High-spatial resolution mapping and three-dimensional architecture of the C1, C2, and C3 presumptive adrenergic neuronal groups in the medulla oblongata of the rat brainstem

Putative adrenergic C1, C2, and C3 neurons in the medulla oblongata have been implicated in nutrient sensing, respiration, and cardiovascular and sympathetic nervous system regulation. Structural characterestics of these neurons have previously been studied individually and as distinct groups by immunostaining catecholamine-synthesizing enzymes, especially phenylethanolamine N -methyltransferase (PNMT) (EC 2.1.1.28). However, the potential network that is formed by these neuron groups and their projections has not been visualized in 3-D or described in a systematic manner. In this study, we used optical tissue clearing, immunofluorescence, and light sheet fluorescence microscopy to visualize PNMT-immunopositive C1, C2, and C3 neuron groups and their potential connectional network in 3-D. These 3-D visualizations have been further validated using atlas-based mapping methods on separate immunostained preparations of brainstem sections. Formaldehyde-fixed Sprague-Dawley rat lower brainstems (n=5) were hydrogel-protected and delipidated using aqueous delipidation methods. These brainstems were immunostained with a polyclonal anti-PNMT antibody and imaged using a SmartSPIM light sheet fluorescence microscope. Acquired images were converted to volume renders using Imaris software. A separate batch of preserved and frozen lower brainstems were sectioned and stained with the same antibody using immunoperoxidase histochemistry. PNMT-immunopositive structural features from these sections were mapped onto Brain Maps 4.0 ( BM 4.0, L. W. Swanson, J. Comp. Neurol., 2018 ) open-access rat brain digital atlas templates using standardized mapping methods (n=4). In our 3-D spatial exploration of volume renders, we observed dense clusters of ventral C1 neurons and their neurite extensions interacting with the dorsal C2 neurons on either side of the midline. C1 and C2 neurons formed finely branched extensions that crossed the medullary midline where the C3 neurons are located, appearing to link the two hemispheres in a putative adrenergic network, which could be a basis for coordinated and perhaps synchronized physiological control. Rostrocaudally oriented PNMT-positive neurite bundles extending from this network were also observed bilaterally in the region of the dorsal reticular nucleus. In conclusion, complete volumetric renders allowed us to visualize and identify what appears to be a mesoscopic-scale connectional network formed by the C1, C2, and C3 putative adrenergic neuronal groups and their rostrocaudal extensions. Future studies are needed to investigate the functional significance of these newly visualized network-like interactions. This work is supported by NIH Grant GM127251 awarded to AMK (PI), C06OD030148 (AMK-co-PI, MJK-PI), UTEP Research & Innovation, and the College of Science. 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.