Sex-related differences in the behavioral phenotype and associated impairments in the dopaminergic, endocannabinoid and PPAR alpha systems in a rat model of autism

ASD is a group of neurodevelopmental conditions characterized by persistent impairments in social interaction and communication, restricted and repetitive patterns of behavior. Although ASD is diagnosed more frequently in males, growing evidence suggests that females may be underdiagnosed due to sex-related differences in symptoms presentation. Nevertheless, most preclinical studies still focus primarily on males. Prenatal exposure to valproic acid is a well-established environmental risk factor for ASD and represents a validated animal model that mimics several behavioral and neurobiological features of the disorder. The first aim of this thesis was to characterize autistic-like phenotypes in both male and female rats prenatally exposed to VPA, from early postnatal life to young adulthood, to identify early alterations and possible sex-dependent differences. The second major aim was to test a novel therapeutic strategy targeting deficits in social motivation. Based on the hypothesis that ASD may involve an early impairment in the perception and processing of social reward, fenofibrate (FBR), a clinically approved PPARα agonist, was administered for 4 weeks starting from weaning. In parallel, this thesis aimed to investigate whether prenatal VPA exposure and FBR treatment modulate dopaminergic and endocannabinoid signaling in reward-related regions by analyzing DARPP-32 phosphorylation, endocannabinoid-related enzymes and receptors, and N-acylethanolamine levels. The VPA model was induced by a single i.p. injection of VPA to pregnant dams at gestational day 12.5. Offspring were evaluated from early postnatal life to young adulthood. Early neurodevelopmental milestones, sensorimotor development, olfactory discrimination and reciprocal social behavior were assessed between postnatal day 7 and 28. At weaning (PND21), male and female rats were assigned to four experimental groups: Saline-exposed rats treated with standard diet (Saline-SD) or FBR-enriched diet (Saline-FBR), and VPA-exposed rats treated with standard diet (VPA-SD) or FBR-enriched diet (VPA-FBR). In young adulthood, animals were tested for social interaction, social novelty, social reward by using the CPP protocol, and repetitive behaviors. Neurochemical analyses were performed in the mPFC and NAc. Dopaminergic activation was assessed by DARPP-32 phosphorylation, and endocannabinoid system (ECS) markers, such as FAAH, NAPE-PLD, CB1R, and PPARα, were analyzed by immunoblotting, while endocannabinoid-related lipids (AEA, OEA, and PEA) were quantified by UHPLC/MS-MS. Prenatal VPA exposure induced early delays in sensorimotor maturation and alterations in olfactory discrimination in both sexes. From the second postnatal week, VPA-exposed pups displayed early impairments in social behavior, including increased latency to initiate social interaction and reduced social play, suggesting an early reduction in social interest. These behavioral alterations were associated with age-dependent modifications in endocannabinoid and PPARα signaling in mPFC and NAc, suggesting earl dysfunction of motivational circuits. At young adulthood, male rats prenatally exposed to VPA showed marked social deficits, reduced preference for social novelty, and increased repetitive behaviors, as well as reduced sensibility to the rewarding value of social interaction. In female rats exposed to VPA the expression of ASD-like behavioral modifications were minimal. Notably, chronic FBR treatment ameliorated social and repetitive behavioral alterations, with particularly robust effects in male rats. At the neurobiological level, prenatal VPA exposure was associated with dysregulation of dopaminergic and endocannabinoid signaling in the mPFC and NAc. In males, reduced DARPP-32 phosphorylation indicated a blunted dopaminergic response to social stimulus, which was restored by FBR. Moreover, VPA increased FAAH expression and reduced anandamide levels, suggesting enhanced degradation of endocannabinoids, whereas FBR normalized FAAH expression and selectively increased PEA levels in the NAc. In females, VPA-induced changes were more limited and region-specific, and were not significantly counteracted by FBR. In addition, prenatal VPA exposure produced region-specific alterations in PPARα expression, supporting a potential involvement of this receptor in the regulation of motivational and reward processes. Overall, these findings show that prenatal VPA exposure induces early onset of social impairments associated with developmental dysregulation of dopaminergic and endocannabinoid signaling, selectively in male rats. Importantly, long-term FBR treatment improved social motivation and reduced repetitive behaviors, supporting the potential of FBR as a promising pharmacological approach to target social deficits associated with ASD.