Virginia Via Research Day Book 2026

Medical Student Research Biomedical

01 DEMONSTRATION OF THE CO-LOCALIZATION OF M3 AND CB1 RECEPTORS IN THE POSTERIOR HYPOTHALAMIC NUCLEUS OF THE RAT AND ITS IMPLICATION IN CONTROL OF BLOOD PRESSURE

Bryce Gambino' OMS-II; Lillia Lucas, OMS-II; Long Nguyen, OMS-II; Simon Casa,s OMS-II; John Martin, PhD Corresponding author: bjgambino1@liberty.edu

Liberty University College of Osteopathic Medicine, Lynchburg, Virginia

This study investigates central mechanisms by which serotonergic and muscarinic receptor activation evokes an increase in mean arterial pressure (MAP) via a connection from the anterior hypothalamic nucleus (AHN) to the posterior hypothalamic nucleus (PHN) and the potential modification of these changes by cannabinoids. The serotonin 2A/2C receptor agonist DOI activates a neural pathway originating in the AHN that likely leads to the release of acetylcholine (ACh) in the PHN as the muscarinic receptor antagonist methyl atropine (MeATR) present in the PHN blocks the AHN DOI-evoked increase in MAP while having no effect on the DOI-evoked pressor response when present in the AHN. This cholinergic activation is hypothesized to elevate MAP and heart rate (HR) through sympathetic activation combined with activation of the renin angiotensin-aldosterone system (RAAS), which is like the mechanism involved in the pressor response evoked by the cholinergic agonist carbachol (CCh) given into the PHN. The cannabinoid receptor agonist CP-55940,

which selectively targets CB1 receptors, given into the PHN prior to the administration of DOI into the AHN blocks the pressor response evoked by DOI while able to only partially block the pressor response evoked by CCh given into the PHN. These results suggest that CB1 receptors are co-localized in the PHN with cholinergic M3 receptors (whose activation was shown in earlier studies to be responsible for the cholinergic evoked pressor response), so the site of interaction between the endocannabinoid and cholinergic systems in the PHN is presumably presynaptic, postsynaptic, or both. In the present study, male Sprague-Dawley rats (280–320 g) were anesthetized with urethane and then euthanized by transcardial perfusion with saline, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS). Brains were post-fixed in 4% PFA overnight, then cryoprotected in 30% sucrose with NaN3. Coronal brain sections (40 µm) through the PHN were made using a cryostat. Free-floating sections underwent immunohistochemistry staining for

CB1 receptor and cholinergic muscarinic receptor M3 using Alexa Fluor 488 and 647-conjugated secondary antibodies, respectively. Nuclei were counterstained with DAPI, and images were acquired using a Keyence fluorescence microscope. Analysis of the staining of the neurons within the slides currently suggests that there is a separation of the CB1 and M3 receptors, which would seem to indicate a presynaptic location for the CB1 receptors and a postsynaptic location for the M3 receptors. The presynaptic location of the CB1 receptors would be in agreement with other studies showing that central CB1 receptors are primarily located presynaptically. A presynaptic location would indicate that the stimulation of CB1 receptors in the PHN would inhibit the release of ACh from cholinergic neurons.

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90 Edward Via College of Osteopathic Medicine (VCOM)