Genetically defined nucleus incertus neurons differ in connectivity and function

Abstract

The nucleus incertus (NI), an understudied hindbrain structure implicated in the stress response, arousal, and memory, is a major site for production of the neuropeptide relaxin-3. On the basis of goosecoid homeobox 2 (gsc2) expression, we identified a neuronal cluster that lies adjacent to relaxin 3a (rln3a) neurons in the zebrafish analogue of the NI. To delineate properties of the gsc2 and rln3a neurons, we used CRISPR/Cas9 targeted integration to drive gene expression in each group, and showed that they differ in afferent input, efferent connections and functional properties. gsc2 and rln3a NI neurons innervate distinct subregions of the interpeduncular nucleus (IPN), but only gsc2 neurons receive reciprocal innervation from the IPN. Whereas gsc2 neurons are activated by electric shock, rln3a neurons exhibit spontaneous fluctuations in calcium signaling and regulate locomotor activity. Our findings define heterogeneous neurons in the NI and provide new tools to probe its diverse functions.