Citation: Kim, H.J.J.; Zagzoog, A.; Ceni, C.; Ferrisi, R.; Janz, N.; Laprairie, R.B. Dual Cannabinoid and Orexin Regulation of Anhedonic Behaviour Caused by Prolonged Restraint Stress. Brain Sci. 2023, 13, 314. https://doi.org/10.3390/ brainsci13020314 Academic Editors: Justin Matheson, Catharine Mielnik and Matteo Marti Received: 28 December 2022 Revised: 28 January 2023 Accepted: 8 February 2023 Published: 13 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). brain sciences Article Dual Cannabinoid and Orexin Regulation of Anhedonic Behaviour Caused by Prolonged Restraint Stress Hye Ji J. Kim 1 , Ayat Zagzoog 1 , Costanza Ceni 1,2,3 , Rebecca Ferrisi 1,2 , Nicola Janz 1 and Robert B. Laprairie 1,4, * 1 College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada 2 Department of Pharmacy, University of Pisa, 56126 Pisa, Italy 3 Doctoral School in Life Sciences, University of Siena, 53100 Siena, Italy 4 Department of Pharmacology, College of Medicine, Dalhousie University, Halifax, NS B3H 4R2, Canada * Correspondence: robert.laprairie@usask.ca Abstract: The endocannabinoid and orexin systems share many biological functions, including wakefulness, stress response, reward processing, and mood. While these systems work against one another with respect to arousal, chronic stress-induced downregulation of both systems often leads to anhedonia or the inability to experience pleasure from natural rewards. In the current study, a 24 h restraint stress test (24 h RST) reduced sucrose preference in adult male and female C57BL/6 mice. Prior to the stressor, subsets of mice were intraperitoneally administered cannabinoid and orexin receptor agonists, antagonists, and combinations of these drugs. Restraint mice that received the cannabinoid receptor type 1 (CB1R) antagonist SR141716A, orexin receptor type 2 (OX2R) agonist YNT-185, and the combination of SR141716A and YNT-185, exhibited less anhedonia compared to vehicle/control mice. Thus, the 24 h RST likely decreased orexin signaling, which was then restored by YNT-185. Receptor colocalization analysis throughout mesocorticolimbic brain regions revealed increased CB1R-OX1R colocalization from SR141716A and YNT-185 treatments. Although a previous study from our group showed additive cataleptic effects between CP55,940 and the dual orexin receptor antagonist (TCS-1102), the opposite combination of pharmacological agents proved additive for sucrose preference. Taken together, these results reveal more of the complex interactions between the endocannabinoid and orexin systems. Keywords: anhedonia; restraint stress; endocannabinoid system; cannabinoid; orexin 1. Introduction Anhedonia is a core symptom in mood disorders such as major depression [1,2] and anxiety [3] and is described as the inability to feel pleasure from natural, non-drug sources [1]. As a form of dopaminergic dysregulation, anhedonia may also appear in substance use disorders (SUD) [4]. The mesocorticolimbic circuit that regulates hedonic and anhedonic behaviours involves dopaminergic projections from the ventral tegmental area (VTA) to subcortical regions including the nucleus accumbens (NAC), medial prefrontal cortex (MPFC), and cingulate cortex [5,6]. These brain regions process wanting, liking, and learning with respect to rewarding stimuli [7,8]. Anhedonia may emerge in times of stress, as wanting or choosing to consume rewarding drugs over natural rewards is a form of coping in humans [4,9,10]. Preclinically, both chronic and acute patterns of homo- and/or heterotypic physiological and psychological stress produce anhedonia [11–13]. The endocannabinoid and orexin systems are among the many neuromodulatory systems involved in anhedonia [14,15]. The endocannabinoid system (ECS) includes 2 G- protein coupled receptors (GPCRs), the type 1 (CB1R) and type 2 (CB2R) cannabinoid recep- tors [16]. These receptors are activated by the endogenous ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA) [17]. CB1R and CB2R are expressed throughout the central Brain Sci. 2023, 13, 314. https://doi.org/10.3390/brainsci13020314 https://www.mdpi.com/journal/brainsci