Migration Of GABAergic Interneurons Research Articles

Both GEF domains of the autism and developmental epileptic encephalopathy-associated Trio protein are required for proper tangential migration of GABAergic interneurons

Both GEF domains of the autism and developmental epileptic encephalopathy-associated Trio protein are required for proper tangential migration of GABAergic interneurons

CNSC-03. LINKING CELL FATE DETERMINATION IN THE EMBRYONIC FOREBRAIN TO PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric high-grade gliomas are treatment resistant, usually fatal cancers encompassing diffuse midline glioma, K27-altered (DMG), and diffuse hemispheric glioma, G34R/V (DHG). Most patients harbour canonical and non-canonical mutations in histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and stalled differentiation states as oligodendroglial progenitor cells (OPC) in DMG and GABAergic interneuron precursors in DHG. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes GABAergic interneuron cell fate and migration during early neurogenesis. Methods & Results Our lab has shown DLX2 promoter occupancy of Gad1/Gad2, and early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo. Co-expression of DLX2 with these target sequences reduces reporter gene expression in vitro, and the Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3 and loss of H3K27 acetylation, as well as a reduction in migration, invasion, and colony formation in vitro. To validate in human pHGG, we first probed an RNA-seq database containing 62 patient-derived pHGG cell lines which demonstrated an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DHG cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2-expressing DMG cell lines underwent stable transfection of a DLX2-mCherry overexpression construct in conjunction with a SOX2-BFP overexpression construct, confirmed by qPCR and immunoblot. Analysis of histone H3 marks, K27M, and OPC signature genes will be compared to OPC genes from Dlx1/Dlx2 DKO mouse ganglionic eminences. CONCLUSIONS Manipulation of DLX2 in pHGG provides a model system for assessing the contribution of a homeobox factor to cell fate decisions regulating differentiation and tumorigenicity.

Interneuron odyssey: molecular mechanisms of tangential migration.

Cortical GABAergic interneurons are critical components of neural networks. They provide local and long-range inhibition and help coordinate network activities involved in various brain functions, including signal processing, learning, memory and adaptative responses. Disruption of cortical GABAergic interneuron migration thus induces profound deficits in neural network organization and function, and results in a variety of neurodevelopmental and neuropsychiatric disorders including epilepsy, intellectual disability, autism spectrum disorders and schizophrenia. It is thus of paramount importance to elucidate the specific mechanisms that govern the migration of interneurons to clarify some of the underlying disease mechanisms. GABAergic interneurons destined to populate the cortex arise from multipotent ventral progenitor cells located in the ganglionic eminences and pre-optic area. Post-mitotic interneurons exit their place of origin in the ventral forebrain and migrate dorsally using defined migratory streams to reach the cortical plate, which they enter through radial migration before dispersing to settle in their final laminar allocation. While migrating, cortical interneurons constantly change their morphology through the dynamic remodeling of actomyosin and microtubule cytoskeleton as they detect and integrate extracellular guidance cues generated by neuronal and non-neuronal sources distributed along their migratory routes. These processes ensure proper distribution of GABAergic interneurons across cortical areas and lamina, supporting the development of adequate network connectivity and brain function. This short review summarizes current knowledge on the cellular and molecular mechanisms controlling cortical GABAergic interneuron migration, with a focus on tangential migration, and addresses potential avenues for cell-based interneuron progenitor transplants in the treatment of neurodevelopmental disorders and epilepsy.

Sonic hedgehog-dependent recruitment of GABAergic interneurons into the developing visual thalamus.

Axons of retinal ganglion cells (RGCs) play critical roles in the development of inhibitory circuits in visual thalamus. We previously reported that RGC axons signal astrocytes to induce the expression of fibroblast growth factor 15 (FGF15), a motogen required for GABAergic interneuron migration into visual thalamus. However, how retinal axons induce thalamic astrocytes to generate Fgf15 and influence interneuron migration remains unknown. Here, we demonstrate that impairing RGC activity had little impact on interneuron recruitment into mouse visual thalamus. Instead, our data show that retinal-derived sonic hedgehog (SHH) is essential for interneuron recruitment. Specifically, we show that thalamus-projecting RGCs express SHH and thalamic astrocytes generate downstream components of SHH signaling. Deletion of RGC-derived SHH leads to a significant decrease in Fgf15 expression, as well as in the percentage of interneurons recruited into visual thalamus. Overall, our findings identify a morphogen-dependent neuron-astrocyte signaling mechanism essential for the migration of thalamic interneurons.

ZEB2 haploinsufficient Mowat-Wilson syndrome induced pluripotent stem cells show disrupted GABAergic transcriptional regulation and function.

Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.

Developmental, neurochemical, and behavioral analyses of ErbB4 Cyt-1 knockout mice.

Neuregulins (NRGs) and their cognate neuronal receptor ERBB4, which is expressed in GABAergic and dopaminergic neurons, regulate numerous behaviors in rodents and have been identified as schizophrenia at-risk genes. ErbB4 transcripts are alternatively spliced to generate isoforms that either include (Cyt-1) or exclude (Cyt-2) exon 26, which encodes a cytoplasmic domain that imparts ErbB4 receptors the ability to signal via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although ErbB4 Cyt-1/2 isoforms have been studied in transfected cultured cells, their functions in vivo remain unknown. Here, we generated ErbB4-floxed (ErbB4-Cyt1fl/fl ) mice to investigate the effects of germline (constitutive) and conditional (acute) deletions of the Cyt-1 exon. Overall receptor mRNA levels remain unchanged in germline ErbB4 Cyt-1 knockouts (Cyt-1 KOs), with all transcripts encoding Cyt-2 variants. In contrast to mice lacking all ErbB4 receptor function, GABAergic interneuron migration and number are unaltered in Cyt-1 KOs. However, basal extracellular dopamine (DA) levels in the medial prefrontal cortex are increased in Cyt-1 heterozygotes. Despite these neurochemical changes, Cyt-1 heterozygous and homozygous mice do not manifest behavioral abnormalities previously reported to be altered in ErbB4 null mice. To address the possibility that Cyt-2 variants compensate for the lack of Cyt-1 during development, we microinjected an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the DA-rich ventral tegmental area of adult ErbB4-Cyt1fl/fl mice to acutely target exon 26. These conditional Cyt-1 KOs were found to exhibit behavioral abnormalities in the elevated plus maze and startle response, consistent with the idea that late exon 26 ablations may circumvent compensation by Cyt-2 variants. Taken together, our observations indicate that ErbB4 Cyt-1 function in vivo is important for DA balance and behaviors in adults.

Quickly moving too slowly: Interneuron migration in Timothy Syndrome.

Aberrant migration of GABAergic interneurons during cortical neurodevelopment is implicated in Timothy Syndrome, yet the underlying mechanisms remain elusive. In this issue of Cell Stem Cell, Birey etal. model developing brain circuitry using "assembloids" from patients, characterizing a bimodal mechanism of mechano-chemically driven interneuron migration inefficiencies.

L-Type Calcium Channels Contribute to Ethanol-Induced Aberrant Tangential Migration of Primordial Cortical GABAergic Interneurons in the Embryonic Medial Prefrontal Cortex.

Exposure of the fetus to alcohol (ethanol) via maternal consumption during pregnancy can result in fetal alcohol spectrum disorders (FASD), hallmarked by long-term physical, behavioral, and intellectual abnormalities. In our preclinical mouse model of FASD, prenatal ethanol exposure disrupts tangential migration of corticopetal GABAergic interneurons (GINs) in the embryonic medial prefrontal cortex (mPFC). We postulated that ethanol perturbed the normal pattern of tangential migration via enhancing GABAA receptor-mediated membrane depolarization that prevails during embryonic development in GABAergic cortical interneurons. However, beyond this, our understanding of the underlying mechanisms is incomplete. Here, we tested the hypothesis that the ethanol-enhanced depolarization triggers downstream an increase in high-voltage-activated nifedipine-sensitive L-type calcium channel (LTCC) activity and provide evidence implicating calcium dynamics in the signaling scheme underlying the migration of embryonic GINs and its aberrance. Tangentially migrating Nkx2.1+ GINs expressed immunoreactivity to Cav1.2, the canonical neuronal isoform of the L-type calcium channel. Prenatal ethanol exposure did not alter its protein expression profile in the embryonic mPFC. However, exposing ethanol concomitantly with the LTCC blocker nifedipine prevented the ethanol-induced aberrant migration both in vitro and in vivo. In addition, whole-cell patch clamp recording of LTCCs in GINs migrating in embryonic mPFC slices revealed that acutely applied ethanol potentiated LTCC activity in migrating GINs. Based on evidence reported in the present study, we conclude that calcium is an important intracellular intermediary downstream of GABAA receptor-mediated depolarization in the mechanistic scheme of an ethanol-induced aberrant tangential migration of embryonic GABAergic cortical interneurons.

Reduced hippocampal inhibition and enhanced autism-epilepsy comorbidity in mice lacking neuropilin 2

The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2-deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2−/− mice have a reduced number of parvalbumin, somatostatin, and neuropeptide Y cells, mainly in CA1. Whole-cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/− and Nrp2−/− mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2-deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration, and higher severity in Nrp2−/− compared to Nrp2+/+ animals. Finally, Nrp2+/− and Nrp2−/− but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2−/− mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

Altered Cl− homeostasis hinders forebrain GABAergic interneuron migration in a mouse model of intellectual disability

Impairments of inhibitory circuits are at the basis of most, if not all, cognitive deficits. The impact of OPHN1, a gene associate with intellectual disability (ID), on inhibitory neurons remains elusive. We addressed this issue by analyzing the postnatal migration of inhibitory interneurons derived from the subventricular zone in a validated mouse model of ID (OPHN1-/y mice). We found that the speed and directionality of migrating neuroblasts were deeply perturbed in OPHN1-/y mice. The significant reduction in speed was due to altered chloride (Cl-) homeostasis, while the overactivation of the OPHN1 downstream signaling pathway, RhoA kinase (ROCK), caused abnormalities in the directionality of the neuroblast progression in mutants. Blocking the cation-Cl- cotransporter KCC2 almost completely rescued the migration speed while proper directionality was restored upon ROCK inhibition. Our data unveil a strong impact of OPHN1 on GABAergic inhibitory interneurons and identify putative targets for successful therapeutic approaches.

Prenatal alcohol exposure is a leading cause of interneuronopathy in humans

Alcohol affects multiple neurotransmitter systems, notably the GABAergic system and has been recognised for a long time as particularly damaging during critical stages of brain development. Nevertheless, data from the literature are most often derived from animal or in vitro models. In order to study the production, migration and cortical density disturbances of GABAergic interneurons upon prenatal alcohol exposure, we performed immunohistochemical studies by means of the proliferation marker Ki67, GABA and calretinin antibodies in the frontal cortical plate of 17 foetal and infant brains antenatally exposed to alcohol, aged 15 weeks’ gestation to 22 postnatal months and in the ganglionic eminences and the subventricular zone of the dorsal telencephalon until their regression, i.e., 34 weeks’ gestation. Results were compared with those obtained in 17 control brains aged 14 weeks of gestation to 35 postnatal months. We also focused on interneuron vascular migration along the cortical microvessels by confocal microscopy with double immunolabellings using Glut1, GABA and calretinin. Semi-quantitative and quantitative analyses of GABAergic and calretininergic interneuron density allowed us to identify an insufficient and delayed production of GABAergic interneurons in the ganglionic eminences during the two first trimesters of the pregnancy and a delayed incorporation into the laminar structures of the frontal cortex. Moreover, a mispositioning of GABAergic and calretininergic interneurons persisted throughout the foetal life, these cells being located in the deep layers instead of the superficial layers II and III. Moreover, vascular migration of calretininergic interneurons within the cortical plate was impaired, as reflected by low numbers of interneurons observed close to the cortical perforating vessel walls that may in part explain their abnormal intracortical distribution. Our results are globally concordant with those previously obtained in mouse models, in which alcohol has been shown to induce an interneuronopathy by affecting interneuron density and positioning within the cortical plate, and which could account for the neurological disabilities observed in children with foetal alcohol disorder spectrum.

Prenatal disruption of D1R-SynGAP complex causes cognitive deficits in adulthood

γ-aminobutyric acid (GABA)-ergic interneurons are essential for the physiological function of the mammalian central nervous system. Dysregulated GABAergic interneuron function has been implicated in the pathophysiology of a number of neurodevelopmental disorders including schizophrenia and autism spectrum disorder. Tangential migration is an important process to ensure the proper localization of GABAergic interneurons. Previously we found that disrupting the interaction between dopamine D1 receptor (D1R) and synaptic Ras GTPase- activating protein (SynGAP) using an interfering peptide (TAT-D1Rpep) during embryonic development impaired tangential migration. Here, we assessed the effects of prenatal disruption of D1R-SynGAP complex with the TAT-D1Rpep on the expression of several behaviours during adulthood. Mice with prenatal D1R-SynGAP disruption exhibited transiently reduced locomotor activity, abnormal sensorimotor gating, impaired sociability and deficits in visual discrimination associative learning compared to their control counterparts. Our findings reinforce the importance of GABAergic interneuron migration in the manifestation of normal motor, sensory, and cognitive behaviours of animals during adulthood.

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

The NKCC1 antagonist bumetanide mitigates interneuronopathy associated with ethanol exposure in utero.

Prenatal exposure to ethanol induces aberrant tangential migration of corticopetal GABAergic interneurons, and long-term alterations in the form and function of the prefrontal cortex. We have hypothesized that interneuronopathy contributes significantly to the pathoetiology of fetal alcohol spectrum disorders (FASD). Activity-dependent tangential migration of GABAergic cortical neurons is driven by depolarizing responses to ambient GABA present in the cortical enclave. We found that ethanol exposure potentiates the depolarizing action of GABA in GABAergic cortical interneurons of the embryonic mouse brain. Pharmacological antagonism of the cotransporter NKCC1 mitigated ethanol-induced potentiation of GABA depolarization and prevented aberrant patterns of tangential migration induced by ethanol in vitro. In a model of FASD, maternal bumetanide treatment prevented interneuronopathy in the prefrontal cortex of ethanol exposed offspring, including deficits in behavioral flexibility. These findings position interneuronopathy as a mechanism of FASD symptomatology, and posit NKCC1 as a pharmacological target for the management of FASD.

JAK3 modulates the migration of gabaergic interneurons during development of murine cortex

JAK3 modulates the migration of gabaergic interneurons during development of murine cortex

A Brief Boost of Positive Energy When Young Makes for a Healthy Adult Interneuron.

Tonic Activation of GluN2C/GluN2D-Containing NMDA Receptors by Ambient Glutamate Facilitates Cortical Interneuron Maturation Hanson E, Armbruster M, Lau LA, Sommer ME, Klaft ZJ, Swanger SA, Traynelis SF, Moss SJ, Noubary F, Chadchankar J, Dulla CG. J Neurosci. 2019;39(19):3611-3626. doi:10.1523/JNEUROSCI.1392-18.2019. PMID: 30846615. Epub Mar 7, 2019.Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and environmental cues to construct inhibitory circuits during early postnatal development. Disruption of these events can cause long-term changes in cortical inhibition and may be involved in neurological disorders associated with inhibitory circuit dysfunction. We hypothesized that tonic glutamate signaling in the neonatal cortex contributes to, and is necessary for, the maturation of cortical interneurons. To test this hypothesis, we used mice of both sexes to quantify extracellular glutamate concentrations in the cortex during development, measure ambient glutamate-mediated activation of developing cortical interneurons, and manipulate tonic glutamate signaling using subtype-specific N-methyl-d-aspartic acid (NMDA) receptor antagonists in vitro and in vivo. We report that ambient glutamate levels are high (≈100 nm) in the neonatal cortex and decrease (to ≈50 nm) during the first weeks of life, coincident with increases in astrocytic glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin-positive interneurons in the cortex (identified using G42: GAD1-eGFP reporter mice) exhibit a transient, tonic NMDA current at the end of the first postnatal week. GluN2C/GluN2D-containing NMDA receptors mediate the majority of this current and contribute to the resting membrane potential and intrinsic properties of developing putative parvalbumin interneurons. Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptors in vivo during the period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron morphological complexity and causes deficits in cortical inhibition later in life. These results demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron maturation via tonic activation of GluN2C/GluN2D-containing NMDA receptors. Significance statement: Inhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple neurological disorders, including epilepsy. Establishing inhibitory cortical networks requires in utero proliferation, differentiation, and migration of immature GABAergic interneurons and subsequent postnatal morphological maturation and circuit integration. Here, we demonstrate that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of developing cortical interneurons by ambient glutamate is critically important for establishing normal cortical inhibition.

Inhibitory neurons in the right place

Neurodevelopment Disruption of γ-aminobutyric acid–mediated (GABAergic) interneuron migration is implicated in neurodevelopmental disorders such as autism spectrum disorder and schizophrenia. Su et al. found that dopamine induced the association of the dopamine receptor D1R with the guanosine triphosphatase–activating protein SynGAP. During embryonic and early postnatal development in mice, this complex promoted dopamine-induced migration of GABAergic interneurons but not glutamatergic interneurons. These findings may explain why mutations in SYNGAP1 give rise to neurodevelopmental disorders. Sci. Signal. 12 , eaau9122 (2019).

Disruption of SynGAP-dopamine D1 receptor complexes alters actin and microtubule dynamics and impairs GABAergic interneuron migration.

Disruption of γ-aminobutyric acid (GABA)-ergic interneuron migration is implicated in various neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. The dopamine D1 receptor (D1R) promotes GABAergic interneuron migration, which is disrupted in various neurological disorders, some of which are also associated with mutations in the gene encoding synaptic Ras-guanosine triphosphatase-activating protein (SynGAP). Here, we explored the mechanisms underlying these associations and their possible connection. In prenatal mouse brain tissue, we found a previously unknown interaction between the D1R and SynGAP. This D1R-SynGAP interaction facilitated D1R localization to the plasma membrane and promoted D1R-mediated downstream signaling pathways, including phosphorylation of protein kinase A and p38 mitogen-activated protein kinase. These effects were blocked by a peptide (TAT-D1Rpep) that disrupted the D1R-SynGAP interaction. Furthermore, disrupting this complex in mice during embryonic development resulted in pronounced and selective deficits in the tangential migration of GABAergic interneurons, possibly due to altered actin and microtubule dynamics. Our results provide insights into the molecular mechanisms regulating interneuron development and suggest that disruption of the D1R-SynGAP interaction may underlie SYNGAP1 mutation-related neurodevelopmental disorders.

Nanoparticle Design and Characterization for Catalytic Applications in Sustainable Chemistry

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common movement disorder. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. However, mechanisms underlying this deficit are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18 days of age) and at age of highest severity of dystonia (33 days of age) in this hamster model. In line with previous findings, the density of GAD67-positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in the dtsz mutant than in control hamsters. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level suggested that the migration of GABAergic IN into the striatum was not retarded. Therefore, different factors that indicate maturation of GABAergic IN were determined. While mRNA of the KCC2 cation/chloride transporters and the cytosolic carboanhydrase VII, used as markers for the so called GABA switch, as well as BDNF were unaltered, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33 days, but not after spontaneous remission of dystonia at an age of 90 days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity.

Tonic Activation of GluN2C/GluN2D-Containing NMDA Receptors by Ambient Glutamate Facilitates Cortical Interneuron Maturation.

Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and environmental cues to construct inhibitory circuits during early postnatal development. Disruption of these events can cause long-term changes in cortical inhibition and may be involved in neurological disorders associated with inhibitory circuit dysfunction. We hypothesized that tonic glutamate signaling in the neonatal cortex contributes to, and is necessary for, the maturation of cortical interneurons. To test this hypothesis, we used mice of both sexes to quantify extracellular glutamate concentrations in the cortex during development, measure ambient glutamate-mediated activation of developing cortical interneurons, and manipulate tonic glutamate signaling using subtype-specific NMDA receptor antagonists in vitro and in vivo We report that ambient glutamate levels are high (≈100 nm) in the neonatal cortex and decrease (to ≈50 nm) during the first weeks of life, coincident with increases in astrocytic glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin-positive interneurons in the cortex (identified using G42:GAD1-eGFP reporter mice) exhibit a transient, tonic NMDA current at the end of the first postnatal week. GluN2C/GluN2D-containing NMDA receptors mediate the majority of this current and contribute to the resting membrane potential and intrinsic properties of developing putative parvalbumin interneurons. Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptors in vivo during the period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron morphological complexity and causes deficits in cortical inhibition later in life. These results demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron maturation via tonic activation of GluN2C/GluN2D-containing NMDA receptors.SIGNIFICANCE STATEMENT Inhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple neurological disorders, including epilepsy. Establishing inhibitory cortical networks requires in utero proliferation, differentiation, and migration of immature GABAergic interneurons, and subsequent postnatal morphological maturation and circuit integration. Here, we demonstrate that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of developing cortical interneurons by ambient glutamate is critically important for establishing normal cortical inhibition.