Serotonin receptor 4 in the ventral CA3 regulates agomelatine-induced anxiolytic-like behaviors

Agomelatine, an atypical antidepressant, offers combined effects of sleep regulation, antidepressant action, and anxiolysis, making it particularly suitable for patients with insomnia and mood disorders. In this study, we investigated the mechanisms underlying the anxiolytic effects of agomelatine in mice. Mice were subjected to chronic restraint stress (CRS) for 14 consecutive days. The mice were treated with agomelatine (10 mg·kg^-1·d^-1, i.p.) for 30 days. Afterward, anxiety-related behavioral tests (OFT, EPM, TST, and FST) or c-Fos immunohistochemical analysis were conducted. The results from c-Fos immunofluorescence combined with in vivo two-photon (2P) calcium imaging demonstrated that the neuronal activity of pyramidal neurons in the ventral CA3 (vCA3) region of the hippocampus was critical for both the expression of anxiety-like behaviors and the anxiolytic effects of agomelatine. Transcriptome sequencing analysis revealed that agomelatine specifically downregulated 5-HTR4 expression and its downstream signaling pathway in the vCA3, but not in the dorsal CA3 (dCA3). Using CRISPR-SpCas9 technology to knock out 5-HTR4 in the vCA3 resulted in increased anxiety-like behaviors in mice, highlighting the essential role of 5-HTR4 in the anxiolytic effects of agomelatine. Together, these results suggest that agomelatine's anxiolytic effects are closely associated with the suppression of 5-HTR4 expression in the vCA3, along with modulation of neuronal activity and GPCR signaling. This study not only uncovers the molecular mechanisms underlying agomelatine's anxiolytic mechanism but also identifies 5-HTR4 as a potential target for anxiety disorders.