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Emerging findings suggest the existence of a cross-talk between hypocretinergic and endocannabinoid systems. Although few studies have examined this relationship, the apparent overlap observed in the neuroanatomical distribution of both systems as well as their putative functions strongly point to the existence of such cross-modulation. In agreement, biochemical and functional studies have revealed the existence of heterodimers between CB1 cannabinoid receptor and hypocretin receptor-1, which modulates the cellular localization and downstream signaling of both receptors. Moreover, the activation of hypocretin receptor-1 stimulates the synthesis of 2-arachidonoyl glycerol culminating in the retrograde inhibition of neighboring cells and suggesting that endocannabinoids could contribute to some hypocretin effects. Pharmacological data indicate that endocannabinoids and hypocretins might have common physiological functions in the regulation of appetite, reward and analgesia. In contrast, these neuromodulatory systems seem to play antagonistic roles in the regulation of sleep/wake cycle and anxiety-like responses. The present review attempts to piece together what is known about this interesting interaction and describes its potential therapeutic implications. ; This work was supported by the Instituto de Salud CarlosIII grants, #PI07/0559, #PI10/00316 and #RD06/001/001 (RTA-RETICS), by the Spanish Ministry of Science and Technology, Consolider-C #SAF2007-64062 and #SAF2011-29864, the Catalan Government (SGR2009-00731), and by the Catalan Institution for Research and Advanced Studies (ICREA Academia program). África Flores is recipient of a predoctoral fellowship from the Spanish Ministry of Education

BACKGROUND: Cannabis is the most widely used illicit drug in the world. Although there is a high prevalence of users who seek treatment for cannabis dependence, no accepted pharmacologic treatment is available to facilitate and maintain abstinence. The hypocretin/orexin system plays a critical role in drug addiction, but the potential participation of this system in the addictive properties of cannabinoids is unknown. METHODS: We investigated the effects of hypocretins in the intravenous self-administration of the synthetic cannabinoid agonist WIN55,212-2 using hypocretin receptor-1 (Hcrtr-1) and hypocretin receptor-2 antagonists and Hcrtr-1 knockout mice. Additional groups of mice were trained to obtain water to rule out operant responding impairments. Activation of hypocretin neurons was analyzed by using double-label immunofluorescence of FosB/ΔFosB with hypocretin-1. Microdialysis studies were performed to evaluate dopamine extracellular levels in the nucleus accumbens after acute Δ(9)-tetrahydrocannabinol administration.RESULTS: Systemic administration of the Hcrtr-1 antagonist SB334867 reduced intravenous self-administration of WIN55,212-2, as well as the maximum effort to obtain a WIN55,212-2 infusion, as revealed under a progressive ratio schedule. This role of Hcrtr-1 in the reinforcing and motivational properties of WIN55,212-2 was confirmed in Hcrtr-1 knockout mice. Contingent, but not noncontingent, WIN55,212-2 self-administration increased the percentage of hypocretin cells expressing FosB/ΔFosB in the lateral hypothalamus. The enhancement in dopamine extracellular levels in the nucleus accumbens induced by Δ(9)-tetrahydrocannabinol was blocked in mice lacking the Hcrtr-1. CONCLUSIONS: /nThese findings demonstrate that Hcrtr-1 modulates the reinforcing properties of cannabinoids, which could have a clear therapeutic interest. ; This work was supported by the Instituto de Salud Carlos III grants, #PI07/0559, #PI10/00316 and #RD06/001/001 (RTA-RETICS), by the Spanish Ministry of Science and Technology, Consolider-C #SAF2007-64062 and #SAF2011-29864, the Catalan Government (SGR2009-00731), and by the Catalan Institution for Research and Advanced Studies (ICREA Academia program). África Flores is recipient of a predoctoral fellowship from the Spanish Ministry of Education

A growing body of evidence suggests the existence of biochemical and functional interactions between the endocannabinoid and orexin systems. Cannabinoid and orexin receptors have been shown to form heterodimers in agreement with the overlapping distribution of both receptors in several brain areas, and the activation of common intracellular signaling pathways, such as the MAP kinase cascade. The activation of orexin receptors induces the synthesis of the endocannabinoid 2-arachidonoyl glycerol suggesting that the endocannabinoid system participates in some physiological functions of orexins. Indeed, functional interactions between these two systems have been demonstrated in several behavioral responses including nociception, reward and food intake. The present review is focused on the latest developments in cannabinoid-orexin cross-modulation and the implications of this interesting interaction. ; This work was supported by Instituto de Salud Carlos III (PI14/00210),the Fundació la Marató de TV3 (231/C/2014) to PR,"Plan Nacional sobre Drogas"(#2014I019) and "Ministerio de Economía y Competitividad"(SAF2017-85299-R) to FB, and the Catalunya Government AGAUR (2017SGR210). We would like to thank Carla Ramón Duaso for her help with reference editing.

BACKGROUND AND PURPOSE: Anatomical, biochemical and pharmacological evidence suggest the existence of a crosstalk between the orexinergic and endocannabinoid systems. While the orexin receptor 1 (OX1 receptor) modulates the reinforcing properties of cannabinoids, the participation of orexins in the acute pharmacological effects of Δ(9) -tetrahydrocannabinol (THC) remains unexplored. EXPERIMENTAL APPROACH: We assessed the possible role of orexins in THC-induced hypolocomotion, hypothermia, antinociception, anxiolytic- and anxiogenic-like effects and memory impairment. Selective OX1 and OX2 receptor antagonists and OX1 knockout (KO) mice as well as prepro-orexin (PPO) KO mice were used as pharmacological and genetic approaches. CB1 receptor levels in control and PPO KO mice were evaluated by immunoblot analysis. The expression of c-Fos after THC treatment was analysed in several brain areas in wild-type mice and in mice lacking the PPO gene. KEY RESULTS: The hypothermia, supraspinal antinociception and anxiolytic-like effects induced by THC were modulated by orexins through OX2 receptor signalling. OX1 receptors did not seem to be involved in these THC responses. No differences in CB1 receptor levels were found between wild-type and PPO KO mice. THC-induced increase in c-Fos expression was reduced in the central amygdala, medial preoptic area and lateral septum in these mutant mice. CONCLUSIONS AND IMPLICATIONS: Our results provide new findings to further clarify the interaction between orexins and cannabinoids. OX1 and OX2 receptors are differently implicated in the pharmacological effects of cannabinoids. ; This work was supported by the Instituto de Salud Carlos III grants [#PI13/00042 and #RD12/0028/0023 (RTA-RETICS)], by the Spanish Ministry of Science (#SAF2011-29864 and #SAF2014-59648-P), National Plan on Drugs (#2014I019) and the Catalan Government (SGR2014-1547)

BACKGROUND: Hypocretin (orexin) signaling is involved in drug addiction. In this study, we investigated the role of these hypothalamic neuropeptides in nicotine withdrawal by using behavioral and neuroanatomical approaches. METHODS: Nicotine withdrawal syndrome was precipitated by mecamylamine (2 mg/kg, subcutaneous) in C57BL/6J nicotine-dependent mice (25 mg/kg/day for 14 days) pretreated with the hypocretin receptor 1 (Hcrtr-1) antagonist SB334867 (5 and 10 mg/kg, intraperitoneal), the hypocretin receptor 2 antagonist TCSOX229 (5 and 10 mg/kg, intraperitoneal), and in preprohypocretin knockout mice. c-Fos expression was analyzed in several brain areas related to nicotine dependence by immunofluorescence techniques. Retrograde tracing with rhodamine-labeled fluorescent latex microspheres was used to determine whether the hypocretin neurons project directly to the paraventricular nucleus of the hypothalamus (PVN), and SB334867 was locally administered intra-PVN (10 nmol/side) to test the specific involvement of Hcrtr-1 in this brain area during nicotine withdrawal./nRESULTS: Somatic signs of nicotine withdrawal were attenuated in mice pretreated with SB334867 and in preprohypocretin knockout mice. No changes were found in TCSOX229 pretreated animals. Nicotine withdrawal increased the percentage of hypocretin cells expressing c-Fos in the perifornical, dorsomedial, and lateral hypothalamus. In addition, the increased c-Fos expression in the PVN during withdrawal was dependent on hypocretin transmission through Hcrtr-1 activation. Hypocretin neurons directly innervate the PVN and the local infusion of SB334867 into the PVN decreased the expression of nicotine withdrawal. CONCLUSIONS: These data demonstrate that hypocretin signaling acting on Hcrtr-1 in the PVN plays a crucial role in the expression of nicotine withdrawal. ; This work was supported by the Instituto de Salud Carlos III grants #PI07/0559,/n#PI10/00316, and #RD06/001/001 (RTA-RETICS), by the Spanish Ministry of Science/nand Technology (Consolider-C, #SAF2007-64062), the Catalan Government/n(SGR2009-00731), and by the Catalan Institution for Research and Advanced Studies/n(ICREA Academia program). A Plaza-Zabala is a recipient of a predoctoral fellowship/nfrom the Spanish Ministry of Education

An understanding of the neurobiological mechanisms involved in the regulation of fear is essential for the development of new treatments for anxiety disorders, such as phobias, panic, and post-traumatic stress disorders (PTSD). Orexins, also known as hypocretins, are neuropeptides located exclusively in hypothalamic neurons that have extensive projections throughout the central nervous system. Although this system was initially believed to be primarily involved in the regulation of feeding behavior, recent studies have shown that orexins also modulate neural circuits implicated in the expression and extinction of fear memories. Here, we discuss recent findings involving orexins in anxiety disorders and current clinical trials using orexin ligands that could be applied to identify new therapies for diseases characterized by pathological fear. ; This work was supported by the Instituto de Salud Carlos III grants, #PI13/00042 and #RD12/0028/0023 (RTA-RETICS), by the Spanish Ministry of Science #SAF2011-29864 and #SAF2014-59648-P, National Plan on Drugs #2014I019, and the Catalan Government (SGR2014-1547).

Anxiety disorders are often associated with an inability to extinguish learned fear responses. The hypocretin/orexin system is involved in the regulation of emotional states and could also participate in the consolidation and extinction of aversive memories. Using hypocretin receptor-1 and hypocretin receptor-2 antagonists, hypocretin-1 and hypocretin-2 peptides, and hypocretin receptor-1 knockout mice, we investigated the role of the hypocretin system in cue- and context-dependent fear conditioning and extinction. Hypocretins were crucial for the consolidation of fear conditioning, and this effect was mainly observed in memories with a high emotional component. Notably, after the acquisition of fear memory, hypocretin receptor-1 blockade facilitated fear extinction, whereas hypocretin-1 administration impaired this extinction process. The extinction-facilitating effects of the hypocretin receptor-1 antagonist SB334867 were associated with increased expression of cFos in the basolateral amygdala and the infralimbic cortex. Intra-amygdala, but neither intra-infralimbic prefrontal cortex nor intra-dorsohippocampal infusion of SB334867 enhanced fear extinction. These results reveal a key role for hypocretins in the extinction of aversive memories and suggest that hypocretin receptor-1 blockade could represent a novel therapeutic target for the treatment of diseases associated with inappropriate retention of fear, such as post-traumatic stress disorder and phobias. ; This work was supported by the Instituto de Salud Carlos III grants, #PI13/00042 and #RD12/0028/0023 (RTA-RETICS), by the Spanish Ministry of Science #SAF2011-29864, the/nCatalan Government (SGR2009-00731), and by the Catalan Institution for Research and Advanced Studies (ICREA Academia program). África Flores is a recipient of a predoctoral fellowship from the Spanish Ministry of Education.

Hypocretin/orexin signaling is critically involved in relapse to drug-seeking behaviors. In this study, we investigated the involvement of the hypocretin system in the reinstatement of nicotine-seeking behavior induced by nicotine-associated cues. Pretreatment with the hypocretin receptor-1 antagonist SB334867, but not with the hypocretin receptor-2 antagonist TCSOX229, attenuated cue-induced reinstatement of nicotine-seeking, which was associated with an activation of hypocretin neurons of the lateral and perifornical hypothalamic areas. In addition, relapse to nicotine-seeking increased the phosphorylation levels of GluR2-Ser880, NR1-Ser890, and p38 MAPK in the nucleus accumbens (NAc), but not in the prefrontal cortex. Notably, phosphorylation levels of NR1-Ser890 and p38 MAPK, but not GluR2-Ser880, were dependent on hypocretin receptor-1 activation. The intra-accumbens infusion of the protein kinase C (PKC) inhibitor NPC-15437 reduced nicotine-seeking behavior elicited by drug-paired cues consistent with the PKC-dependent phosphorylations of GluR2-Ser880 and NR1-Ser890. SB334867 failed to modify cue-induced reinstatement of food-seeking, which did not produce any biochemical changes in the NAc. These data identify hypocretin receptor-1 and PKC signaling as potential targets for the treatment of relapse to nicotine-seeking induced by nicotine-associated cues. ; This work was supported by the Instituto de Salud Carlos III grants, #PI07/0559, #PI10/00316 and #RD06/001/001 (RTA-RETICS), by the Spanish Ministry of Science and Technology, Consolider-C #SAF2007-64062 and #SAF2011-29864, the Catalan Government (SGR2009-00731), and by the Catalan Institution for Research and Advanced Studies (ICREA Academia program). Ainhoa Plaza-Zabala and África Flores are recipients of a predoctoral fellowship from the Spanish Ministry of Education

Multiple studies in animal models and humans suggest that the endogenous opioid system is an important neurobiological substrate for nicotine addictive properties. In this study, we evaluated the participation of δ-opioid receptors in different behavioral responses of nicotine by using δ-opioid receptor knockout mice. Acute nicotine administration induced hypolocomotion and antinociception in wild-type mice, which were similar in knockout animals. The development of tolerance to nicotine-induced antinociception was also similar in both genotypes. In agreement, the expression and functional activity of δ-opioid receptors were not modified in the different layers of the spinal cord and brain areas evaluated after chronic nicotine treatment. The somatic manifestation of the nicotine withdrawal syndrome precipitated by mecamylamine was also similar in wild-type and δ-opioid receptor knockout mice. In contrast, nicotine induced a conditioned place preference in wild-type animals that was abolished in knockout mice. Moreover, a lower percentage of acquisition of intravenous nicotine self-administration was observed in mice lacking δ-opioid receptors as well as in wild-type mice treated with the selective δ-opioid receptor antagonist naltrindole. Accordingly, in-vivo microdialysis studies revealed that the enhancement in dopamine extracellular levels induced by nicotine in the nucleus accumbens was reduced in mutant mice. In summary, the present results show that δ-opioid receptors are involved in the modulation of nicotine rewarding effects. However, this opioid receptor does not participate either in several acute effects of nicotine or in the development of tolerance and physical dependence induced by chronic nicotine administration. ; This work was supported by the Spanish "Ministerio de Ciencia e Innovación"/n(#SAF2007-64062 and #SAF-2011-29864), the "Instituto de Salud Carlos III" grants/n#PI07/0559, #PI10/00316 and #RD06/001/001, the "Plan Nacional Sobre Drogas"/n(#2009/026), the Catalan Government (SGR2009-00131), and the ICREA Foundation/n(ICREA Academia Award-2008) to R.M. Partial support of FEDER funds is also acknowledged.

Nicotine exerts its psychopharmacological effects by activating the nicotinic acetylcholine receptor (nAChR), composed of alpha and/or beta subunits, giving rise to a diverse population of receptors with a distinct pharmacology. β4-containing (β4*) nAChRs are located almost exclusively in the habenulo-interpeduncular pathway. We examined the role of β4* nAChRs in the medial habenula (MHb) and the interpeduncular nucleus (IPN) in nicotine reinforcement using behavioral, electrophysiological, and molecular techniques in transgenic mice. Nicotine intravenous self-administration (IVSA) was lower in constitutive β4 knockout (KO) mice at all doses tested (7.5, 15, 30, and 60 μg/kg/infusion) compared with wild-type (WT) mice. In vivo microdialysis showed that β4KO mice have higher extracellular dopamine (DA) levels in the nucleus accumbens than in WT mice, and exhibit a differential sensitivity to nicotine-induced DA outflow. Furthermore, electrophysiological recordings in the ventral tegmental area (VTA) demonstrated that DA neurons of β4KO mice are more sensitive to lower doses of nicotine than that of WT mice. Re-expression of β4* nAChRs in IPN neurons fully restored nicotine IVSA, and attenuated the increased sensitivity of VTA DA neurons to nicotine. These findings suggest that β4* nAChRs in the IPN have a role in maintaining nicotine IVSA ; This work was supported by the Spanish Instituto de Salud Carlos III (RD06/001/001 and PI10/01708; PI14/00210), FEDER funds, Ministerio de Ciencia e Innovación (#SAF2014-59648-P), the Catalan Government AGAUR (#2014-SGR-1547), Plan Nacional Sobre Drogas, Ministerio de Sanidad, Asuntos Sociales e Igualdad-MSASI (#PNSD- 2013-0068), FP7 ERANET program (NICO-GENE), the ICREA Foundation (ICREA Academia-2008), and a postdoctoral fellowship from CONACyT to AHS. The work in Paris was supported by the Institut Pasteur, Centre National de la Recherche Scientifique CNRS UMR 3571 (UM) and CNRS UMR 8246 (PF), the INSERM U1130 (PF), the Pierre et Marie Curie University (UM119), the Agence Nationale pour la Recherche (ANR Neuroscience), and FP7 ERANET program (NICO-GENE), Grant agreement n009 BLANC 20092009BLANC 20 NeuroCypres" project), Fondation EDF, the Fondation des Treilles, and the Foundation for Medical Research FMR (Equipe FRMDEQ20130326488 to PF). The groups of UM and PF are members of the Bio-Psy Labex. As such this work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02. The teams of UM and PF are part of the École des Neurosciences de Paris Ile-de-France Network. We would like to thank Martine Soudant, Stephanie Pons, and Dulce Real for technical support, and Inés Ibañes-Tallon and Jessica/nAbles for providing the Tg(Chrnb4-cre)OL57Gsat/+ transgenic mice