GHRELIN SIGNALLING MEDIATES SEX DIFFERENCES IN MIDBRAIN CONTROL OF BINGE DRINKING

Abstract Background The circuitry mediating excessive alcohol consumption includes the understudied central projecting Edinger-Westphal (EWcp); a structure dense in neuropeptide expression, including cocaine and amphetamine regulated transcript (CART). While studies have shown a critical role for this nucleus in alcohol consumption, few studies have interrogated the contributions of distinct EWcp populations. Aims We aimed to understand the contribution of peptidergic EWcp cells in alcohol consumption, and hypothesized sex differences may be present. Methods To examine a functional role of these cells in binge drinking we used chemogenetics to inhibit EWcppeptidergic cells in male and female CART-Cre mice. We next used RNAscope to examined the neurochemistry of EWcppeptidergic cells observing strong overlap with the ghrelin receptor (GHSR) and using whole cell patch clamp electrophysiology, we assessed the response of EW peptidergic cells to bath application of ghrelin in male and female mice. Next, we knocked down Ghsr expression non-specifically in the EWcp of male and female mice using a shRNA and specifically from peptidergic (CART-Cre) and glutamatergic (vGlut2-Cre) populations using Cre-dependent Ghsr-shRNA. Finally, given ghrelin has high constitutive activity, we assessed whether a ghrelin antagonist (JMV2959) or inverse agonist (LEAP2) could reduce binge drinking when administered directly within the EW nucleus. Results Chemogenetic inhibition of EWcppeptidergic cells had no effect on binge drinking, anxiety behaviour or other consummatory behaviours in male mice; however, a specific reduction in alcohol binge drinking was observed in female mice. Ghsr mRNA was strongly expressed in EWcppeptidergic, and to a lesser extent in EWcpglutamatergic cells. Further, bath application of ghrelin increased firing frequency in EWcppeptidergic cells in both male and female mice, however, the change in response was significantly higher in females. Systemic ghrelin administration increased binge drinking in female mice, which was reduced by chemogenetic inhibition of EWcppeptidergic cells. In addition, non-specific EWcp Ghsr-shRNA knockdown reduced binge drinking specifically in female, but not male mice compared to scram-ShRNA controls. Further in female mice, Ghsr-shRNA localised to EWcppeptidergic, but not EWcpvGlut2 cells reduced binge drinking. Finally, infusion of both a GHSR antagonist and inverse agonist directly within the EWcp reduced binge drinking in female mice. Conclusions Together, our results suggest the EWcp is a region mediating excessive alcohol bingeing through GHSR actions on peptidergic cells in female mice. Given the recent interest in GHSR1a inverse agonists for the treatment of alcohol use disorder, understanding the neural mechanism(s) underpinning how the ghrelin system mediates alcohol consumption are critical.