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.

Diverse neurons in the parabrachial nucleus (PB) communicate with widespread brain regions. Despite evidence linking them to a variety of homeostatic functions, it remains difficult to determine which PB neurons influence which functions because their subpopulations intermingle extensively. An improved framework for identifying these intermingled subpopulations would help advance our understanding of neural circuit functions linked to this region. Here, we present the foundation of a developmental-genetic ontology that classifies PB neurons based on their intrinsic, molecular features. By combining transcription factor labeling with Cre fate-mapping, we find that the PB is a blend of two, developmentally distinct macropopulations of glutamatergic neurons. Neurons in the first macropopulation express Lmx1b (and, to a lesser extent, Lmx1a) and are mutually exclusive with those in a second macropopulation, which derive from precursors expressing Atoh1. This second, Atoh1-derived macropopulation includes many Foxp2-expressing neurons, but Foxp2 also identifies a subset of Lmx1b-expressing neurons in the Kölliker-Fuse nucleus (KF) and a population of GABAergic neurons ventrolateral to the PB ("caudal KF"). Immediately ventral to the PB, Phox2b-expressing glutamatergic neurons (some coexpressing Lmx1b) occupy the KF, supratrigeminal nucleus, and reticular formation. We show that this molecular framework organizes subsidiary patterns of adult gene expression (including Satb2, Calca, Grp, and Pdyn) and predicts output projections to the amygdala (Lmx1b), hypothalamus (Atoh1), and hindbrain (Phox2b/Lmx1b). Using this molecular ontology to organize, interpret, and communicate PB-related information could accelerate the translation of experimental findings from animal models to human patients.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

The nucleus of the solitary tract (NTS) regulates life-sustaining functions ranging from appetite and digestion to heart rate and breathing. It is also the brain's primary sensory nucleus for visceral sensations relevant to symptoms in medical and psychiatric disorders. To better understand which neurons may exert top-down control over the NTS, here we provide a brain-wide map of all neurons that project axons directly to the caudal, viscerosensory NTS, focusing on a medial subregion with aldosterone-sensitive HSD2 neurons. Injecting an axonal tracer (cholera toxin b) into the NTS produces a similar pattern of retrograde labeling in rats and mice. The paraventricular hypothalamic nucleus (PVH), lateral hypothalamic area, and central nucleus of the amygdala (CeA) contain the densest concentrations of NTS-projecting neurons. PVH afferents are glutamatergic (express Slc17a6/Vglut2) and are distinct from neuroendocrine PVH neurons. CeA afferents are GABAergic (express Slc32a1/Vgat) and are distributed largely in the medial CeA subdivision. Other retrogradely labeled neurons are located in a variety of brain regions, including the cerebral cortex (insular and infralimbic areas), bed nucleus of the stria terminalis, periaqueductal gray, Barrington's nucleus, Kölliker-Fuse nucleus, hindbrain reticular formation, and rostral NTS. Similar patterns of retrograde labeling result from tracer injections into different NTS subdivisions, with dual retrograde tracing revealing that many afferent neurons project axon collaterals to both the lateral and medial NTS subdivisions. This information provides a roadmap for studying descending axonal projections that may influence visceromotor systems and visceral "mind-body" symptoms.

Neuropeptide S (NPS) increases wakefulness. A small number of neurons in the brainstem express Nps. These neurons are located in or near the parabrachial nucleus (PB), but we know very little about their ontogeny, connectivity, and function. To identify Nps-expressing neurons within the molecular framework of the PB region, we used in situ hybridization, immunofluorescence, and Cre-reporter labeling in mice. The primary concentration of Nps-expressing neurons borders the lateral lemniscus at far-rostral levels of the lateral PB. Caudal to this main cluster, Nps-expressing neurons scatter through the PB and form a secondary concentration medial to the locus coeruleus (LC). Most Nps-expressing neurons in the PB region are Atoh1-derived, Foxp2-expressing, and mutually exclusive with neurons expressing Calca or Lmx1b. Among Foxp2-expressing PB neurons, those expressing Nps are distinct from intermingled subsets expressing Cck or Pdyn. Examining Nps Cre-reporter expression throughout the brain identified novel populations of neurons in the nucleus incertus, anterior hypothalamus, and lateral habenula. This information will help focus experimental questions about the connectivity and function of NPS neurons.