Background and purpose: Neuropeptide S (NPS) was recently identified as the endogenous ligand of an orphan receptor, now referred to as the NPS receptor. In vivo, NPS produces a unique behavioural profile by increasing wakefulness and exerting anxiolytic-like effects. In the present study, we further evaluated the effects of in vivo supraspinal NPS in mice. Experimental approach: Effects of NPS, injected intracerebroventricularly (i.c.v.), on locomotor activity (LA), righting reflex (RR) recovery and on anxiety states (measured with the elevated plus maze (EPM) and stress-induced hyperthermia (SIH) tests) were assessed in Swiss mice. Key results: NPS (0.01-1 nmol per mouse) caused a significant increase in LA in naive mice, in mice habituated to the test cages and in animals sedated with diazepam (5 mg kg À1 ). In the RR assay, NPS dose dependently reduced the proportion of animals losing the RR in response to diazepam (15 mg kg À1 ) and their sleeping time. In the EPM and SIH test, NPS dose dependently evoked anxiolytic-like effects by increasing the time spent by animals in the open arms and reducing the SIH response, respectively. Conclusions and implications:We provide further evidence that NPS acts as a novel modulator of arousal and anxiety-related behaviours by promoting a unique pattern of effects: stimulation associated with anxiolysis. Therefore, NPS receptor ligands may represent innovative drugs for the treatment of sleep and anxiety disorders.

1 Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a speci®c G-protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identi®cation and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe 1 ]nociceptin(1-13)NH 2 acts as the ®rst truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. ]nociceptin(1-13)NH 2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe 1 ]nociceptin(1-13)NH 2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine-induced analgesia. 4 Collectively our data indicate that [Nphe 1 ]nociceptin(1-13)NH 2 , acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

2 UFP-101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high a nity (pK i 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP-101 competitively antagonizes the e ects of N/OFQ on GTPg 35 S binding in CHO hNOP cell membranes (pA 2 9.1) and on cyclic AMP accumulation in CHO hNOP cells (pA 2 7.1), being per se inactive at concentrations up to 10 mM. 3 In isolated peripheral tissues of mice, rats and guinea-pigs, and in rat cerebral cortex synaptosomes preloaded with [ 3 H]-5-HT, UFP-101 competitively antagonized the e ects of N/OFQ with pA 2 values in the range of 7.3 ± 7.7. In the same preparations, the peptide was inactive alone and did not modify the e ects of classical opioid receptor agonists. 4 UFP-101 is also active in vivo where it prevented the depressant action on locomotor activity and the pronociceptive e ect induced by 1 nmol N/OFQ i.c.v. in the mouse. In the tail withdrawal assay, UFP-101 at 10 nmol produces per se a robust and long lasting antinociceptive e ect. 5 UFP-101 is a novel, potent and selective NOP receptor antagonist which appears to be a useful tool for future investigations of the N/OFQ-NOP receptor system.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, regulates several central functions such as pain transmission, learning and memory, fear and anxiety and feeding and locomotor activity. It has been recently reported that NOP receptor antagonists induce antidepressant-like effects in the mouse forced swimming test (FST), i.e. reduce immobility time. This assay was used in the present study for further investigating the involvement of the NOP receptor in depression states. In male Swiss mice, intracerebroventricular injection (i.c.v) of the novel NOP receptor antagonist, UFP-101 (1-10 nmol) dose-dependently reduced the immobility time (control 192 +/- 14 s, UFP-101 91 +/- 15 s). The effect of 3 or 10 nmol UFP-101 was fully or partially reversed, respectively, by the coadministration of 1 nmol N/OFQ, which was inactive per se. NOP receptor knockout mice showed a reduced immobility time compared with their wild-type littermates (wild-type 215 +/- 10 s, knockout 143 +/- 12 s). Moreover, i.c.v. injected UFP-101 (10 nmol) significantly reduced immobility time in wild-type mice but not in NOP receptor knockout animals. In conclusion, these results, obtained using a combined pharmacological and genetic approach, indicate that blockade of the N/OFQ-NOP receptor signalling in the brain produces antidepressant-like effects in the mouse FST. These findings support the NOP receptor as a candidate target for the development of innovative antidepressant drugs.

1 The newly discovered neuropeptide nociceptin (NC) has recently been reported to be the endogenous ligand of the opioid-like orphan receptor. Despite its structural similarity to opioids, when injected intracerebroventricularly (i.c.v.) in the mouse, NC exerts a direct hyperalgesic e ect and reverses opioidinduced analgesia. In the present investigation, these two e ects of NC were evaluated under the same experimental conditions; in addition, a pharmacological characterization of the receptor mediating these central e ects of NC was attempted. 2 NC caused a dose dependent (0.1 ± 10 nmol/mouse), naloxone-insensitive reduction of tail withdrawal latency with a maximal e ect of about 50% of the reaction time observed in saline injected mice. In the same range of doses, NC inhibited morphine (1 nmol/mouse) induced analgesia.3 The e ects of the natural peptide were mimicked by NCNH 2 and NC(1 ± 13)NH 2 (all tested at 1 nmol/mouse) while 1 nmol NC(1 ± 9)NH 2 was found to be inactive either in reducing tail withdrawal latency or in preventing morphine analgesia. 4 [Phe 1 c(CH 2 -NH)Gly 2 ]NC(1 ± 13)NH 2 ([F/G]NC(1 ± 13)NH 2 ), which has been shown to antagonize NC e ects in the mouse vas deferens, acted as an agonist, mimicking NC e ects in both the experimental paradigms. In addition, when NC and [F/G]NC(1 ± 13)NH 2 were given together, their e ects were additive. 5 These results demonstrate that both the direct hyperalgesic action and the anti-morphine e ect of NC can be studied under the same experimental conditions in the mouse tail withdrawal assay. Moreover, the pharmacological characterization of the NC functional site responsible for these actions compared with the peripherally active site, indicates the existence of important di erences between peripheral and central NC receptors.