@article {NasirovaENEURO.0272-20.2020, author = {Nailyam Nasirova and Lely A. Quina and Glenn Morton and Andrew Walker and Eric E. Turner}, title = {Mapping Cell Types and Efferent Pathways in the Ascending Relaxin-3 System of the Nucleus Incertus}, elocation-id = {ENEURO.0272-20.2020}, year = {2020}, doi = {10.1523/ENEURO.0272-20.2020}, publisher = {Society for Neuroscience}, abstract = {Relaxin-3 (Rln3) is an insulin-family peptide neurotransmitter expressed primarily in neurons of the nucleus incertus (NI) of the pontine tegmentum, with smaller populations located in the deep mesencephalon (DpMe) and periaqueductal gray (PAG). Here, we have used targeted recombination at the Rln3 gene locus to generate an Rln3Cre transgenic mouse line, and characterize the molecular identity and axonal projections of Rln3-expressing neurons. Expression of Cre recombinase in Rln3Cre mice, and the expression of Cre-mediated reporters, accurately reflect the expression of Rln3 mRNA in all brain regions. In the NI, Rln3 mRNA is expressed in a subset of a larger population of tegmental neurons that express the neuropeptide neuromedin-b (NMB). These Rln3-expressing and NMB-expressing neurons also express the GABAergic marker GAD2 but not the glutamatergic marker Slc17a6 (VGluT2). Cre-mediated anterograde tracing with adeno-associated viruses (AVVs) shows that the efferents of the Rln3-expressing neurons in the DpMe and PAG are largely confined to the brain regions in which they originate, while the NI-Rln3 neurons form an extensive ascending system innervating the limbic cortex, septum, hippocampus, and hypothalamus. Viral anterograde tracing also reveals the potential synaptic targets of NI-Rln3 neurons in several brain regions, and the distinct projections of Rln3-expressing and non-expressing neurons in the pontine tegmentum. Rabies virus (RV)-mediated transsynaptic retrograde tracing demonstrates a probable synaptic link between NI-Rln3 neurons and GABAergic neurons in the septum, with implications for the modulation of neural activity in the septo-hippocampal system. Together, these results form the basis for functional studies of the NI-Rln3 system.Significance Statement: Relaxin-3 (Rln3) is a peptide neurotransmitter expressed mainly in the nucleus incertus (NI) of the pons. Rln3 neurons project to the limbic cortex, septum, hippocampus, and hypothalamus, in a way that resembles the ascending brain systems expressing dopamine, serotonin, and norepinephrine. Roles in arousal and stress responses have been proposed for NI-Rln3 neurons, but their function is not well understood. We have defined the molecular signature of NI-Rln3 neurons in terms of the expression markers for GABA, the principal inhibitory neurotransmitter of the CNS, and neuromedin-b (NMB), another neuropeptide expressed in the pons. A transgenic mouse model for the Rln3 system has allowed the projections of the Rln3 neurons to be mapped in detail, and will facilitate functional studies of this pathway.}, URL = {https://www.eneuro.org/content/early/2020/10/14/ENEURO.0272-20.2020}, eprint = {https://www.eneuro.org/content/early/2020/10/14/ENEURO.0272-20.2020.full.pdf}, journal = {eNeuro} }