Investigating the Existence and Implications of a PACAP-mediated Circuit from the Medial Habenula (MHb) to the Interpeduncular Nucleus (IPN)

Chronic stress-induced psychiatric disorders, such as anxiety and depression, are some of the most widespread pathologies across the globe. Nevertheless, first-line treatments such as selective serotonin reuptake inhibitors (SSRIs) have limited efficacy. Recent evidence suggests that dysregulation of neuropeptidergic signaling pathways, particularly those involving pituitary adenylate cyclase-activating polypeptide (PACAP), may play a key role in the pathophysiology of anxiety and depression. This study investigates the existence and functional relevance of a novel PACAP-sensitive circuit extending from the bed nucleus of the stria terminalis (BNST) to the medial habenula (MHb), and from the MHb to the interpeduncular nucleus (IPN), a pathway hypothesized to mediate the generation of chronic stress-induced psychopathologies. To assess this circuitry, we conducted two experiments. In Experiment 1, PAC1-Ires-Cre mice received stereotaxic injections of a Cre-dependent AAV vector encoding mCherry into the MHb to label PAC1R-expressing neurons. Subsequent imaging revealed that these neurons terminate in the IPN, validating the existence of a PACAP-sensitive MHb-IPN projection. In Experiment 2, retrograde tracing with Fluoro-Gold infusions into the IPN of C57BL/6 mice was used to identify upstream afferents. While technical limitations affected signal detection in the MHb, a fluorescent signal detected in the lateral habenula (LHb) and rostromedial tegmental nucleus (RMTg) suggests anatomical overlap and possible off-target tracer diffusion. These findings support the existence of a BNST-MHb-IPN projection in which PACAP-secreting neurons in the BNST activate PAC1R-expressing and Substance P (SP) secreting neurons in the dorsal MHb, which in turn synapse onto the IPN. Dysregulation of this pathway likely contributes to the emergence of stress-induced anxious and depressive behavior. Given the downstream connectivity of the IPN with serotonergic nuclei such as the dorsal raphe, this pathway may represent the mechanism through which chronic stress disrupts systemic serotonergic tone and generates psychopathology. The anatomical and functional insights gained from this study emphasize the therapeutic potential of targeting PACAP and SP signaling within this circuitry to treat chronic stress-related disorders such as depression and anxiety.