Drug relapse can be initiated by environmental cues and stress even after extended periods of abstinence, leading to the failure of drug rehabilitation programs. Currently, there are few effective treatments to prevent drug relapse. Therefore, drug relapse is not only an unmet medical need but also an important socioeconomic concern, especially stress-induced drug relapse. The mechanisms by which stress initiates drug relapse remain unclear. The study group led by Prof. Lih-Chu Chiou at the Department of Pharmacology at the Medical College of National Taiwan University revealed a novel mechanism for stress-induced cocaine relapse. This may shed light on the prevention of stress-related drug relapse. This work was mainly performed by Dr. Li-Wi Tung during his PhD study period at the Graduate Institute of Pharmacology at NTU. The research group found that stress can reinstate extinguished cocaine preference in mice in a conditioned place preference (CPP) training task. The CPP score in this animal model is usually used to evaluate the degree of drug craving. Their results suggest that this stress-reinstated cocaine relapse is mediated by a sequential cascade involving orexins and endocannabinoids. Orexins consist of a pair of neuropeptides, orexin A and B (also named hypocretin 1 and 2). They are generated by a group of neurons in the hypothalamus, especially in the perifornical area (PeF) and lateral hypothalamus (LH). Their receptors, OX1R and OX2R, are widely distributed throughout the brain, including the ventral tegmental area (VTA), an important brain region for reward processing where dopamine neuron plasticity can be altered by addictive substances. Orexins have been known to play a role in drug seeking behavior in animals. However, the mechanisms by which orexins regulate the VTA dopaminergic activity to provoke drug craving, especially under stressful conditions, remain unclear. Previously, Prof. Chiou’s group found that orexins can be released under stress from the LH to activate a pain-regulating region, the periaqueductal gray (PAG), leading to analgesia. This stress-induced analgesic effect is mediated by a signaling cascade in the PAG, i.e., orexins activate postsynaptic OX1Rs, a family of Gq-protein coupled receptors, resulting in the synthesis of 2-arachydonoylglycerol (2-AG) via the phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic pathway. 2-AG is an endogenous cannabinoid that can produce retrograde inhibition of GABA release (disinhibition) by activating presynaptic cannabinoid 1 receptors (CB1Rs). Inspired by the previous finding in the PAG, Tung et al. validated a hypothesis that this OX1R-PLC-DAGL-2-AG retrograde disinhibition signaling also exists in VTA dopamine neurons and can contribute to stress-induced extinguished cocaine seeking behavior in mice using both electrophysiological and behavioral approaches. Indeed, they found that orexin A inhibited GABAergic transmission via a presynaptic mechanism in dopamine neurons of VTA slices. This effect of orexin A was prevented by internal inhibition of G protein or the antagonist of OX1Rs or CB1Rs. It was also prevented by inhibiting the enzymes involved in 2-AG synthesis, PLC and DAGL, and potentiated by inhibiting 2-AG degradation. Furthermore, they found that acute restraint stress activated orexin neurons in the LH, increased both orexin A and 2-AG levels in the VTA, and reinstated extinguished cocaine CPP in mice. This stress-reinstated cocaine CPP was prevented by intra-VTA injection of the antagonist of OX1Rs, CB1Rs or DAGL and was abolished in CB1R-knockout mice. These results suggest a novel mechanism for stress-induced cocaine relapse: acute restraint stress activates LH orexin neurons, releasing orexins to activate postsynaptic OX1Rs on VTA dopaminergic neurons and, through a Gq-protein-PLC-DAGL cascade, generating 2-AG that retrogradely inhibits GABA release through presynaptic CB1Rs, leading to VTA dopaminergic disinhibition and reinstating cocaine CPP. This finding provides a prospective in the development of OX1R antagonists as a novel therapeutic approach in the prevention of stress-related cocaine relapse. Li-Wei Tung, Guan-Ling Lu, Yen-Hsien Lee, Lung Yu, Hsin-Jung Lee, Emma Leishman, Heather Bradshaw, Ling-Ling Hwang, Ming-Shiu Hung, Ken Mackie, Andreas Zimmer & Lih-Chu Chiou. Orexins contribute to restraint stress-induced cocaine relapse by endocannabinoid-mediated disinhibition of dopaminergic neurons. Nat. Commun. 7:12199 doi: 10.1038/ncomms12199 (2016) Professor Lih-Chu Chiou Department of Pharmacology, School of Medicine Graduate Institute of Pharmacology Graduate Institute of Brain and Mind Sciences lcchiou@ntu.edu.tw References 1. Yu-Cheng Ho, Hsin-Jung Lee, Li-Wei Tung, Yan-Yu Liao, SzuYing Fu, Shu-Fang Teng, HsinTzu Liao, Ken Mackie, and LihChu Chiou. (2011). Activation of orexin 1 receptors in the periaqueductal gray of male rats leads to antinociception via retrograde endocannabinoid (2-arachidonoylglycerol)induced disinhibition. Journal of Neuroscience, 31, 14600 14610. DOI: 10.1523/ JNEUROSCI.2671-11.2011 2. Hsin-Jung Lee, Lu-Yang Chang, Yu-Cheng Ho, ShuFang Teng, Ling-Ling Hwang, Ken Mackie, Lih-Chu Chiou. (2016). Stress induces analgesia via orexin 1 receptor-initiated endocannabinoid/CB1 signaling in the mouse periaqueductal gray. Neuropharmacology, 105, 577-586. DOI: 10.1016/ j.neuropharm.2016.02.018 3. Li-Wei Tung, Guan-Ling Lu, YenHsien Lee, Lung Yu, Hsin-Jung Lee, Emma Leishman, Heather Bradshaw, Ling-Ling Hwang, Ming-Shiu Hung, Ken Mackie, Andreas Zimmer and Lih-Chu Chiou. (2016). Orexins contribute to restraint stress-induced cocaine relapse by endocannabinoidmediated disinhibition of dopaminergic neurons. Nature Communications, 7:12199. DOI: 10.1038/ncomms12199 Professor Lih-Chu Chiou Department of Pharmacology, School of Medicine Graduate Institute of Pharmacology Graduate Institute of Brain and Mind Sciences lcchiou@ntu.edu.tw