Molecular and Anatomical Characterization of Parabrachial Neurons and Their Axonal Projections

The parabrachial nucleus (PBN) predominantly relays threatening signals and learned sensory cues that predict threats to forebrain regions. Viral-mediated expression of Cre-dependent effector genes in Cre-driver lines of mice has demonstrated that specific populations of PBN neurons are necessary and sufficient for learning about threats. However, these neurons account for only a fraction of the total population. To further our understanding of the complexity of the PBN neuronal populations, we used single-cell RNA-sequencing technologies, which revealed 21 clusters of neurons (19 glutamatergic, 2 GABAergic) in the PBN and neighboring regions. RNAscope HiPlex <em>in situ</em> hybridization located 12 of these clusters within subregions of the PBN. Viral expression of fluorescent proteins in 21 Cre-driver lines of mice was used to map their axonal projections throughout the brain; they constitute two pathways innervating distinct brain regions. These results are a resource for further interrogation of PBN functions.