Cholecystokinin upregulation in mPFC leads to social defeat-induced anxiety susceptibility in neuroligin 3 R451C knockin mice

Abstract

Background: Anxiety is a prevalent and clinically debilitating comorbidity in autism spectrum disorder (ASD), yet its underlying neurobiological mechanisms remain incompletely elucidated. This study aimed to investigate the role of dysregulated cholecystokinin (CCK) signaling within the medial prefrontal cortex (mPFC) in mediating stress-induced anxiety susceptibility in ASD. Methods: Using Neuroligin 3 R451C knockin (KI) mice, a genetically validated ASD model, we examined the developmental trajectory of CCK-expressing neurons in the mPFC through immunohistochemistry. Elevated plus maze tests were conducted to assess baseline anxiety and susceptibility to social defeat stress (SDS) across developmental stages. In vivo fiber photometry was employed to monitor SDS-induced mPFC CCK secretion dynamics. Functional manipulations included targeted knockdown and overexpression of Cck in mPFC excitatory neurons via viral-mediated gene transfer. Results: We demonstrate a developmental perturbation in CCK-expressing excitatory neurons within the mPFC: their density increases sharply in early postnatal life, stabilizes during adolescence, and typically declines in adulthood in wild-type (WT) mice, yet remains persistently elevated in adult KI counterparts. In line with this anomaly, adult WT mice exhibit greater resilience to social defeat stress (SDS) than their adolescent counterparts, whereas KI mice sustain heightened anxiety susceptibility throughout adulthood—despite showing no baseline anxiety. Remarkably, SDS elicits a rapid and exaggerated surge in mPFC CCK secretion in adult KI mice, whereas WT controls exhibit a delayed and blunted response. Targeted knockdown of Cck in mPFC excitatory neurons significantly ameliorates SDS-induced anxiety-like behaviors in adult KI mice, whereas Cck overexpression in adult WT animals recapitulates the anxiogenic phenotype. Limitations: All experiments were conducted in male mice. Given the well-documented sex differences in the prevalence of ASD and anxiety disorders, further studies are warranted to determine whether similar effects occur in females. Conclusions: These findings implicate dysregulated CCK signaling within the mPFC as a mechanistic substrate for stress-induced anxiety in ASD, and provide a neurobiological framework for understanding the heightened affective vulnerability associated with this condition.