A New Insight into the Role of CART Peptide in Serotonergic Function and Anxiety

Cocaine- and amphetamine-regulated transcript (CART) peptide has been implicated in stress-related behaviors that are regulated by central serotonergic (5-HT) systems in the dorsal raphe nucleus (DRN). Here, we aimed to investigate the interaction between CART and DRN 5-HTergic systems after initially observing CART axonal terminals in the DRN. We found that microinfusion of CART peptide _(55-102) into the DRN-induced anxiogenic effects in male C57BL/6J mice, while central administration of CART reduced c-Fos in 5-HT^DRN neurons. This inhibitory effect of exogenous CART on 5-HT^DRN activity and local 5-HT release was also demonstrated via in vivo fiber photometry coupled with calcium and 5-HT biosensors. CART inputs to the DRN were observed in various subcortical nuclei, but only those in the centrally projecting Edinger-Westphal nucleus (EWcp) were highly responsive to stress. Chemogenetic activation of these DRN-projecting CART^EWcp neurons recapitulated the effects of intra-DRN CART infusion on anxiety-like behavior in males, but not in females, suggesting a sex-specific role for this pathway. Interestingly, CART^EWcp projections to the DRN made direct synaptic contact primarily with non-5-HT neurons, which were also found to express putative CART receptors. Furthermore, chemogenetic stimulation of this CART^EWcp→DRN pathway inhibited 5-HT neurons while increasing activity in local GABAergic neurons. In summary, this study establishes for the first time a neuromodulatory role for CART^EWcp neurons in 5-HT^DRN neurotransmission and suggests that CART may drive anxiety-like behavior by promoting feedforward inhibition of 5-HT neurons.