Identification of MC4R-expressing Neuronal Populations with Projections to the Periaqueductal Gray

Background: More than half of obese individuals develop severe breathing disorders, characterized by a reduction in chemosensitivity. Yet, the neuronal mechanisms that link obesity to the control of breathing remain unclear. The periaqueductal gray (PAG) has been implicated in chemosensitivity, suggesting it may play a key role in this connection. We hypothesized that melanocortin-4 receptor (MC4R)-expressing neurons, which are critical regulators of body weight, project to the PAG and may contribute to its role in chemosensitivity regulation. Methods: We used transgenic mice and retrograde viral injections to determine the extent to which Melanocortin-4 Receptor (MC4R)-expressing populations project to the dorsal medial PAG (dmPAG). MC4R-cre mice were mated with a commercially available transgenic line (RC:FLTG mouse) which expresses TdTomato in the presence of flp-recombinase and GFP in the dual presence of flp- and cre-recombinase. The resulting mice had flp-recombinase stereotaxically delivered to the dmPAG via a retrograde AAV. After 5 weeks, brains were removed and later imaged with a Keyence Fluorescence microscope. Placement of virus into the dmPAG was verified by the presence of a fluorescent marker virus co-delivered with the retrograde AAV. Using this approach (n = 2 brains), red fluorescence identified cells projecting to the dmPAG whereas green fluorescence identified cells that both express MC4R and project to the dmPAG. Results: As expected, we observed several brain regions with labeled projections to the dmPAG but few consisted of cells that also expressed MC4R. The superior colliculus and the tegmental reticular nucleus contained a high percentage of dmPAG afferents that also expressed MC4R. The retrosplienal area, the pontine reticular nucleus, the parabrachial nucleus, and the gigantocellular reticular nucleus contained a relatively small percentage of dmPAG afferents expressing MC4R. With visual inspection, we did not find any region that exclusively innervated the dmPAG with MC4R-expressing cells. Conclusions: While previous studies have separately demonstrated a role for MC4Rs and the PAG in the regulation of chemosensitivity, these results link those components and implicate several key regions. Although the precise mechanism underlying the interaction of these components remains unclear, these findings bring us a step further in characterizing the neuronal network regulating the interaction between obesity and chemosensitivity. This work was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R15HL170276. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.