Relaxin-3/RXFP3 signalling control of alcohol and sucrose consumption: Studies in Rxfp3 knockout mice

© 2015 Dr. Andrew Walter WalkerRelaxin-3 is a highly conserved neuropeptide with putative roles in behavioural arousal, motivation and stress responses. Within the rodent brain, relaxin-3 is primarily expressed by neurons in the pontine nucleus incertus (NI) that project to a number of forebrain regions containing target neurons expressing its cognate Gprotein coupled receptor, relaxin-family peptide 3 receptor (RXFP3). Recent studies in rats have demonstrated that central injection of an RXFP3 antagonist reduces operant responding for alcohol and stress-induced reinstatement of alcohol-seeking; while in mice, similar pharmacological blockade of RXFP3 reduces the motivation to seek and consume food. The present studies used Rxfp3 gene deletion (knockout, KO) mice to further investigate the role of relaxin-3/RXFP3 signalling in alcohol- and sucrose-seeking. Firstly, due to potential interactions between RXFP3 signalling and GABAA receptor function, it was important to determine if Rxfp3 KO mice had altered sensitivity to low doses of ethanol, as this could confound subsequent testing. Automated locomotor cell testing revealed that Rxfp3 KO mice display a similar sensitivity to acute administration of low doses of ethanol as wild-type (WT) control mice. Their relative sensitivity to moderate-dose alcohol- and pentobarbital-induced ataxia was subsequently assessed using rotarod paradigms. However, these studies failed to yield consistent findings across multiple cohorts. In light of these findings, an alternative assay using a ‘DigiGait’ protocol was developed, which reliably detected consistent changes in gait in WT mice following moderate-dose alcohol treatment, which will allow a more accurate reassessment of motor performance in Rxfp3 KO mice. Secondly, the impact of Rxfp3 gene deletion on baseline and stress-related alcohol consumption was investigated. Male WT and Rxfp3 KO littermate mice were tested for baseline saccharin and alcohol consumption and preference over water in a continuous access two-bottle free-choice paradigm. Another cohort of mice was subjected to repeated restraint followed by swim stress to examine stress-related alcohol preference. Hepatic alcohol and aldehyde dehydrogenase activity was assessed in WT and Rxfp3 KO mice following chronic alcohol intake, and in naive controls. These studies revealed that WT and Rxfp3 KO mice had similar baseline saccharin and alcohol preference, and similar hepatic alcohol metabolism. However, Rxfp3 KO mice displayed stress-induced reductions in alcohol preference that were not observed in WT littermates. Notably, this phenotype, once established, persisted for at least six weeks after cessation of stress exposure. These findings suggest that in C57BL/6J background mice, relaxin-3/RXFP3 signalling is involved in maintaining high alcohol preference during and after stress, but does not appear to strongly regulate the primary reinforcing properties of alcohol. Finally, the relationship between motivated appetitive behaviours and relaxin- 3/RXFP3 signalling was further explored by examining the impact of Rxfp3 gene deletion on sucrose and alcohol self-administration and cue-induced reinstatement of sucrose- and alcohol-seeking. Acquisition and maintenance of sucrose/alcohol selfadministration was assessed in male WT and Rxfp3 KO littermate mice using fixedratio (FR) schedules of reinforcement. Mice were subsequently challenged with a progressive-ratio (PR) test to measure motivation and, following extinction training, re-exposed to reward-associated cues to evaluate reinstatement of active leverresponding. WT and Rxfp3 KO mice displayed similar acquisition of FR1 sucrose self-administration, but Rxfp3 KO mice responded less when the instrumental requirement was increased to FR3. These mice also demonstrated a lower breakpoint for sucrose-responding and attenuated cue-induced reinstatement of sucrose-seeking. Notably, no marked genotype differences in alcohol-responding were observed. These data suggest that in mice, endogenous relaxin-3/RXFP3 signalling promotes selfadministration of sucrose under high response requirements and cue-induced reinstatement of sucrose-seeking, but does not strongly regulate motivation to consume alcohol or alcohol-seeking behaviour. Together, these findings provide solid evidence that relaxin-3/RXFP3 signalling can increase motivational drive and regulate alcohol intake under conditions of neurogenic stress. Due to the importance of these processes in overall cognitive function and mental health, these studies represent an important contribution to the field, and provide a foundation for further studies to characterise the role of relaxin- 3/RXFP3 signalling in alcohol consumption, motivation and stress responses, and associated cellular and neural circuit mechanisms