RESUMEN
Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Canales de Potasio Shaw/metabolismo , Ataxias Espinocerebelosas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Células Madre Pluripotentes/metabolismo , Canales de Potasio Shaw/química , Canales de Potasio Shaw/genética , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.
Asunto(s)
Trastornos Generalizados del Desarrollo Infantil/genética , Trastornos Generalizados del Desarrollo Infantil/patología , Factores de Transcripción Forkhead/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Telencéfalo/embriología , Femenino , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Megalencefalia/genética , Megalencefalia/patología , Modelos Biológicos , Neuronas/citología , Neuronas/metabolismo , Organoides/patología , Telencéfalo/patologíaRESUMEN
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.
Asunto(s)
Consenso , Sistema Nervioso , Organoides , Terminología como Asunto , Humanos , Modelos Biológicos , Sistema Nervioso/citología , Sistema Nervioso/patología , Organoides/citología , Organoides/patología , Células Madre Pluripotentes/citologíaRESUMEN
Mosaic mutations can be used to track cell ancestries and reconstruct high-resolution lineage trees during cancer progression and during development, starting from the first cell divisions of the zygote. However, this approach requires sampling and analyzing the genomes of multiple cells, which can be redundant in lineage representation, limiting the scalability of the approach. We describe a strategy for cost- and time-efficient lineage reconstruction using clonal induced pluripotent stem cell lines from human skin fibroblasts. The approach leverages shallow sequencing coverage to assess the clonality of the lines, clusters redundant lines and sums their coverage to accurately discover mutations in the corresponding lineages. Only a fraction of lines needs to be sequenced to high coverage. We demonstrate the effectiveness of this approach for reconstructing lineage trees during development and in hematologic malignancies. We discuss and propose an optimal experimental design for reconstructing lineage trees.
Asunto(s)
Linaje de la Célula , Neoplasias , Programas Informáticos , Humanos , Células Germinativas , Mutación , Neoplasias/patologíaRESUMEN
Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) is characterized by the abrupt onset of significant obsessive-compulsive symptoms (OCS) and/or severe food restriction, together with other neuropsychiatric manifestations. An autoimmune pathogenesis triggered by infection has been proposed for at least a subset of PANS. The older diagnosis of Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS) describes rapid onset of OCD and/or tics associated with infection with Group A Streptococcus. The pathophysiology of PANS and PANDAS remains incompletely understood. We recently found serum antibodies from children with rigorously defined PANDAS to selectively bind to cholinergic interneurons (CINs) in the striatum. Here we examine this binding in children with relapsing and remitting PANS, a more heterogeneous condition, collected in a distinct clinical context from those examined in our previous work, from children with a clinical history of Streptococcus infection. IgG from PANS cases showed elevated binding to striatal CINs in both mouse and human brain. Patient plasma collected during symptom flare decreased a molecular marker of CIN activity, phospho-riboprotein S6, in ex vivo brain slices; control plasma did not. Neither elevated antibody binding to CINs nor diminished CIN activity was seen with plasma collected from the same children during remission. These findings replicate what we have seen previously in PANDAS and support the hypothesis that at least a subset of PANS cases have a neuroimmune pathogenesis. Given the critical role of CINs in modulating basal ganglia function, these findings confirm striatal CINs as a locus of interest in the pathophysiology of both PANS and PANDAS.
RESUMEN
Tracing cell lineages is fundamental for understanding the rules governing development in multicellular organisms and delineating complex biological processes involving the differentiation of multiple cell types with distinct lineage hierarchies. In humans, experimental lineage tracing is unethical, and one has to rely on natural-mutation markers that are created within cells as they proliferate and age. Recent studies have demonstrated that it is now possible to trace lineages in normal, noncancerous cells with a variety of data types using natural variations in the nuclear and mitochondrial DNA as well as variations in DNA methylation status. It is also apparent that the scientific community is on the verge of being able to make a comprehensive and detailed cell lineage map of human embryonic and fetal development. In this review, we discuss the advantages and disadvantages of different approaches and markers for lineage tracing. We also describe the general conceptual design for how to derive a lineage map for humans.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Núcleo Celular/genética , Metilación de ADN , ADN Mitocondrial/análisis , Embrión de Mamíferos/citología , ADN Mitocondrial/genética , Biología Evolutiva , Embrión de Mamíferos/metabolismo , Humanos , Análisis de la Célula IndividualRESUMEN
Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200-400 mosaic SNVs per cell in three human fetal brains (15-21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to â¼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in â¼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (â¼6200 vs. â¼4000 bp), implying that they may have similar functional consequences.
Asunto(s)
Encéfalo/embriología , ADN Circular/genética , Variación Estructural del Genoma , Análisis de Secuencia de ADN/métodos , Evolución Clonal , Femenino , Técnicas de Genotipaje , Edad Gestacional , Humanos , Mosaicismo , Neurogénesis , EmbarazoRESUMEN
Tourette Syndrome (TS) is a neuropsychiatric disorder thought to involve a reduction of basal ganglia (BG) interneurons and malfunctioning of the BG circuitry. However, whether interneurons fail to develop or are lost postnatally remains unknown. To investigate the pathophysiology of early development in TS, induced pluripotent stem cell (iPSC)-derived BG organoids from TS patients and healthy controls were compared on multiple levels of measurement and analysis. BG organoids from TS individuals manifested an impaired medial ganglionic eminence fate and a decreased differentiation of cholinergic and GABAergic interneurons. Transcriptome analyses revealed organoid mispatterning in TS, with a preference for dorsolateral at the expense of ventromedial fates. Our results point to altered expression of GLI transcription factors downstream of the Sonic Hedgehog signaling pathway with cilia disruption at the earliest stages of BG organoid differentiation as a potential mechanism for the BG mispatterning in TS. This study uncovers early neurodevelopmental underpinnings of TS neuropathological deficits using organoids as a model system.
Asunto(s)
Síndrome de Tourette , Humanos , Síndrome de Tourette/metabolismo , Proteínas Hedgehog/metabolismo , Ganglios Basales/patología , Interneuronas/metabolismo , Organoides/metabolismoRESUMEN
Accurate discovery of somatic mutations in a cell is a challenge that partially lays in immaturity of dedicated analytical approaches. Approaches comparing a cell's genome to a control bulk sample miss common mutations, while approaches to find such mutations from bulk suffer from low sensitivity. We developed a tool, All2, which enables accurate filtering of mutations in a cell without the need for data from bulk(s). It is based on pair-wise comparisons of all cells to each other where every call for base pair substitution and indel is classified as either a germline variant, mosaic mutation, or false positive. As All2 allows for considering dropped-out regions, it is applicable to whole genome and exome analysis of cloned and amplified cells. By applying the approach to a variety of available data, we showed that its application reduces false positives, enables sensitive discovery of high frequency mutations, and is indispensable for conducting high resolution cell lineage tracing.
Asunto(s)
Exoma , Programas Informáticos , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación INDEL/genética , Mutación/genética , Secuenciación del ExomaRESUMEN
[This corrects the article DOI: 10.1371/journal.pcbi.1009487.].
RESUMEN
Few studies have been conducted to understand post-zygotic accumulation of mutations in cells of the healthy human body. We reprogrammed 32 skin fibroblast cells from families of donors into human induced pluripotent stem cell (hiPSC) lines. The clonal nature of hiPSC lines allows a high-resolution analysis of the genomes of the founder fibroblast cells without being confounded by the artifacts of single-cell whole-genome amplification. We estimate that on average a fibroblast cell in children has 1035 mostly benign mosaic SNVs. On average, 235 SNVs could be directly confirmed in the original fibroblast population by ultradeep sequencing, down to an allele frequency (AF) of 0.1%. More sensitive droplet digital PCR experiments confirmed more SNVs as mosaic with AF as low as 0.01%, suggesting that 1035 mosaic SNVs per fibroblast cell is the true average. Similar analyses in adults revealed no significant increase in the number of SNVs per cell, suggesting that a major fraction of mosaic SNVs in fibroblasts arises during development. Mosaic SNVs were distributed uniformly across the genome and were enriched in a mutational signature previously observed in cancers and in de novo variants and which, we hypothesize, is a hallmark of normal cell proliferation. Finally, AF distribution of mosaic SNVs had distinct narrow peaks, which could be a characteristic of clonal cell selection, clonal expansion, or both. These findings reveal a large degree of somatic mosaicism in healthy human tissues, link de novo and cancer mutations to somatic mosaicism, and couple somatic mosaicism with cell proliferation.
Asunto(s)
Evolución Clonal , Variaciones en el Número de Copia de ADN , Fibroblastos/citología , Mosaicismo , Acumulación de Mutaciones , Proliferación Celular , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Piel/citologíaRESUMEN
Mutations in MECP2 gene have been identified in more than 95% of patients with classic Rett syndrome, one of the most common neurodevelopmental disorders in females. Taking advantage of the breakthrough technology of genetic reprogramming, we investigated transcriptome changes in neurons differentiated from induced Pluripotent Stem Cells (iPSCs) derived from patients with different mutations. Profiling by RNA-seq in terminally differentiated neurons revealed a prominent GABAergic circuit disruption along with a perturbation of cytoskeleton dynamics. In particular, in mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with selective inhibitors of HDAC6, the main α-tubulin deacetylase. These findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the treatment of Rett-associated epileptic behavior as well as for the definition of new therapeutic strategies for Rett syndrome.
Asunto(s)
Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/fisiología , Histona Desacetilasa 6/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatología , Tubulina (Proteína)/metabolismo , Acetilación , Diferenciación Celular/fisiología , Femenino , Humanos , MasculinoRESUMEN
The GABAergic system is regulated by the brain-derived neurotrophic factor (BDNF)/Tropomyosin-related kinase B (TrkB) pathway, but the cell-intrinsic role of TrkB signaling in parvalbumin cortical interneuron development and function is unclear. We performed conditional ablation of the TrkB receptor in parvalbumin-expressing (PV) interneurons to study whether postnatal loss of TrkB in parvalbumin cells affects their survival, connectivity, spontaneous and evoked neuronal activity and behavior. Using in vivo recordings of local field potentials, we found reduced gamma oscillations in the sensory cortex of PVcre+; TrkBF/F conditional knockout mice (TrkB cKO), along with increased firing of putative excitatory neurons. There was a significant downregulation in parvalbumin neuron number in cerebral and cerebellar cortices of TrkB cKO mice. In addition, inhibitory synaptic connections between basket cells and pyramidal neurons were profoundly reduced in the neocortex of TrkB cKO mice and there was a loss of cortical volume. TrkB cKO mice also showed profound hyperactivity, stereotypies, motor deficits and learning/memory defects. Our findings demonstrate that the targeting and/or synapse formation of PV-expressing basket cells with principal excitatory neurons require TrkB signaling in parvalbumin cells. Disruption of this signaling has major consequences for parvalbumin interneuron connectivity, network dynamics, cognitive and motor behavior.
Asunto(s)
Conducta Animal , Corteza Cerebral/citología , Corteza Cerebral/fisiopatología , Interneuronas , Glicoproteínas de Membrana/genética , Neuronas , Proteínas Tirosina Quinasas/genética , Animales , Fenómenos Electrofisiológicos/genética , Potenciales Evocados/fisiología , Discapacidades para el Aprendizaje/genética , Discapacidades para el Aprendizaje/psicología , Glicoproteínas de Membrana/deficiencia , Trastornos de la Memoria/genética , Trastornos de la Memoria/psicología , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos del Movimiento/genética , Trastornos del Movimiento/psicología , Neocórtex/citología , Parvalbúminas/biosíntesis , Parvalbúminas/genética , Proteínas Tirosina Quinasas/deficiencia , Células Piramidales , Análisis de SupervivenciaRESUMEN
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Células Madre Pluripotentes Inducidas/metabolismo , Mosaicismo , Piel/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Células Clonales , Fibroblastos/citología , Perfilación de la Expresión Génica , Genoma Humano/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Neuronas/citología , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Piel/citologíaRESUMEN
Gilles de la Tourette syndrome (TS) is characterized by tics, which are transiently worsened by stress, acute administration of dopaminergic drugs, and by subtle deficits in motor coordination and sensorimotor gating. It represents the most severe end of a spectrum of tic disorders that, in aggregate, affect â¼ 5% of the population. Available treatments are frequently inadequate, and the pathophysiology is poorly understood. Postmortem studies have revealed a reduction in specific striatal interneurons, including the large cholinergic interneurons, in severe disease. We tested the hypothesis that this deficit is sufficient to produce aspects of the phenomenology of TS, using a strategy for targeted, specific cell ablation in mice. We achieved â¼ 50% ablation of the cholinergic interneurons of the striatum, recapitulating the deficit observed in patients postmortem, without any effect on GABAergic markers or on parvalbumin-expressing fast-spiking interneurons. Interneuron ablation in the dorsolateral striatum (DLS), corresponding roughly to the human putamen, led to tic-like stereotypies after either acute stress or d-amphetamine challenge; ablation in the dorsomedial striatum, in contrast, did not. DLS interneuron ablation also led to a deficit in coordination on the rotorod, but not to any abnormalities in prepulse inhibition, a measure of sensorimotor gating. These results support the causal sufficiency of cholinergic interneuron deficits in the DLS to produce some, but not all, of the characteristic symptoms of TS.
Asunto(s)
Cuerpo Estriado/patología , Toxina Diftérica/farmacología , Interneuronas/citología , Receptores Colinérgicos/metabolismo , Síndrome de Tourette/patología , Potenciales de Acción , Animales , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Síndrome de Tourette/psicologíaRESUMEN
Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance.
Asunto(s)
Diferenciación Celular , Genoma Humano , Neuronas/citología , Células Madre/citología , Trastorno Autístico/genética , Silenciador del Gen , Marcación de Gen , Humanos , Discapacidad Intelectual/genética , Neuronas/metabolismo , ARN/metabolismo , Células Madre/metabolismoRESUMEN
Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8-10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex.
Asunto(s)
Corteza Cerebral , Células Madre Pluripotentes Inducidas , Modelos Biológicos , Neuronas , Transcriptoma/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Gyrification allows an expanded cortex with greater functionality to fit into a smaller cranium. However, the mechanisms of gyrus formation have been elusive. We show that ventricular injection of FGF2 protein at embryonic day 11.5-before neurogenesis and before the formation of intrahemispheric axonal connections-altered the overall size and shape of the cortex and induced the formation of prominent, bilateral gyri and sulci in the rostrolateral neocortex. We show increased tangential growth of the rostral ventricular zone (VZ) but decreased Wnt3a and Lef1 expression in the cortical hem and adjacent hippocampal promordium and consequent impaired growth of the caudal cortical primordium, including the hippocampus. At the same time, we observed ectopic Er81 expression, increased proliferation of Tbr2-expressing (Tbr2(+)) intermediate neuronal progenitors (INPs), and elevated Tbr1(+) neurogenesis in the regions that undergo gyrification, indicating region-specific actions of FGF2 on the VZ and subventricular zone (SVZ). However, the relative number of basal radial glia-recently proposed to be important in gyrification-appeared to be unchanged. These findings are consistent with the hypothesis that increased radial unit production together with rapid SVZ growth and heightened localized neurogenesis can cause cortical gyrification in lissencephalic species. These data also suggest that the position of cortical gyri can be molecularly specified in mice. In contrast, a different ligand, FGF8b, elicited surface area expansion throughout the cortical primordium but no gyrification. Our findings demonstrate that individual members of the diverse Fgf gene family differentially regulate global as well as regional cortical growth rates while maintaining cortical layer structure.
Asunto(s)
Corteza Cerebral/anatomía & histología , Corteza Cerebral/crecimiento & desarrollo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Animales , Antimetabolitos/farmacología , Axones/fisiología , Química Encefálica/efectos de los fármacos , Bromodesoxiuridina/farmacología , Recuento de Células , Corteza Cerebral/efectos de los fármacos , Ventrículos Cerebrales/metabolismo , Ventrículos Cerebrales/fisiología , ADN Complementario/biosíntesis , ADN Complementario/genética , Densitometría , Dependovirus , Femenino , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Hibridación in Situ , Factor de Unión 1 al Potenciador Linfoide/biosíntesis , Factor de Unión 1 al Potenciador Linfoide/genética , Ratones , Neocórtex/anatomía & histología , Neocórtex/crecimiento & desarrollo , Embarazo , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Wnt3A/biosíntesis , Proteína Wnt3A/genéticaRESUMEN
Infants born premature experience hypoxic episodes due to immaturity of their respiratory and central nervous systems. This profoundly affects brain development and results in cognitive impairments. We used a mouse model to examine the impact of hypoxic rearing (9.5-10.5% O2) from postnatal day 3 to 11 (P3-P11) on GABAergic interneurons and the potential for environmental enrichment to ameliorate these developmental abnormalities. At P15 the numbers of cortical interneurons expressing immunohistochemically detectable levels of parvalbumin (PV), somatostatin (SST), and vasoactive intestinal peptide were decreased in hypoxic-reared mice by 59%, 32%, and 38%, respectively, compared with normoxic controls. Hypoxia also decreased total GABA content in frontal neocortex by 31%. However, GAD67-EGFP knock-in mice reared under hypoxic conditions showed no changes in total number of GAD67-EGFP(+) cells and no evidence of increased interneuron death, suggesting that the total number of interneurons was not decreased, but rather, that hypoxic-rearing decreased interneuron marker expression in these cells. In adulthood, PV and SST expression levels were decreased in hypoxic-reared mice. In contrast, intensity of reelin (RLN) expression was significantly increased in adult hypoxic-reared mice compared with normoxic controls. Housing mice in an enriched environment from P21 until adulthood normalized phenotypic interneuron marker expression without affecting total interneuron numbers or leading to increased neurogenesis. Our data show that (1) hypoxia decreases PV and SST and increases RLN expression in cortical interneurons during postnatal cortical development and (2) enriched environment has the capacity to normalize the interneuron abnormalities in cortex.