Orexins are associated with drug relapse in rodents. Here, we show that acute restraint stress in mice activates lateral hypothalamic (LH) orexin neurons, increases levels of orexin A and 2-arachidonoylglycerol (2-AG) in the ventral tegmental area (VTA), and reinstates extinguished cocaine-conditioned place preference (CPP). This stress-induced reinstatement of cocaine CPP depends on type 1 orexin receptors (OX1Rs), type 1 cannabinoid receptors (CB1Rs) and diacylglycerol lipase (DAGL) in the VTA. In dopaminergic neurons of VTA slices, orexin A presynaptically inhibits GABAergic transmission. This effect is prevented by internal GDP-β-S or inhibiting OX1Rs, CB1Rs, phospholipase C or DAGL, and potentiated by inhibiting 2-AG degradation. These results suggest that restraint stress activates LH orexin neurons, releasing orexins into the VTA to activate postsynaptic OX1Rs of dopaminergic neurons and generate 2-AG through a Gq-protein-phospholipase C-DAGL cascade. 2-AG retrogradely inhibits GABA release through presynaptic CB1Rs, leading to VTA dopaminergic disinhibition and reinstatement of cocaine CPP.

An injection of orexin A or B into the cisterna magna or the rostral ventrolateral medulla (RVLM), where bulbospinal vasomotor neurons are located, elevated arterial pressure (AP) and heart rate (HR). We examined how orexins affected RVLM neurons to regulate cardiovascular functions by using in vitro recordings of neuronal activity of the RVLM and in vivo measurement of cardiovascular functions in rats. Orexin A and B concentration-dependently depolarized RVLM neurons. At 100 nM, both peptides excited 42% of RVLM neurons. Tetrodotoxin failed to block orexin-induced depolarization. In the presence of N-(2-methyl-6-benzoxazolyl)-NЈ-1, 5-naphthyridin-4-yl urea (SB-334867), an orexin 1 receptor (OX 1 R) antagonist, orexin A depolarized 42% of RVLM neurons with a smaller, but not significantly different, amplitude (4.9 Ϯ 0.8 versus 7.2 Ϯ 1.1 mV). In the presence of (2S)-1-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3,3-dimethyl-2-[(4-pyridinylmethyl)amino]-1-butanone hydrochloride (TCS OX2 29), an orexin 2 receptor (OX 2 R) antagonist, orexin A depolarized 25% of RVLM neurons with a significantly smaller amplitude (1.7 Ϯ 0.5 mV). Coapplication of both antagonists completely eliminated orexin A-induced depolarization. An OX 2 R agonist, [Ala 11 ,D-Leu 15 ]-orexin B, concentration-dependently depolarized RVLM neurons. Regarding neuronal phenotypes, orexins depolarized 88% of adrenergic, 43% of nonadrenergic, and 36 to 41% of rhythmically firing RVLM neurons. Intracisternal TCS OX2 29 (3 and 10 nmol) suppressed intracisternal orexin A-induced increases of AP and HR, whereas intracisternal SB-334867 (3 and 10 nmol) had no effect on the orexin A-induced increase of HR but suppressed the orexin A-induced pressor response at 10 nmol. We concluded that orexins directly excite RVLM neurons, which include bulbospinal vasomotor neurons, and regulate cardiovascular function mainly via the OX 2 R, with a smaller contribution from the OX 1 R.Orexin A and orexin B (also known as hypocretin 1 and hypocretin 2) increase arterial pressure (AP), heart rate (HR), and sympathetic activity in rats and rabbits, when given by an intracerebroventricular (Samson et al., 1999;Shirasaka et al., 1999;Matsumura et al., 2001), intracisternal (Chen et al., 2000, or intrathecal (Antunes et al., 2001) injection. Regarding the sites of action of orexins in regulating cardiovascular and autonomic functions, the hypothalamic paraventricular nucleus was proposed to be one of the critical sites (reviewed in Kannan et al., 2007). In addition, the medullary structures essential for cardiovascular and autonomic regulation may be responsible for orexin-induced cardiovascular alterations. To explore the role of orexins in medullary control of the AP and HR, Chen et al. (2000) found that an intracisternal injection of orexins induced long-lasting pressor and positive chronotropic responses, and microinjection of orexin A into the rostral ventrolateral medulla (RVLM) caused similar responses in rats. The cardiovascular effects of orexins were also ex...

Adequate pain management remains an unmet medical need. We previously revealed an opioid-independent analgesic mechanism mediated by orexin 1 receptor (OX1R)-initiated 2-arachidonoylglycerol (2-AG) signaling in the ventrolateral periaqueductal gray (vlPAG). Here, we found that low-frequency median nerve stimulation (MNS) through acupuncture needles at the PC6 (Neiguan) acupoint (MNS-PC6) induced an antinociceptive effect that engaged this mechanism. In mice, MNS-PC6 reduced acute thermal nociceptive responses and neuropathy-induced mechanical allodynia, increased the number of c-Fos–immunoreactive hypothalamic orexin neurons, and led to higher orexin A and lower GABA levels in the vlPAG. Such responses were not seen in mice with PC6 needle insertion only or electrical stimulation of the lateral deltoid, a nonmedian nerve-innervated location. Directly stimulating the surgically exposed median nerve also increased vlPAG orexin A levels. MNS-PC6–induced antinociception (MNS-PC6-IA) was prevented by proximal block of the median nerve with lidocaine as well as by systemic or intravlPAG injection of an antagonist of OX1Rs or cannabinoid 1 receptors (CB1Rs) but not by opioid receptor antagonists. Systemic blockade of OX1Rs or CB1Rs also restored vlPAG GABA levels after MNS-PC6. A cannabinoid (2-AG)-dependent mechanism was also implicated by the observations that MNS-PC6-IA was prevented by intravlPAG inhibition of 2-AG synthesis and was attenuated inCnr1−/−mice. These findings suggest that PC6-targeting low-frequency MNS activates hypothalamic orexin neurons, releasing orexins to induce analgesia through a CB1R-dependent cascade mediated by OX1R-initiated 2-AG retrograde disinhibition in the vlPAG. The opioid-independent characteristic of MNS-PC6–induced analgesia may provide a strategy for pain management in opioid-tolerant patients.

New Findings r What is the central question of this study?Central orexinergic activity is involved in tonic and phasic control of cardiovascular homeostasis. A potential role for elevated central orexinergic activity in the maintenance of hypertension in spontaneously hypertensive rats (SHRs) has not previously been explored. r What is the main finding and what is its importance?We show that central or intra-rostral ventrolateral medulla blockade of orexin 2 receptors produces a significant reduction of arterial pressure in SHRs, but not Wistar-Kyoto rats. This study demonstrates a previously unrecognized role of orexin 2 receptors in maintaining hypertension in SHRs.Orexins can raise arterial pressure and sympathetic activity and are involved in tonic and phasic control of cardiovascular homeostasis. We hypothesized that elevated central orexinergic activity contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). We examined this hypothesis by suppressing central orexinergic activity in SHRs and Wistar-Kyoto rats (WKYs) with specific antagonists or antibodies against orexin 1 (OX1R) and 2 receptors (OX2R). Intracerebroventricular administration of an OX1R antagonist, SB-334867 (30 and 100 nmol), induced no significant change in mean arterial pressure (MAP) and heart rate (HR) in SHRs and WKYs except that at 100 nmol it reduced HR in WKYs. In contrast, an OX2R antagonist, TCS-OX2-29 (3-30 nmol) induced long-lasting reductions of MAP and HR in SHRs (21 ± 3 mmHg and 22 ± 2 beats min −1 at 30 nmol), but not in WKYs. Intracerebroventricular anti-OX2R IgG, but not anti-OX1R IgG or non-immune goat IgG, significantly lowered MAP and HR in SHRs. None of the three IgGs affected MAP or HR in WKYs. The OX2R protein level in the rostral ventrolateral medulla (RVLM) was lower in SHRs than in WKYs, whereas no differences were found between SHRs and WKYs in the paraventricular hypothalamic nucleus, dorsomedialperifornical hypothalamic area or caudal nucleus tractus solitarii. The OX1R protein levels in these four regions did not differ between SHRs and WKYs. Injection of TCS-OX2-29 (50 pmol) into the RVLM produced a larger reduction of MAP in SHRs than in WKYs. We conclude that elevated OX2R-mediated activity in the brain, especially in the RVLM, may contribute to hypertension in SHRs.

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