Crispr/Cas9-Mediated Loss Of Vesicular Glutamate Transporter In Serotonin Neurons Of The Dorsal Raphe Nucleus Leads To Synaptic Changes And Anxiety-Like Behaviors

Vesicular glutamate transporter 3 (VGluT3) is expressed in a large subset of serotonin (5-HT) neurons of the dorsal raphe nucleus (DRN), suggesting a potential for glutamate co-transmission. Although VGluT3 has been implicated in the physiology of several non-glutamatergic neuronal populations, its specific role in the organization and function of 5-HT axons remains unclear. Here, we used CRISPR-Cas9 mediated knockdown and viral overexpression of VGluT3 in DRN 5-HT neurons of adult mice to assess its contribution to synaptic architecture in the lateral hypothalamus (LHA) and to 5-HT-related behaviors. VGluT3 depletion did not significantly alter synaptic incidence or organization of 5-HT DRN terminals in the LHA. In contrast, VGluT3 overexpression increased the proportion of asymmetric synapses without changing the overall synaptic incidence. In behavioral assays, VGluT3 depletion impaired motor coordination and increased anxiety-like, repetitive, and social behavior, whereas VGluT3 overexpression selectively reduced repetitive behavior. Basal locomotion and depressive-like behaviors were unchanged by either manipulation. Together, these findings indicate that VGluT3 modulates both the structural organization and behavioral output of DRN 5-HT neurons, supporting a modulatory role for VGluT3-dependent signaling within 5-HT circuits.