AIMS: Anxiety and depression affect 35-50% of patients with Parkinson's disease (PD). In the serotonin (5-HT) system alpha-synuclein (a-Syn) aggregates are present and functional changes are involved in the PD prodromal phase and contribute to non-motor symptoms​. Using a mouse model of depression and synucleinopathy in raphe 5-HT neurons, we aim to evaluate: 1- behavioral phenotype, 2- synaptic changes in prefrontal cortex and striatum, and 3- possible sex-based differences. METHODS: Recombinant adeno-associated viral vectors (AAV2/5) encoding wild-type human a-Syn (h-a-Syn) were infused in raphe nuclei of male/female mice. The behavioral phenotype was assessed at 4- and 8-weeks post-infusion. Confocal microscopy was used to examine cortical and striatal brain sections immunostained for synapse-related proteins. Fluorescence activated synaptosome sorting (FASS) was performed to obtain purified and enriched cortical and striatal synaptosomes from VGLUT1-Venus mice infused with AAV2/5, in which 5-HT markers were evaluated in association with both excitatory and inhibitory partners. Statistical significance was determined by t-tests or one-way ANOVA. RESULTS: Overexpression of raphe h-a-Syn induced a sex-dependent depressive-like phenotype and anxiety-like behavior in female, which was absent in male mice. We found a progressive increase in axonal swellings in the striatum and prefrontal cortex, where h-a-Syn colocalized with serotonin transporter (SERT). In parallel, we measured decreased microtubule-associated protein 2 (MAP2) and increased synaptic vesicle 2A (SV2A) and synaptophysin (SYP) protein levels in the same brain areas of 5-HT projection. CONCLUSIONS: Overexpression of h-a-Syn in the mouse 5-HT system induces a sex-specific behavioral phenotype and synaptic pathology in interconnected brain areas.