Background: Our previous research has showed that increased sympathetic nervous activity and decreased cannabinoid receptor 1 (CB1) were associated with increased energy expenditure and weight loss following Roux-en-Y gastric bypass (RYGB) in mice. Our study aims to explore the neurological pathways responsible for these effects. Methods: We localized CB1 receptors within the small intestine to specific neuronal cells using immunofluorescence. We administered CB1 receptor agonists anandamide (AEA), and a whole-body inverse agonist rimonabant (Rim), to obese C57Blk/6 mice and measured afferent nerve activity of vagal vs splanchnic fibers. We administered a whole-body vs a peripherally-restricted CB1 antagonist to CB1 sensory neuron-deleted mice vs wild-type controls and measured elements of energy. Results: Immunofluorescence revealed the presence of CB1 receptors in sensory afferent neurons using markers such as calcitonin gene-related peptide (CGRP) and monocyte chemoattractant protein-1 (MCP1) (Fig. 1 A, B). CB1 receptors were highly expressed in the superior mesenteric, celiac ganglion, and nodose ganglion, with a higher abundance observed in the former two (Fig. 1C). Furthermore, using Nav 1.8-tdTomato mice, we observed a greater co-localization of CB1 receptors with Nav 1.8 positive cells in the celiac ganglion and dorsal root ganglion compared to the nodose ganglion (Fig. 1D). This confirmed the localization of CB1 receptors in both splanchnic and vagal sensory neurons, with a higher prevalence in splanchnic neurons. Subsequent measurements of splanchnic and vagal afferent nerves’ activity following local luminal administration of AEA and Rim revealed significant changes in splanchnic nerve activity but not the vagal one (data not shown). We developed a CB1 specific sensory neuron-specific deletion by crossing Nav1.8Cre mice with CB1^flx/flxand we confirmed the model by obtaining CB1 immunofluorescence imaging that showed deletion in vagal and spinal sensory neurons but not in the central nervous system (Fig. 2 A). Mice lacking CB1 receptors exhibited attenuated weight gain and food intake in response to high-fat diet feeding (Fig.2 B). When administered JD5037, a peripherally-restricted CB1 antagonist, these mice had further attenuation of food intake and weight changes experienced on high fat diet (Fig. 2 C, D). Conclusion: Our findings suggest that the endocannabinoid signaling system within afferent splanchnic neurons, play a key role in energy regulation and feeding behavior. This potentially opens the door for further exploration of the pathways involved.

Figure 1. Strong co-localization of CB1 with markers of sensory/afferent neurons (calcitonin gene-related peptide [CGRP] and monocyte chemoattractant protein-1 [MCP1]) within the small intestine of HF diet-induced obese male C57Blk/6J mice. Co-immunostaining of CGRP or MCP1 (Green) with CB1 (Red) within the small intestine of HF diet-induced obese male C57Blk/6J mice at magnification of (A) 20X and (B) 63X. (C) CB1 immunostaining of cross-sections of the superior mesenteric ganglion, the celiac ganglion, and the nodose ganglion (CB1 = Green, DAPI= Blue). (D) CB1 immunostaining (Green) of cross-sections of the celiac ganglion, nodose ganglion, and dorsal root ganglion of Nav.1.8-tdTomato(Red) mice , DAPI= Blue. n=4-5 mice.

<b>Figure 2. </b>(<b>A</b> ) CB1 immunostaining (Red) in Dorsal root ganglion (DRG-T10), nodose ganglion, and hippocampus of 12 weeks old male Nav1.8-CB1-/- and Nav1.8-CB1 -/- mice. (<b>B</b>) body weight in (g) over time in (days) , average daily food intake in (Kcal/day), % weight change from baseline over time in (days), and % weight gain after 3 weeks (21 days) of HF diet feeding in Nav1.8-CB1 +/+ and Nav1.8-CB1 -/- male mice. (<b>C, D)</b> % total weight change and average daily food intake (measured over 3 days) in HF diet-fed Nav1.8-CB1 +/+ and Nav1.8-CB1 -/- male mice after 15 days of administration of peripherally-restricted CB1 antagonist (JD) vs non-selective CB1 antagonist (RIM) vs vehicle (VEH). n=8-10.

Figure 2. (A ) CB1 immunostaining (Red) in Dorsal root ganglion (DRG-T10), nodose ganglion, and hippocampus of 12 weeks old male Nav1.8-CB1-/- and Nav1.8-CB1 -/- mice. (B) body weight in (g) over time in (days) , average daily food intake in (Kcal/day), % weight change from baseline over time in (days), and % weight gain after 3 weeks (21 days) of HF diet feeding in Nav1.8-CB1 +/+ and Nav1.8-CB1 -/- male mice. (C, D) % total weight change and average daily food intake (measured over 3 days) in HF diet-fed Nav1.8-CB1 +/+ and Nav1.8-CB1 -/- male mice after 15 days of administration of peripherally-restricted CB1 antagonist (JD) vs non-selective CB1 antagonist (RIM) vs vehicle (VEH). n=8-10.

Dana Tasabehji

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