Efferent projections of CGRP/Calca-expressing parabrachial neurons in mice

The parabrachial nucleus (PB) is composed of glutamatergic neurons at the midbrain-hindbrain junction. These neurons form many subpopulations, one of which expresses Calca, which encodes the neuropeptide calcitonin gene-related peptide (CGRP). This Calca-expressing subpopulation has been implicated in a variety of homeostatic functions, but the overall distribution of Calca-expressing neurons in this region remains unclear. Also, while previous studies in rats and mice have identified output projections from CGRP-immunoreactive or Calca-expressing neurons, we lack a comprehensive understanding of their efferent projections. We began by identifying neurons with Calca mRNA and CGRP immunoreactivity in and around the PB, including populations in the locus coeruleus and motor trigeminal nucleus. Calca-expressing neurons in the PB prominently express the mu opioid receptor (Oprm1) and are distinct from neighboring neurons that express Foxp2 and Pdyn. Next, we used Cre-dependent anterograde tracing with synaptophysin-mCherry to map the efferent projections of these neurons. Calca-expressing PB neurons heavily target subregions of the amygdala, bed nucleus of the stria terminalis, basal forebrain, thalamic intralaminar and ventral posterior parvicellular nuclei, and hindbrain, in different patterns depending on the injection site location within the PB region. Retrograde axonal tracing revealed that the previously unreported hindbrain projections arise from a rostral-ventral subset of CGRP/Calca neurons. Finally, we show that these efferent projections of Calca-expressing neurons are distinct from those of neighboring PB neurons that express Pdyn. This information provides a detailed neuroanatomical framework for interpreting experimental work involving CGRP/Calca-expressing neurons and opioid action in the PB region.