = "en"
}

Radial glia, the neuronal stem cells of the embryonic cerebral cortex, reside deep within the developing brain and extend radial fibres to the pial surface, along which embryonic neurons migrate to reach the cortical plate. Here we show that the gap junction subunits connexin 26 (Cx26) and connexin 43 (Cx43) are expressed at the contact points between radial fibres and migrating neurons, and acute downregulation of Cx26 or Cx43 impairs the migration of neurons to the cortical plate. Unexpectedly, gap junctions do not mediate neuronal migration by acting in the classical manner to provide an aqueous channel for cell-cell communication. Instead, gap junctions provide dynamic adhesive contacts that interact with the internal cytoskeleton to enable leading process stabilization along radial fibres as well as the subsequent translocation of the nucleus. These results indicate that gap junction adhesions are necessary for glial-guided neuronal migration, raising the possibility that the adhesive properties of gap junctions may have an important role in other physiological processes and diseases associated with gap junction function.

@ARTICLE{Martinez-Cerdeno2006-ul,
  title    = "The role of intermediate progenitor cells in the evolutionary
              expansion of the cerebral cortex",
  author   = "Mart{\'\i}nez-Cerde{\~n}o, Ver{\'o}nica and Noctor, Stephen C and
              Kriegstein, Arnold R",
  abstract = "The vertebrate cerebral cortex varies from the 3-layered dorsal
              cortex of reptiles to the 6-layered lissencephalic cortex
              characteristic of rodents and to the 6-layered gyrencephalic
              cortex typical of carnivores and primates. Distinct developmental
              mechanisms may have evolved independently to account for the
              radial expansion that produced the multilayered cortex of mammals
              and for the tangential expansion of cortical surface area that
              resulted in gyrencephalic cortex. Recent evidence shows that
              during the late stages of cortical development, radial glial
              cells divide asymmetrically in the ventricular zone to generate
              radial glial cells and intermediate progenitor (IP) cells and
              that IP cells subsequently divide symmetrically in the
              subventricular zone to produce multiple neurons. We propose that
              the evolution of this two-step pattern of neurogenesis played an
              important role in the amplification of cell numbers underlying
              the radial and tangential expansion of the cerebral cortex.",
  journal  = "Cereb Cortex",
  volume   = "16 Suppl 1",
  pages    = "i152--61",
  month    =  jul,
  year     =  2006,
  address  = "United States",
  language = "en"
}

The vertebrate cerebral cortex varies from the 3-layered dorsal cortex of reptiles to the 6-layered lissencephalic cortex characteristic of rodents and to the 6-layered gyrencephalic cortex typical of carnivores and primates. Distinct developmental mechanisms may have evolved independently to account for the radial expansion that produced the multilayered cortex of mammals and for the tangential expansion of cortical surface area that resulted in gyrencephalic cortex. Recent evidence shows that during the late stages of cortical development, radial glial cells divide asymmetrically in the ventricular zone to generate radial glial cells and intermediate progenitor (IP) cells and that IP cells subsequently divide symmetrically in the subventricular zone to produce multiple neurons. We propose that the evolution of this two-step pattern of neurogenesis played an important role in the amplification of cell numbers underlying the radial and tangential expansion of the cerebral cortex.

@ARTICLE{Young-Pearse2006-oj,
  title    = "Characterization of mice with targeted deletion of glycine
              receptor alpha 2",
  author   = "Young-Pearse, T L and Ivic, L and Kriegstein, A R and Cepko, C L",
  abstract = "Glycine receptors are ligand-gated chloride channels that mediate
              inhibitory neurotransmission in the adult nervous system. During
              development, glycine receptor alpha 2 (GlyRalpha2) is expressed
              in the retina, in the spinal cord, and throughout the brain.
              Within the cortex, GlyRalpha2 is expressed in immature cells and
              these receptors have been shown to be active and excitatory. In
              the developing retina, inhibition of glycine receptor activity
              prevents proper rod photoreceptor development. These data suggest
              that GlyRalpha2, the developmentally expressed glycine receptor,
              may play an important role in neuronal development. We have
              generated mice with a targeted deletion of glycine receptor alpha
              2 (Glra2). Although these mice lack expression of GlyRalpha2, no
              gross morphological or molecular alterations were observed in the
              nervous system. In addition, the cerebral cortex does not appear
              to require glycine receptor activity for proper development, as
              Glra2 knockout mice did not show any electrophysiological
              responses to glycine.",
  journal  = "Mol Cell Biol",
  volume   =  26,
  number   =  15,
  pages    = "5728--5734",
  month    =  aug,
  year     =  2006,
  language = "en"
}

Glycine receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system. During development, glycine receptor alpha 2 (GlyRalpha2) is expressed in the retina, in the spinal cord, and throughout the brain. Within the cortex, GlyRalpha2 is expressed in immature cells and these receptors have been shown to be active and excitatory. In the developing retina, inhibition of glycine receptor activity prevents proper rod photoreceptor development. These data suggest that GlyRalpha2, the developmentally expressed glycine receptor, may play an important role in neuronal development. We have generated mice with a targeted deletion of glycine receptor alpha 2 (Glra2). Although these mice lack expression of GlyRalpha2, no gross morphological or molecular alterations were observed in the nervous system. In addition, the cerebral cortex does not appear to require glycine receptor activity for proper development, as Glra2 knockout mice did not show any electrophysiological responses to glycine.

@ARTICLE{Castaneda-Castellanos2006-we,
  title    = "Blind patch clamp recordings in embryonic and adult mammalian
              brain slices",
  author   = "Casta{\~n}eda-Castellanos, David R and Flint, Alexander C and
              Kriegstein, Arnold R",
  abstract = "To obtain electrophysiological recordings in brain slices,
              sophisticated and expensive pieces of equipment can be used.
              However, costly microscope equipment with infrared differential
              interference contrast optics is not always necessary or even
              desirable. For instance, obtaining a randomized unbiased sample
              in a given preparation would be better accomplished if cells were
              not directly visualized before recording. In addition, some
              preparations require thick slices, and direct visualization is
              not possible. Here we describe a protocol for the 'blind patch
              clamp method' that we developed several years ago to perform
              electrophysiological recordings in mammalian brain slices using a
              standard patch clamp amplifier, dissecting microscope and
              recording chamber. Overall, it takes approximately 3-4 h to set
              up this procedure.",
  journal  = "Nat Protoc",
  volume   =  1,
  number   =  2,
  pages    = "532--542",
  year     =  2006,
  address  = "England",
  language = "en"
}

To obtain electrophysiological recordings in brain slices, sophisticated and expensive pieces of equipment can be used. However, costly microscope equipment with infrared differential interference contrast optics is not always necessary or even desirable. For instance, obtaining a randomized unbiased sample in a given preparation would be better accomplished if cells were not directly visualized before recording. In addition, some preparations require thick slices, and direct visualization is not possible. Here we describe a protocol for the 'blind patch clamp method' that we developed several years ago to perform electrophysiological recordings in mammalian brain slices using a standard patch clamp amplifier, dissecting microscope and recording chamber. Overall, it takes approximately 3-4 h to set up this procedure.

@ARTICLE{Martinez-Cerdeno2006-md,
  title    = "Estradiol stimulates progenitor cell division in the ventricular
              and subventricular zones of the embryonic neocortex",
  author   = "Mart{\'\i}nez-Cerde{\~n}o, Ver{\'o}nica and Noctor, Stephen C and
              Kriegstein, Arnold R",
  abstract = "Two distinct populations of cerebral cortical progenitor cells
              that generate neurons during embryogenesis have been identified:
              radial glial cells and intermediate progenitor cells. Despite
              advances in our understanding of progenitor cell populations, we
              know relatively little about factors that regulate their
              proliferative behaviour. 17-beta-Estradiol (E2) is present in the
              adult and developing mammalian brain, and plays an important role
              in central nervous system processes such as neuronal
              differentiation, survival and plasticity. E2 also stimulates
              neurogenesis in the adult dentate gyrus. We examined the role of
              E2 during embryonic cortical neurogenesis through
              immunohistochemistry, in situ hybridization, functional enzyme
              assay, organotypic culture and in utero administration of
              estradiol-blocking agents in mice. We show that aromatase, the E2
              synthesizing enzyme, is present in the embryonic neocortex, that
              estrogen receptor-alpha is present in progenitor cells during
              cortical neurogenesis, that in vitro E2 administration rapidly
              promotes proliferation, and that in utero blockade of estrogen
              receptors decreases proliferation of embryonic cortical
              progenitor cells. Furthermore, the E2 inhibitor alpha-fetoprotein
              is expressed at high levels by radial glial cells but at lower
              levels by intermediate progenitor cells, suggesting that E2
              differentially influences the proliferation of these cortical
              progenitor cell types. These findings demonstrate a new
              functional role for E2 as a proliferative agent during critical
              stages of cerebral cortex development.",
  journal  = "Eur J Neurosci",
  volume   =  24,
  number   =  12,
  pages    = "3475--3488",
  month    =  dec,
  year     =  2006,
  address  = "France",
  language = "en"
}

Two distinct populations of cerebral cortical progenitor cells that generate neurons during embryogenesis have been identified: radial glial cells and intermediate progenitor cells. Despite advances in our understanding of progenitor cell populations, we know relatively little about factors that regulate their proliferative behaviour. 17-beta-Estradiol (E2) is present in the adult and developing mammalian brain, and plays an important role in central nervous system processes such as neuronal differentiation, survival and plasticity. E2 also stimulates neurogenesis in the adult dentate gyrus. We examined the role of E2 during embryonic cortical neurogenesis through immunohistochemistry, in situ hybridization, functional enzyme assay, organotypic culture and in utero administration of estradiol-blocking agents in mice. We show that aromatase, the E2 synthesizing enzyme, is present in the embryonic neocortex, that estrogen receptor-alpha is present in progenitor cells during cortical neurogenesis, that in vitro E2 administration rapidly promotes proliferation, and that in utero blockade of estrogen receptors decreases proliferation of embryonic cortical progenitor cells. Furthermore, the E2 inhibitor alpha-fetoprotein is expressed at high levels by radial glial cells but at lower levels by intermediate progenitor cells, suggesting that E2 differentially influences the proliferation of these cortical progenitor cell types. These findings demonstrate a new functional role for E2 as a proliferative agent during critical stages of cerebral cortex development.

@ARTICLE{Kriegstein2005-gu,
  title    = "{GABA} puts the brake on stem cells",
  author   = "Kriegstein, Arnold R",
  journal  = "Nat Neurosci",
  volume   =  8,
  number   =  9,
  pages    = "1132--1133",
  month    =  sep,
  year     =  2005,
  address  = "United States",
  language = "en"
}

@ARTICLE{Kriegstein2005-pq,
  title    = "Constructing circuits: neurogenesis and migration in the
              developing neocortex",
  author   = "Kriegstein, Arnold R",
  abstract = "Our knowledge of the proliferation, migration, and
              differentiation of neurons has changed dramatically over the last
              10 years. Whereas traditionally it was thought that glial and
              neuronal cells were separate cell lines with different lineages,
              we now know that this is not true. Radial glia are a type of
              neural stem cell that generate excitatory pyramidal neurons
              directly through asymmetric cell division in the ventricular zone
              (VZ) of the telencephalon and indirectly through the symmetric
              division of daughter intermediate precursor cells that divide in
              the subventricular zone (SVZ). Moreover, pyramidal neurons, once
              thought to migrate only along radial guide fibers to the
              developing layers of the cortex, have been shown to proceed
              through four distinct stages of migration during which they
              change shape, direction, and speed. Gamma-aminobutyric acid
              (GABAergic) inhibitory interneurons, on the other hand, are
              generated not in the cortex, but in the medial ganglionic
              eminence and migrate tangentially to their final cortical
              destinations. Evidence suggests that GABA activation may play a
              role in coordinating the generation and migration of both
              pyramidal and interneuron populations. At the end of
              neurogenesis, radial glial cells translocate to the cortex and
              transform into astrocytes. Although they do not actively divide
              in the adult brain, astrocytes may retain the potential to
              generate new neurons. These new findings have increased our
              understanding of the mechanisms underlying certain developmental
              disorders and, in doing so, reveal potentially useful modes of
              therapeutic intervention.",
  journal  = "Epilepsia",
  volume   = "46 Suppl 7",
  pages    = "15--21",
  year     =  2005,
  address  = "United States",
  language = "en"
}

Our knowledge of the proliferation, migration, and differentiation of neurons has changed dramatically over the last 10 years. Whereas traditionally it was thought that glial and neuronal cells were separate cell lines with different lineages, we now know that this is not true. Radial glia are a type of neural stem cell that generate excitatory pyramidal neurons directly through asymmetric cell division in the ventricular zone (VZ) of the telencephalon and indirectly through the symmetric division of daughter intermediate precursor cells that divide in the subventricular zone (SVZ). Moreover, pyramidal neurons, once thought to migrate only along radial guide fibers to the developing layers of the cortex, have been shown to proceed through four distinct stages of migration during which they change shape, direction, and speed. Gamma-aminobutyric acid (GABAergic) inhibitory interneurons, on the other hand, are generated not in the cortex, but in the medial ganglionic eminence and migrate tangentially to their final cortical destinations. Evidence suggests that GABA activation may play a role in coordinating the generation and migration of both pyramidal and interneuron populations. At the end of neurogenesis, radial glial cells translocate to the cortex and transform into astrocytes. Although they do not actively divide in the adult brain, astrocytes may retain the potential to generate new neurons. These new findings have increased our understanding of the mechanisms underlying certain developmental disorders and, in doing so, reveal potentially useful modes of therapeutic intervention.

@ARTICLE{Lo2005-oe,
  title    = "A new era in the ethics of human embryonic stem cell research",
  author   = "Lo, Bernard and Zettler, Patricia and Cedars, Marcelle I and
              Gates, Elena and Kriegstein, Arnold R and Oberman, Michelle and
              Reijo Pera, Renee and Wagner, Richard M and Wuerth, Mary T and
              Wolf, Leslie E and Yamamoto, Keith R",
  abstract = "Scientific progress in human embryonic stem cell (hESC) research
              and increased funding make it imperative to look ahead to the
              ethical issues generated by the expected use of hESCs for
              transplantation. Several issues should be addressed now, even
              though phase I clinical trials of hESC transplantation are still
              in the future. To minimize the risk of hESC transplantation,
              donors of materials used to derive hESC lines will need to be
              recontacted to update their medical history and screening.
              Because of privacy concerns, such recontact needs to be discussed
              and agreed to at the time of donation, before new hESC lines are
              derived. Informed consent for phase I clinical trials of hESC
              transplantation also raises ethical concerns. In previous phase I
              trials of highly innovative interventions, allegations that trial
              participants had not really understood the risk and benefits
              caused delays in subsequent trials. Thus, researchers should
              consider what information needs to be discussed during the
              consent process for hESC clinical trials and how to verify that
              participants have a realistic understanding of the study. Lack of
              attention to the special ethical concerns raised by clinical
              trials of hESC transplantation and their implications for the
              derivation of new hESC lines may undermine or delay progress
              toward stem cell therapies.",
  journal  = "Stem Cells",
  volume   =  23,
  number   =  10,
  pages    = "1454--1459",
  month    =  nov,
  year     =  2005,
  address  = "England",
  language = "en"
}

Scientific progress in human embryonic stem cell (hESC) research and increased funding make it imperative to look ahead to the ethical issues generated by the expected use of hESCs for transplantation. Several issues should be addressed now, even though phase I clinical trials of hESC transplantation are still in the future. To minimize the risk of hESC transplantation, donors of materials used to derive hESC lines will need to be recontacted to update their medical history and screening. Because of privacy concerns, such recontact needs to be discussed and agreed to at the time of donation, before new hESC lines are derived. Informed consent for phase I clinical trials of hESC transplantation also raises ethical concerns. In previous phase I trials of highly innovative interventions, allegations that trial participants had not really understood the risk and benefits caused delays in subsequent trials. Thus, researchers should consider what information needs to be discussed during the consent process for hESC clinical trials and how to verify that participants have a realistic understanding of the study. Lack of attention to the special ethical concerns raised by clinical trials of hESC transplantation and their implications for the derivation of new hESC lines may undermine or delay progress toward stem cell therapies.

@ARTICLE{Tsai2005-ei,
  title    = "{LIS1} {RNA} interference blocks neural stem cell division,
              morphogenesis, and motility at multiple stages",
  author   = "Tsai, Jin-Wu and Chen, Yu and Kriegstein, Arnold R and Vallee,
              Richard B",
  abstract = "Mutations in the human LIS1 gene cause the smooth brain disease
              classical lissencephaly. To understand the underlying mechanisms,
              we conducted in situ live cell imaging analysis of LIS1 function
              throughout the entire radial migration pathway. In utero
              electroporation of LIS1 small interference RNA and short hairpin
              dominant negative LIS1 and dynactin cDNAs caused a dramatic
              accumulation of multipolar progenitor cells within the
              subventricular zone of embryonic rat brains. This effect resulted
              from a complete failure in progression from the multipolar to the
              migratory bipolar state, as revealed by time-lapse analysis of
              brain slices. Surprisingly, interkinetic nuclear oscillations in
              the radial glial progenitors were also abolished, as were cell
              divisions at the ventricular surface. Those few bipolar cells
              that reached the intermediate zone also exhibited a complete
              block in somal translocation, although, remarkably, process
              extension persisted. Finally, axonal growth also ceased. These
              results identify multiple distinct and novel roles for LIS1 in
              nucleokinesis and process dynamics and suggest that nuclear
              position controls neural progenitor cell division.",
  journal  = "J Cell Biol",
  volume   =  170,
  number   =  6,
  pages    = "935--945",
  month    =  sep,
  year     =  2005,
  language = "en"
}

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.

@ARTICLE{Kriegstein2004-vm,
  title    = "Patterns of neuronal migration in the embryonic cortex",
  author   = "Kriegstein, Arnold R and Noctor, Stephen C",
  abstract = "Real-time imaging of migrating neurons has changed our
              understanding of how newborn neurons reach their final positions
              in the developing cerebral cortex. The migratory routes and modes
              of migration are more diverse and complex than previously
              thought. The finding that cortical interneurons migrate to the
              cortex from origins in the ventral telencephalon has already
              markedly altered our view of cortical migration. More recent
              findings have demonstrated additional nuances in the migratory
              pattern and highlighted differences between subsets of
              interneurons. Moreover, radial migration of pyramidal neurons
              does not progress smoothly from ventricle to cortical plate, but
              is instead characterized by distinct migratory phases in which
              neurons change shape and direction of movement. Integrating these
              findings with the molecular machinery underlying migration will
              provide a more complete picture of how the cerebral cortex is
              assembled.",
  journal  = "Trends Neurosci",
  volume   =  27,
  number   =  7,
  pages    = "392--399",
  month    =  jul,
  year     =  2004,
  address  = "England",
  language = "en"
}

Real-time imaging of migrating neurons has changed our understanding of how newborn neurons reach their final positions in the developing cerebral cortex. The migratory routes and modes of migration are more diverse and complex than previously thought. The finding that cortical interneurons migrate to the cortex from origins in the ventral telencephalon has already markedly altered our view of cortical migration. More recent findings have demonstrated additional nuances in the migratory pattern and highlighted differences between subsets of interneurons. Moreover, radial migration of pyramidal neurons does not progress smoothly from ventricle to cortical plate, but is instead characterized by distinct migratory phases in which neurons change shape and direction of movement. Integrating these findings with the molecular machinery underlying migration will provide a more complete picture of how the cerebral cortex is assembled.

@ARTICLE{Castaneda-Castellanos2004-zj,
  title    = "Controlling neuron number: does Numb do the math?",
  author   = "Casta{\~n}eda-Castellanos, David R and Kriegstein, Arnold R",
  journal  = "Nat Neurosci",
  volume   =  7,
  number   =  8,
  pages    = "793--794",
  month    =  aug,
  year     =  2004,
  address  = "United States",
  language = "en"
}

@ARTICLE{Weissman2004-ov,
  title    = "Calcium waves propagate through radial glial cells and modulate
              proliferation in the developing neocortex",
  author   = "Weissman, Tamily A and Riquelme, Patricio A and Ivic, Lidija and
              Flint, Alexander C and Kriegstein, Arnold R",
  abstract = "The majority of neurons in the adult neocortex are produced
              embryonically during a brief but intense period of neuronal
              proliferation. The radial glial cell, a transient embryonic cell
              type known for its crucial role in neuronal migration, has
              recently been shown to function as a neuronal progenitor cell and
              appears to produce most cortical pyramidal neurons. Radial glial
              cell modulation could thus affect neuron production, neuronal
              migration, and overall cortical architecture; however, signaling
              mechanisms among radial glia have not been studied directly. We
              demonstrate here that calcium waves propagate through radial
              glial cells in the proliferative cortical ventricular zone (VZ).
              Radial glial calcium waves occur spontaneously and require
              connexin hemichannels, P2Y1 ATP receptors, and intracellular
              IP3-mediated calcium release. Furthermore, we show that wave
              disruption decreases VZ proliferation during the peak of
              embryonic neurogenesis. Taken together, these results demonstrate
              a radial glial signaling mechanism that may regulate cortical
              neuronal production.",
  journal  = "Neuron",
  volume   =  43,
  number   =  5,
  pages    = "647--661",
  month    =  sep,
  year     =  2004,
  address  = "United States",
  language = "en"
}

The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.

@ARTICLE{Noctor2004-il,
  title    = "Cortical neurons arise in symmetric and asymmetric division zones
              and migrate through specific phases",
  author   = "Noctor, Stephen C and Mart{\'\i}nez-Cerde{\~n}o, Ver{\'o}nica and
              Ivic, Lidija and Kriegstein, Arnold R",
  abstract = "Precise patterns of cell division and migration are crucial to
              transform the neuroepithelium of the embryonic forebrain into the
              adult cerebral cortex. Using time-lapse imaging of clonal cells
              in rat cortex over several generations, we show here that neurons
              are generated in two proliferative zones by distinct patterns of
              division. Neurons arise directly from radial glial cells in the
              ventricular zone (VZ) and indirectly from intermediate progenitor
              cells in the subventricular zone (SVZ). Furthermore, newborn
              neurons do not migrate directly to the cortex; instead, most
              exhibit four distinct phases of migration, including a phase of
              retrograde movement toward the ventricle before migration to the
              cortical plate. These findings provide a comprehensive and new
              view of the dynamics of cortical neurogenesis and migration.",
  journal  = "Nat Neurosci",
  volume   =  7,
  number   =  2,
  pages    = "136--144",
  month    =  jan,
  year     =  2004,
  address  = "United States",
  language = "en"
}

Precise patterns of cell division and migration are crucial to transform the neuroepithelium of the embryonic forebrain into the adult cerebral cortex. Using time-lapse imaging of clonal cells in rat cortex over several generations, we show here that neurons are generated in two proliferative zones by distinct patterns of division. Neurons arise directly from radial glial cells in the ventricular zone (VZ) and indirectly from intermediate progenitor cells in the subventricular zone (SVZ). Furthermore, newborn neurons do not migrate directly to the cortex; instead, most exhibit four distinct phases of migration, including a phase of retrograde movement toward the ventricle before migration to the cortical plate. These findings provide a comprehensive and new view of the dynamics of cortical neurogenesis and migration.

@ARTICLE{Fishell2003-nr,
  title    = "Neurons from radial glia: the consequences of asymmetric
              inheritance",
  author   = "Fishell, Gord and Kriegstein, Arnold R",
  abstract = "Recent work suggests that radial glial cells represent many, if
              not most, of the neuronal progenitors in the developing cortex.
              Asymmetric cell division of radial glia results in the
              self-renewal of the radial glial cell and the birth of a neuron.
              Among the proteins that direct cell fate in Drosophila
              melanogaster that have known mammalian homologs, Numb is the best
              candidate to have a similar function in radial glia. During
              asymmetric divisions of radial glial cells, the basal cell may
              inherit the radial glial fibre, while the apical cell sequesters
              the majority of the Numb protein. We suggest two models that make
              opposite predictions as to whether the radial glia or nascent
              neuron inherit the radial glial fiber or the majority of the Numb
              protein.",
  journal  = "Curr Opin Neurobiol",
  volume   =  13,
  number   =  1,
  pages    = "34--41",
  month    =  feb,
  year     =  2003,
  address  = "England",
  language = "en"
}

Recent work suggests that radial glial cells represent many, if not most, of the neuronal progenitors in the developing cortex. Asymmetric cell division of radial glia results in the self-renewal of the radial glial cell and the birth of a neuron. Among the proteins that direct cell fate in Drosophila melanogaster that have known mammalian homologs, Numb is the best candidate to have a similar function in radial glia. During asymmetric divisions of radial glial cells, the basal cell may inherit the radial glial fibre, while the apical cell sequesters the majority of the Numb protein. We suggest two models that make opposite predictions as to whether the radial glia or nascent neuron inherit the radial glial fiber or the majority of the Numb protein.

@ARTICLE{Kriegstein2003-qp,
  title    = "Radial glia diversity: a matter of cell fate",
  author   = "Kriegstein, Arnold R and G{\"o}tz, Magdalena",
  abstract = "Early in development of the central nervous system, radial glial
              cells arise from the neuroepithelial cells lining the ventricles
              around the time that neurons begin to appear. The transition of
              neuroepithelial cells to radial glia is accompanied by a series
              of structural and functional changes, including the appearance of
              ``glial'' features, as well as the appearance of new signaling
              molecules and junctional proteins. However, not all radial glia
              are alike. Radial glial lineages appear to be heterogeneous both
              within and across different brain regions. Subtypes of neurogenic
              radial glia within the cortex, for example, may have restricted
              potential in terms of the cell types they are able to generate.
              Radial glia located in different brain regions also differ in
              their expression of growth factors, a diverse number of
              transcription factors, and the cell types they generate,
              suggesting that they are involved in regionalization of the
              developing nervous system in several aspects. These findings
              highlight the important but complex role of radial glia as
              participants in key steps of brain development.",
  journal  = "Glia",
  volume   =  43,
  number   =  1,
  pages    = "37--43",
  month    =  jul,
  year     =  2003,
  address  = "United States",
  language = "en"
}

Early in development of the central nervous system, radial glial cells arise from the neuroepithelial cells lining the ventricles around the time that neurons begin to appear. The transition of neuroepithelial cells to radial glia is accompanied by a series of structural and functional changes, including the appearance of ``glial'' features, as well as the appearance of new signaling molecules and junctional proteins. However, not all radial glia are alike. Radial glial lineages appear to be heterogeneous both within and across different brain regions. Subtypes of neurogenic radial glia within the cortex, for example, may have restricted potential in terms of the cell types they are able to generate. Radial glia located in different brain regions also differ in their expression of growth factors, a diverse number of transcription factors, and the cell types they generate, suggesting that they are involved in regionalization of the developing nervous system in several aspects. These findings highlight the important but complex role of radial glia as participants in key steps of brain development.

@ARTICLE{Ivic2003-fv,
  title    = "Terpene trilactones from Ginkgo biloba are antagonists of
              cortical glycine and {GABA(A}) receptors",
  author   = "Ivic, Lidija and Sands, Tristan T J and Fishkin, Nathan and
              Nakanishi, Koji and Kriegstein, Arnold R and Str{\o}mgaard,
              Kristian",
  abstract = "Glycine and gamma-aminobutyric acid, type A (GABA(A)) receptors
              are members of the ligand-gated ion channel superfamily that
              mediate inhibitory synaptic transmission in the adult central
              nervous system. During development, the activation of these
              receptors leads to membrane depolarization. Ligands for the two
              receptors have important implications both in disease therapy and
              as pharmacological tools. Terpene trilactones (ginkgolides and
              bilobalide) are unique constituents of Ginkgo biloba extracts
              that have various effects on the central nervous system. We have
              investigated the relative potency of these compounds on glycine
              and GABA(A) receptors. We find that most of the ginkgolides are
              selective and potent antagonists of the glycine receptor.
              Bilobalide, the single major component in G. biloba extracts,
              also reduces glycine-induced currents, although to a lesser
              extent. Both ginkgolides and bilobalide inhibit GABA(A)
              receptors, with bilobalide demonstrating a more potent effect.
              Additionally, we provide evidence that open channels are required
              for glycine receptor inhibition by ginkgolides. Finally, we
              employ molecular modeling to elucidate the similarities and
              differences in the structure of the terpene trilactones to
              account for the pharmacological properties of these compounds and
              demonstrate a striking similarity between ginkgolides and
              picrotoxinin, a GABA(A) and recombinant glycine alpha-homomeric
              receptor antagonist.",
  journal  = "J Biol Chem",
  volume   =  278,
  number   =  49,
  pages    = "49279--49285",
  month    =  sep,
  year     =  2003,
  address  = "United States",
  language = "en"
}

Glycine and gamma-aminobutyric acid, type A (GABA(A)) receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory synaptic transmission in the adult central nervous system. During development, the activation of these receptors leads to membrane depolarization. Ligands for the two receptors have important implications both in disease therapy and as pharmacological tools. Terpene trilactones (ginkgolides and bilobalide) are unique constituents of Ginkgo biloba extracts that have various effects on the central nervous system. We have investigated the relative potency of these compounds on glycine and GABA(A) receptors. We find that most of the ginkgolides are selective and potent antagonists of the glycine receptor. Bilobalide, the single major component in G. biloba extracts, also reduces glycine-induced currents, although to a lesser extent. Both ginkgolides and bilobalide inhibit GABA(A) receptors, with bilobalide demonstrating a more potent effect. Additionally, we provide evidence that open channels are required for glycine receptor inhibition by ginkgolides. Finally, we employ molecular modeling to elucidate the similarities and differences in the structure of the terpene trilactones to account for the pharmacological properties of these compounds and demonstrate a striking similarity between ginkgolides and picrotoxinin, a GABA(A) and recombinant glycine alpha-homomeric receptor antagonist.

@ARTICLE{Weissman2003-hg,
  title    = "Neurogenic radial glial cells in reptile, rodent and human: from
              mitosis to migration",
  author   = "Weissman, Tamily and Noctor, Stephen C and Clinton, Brian K and
              Honig, Lawrence S and Kriegstein, Arnold R",
  abstract = "Radial glial cells play at least two crucial roles in cortical
              development: neuronal production in the ventricular zone (VZ) and
              the subsequent guidance of neuronal migration. There is evidence
              that radial glia-like cells are present not only during
              development but in the adult mammalian brain as well. In
              addition, radial glial cells appear to be neurogenic in the
              central nervous system of a number of vertebrate species. We
              demonstrate here that most dividing progenitor cells in the
              embryonic human VZ express radial glial proteins. Furthermore, we
              provide evidence that radial glial cells maintain a
              vimentin-positive radial fiber throughout each stage of cell
              division. Asymmetric inheritance of this fiber may be an
              important factor in determining how neuronal progeny will migrate
              into the developing cortical plate. Although radial glial cells
              have traditionally been characterized by their role in guiding
              migration, their role as neuronal progenitors may represent their
              defining characteristic throughout the vertebrate CNS.",
  journal  = "Cereb Cortex",
  volume   =  13,
  number   =  6,
  pages    = "550--559",
  month    =  jun,
  year     =  2003,
  address  = "United States",
  language = "en"
}

Radial glial cells play at least two crucial roles in cortical development: neuronal production in the ventricular zone (VZ) and the subsequent guidance of neuronal migration. There is evidence that radial glia-like cells are present not only during development but in the adult mammalian brain as well. In addition, radial glial cells appear to be neurogenic in the central nervous system of a number of vertebrate species. We demonstrate here that most dividing progenitor cells in the embryonic human VZ express radial glial proteins. Furthermore, we provide evidence that radial glial cells maintain a vimentin-positive radial fiber throughout each stage of cell division. Asymmetric inheritance of this fiber may be an important factor in determining how neuronal progeny will migrate into the developing cortical plate. Although radial glial cells have traditionally been characterized by their role in guiding migration, their role as neuronal progenitors may represent their defining characteristic throughout the vertebrate CNS.

@ARTICLE{Owens2002-eu,
  title    = "Developmental neurotransmitters?",
  author   = "Owens, David F and Kriegstein, Arnold R",
  abstract = "Previous studies support an early role for neurotransmitter
              signaling before synaptogenesis, but puzzlingly, a neurological
              phenotype is absent in embryonic mice that lack vesicular
              release. Demarque et al. (in this issue of Neuron) now report
              that early release of transmitter is unconventional in not
              requiring action potentials, Ca(2+) entry, or vesicle fusion,
              thus potentially reconciling the discrepancy.",
  journal  = "Neuron",
  volume   =  36,
  number   =  6,
  pages    = "989--991",
  month    =  dec,
  year     =  2002,
  address  = "United States",
  language = "en"
}

Previous studies support an early role for neurotransmitter signaling before synaptogenesis, but puzzlingly, a neurological phenotype is absent in embryonic mice that lack vesicular release. Demarque et al. (in this issue of Neuron) now report that early release of transmitter is unconventional in not requiring action potentials, Ca(2+) entry, or vesicle fusion, thus potentially reconciling the discrepancy.

@ARTICLE{Owens2002-oj,
  title    = "Is there more to {GABA} than synaptic inhibition?",
  author   = "Owens, David F and Kriegstein, Arnold R",
  abstract = "In the mature brain, GABA (gamma-aminobutyric acid) functions
              primarily as an inhibitory neurotransmitter. But it can also act
              as a trophic factor during nervous system development to
              influence events such as proliferation, migration,
              differentiation, synapse maturation and cell death. GABA mediates
              these processes by the activation of traditional ionotropic and
              metabotropic receptors, and probably by both synaptic and
              non-synaptic mechanisms. However, the functional properties of
              GABA receptor signalling in the immature brain are significantly
              different from, and in some ways opposite to, those found in the
              adult brain. The unique features of the early-appearing GABA
              signalling systems might help to explain how GABA acts as a
              developmental signal.",
  journal  = "Nat Rev Neurosci",
  volume   =  3,
  number   =  9,
  pages    = "715--727",
  month    =  sep,
  year     =  2002,
  address  = "England",
  language = "en"
}

In the mature brain, GABA (gamma-aminobutyric acid) functions primarily as an inhibitory neurotransmitter. But it can also act as a trophic factor during nervous system development to influence events such as proliferation, migration, differentiation, synapse maturation and cell death. GABA mediates these processes by the activation of traditional ionotropic and metabotropic receptors, and probably by both synaptic and non-synaptic mechanisms. However, the functional properties of GABA receptor signalling in the immature brain are significantly different from, and in some ways opposite to, those found in the adult brain. The unique features of the early-appearing GABA signalling systems might help to explain how GABA acts as a developmental signal.

@ARTICLE{Benardete2002-kl,
  title    = "Increased excitability and decreased sensitivity to {GABA} in an
              animal model of dysplastic cortex",
  author   = "Benardete, Ethan A and Kriegstein, Arnold R",
  abstract = "PURPOSE: Cortical dysplasia (CD) is associated with epilepsy in
              both the pediatric and adult populations. The mechanism
              underlying seizures with cortical malformations is still poorly
              understood. To study the physiology of dysplastic cortex, we
              developed an experimental model of CD. METHODS: Pregnant rats
              were given intraperitoneal injections of carmustine
              (1-3-bis-chloroethyl-nitrosourea; BCNU) on embryonic day 15
              (E15). Cortical histology was examined in the resulting pups at
              P0, P28, and P60. In addition, evoked and spontaneous field
              potential recordings were obtained in cortical slices from adult
              control and BCNU-exposed rats. Finally, we used whole-cell
              recordings to compare physiologic properties of pyramidal neurons
              and gamma-aminobutyric acid (GABA) responses in control and
              BCNU-treated animals. RESULTS: Features characteristic of CD were
              found in the offspring, including laminar disorganization,
              cytomegalic neurons, and neuronal heterotopias. Dysplastic cortex
              also contained abnormal clusters of Cajal-Retzius (CR) cells and
              disruption of radial glial fibers, as demonstrated with
              immunohistochemistry. Under conditions of partial GABAA-receptor
              blockade with 10 microM bicuculline methiodide (BMI), slices of
              dysplastic cortex demonstrated a significant increase in the
              number of spontaneous and evoked epileptiform discharges.
              Individual pyramidal neurons in dysplastic cortex were less
              sensitive to application of GABA compared with controls.
              CONCLUSIONS: BCNU exposure in utero produces histologic
              alterations suggestive of CD in rat offspring. Dysplastic cortex
              from this model demonstrates features of hyperexcitability and
              decreased neuronal sensitivity to GABA. Such physiologic
              alterations may underlie the increased epileptogenicity of
              dysplastic cortex.",
  journal  = "Epilepsia",
  volume   =  43,
  number   =  9,
  pages    = "970--982",
  month    =  sep,
  year     =  2002,
  address  = "United States",
  language = "en"
}

PURPOSE: Cortical dysplasia (CD) is associated with epilepsy in both the pediatric and adult populations. The mechanism underlying seizures with cortical malformations is still poorly understood. To study the physiology of dysplastic cortex, we developed an experimental model of CD. METHODS: Pregnant rats were given intraperitoneal injections of carmustine (1-3-bis-chloroethyl-nitrosourea; BCNU) on embryonic day 15 (E15). Cortical histology was examined in the resulting pups at P0, P28, and P60. In addition, evoked and spontaneous field potential recordings were obtained in cortical slices from adult control and BCNU-exposed rats. Finally, we used whole-cell recordings to compare physiologic properties of pyramidal neurons and gamma-aminobutyric acid (GABA) responses in control and BCNU-treated animals. RESULTS: Features characteristic of CD were found in the offspring, including laminar disorganization, cytomegalic neurons, and neuronal heterotopias. Dysplastic cortex also contained abnormal clusters of Cajal-Retzius (CR) cells and disruption of radial glial fibers, as demonstrated with immunohistochemistry. Under conditions of partial GABAA-receptor blockade with 10 microM bicuculline methiodide (BMI), slices of dysplastic cortex demonstrated a significant increase in the number of spontaneous and evoked epileptiform discharges. Individual pyramidal neurons in dysplastic cortex were less sensitive to application of GABA compared with controls. CONCLUSIONS: BCNU exposure in utero produces histologic alterations suggestive of CD in rat offspring. Dysplastic cortex from this model demonstrates features of hyperexcitability and decreased neuronal sensitivity to GABA. Such physiologic alterations may underlie the increased epileptogenicity of dysplastic cortex.

@ARTICLE{Noctor2002-kh,
  title    = "Dividing precursor cells of the embryonic cortical ventricular
              zone have morphological and molecular characteristics of radial
              glia",
  author   = "Noctor, Stephen C and Flint, Alexander C and Weissman, Tamily A
              and Wong, Winston S and Clinton, Brian K and Kriegstein, Arnold R",
  abstract = "The embryonic ventricular zone (VZ) of the cerebral cortex
              contains migrating neurons, radial glial cells, and a large
              population of cycling progenitor cells that generate newborn
              neurons. The latter two cell classes have been assumed for some
              time to be distinct in both function and anatomy, but the
              cellular anatomy of the progenitor cell type has remained poorly
              defined. Several recent reports have raised doubts about the
              distinction between radial glial and precursor cells by
              demonstrating that radial glial cells are themselves neuronal
              progenitor cells (Malatesta et al., 2000; Hartfuss et al., 2001;
              Miyata et al., 2001; Noctor et al., 2001). This discovery raises
              the possibility that radial glia and the population of VZ
              progenitor cells may be one anatomical and functional cell class.
              Such a hypothesis predicts that throughout neurogenesis almost
              all mitotically active VZ cells and a substantial percentage of
              VZ cells overall are radial glia. We have therefore used various
              anatomical, immunohistochemical, and electrophysiological
              techniques to test these predictions. Our data demonstrate that
              the majority of VZ cells, and nearly all mitotically active VZ
              cells during neurogenesis, both have radial glial morphology and
              express radial glial markers. In addition, intracellular dye
              filling of electrophysiologically characterized progenitor cells
              in the VZ demonstrates that these cells have the morphology of
              radial glia. Because the vast majority cycling cells in the
              cortical VZ have characteristics of radial glia, the radial glial
              precursor cell may be responsible for both the production of
              newborn neurons and the guidance of daughter neurons to their
              destinations in the developing cortex.",
  journal  = "J Neurosci",
  volume   =  22,
  number   =  8,
  pages    = "3161--3173",
  month    =  apr,
  year     =  2002,
  language = "en"
}

The embryonic ventricular zone (VZ) of the cerebral cortex contains migrating neurons, radial glial cells, and a large population of cycling progenitor cells that generate newborn neurons. The latter two cell classes have been assumed for some time to be distinct in both function and anatomy, but the cellular anatomy of the progenitor cell type has remained poorly defined. Several recent reports have raised doubts about the distinction between radial glial and precursor cells by demonstrating that radial glial cells are themselves neuronal progenitor cells (Malatesta et al., 2000; Hartfuss et al., 2001; Miyata et al., 2001; Noctor et al., 2001). This discovery raises the possibility that radial glia and the population of VZ progenitor cells may be one anatomical and functional cell class. Such a hypothesis predicts that throughout neurogenesis almost all mitotically active VZ cells and a substantial percentage of VZ cells overall are radial glia. We have therefore used various anatomical, immunohistochemical, and electrophysiological techniques to test these predictions. Our data demonstrate that the majority of VZ cells, and nearly all mitotically active VZ cells during neurogenesis, both have radial glial morphology and express radial glial markers. In addition, intracellular dye filling of electrophysiologically characterized progenitor cells in the VZ demonstrates that these cells have the morphology of radial glia. Because the vast majority cycling cells in the cortical VZ have characteristics of radial glia, the radial glial precursor cell may be responsible for both the production of newborn neurons and the guidance of daughter neurons to their destinations in the developing cortex.

@ARTICLE{Kriegstein2001-qw,
  title    = "{GABA} may act as a self-limiting trophic factor at developing
              synapses",
  author   = "Kriegstein, A R and Owens, D F",
  abstract = "Early in development, synapses with glycine or gamma-aminobutyric
              acid (GABA)-gated chloride channels exhibit the ability to
              depolarize postsynaptic cells. As the synapses mature and the
              gradient of chloride ions across the cell membrane is altered,
              these neurotransmitters signal an inhibitory response,
              hyperpolarizing the membrane and decreasing neuronal
              excitability. Kriegstein and Owens discuss how GABA-stimulated
              up-regulation of the expression of the potassium chloride
              cotransporter KCC2 may be the mechanism underlying this synaptic
              switch.",
  journal  = "Sci STKE",
  volume   =  2001,
  number   =  95,
  pages    = "e1",
  month    =  aug,
  year     =  2001,
  address  = "United States",
  language = "en"
}

Early in development, synapses with glycine or gamma-aminobutyric acid (GABA)-gated chloride channels exhibit the ability to depolarize postsynaptic cells. As the synapses mature and the gradient of chloride ions across the cell membrane is altered, these neurotransmitters signal an inhibitory response, hyperpolarizing the membrane and decreasing neuronal excitability. Kriegstein and Owens discuss how GABA-stimulated up-regulation of the expression of the potassium chloride cotransporter KCC2 may be the mechanism underlying this synaptic switch.

@ARTICLE{Owens2001-yb,
  title    = "Maturation of channels and receptors: consequences for
              excitability",
  author   = "Owens, D F and Kriegstein, A R",
  journal  = "Int Rev Neurobiol",
  volume   =  45,
  pages    = "43--87",
  year     =  2001,
  address  = "United States",
  language = "en"
}

@ARTICLE{Noctor2001-kn,
  title    = "Neurons derived from radial glial cells establish radial units in
              neocortex",
  author   = "Noctor, S C and Flint, A C and Weissman, T A and Dammerman, R S
              and Kriegstein, A R",
  abstract = "The neocortex of the adult brain consists of neurons and glia
              that are generated by precursor cells of the embryonic
              ventricular zone. In general, glia are generated after neurons
              during development, but radial glia are an exception to this
              rule. Radial glia are generated before neurogenesis and guide
              neuronal migration. Radial glia are mitotically active throughout
              neurogenesis, and disappear or become astrocytes when neuronal
              migration is complete. Although the lineage relationships of
              cortical neurons and glia have been explored, the clonal
              relationship of radial glia to other cortical cells remains
              unknown. It has been suggested that radial glia may be neuronal
              precursors, but this has not been demonstrated in vivo. We have
              used a retroviral vector encoding enhanced green fluorescent
              protein to label precursor cells in vivo and have examined clones
              1-3 days later using morphological, immunohistochemical and
              electrophysiological techniques. Here we show that clones consist
              of mitotic radial glia and postmitotic neurons, and that neurons
              migrate along clonally related radial glia. Time-lapse images
              show that proliferative radial glia generate neurons. Our results
              support the concept that a lineage relationship between neurons
              and proliferative radial glia may underlie the radial
              organization of neocortex.",
  journal  = "Nature",
  volume   =  409,
  number   =  6821,
  pages    = "714--720",
  month    =  feb,
  year     =  2001,
  address  = "England",
  language = "en"
}

The neocortex of the adult brain consists of neurons and glia that are generated by precursor cells of the embryonic ventricular zone. In general, glia are generated after neurons during development, but radial glia are an exception to this rule. Radial glia are generated before neurogenesis and guide neuronal migration. Radial glia are mitotically active throughout neurogenesis, and disappear or become astrocytes when neuronal migration is complete. Although the lineage relationships of cortical neurons and glia have been explored, the clonal relationship of radial glia to other cortical cells remains unknown. It has been suggested that radial glia may be neuronal precursors, but this has not been demonstrated in vivo. We have used a retroviral vector encoding enhanced green fluorescent protein to label precursor cells in vivo and have examined clones 1-3 days later using morphological, immunohistochemical and electrophysiological techniques. Here we show that clones consist of mitotic radial glia and postmitotic neurons, and that neurons migrate along clonally related radial glia. Time-lapse images show that proliferative radial glia generate neurons. Our results support the concept that a lineage relationship between neurons and proliferative radial glia may underlie the radial organization of neocortex.

@ARTICLE{Dammerman2000-mo,
  title    = "Transient actions of neurotransmitters during neocortical
              development",
  author   = "Dammerman, R S and Kriegstein, A R",
  journal  = "Epilepsia",
  volume   =  41,
  number   =  8,
  pages    = "1080--1081",
  month    =  aug,
  year     =  2000,
  address  = "United States",
  language = "en"
}

@ARTICLE{Owens2000-nl,
  title    = "Calcium dynamics of neocortical ventricular zone cells",
  author   = "Owens, D F and Flint, A C and Dammerman, R S and Kriegstein, A R",
  abstract = "Cell-cell signaling within the neocortical ventricular zone (VZ)
              has been shown to influence the proliferation of VZ precursor
              cells and the subsequent differentiation and fate of postmitotic
              neurons. Calcium (Ca(2+)), a ubiquitous second messenger
              implicated in the regulation of many aspects of development, may
              play a role in these signaling events. Accordingly, we have
              examined the spatiotemporal patterns of spontaneous intracellular
              free Ca(2+) ([Ca(2+)](i)) fluctuations of cells within the intact
              neocortical VZ. Previous observations have demonstrated that
              similar patterns of spontaneous [Ca(2+)](i) increase occur in
              both proliferative and postmitotic cortical cells, suggesting
              that they may be mechanistically similar. Our results suggest
              that the changes in [Ca(2+)](i) in VZ cells and cortical plate
              neurons are likely triggered by different mechansims, and imply
              that similar changes in [Ca(2+)](i) may underlie different
              signaling events during distinct phases of neocortical
              development.",
  journal  = "Dev Neurosci",
  volume   =  22,
  number   = "1-2",
  pages    = "25--33",
  year     =  2000,
  address  = "Switzerland",
  language = "en"
}

Cell-cell signaling within the neocortical ventricular zone (VZ) has been shown to influence the proliferation of VZ precursor cells and the subsequent differentiation and fate of postmitotic neurons. Calcium (Ca(2+)), a ubiquitous second messenger implicated in the regulation of many aspects of development, may play a role in these signaling events. Accordingly, we have examined the spatiotemporal patterns of spontaneous intracellular free Ca(2+) ([Ca(2+)](i)) fluctuations of cells within the intact neocortical VZ. Previous observations have demonstrated that similar patterns of spontaneous [Ca(2+)](i) increase occur in both proliferative and postmitotic cortical cells, suggesting that they may be mechanistically similar. Our results suggest that the changes in [Ca(2+)](i) in VZ cells and cortical plate neurons are likely triggered by different mechansims, and imply that similar changes in [Ca(2+)](i) may underlie different signaling events during distinct phases of neocortical development.

@ARTICLE{Dammerman2000-yv,
  title    = "An excitatory {GABAergic} plexus in developing neocortical layer
              1",
  author   = "Dammerman, R S and Flint, A C and Noctor, S and Kriegstein, A R",
  abstract = "Layer 1 of the developing rodent somatosensory cortex contains a
              dense, transient GABAergic fiber plexus. Axons arising from the
              zona incerta (ZI) of the ventral thalamus contribute to this
              plexus, as do axons of intrinsic GABAergic cells of layer 1. The
              function of this early-appearing fiber plexus is not known, but
              these fibers are positioned to contact the apical dendrites of
              most postmigratory neurons. Here we show that electrical
              stimulation of layer 1 results in a GABA(A)-mediated postsynaptic
              current (PSC) in pyramidal neurons. Gramicidin perforated patch
              recording demonstrates that the GABAergic layer 1 synapse is
              excitatory and can trigger action potentials in cortical neurons.
              In contrast to electrical stimulation, activation of intrinsic
              layer 1 neurons with a glutamate agonist fails to produce PSCs in
              pyramidal cells. In addition, responses can be evoked by
              stimulation of layer 1 at relatively large distances from the
              recording site. These findings are consistent with a contribution
              of the widely projecting incertocortical pathway, the only
              described GABAergic projection to neonatal cortex. Recording of
              identified neonatal incertocortical neurons reveals a population
              of active cells that exhibit high frequencies of spontaneous
              action potentials and are capable of robustly activating neonatal
              cortical neurons. Because the fiber plexus is confined to layer
              1, this pathway provides a spatially restricted excitatory
              GABAergic innervation of the distal apical dendrites of pyramidal
              neurons during the peak period of cortical synaptogenesis.",
  journal  = "J Neurophysiol",
  volume   =  84,
  number   =  1,
  pages    = "428--434",
  month    =  jul,
  year     =  2000,
  address  = "United States",
  language = "en"
}

Layer 1 of the developing rodent somatosensory cortex contains a dense, transient GABAergic fiber plexus. Axons arising from the zona incerta (ZI) of the ventral thalamus contribute to this plexus, as do axons of intrinsic GABAergic cells of layer 1. The function of this early-appearing fiber plexus is not known, but these fibers are positioned to contact the apical dendrites of most postmigratory neurons. Here we show that electrical stimulation of layer 1 results in a GABA(A)-mediated postsynaptic current (PSC) in pyramidal neurons. Gramicidin perforated patch recording demonstrates that the GABAergic layer 1 synapse is excitatory and can trigger action potentials in cortical neurons. In contrast to electrical stimulation, activation of intrinsic layer 1 neurons with a glutamate agonist fails to produce PSCs in pyramidal cells. In addition, responses can be evoked by stimulation of layer 1 at relatively large distances from the recording site. These findings are consistent with a contribution of the widely projecting incertocortical pathway, the only described GABAergic projection to neonatal cortex. Recording of identified neonatal incertocortical neurons reveals a population of active cells that exhibit high frequencies of spontaneous action potentials and are capable of robustly activating neonatal cortical neurons. Because the fiber plexus is confined to layer 1, this pathway provides a spatially restricted excitatory GABAergic innervation of the distal apical dendrites of pyramidal neurons during the peak period of cortical synaptogenesis.

@ARTICLE{Dammerman2000-jq,
  title    = "Extrinsic {GABAergic} innervation of developing neocortical layer
              1 in organotypic slice co-cultures",
  author   = "Dammerman, R S and Noctor, S C and Kriegstein, A R",
  abstract = "Afferents from the zona incerta (ZI) of the ventral thalamus
              contribute to the dense, transient gamma-aminobutyric acid
              (GABA)ergic fiber plexus in layer 1 of the developing rodent
              somatosensory cortex. Incertocortical axons contact the distal
              apical dendrites of postmigratory cortical pyramidal cells.
              Although recent work has shown that these GABAergic
              incertocortical fibers are likely to provide widespread fast
              synaptic excitation of pyramidal cells in layers 2-6 during peak
              periods of cortical synaptogenesis, little is known about the
              mechanisms by which these axons project to the neocortex and are
              confined to layer 1. Here we characterize organotypic slice
              co-cultures in which a region of embryonic diencephalon
              containing the ZI is maintained adjacent to a region of embryonic
              somatosensory cortex. Diencephalic explants from transgenic mice
              expressing enhanced green fluorescent protein (EGFP) enabled
              direct visualization of diencephalocortical connections.
              Isochronic co-cultures exhibited diencephalocortical fiber
              ingrowth immunoreactive for both GABA and the presynaptic
              vesicle-associated protein synaptophysin that was restricted to
              neocortical layer 1. This pattern of lamina-specific
              diencephalocortical ingrowth occurred irrespective of placement
              of the afferent explant, and persisted in the absence of action
              potential activity and GABA(A) receptor activation. Heterochronic
              co-cultures containing older cortex demonstrated that the
              cortical explants remain permissive for lamina-specific ingrowth
              through the first postnatal week. Organotypic slice cocultures
              provide a system in which to study the mechanisms underlying the
              layer 1-specific ingrowth of extrinsic GABAergic inputs to the
              perinatal neocortex.",
  journal  = "J Comp Neurol",
  volume   =  423,
  number   =  1,
  pages    = "112--120",
  month    =  jul,
  year     =  2000,
  address  = "United States",
  language = "en"
}

Afferents from the zona incerta (ZI) of the ventral thalamus contribute to the dense, transient gamma-aminobutyric acid (GABA)ergic fiber plexus in layer 1 of the developing rodent somatosensory cortex. Incertocortical axons contact the distal apical dendrites of postmigratory cortical pyramidal cells. Although recent work has shown that these GABAergic incertocortical fibers are likely to provide widespread fast synaptic excitation of pyramidal cells in layers 2-6 during peak periods of cortical synaptogenesis, little is known about the mechanisms by which these axons project to the neocortex and are confined to layer 1. Here we characterize organotypic slice co-cultures in which a region of embryonic diencephalon containing the ZI is maintained adjacent to a region of embryonic somatosensory cortex. Diencephalic explants from transgenic mice expressing enhanced green fluorescent protein (EGFP) enabled direct visualization of diencephalocortical connections. Isochronic co-cultures exhibited diencephalocortical fiber ingrowth immunoreactive for both GABA and the presynaptic vesicle-associated protein synaptophysin that was restricted to neocortical layer 1. This pattern of lamina-specific diencephalocortical ingrowth occurred irrespective of placement of the afferent explant, and persisted in the absence of action potential activity and GABA(A) receptor activation. Heterochronic co-cultures containing older cortex demonstrated that the cortical explants remain permissive for lamina-specific ingrowth through the first postnatal week. Organotypic slice cocultures provide a system in which to study the mechanisms underlying the layer 1-specific ingrowth of extrinsic GABAergic inputs to the perinatal neocortex.

@ARTICLE{Kriegstein1999-fb,
  title    = "Ontogeny of channels, transmitters and epileptogenesis",
  author   = "Kriegstein, A R and Owens, D F and Avoli, M",
  journal  = "Adv Neurol",
  volume   =  79,
  pages    = "145--159",
  year     =  1999,
  address  = "United States",
  language = "en"
}

@ARTICLE{Owens1999-bd,
  title    = "Changing properties of {GABA(A}) receptor-mediated signaling
              during early neocortical development",
  author   = "Owens, D F and Liu, X and Kriegstein, A R",
  abstract = "Evidence from several brain regions suggests gamma-aminobutyric
              acid (GABA) can exert a trophic influence during development,
              expanding the role of this amino acid beyond its function as an
              inhibitory neurotransmitter. Proliferating precursor cells in the
              neocortical ventricular zone (VZ) express functional GABA(A)
              receptors as do immature postmigratory neurons in the developing
              cortical plate (CP); however, GABA(A) receptor properties in
              these distinct cell populations have not been compared. Using
              electrophysiological techniques in embryonic and early postnatal
              neocortex, we find that GABA(A) receptors expressed by VZ cells
              have a higher apparent affinity for GABA and are relatively
              insensitive to receptor desensitization compared with neurons in
              the CP. GABA-induced current magnitude increases with maturation
              with the smallest responses found in recordings from precursor
              cells in the VZ. No evidence was found that GABA(A) receptors on
              VZ cells are activated synaptically, consistent with previous
              data suggesting that these receptors are activated in a paracrine
              fashion by nonsynaptically released ligand. After neurons are
              born and migrate to the CP, they begin to demonstrate spontaneous
              synaptic activity, the majority of which is GABA(A) mediated.
              These spontaneous GABA(A) postsynaptic currents (sPSCs) first
              were detected at embryonic day 18 (E18). At birth, approximately
              50\% of recordings from cortical neurons demonstrated
              GABA(A)-mediated sPSCs, and this value increased with age.
              GABA(A)-mediated sPSCs were action potential dependent and arose
              from local GABAergic interneurons. GABA application could evoke
              action potential-dependent PSCs in neonatal cortical neurons,
              suggesting that during the first few postnatal days, GABA can act
              as an excitatory neurotransmitter. Finally, N-methyl-D-aspartate
              (NMDA)- but not non-NMDA-mediated sPSCs were also present in
              early postnatal neurons. These events were not observed in cells
              voltage clamped at negative holding potentials (-60 to -70 mV)
              but were evident when the holding potential was set at positive
              values (+30 to +60 mV). Together these results provide evidence
              for the early maturation of GABAergic communication in the
              neocortex and a functional change in GABA(A)-receptor properties
              between precursor cells and early postmitotic neurons. The change
              in GABA(A)-receptor properties may reflect the shift from
              paracrine to synaptic receptor activation.",
  journal  = "J Neurophysiol",
  volume   =  82,
  number   =  2,
  pages    = "570--583",
  month    =  aug,
  year     =  1999,
  address  = "United States",
  language = "en"
}

Evidence from several brain regions suggests gamma-aminobutyric acid (GABA) can exert a trophic influence during development, expanding the role of this amino acid beyond its function as an inhibitory neurotransmitter. Proliferating precursor cells in the neocortical ventricular zone (VZ) express functional GABA(A) receptors as do immature postmigratory neurons in the developing cortical plate (CP); however, GABA(A) receptor properties in these distinct cell populations have not been compared. Using electrophysiological techniques in embryonic and early postnatal neocortex, we find that GABA(A) receptors expressed by VZ cells have a higher apparent affinity for GABA and are relatively insensitive to receptor desensitization compared with neurons in the CP. GABA-induced current magnitude increases with maturation with the smallest responses found in recordings from precursor cells in the VZ. No evidence was found that GABA(A) receptors on VZ cells are activated synaptically, consistent with previous data suggesting that these receptors are activated in a paracrine fashion by nonsynaptically released ligand. After neurons are born and migrate to the CP, they begin to demonstrate spontaneous synaptic activity, the majority of which is GABA(A) mediated. These spontaneous GABA(A) postsynaptic currents (sPSCs) first were detected at embryonic day 18 (E18). At birth, approximately 50% of recordings from cortical neurons demonstrated GABA(A)-mediated sPSCs, and this value increased with age. GABA(A)-mediated sPSCs were action potential dependent and arose from local GABAergic interneurons. GABA application could evoke action potential-dependent PSCs in neonatal cortical neurons, suggesting that during the first few postnatal days, GABA can act as an excitatory neurotransmitter. Finally, N-methyl-D-aspartate (NMDA)- but not non-NMDA-mediated sPSCs were also present in early postnatal neurons. These events were not observed in cells voltage clamped at negative holding potentials (-60 to -70 mV) but were evident when the holding potential was set at positive values (+30 to +60 mV). Together these results provide evidence for the early maturation of GABAergic communication in the neocortex and a functional change in GABA(A)-receptor properties between precursor cells and early postmitotic neurons. The change in GABA(A)-receptor properties may reflect the shift from paracrine to synaptic receptor activation.

@ARTICLE{Flint1999-ol,
  title    = "Endogenous activation of metabotropic glutamate receptors in
              neocortical development causes neuronal calcium oscillations",
  author   = "Flint, A C and Dammerman, R S and Kriegstein, A R",
  abstract = "Oscillations in intracellular free calcium concentration
              ([Ca(2+)](i)) occur spontaneously in immature neurons of the
              developing cerebral cortex. Here, we show that developing murine
              cortical neurons exhibit calcium oscillations in response to
              direct activation of the mGluR5 subtype of the group I
              metabotropic glutamate receptor (mGluR). In contrast, other
              manipulations that elicit [Ca(2+)](i) increases produce simple,
              nonoscillatory changes. Furthermore, we find that spontaneous
              oscillatory [Ca(2+)](i) activity is blocked by antagonists of
              group I mGluRs, suggesting a specific role for mGluR activation
              in the promotion of oscillatory [Ca(2+)](i) dynamics in immature
              cortical neurons. The oscillatory pattern of [Ca(2+)](i)
              increases produced by mGluR activation might play a role in the
              regulation of gene expression and the control of developmental
              events.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  96,
  number   =  21,
  pages    = "12144--12149",
  month    =  oct,
  year     =  1999,
  language = "en"
}

Oscillations in intracellular free calcium concentration ([Ca(2+)](i)) occur spontaneously in immature neurons of the developing cerebral cortex. Here, we show that developing murine cortical neurons exhibit calcium oscillations in response to direct activation of the mGluR5 subtype of the group I metabotropic glutamate receptor (mGluR). In contrast, other manipulations that elicit [Ca(2+)](i) increases produce simple, nonoscillatory changes. Furthermore, we find that spontaneous oscillatory [Ca(2+)](i) activity is blocked by antagonists of group I mGluRs, suggesting a specific role for mGluR activation in the promotion of oscillatory [Ca(2+)](i) dynamics in immature cortical neurons. The oscillatory pattern of [Ca(2+)](i) increases produced by mGluR activation might play a role in the regulation of gene expression and the control of developmental events.

@ARTICLE{Kriegstein1999-xf,
  title    = "Leukoencephalopathy and raised brain lactate from heroin vapor
              inhalation (``chasing the dragon'')",
  author   = "Kriegstein, A R and Shungu, D C and Millar, W S and Armitage, B A
              and Brust, J C and Chillrud, S and Goldman, J and Lynch, T",
  abstract = "BACKGROUND: Inhalation of heated heroin vapor (``chasing the
              dragon''), which is gaining popularity among drug users seeking
              to avoid the risks of parenteral drug administration, can produce
              progressive spongiform leukoencephalopathy. METHODS: We studied
              the clinical phenotype and course, MRI, MRS, and brain pathology
              in the first American patients described with this syndrome.
              RESULTS: Two of the three heroin users studied inhaled heroin
              pyrolysate together daily over the course of 2 weeks. They
              developed ataxia, dysmetria, and dysarthria. Patient 1 progressed
              to an akinetic mute state with decorticate posture and subsequent
              spastic quadriparesis. Patient 2 developed a mild spastic
              quadriparesis and gait freezing. Patient 3 was asymptomatic
              following less heroin exposure. Brain MRI showed diffuse,
              symmetrical white matter hyperintensities in the cerebellum,
              posterior cerebrum, posterior limbs of the internal capsule,
              splenium of the corpus callosum, medial lemniscus, and lateral
              brainstem. MRS showed elevated lactate. Brain biopsy (Patient 1)
              showed white matter spongiform degeneration with relative sparing
              of U-fibers; electron microscopy revealed intramyelinic
              vacuolation with splitting of intraperiod lines. Progressive
              deterioration occurred in Patients 1 and 2 over 4 weeks. Both
              were treated with antioxidants including oral coenzyme Q, and
              clinical improvement occurred. Patient 1 recovered nearly
              completely over 24 months. Patient 2 improved, but developed a
              delayed-onset cerebellar hand tremor. Both still have white
              matter abnormalities on MRI and MRS. CONCLUSIONS: Elevated
              lactate in white matter and the possible response to antioxidants
              suggests mitochondrial dysfunction in progressive spongiform
              leukoencephalopathy following inhalation of heated heroin vapor.",
  journal  = "Neurology",
  volume   =  53,
  number   =  8,
  pages    = "1765--1773",
  month    =  nov,
  year     =  1999,
  address  = "United States",
  language = "en"
}

BACKGROUND: Inhalation of heated heroin vapor (``chasing the dragon''), which is gaining popularity among drug users seeking to avoid the risks of parenteral drug administration, can produce progressive spongiform leukoencephalopathy. METHODS: We studied the clinical phenotype and course, MRI, MRS, and brain pathology in the first American patients described with this syndrome. RESULTS: Two of the three heroin users studied inhaled heroin pyrolysate together daily over the course of 2 weeks. They developed ataxia, dysmetria, and dysarthria. Patient 1 progressed to an akinetic mute state with decorticate posture and subsequent spastic quadriparesis. Patient 2 developed a mild spastic quadriparesis and gait freezing. Patient 3 was asymptomatic following less heroin exposure. Brain MRI showed diffuse, symmetrical white matter hyperintensities in the cerebellum, posterior cerebrum, posterior limbs of the internal capsule, splenium of the corpus callosum, medial lemniscus, and lateral brainstem. MRS showed elevated lactate. Brain biopsy (Patient 1) showed white matter spongiform degeneration with relative sparing of U-fibers; electron microscopy revealed intramyelinic vacuolation with splitting of intraperiod lines. Progressive deterioration occurred in Patients 1 and 2 over 4 weeks. Both were treated with antioxidants including oral coenzyme Q, and clinical improvement occurred. Patient 1 recovered nearly completely over 24 months. Patient 2 improved, but developed a delayed-onset cerebellar hand tremor. Both still have white matter abnormalities on MRI and MRS. CONCLUSIONS: Elevated lactate in white matter and the possible response to antioxidants suggests mitochondrial dysfunction in progressive spongiform leukoencephalopathy following inhalation of heated heroin vapor.

@ARTICLE{Flint1998-np,
  title    = "Nonsynaptic glycine receptor activation during early neocortical
              development",
  author   = "Flint, A C and Liu, X and Kriegstein, A R",
  abstract = "Glycine receptors (GlyRs) contribute to fast inhibitory synaptic
              transmission in the brain stem and spinal cord. GlyR subunits are
              expressed in the developing neocortex, but a neurotransmitter
              system involving cortical GlyRs has yet to be demonstrated. Here,
              we show that GlyRs in immature neocortex are excitatory and
              activated by a nonsynaptically released endogenous ligand. Of the
              potential ligands for cortical GlyRs, taurine is by far the most
              abundant in the developing neocortex. We found that taurine is
              stored in immature cortical neurons and that manipulations known
              to elevate extracellular taurine cause GlyR activation. These
              data indicate that nonsynaptically released taurine activates
              GlyRs during neocortical development. As fetal taurine
              deprivation can cause cortical dysgenesis, it is possible that
              taurine influences neocortical development by activating GlyRs.",
  journal  = "Neuron",
  volume   =  20,
  number   =  1,
  pages    = "43--53",
  month    =  jan,
  year     =  1998,
  address  = "United States",
  language = "en"
}

Glycine receptors (GlyRs) contribute to fast inhibitory synaptic transmission in the brain stem and spinal cord. GlyR subunits are expressed in the developing neocortex, but a neurotransmitter system involving cortical GlyRs has yet to be demonstrated. Here, we show that GlyRs in immature neocortex are excitatory and activated by a nonsynaptically released endogenous ligand. Of the potential ligands for cortical GlyRs, taurine is by far the most abundant in the developing neocortex. We found that taurine is stored in immature cortical neurons and that manipulations known to elevate extracellular taurine cause GlyR activation. These data indicate that nonsynaptically released taurine activates GlyRs during neocortical development. As fetal taurine deprivation can cause cortical dysgenesis, it is possible that taurine influences neocortical development by activating GlyRs.

@ARTICLE{Owens1998-hc,
  title    = "Patterns of intracellular calcium fluctuation in precursor cells
              of the neocortical ventricular zone",
  author   = "Owens, D F and Kriegstein, A R",
  abstract = "Changes in intracellular free calcium concentration ([Ca2+]i) are
              known to influence a variety of events in developing neurons.
              Although spontaneous changes of [Ca2+]i have been examined in
              immature cortical neurons, the calcium dynamics of cortical
              precursor cells have received less attention. Using an intact
              cortical mantle and confocal laser microscopy, we examined the
              spatiotemporal patterns of spontaneous [Ca2+]i fluctuations in
              neocortical ventricular zone (VZ) cells in situ. The majority of
              activity consisted of single cells that displayed independent
              [Ca2+]i fluctuations. These events occurred in cells throughout
              the depth of the VZ. Immunohistochemical staining confirmed that
              these events occurred primarily in precursor cells rather than in
              postmitotic neurons. When imaging near the ventricular surface,
              synchronous spontaneous [Ca2+]i increases were frequently
              observed in pairs of adjacent cells. Cellular morphology,
              time-lapse imaging, and nuclear staining demonstrated that this
              activity occurred in mitotically active cells. A third and
              infrequently encountered pattern of activity consisted of
              coordinated spontaneous increases in [Ca2+]i in groups of
              neighboring VZ cells. The morphological characteristics of these
              cells and immunohistochemical staining suggested that the
              coordinated events occurred in gap junction-coupled precursor
              cells. All three patterns of activity were dependent on the
              release of Ca2+ from intracellular stores. These results
              demonstrate distinct patterns of spontaneous [Ca2+]i change in
              cortical precursor cells and raise the possibility that these
              dynamics may contribute to the regulation of neurogenesis.",
  journal  = "J Neurosci",
  volume   =  18,
  number   =  14,
  pages    = "5374--5388",
  month    =  jul,
  year     =  1998,
  language = "en"
}

Changes in intracellular free calcium concentration ([Ca2+]i) are known to influence a variety of events in developing neurons. Although spontaneous changes of [Ca2+]i have been examined in immature cortical neurons, the calcium dynamics of cortical precursor cells have received less attention. Using an intact cortical mantle and confocal laser microscopy, we examined the spatiotemporal patterns of spontaneous [Ca2+]i fluctuations in neocortical ventricular zone (VZ) cells in situ. The majority of activity consisted of single cells that displayed independent [Ca2+]i fluctuations. These events occurred in cells throughout the depth of the VZ. Immunohistochemical staining confirmed that these events occurred primarily in precursor cells rather than in postmitotic neurons. When imaging near the ventricular surface, synchronous spontaneous [Ca2+]i increases were frequently observed in pairs of adjacent cells. Cellular morphology, time-lapse imaging, and nuclear staining demonstrated that this activity occurred in mitotically active cells. A third and infrequently encountered pattern of activity consisted of coordinated spontaneous increases in [Ca2+]i in groups of neighboring VZ cells. The morphological characteristics of these cells and immunohistochemical staining suggested that the coordinated events occurred in gap junction-coupled precursor cells. All three patterns of activity were dependent on the release of Ca2+ from intracellular stores. These results demonstrate distinct patterns of spontaneous [Ca2+]i change in cortical precursor cells and raise the possibility that these dynamics may contribute to the regulation of neurogenesis.

@ARTICLE{Flint1997-is,
  title    = "Mechanisms underlying neuronal migration disorders and epilepsy",
  author   = "Flint, A C and Kriegstein, A R",
  abstract = "Neuronal migration disorders are often associated with
              intractable epilepsy. These cortical malformations are quite
              heterogeneous, suggesting that they may result from interference
              with a diverse set of processes during corticogenesis. Progress
              toward understanding the pathophysiologic basis of these
              disorders is coming from research into the basic mechanisms of
              corticogenesis, animal models of cortical malformations, and
              molecular genetic approaches to migration disorders.",
  journal  = "Curr Opin Neurol",
  volume   =  10,
  number   =  2,
  pages    = "92--97",
  month    =  apr,
  year     =  1997,
  address  = "England",
  language = "en"
}

Neuronal migration disorders are often associated with intractable epilepsy. These cortical malformations are quite heterogeneous, suggesting that they may result from interference with a diverse set of processes during corticogenesis. Progress toward understanding the pathophysiologic basis of these disorders is coming from research into the basic mechanisms of corticogenesis, animal models of cortical malformations, and molecular genetic approaches to migration disorders.

@ARTICLE{Kriegstein1997-wn,
  title    = "Heroin inhalation and progressive spongiform leukoencephalopathy",
  author   = "Kriegstein, A R and Armitage, B A and Kim, P Y",
  journal  = "N Engl J Med",
  volume   =  336,
  number   =  8,
  pages    = "589--590",
  month    =  feb,
  year     =  1997,
  address  = "United States",
  language = "en"
}

@ARTICLE{Flint1997-iq,
  title    = "{NR2A} subunit expression shortens {NMDA} receptor synaptic
              currents in developing neocortex",
  author   = "Flint, A C and Maisch, U S and Weishaupt, J H and Kriegstein, A R
              and Monyer, H",
  abstract = "NMDA receptors play important roles in learning and memory and in
              sculpting neural connections during development. After the period
              of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR
              EPSCs) decrease in duration. A likely mechanism for this change
              in NMDA receptor properties is the molecular alteration of NMDA
              receptor structure by regulation of NMDA receptor subunit gene
              expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor
              subunits are known to alter NMDA receptor properties, and the
              expression of these subunits is regulated developmentally. It is
              unclear, however, how the four NR2 subunits are expressed in
              individual neurons and which NR2 subunits are important to the
              regulation of NMDA receptor properties during development in
              vivo. Analysis of NR2 subunit gene expression in single
              characterized neurons of postnatal neocortex revealed that cells
              expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells
              not expressing this subunit, regardless of postnatal age.
              Expression of NR2A subunit mRNA in cortical neurons at even low
              levels seemed sufficient to alter the NMDA receptor time course.
              The proportion of cells expressing NR2A and displaying fast NMDAR
              EPSCs increased developmentally, thus providing a molecular basis
              for the developmental change in mean NMDAR EPSC duration.",
  journal  = "J Neurosci",
  volume   =  17,
  number   =  7,
  pages    = "2469--2476",
  month    =  apr,
  year     =  1997,
  language = "en"
}

NMDA receptors play important roles in learning and memory and in sculpting neural connections during development. After the period of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR EPSCs) decrease in duration. A likely mechanism for this change in NMDA receptor properties is the molecular alteration of NMDA receptor structure by regulation of NMDA receptor subunit gene expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor subunits are known to alter NMDA receptor properties, and the expression of these subunits is regulated developmentally. It is unclear, however, how the four NR2 subunits are expressed in individual neurons and which NR2 subunits are important to the regulation of NMDA receptor properties during development in vivo. Analysis of NR2 subunit gene expression in single characterized neurons of postnatal neocortex revealed that cells expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells not expressing this subunit, regardless of postnatal age. Expression of NR2A subunit mRNA in cortical neurons at even low levels seemed sufficient to alter the NMDA receptor time course. The proportion of cells expressing NR2A and displaying fast NMDAR EPSCs increased developmentally, thus providing a molecular basis for the developmental change in mean NMDAR EPSC duration.

@ARTICLE{Flint1997-um,
  title    = "Postnatal development of low [Mg2+] oscillations in neocortex",
  author   = "Flint, A C and Maisch, U S and Kriegstein, A R",
  abstract = "One form of rhythmic activity intrinsic to neocortex can be
              induced in slices of adult somatosensory cortex by lowering
              [Mg2+]o to unblock N-methyl--aspartate (NMDA) receptors. It has
              been suggested that a population of intrinsically burst-firing
              (IB) neurons that are unique to cortical layer 5 may play a role
              in the rhythmic activity seen under these conditions. Whole cell
              patch-clamp and field-potential recordings in slices of
              somatosensory cortex from neonatal rats were used to study the
              development of IB cells and the development of 0 [Mg2+]
              oscillations. IB cells were not encountered before postnatal day
              12 (P12) in layer 5, but from P13 to P19 an increasing proportion
              of cells had IB properties. Recordings from cells at P7, P17, and
              P19 in 0 [Mg2+] indicate that dramatic changes occur postnatally
              in 0 [Mg2+]-induced activity. At P7, cells largely showed trains
              of single action potentials. In contrast, at P19, cells showed
              organized bursts of rhythmic activity lasting 0.5-5 s separated
              by periods of relative quiescence. Cells recorded at P17 were
              found to have less organized rhythmic activity than cells from
              P19 cortex. Field-potential recordings in 0 [Mg2+] made at P7
              showed infrequent and slowly occurring field depolarizations,
              whereas field-potential recordings at P19 consisted of
              spontaneous bursts of 4-12 Hz oscillations identical to those
              observed in the adult. Application of NE, which inhibits
              burst-firing of layer 5 IB cells, significantly altered the
              discharge pattern of 0 [Mg2+] oscillations at P19. These data
              suggest that the maturation of one type of rhythmic network
              activity intrinsic to neocortex is influenced by the development
              of the membrane properties of a single cell type.",
  journal  = "J Neurophysiol",
  volume   =  78,
  number   =  4,
  pages    = "1990--1996",
  month    =  oct,
  year     =  1997,
  address  = "United States",
  language = "en"
}

One form of rhythmic activity intrinsic to neocortex can be induced in slices of adult somatosensory cortex by lowering [Mg2+]o to unblock N-methyl–aspartate (NMDA) receptors. It has been suggested that a population of intrinsically burst-firing (IB) neurons that are unique to cortical layer 5 may play a role in the rhythmic activity seen under these conditions. Whole cell patch-clamp and field-potential recordings in slices of somatosensory cortex from neonatal rats were used to study the development of IB cells and the development of 0 [Mg2+] oscillations. IB cells were not encountered before postnatal day 12 (P12) in layer 5, but from P13 to P19 an increasing proportion of cells had IB properties. Recordings from cells at P7, P17, and P19 in 0 [Mg2+] indicate that dramatic changes occur postnatally in 0 [Mg2+]-induced activity. At P7, cells largely showed trains of single action potentials. In contrast, at P19, cells showed organized bursts of rhythmic activity lasting 0.5-5 s separated by periods of relative quiescence. Cells recorded at P17 were found to have less organized rhythmic activity than cells from P19 cortex. Field-potential recordings in 0 [Mg2+] made at P7 showed infrequent and slowly occurring field depolarizations, whereas field-potential recordings at P19 consisted of spontaneous bursts of 4-12 Hz oscillations identical to those observed in the adult. Application of NE, which inhibits burst-firing of layer 5 IB cells, significantly altered the discharge pattern of 0 [Mg2+] oscillations at P19. These data suggest that the maturation of one type of rhythmic network activity intrinsic to neocortex is influenced by the development of the membrane properties of a single cell type.

@ARTICLE{Bittman1997-ow,
  title    = "Cell coupling and uncoupling in the ventricular zone of
              developing neocortex",
  author   = "Bittman, K and Owens, D F and Kriegstein, A R and LoTurco, J J",
  abstract = "Cells within the ventricular zone (VZ) of developing neocortex
              are coupled together into clusters by gap junction channels. The
              specific role of clustering in cortical neurogenesis is unknown;
              however, clustering provides a means for spatially restricted
              local interactions between subsets of precursors and other cells
              within the VZ. In the present study, we have used a combination
              of 5-bromo-2'-deoxyuridine (BrDU) pulse labeling, intracellular
              biocytin labeling, and immunocytochemistry to determine when in
              the cell cycle VZ cells couple and uncouple from clusters and to
              determine what cell types within the VZ are coupled to clusters.
              Our results indicate that clusters contain radial glia and neural
              precursors but do not contain differentiating or migrating
              neurons. In early neurogenesis, all precursors in S and G2 phases
              of the cell cycle are coupled, and approximately half of the
              cells in G1 are coupled. In late neurogenesis, however, over half
              of the cells in both G1 and S phases are not coupled to VZ
              clusters, whereas all cells in G2 are coupled to clusters.
              Increased uncoupling in S phase during late neurogenesis may
              contribute to the greater percentage of VZ cells exiting the cell
              cycle at this time. Consistent with this hypothesis, we found
              that pharmacologically uncoupling VZ cells with octanol decreases
              the percentage of VZ cells that enter S phase. These results
              demonstrate that cell clustering in the VZ is restricted to
              neural precursors and radial glia, is dynamic through the cell
              cycle, and may play a role in regulating neurogenesis.",
  journal  = "J Neurosci",
  volume   =  17,
  number   =  18,
  pages    = "7037--7044",
  month    =  sep,
  year     =  1997,
  language = "en"
}

Cells within the ventricular zone (VZ) of developing neocortex are coupled together into clusters by gap junction channels. The specific role of clustering in cortical neurogenesis is unknown; however, clustering provides a means for spatially restricted local interactions between subsets of precursors and other cells within the VZ. In the present study, we have used a combination of 5-bromo-2'-deoxyuridine (BrDU) pulse labeling, intracellular biocytin labeling, and immunocytochemistry to determine when in the cell cycle VZ cells couple and uncouple from clusters and to determine what cell types within the VZ are coupled to clusters. Our results indicate that clusters contain radial glia and neural precursors but do not contain differentiating or migrating neurons. In early neurogenesis, all precursors in S and G2 phases of the cell cycle are coupled, and approximately half of the cells in G1 are coupled. In late neurogenesis, however, over half of the cells in both G1 and S phases are not coupled to VZ clusters, whereas all cells in G2 are coupled to clusters. Increased uncoupling in S phase during late neurogenesis may contribute to the greater percentage of VZ cells exiting the cell cycle at this time. Consistent with this hypothesis, we found that pharmacologically uncoupling VZ cells with octanol decreases the percentage of VZ cells that enter S phase. These results demonstrate that cell clustering in the VZ is restricted to neural precursors and radial glia, is dynamic through the cell cycle, and may play a role in regulating neurogenesis.

@ARTICLE{Kriegstein1996-tm,
  title    = "Cortical neurogenesis and its disorders",
  author   = "Kriegstein, A R",
  abstract = "Proliferative cells lining the cerebral ventricles generate all
              of the phenotypically diverse neurons of the adult cortex. Recent
              evidence indicates that cell cycle events of neuronal precursor
              cells are under the influence of neurotransmitters and a variety
              of signaling factors. The newly discovered environmental factors
              that regulate neurogenesis promise to expand our understanding of
              the mechanisms responsible for cerebral malformations and
              disorders of cortical organization.",
  journal  = "Curr Opin Neurol",
  volume   =  9,
  number   =  2,
  pages    = "113--117",
  month    =  apr,
  year     =  1996,
  address  = "England",
  language = "en"
}

Proliferative cells lining the cerebral ventricles generate all of the phenotypically diverse neurons of the adult cortex. Recent evidence indicates that cell cycle events of neuronal precursor cells are under the influence of neurotransmitters and a variety of signaling factors. The newly discovered environmental factors that regulate neurogenesis promise to expand our understanding of the mechanisms responsible for cerebral malformations and disorders of cortical organization.

@ARTICLE{Owens1996-gh,
  title    = "Excitatory {GABA} responses in embryonic and neonatal cortical
              slices demonstrated by gramicidin perforated-patch recordings and
              calcium imaging",
  author   = "Owens, D F and Boyce, L H and Davis, M B and Kriegstein, A R",
  abstract = "Gramicidin perforated-patch-clamp recordings in brain slices were
              used to obtain an accurate assessment of the developmental change
              in the GABAA receptor reversal potential (EGABAA) in embryonic
              and early postnatal rat neocortical cells including
              neuroepithelial precursor cells, cortical plate neurons, and
              postnatal neocortical neurons. Our results demonstrate that there
              is a progressive negative shift in EGABAA with the most positive
              values found in the youngest cortical precursor cells. At the
              early stages of neocortical development, EGABAA is determined by
              the chloride (Cl-) gradient, and the internal chloride
              concentration ([Cl-]i) decreases with development. EGABAA is
              positive to the resting potential, indicating that GABA serves to
              depolarize developing neocortical cells. Consistent with this
              conclusion, GABAA receptor activation with muscimol was found-to
              increase the internal calcium concentration ([Ca2+]i) in both
              embryonic and early postnatal neocortical cells through the
              activation of voltage-gated calcium channels (VGCCs). Postnatal
              cells exhibit spontaneous postsynaptic synaptic currents, which
              are eliminated by bicuculline methiodide (BMI) but not glutamate
              receptor antagonists and reverse at the Cl- equilibrium
              potential. Likewise, brief spontaneous increases in [Ca2+]i,
              sensitive to BMI and TTX, are observed at the same ages,
              suggesting that endogenous synaptic GABAA receptor activation can
              depolarize cells and activate VGCCs. These results suggest that
              GABAA receptor-mediated depolarization may influence early
              neocortical developmental events, including neurogenesis and
              synaptogenesis, through the activation of Ca(2+)-dependent signal
              transduction pathways.",
  journal  = "J Neurosci",
  volume   =  16,
  number   =  20,
  pages    = "6414--6423",
  month    =  oct,
  year     =  1996,
  language = "en"
}

Gramicidin perforated-patch-clamp recordings in brain slices were used to obtain an accurate assessment of the developmental change in the GABAA receptor reversal potential (EGABAA) in embryonic and early postnatal rat neocortical cells including neuroepithelial precursor cells, cortical plate neurons, and postnatal neocortical neurons. Our results demonstrate that there is a progressive negative shift in EGABAA with the most positive values found in the youngest cortical precursor cells. At the early stages of neocortical development, EGABAA is determined by the chloride (Cl-) gradient, and the internal chloride concentration ([Cl-]i) decreases with development. EGABAA is positive to the resting potential, indicating that GABA serves to depolarize developing neocortical cells. Consistent with this conclusion, GABAA receptor activation with muscimol was found-to increase the internal calcium concentration ([Ca2+]i) in both embryonic and early postnatal neocortical cells through the activation of voltage-gated calcium channels (VGCCs). Postnatal cells exhibit spontaneous postsynaptic synaptic currents, which are eliminated by bicuculline methiodide (BMI) but not glutamate receptor antagonists and reverse at the Cl- equilibrium potential. Likewise, brief spontaneous increases in [Ca2+]i, sensitive to BMI and TTX, are observed at the same ages, suggesting that endogenous synaptic GABAA receptor activation can depolarize cells and activate VGCCs. These results suggest that GABAA receptor-mediated depolarization may influence early neocortical developmental events, including neurogenesis and synaptogenesis, through the activation of Ca(2+)-dependent signal transduction pathways.

@ARTICLE{LoTurco1995-qf,
  title    = "{GABA} and glutamate depolarize cortical progenitor cells and
              inhibit {DNA} synthesis",
  author   = "LoTurco, J J and Owens, D F and Heath, M J and Davis, M B and
              Kriegstein, A R",
  abstract = "We have found that, during the early stages of cortical
              neurogenesis, both GABA and glutamate depolarize cells in the
              ventricular zone of rat embryonic neocortex. In the ventricular
              zone, glutamate acts on AMPA/kainate receptors, while GABA acts
              on GABAA receptors. GABA induces an inward current at resting
              membrane potentials, presumably owing to a high intracellular Cl-
              concentration maintained by furosemide-sensitive Cl- transport.
              GABA and glutamate also produce increases in intracellular Ca2+
              in ventricular zone cells, in part through activation of
              voltage-gated Ca2+ channels. Furthermore, GABA and glutamate
              decrease the number of embryonic cortical cells synthesizing DNA.
              Depolarization with K+ similarly decreases DNA synthesis,
              suggesting that the neurotransmitters act via membrane
              depolarization. Applied alone, GABAA and AMPA/kainate receptor
              antagonists increase DNA synthesis, indicating that endogenously
              released amino acids influence neocortical progenitors in the
              cell cycle. These results demonstrate a novel role for amino acid
              neurotransmitters in regulating neocortical neurogenesis.",
  journal  = "Neuron",
  volume   =  15,
  number   =  6,
  pages    = "1287--1298",
  month    =  dec,
  year     =  1995,
  address  = "United States",
  language = "en"
}

We have found that, during the early stages of cortical neurogenesis, both GABA and glutamate depolarize cells in the ventricular zone of rat embryonic neocortex. In the ventricular zone, glutamate acts on AMPA/kainate receptors, while GABA acts on GABAA receptors. GABA induces an inward current at resting membrane potentials, presumably owing to a high intracellular Cl- concentration maintained by furosemide-sensitive Cl- transport. GABA and glutamate also produce increases in intracellular Ca2+ in ventricular zone cells, in part through activation of voltage-gated Ca2+ channels. Furthermore, GABA and glutamate decrease the number of embryonic cortical cells synthesizing DNA. Depolarization with K+ similarly decreases DNA synthesis, suggesting that the neurotransmitters act via membrane depolarization. Applied alone, GABAA and AMPA/kainate receptor antagonists increase DNA synthesis, indicating that endogenously released amino acids influence neocortical progenitors in the cell cycle. These results demonstrate a novel role for amino acid neurotransmitters in regulating neocortical neurogenesis.

@ARTICLE{Troyer1992-ro,
  title    = "Abnormal action-potential bursts and synchronized,
              {GABA-mediated} inhibitory potentials in an in vitro model of
              focal epilepsy",
  author   = "Troyer, M D and Blanton, M G and Kriegstein, A R",
  abstract = "Focal, freeze-induced lesions were made in isolated hemispheres
              of turtle cerebral cortex in vitro, permitting the investigation
              of epileptiform discharges in a preparation with preserved
              intracortical circuitry. Freeze lesions resulted in interictal
              discharges and occasional ictal-like events. The interictal
              discharges were dependent upon activation of non-NMDA excitatory
              amino acid receptors and were affected by but did not require
              NMDA receptor activation. Voltage clamp and current clamp
              recordings revealed abnormal bursts of low-amplitude action
              potentials in 36\% of recorded neurons, while large, repetitive
              inhibitory potentials, mediated by GABAA receptors, were recorded
              in 90\% of the neurons. Thus, prominent findings in this model
              include abnormalities of both excitatory and inhibitory activity.
              Since these changes in neuronal excitability resulted from a
              localized physical injury, they may resemble the changes that
              occur in acute posttraumatic epilepsy.",
  journal  = "Epilepsia",
  volume   =  33,
  number   =  2,
  pages    = "199--212",
  month    =  mar,
  year     =  1992,
  address  = "United States",
  language = "en"
}

Focal, freeze-induced lesions were made in isolated hemispheres of turtle cerebral cortex in vitro, permitting the investigation of epileptiform discharges in a preparation with preserved intracortical circuitry. Freeze lesions resulted in interictal discharges and occasional ictal-like events. The interictal discharges were dependent upon activation of non-NMDA excitatory amino acid receptors and were affected by but did not require NMDA receptor activation. Voltage clamp and current clamp recordings revealed abnormal bursts of low-amplitude action potentials in 36% of recorded neurons, while large, repetitive inhibitory potentials, mediated by GABAA receptors, were recorded in 90% of the neurons. Thus, prominent findings in this model include abnormalities of both excitatory and inhibitory activity. Since these changes in neuronal excitability resulted from a localized physical injury, they may resemble the changes that occur in acute posttraumatic epilepsy.

@ARTICLE{Blanton1992-as,
  title    = "Norepinephrine activates potassium conductance in neurons of the
              turtle cerebral cortex",
  author   = "Blanton, K J and Kriegstein, A R",
  abstract = "Whole-cell voltage and current clamp recordings were obtained
              from cortical neurons of the pond turtle, Pseudemys scripta
              elegans. Norepinephrine (NE) induced an outward current in 50\%
              of pyramidal neurons. This current had a reversal potential of
              -88.3 +/- 3.2 mV, consistent with a K+ conductance increase, and
              had a mean amplitude of 18.3 +/- 7.2 pA at -40 mV. The ionic
              dependence and pharmacological analyses are both consistent with
              alpha 2 adrenergic receptor stimulation. Inhibition of
              Na(+)-dependent action potentials with TTX did not diminish the
              NE-induced K+ conductance, indicating that NE acts directly on
              the postsynaptic neuron. In addition to effects on postsynaptic
              conductance, NE dramatically decreased the amplitude of
              spontaneous inhibitory postsynaptic currents (IPSCs) in 55\% of
              pyramidal neurons. The decrease in spontaneous IPSCs was observed
              both in those neurons which exhibited an increase in K+
              conductance in response to NE administration (81\%) and in those
              which did not (33\%). Thus, NE modulates neuronal excitability
              both directly by activating a postsynaptic K+ conductance and
              indirectly by decreasing spontaneous IPSCs.",
  journal  = "Brain Res",
  volume   =  570,
  number   = "1-2",
  pages    = "42--48",
  month    =  jan,
  year     =  1992,
  address  = "Netherlands",
  language = "en"
}

Whole-cell voltage and current clamp recordings were obtained from cortical neurons of the pond turtle, Pseudemys scripta elegans. Norepinephrine (NE) induced an outward current in 50% of pyramidal neurons. This current had a reversal potential of -88.3 +/- 3.2 mV, consistent with a K+ conductance increase, and had a mean amplitude of 18.3 +/- 7.2 pA at -40 mV. The ionic dependence and pharmacological analyses are both consistent with alpha 2 adrenergic receptor stimulation. Inhibition of Na(+)-dependent action potentials with TTX did not diminish the NE-induced K+ conductance, indicating that NE acts directly on the postsynaptic neuron. In addition to effects on postsynaptic conductance, NE dramatically decreased the amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in 55% of pyramidal neurons. The decrease in spontaneous IPSCs was observed both in those neurons which exhibited an increase in K+ conductance in response to NE administration (81%) and in those which did not (33%). Thus, NE modulates neuronal excitability both directly by activating a postsynaptic K+ conductance and indirectly by decreasing spontaneous IPSCs.

@ARTICLE{Blanton1992-az,
  title    = "Properties of amino acid neurotransmitter receptors of embryonic
              cortical neurons when activated by exogenous and endogenous
              agonists",
  author   = "Blanton, M G and Kriegstein, A R",
  abstract = "1. The properties of receptors for amino acid neurotransmitters
              expressed by developing cortical neurons were studied with the
              use of whole-cell recording in the intact cerebral cortex of
              embryonic turtles in vitro. The inhibitory agonist
              gamma-aminobutyric acid (GABA) and the excitatory agonist
              glutamate were focally applied to single cells under voltage
              clamp, and the ionic dependence, voltage dependence, and
              pharmacological sensitivity of the responses were characterized.
              The responses mediated by a glutamate receptor subtype, the
              N-methyl-D-aspartate (NMDA) receptor, produced by glutamate and
              by evoked release of an endogenous excitatory agonist, were
              compared further. Fluctuation analysis was used to characterize
              the properties of the NMDA channels and the mechanism of action
              of receptor antagonists. 2. When postmitotic neurons first
              appeared at stage 15, all neurons tested responded to GABA with a
              current that reversed at the equilibrium potential for chloride
              ions and that was sensitive to the GABAA receptor antagonist
              bicuculline methiodide (BMI). As development proceeded, an
              increasing proportion of neurons also responded with a
              BMI-insensitive current that reversed near the equilibrium
              potential for potassium ions. This current was blocked by the
              GABAB receptor antagonist 3-amino-2-propyl phosponic acid
              (phaclofen). The GABAB agonist baclofen, however, failed to
              produce a detectable postsynaptic current. 3. Neurons at stage 15
              showed a biphasic response to glutamate that reversed at the
              equilibrium potential for cations. All neurons tested showed a
              slow, sustained response associated with an increase in current
              variance compared with background, and, as development proceeded,
              an increasing proportion also exhibited a fast, transient
              response. Both fast and slow responses varied linearly with
              voltage in the absence of Mg2+ ions, but the addition of Mg2+
              ions to the bathing medium attenuated the slow response at
              hyperpolarized potentials. As a result, the current-voltage
              relation of the slow response in the presence of Mg2+ ions
              exhibited a region of negative slope conductance, like that of
              currents mediated by NMDA receptors. 4. The fast and slow
              responses to glutamate differed in their pharmacological
              sensitivity. The fast responses were sensitive to the non-NMDA
              receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
              whereas the slow responses were sensitive to the NMDA receptor
              antagonist D(-)-2-amino-5-phosphonovalerate (D-APV). 5. When
              cells were held at -70 mV, glutamate evoked a fluctuating current
              consisting of channel currents with a mean open time, tau, of
              4.42 +/- 0.47 (SE) ms in early postmitotic neurons at stage 15
              and 4.99 +/- 0.38 ms at stages 17-20.(ABSTRACT TRUNCATED AT 400
              WORDS)",
  journal  = "J Neurophysiol",
  volume   =  67,
  number   =  5,
  pages    = "1185--1200",
  month    =  may,
  year     =  1992,
  address  = "United States",
  language = "en"
}

1. The properties of receptors for amino acid neurotransmitters expressed by developing cortical neurons were studied with the use of whole-cell recording in the intact cerebral cortex of embryonic turtles in vitro. The inhibitory agonist gamma-aminobutyric acid (GABA) and the excitatory agonist glutamate were focally applied to single cells under voltage clamp, and the ionic dependence, voltage dependence, and pharmacological sensitivity of the responses were characterized. The responses mediated by a glutamate receptor subtype, the N-methyl-D-aspartate (NMDA) receptor, produced by glutamate and by evoked release of an endogenous excitatory agonist, were compared further. Fluctuation analysis was used to characterize the properties of the NMDA channels and the mechanism of action of receptor antagonists. 2. When postmitotic neurons first appeared at stage 15, all neurons tested responded to GABA with a current that reversed at the equilibrium potential for chloride ions and that was sensitive to the GABAA receptor antagonist bicuculline methiodide (BMI). As development proceeded, an increasing proportion of neurons also responded with a BMI-insensitive current that reversed near the equilibrium potential for potassium ions. This current was blocked by the GABAB receptor antagonist 3-amino-2-propyl phosponic acid (phaclofen). The GABAB agonist baclofen, however, failed to produce a detectable postsynaptic current. 3. Neurons at stage 15 showed a biphasic response to glutamate that reversed at the equilibrium potential for cations. All neurons tested showed a slow, sustained response associated with an increase in current variance compared with background, and, as development proceeded, an increasing proportion also exhibited a fast, transient response. Both fast and slow responses varied linearly with voltage in the absence of Mg2+ ions, but the addition of Mg2+ ions to the bathing medium attenuated the slow response at hyperpolarized potentials. As a result, the current-voltage relation of the slow response in the presence of Mg2+ ions exhibited a region of negative slope conductance, like that of currents mediated by NMDA receptors. 4. The fast and slow responses to glutamate differed in their pharmacological sensitivity. The fast responses were sensitive to the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the slow responses were sensitive to the NMDA receptor antagonist D(-)-2-amino-5-phosphonovalerate (D-APV). 5. When cells were held at -70 mV, glutamate evoked a fluctuating current consisting of channel currents with a mean open time, tau, of 4.42 +/- 0.47 (SE) ms in early postmitotic neurons at stage 15 and 4.99 +/- 0.38 ms at stages 17-20.(ABSTRACT TRUNCATED AT 400 WORDS)

@ARTICLE{Lo_Turco1991-bz,
  title    = "Clusters of coupled neuroblasts in embryonic neocortex",
  author   = "Lo Turco, J J and Kriegstein, A R",
  abstract = "The neocortex of the brain develops from a simple germinal layer
              into a complex multilayer structure. To investigate cellular
              interactions during early neocortical development, whole-cell
              patch clamp recordings were made from neuroblasts in the
              ventricular zone of fetal rats. During early corticogenesis,
              neuroblasts are physiologically coupled by gap junctions into
              clusters of 15 to 90 cells. The coupled cells form columns within
              the ventricular zone and, by virtue of their membership in
              clusters, have low apparent membrane resistances and generate
              large responses to the inhibitory neurotransmitter
              gamma-aminobutyric acid. As neuronal migration out of the
              ventricular zone progresses, the number of cells within the
              clusters decreases. These clusters allow direct cell to cell
              interaction at the earliest stages of corticogenesis.",
  journal  = "Science",
  volume   =  252,
  number   =  5005,
  pages    = "563--566",
  month    =  apr,
  year     =  1991,
  address  = "United States",
  language = "en"
}

The neocortex of the brain develops from a simple germinal layer into a complex multilayer structure. To investigate cellular interactions during early neocortical development, whole-cell patch clamp recordings were made from neuroblasts in the ventricular zone of fetal rats. During early corticogenesis, neuroblasts are physiologically coupled by gap junctions into clusters of 15 to 90 cells. The coupled cells form columns within the ventricular zone and, by virtue of their membership in clusters, have low apparent membrane resistances and generate large responses to the inhibitory neurotransmitter gamma-aminobutyric acid. As neuronal migration out of the ventricular zone progresses, the number of cells within the clusters decreases. These clusters allow direct cell to cell interaction at the earliest stages of corticogenesis.

@ARTICLE{Blanton1991-at,
  title    = "Morphological differentiation of distinct neuronal classes in
              embryonic turtle cerebral cortex",
  author   = "Blanton, M G and Kriegstein, A R",
  abstract = "As a starting point for understanding the development of the
              cerebral cortex in reptiles and for determining how reptilian
              cortical development compares to that in other vertebrate
              classes, we studied the appearance and morphological
              differentiation of cerebral cortical neurons in embryonic
              turtles. 3H-thymidine birthdate labeling and focal injections of
              horseradish peroxidase (HRP) in in vitro cortical slices revealed
              that replicating cells occupy the outer ventricular zone, and
              subsequently migrate to the ventricular surface where they
              divide. Postmitotic neurons begin differentiating and elaborating
              neurites while migrating back through the ventricular zone. On
              their arrival at the top of the ventricular zone, pyramidal and
              nonpyramidal neurons can be distinguished morphologically. Cells
              with multipolar apical dendritic tufts ascending in the marginal
              zone resemble immature pyramidal neurons. Neurons morphologically
              similar to these early pyramidal cells were retrogradely labeled
              by injections of the lipophilic tracer
              1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine
              perchlorate (diI) in a known pyramidal cell target, the thalamus.
              Nonpyramidal neurons, resembling Cajal-Retzius cells, had
              horizontally oriented long axons and dendrites coursing in the
              plexiform primordium, the future marginal zone. With further
              development morphological differences between cell types became
              accentuated, and pyramidal cell somata were segregated into a
              single cellular layer flanked by zones containing predominantly
              nonpyramidal cells. Axon elaboration occurred early in embryonic
              development, as pyramidal cells sent axonal branches to the
              septum, thalamus, and cortical targets soon after their
              generation, and the intracortical axonal plexus became
              increasingly dense during embryonic life. Over a similar time
              course the distribution of projecting neurons labeled by thalamic
              diI injections changed from an initial homogeneous distribution
              to a preferential location in the superficial half of the
              cellular layer. Results from this study demonstrate several
              features of cortical differentiation that are conserved in
              reptiles and mammals, including similar early morphological
              differentiation events, the early distinction of principal cell
              types, and the parallel development of pyramidal and nonpyramidal
              neurons. The context in which these similar developmental events
              occur, however, differs profoundly in reptiles and mammals, with
              differences in the timing and location of neurite elaboration and
              differences in the appearance and architectonic organization of
              the cortex. Comparison of cortical developmental patterns between
              reptiles and mammals shows that similar functional cortical
              circuits with balanced excitation and inhibition can emerge in
              diverse cortical structures.",
  journal  = "J Comp Neurol",
  volume   =  310,
  number   =  4,
  pages    = "558--570",
  month    =  aug,
  year     =  1991,
  address  = "United States",
  language = "en"
}

As a starting point for understanding the development of the cerebral cortex in reptiles and for determining how reptilian cortical development compares to that in other vertebrate classes, we studied the appearance and morphological differentiation of cerebral cortical neurons in embryonic turtles. 3H-thymidine birthdate labeling and focal injections of horseradish peroxidase (HRP) in in vitro cortical slices revealed that replicating cells occupy the outer ventricular zone, and subsequently migrate to the ventricular surface where they divide. Postmitotic neurons begin differentiating and elaborating neurites while migrating back through the ventricular zone. On their arrival at the top of the ventricular zone, pyramidal and nonpyramidal neurons can be distinguished morphologically. Cells with multipolar apical dendritic tufts ascending in the marginal zone resemble immature pyramidal neurons. Neurons morphologically similar to these early pyramidal cells were retrogradely labeled by injections of the lipophilic tracer 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (diI) in a known pyramidal cell target, the thalamus. Nonpyramidal neurons, resembling Cajal-Retzius cells, had horizontally oriented long axons and dendrites coursing in the plexiform primordium, the future marginal zone. With further development morphological differences between cell types became accentuated, and pyramidal cell somata were segregated into a single cellular layer flanked by zones containing predominantly nonpyramidal cells. Axon elaboration occurred early in embryonic development, as pyramidal cells sent axonal branches to the septum, thalamus, and cortical targets soon after their generation, and the intracortical axonal plexus became increasingly dense during embryonic life. Over a similar time course the distribution of projecting neurons labeled by thalamic diI injections changed from an initial homogeneous distribution to a preferential location in the superficial half of the cellular layer. Results from this study demonstrate several features of cortical differentiation that are conserved in reptiles and mammals, including similar early morphological differentiation events, the early distinction of principal cell types, and the parallel development of pyramidal and nonpyramidal neurons. The context in which these similar developmental events occur, however, differs profoundly in reptiles and mammals, with differences in the timing and location of neurite elaboration and differences in the appearance and architectonic organization of the cortex. Comparison of cortical developmental patterns between reptiles and mammals shows that similar functional cortical circuits with balanced excitation and inhibition can emerge in diverse cortical structures.

@ARTICLE{LoTurco1991-xv,
  title    = "Initial expression and endogenous activation of {NMDA} channels
              in early neocortical development",
  author   = "LoTurco, J J and Blanton, M G and Kriegstein, A R",
  abstract = "We have made patch-clamp recordings from slices of fetal and
              postnatal rat neocortex in order to study the initial expression
              and activation of NMDA channels. Recordings from both whole cells
              and outside-out patches indicated that functional NMDA channels
              are expressed on neurons within the cortical plate, but not on
              younger cells within the ventricular zone. The NMDA channels on
              cortical plate neurons had a unitary conductance of approximately
              40 pS, had a mean open time of approximately 6 msec, required
              glycine to open, and were blocked in a voltage-dependent manner
              by magnesium. These precocious channels were present before the
              appearance of functional synaptic activity, yet like NMDA
              channels in the mature neocortex, they were spontaneously
              activated by an agonist within brain slices. These results
              demonstrate that NMDA channels are initially expressed on
              neocortical neurons some time between the last mitotic division
              within the ventricular zone and completion of migration into the
              cortical plate. These early NMDA channels have properties
              characteristic of NMDA channels on more mature neurons and are
              similarly activated by an endogenous agonist in situ. Their early
              appearance and activation indicate that NMDA channels may play a
              role during early stages of cortical development.",
  journal  = "J Neurosci",
  volume   =  11,
  number   =  3,
  pages    = "792--799",
  month    =  mar,
  year     =  1991,
  language = "en"
}

We have made patch-clamp recordings from slices of fetal and postnatal rat neocortex in order to study the initial expression and activation of NMDA channels. Recordings from both whole cells and outside-out patches indicated that functional NMDA channels are expressed on neurons within the cortical plate, but not on younger cells within the ventricular zone. The NMDA channels on cortical plate neurons had a unitary conductance of approximately 40 pS, had a mean open time of approximately 6 msec, required glycine to open, and were blocked in a voltage-dependent manner by magnesium. These precocious channels were present before the appearance of functional synaptic activity, yet like NMDA channels in the mature neocortex, they were spontaneously activated by an agonist within brain slices. These results demonstrate that NMDA channels are initially expressed on neocortical neurons some time between the last mitotic division within the ventricular zone and completion of migration into the cortical plate. These early NMDA channels have properties characteristic of NMDA channels on more mature neurons and are similarly activated by an endogenous agonist in situ. Their early appearance and activation indicate that NMDA channels may play a role during early stages of cortical development.

@ARTICLE{Blanton1991-uj,
  title    = "Spontaneous action potential activity and synaptic currents in
              the embryonic turtle cerebral cortex",
  author   = "Blanton, M G and Kriegstein, A R",
  abstract = "We used loose-patch and whole-cell recording techniques to study
              the development of spontaneous action potential activity and
              spontaneous excitatory and inhibitory synaptic currents in
              embryonic neurons in the cerebral hemispheres of turtles.
              Sporadic action potential activity appeared early in development
              at stage 17, soon after morphologically identifiable pyramidal
              and nonpyramidal neurons were first observed in the cortex. As
              the cortical plate matured in midembryonic stages, action
              potential activity became more regular and fell into one of two
              distinct patterns, tonic and intermittent high-frequency firing.
              Spontaneous excitatory and inhibitory postsynaptic currents
              (EPSCs and IPSCs) appeared at developmental stages 18 and 20,
              respectively, after action potential activity was established.
              EPSCs and IPSCs exhibited characteristic ionic dependence and
              pharmacology throughout development. EPSCs reversed in direction
              at the equilibrium potential for cations and were sensitive to
              6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the
              non-NMDA type of glutamate receptor. IPSCs reversed at the
              equilibrium potential for chloride and were sensitive to
              bicuculline methiodide, a GABAA receptor antagonist. Spontaneous
              synaptic currents differed in their time course of development
              and in waveform parameters. Spontaneous synaptic currents
              differed in their time course of development and in waveform
              parameters. Spontaneous EPSCs appeared at stage 18 and increased
              progressively in frequency, from 0.2 +/- 0.1 Hz at stage 20 to
              3.2 +/- 2.0 Hz at stage 26 (hatching), while spontaneous IPSCs
              appeared at stage 20 and surpassed EPSCs in frequency, increasing
              to 7.1 +/- 1.6 Hz at stage 26. EPSCs exhibited stable amplitudes
              during development, with a mean conductance of 126 +/- 20 pS at
              stage 26, while IPSCs increased in mean amplitude, from 180 +/-
              12 pS at stage 18 to 260 +/- 44 pS at stage 26. The rise time to
              peak conductance of both types of synaptic currents increased
              with developmental time, for EPSCs increasing from 1.5 +/- 0.5
              msec at stage 20 to 2.7 +/- 0.6 msec at stage 26 and for IPSCs
              increasing from 2.9 +/- 0.2 msec at stage 18 to 6.2 +/- 0.8 msec
              at stage 26. While the decay time constants increased for EPSCs,
              from 3.9 +/- 1.2 msec at stage 20 to 8.7 +/- 2.3 msec at stage
              26, decay time constants for IPSCs showed a decreasing trend from
              24.0 +/- 5.2 msec at stage 18 to 18.4 +/- 5.3 msec at stage 26.
              The excitatory and inhibitory synaptic currents were sensitive to
              the sodium channel blocker TTX and were thus dependent, in part,
              on spontaneous action potential activity.(ABSTRACT TRUNCATED AT
              400 WORDS)",
  journal  = "J Neurosci",
  volume   =  11,
  number   =  12,
  pages    = "3907--3923",
  month    =  dec,
  year     =  1991,
  language = "en"
}

We used loose-patch and whole-cell recording techniques to study the development of spontaneous action potential activity and spontaneous excitatory and inhibitory synaptic currents in embryonic neurons in the cerebral hemispheres of turtles. Sporadic action potential activity appeared early in development at stage 17, soon after morphologically identifiable pyramidal and nonpyramidal neurons were first observed in the cortex. As the cortical plate matured in midembryonic stages, action potential activity became more regular and fell into one of two distinct patterns, tonic and intermittent high-frequency firing. Spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) appeared at developmental stages 18 and 20, respectively, after action potential activity was established. EPSCs and IPSCs exhibited characteristic ionic dependence and pharmacology throughout development. EPSCs reversed in direction at the equilibrium potential for cations and were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the non-NMDA type of glutamate receptor. IPSCs reversed at the equilibrium potential for chloride and were sensitive to bicuculline methiodide, a GABAA receptor antagonist. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous EPSCs appeared at stage 18 and increased progressively in frequency, from 0.2 +/- 0.1 Hz at stage 20 to 3.2 +/- 2.0 Hz at stage 26 (hatching), while spontaneous IPSCs appeared at stage 20 and surpassed EPSCs in frequency, increasing to 7.1 +/- 1.6 Hz at stage 26. EPSCs exhibited stable amplitudes during development, with a mean conductance of 126 +/- 20 pS at stage 26, while IPSCs increased in mean amplitude, from 180 +/- 12 pS at stage 18 to 260 +/- 44 pS at stage 26. The rise time to peak conductance of both types of synaptic currents increased with developmental time, for EPSCs increasing from 1.5 +/- 0.5 msec at stage 20 to 2.7 +/- 0.6 msec at stage 26 and for IPSCs increasing from 2.9 +/- 0.2 msec at stage 18 to 6.2 +/- 0.8 msec at stage 26. While the decay time constants increased for EPSCs, from 3.9 +/- 1.2 msec at stage 20 to 8.7 +/- 2.3 msec at stage 26, decay time constants for IPSCs showed a decreasing trend from 24.0 +/- 5.2 msec at stage 18 to 18.4 +/- 5.3 msec at stage 26. The excitatory and inhibitory synaptic currents were sensitive to the sodium channel blocker TTX and were thus dependent, in part, on spontaneous action potential activity.(ABSTRACT TRUNCATED AT 400 WORDS)

@ARTICLE{Wilson1991-wi,
  title    = "Turtle cortical neurons survive glutamate exposures that are
              lethal to mammalian neurons",
  author   = "Wilson, A M and Kriegstein, A R",
  abstract = "Glutamate is an excitatory neurotransmitter in turtle and
              mammalian cortex. In high concentrations it is toxic to mammalian
              neurons and is an important mediator in the pathway that leads to
              neuronal death from anoxia. Turtle neurons are remarkably
              resistant to anoxic injury and we sought to determine whether
              part of this resistance could be attributed to the sensitivity of
              turtle neurons to glutamate toxicity. Embryonic turtle cortical
              neurons were grown for 25 days in dissociated cell culture using
              a modification of a method developed for murine cortical cell
              culture. Turtle neurons in dissociated culture were found to
              express glutamate receptors which include both
              N-methyl-D-aspartate (NMDA) and non-NMDA receptor types.
              Remarkably, these neurons survive 5 minute exposures to glutamate
              in concentrations up to 3 mM, doses 30 times the LD50 and 6 times
              the LD100 for mouse cortical neurons. Elucidating the mechanism
              for this resistance may suggest new strategies for brain
              protection.",
  journal  = "Brain Res",
  volume   =  540,
  number   = "1-2",
  pages    = "297--301",
  month    =  feb,
  year     =  1991,
  address  = "Netherlands",
  language = "en"
}

Glutamate is an excitatory neurotransmitter in turtle and mammalian cortex. In high concentrations it is toxic to mammalian neurons and is an important mediator in the pathway that leads to neuronal death from anoxia. Turtle neurons are remarkably resistant to anoxic injury and we sought to determine whether part of this resistance could be attributed to the sensitivity of turtle neurons to glutamate toxicity. Embryonic turtle cortical neurons were grown for 25 days in dissociated cell culture using a modification of a method developed for murine cortical cell culture. Turtle neurons in dissociated culture were found to express glutamate receptors which include both N-methyl-D-aspartate (NMDA) and non-NMDA receptor types. Remarkably, these neurons survive 5 minute exposures to glutamate in concentrations up to 3 mM, doses 30 times the LD50 and 6 times the LD100 for mouse cortical neurons. Elucidating the mechanism for this resistance may suggest new strategies for brain protection.

@ARTICLE{Blanton1991-lg,
  title    = "Appearance of putative amino acid neurotransmitters during
              differentiation of neurons in embryonic turtle cerebral cortex",
  author   = "Blanton, M G and Kriegstein, A R",
  abstract = "Pyramidal and nonpyramidal neurons can be recognized early in the
              development of the cerebral cortex in both reptiles and mammals,
              and the neurotransmitters likely utilized by these cells,
              glutamate and gamma-aminobutyric acid, or GABA, have been
              suggested to play critical developmental roles. Information
              concerning the timing and topography of neurotransmitter
              synthesis by specific classes of cortical neurons is important
              for understanding developmental roles of neurotransmitters and
              for identifying potential zones of neurotransmitter action in the
              developing brain. We therefore analyzed the appearance of GABA
              and glutamate in the cerebral cortex of embryonic turtles using
              polyclonal antisera raised against GABA and glutamate. Neuronal
              subtypes become immunoreactive for the putative amino acid
              neurotransmitters GABA and glutamate early in the embryonic
              development of turtle cerebral cortex, with nonpyramidal cells
              immunoreactive for GABA and pyramidal cells immunoreactive for
              glutamate. The results of controls strongly suggest that the
              immunocytochemical staining in tissue sections by the GABA and
              glutamate antisera corresponds to fixed endogenous GABA and
              glutamate. Horizontally oriented cells in the early marginal zone
              (stages 15-16) that are GABA-immunoreactive (GABA-IR) resemble
              nonpyramidal cells in morphology and distribution. GABA-IR
              neurons exhibit increasingly diverse morphologies and become
              distributed in all cortical layers as the cortex matures.
              Glutamate-immunoreactive (Glu-IR) cells dominate the cellular
              layer throughout development and are also common in the
              subcellular layer at early stages, a distribution like that of
              pyramidal neurons and distinct from that of GABA-IR nonpyramidal
              cells. The early organization of embryonic turtle cortex in
              reptiles resembles that of embryonic mammalian cortex, and the
              immunocytochemical results underline several shared as well as
              distinguishing features. Early GABA-IR nonpyramidal cells flank
              the developing cortical plate, composed primarily of pyramidal
              cells, shown here to be Glu-IR. The earliest GABA-IR cells in
              turtles likely correspond to Cajal-Retzius cells, a ubiquitous
              and precocious cell type in vertebrate cortex. Glutamate-IR
              projection neurons in vertebrates may also be related. The
              distinctly different topographies of GABA and glutamate
              containing cells in reptiles and mammals indicate that even if
              the basic amino acid transmitter-containing cell types are
              conserved in higher vertebrates, the local interactions mediated
              by these transmitters may differ. The potential role of GABA and
              glutamate in nonsynaptic interactions early in cortical
              development is reinforced by the precocious expression of these
              neurotransmitters in turtles, well before they are required for
              synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)",
  journal  = "J Comp Neurol",
  volume   =  310,
  number   =  4,
  pages    = "571--592",
  month    =  aug,
  year     =  1991,
  address  = "United States",
  language = "en"
}

Pyramidal and nonpyramidal neurons can be recognized early in the development of the cerebral cortex in both reptiles and mammals, and the neurotransmitters likely utilized by these cells, glutamate and gamma-aminobutyric acid, or GABA, have been suggested to play critical developmental roles. Information concerning the timing and topography of neurotransmitter synthesis by specific classes of cortical neurons is important for understanding developmental roles of neurotransmitters and for identifying potential zones of neurotransmitter action in the developing brain. We therefore analyzed the appearance of GABA and glutamate in the cerebral cortex of embryonic turtles using polyclonal antisera raised against GABA and glutamate. Neuronal subtypes become immunoreactive for the putative amino acid neurotransmitters GABA and glutamate early in the embryonic development of turtle cerebral cortex, with nonpyramidal cells immunoreactive for GABA and pyramidal cells immunoreactive for glutamate. The results of controls strongly suggest that the immunocytochemical staining in tissue sections by the GABA and glutamate antisera corresponds to fixed endogenous GABA and glutamate. Horizontally oriented cells in the early marginal zone (stages 15-16) that are GABA-immunoreactive (GABA-IR) resemble nonpyramidal cells in morphology and distribution. GABA-IR neurons exhibit increasingly diverse morphologies and become distributed in all cortical layers as the cortex matures. Glutamate-immunoreactive (Glu-IR) cells dominate the cellular layer throughout development and are also common in the subcellular layer at early stages, a distribution like that of pyramidal neurons and distinct from that of GABA-IR nonpyramidal cells. The early organization of embryonic turtle cortex in reptiles resembles that of embryonic mammalian cortex, and the immunocytochemical results underline several shared as well as distinguishing features. Early GABA-IR nonpyramidal cells flank the developing cortical plate, composed primarily of pyramidal cells, shown here to be Glu-IR. The earliest GABA-IR cells in turtles likely correspond to Cajal-Retzius cells, a ubiquitous and precocious cell type in vertebrate cortex. Glutamate-IR projection neurons in vertebrates may also be related. The distinctly different topographies of GABA and glutamate containing cells in reptiles and mammals indicate that even if the basic amino acid transmitter-containing cell types are conserved in higher vertebrates, the local interactions mediated by these transmitters may differ. The potential role of GABA and glutamate in nonsynaptic interactions early in cortical development is reinforced by the precocious expression of these neurotransmitters in turtles, well before they are required for synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

@ARTICLE{LoTurco1990-ho,
  title    = "Differential activation of glutamate receptors by spontaneously
              released transmitter in slices of neocortex",
  author   = "LoTurco, J J and Mody, I and Kriegstein, A R",
  abstract = "Whole-cell recordings were made from neurons in neocortical brain
              slices in order to characterize excitatory synaptic currents
              mediated by glutamate receptors. Glutamate receptor antagonists,
              D-aminophosphonovalerate (D-APV) and CNQX, selectively attenuated
              distinct components in evoked synaptic currents, and were used to
              differentiate spontaneous synaptic currents mediated by
              N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Spontaneous
              excitatory synaptic currents were independent of action
              potentials, varied linearly with voltage, and were blocked by the
              non-NMDA receptor antagonist CNQX. An NMDA receptor-mediated
              component was not apparent in these spontaneous synaptic
              currents, however, when magnesium was omitted from the recording
              medium, fluctuations in current and sustained inward current
              became apparent, and these were blocked by the NMDA receptor
              antagonist D-APV. Based on these findings, we conclude that NMDA
              and non-NMDA receptors are activated differentially by
              transmitter released independently of action potentials.",
  journal  = "Neurosci Lett",
  volume   =  114,
  number   =  3,
  pages    = "265--271",
  month    =  jul,
  year     =  1990,
  address  = "Ireland",
  language = "en"
}

Whole-cell recordings were made from neurons in neocortical brain slices in order to characterize excitatory synaptic currents mediated by glutamate receptors. Glutamate receptor antagonists, D-aminophosphonovalerate (D-APV) and CNQX, selectively attenuated distinct components in evoked synaptic currents, and were used to differentiate spontaneous synaptic currents mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Spontaneous excitatory synaptic currents were independent of action potentials, varied linearly with voltage, and were blocked by the non-NMDA receptor antagonist CNQX. An NMDA receptor-mediated component was not apparent in these spontaneous synaptic currents, however, when magnesium was omitted from the recording medium, fluctuations in current and sustained inward current became apparent, and these were blocked by the NMDA receptor antagonist D-APV. Based on these findings, we conclude that NMDA and non-NMDA receptors are activated differentially by transmitter released independently of action potentials.

@ARTICLE{Blanton1990-pg,
  title    = "Endogenous neurotransmitter activates {N-methyl-D-aspartate}
              receptors on differentiating neurons in embryonic cortex",
  author   = "Blanton, M G and Lo Turco, J J and Kriegstein, A R",
  abstract = "Before synapses form in embryonic turtle cerebral cortex, an
              endogenous neurotransmitter activates N-methyl-D-aspartate (NMDA)
              channels on neurons in the cortical plate. Throughout cortical
              development, these channels exhibit voltage-dependent Mg2+
              blockade and are antagonized by D-2-amino-5-phosphonovaleric
              acid, a selective NMDA receptor antagonist. The activation in
              situ of these nonsynaptic NMDA channels demonstrates a potential
              physiological substrate for control of early neuronal
              differentiation.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  87,
  number   =  20,
  pages    = "8027--8030",
  month    =  oct,
  year     =  1990,
  language = "en"
}

Before synapses form in embryonic turtle cerebral cortex, an endogenous neurotransmitter activates N-methyl-D-aspartate (NMDA) channels on neurons in the cortical plate. Throughout cortical development, these channels exhibit voltage-dependent Mg2+ blockade and are antagonized by D-2-amino-5-phosphonovaleric acid, a selective NMDA receptor antagonist. The activation in situ of these nonsynaptic NMDA channels demonstrates a potential physiological substrate for control of early neuronal differentiation.

@ARTICLE{Blanton1989-tq,
  title    = "Whole cell recording from neurons in slices of reptilian and
              mammalian cerebral cortex",
  author   = "Blanton, M G and Lo Turco, J J and Kriegstein, A R",
  abstract = "We describe methods for obtaining stable, whole-cell recordings
              from neurons in brain hemispheres from turtles and in brain
              slices from rats and turtles. Synaptic currents and membrane
              properties of central neurons can be studied in voltage and
              current clamp in cells maintained within their endogenous
              synaptic circuits. The methods described here are compatible with
              unmodified dissecting microscopes and recording chambers, and
              with brain slices of standard thickness (400-500 microns).",
  journal  = "J Neurosci Methods",
  volume   =  30,
  number   =  3,
  pages    = "203--210",
  month    =  dec,
  year     =  1989,
  address  = "Netherlands",
  language = "en"
}

We describe methods for obtaining stable, whole-cell recordings from neurons in brain hemispheres from turtles and in brain slices from rats and turtles. Synaptic currents and membrane properties of central neurons can be studied in voltage and current clamp in cells maintained within their endogenous synaptic circuits. The methods described here are compatible with unmodified dissecting microscopes and recording chambers, and with brain slices of standard thickness (400-500 microns).

@ARTICLE{Shen1989-wn,
  title    = "The development of bicuculline-induced epileptiform discharges in
              embryonic turtle cortex",
  author   = "Shen, J M and Kriegstein, A R",
  abstract = "The appearance of bicuculline-induced epileptiform discharges was
              studied in embryonic turtle cortex using extracellular recording
              techniques. Drug-induced discharges occurred at an early stage of
              cortical plate formation, suggesting that mechanisms for
              synchronizing neuronal discharges are functional at this stage.
              Discharges originated in the medial cortex and increased in
              amplitude and decreased in frequency with development.
              gamma-Aminobutyric acid (GABA)-containing neurons and functional
              GABA receptors are present in advance of excitatory synchronizing
              mechanisms and may have a non-synaptic role in corticogenesis.",
  journal  = "Neurosci Lett",
  volume   =  98,
  number   =  2,
  pages    = "184--188",
  month    =  mar,
  year     =  1989,
  address  = "Ireland",
  language = "en"
}

The appearance of bicuculline-induced epileptiform discharges was studied in embryonic turtle cortex using extracellular recording techniques. Drug-induced discharges occurred at an early stage of cortical plate formation, suggesting that mechanisms for synchronizing neuronal discharges are functional at this stage. Discharges originated in the medial cortex and increased in amplitude and decreased in frequency with development. gamma-Aminobutyric acid (GABA)-containing neurons and functional GABA receptors are present in advance of excitatory synchronizing mechanisms and may have a non-synaptic role in corticogenesis.

@ARTICLE{Shen1988-bf,
  title    = "Development of {GABA} responsiveness in embryonic turtle cortical
              neurons",
  author   = "Shen, J M and Huguenard, J R and Kriegstein, A R",
  abstract = "The whole-cell patch-clamp method was used to study the
              development of functional GABA receptors in cortical neurons
              dissociated from embryonic turtles. GABA elicited an increase in
              membrane conductance, even from cells obtained from the earliest
              stages of corticogenesis. The GABA-mediated conductance had a
              mean value 7.4 times greater than membrane 'leak' conductance and
              increased with developmental age. In all stages studied, the
              response inverted polarity at a value approximating ECl- and was
              blocked by applications of bicuculline, suggesting that it was
              mediated by GABAA receptors. GABA receptors are thus present and
              functional very early in corticogenesis, preceding
              electrogenesis, synaptogenesis, and full neuronal
              differentiation.",
  journal  = "Neurosci Lett",
  volume   =  89,
  number   =  3,
  pages    = "335--341",
  month    =  jul,
  year     =  1988,
  address  = "Ireland",
  language = "en"
}

The whole-cell patch-clamp method was used to study the development of functional GABA receptors in cortical neurons dissociated from embryonic turtles. GABA elicited an increase in membrane conductance, even from cells obtained from the earliest stages of corticogenesis. The GABA-mediated conductance had a mean value 7.4 times greater than membrane 'leak' conductance and increased with developmental age. In all stages studied, the response inverted polarity at a value approximating ECl- and was blocked by applications of bicuculline, suggesting that it was mediated by GABAA receptors. GABA receptors are thus present and functional very early in corticogenesis, preceding electrogenesis, synaptogenesis, and full neuronal differentiation.

@ARTICLE{Choi1987-kh,
  title    = "Glutamate neurotoxicity in cortical cell culture",
  author   = "Choi, D W and Maulucci-Gedde, M and Kriegstein, A R",
  abstract = "The central neurotoxicity of the excitatory amino acid
              neurotransmitter glutamate has been postulated to participate in
              the pathogenesis of the neuronal cell loss associated with
              several neurological disease states, but the complexity of the
              intact nervous system has impeded detailed analysis of the
              phenomenon. In the present study, glutamate neurotoxicity was
              studied with novel precision in dissociated cell cultures
              prepared from the fetal mouse neocortex. Brief exposure to
              glutamate was found to produce morphological changes in mature
              cortical neurons beginning as quickly as 90 sec after exposure,
              followed by widespread neuronal degeneration over the next hours.
              Quantitative dose-toxicity study suggested an ED50 of 50-100
              microM for a 5 min exposure to glutamate. Immature cortical
              neurons and glia were not injured by such exposures to glutamate.
              Uptake processes probably do not limit GNT in culture, as the
              uptake inhibitor dihydrokainate did not potentiate GNT. Possibly
              reflecting the lack of uptake limitation, glutamate was found to
              be actually more potent than kainate as a neurotoxin in these
              cultures, a dramatic reversal of the in vivo potency rank order.
              Some neurons regularly survived brief glutamate exposure; these
              possibly glutamate-resistant neurons had electrophysiologic
              properties, including chemosensitivity to glutamate, that were
              grossly similar to those of the original population.",
  journal  = "J Neurosci",
  volume   =  7,
  number   =  2,
  pages    = "357--368",
  month    =  feb,
  year     =  1987,
  language = "en"
}

The central neurotoxicity of the excitatory amino acid neurotransmitter glutamate has been postulated to participate in the pathogenesis of the neuronal cell loss associated with several neurological disease states, but the complexity of the intact nervous system has impeded detailed analysis of the phenomenon. In the present study, glutamate neurotoxicity was studied with novel precision in dissociated cell cultures prepared from the fetal mouse neocortex. Brief exposure to glutamate was found to produce morphological changes in mature cortical neurons beginning as quickly as 90 sec after exposure, followed by widespread neuronal degeneration over the next hours. Quantitative dose-toxicity study suggested an ED50 of 50-100 microM for a 5 min exposure to glutamate. Immature cortical neurons and glia were not injured by such exposures to glutamate. Uptake processes probably do not limit GNT in culture, as the uptake inhibitor dihydrokainate did not potentiate GNT. Possibly reflecting the lack of uptake limitation, glutamate was found to be actually more potent than kainate as a neurotoxin in these cultures, a dramatic reversal of the in vivo potency rank order. Some neurons regularly survived brief glutamate exposure; these possibly glutamate-resistant neurons had electrophysiologic properties, including chemosensitivity to glutamate, that were grossly similar to those of the original population.

@ARTICLE{Kriegstein1987-ra,
  title    = "Synaptic responses of cortical pyramidal neurons to light
              stimulation in the isolated turtle visual system",
  author   = "Kriegstein, A R",
  abstract = "The resistance of the turtle brain to hypoxic injury permits a
              unique in vitro preparation in which the organization and
              function of visual cortex can be explored. Intracellular
              recordings from cortical pyramidal neurons revealed biphasic
              responses to flashes of light, consisting of an early phase
              (50-100 msec) of concurrent inhibitory and excitatory activation,
              followed by a longer, inhibitory phase (250-600 msec) composed of
              summated Cl- -dependent postsynaptic potentials mediated by GABA.
              This response sequence results from the coactivation of pyramidal
              and GABAergic non-pyramidal cells, followed by feed-forward and
              possibly feed-back pyramidal cell inhibition, and is partly
              dependent on differences in the membrane properties of pyramidal
              and non-pyramidal neurons.",
  journal  = "J Neurosci",
  volume   =  7,
  number   =  8,
  pages    = "2488--2492",
  month    =  aug,
  year     =  1987,
  language = "en"
}

The resistance of the turtle brain to hypoxic injury permits a unique in vitro preparation in which the organization and function of visual cortex can be explored. Intracellular recordings from cortical pyramidal neurons revealed biphasic responses to flashes of light, consisting of an early phase (50-100 msec) of concurrent inhibitory and excitatory activation, followed by a longer, inhibitory phase (250-600 msec) composed of summated Cl- -dependent postsynaptic potentials mediated by GABA. This response sequence results from the coactivation of pyramidal and GABAergic non-pyramidal cells, followed by feed-forward and possibly feed-back pyramidal cell inhibition, and is partly dependent on differences in the membrane properties of pyramidal and non-pyramidal neurons.

@ARTICLE{Schlegel1987-og,
  title    = "Quantitative autoradiography of muscarinic and benzodiazepine
              receptors in the forebrain of the turtle, Pseudemys scripta",
  author   = "Schlegel, J R and Kriegstein, A R",
  abstract = "The distribution of muscarinic and benzodiazepine receptors was
              investigated in the turtle forebrain by the technique of in vitro
              receptor autoradiography. Muscarinic binding sites were labeled
              with 1 nM 3H-quinuclidinyl benzilate (3H-QNB), and benzodiazepine
              sites were demonstrated with the aid of 1 nM 3H-flunitrazepam
              (3H-FLU). Autoradiograms generated on 3H-Ultrofilm apposed to
              tissue slices revealed regionally specific distributions of
              muscarinic and benzodiazepine binding sites that are comparable
              with those for mammalian brain. Dense benzodiazepine binding was
              found in the anterior olfactory nucleus, the lateral and dorsal
              cortices, and the dorsal ventricular ridge (DVR), a structure
              with no clear mammalian homologue. Muscarinic binding sites were
              most dense in the striatum, accumbens, DVR, lateral geniculate,
              and the anterior olfactory nucleus. Cortical binding sites were
              studied in greater detail by quantitative analysis of
              autoradiograms generated by using emulsion-coated coverslips.
              Laminar gradients of binding were observed that were specific for
              each radioligand; 3H-QNB sites were most dense in the inner
              molecular layer in all cortical regions, whereas 3H-FLU binding
              was generally most concentrated in the outer molecular layer and
              was least dense through all layers in the dorsomedial cortex.
              Because pyramidal cells are arranged in register in turtle
              cortex, the laminar patterns of receptor binding may reflect
              different receptor density gradients along pyramidal cell
              dendrites.",
  journal  = "J Comp Neurol",
  volume   =  265,
  number   =  4,
  pages    = "521--529",
  month    =  nov,
  year     =  1987,
  address  = "United States",
  language = "en"
}

The distribution of muscarinic and benzodiazepine receptors was investigated in the turtle forebrain by the technique of in vitro receptor autoradiography. Muscarinic binding sites were labeled with 1 nM 3H-quinuclidinyl benzilate (3H-QNB), and benzodiazepine sites were demonstrated with the aid of 1 nM 3H-flunitrazepam (3H-FLU). Autoradiograms generated on 3H-Ultrofilm apposed to tissue slices revealed regionally specific distributions of muscarinic and benzodiazepine binding sites that are comparable with those for mammalian brain. Dense benzodiazepine binding was found in the anterior olfactory nucleus, the lateral and dorsal cortices, and the dorsal ventricular ridge (DVR), a structure with no clear mammalian homologue. Muscarinic binding sites were most dense in the striatum, accumbens, DVR, lateral geniculate, and the anterior olfactory nucleus. Cortical binding sites were studied in greater detail by quantitative analysis of autoradiograms generated by using emulsion-coated coverslips. Laminar gradients of binding were observed that were specific for each radioligand; 3H-QNB sites were most dense in the inner molecular layer in all cortical regions, whereas 3H-FLU binding was generally most concentrated in the outer molecular layer and was least dense through all layers in the dorsomedial cortex. Because pyramidal cells are arranged in register in turtle cortex, the laminar patterns of receptor binding may reflect different receptor density gradients along pyramidal cell dendrites.

@ARTICLE{Blanton1987-oz,
  title    = "Evidence for the inhibitory neurotransmitter gamma-aminobutyric
              acid in aspiny and sparsely spiny nonpyramidal neurons of the
              turtle dorsal cortex",
  author   = "Blanton, M G and Shen, J M and Kriegstein, A R",
  abstract = "In order to learn more about the anatomical substrate for
              gamma-aminobutyric acid (GABA)-mediated inhibition in cortical
              structures, the intrinsic neuronal organization of turtle dorsal
              cortex was studied by using Golgi impregnation,
              immunohistochemical localization of GABA and its synthetic enzyme
              glutamic acid decarboxylase (GAD), and histochemical localization
              of the presynaptic GABA-degrading enzyme GABA-transaminase
              (GABA-T). GABAergic markers are found in neurons identical in
              morphology and distribution to Golgi-impregnated aspiny and
              sparsely spiny nonpyramidal neurons with locally arborizing axons
              and appear to label most if not all of the nonpyramidal neurons.
              In addition, the GABAergic markers are found in punctate
              structures in a distribution characteristic of presumed
              inhibitory terminals. The spine-laden pyramidal neurons, the
              principal projecting cell type in the dorsal cortex, are devoid
              of labelling for GABAergic markers but are surrounded by presumed
              GABAergic terminals. The data complement previous physiological
              and ultrastructural studies that implicate aspiny and sparsely
              spiny nonpyramidal neurons as mediators of intrinsic inhibition
              of pyramidal neurons in turtle cortex. The results also suggest
              similarities in the functional organization of intrinsic
              inhibitory elements in turtle and mammalian cortex.",
  journal  = "J Comp Neurol",
  volume   =  259,
  number   =  2,
  pages    = "277--297",
  month    =  may,
  year     =  1987,
  address  = "United States",
  language = "en"
}

In order to learn more about the anatomical substrate for gamma-aminobutyric acid (GABA)-mediated inhibition in cortical structures, the intrinsic neuronal organization of turtle dorsal cortex was studied by using Golgi impregnation, immunohistochemical localization of GABA and its synthetic enzyme glutamic acid decarboxylase (GAD), and histochemical localization of the presynaptic GABA-degrading enzyme GABA-transaminase (GABA-T). GABAergic markers are found in neurons identical in morphology and distribution to Golgi-impregnated aspiny and sparsely spiny nonpyramidal neurons with locally arborizing axons and appear to label most if not all of the nonpyramidal neurons. In addition, the GABAergic markers are found in punctate structures in a distribution characteristic of presumed inhibitory terminals. The spine-laden pyramidal neurons, the principal projecting cell type in the dorsal cortex, are devoid of labelling for GABAergic markers but are surrounded by presumed GABAergic terminals. The data complement previous physiological and ultrastructural studies that implicate aspiny and sparsely spiny nonpyramidal neurons as mediators of intrinsic inhibition of pyramidal neurons in turtle cortex. The results also suggest similarities in the functional organization of intrinsic inhibitory elements in turtle and mammalian cortex.

@ARTICLE{Kriegstein1987-jd,
  title    = "Cellular and synaptic physiology and epileptogenesis of
              developing rat neocortical neurons in vitro",
  author   = "Kriegstein, A R and Suppes, T and Prince, D A",
  abstract = "The cellular and synaptic physiology of developing rat
              neocortical neurons was studied using the in vitro slice method.
              Rats aged 1-28 days were used for analysis. During the first two
              postnatal weeks several sequential changes occur in membrane
              properties and evoked synaptic potentials. Immature neurons had
              higher input resistances, more linear I-V characteristics, longer
              membrane time constants, and slower rising and falling phases of
              action potentials. The developmental increase in rate of rise of
              the action potential suggests an increasing density of
              voltage-dependent Na+-channels are inserted in neuronal membranes
              during postnatal development. The higher input resistance of
              young cells might be due to their small size and differences in
              membrane properties. The long time constant indicates a higher
              specific membrane resistivity of immature neurons. Postsynaptic
              potentials (PSPs) recorded in young neurons were longer in
              latency, longer in duration, and more fragile during repetitive
              activation than their mature counterparts. In addition, PSPs
              evoked in neurons of animals less than 1 week old did not contain
              inhibitory postsynaptic components. These physiological features
              of immature neocortical neurons help explain the pattern of
              epileptogenesis in young animals. When neonatal cortical slices
              were exposed to the gamma-aminobutyric acid (GABA) antagonists
              penicillin or bicuculline, the frequency of occurrence of
              discharges resembling epileptiform depolarization shifts
              approached that found in mature slices only during the second
              postnatal week. Depolarization shifts at younger ages were less
              stereotyped and more sensitive to stimulus parameters than those
              in mature neurons.",
  journal  = "Brain Res",
  volume   =  431,
  number   =  2,
  pages    = "161--171",
  month    =  aug,
  year     =  1987,
  address  = "Netherlands",
  language = "en"
}

The cellular and synaptic physiology of developing rat neocortical neurons was studied using the in vitro slice method. Rats aged 1-28 days were used for analysis. During the first two postnatal weeks several sequential changes occur in membrane properties and evoked synaptic potentials. Immature neurons had higher input resistances, more linear I-V characteristics, longer membrane time constants, and slower rising and falling phases of action potentials. The developmental increase in rate of rise of the action potential suggests an increasing density of voltage-dependent Na+-channels are inserted in neuronal membranes during postnatal development. The higher input resistance of young cells might be due to their small size and differences in membrane properties. The long time constant indicates a higher specific membrane resistivity of immature neurons. Postsynaptic potentials (PSPs) recorded in young neurons were longer in latency, longer in duration, and more fragile during repetitive activation than their mature counterparts. In addition, PSPs evoked in neurons of animals less than 1 week old did not contain inhibitory postsynaptic components. These physiological features of immature neocortical neurons help explain the pattern of epileptogenesis in young animals. When neonatal cortical slices were exposed to the gamma-aminobutyric acid (GABA) antagonists penicillin or bicuculline, the frequency of occurrence of discharges resembling epileptiform depolarization shifts approached that found in mature slices only during the second postnatal week. Depolarization shifts at younger ages were less stereotyped and more sensitive to stimulus parameters than those in mature neurons.

@ARTICLE{Shen1986-fm,
  title    = "Turtle hippocampal cortex contains distinct cell types,
              burst-firing neurons, and an epileptogenic subfield",
  author   = "Shen, J M and Kriegstein, A R",
  abstract = "The dorsal and medial telencephalon of reptiles consists of a
              simple trilaminar cortex. The turtle dorsal cortex has been
              identified as a favorable physiological preparation that may bear
              a phylogenetic relationship to mammalian neocortex. While
              anatomical studies have likened the reptilian medial cortical
              region to mammalian hippocampus, its physiological properties
              have not been explored. We therefore used intracellular and
              extracellular recording techniques to examine the cellular and
              synaptic physiology of turtle ``hippocampal'' or medial cortex.
              Turtle medial cortex contains two principal classes of neurons,
              pyramidal cells and stellate neurons. Recordings with Lucifer
              yellow CH (LY)-filled microelectrodes allowed us to correlate the
              physiological properties of medial cortical neurons with their
              cellular morphology. Pyramidal neurons were situated in a single
              cellular layer and had spiny apical dendrites extending into the
              molecular layer. These cells fired relatively long-duration
              action potentials (APs) and showed frequency adaptation to
              suprathreshold current pulse injections. Stellate cells were
              usually found in the subcellular and molecular layers and had
              aspiny dendrites. In contrast to pyramidal cells, they fired
              brief APs and displayed no frequency adaptation. A discrete
              population of cells in the dorsal portion of medial cortex (DMC)
              was capable of bursting endogenously or in response to synaptic
              activation. Bursts usually contained an underlying slow
              depolarization and often occurred at regular intervals.
              Intracellular LY injections confirmed that these cells were
              pyramidal in morphology. Electrical stimulation of afferent
              fibers revealed that pyramidal cells and stellate neurons
              differed in their synaptic responses. In ventral medial cortex
              (VMC), afferent stimulation evoked a multiphasic response in most
              pyramidal cells, whereas stellate cells were synaptically
              excited. Orthodromic activation of DMC bursting cells resulted in
              a powerful excitation--often a short burst--and subsequent
              inhibition. Stellate neurons in DMC also had a biphasic synaptic
              response consisting of both an early excitation and a late
              inhibition. Experiments using intracellular chloride (Cl-)
              injection or focal bicuculline application suggested that part of
              the inhibitory component of the pyramidal cell synaptic response
              was dependent on a gamma-aminobutyric acid (GABA)-mediated
              increase in Cl- conductance. These results correlated with our
              immunohistochemical studies that revealed the presence of
              GABAergic neurons in medial cortex.(ABSTRACT TRUNCATED AT 400
              WORDS)",
  journal  = "J Neurophysiol",
  volume   =  56,
  number   =  6,
  pages    = "1626--1649",
  month    =  dec,
  year     =  1986,
  address  = "United States",
  language = "en"
}

The dorsal and medial telencephalon of reptiles consists of a simple trilaminar cortex. The turtle dorsal cortex has been identified as a favorable physiological preparation that may bear a phylogenetic relationship to mammalian neocortex. While anatomical studies have likened the reptilian medial cortical region to mammalian hippocampus, its physiological properties have not been explored. We therefore used intracellular and extracellular recording techniques to examine the cellular and synaptic physiology of turtle ``hippocampal'' or medial cortex. Turtle medial cortex contains two principal classes of neurons, pyramidal cells and stellate neurons. Recordings with Lucifer yellow CH (LY)-filled microelectrodes allowed us to correlate the physiological properties of medial cortical neurons with their cellular morphology. Pyramidal neurons were situated in a single cellular layer and had spiny apical dendrites extending into the molecular layer. These cells fired relatively long-duration action potentials (APs) and showed frequency adaptation to suprathreshold current pulse injections. Stellate cells were usually found in the subcellular and molecular layers and had aspiny dendrites. In contrast to pyramidal cells, they fired brief APs and displayed no frequency adaptation. A discrete population of cells in the dorsal portion of medial cortex (DMC) was capable of bursting endogenously or in response to synaptic activation. Bursts usually contained an underlying slow depolarization and often occurred at regular intervals. Intracellular LY injections confirmed that these cells were pyramidal in morphology. Electrical stimulation of afferent fibers revealed that pyramidal cells and stellate neurons differed in their synaptic responses. In ventral medial cortex (VMC), afferent stimulation evoked a multiphasic response in most pyramidal cells, whereas stellate cells were synaptically excited. Orthodromic activation of DMC bursting cells resulted in a powerful excitation–often a short burst–and subsequent inhibition. Stellate neurons in DMC also had a biphasic synaptic response consisting of both an early excitation and a late inhibition. Experiments using intracellular chloride (Cl-) injection or focal bicuculline application suggested that part of the inhibitory component of the pyramidal cell synaptic response was dependent on a gamma-aminobutyric acid (GABA)-mediated increase in Cl- conductance. These results correlated with our immunohistochemical studies that revealed the presence of GABAergic neurons in medial cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

@ARTICLE{Kriegstein1986-rp,
  title    = "Cellular physiology of the turtle visual cortex: synaptic
              properties and intrinsic circuitry",
  author   = "Kriegstein, A R and Connors, B W",
  abstract = "We have examined the synaptic physiology of the isolated dorsal
              cortex of the turtle, Pseudemys scripta elegans. Electrical
              stimulation of afferent pathways elicited distinct, stereotyped
              responses in pyramidal and stellate neurons. Single shocks evoked
              a long-lasting barrage of excitatory postsynaptic potentials
              (EPSPs) in stellate cells, and led to a burst of several action
              potentials. Under the same circumstances, pyramidal cells
              displayed a small amount of short-latency excitation, but this
              was accompanied by a profound and prolonged set of inhibitory
              post-synaptic potentials (IPSPs). Synaptic excitation of the
              distal dendrites of pyramidal cells could evoke dendritic action
              potentials that were visible at the soma as small all-or-none
              spikes rising from the hyperpolarized level of the IPSP. There
              appeared to be two mechanistically different types of IPSPs in
              pyramidal cells. The first occurred at short latency, could
              produce a very large conductance increase, reversed polarity at
              -71 mV, and was chloride-dependent. The second was generally
              smaller and more protracted, had a relatively negative reversal
              potential of -85 to -95 mV, and was insensitive to chloride
              injection. Focal application of small doses of the putative
              inhibitory neurotransmitter gamma-aminobutyric acid (GABA) onto
              the somata of pyramidal cells caused a conductance increase and
              hyperpolarization. This response had features in common with the
              short-latency IPSP, including an identical reversal potential.
              Application of large doses of GABA to the somata of pyramidal
              cells or smaller doses to their dendrites elicited multiphasic or
              purely depolarizing responses that were at least partly due to
              time- or space-dependent shifts of the equilibrium potential of
              the response. Bicuculline methiodide, a potent GABA antagonist,
              depressed both the responses to GABA and the short-latency IPSP,
              but not the long-latency IPSP; synchronized epileptiform burst
              discharges also resulted. These findings, together with responses
              to locally applied electric shocks and the excitatory amino acid
              glutamate, suggested that inhibition of pyramidal cells was
              generated intrinsically by stellate cells, and that the cortical
              circuit provides pathways for both feedforward and feedback
              GABAergic inhibition. The data also suggest that pyramidal cells
              are mutually excitatory. These features are similar to the basic
              intrinsic circuitry in the telencephalic cortices of mammals.",
  journal  = "J Neurosci",
  volume   =  6,
  number   =  1,
  pages    = "178--191",
  month    =  jan,
  year     =  1986,
  language = "en"
}

We have examined the synaptic physiology of the isolated dorsal cortex of the turtle, Pseudemys scripta elegans. Electrical stimulation of afferent pathways elicited distinct, stereotyped responses in pyramidal and stellate neurons. Single shocks evoked a long-lasting barrage of excitatory postsynaptic potentials (EPSPs) in stellate cells, and led to a burst of several action potentials. Under the same circumstances, pyramidal cells displayed a small amount of short-latency excitation, but this was accompanied by a profound and prolonged set of inhibitory post-synaptic potentials (IPSPs). Synaptic excitation of the distal dendrites of pyramidal cells could evoke dendritic action potentials that were visible at the soma as small all-or-none spikes rising from the hyperpolarized level of the IPSP. There appeared to be two mechanistically different types of IPSPs in pyramidal cells. The first occurred at short latency, could produce a very large conductance increase, reversed polarity at -71 mV, and was chloride-dependent. The second was generally smaller and more protracted, had a relatively negative reversal potential of -85 to -95 mV, and was insensitive to chloride injection. Focal application of small doses of the putative inhibitory neurotransmitter gamma-aminobutyric acid (GABA) onto the somata of pyramidal cells caused a conductance increase and hyperpolarization. This response had features in common with the short-latency IPSP, including an identical reversal potential. Application of large doses of GABA to the somata of pyramidal cells or smaller doses to their dendrites elicited multiphasic or purely depolarizing responses that were at least partly due to time- or space-dependent shifts of the equilibrium potential of the response. Bicuculline methiodide, a potent GABA antagonist, depressed both the responses to GABA and the short-latency IPSP, but not the long-latency IPSP; synchronized epileptiform burst discharges also resulted. These findings, together with responses to locally applied electric shocks and the excitatory amino acid glutamate, suggested that inhibition of pyramidal cells was generated intrinsically by stellate cells, and that the cortical circuit provides pathways for both feedforward and feedback GABAergic inhibition. The data also suggest that pyramidal cells are mutually excitatory. These features are similar to the basic intrinsic circuitry in the telencephalic cortices of mammals.

@ARTICLE{Connors1986-ue,
  title    = "Cellular physiology of the turtle visual cortex: distinctive
              properties of pyramidal and stellate neurons",
  author   = "Connors, B W and Kriegstein, A R",
  abstract = "The electrophysiological properties of neurons in the
              three-layered dorsal cortex of the turtle, Pseudemys scripta
              elegans, have been studied in vitro. Intracellular recordings
              suggested two distinct classes of neuronal behavior. Cell
              labeling with either Lucifer Yellow or horseradish peroxidase
              revealed that these behaviors correlated with the two
              morphological classes of cortical neurons: pyramidal cells and
              stellate cells. Examination of Golgi-stained neurons of dorsal
              cortex did not uncover any other obvious classes. Pyramidal cells
              had their somata in the cell layer, and extended several densely
              spined apical dendrites through the molecular layer to the pia.
              They also had spiny basilar dendrites directed through the
              subcellular layer toward the ependymal border. Physiologically,
              pyramidal cells had relatively prolonged action potentials that
              showed marked frequency adaptation during a sustained
              suprathreshold current pulse. Their most striking characteristic
              was a tendency to fire two discrete sizes of action potential,
              one small (mean = 34 mV) and of relatively low threshold, the
              other large (mean = 76 mV) and of higher threshold. We
              hypothesize that at least some small spikes arise from distal
              dendritic sites, whereas large spikes are somatically generated.
              Both spikes were tetrodotoxin-sensitive, although
              calcium-dependent electrogenesis occurred when potassium channels
              were blocked. In contrast to pyramidal cells, the somata of
              stellate cells were found in the molecular and subcellular zones.
              Their dendrites tended to be horizontally oriented and
              spine-free. Stellate cells had relatively brief action
              potentials, each of which was followed by a large but
              short-lasting undershoot of membrane potential. Stellate cells
              showed little or no spike frequency adaptation. Spike amplitudes
              were always relatively uniform and large (mean = 73 mV). Thus, in
              the dorsal cortex of turtles, the pyramidal cells, which are
              projection neurons, and stellate cells, which are local GABAergic
              inhibitory neurons, have distinctly different membrane
              characteristics. The physiological properties of the two types of
              turtle cortical neurons are very similar to their counterparts in
              cortical structures of the mammalian telencephalon.",
  journal  = "J Neurosci",
  volume   =  6,
  number   =  1,
  pages    = "164--177",
  month    =  jan,
  year     =  1986,
  language = "en"
}

The electrophysiological properties of neurons in the three-layered dorsal cortex of the turtle, Pseudemys scripta elegans, have been studied in vitro. Intracellular recordings suggested two distinct classes of neuronal behavior. Cell labeling with either Lucifer Yellow or horseradish peroxidase revealed that these behaviors correlated with the two morphological classes of cortical neurons: pyramidal cells and stellate cells. Examination of Golgi-stained neurons of dorsal cortex did not uncover any other obvious classes. Pyramidal cells had their somata in the cell layer, and extended several densely spined apical dendrites through the molecular layer to the pia. They also had spiny basilar dendrites directed through the subcellular layer toward the ependymal border. Physiologically, pyramidal cells had relatively prolonged action potentials that showed marked frequency adaptation during a sustained suprathreshold current pulse. Their most striking characteristic was a tendency to fire two discrete sizes of action potential, one small (mean = 34 mV) and of relatively low threshold, the other large (mean = 76 mV) and of higher threshold. We hypothesize that at least some small spikes arise from distal dendritic sites, whereas large spikes are somatically generated. Both spikes were tetrodotoxin-sensitive, although calcium-dependent electrogenesis occurred when potassium channels were blocked. In contrast to pyramidal cells, the somata of stellate cells were found in the molecular and subcellular zones. Their dendrites tended to be horizontally oriented and spine-free. Stellate cells had relatively brief action potentials, each of which was followed by a large but short-lasting undershoot of membrane potential. Stellate cells showed little or no spike frequency adaptation. Spike amplitudes were always relatively uniform and large (mean = 73 mV). Thus, in the dorsal cortex of turtles, the pyramidal cells, which are projection neurons, and stellate cells, which are local GABAergic inhibitory neurons, have distinctly different membrane characteristics. The physiological properties of the two types of turtle cortical neurons are very similar to their counterparts in cortical structures of the mammalian telencephalon.

@ARTICLE{Kriegstein1986-xj,
  title    = "Monoclonal antibodies to the turtle cortex reveal neuronal
              subsets, antigenic cross-reactivity with the mammalian neocortex,
              and forebrain structures sharing a pallial derivation",
  author   = "Kriegstein, A R and Shen, J M and Eshhar, N",
  abstract = "The dorsal cortex of the pond turtle (Pseudemys scripta) is a
              relatively simple structure consisting of two principal classes
              of neurons that occupy three distinct layers. Morphological,
              pharmacological, and physiological data suggest many similarities
              to the mammalian neocortex, rendering it an interesting
              preparation for comparative studies. We prepared monoclonal
              antibodies to the turtle dorsal cortex by immunizing mice with
              cortical tissue from adult turtles. Twelve antibodies were
              generated that recognize specific components of the turtle
              cortex. Among these, eight antibodies label only neurons and four
              label only ependymal glial cells. Differences in tissue staining
              pattern and immunoglobulin class suggest a heterogeneity of
              antigenic specificity among the antibodies. The staining patterns
              of three of our antibodies are described. TC3, like all other
              neuron-marking antibodies generated, labels a subset of both
              pyramidal and stellate cell types. It also cross-reacts with a
              subset of mammalian cortical neurons and labels them with a
              pattern similar to that observed in the turtle cortex. TC5 stains
              ependymal cells and their glial processes in the turtle cortex,
              and cross-reacts with fibrous astrocytelike processes in
              mammalian neocortical white matter. TC9 appears to recognize
              antigens of neurons sharing a pallial derivation in turtle.",
  journal  = "J Comp Neurol",
  volume   =  254,
  number   =  3,
  pages    = "330--340",
  month    =  dec,
  year     =  1986,
  address  = "United States",
  language = "en"
}

The dorsal cortex of the pond turtle (Pseudemys scripta) is a relatively simple structure consisting of two principal classes of neurons that occupy three distinct layers. Morphological, pharmacological, and physiological data suggest many similarities to the mammalian neocortex, rendering it an interesting preparation for comparative studies. We prepared monoclonal antibodies to the turtle dorsal cortex by immunizing mice with cortical tissue from adult turtles. Twelve antibodies were generated that recognize specific components of the turtle cortex. Among these, eight antibodies label only neurons and four label only ependymal glial cells. Differences in tissue staining pattern and immunoglobulin class suggest a heterogeneity of antigenic specificity among the antibodies. The staining patterns of three of our antibodies are described. TC3, like all other neuron-marking antibodies generated, labels a subset of both pyramidal and stellate cell types. It also cross-reacts with a subset of mammalian cortical neurons and labels them with a pattern similar to that observed in the turtle cortex. TC5 stains ependymal cells and their glial processes in the turtle cortex, and cross-reacts with fibrous astrocytelike processes in mammalian neocortical white matter. TC9 appears to recognize antigens of neurons sharing a pallial derivation in turtle.

@ARTICLE{Suppes1985-pm,
  title    = "The influence of dopamine on epileptiform burst activity in
              hippocampal pyramidal neurons",
  author   = "Suppes, T and Kriegstein, A R and Prince, D A",
  abstract = "Dopamine (DA) application to guinea pig hippocampal CA1 neurons
              in vitro causes hyperpolarization of the resting potential,
              increase in conductance, and increase in amplitude and duration
              of the afterhyperpolarization (AHP). Since these changes could
              influence repetitive firing, we performed experiments to
              determine whether DA-induced effects would suppress
              epileptogenesis in the hippocampus. Epileptiform bursts were
              induced by adding penicillin (3.4 mM) to the perfusion medium.
              Focal application of DA (40-160 microns) onto CA1 cells (n = 15)
              produced a hyperpolarization averaging 4.5 mV beginning in 5-20 s
              and lasting up to 3 min. DA also caused an increase in the
              amplitude and duration of slow AHPs. The frequency of spontaneous
              epileptiform events however was not affected. CA3 neurons (n = 6)
              responded to DA application with an initial 1-3 mV depolarization
              beginning within 5-30 s and lasting 1-2 min. In 3 cases a small
              hyperpolarization lasting several minutes subsequently developed.
              AHP duration increased 70\% and amplitude increased 35\% (n = 4).
              Along with these membrane changes the frequency of epileptiform
              bursting in CA3 cells slowed for 1-3 min. We added DA (10-80
              microM) to the perfusion medium to see whether a significant
              decrease in epileptiform burst frequency might occur in the
              follower CA1 region if greater numbers of pacemaker CA2 and CA3
              cells were exposed to DA. Spontaneous CA1 bursting was reversibly
              slowed, the interburst interval became variable and increased
              from a mean of 4 to a mean of 5-7 s (n = 6). These results
              suggest that DA may play a role in decreasing the incidence or
              frequency of epileptogenic discharges in vivo.",
  journal  = "Brain Res",
  volume   =  326,
  number   =  2,
  pages    = "273--280",
  month    =  feb,
  year     =  1985,
  address  = "Netherlands",
  language = "en"
}

Dopamine (DA) application to guinea pig hippocampal CA1 neurons in vitro causes hyperpolarization of the resting potential, increase in conductance, and increase in amplitude and duration of the afterhyperpolarization (AHP). Since these changes could influence repetitive firing, we performed experiments to determine whether DA-induced effects would suppress epileptogenesis in the hippocampus. Epileptiform bursts were induced by adding penicillin (3.4 mM) to the perfusion medium. Focal application of DA (40-160 microns) onto CA1 cells (n = 15) produced a hyperpolarization averaging 4.5 mV beginning in 5-20 s and lasting up to 3 min. DA also caused an increase in the amplitude and duration of slow AHPs. The frequency of spontaneous epileptiform events however was not affected. CA3 neurons (n = 6) responded to DA application with an initial 1-3 mV depolarization beginning within 5-30 s and lasting 1-2 min. In 3 cases a small hyperpolarization lasting several minutes subsequently developed. AHP duration increased 70% and amplitude increased 35% (n = 4). Along with these membrane changes the frequency of epileptiform bursting in CA3 cells slowed for 1-3 min. We added DA (10-80 microM) to the perfusion medium to see whether a significant decrease in epileptiform burst frequency might occur in the follower CA1 region if greater numbers of pacemaker CA2 and CA3 cells were exposed to DA. Spontaneous CA1 bursting was reversibly slowed, the interburst interval became variable and increased from a mean of 4 to a mean of 5-7 s (n = 6). These results suggest that DA may play a role in decreasing the incidence or frequency of epileptogenic discharges in vivo.

@ARTICLE{Kriegstein1983-gx,
  title    = "Morphological classification of rat cortical neurons in cell
              culture",
  author   = "Kriegstein, A R and Dichter, M A",
  abstract = "Neurons in ``mature'' (4- to 6-week-old) dissociated cell
              cultures of 15-day gestational age rat fetal cortex were injected
              with Lucifer Yellow in order to compare their detailed
              morphological features with those of cortical neurons in situ,
              and in order to determine which features of cellular morphology
              were dependent on local environmental conditions. Neurons were
              characterized by their cell form (pyramidal, multipolar,
              fusiform, etc.), dendritic branching pattern, spine density, and
              axonal projections. The neurons in culture appeared to display
              all the morphological features seen in cortical neurons in situ.
              These characteristics appeared to be independent of whether an
              individual neuron grew in a dense or sparse region of the
              culture. In addition, examination of neurons during early
              differentiation indicated that many of their morphological
              features developed as soon as the neurons could be recognized and
              before extensive synapse formation occurred.",
  journal  = "J Neurosci",
  volume   =  3,
  number   =  8,
  pages    = "1634--1647",
  month    =  aug,
  year     =  1983,
  language = "en"
}

Neurons in ``mature'' (4- to 6-week-old) dissociated cell cultures of 15-day gestational age rat fetal cortex were injected with Lucifer Yellow in order to compare their detailed morphological features with those of cortical neurons in situ, and in order to determine which features of cellular morphology were dependent on local environmental conditions. Neurons were characterized by their cell form (pyramidal, multipolar, fusiform, etc.), dendritic branching pattern, spine density, and axonal projections. The neurons in culture appeared to display all the morphological features seen in cortical neurons in situ. These characteristics appeared to be independent of whether an individual neuron grew in a dense or sparse region of the culture. In addition, examination of neurons during early differentiation indicated that many of their morphological features developed as soon as the neurons could be recognized and before extensive synapse formation occurred.

@ARTICLE{Kriegstein1983-pi,
  title    = "Cholinergic enhancement of penicillin-induced epileptiform
              discharges in pyramidal neurons of the guinea pig hippocampus",
  author   = "Kriegstein, A R and Suppes, T and Prince, D A",
  abstract = "Acetylcholine (1-20 mM) was applied to guinea pig hippocampal
              slices bathed in normal and penicillin-containing media.
              Recordings in the CA 1 pyramidal cell layer in the presence of
              penicillin showed that acetylcholine caused a prolonged
              enhancement of the extracellular field potential. Intracellular
              recordings documented an increase in duration of cell bursting, a
              decrease in burst afterhyperpolarization, and a membrane
              depolarization lasting 1-5 min. These results suggest that the
              actions of acetylcholine to increase membrane excitability
              interact with penicillin-induced disinhibition to enhance
              hippocampal epileptogenesis.",
  journal  = "Brain Res",
  volume   =  266,
  number   =  1,
  pages    = "137--142",
  month    =  apr,
  year     =  1983,
  address  = "Netherlands",
  language = "en"
}

Acetylcholine (1-20 mM) was applied to guinea pig hippocampal slices bathed in normal and penicillin-containing media. Recordings in the CA 1 pyramidal cell layer in the presence of penicillin showed that acetylcholine caused a prolonged enhancement of the extracellular field potential. Intracellular recordings documented an increase in duration of cell bursting, a decrease in burst afterhyperpolarization, and a membrane depolarization lasting 1-5 min. These results suggest that the actions of acetylcholine to increase membrane excitability interact with penicillin-induced disinhibition to enhance hippocampal epileptogenesis.

@ARTICLE{Kriegstein1982-sq,
  title    = "Neurology-important advances in clinical medicine: treatment of
              febrile convulsions",
  author   = "Kriegstein, A R",
  journal  = "West J Med",
  volume   =  137,
  number   =  4,
  pages    = "313--314",
  month    =  oct,
  year     =  1982,
  language = "en"
}

@ARTICLE{Kriegstein1977-bg,
  title    = "Stages in the post-hatching development of Aplysia californica",
  author   = "Kriegstein, A R",
  abstract = "In order to study the development of the nervous system of the
              marine mollusc, Aplysia californica, it is necessary objectively
              to assess the maturity of individual specimens. This can be done
              by defining stages in the life cycle. The post-hatching
              development can be divided into four phases: planktonic,
              metamorphic, juvenile, and adult. These phases can be further
              subdivided into 13 stages on the basis of behavioral and
              morphological characteristics visible in living specimens: Stage
              1, newly hatched; Stage 2, eyes develop; Stage 3, the larval
              heart beats; Stage 4, maximum shell size is reached; Stage 5, the
              propodium develops; Stage 6, red spots appear; Stage 7, the velum
              is shed; Stage 8, eyebrows appear; Stage 9, pink color develops;
              Stage 10, white spots appear; Stage 11, rhinophores grow; Stage
              12, the genital groove forms; Stage 13, egg laying begins.
              Reconstructions from serial sections taken from specimens fixed
              at each of these stages reveal the sequence of formation of the
              major organ systems. The nervous system develops gradually. The
              cerebral and pedal ganglia are present at Stage 1, the optic
              ganglia develop at Stage 2, the abdominal, pleural, and
              osphradial ganglia at Stage 3, the buccal ganglia at Stage 5, and
              the genital ganglion at Stage 13. Because Aplysia develops
              gradually, it is possible to analyze the contribution which
              gastropod torsion makes to the different phases of the life
              cycle. The Aplysia embryo undergoes 120 degrees torsion prior to
              Stage 1. The major visceral organs, the digestive system, heart,
              gill, and visceral nervous system, develop sybsequently in their
              post-torsional positions. After metamorphosis, there is a partial
              de-torsion which involves only the digestive system. Torsion of
              the digestive system may therefore be beneficial only to the
              pre-metamorphic larva, and not to the postmetamorphic juvenile.",
  journal  = "J Exp Zool",
  volume   =  199,
  number   =  2,
  pages    = "275--288",
  month    =  feb,
  year     =  1977,
  address  = "United States",
  language = "en"
}

In order to study the development of the nervous system of the marine mollusc, Aplysia californica, it is necessary objectively to assess the maturity of individual specimens. This can be done by defining stages in the life cycle. The post-hatching development can be divided into four phases: planktonic, metamorphic, juvenile, and adult. These phases can be further subdivided into 13 stages on the basis of behavioral and morphological characteristics visible in living specimens: Stage 1, newly hatched; Stage 2, eyes develop; Stage 3, the larval heart beats; Stage 4, maximum shell size is reached; Stage 5, the propodium develops; Stage 6, red spots appear; Stage 7, the velum is shed; Stage 8, eyebrows appear; Stage 9, pink color develops; Stage 10, white spots appear; Stage 11, rhinophores grow; Stage 12, the genital groove forms; Stage 13, egg laying begins. Reconstructions from serial sections taken from specimens fixed at each of these stages reveal the sequence of formation of the major organ systems. The nervous system develops gradually. The cerebral and pedal ganglia are present at Stage 1, the optic ganglia develop at Stage 2, the abdominal, pleural, and osphradial ganglia at Stage 3, the buccal ganglia at Stage 5, and the genital ganglion at Stage 13. Because Aplysia develops gradually, it is possible to analyze the contribution which gastropod torsion makes to the different phases of the life cycle. The Aplysia embryo undergoes 120 degrees torsion prior to Stage 1. The major visceral organs, the digestive system, heart, gill, and visceral nervous system, develop sybsequently in their post-torsional positions. After metamorphosis, there is a partial de-torsion which involves only the digestive system. Torsion of the digestive system may therefore be beneficial only to the pre-metamorphic larva, and not to the postmetamorphic juvenile.

@ARTICLE{Kriegstein1977-vv,
  title    = "Development of the nervous system of Aplysia californica",
  author   = "Kriegstein, A R",
  abstract = "The ability to grow the marine molluse Aplysia under laboratory
              conditions allows a detailed study of the formation of the
              nervous system and of the development of specific identified
              cells. I have found that the ganglia develop in a specific
              temporal order. Cerebral and pedal ganglia develop at hatching,
              the abdominal, pleural, and osphradial ganglia 3 weeks after
              hatching, and the buccal ganglia at 4 weeks. The origin of the
              abdominal ganglion is complex; its anlage forms at 3 weeks from
              three larval ganglia that fuse to form the abdominal ganglion.
              Individual cells cannot be distinguished from one another by
              their location within the ganglion or by their appearance alone
              until metamorphosis at 5 weeks. After metamorphosis, the
              identified neuron, R2, suddenly becomes recognizable because of a
              significant increase in its size.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  74,
  number   =  1,
  pages    = "375--378",
  month    =  jan,
  year     =  1977,
  language = "en"
}

The ability to grow the marine molluse Aplysia under laboratory conditions allows a detailed study of the formation of the nervous system and of the development of specific identified cells. I have found that the ganglia develop in a specific temporal order. Cerebral and pedal ganglia develop at hatching, the abdominal, pleural, and osphradial ganglia 3 weeks after hatching, and the buccal ganglia at 4 weeks. The origin of the abdominal ganglion is complex; its anlage forms at 3 weeks from three larval ganglia that fuse to form the abdominal ganglion. Individual cells cannot be distinguished from one another by their location within the ganglion or by their appearance alone until metamorphosis at 5 weeks. After metamorphosis, the identified neuron, R2, suddenly becomes recognizable because of a significant increase in its size.

@ARTICLE{Kriegstein1974-xu,
  title    = "Metamorphosis of Aplysia californica in laboratory culture",
  author   = "Kriegstein, A R and Castellucci, V and Kandel, E R",
  abstract = "To utilize the advantages offered by the large identified nerve
              cells of the marine mollusc Aplysia californica for cellular
              biological studies of development, we have devised simple
              techniques for growing this species in the laboratory in large
              number with a generation time as short as nineteen weeks. We have
              used the cultured animals to study the life cycle from fertilized
              egg to reproductive adult. The major developmental and behavioral
              changes occur at metamorphosis, when the larvae settle on the
              seaweed Laurencia pacifica and the locomotor and feeding
              behaviors are transformed into their adult forms. We have
              examined the timetable for the abandonment of larval behaviors
              and the emergence of adult ones and found that the transition
              from swimming to crawling occurs first and marks the onset of
              metamorphosis. The change from ciliary feeding to radular feeding
              occurs later and signals the end of metamorphosis. Other adult
              behaviors, such as the reflex responses and fixed-action patterns
              of the mantle organs, appear after metamorphosis.",
  journal  = "Proc Natl Acad Sci U S A",
  volume   =  71,
  number   =  9,
  pages    = "3654--3658",
  month    =  sep,
  year     =  1974,
  language = "en"
}

To utilize the advantages offered by the large identified nerve cells of the marine mollusc Aplysia californica for cellular biological studies of development, we have devised simple techniques for growing this species in the laboratory in large number with a generation time as short as nineteen weeks. We have used the cultured animals to study the life cycle from fertilized egg to reproductive adult. The major developmental and behavioral changes occur at metamorphosis, when the larvae settle on the seaweed Laurencia pacifica and the locomotor and feeding behaviors are transformed into their adult forms. We have examined the timetable for the abandonment of larval behaviors and the emergence of adult ones and found that the transition from swimming to crawling occurs first and marks the onset of metamorphosis. The change from ciliary feeding to radular feeding occurs later and signals the end of metamorphosis. Other adult behaviors, such as the reflex responses and fixed-action patterns of the mantle organs, appear after metamorphosis.