Afferent Projections to the Calca/CGRP-Expressing Parabrachial Neurons in Mice

The parabrachial nucleus (PB), located in the dorsolateral pons, contains primarily glutamatergic neurons that regulate responses to a variety of interoceptive and cutaneous sensory signals. One lateral PB subpopulation expresses the Calca gene, which codes for the neuropeptide calcitonin gene-related peptide (CGRP). These PB^{Calca /CGRP} neurons relay signals related to threatening stimuli such as hypercarbia, pain, and nausea, yet their inputs and their neurochemical identity are only partially understood. We mapped the afferent projections to the lateral part of the PB in mice using conventional cholera toxin B subunit (CTb) retrograde tracing and then used conditional rabies virus retrograde tracing to map monosynaptic inputs specifically targeting the PB^{Calca /CGRP} neurons. Using vesicular GABA (vGAT) and glutamate (vGLUT2) transporter reporter mice, we found that lateral PB neurons receive GABAergic afferents from regions such as the lateral part of the central nucleus of the amygdala, lateral dorsal subnucleus of the bed nucleus of the stria terminalis, substantia innominata, and ventrolateral periaqueductal gray. Additionally, they receive glutamatergic afferents from the infralimbic and insular cortex, paraventricular nucleus, parasubthalamic nucleus, trigeminal complex, medullary reticular nucleus, and nucleus of the solitary tract. Using anterograde tracing and confocal microscopy, we then identified close axonal appositions between these afferents and PB^{Calca /CGRP} neurons. Finally, we used channelrhodopsin-assisted circuit mapping and found that GABAergic neurons of the central nucleus of the amygdala directly inhibit the PB^{Calca /CGRP} neurons. These findings provide a comprehensive neuroanatomical framework for understanding the afferent projections regulating the PB^{Calca /CGRP} neurons.