Pharmacological modulation of GABAergic function and behavior in FMR1 knockout mice

Background: Autism spectrum disorders (ASD) are highly heterogeneous neurodevelopmental conditions characterized by deficits in social communication and the presence of restricted, repetitive behaviors. Dysregulation of the GABAergic system, the primary inhibitory neurotransmitter network in the CNS is strongly implicated in ASD pathophysiology and contributes to the manifestation of core ASD phenotypes. This association is supported by evidence from genetic and neuroimaging studies, as well as postmortem analyses, which have revealed abnormalities in GABA receptor expression and GABA synthesis pathways. The FMR1 knockout mouse is a widely used preclinical model of ASD that exhibits core ASD-like behaviors alongside pronounced GABAergic system alterations, including dysbalance in excitatory–inhibitory balance.

Aim: The aim of the current study was to evaluate the effects of GABA-A receptor agonist Isoguvacine and cap-dependent translational inhibitor 4EGI-1 on behavioral outcomes associated with an ASD-like phenotype, and their effects on the GABAergic system in the prefrontal cortex of FMR1 knockout mice.

Methods: The experiments were performed from postnatal day PND 7 on male C57Bl/6 Fmr1 knock-out (k/o; n=6) and C57Bl/6 wild-type control mice (WT; n=6) mice. Animals were treated i.p. with Isoguvacine (Isog; 2 mg/kg) as peripherally restricted GABA-A receptor agonist, 4EGI (50 mg/kg) as eIF4E inhibitor or saline for 27 consecutive days followed by behavioural evaluation (open field test, zero-maze test, and three-chamber test). Mice were sacrificed on PND 47 and prefrontal cortices were dissected for subsequent Western blot analysis of GABA-A​ receptor and GAD65/67 protein levels.

Results: FMR1 k/o mice demonstrated increased locomotor activity in the open field test, with higher total distance traveled, mean and maximal speed, while time in the center remained unchanged. An increased number of entries in the closed arms and elevated total distance moved was also observed in FMR1 k/o mice in the elevated zero-maze. In the three-chamber test, FMR1 k/o mice exhibited impaired social preference, whereas wild-type mice displayed the expected hierarchy of social preference, reflecting motivation and social cognition. These behavioral alteration of the FRM1 k/o mice were accompanied by elevated GAD65/67 protein levels in the prefrontal cortex, however no changes were detected in overall GABA-A​ receptor protein expression. Neither isoguvacine nor 4EGI produced significant behavioral effects in FMR1 knockout mice, although 4EGI per se increased locomotor activity, indicating a genotype-independent behavioral effect. Although total GABA-A receptor protein levels remained unchanged, alterations in specific subunits may underlie functional impairments and warrant further investigation. The limitations of this study include the relatively small number of mice used for behavioural analyses and the absence of assessment of treatment effects in both sexes. Conclusion: Overall, these findings highlighting GABAergic system dysregulation in FMR1 knockout mice, and although the current study did not demonstrate significant pharmacological effects of Isoguvacine or 4EGI, continued investigation into novel compounds remains crucial to identify potential therapeutic candidates for the treatment of ASD and elucidate underlying mechanisms.

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