Differentiation Of GABAergic Neurons Research Articles

Human-Induced Pluripotent Stem Cell (iPSC)-Derived GABAergic Neuron Differentiation in Bipolar Disorder.

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

DLX genes and proteins in mammalian forebrain development.

The vertebrate Dlx gene family encode homeobox transcription factors that are related to the Drosophila Distal-less (Dll) gene and are crucial for development. Over the last ∼35 years detailed information has accrued about the redundant and unique expression and function of the six mammalian Dlx family genes. DLX proteins interact with general transcriptional regulators, and co-bind with other transcription factors to enhancer elements with highly specific activity in the developing forebrain. Integration of the genetic and biochemical data has yielded a foundation for a gene regulatory network governing the differentiation of forebrain GABAergic neurons. In this Primer, we describe the discovery of vertebrate Dlx genes and their crucial roles in embryonic development. We largely focus on the role of Dlx family genes in mammalian forebrain development revealed through studies in mice. Finally, we highlight questions that remain unanswered regarding vertebrate Dlx genes despite over 30 years of research.

The proneural factors Ascl1a and Ascl1b contribute to the terminal differentiation of dopaminergic GABAergic dual transmitter neurons in zebrafish

The proneural factor Ascl1 is involved in several steps of neurogenesis, from neural progenitor maintenance to initiation of terminal differentiation and neuronal subtype specification. In neural progenitor cells, Ascl1 initiates the cell-cycle exit of progenitors, and contributes to their differentiation into mainly GABAergic neurons. Several catecholaminergic neuron groups in the forebrain of zebrafish use GABA as co-transmitter, but a potential role of the two paralogues Ascl1a and Ascl1b in their neurogenesis is not understood. Here, we show that ascl1a, ascl1b double mutant embryos develop a significantly reduced number of neurons in all GABAergic and catecholaminergic dual transmitter neuron anatomical clusters in the fore- and hindbrain, while glutamatergic catecholaminergic clusters develop normally. However, none of the affected catecholaminergic cell clusters are lost completely, suggesting an impairment in progenitor pools, or a requirement of Ascl1a/b for differentiation of a subset of neurons in each cluster. Early progenitors which are dlx2a+, fezf2 + or emx2 + are not reduced whereas late progenitors and differentiating neurons marked by the expression of dlx5a, isl1 and arxa are severely reduced in ascl1a, ascl1b double mutant embryos. This suggests that Ascl1a and Ascl1b play only a minor or no role in the maintenance of their progenitor pools, but rather contribute to the initiation of terminal differentiation of GABAergic catecholaminergic neurons.

Perinatal mood and anxiety disorders: biomarker discovery using plasma proteomics

Perinatal mood and anxiety disorders encompass a range of mental health disorders that occur during pregnancy and up to 1 year postpartum, affecting approximately 20% of women. Traditional risk factors, such as a history of depression and pregnancy complications including preeclampsia, are known. Their predictive utility, however, is not specific or sensitive enough to inform clinical decision-making or prevention strategies for perinatal mood and anxiety disorders. Better diagnostic and prognostic models are needed for early identification and referral to treatment. This study aimed to determine if a panel of novel third-trimester plasma protein biomarkers in pregnant women can be used to identify those who have a high predisposed risk for perinatal mood and anxiety disorders within 3 months postpartum. We studied 52 women (n=34 with a risk for perinatal mood and anxiety disorders and n=18 controls) among whom mental health screening was conducted at 2 time points, namely in the third trimester and again at 3 months postdelivery. An elevated perinatal mood and anxiety disorder risk was identified by screening individuals with above-validated cutoffs for depression (Edinburgh Postnatal Depression Scale ≥12), anxiety (Overall Anxiety Severity and Impairment Scale ≥7), and/or posttraumatic stress disorder (Impact of Events Scale >26) at both time points. Plasma samples collected in the third trimester were screened using the aptamer-based SomaLogic SomaScan proteomic assay technology to evaluate perinatal mood and anxiety disorder-associated changes in the expression of 1305 protein analytes. Ingenuity Pathway Analysis was conducted to highlight pathophysiological relationships between perinatal mood and anxiety disorder-specific proteins found to be significantly up- or down-regulated in all subjects with perinatal mood and anxiety disorder and in those with perinatal mood and anxiety disorders and no preeclampsia. From a panel of 53 significant perinatal mood and anxiety disorder-associated proteins, a unique 20-protein signature differentiated perinatal mood and anxiety disorder cases from controls in a principal component analysis (P<.05). This protein signature included NCAM1, NRCAM, and NTRK3 that converge around neuronal signaling pathways regulating axonal guidance, astrocyte differentiation, and maintenance of GABAergic neurons. Interestingly, when we restricted the analysis to subjects without preeclampsia, a 30-protein signature differentiated perinatal mood and anxiety disorder cases from all controls without overlap on the principal component analysis (P<.001). In the nonpreeclamptic perinatal mood and anxiety disorder group, we observed increased expression of proteins, such as CXCL11, CXCL6, MIC-B, and B2MG, which regulate leucocyte migration, inflammation, and immune function. Participants with perinatal mood and anxiety disorders had a unique and distinct plasma protein signature that regulated a variety of neuronal signaling and proinflammatory pathways. Additional validation studies with larger sample sizes are needed to determine whether some of these molecules can be used in conjunction with traditional risk factors for the early detection of perinatal mood and anxiety disorders.

LncRNA SOX1-OT V1 acts as a decoy of HDAC10 to promote SOX1-dependent hESC neuronal differentiation.

Long noncoding RNAs (lncRNAs) are abundantly expressed in the nervous system, but their regulatory roles in neuronal differentiation are poorly understood. Using a human embryonic stem cell (hESC)-based 2D neural differentiation approach and a 3D cerebral organoid system, we show that SOX1-OT variant 1 (SOX1-OT V1), a SOX1 overlapping noncoding RNA, plays essential roles in both dorsal cortical neuron differentiation and ventral GABAergic neuron differentiation by facilitating SOX1 expression. SOX1-OT V1 physically interacts with HDAC10 through its 5' region, acts as a decoy to block HDAC10 binding to the SOX1 promoter, and thus maintains histone acetylation levels at the SOX1 promoter. SOX1 in turn activates ASCL1 expression and promotes neuronal differentiation. Taken together, we identify a SOX1-OT V1/HDAC10-SOX1-ASCL1 axis, which promotes neurogenesis, highlighting a role for lncRNAs in hESC neuronal differentiation.

Mouse and human share conserved transcriptional programs for interneuron development.

Genetic variation confers susceptibility to neurodevelopmental disorders by affecting the development of specific cell types. Changes in cortical and striatal γ-aminobutyric acid–expressing (GABAergic) neurons are common in autism and schizophrenia. In this study, we used single-cell RNA sequencing to characterize the emergence of cell diversity in the human ganglionic eminences, the transitory structures of the human fetal brain where striatal and cortical GABAergic neurons are generated. We identified regional and temporal diversity among progenitor cells underlying the generation of a variety of projection neurons and interneurons. We found that these cells are specified within the human ganglionic eminences by transcriptional programs similar to those previously identified in rodents. Our findings reveal an evolutionarily conserved regulatory logic controlling the specification, migration, and differentiation of GABAergic neurons in the human telencephalon.

Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons

GABAergic neurons of the hypothalamus regulate many innate behaviors, but little is known about the mechanisms that control their development. We previously identified hypothalamic neurons that express the LIM homeodomain transcription factor Lhx6, a master regulator of cortical interneuron development, as sleep-promoting. In contrast to telencephalic interneurons, hypothalamic Lhx6 neurons do not undergo long-distance tangential migration and do not express cortical interneuronal markers such as Pvalb. Here, we show that Lhx6 is necessary for the survival of hypothalamic neurons. Dlx1/2, Nkx2-2, and Nkx2-1 are each required for specification of spatially distinct subsets of hypothalamic Lhx6 neurons, and that Nkx2-2+/Lhx6+ neurons of the zona incerta are responsive to sleep pressure. We further identify multiple neuropeptides that are enriched in spatially segregated subsets of hypothalamic Lhx6 neurons, and that are distinct from those seen in cortical neurons. These findings identify common and divergent molecular mechanisms by which Lhx6 controls the development of GABAergic neurons in the hypothalamus.

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in post-mitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.

Ethanol and Cannabinoids Regulate Zebrafish GABAergic Neuron Development and Behavior in a Sonic Hedgehog and Fibroblast Growth Factor-Dependent Mechanism.

Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development. Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior. Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids. These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD.

Expression of dlx genes in the normal and regenerating brain of adult zebrafish.

Dysfunctions in the GABAergic system lead to various pathological conditions and impaired inhibitory function is one of the causes behind neuropathies characterized by neuronal hyper excitability. The Dlx homeobox genes are involved in the development of nervous system, neural crest, branchial arches and developing appendages. Dlx genes also take part in neuronal migration and differentiation during development, more precisely, in the migration and differentiation of GABAergic neurons. Functional analysis of dlx genes has mainly been carried out in developing zebrafish embryos and larvae, however information regarding the expression and roles of these genes in the adult zebrafish brain is still lacking. The extensive neurogenesis that takes place in the adult zebrafish brain, makes them a good model for the visualization of mechanisms involving dlx genes during adulthood in physiological conditions and during regeneration of the nervous system. We have identified the adult brain regions where transcripts of dlx1a, dlx2a, dlx5a and dlx6a genes are normally found and have confirmed that within telencephalic domains, there is high overlapping expression of the four dlx paralogs with a marker for GABAergic neurons. Co-localization analyses carried with the Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP) reporter line have also shown that in some areas of the diencephalon, cells expressing the dlx5a/6a bigene may have a neural stem cell identity. Furthermore, investigations in a response to stab wound lesions, have demonstrated a possible participation of the dlx5a/6a bigene, most likely of dlx5a, during regeneration of the adult zebrafish brain. These observations suggest a possible participation of dlx-expressing cells during brain regeneration in adult zebrafish and also provide information on the role of dlx genes under normal physiological conditions in adults.

Impairment of motor but not anxiety-like behavior caused by the increase of dopamine during development is sustained in zebrafish larvae at later stages.

Many neuropsychiatric disorders are associated with both dopaminergic (DAergic) and developmental hypotheses. Since DAergic receptors are expressed in the developing brain, it is possible that alterations in dopamine (DA) signaling may impair brain development and consequent behavior. In our previous study, using a zebrafish model, we showed that an increase of DA during the 3 to 5days postfertilization (dpf) developmental window (an important window for GABAergic neuronal differentiation) affects the motor behavior of 5 dpf larvae. In this study, we set out to determine whether these behavioral alterations were sustained in larvae at older stages (7 and 14 dpf). To test this hypothesis, we chronically treated zebrafish larvae from 3 to 5 dpf with DA. After washing the drug, we recorded and analyzed the first 5 and 30min of the motor behavior of 5, 7, and 14 dpf subjects. We analyzed mobile episodes, distance traveled, time mobile, distance traveled per mobile episode, time in movement per mobile episode, and distance traveled per time mobile. We showed, once again, that an increase of DA during the 3 to 5 dpf developmental window reduces the number of movement episodes initiated by 5 dpf larvae. We also detected a decrease of other motor behavior parameters in 5 dpf DA-treated larvae. We observed that these alterations are sustained in the 7 dpf larvae. However, we did not see these general locomotor alterations in the 14 dpf larvae. Moreover, we detected a decrease of distance traveled and an increase of time of locomotion per episode in the first 5min of behavioral analyses in 14 dpf DA-treated larvae. To test if the alterations in the first 5min were due to anxiety-like behavior, we used a light/dark preference paradigm. We recorded 5dpf, 7dpf, and 14dpf larvae for 5min and analyzed time of freezing, preference for light or dark, number of entries to the dark, percentage of time in the light. We observed that 5dpf larvae treated with DA showed more freezing, less passages to the dark, and more time spent in the light as compared to their control counterparts. But 7dpf and 14dpf larvae did not show these alterations. Taken overall, therefore, our results suggest that DA does play a role in the development of zebrafish motor behavior, and, furthermore, that some behaviors are more sensitive than others to the effects of DAergic imbalances during development.

Modelling monogenic autism spectrum disorder using mouse cortical organoids

Variants of the contactin-associated protein-like 2 (CNTNAP2), which is a member of the neurexin family of proteins, function as cell adhesion molecules. The loss of CNTNAP2 function leads to autism spectrum disorder in humans and to autistic behaviours in mice. However, the functional effects of these mutations at the cellular level during fetal developmental periods remain elusive. Here, we studied mouse cortical organoids (mCOs) derived from Cntnap2−/− (knockout, KO) mouse induced pluripotent stem cells (miPSCs). Our results showed that KO mCOs displayed inhibitory-neuron-specific defects. At the neural progenitor stage, the GABAergic-neurogenesis-governing transcriptional network was dysregulated in the absence of Cntnap2. Our findings suggest that, in the early fetal cortical development, the cell adhesion molecule Cntnap2 plays a crucial role in the regulation of the differentiation of GABAergic neurons in the organoid platform. The reduced number of GABAergic neurons was efficiently restored in KO mCOs by treatment with the antiepileptic drug retigabine, showing the effectiveness of Cntnap2 KO mCOs in the therapeutic targeting of ASD.

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons

SUMMARYDLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.

Neuroprotective effects of creatine treatment on the differentiation of E14 rat cortical stem cells and expression of GABA-ergic neurons.

Along with the promotion of muscle growth and recovery, it has been observed that the supplement, creatine, also contains neuroprotective properties that aid in the developmental process of neuronal cells by increasing developmental competency in early differentiation. In clinical research, addiction treatments are beginning to introduce dietary supplements such as Creatine for patients in early recovery to improve recovery abstinence. Research has shown that supplemental Creatine induces higher cerebral phosphocreatine levels which could help with improved neuron connectivity. In order to examine the neuroprotective effects of Creatine, E14 rat cortical stem cells were differentiated and treated with different dosages of alcohol and creatine. Alcohol was used as a negative control in order to compare the differentiation and expression of GABAergic neurons associated with alcohol exposure and neuronal protection from Creative. We anticipate that Creatine acts as a neuroprotective agent in GABAergic neurons treated with alcohol

NEUROPROTECTIVE EFFECTS OF CREATINE TREATMENT ON THE DIFFERENTIATION OF E14 RAT CORTISOL STEM CELLS AND EXPRESSION OF GABA-ERGIC NEURONS

Along with the promotion of muscle growth and recovery, it has been observed that the supplement, Creatine, also contains neuroprotective properties that aid in the developmental process of neuronal cells by increasing developmental competency in early differentiation. In clinical research, addiction treatments are beginning to introduce dietary supplements such as Creatine for patients in early recovery to improve recovery abstinence. Research has shown that supplemental Creatine induces higher cerebral phosphocreatine levels which could help with improved neuron connectivity. In order to examine the neuroprotective effects of Creatine, E14 rat cortical stem cells were differentiated and treated with different dosages of alcohol and creatine. Alcohol was used as a negative control in order to compare the differentiation and expression of GABAergic neurons associated with alcohol exposure and neuronal protection from Creative. We anticipate that Creatine acts as a neuroprotective agent in GABAergic neurons treated with alcohol.

LHX1-a multifunctional regulator in preoptic area-derived interneuron development.

LHX1-a multifunctional regulator in preoptic area-derived interneuron development.

SU105 - MODELING SCHIZOPHRENIA IN VITRO BY THE GENERATION OF NEURONS FROM PATIENT-SPECIFIC STEM CELLS

Background Schizophrenia is a neurodevelopmental disease. Causes include environmental and genetic factors. Characteristics of schizophrenia include a deregulation of the glutamatergic and the GABAergic neurotransmission and the degeneration of predominantly inhibitory interneurons. Recently, 108 loci referring to Single Nucleotide Polymorphisms (SNPs) and 8 loci containing Copy Number Variations (CNVs) have been associated with schizophrenia suggesting a strong impact of genetic factors on the embryonic development of human brain. Together with other research teams, we identified CNVs in the Neurexin 1 gene (NRXN1) in patients suffering from schizophrenia, which highly recommends the detailed analysis of NRXN1-related disease mechanisms. Neurexins are presynaptic transmembrane proteins. We established patient-specific induced Pluripotent Stem (iPS) cells from schizophrenia patients and healthy donors to differentiate neurons for the investigation of disease mechanisms related to NRXN1 in schizophrenia patients. Patient-specific iPS cells carry the disease-associated genetic background of a patient, which is a CNVs affecting NRXN1. Methods Human iPS cells were generated from B-Lymphoblastoid Cell Lines (B-LCLs) using episomal vectors. B-LCLs were obtained from patients carrying CNVs in NRXN1 and healthy donors. The pluripotency of stem cells was verified by alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers. A neuronal screening protocol was applied to select iPS cell clones with a high differentiation potential towards neuronal cell lineages. Established lines were differentiated into Neural Stem Cells (NSCs) and further differentiated into functional and mature neurons. Terminal differentiated neurons were characterized by morphology, transcript analysis, western blot analysis, immunofluorescence analysis, electrophysiology, and mitochondrial respiration. Results Transcript analysis and western blot analysis of iPS cells verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The induction of neural stem cells was characterized by mRNA and protein analysis of neural stem cells markers including SOX2 and PAX6. Neural induction towards cortical neurons was monitored by a set of neurodevelopmental marker genes including NESTIN, GFAP, and NGN3. The efficient differentiation of mature neurons was shown by the expression of marker genes including glutamate and GABA receptors. Mitochondrial respiration was elevated in differentiated neurons. Patch clamp analysis verified the differentiation of functional GABAergic and glutamatergic neurons. Discussion Human iPS cells provide a powerful tool for the analysis of schizophrenia-associated and patient-specific DNA variations including CNVs. We successfully established mature and functional neuronal cultures suitable to study disease mechanisms related to NRXN1. Accordingly, this approach is an excellent opportunity for the identification of potential therapeutic targets.

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This elucidated the DLX-organized gene regulatory network at genomic, cellular, and spatial level in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.

Impaired Neuronal Differentiation of Neural Stem Cells Lacking the Engrailed-2 Gene

The Engrailed-2 (En2) gene codes for a homeobox-containing transcription factor, involved in midbrain-hindbrain embryonic development. In postnatal brain, En2 is expressed in the ventral mesencephalon, cerebellum, hippocampus and neocortex. Two single-nucleotide polymorphisms (SNPs) that are associated to autism spectrum disorders (ASD) have been identified in the human EN2 gene. Accordingly, mice lacking the En2 homeodomain (En2hd/hd, referred to as En2−/−) show molecular, anatomical and behavioral “ASD-like” features. Among these, we previously showed a partial loss of GABAergic interneurons in the En2−/− postnatal hippocampus and neocortex, accompanied by a marked decrease of brain-derived neurotrophic factor (BDNF) signaling, a crucial determinant of GABAergic differentiation. In order to better investigate the role of En2 in GABAergic interneuron differentiation, we generated and subsequently differentiated neural stem cells (NSCs) from basal ganglia and neocortex of En2+/+ and En2−/− mouse embryos. Wild-type NSCs from both basal ganglia and neocortex express En2, while mutant ones do not, as expected. As compared to En2+/+ NSCs, En2−/− NSCs derived from basal ganglia show impaired GABAergic differentiation accompanied by a reduced expression of the BDNF receptor trkB. Conversely, En2−/− NSCs derived from the neocortex expressed high levels of trkB and readily differentiated into neurons, as En2+/+ NSCs. Our results suggest that En2 contributes to GABAergic neuron differentiation from basal ganglia NSCs through a trkB-dependent BDNF signaling, thus providing a possible explanation for the reduced number of GABAergic interneurons detected in the En2−/− postnatal forebrain.

Exposure to bisphenol A affects GABAergic neuron differentiation in neurosphere cultures.

Endocrine-disrupting chemicals (EDCs) influence not only endocrine functions but also neuronal development and functions. In-vivo studies have suggested the relationship of EDC-induced neurobehavioral disorders with dysfunctions of neurotransmitter mechanisms including γ-aminobutyric acid (GABA)ergic mechanisms. However, whether EDCs affect GABAergic neuron differentiation remains unclear. In the present study, we show that a representative EDC, bisphenol A (BPA), affects GABAergic neuron differentiation. Cortical neurospheres prepared from embryonic mice were exposed to BPA for 7 days, and then neuronal differentiation was induced. We found that BPA exposure resulted in a decrease in the ratio of GABAergic neurons to total neurons. However, the same exposure stimulated the differentiation of neurons expressing calbindin, a calcium-binding protein observed in a subpopulation of GABAergic neurons. These findings suggested that BPA might influence the formation of an inhibitory neuronal network in developing cerebral cortex involved in the occurrence of neurobehavioral disorders.