Dataset Information

Generational synaptic functions of GABA_A receptor β3 subunit deteriorations in an animal model of social deficit.

ABSTRACT:

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 GABA_A receptors (GABA_ARs) 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 GABA_ARs 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 GABA_AR 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 GABA_AR was shown in the F2 generation than the F1 generation. Moreover, dysregulated NMDA-induced enhancements of gephyrin and GABA_AR 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.

SUBMITTER: Chu MC

PROVIDER: S-EPMC9277936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Key pointsCurrent views suggest ?2 subunit-containing GABAA receptors mediate phasic IPSCs while extrasynaptic ? subunits mediate diffusional IPSCs and tonic current. We have re-examined the roles of the two receptor populations using mice with picrotoxin resistance engineered into receptors containing the ? subunit. Using pharmacological separation, we find that in general ? and ? IPSCs are modulated in parallel by manipulations of transmitter output and diffusion, with evidence favouring modestly more diffusional contribution to ? IPSCs. Our findings also reveal that spontaneous ? IPSCs are mainly driven by channel deactivation, rather than by diffusion of GABA. Understanding the functional contributions of the two receptor classes may help us understand the actions of drug therapies with selective effects on one population over the other.AbstractGABAA receptors mediate transmission throughout the central nervous system and typically contain a ? subunit (? receptors) or a ?2 subunit (?2 receptors). ? IPSCs decay slower than ?2 IPSCs, but the reasons are unclear. Transmitter diffusion, rebinding, or slow deactivation kinetics of channels are candidates. We used gene editing to confer picrotoxin resistance on ? receptors in mice, then pharmacologically isolated ? receptors in mouse dentate granule cells to explore IPSCs. ?2 and ? components of IPSCs were modulated similarly by presynaptic manipulations and manipulations of transmitter lifetime, suggesting that GABA release recruits ? receptors proportionally to ?2 receptors. ? IPSCs showed more sensitivity to altered transmitter release and to a rapidly dissociating antagonist, suggesting an additional spillover contribution. Reducing GABA diffusion with 5% dextran increased the peak amplitude and decreased the decay of evoked ? IPSCs but had no effect on ? or dual-component (mainly ?2-driven) spontaneous IPSCs, suggesting that GABA actions can be local for both receptor types. Rapid application of varied [GABA] onto nucleated patches from dentate granule cells demonstrated a deactivation rate of ? receptors similar to that of ? spontaneous IPSCs, consistent with the idea that deactivation and local GABA actions drive ? spontaneous IPSCs. Overall, our results indicate that ? IPSCs are activated by both synaptic and diffusional GABA. Our results are consistent with a functional relationship between ? and ?2 GABAA receptors akin to that of slow NMDA and fast AMPA EPSCs at glutamate synapses.

Project description:Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting γ2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between α2 and γ2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Additional time-series experiments revealed the gephyrin regulating kinase ERK was inactivated by DZP at multiple time points. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes.

Dataset Information

Generational synaptic functions of GABA_A receptor β3 subunit deteriorations in an animal model of social deficit.

Background

Methods

Results

Conclusions

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Dataset Information

Generational synaptic functions of GABAA receptor β3 subunit deteriorations in an animal model of social deficit.

Background

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Generational synaptic functions of GABA_A receptor β3 subunit deteriorations in an animal model of social deficit.