Characterization of the nociceptin receptor (ORL-1) agonist, Ro64-6198, in tests of anxiety across multiple species.

RATIONALE: Previous studies have demonstrated behaviors indicative of anxiolysis in rats pretreated with the nociceptin receptor (opioid receptor like-1, ORL-1) agonist, Ro64-6198. OBJECTIVES: The aim of this study was to examine the effects of Ro64-6198 in anxiety models across three species: rat, guinea pig, and mouse. In addition, the receptor specificity of Ro64-6198 was studied, using the ORL-1 receptor antagonist, J-113397, and ORL-1 receptor knockout (KO) mice. Finally, neurological studies examined potential side effects of Ro64-6198 in the rat and mouse. RESULTS: Ro64-6198 (3-10 mg/kg) increased punished responding in a rat conditioned lick suppression test similarly to chlordiazepoxide (6 mg/kg). This effect of Ro64-6198 was attenuated by J-113397 (10 mg/kg), but not the mu opioid antagonist, naltrexone (3 mg/kg). In addition, Ro64-6198 (1-3 mg/kg) reduced isolation-induced vocalizations in rat and guinea pig pups. Ro64-6198 (3 mg/kg) increased the proportion of punished responding in a mouse Geller-Seifter test in wild-type (WT) but not ORL-1 KO mice, whereas diazepam (1-5.6 mg/kg) was effective in both genotypes. In rats, Ro64-6198 reduced locomotor activity (LMA) and body temperature and impaired rotarod, beam walking, and fixed-ratio (FR) performance at doses of 10-30 mg/kg, i.e., three to ten times higher than an anxiolytic dose. In WT mice, Ro64-6198 (3-10 mg/kg) reduced LMA and rotarod performance, body temperature, and FR responding, but these same measures were unaffected in ORL-1 KO mice. Haloperidol (0.3-3 mg/kg) reduced these measures to a similar extent in both genotypes. These studies confirm the potent, ORL-1 receptor-mediated, anxiolytic-like effects of Ro64-6198, extending the findings across three species. Ro64-6198 has target-based side effects, although the magnitude of these effects varies across species.

Transgenic overexpression of neuromedin U promotes leanness and hypophagia in mice.

Recent work has shown that neuromedin U (NmU), a peptide initially identified as a smooth muscle contractor, may play a role in regulating food intake and energy homeostasis. To further evaluate this putative function, we measured food intake, body weight, energy expenditure and glucose homeostasis in transgenic mice that ubiquitously overexpress murine proNmU. NmU transgenic mice were lighter and had less somatic and liver fat, were hypophagic, and had improved insulin sensitivity as judged by an intraperitoneal insulin tolerance test. Transgenic mice had higher levels of hypothalamic NPY, POMC and MCH mRNA. There was no difference in O2 consumption between genotypes; however, NmU transgenic mice displayed a modest increase in respiratory quotient during food deprivation and refeeding. There were no behavioral disturbances in the NmU transgenic mice that could account for the results (e.g. changes in locomotor activity). When placed on a high-fat diet, transgenic mice remained lighter than wild-type mice and ate less, but gained weight at a rate similar to wild-type mice. Despite the increased weight gain with high-fat feeding, glucose tolerance was significantly improved in the transgenic mice. These findings support the hypothesized role of NmU as an endogenous anorexigenic peptide.

Automated method for the measurement of fentanyl-induced muscular rigidity.

An automated method is described that can accurately and reliably measure weight displacement resulting from hindlimb extension due to muscular rigidity following opioid administration in rats. IV administration of fentanyl (0.035 mg/kg) immediately induced rigidity. Rigidity was dose dependently reversed by the alpha 2-agonist clonidine with an ED50 value equal to 0.011 mg/kg IV. Second, rigidity was restored following administration of the alpha 2-antagonist idazoxan (0.3 mg/kg, IV) thereby confirming an alpha 2-mediated mechanism of action. Previously, reversal of fentanyl-induced muscular rigidity was measured by subjective rating criteria not suitable for quantitative potency comparisons. The new automated rigidity model provides a simple yet precise measurement of the ability of alpha 2-agonists to attenuate opioid-induced muscular rigidity in rats.

An in vivo alpha-2 assay reversal of opioid-induced muscular rigidity and neuroleptic-induced ptosis.

A method is described to detect selective alpha-2 adrenergic agonists in vivo. Palpebral ptosis is induced in rats by the neuroleptic agent haloperidol (Hal), or by tetrabenazine (TBZ) methanesulfonate. Twenty minutes later, test compounds are injected, and ptosis is scored. In a separate test, muscular rigidity is induced by the opioid, fentanyl, and subsequently, test compounds are assessed for their ability to reverse muscular rigidity. Results indicate that only alpha-2 agonists reliably reverse neuroleptic-induced and TBZ-induced ptosis, as well as opioid-induced rigidity. An alpha-1 antagonist reversed only rigidity, whereas, alpha-2 antagonists and beta-agonists were generally ineffective in all tests. Therefore, the ability to reverse neuroleptic and TBZ-induced ptosis along with the ability to reverse opioid-induced muscular rigidity is a characteristic unique to alpha-2 agonists.

The discriminative stimulus effect of MK-801 in ketamine-trained rats.

MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate] was assessed for its discriminative stimulus properties in rats trained to discriminate ketamine (7 mg/kg) from vehicle. MK-801 generalized to ketamine in a dose dependent manner with a maximum effect at 0.2 mg/kg, while ketamine generalized fully at 10 mg/kg. These results indicate that ketamine and MK-801 may share a common mechanism of action, which is related to the phencyclidine recognition site in the brain.