Neurochemical phenotype of relaxin family peptide receptor-3 (RXFP3) lateral hypothalamus/zona incerta cells

The relaxin-3/relaxin family peptide receptor 3 (RXFP3) neuropeptidergic system is emerging as a potential target for treating various neuropsychiatric diseases, particularly those involving dysregulated stress and arousal. RXFP3 is abundantly expressed in several hypothalamic nuclei, and in the zona incerta (ZI). These regions play a central role in the regulation of stress and arousal, however the function of relaxin-3/RXFP3 within these circuits is unknown. The purpose of this study was to begin characterising this function by describing the distribution and genetic signature of neurons that express RXFP3. We used RNAscope fluorescent in situ hybridisation to characterise the spatial expression pattern and neurochemical phenotype of cells expressing Rxfp3 mRNA throughout the mouse lateral hypothalamus (LH) and ZI. We found that Rxfp3 is expressed across the rostrocaudal extent of both the LH and ZI and follows a parabolic pattern of expression, peaking in more rostral areas of each nucleus. Neurochemical phenotyping of Rxfp3+ cells with Gad1, Slc17a6 (vGlut2), Pvalb, Th, and Sst showed that LH/ZI Rxfp3+ cells co-express each marker to varying extents, generally proportional to their overall abundance within each structure. Furthermore, LH/ZI Rxfp3+ cells overlapped with several known populations involved in various facets of fear learning and defensive behaviour, such as the dopaminergic A13 group, somatostatin-expressing rostral ZI neurons, and glutamatergic LH neurons. The neurochemical diversity of these neurons may reflect the overall role of both the LH and ZI as global regulators of behaviour and the role of relaxin-3/RXFP3 signalling in modulating high-vigilance states.