Wheel-Running Exercise Alleviates Anxiety-Like Behavior via Down-Regulating S-Nitrosylation of Gephyrin in the Basolateral Amygdala of Male Rats.

Physical exercise has beneficial effect on anxiety disorders, but the underlying molecular mechanism remains largely unknown. Here, it is demonstrated that physical exercise can downregulate the S-nitrosylation of gephyrin (SNO-gephyrin) in the basolateral amygdala (BLA) to exert anxiolytic effects. It is found that the level of SNO-gephyrin is significantly increased in the BLA of high-anxiety rats and a downregulation of SNO-gephyrin at cysteines 212 and 284 produced anxiolytic effect. Mechanistically, inhibition of SNO-gephyrin by either Cys212 or Cys284 mutations increased the surface expression of GABAAR γ2 and the subsequent GABAergic neurotransmission, exerting anxiolytic effect in male rats. On the other side, overexpression of neuronal nitric oxide synthase in the BLA abolished the anxiolytic-like effects of physical exercise. This study reveals a key role of downregulating SNO-gephyrin in the anxiolytic effects of physical exercise, providing a new explanation for protein post-translational modifications in the brain after exercise.

Gephyrin promotes autonomous assembly and synaptic localization of GABAergic postsynaptic components without presynaptic GABA release.

Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.

Postweaning Development Influences Endogenous VPAC1 Modulation of LTP Induced by Theta-Burst Stimulation: A Link to Maturation of the Hippocampal GABAergic System.

Long-term potentiation (LTP) induced by theta-burst stimulation (TBS) undergoes postweaning developmental changes partially linked to GABAergic circuit maturation. Endogenous vasoactive intestinal peptide (VIP) acting on its VPAC1 receptor strongly influences LTP induced by theta-burst stimulation (TBS), an effect dependent on GABAergic transmission. Although VPAC1 receptor levels are developmentally regulated during embryogenesis, their variation along postweaning development is unknown, as is the VPAC1 modulation of LTP or its relation to hippocampal GABAergic circuit maturation. As such, we investigated how VPAC1 modulation of LTP adjusts from weaning to adulthood along with GABAergic circuit maturation. As described, LTP induced by mild TBS (5 bursts, 4 pulses delivered at 100 Hz) was increasingly greater from weaning to adulthood. The influence of the VPAC1 receptor antagonist PG 97-269 (100 nM) on TBS-induced LTP was much larger in juvenile (3-week-old) than in young adult (6-7-week-old) or adult (12-week-old) rats. This effect was not associated with a developmental decrease in synaptic VPAC1 receptor levels. However, an increase in pre and post-synaptic GABAergic synaptic markers suggests an increase in the number of GABAergic synaptic contacts that is more prominent than the one observed in glutamatergic connections during this period. Conversely, endogenous VPAC2 receptor activation did not significantly influence TBS-induced LTP. VPAC2 receptor levels enhance pronouncedly during postweaning development, but not at synaptic sites. Given the involvement of VIP interneurons in several aspects of hippocampal-dependent learning, neurodevelopmental disorders, and epilepsy, this could provide important insights into the role of VIP modulation of hippocampal synaptic plasticity during normal and altered brain development potentially contributing to epileptogenesis.

KIF5B plays important roles in dendritic spine plasticity and dendritic localization of PSD95 and FMRP in the mouse cortex in vivo.

Kinesin 1 (KIF5) is one major type of motor protein in neurons, but its members' function in the intact brain remains less studied. Using in vivo two-photon imaging, we find that conditional knockout of Kif5b (KIF5B cKO) in CaMKIIα-Cre-expressing neurons shows heightened turnover and lower stability of dendritic spines in layer 2/3 pyramidal neurons with reduced spine postsynaptic density protein 95 acquisition in the mouse cortex. Furthermore, the RNA-binding protein fragile X mental retardation protein (FMRP) is translocated to the proximity of newly formed spines several hours before the spine formation events in vivo in control mice, but this preceding transport of FMRP is abolished in KIF5B cKO mice. We further find that FMRP is localized closer to newly formed spines after fear extinction, but this learning-dependent localization is disrupted in KIF5B cKO mice. Our findings provide the crucial in vivo evidence that KIF5B is involved in the dendritic targeting of synaptic proteins that underlies dendritic spine plasticity.

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Modelling and Molecular Dynamics Predict the Structure and Interactions of the Glycine Receptor Intracellular Domain.

Glycine receptors (GlyRs) are glycine-gated inhibitory pentameric ligand-gated ion channels composed of α or α + ß subunits. A number of structures of these proteins have been reported, but to date, these have only revealed details of the extracellular and transmembrane domains, with the intracellular domain (ICD) remaining uncharacterised due to its high flexibility. The ICD is a region that can modulate function in addition to being critical for receptor localisation and clustering via proteins such as gephyrin. Here, we use modelling and molecular dynamics (MD) to reveal details of the ICDs of both homomeric and heteromeric GlyR. At their N and C ends, both the α and ß subunit ICDs have short helices, which are major sites of stabilising interactions; there is a large flexible loop between them capable of forming transient secondary structures. The α subunit can affect the ß subunit ICD structure, which is more flexible in a 4α2:1ß than in a 4α1:1ß GlyR. We also explore the effects of gephyrin binding by creating GlyR models bound to the gephyrin E domain; MD simulations suggest these are more stable than the unbound forms, and again there are α subunit-dependent differences, despite the fact the gephyrin binds to the ß subunit. The bound models also suggest that gephyrin causes compaction of the ICD. Overall, the data expand our knowledge of this important receptor protein and in particular clarify features of the underexplored ICD.

Acute reorganization of postsynaptic GABAA receptors reveals the functional impact of molecular nanoarchitecture at inhibitory synapses.

Neurotransmitter receptors partition into nanometer-scale subdomains within the postsynaptic membrane that are precisely aligned with presynaptic neurotransmitter release sites. While spatial coordination between pre- and postsynaptic elements is observed at both excitatory and inhibitory synapses, the functional significance of this molecular architecture has been challenging to evaluate experimentally. Here we utilized an optogenetic clustering approach to acutely alter the nanoscale organization of the postsynaptic inhibitory scaffold gephyrin while monitoring synaptic function. Gephyrin clustering rapidly enlarged postsynaptic area, laterally displacing GABAA receptors from their normally precise apposition with presynaptic active zones. Receptor displacement was accompanied by decreased synaptic GABAA receptor currents even though presynaptic release probability and the overall abundance and function of synaptic GABAA receptors remained unperturbed. Thus, acutely repositioning neurotransmitter receptors within the postsynaptic membrane profoundly influences synaptic efficacy, establishing the functional importance of precision pre-/postsynaptic molecular coordination at inhibitory synapses.

Impact of Developmental Changes of GABAA Receptors on Interneuron-NG2 Glia Transmission in the Hippocampus.

NG2 glia receive synaptic input from neurons, but the functional impact of this glial innervation is not well understood. In the developing cerebellum and somatosensory cortex the GABAergic input might regulate NG2 glia differentiation and myelination, and a switch from synaptic to extrasynaptic neuron-glia signaling was reported in the latter region. Myelination in the hippocampus is sparse, and most NG2 glia retain their phenotype throughout adulthood, raising the question of the properties and function of neuron-NG2 glia synapses in that brain region. Here, we compared spontaneous and evoked GABAA receptor-mediated currents of NG2 glia in juvenile and adult hippocampi of mice of either sex and assessed the mode of interneuron-glial signaling changes during development. With patch-clamp and pharmacological analyses, we found a decrease in innervation of hippocampal NG2 glia between postnatal days 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors occurred, indicating a spillover of GABA. This switch from synaptic to extrasynaptic receptor activation was accompanied by downregulation of γ2 and upregulation of the α5 subunit. Molecular analyses and high-resolution expansion microscopy revealed mechanisms of glial GABAA receptor trafficking and clustering. We found that gephyrin and radixin are organized in separate clusters along glial processes. Surprisingly, the developmental loss of γ2 and postsynaptic receptors were not accompanied by altered glial expression of scaffolding proteins, auxiliary receptor subunits or postsynaptic interaction proteins. The GABAergic input to NG2 glia might contribute to the release of neurotrophic factors from these cells and influence neuronal synaptic plasticity.

Adamtsl3 mediates DCC signaling to selectively promote GABAergic synapse function.

The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.

GSK-3ß orchestrates the inhibitory innervation of adult-born dentate granule cells in vivo.

Adult hippocampal neurogenesis enhances brain plasticity and contributes to the cognitive reserve during aging. Adult hippocampal neurogenesis is impaired in neurological disorders, yet the molecular mechanisms regulating the maturation and synaptic integration of new neurons have not been fully elucidated. GABA is a master regulator of adult and developmental neurogenesis. Here we engineered a novel retrovirus encoding the fusion protein Gephyrin:GFP to longitudinally study the formation and maturation of inhibitory synapses during adult hippocampal neurogenesis in vivo. Our data reveal the early assembly of inhibitory postsynaptic densities at 1 week of cell age. Glycogen synthase kinase 3 Beta (GSK-3ß) emerges as a key regulator of inhibitory synapse formation and maturation during adult hippocampal neurogenesis. GSK-3ß-overexpressing newborn neurons show an increased number and altered size of Gephyrin+ postsynaptic clusters, enhanced miniature inhibitory postsynaptic currents, shorter and distanced axon initial segments, reduced synaptic output at the CA3 and CA2 hippocampal regions, and impaired pattern separation. Moreover, GSK-3ß overexpression triggers a depletion of Parvalbumin+ interneuron perineuronal nets. These alterations might be relevant in the context of neurological diseases in which the activity of GSK-3ß is dysregulated.

Intracellular signaling mechanisms that shape postsynaptic GABAergic synapses.

Postsynaptic GABAergic receptors interact with various membrane and intracellular proteins to mediate inhibitory synaptic transmission. They form structural and/or signaling synaptic protein complexes that perform a variety of postsynaptic functions. In particular, the key GABAergic synaptic scaffold, gephyrin, and its interacting partners govern downstream signaling pathways that are essential for GABAergic synapse development, transmission, and plasticity. In this review, we discuss recent researches on GABAergic synaptic signaling pathways. We also outline the main outstanding issues that need to be addressed in this field and highlight the association of dysregulated GABAergic synaptic signaling with the onset of various brain disorders.

Gestational 1-nitropyrene exposure causes anxiety-like behavior partially by altering hippocampal epigenetic reprogramming of synaptic plasticity in male adult offspring.

1-Nitropyrene (1-NP), a typical nitro-polycyclic aromatic hydrocarbon, is a developmental toxicant. This study was to evaluate gestational 1-NP-induced anxiety-like behavior in male adult offspring. Pregnant mice were orally administered to 1-NP daily throughout pregnancy. Anxiety-like behaviors, as determined by Elevated Plus-Maze (EPM) and Open-Field Test (OFT), were showed in male adult offspring whose mothers were exposed to 1-NP. Gestational 1-NP exposure reduced dendritic arborization, dendritic length and dendritic spine density in ventral hippocampus of male adult offspring. Additional experiments showed that gephyrin, an inhibitory synaptic marker, was reduced in fetal forebrain and hippocampus in male adult offspring. Nrg1 and Erbb4, two gephyrin-related genes, were reduced in 1-NP-exposed fetuses. Accordingly, 5hmC contents in two CpG sites (32008909 and 32009239) of Nrg1 gene and three CpG sites (69107743, 69107866 and 69107899) of Erbb4 gene were decreased in 1-NP-exposed fetuses. Mechanistically, ten-eleven translocation (TET) activity and alpha-ketoglutarate (α-KG) content were decreased in 1-NP-exposed fetal forebrain. Supplementation with α-KG alleviated 1-NP-induced downregulation of gephyrin-related genes, prevented hippocampal synaptic damage, and improved anxiety-like behavior in male adult offspring. These results indicate that early-life 1-NP exposure causes anxiety-like behavior in male adulthood partially by altering hippocampal epigenetic reprogramming of synaptic plasticity.

Triptolide injection reduces Alzheimer's disease-like pathology in mice.

Triptolide is an epoxidized diterpene lactone isolated from Tripterygium wilfordii. Studies have shown that triptolide exerts organ-protective effects. However, it remains unknown whether triptolide improves Alzheimer's disease (AD)-like presentations. Thirty healthy 8-week-old male C57BL/6J mice were randomly divided into control (n = 10), model (n = 10), and triptolide (n = 10) groups. Amyloid-ß (Aß)42 was injected bilaterally into the ventricles of mice in the model group. Triptolide was injected intraperitoneally daily after injecting Aß42 (a total of 30 days) in the triptolide group. Learning and memory were tested using the Morris water maze test. The deposition of Aß42 in the hippocampus was detected using immunohistochemical staining. In the hippocampus, three synaptic-associated proteins-gephyrin, collybistin, and GABRA1 -were detected by western blotting. Furthermore, we used ELISA to detect proinflammatory cytokines, including TNF-α and IL-1ß, in the blood and hippocampus. Moreover, superoxide dismutase (SOD), malondialdehyde (MDA), and GSH levels were measured using the corresponding kits. We found that triptolide improved spatial learning and memory in AD-like mice. Additionally, triptolide maintained the expression of gephyrin, collybistin, and GABRA1 and reduced Aß in these mice. Additionally, triptolide reduced the expression of inflammatory cytokines and decreased oxidative damage in AD-like mice. Our study suggests that triptolide attenuates AD-like changes in the mouse brain.

Automated Image Analysis Reveals Different Localization of Synaptic Gephyrin C4 Splice Variants.

Postsynaptic scaffolding proteins function as central organization hubs, ensuring the synaptic localization of neurotransmitter receptors, trans-synaptic adhesion proteins, and signaling molecules. Gephyrin is the major postsynaptic scaffolding protein at glycinergic and a subset of GABAergic inhibitory synapses. In contrast to cells outside the CNS, where one gephyrin isoform is predominantly expressed, neurons express different splice variants. In this study, we characterized the expression and scaffolding of neuronal gephyrin isoforms differing in the inclusion of the C4 cassettes located in the central C-domain. In hippocampal and cortical neuronal populations, gephyrin P1, lacking additional cassettes, is the most abundantly expressed isoform. In addition, alternative splicing generated isoforms carrying predominantly C4a, and minor amounts of C4c or C4d cassettes. We detected no striking difference in C4 isoform expression between different neuron types and a single neuron can likely express all C4 isoforms. To avoid the cytosolic aggregates that are commonly observed upon exogenous gephyrin expression, we used adeno-associated virus (AAV)-mediated expression to analyze the scaffolding behavior of individual C4 isoforms in murine dissociated hippocampal glutamatergic neurons. While all isoforms showed similar clustering at GABAergic synapses, a thorough quantitative analysis revealed localization differences for the C4c isoform (also known as P2). Specifically, synaptic C4c isoform clusters showed a more distal dendritic localization and reduced occurrence at P1-predominating synapses. Additionally, inhibitory currents displayed faster decay kinetics in the presence of gephyrin C4c compared with P1. Therefore, inhibitory synapse heterogeneity may be influenced, at least in part, by mechanisms relating to C4 cassette splicing.

Dually innervated dendritic spines develop in the absence of excitatory activity and resist plasticity through tonic inhibitory crosstalk.

Dendritic spines can be directly connected to both inhibitory and excitatory presynaptic terminals, resulting in nanometer-scale proximity of opposing synaptic functions. While dually innervated spines (DiSs) are observed throughout the central nervous system, their developmental timeline and functional properties remain uncharacterized. Here we used a combination of serial section electron microscopy, live imaging, and local synapse activity manipulations to investigate DiS development and function in rodent hippocampus. Dual innervation occurred early in development, even on spines where the excitatory input was locally silenced. Synaptic NMDA receptor currents were selectively reduced at DiSs through tonic GABAB receptor signaling. Accordingly, spine enlargement normally associated with long-term potentiation on singly innervated spines (SiSs) was blocked at DiSs. Silencing somatostatin interneurons or pharmacologically blocking GABABRs restored NMDA receptor function and structural plasticity to levels comparable to neighboring SiSs. Thus, hippocampal DiSs are stable structures where function and plasticity are potently regulated by nanometer-scale GABAergic signaling.

Correlated Expression of the Opsin Retrogene LWS-R and its Host Gene in Two Poeciliid Fishes.

The important role of retrogenes in genome evolution and species differentiation is becoming increasingly accepted. One synapomorphy among cyprinodontoid fish is a retrotransposed version of a long-wavelength sensitive (LWS) opsin gene, LWS-R, within an intron of the gephyrin (GPHN) gene. These two genes display opposing orientations. It had been speculated that LWS-R hijacks the cis-regulatory elements of GPHN for transcription, but whether their expression is correlated had remained unclear. Here, in silico predictions identified putative promoters upstream of the translation start site of LWS-R, indicating that its transcription is driven by its own promoter rather than by the GPHN promoter. However, consistent expression ratios of LWS-R:GPHN in the eyeball and brain of fishes indicate that the respective gene transcriptions are correlated. Co-expression is potentially modulated by histone exchange during GPHN transcription. Two isoforms were detected in this study, i.e., intron-free and intron-retaining. Intron-free LWS-R was only expressed in the eyeball of fishes, whereas intron-retaining LWS-R occurred in both eyeball and brain. Expression of vision-associated LWS-R beyond the eyeball supports that it is co-expressed with more ubiquitous GPHN.

TFG regulates secretory and endosomal sorting pathways in neurons to promote their activity and maintenance.

Molecular pathways that intrinsically regulate neuronal maintenance are poorly understood, but rare pathogenic mutations that underlie neurodegenerative disease can offer important insights into the mechanisms that facilitate lifelong neuronal function. Here, we leverage a rat model to demonstrate directly that the TFG p.R106C variant implicated previously in complicated forms of hereditary spastic paraplegia (HSP) underlies progressive spastic paraparesis with accompanying ventriculomegaly and thinning of the corpus callosum, consistent with disease phenotypes identified in adolescent patients. Analyses of primary cortical neurons obtained from CRISPR-Cas9-edited animals reveal a kinetic delay in biosynthetic secretory protein transport from the endoplasmic reticulum (ER), in agreement with prior induced pluripotent stem cell-based studies. Moreover, we identify an unexpected role for TFG in the trafficking of Rab4A-positive recycling endosomes specifically within axons and dendrites. Impaired TFG function compromises the transport of at least a subset of endosomal cargoes, which we show results in down-regulated inhibitory receptor signaling that may contribute to excitation-inhibition imbalances. In contrast, the morphology and trafficking of other organelles, including mitochondria and lysosomes, are unaffected by the TFG p.R106C mutation. Our findings demonstrate a multifaceted role for TFG in secretory and endosomal protein sorting that is unique to cells of the central nervous system and highlight the importance of these pathways to maintenance of corticospinal tract motor neurons.

The History of the Molybdenum Cofactor-A Personal View.

The transition element molybdenum (Mo) is an essential micronutrient for plants, animals, and microorganisms, where it forms part of the active center of Mo enzymes. To gain biological activity in the cell, Mo has to be complexed by a pterin scaffold to form the molybdenum cofactor (Moco). Mo enzymes and Moco are found in all kingdoms of life, where they perform vital transformations in the metabolism of nitrogen, sulfur, and carbon compounds. In this review, I recall the history of Moco in a personal view, starting with the genetics of Moco in the 1960s and 1970s, followed by Moco biochemistry and the description of its chemical structure in the 1980s. When I review the elucidation of Moco biosynthesis in the 1990s and the early 2000s, I do it mainly for eukaryotes, as I worked with plants, human cells, and filamentous fungi. Finally, I briefly touch upon human Moco deficiency and whether there is life without Moco.

Differential modulation of collybistin conformational dynamics by the closely related GTPases Cdc42 and TC10.

Interneuronal synaptic transmission relies on the proper spatial organization of presynaptic neurotransmitter release and its reception on the postsynaptic side by cognate neurotransmitter receptors. Neurotransmitter receptors are incorporated into and arranged within the plasma membrane with the assistance of scaffolding and adaptor proteins. At inhibitory GABAergic postsynapses, collybistin, a neuronal adaptor protein, recruits the scaffolding protein gephyrin and interacts with various neuronal factors including cell adhesion proteins of the neuroligin family, the GABA A receptor α2-subunit and the closely related small GTPases Cdc42 and TC10 (RhoQ). Most collybistin splice variants harbor an N-terminal SH3 domain and exist in an autoinhibited/closed state. Cdc42 and TC10, despite sharing 67.4% amino acid sequence identity, interact differently with collybistin. Here, we delineate the molecular basis of the collybistin conformational activation induced by TC10 with the aid of recently developed collybistin FRET sensors. Time-resolved fluorescence-based FRET measurements reveal that TC10 binds to closed/inactive collybistin leading to relief of its autoinhibition, contrary to Cdc42, which only interacts with collybistin when forced into an open state by the introduction of mutations destabilizing the closed state of collybistin. Taken together, our data describe a TC10-driven signaling mechanism in which collybistin switches from its autoinhibited closed state to an open/active state.

Generational synaptic functions of GABAA receptor ß3 subunit deteriorations in an animal model of social deficit.

BACKGROUND: Disruption of normal brain development is implicated in numerous psychiatric disorders with neurodevelopmental origins, including autism spectrum disorder (ASD). Widespread abnormalities in brain structure and functions caused by dysregulations of neurodevelopmental processes has been recently shown to exert adverse effects across generations. An imbalance between excitatory/inhibitory (E/I) transmission is the putative hypothesis of ASD pathogenesis, supporting by the specific implications of inhibitory γ-aminobutyric acid (GABA)ergic system in autistic individuals and animal models of ASD. However, the contribution of GABAergic system in the neuropathophysiology across generations of ASD is still unknown. Here, we uncover profound alterations in the expression and function of GABAA receptors (GABAARs) in the amygdala across generations of the VPA-induced animal model of ASD. METHODS: The F2 generation was produced by mating an F1 VPA-induced male offspring with naïve females after a single injection of VPA on embryonic day (E12.5) in F0. Autism-like behaviors were assessed by animal behavior tests. Expression and functional properties of GABAARs and related proteins were examined by using western blotting and electrophysiological techniques. RESULTS: Social deficit, repetitive behavior, and emotional comorbidities were demonstrated across two generations of the VPA-induced offspring. Decreased synaptic GABAAR and gephyrin levels, and inhibitory transmission were found in the amygdala from two generations of the VPA-induced offspring with greater reductions in the F2 generation. Weaker association of gephyrin with GABAAR was shown in the F2 generation than the F1 generation. Moreover, dysregulated NMDA-induced enhancements of gephyrin and GABAAR at the synapse in the VPA-induced offspring was worsened in the F2 generation than the F1 generation. Elevated glutamatergic modifications were additionally shown across generations of the VPA-induced offspring without generation difference. CONCLUSIONS: Taken together, these findings revealed the E/I synaptic abnormalities in the amygdala from two generations of the VPA-induced offspring with GABAergic deteriorations in the F2 generation, suggesting a potential therapeutic role of the GABAergic system to generational pathophysiology of ASD.