Differential effects of endocannabinoid catabolic inhibitors on morphine withdrawal in mice
Introduction
Opioid abuse and dependence continue to present a serious threat to public health (Johnston et al., 2010). Fear of withdrawal symptoms that include diarrhea, emesis, body aches, anxiety, dysphoria (Farrell, 1994, Gossop, 1988, Jasinski, 1981, Wesson and Ling, 2003) are thought to contribute to the maintenance of drug-taking in opioid dependent individuals. Likewise, continued opioid use alleviates the withdrawal state, thus serving as a negative reinforcer (Koob and Le Moal, 2005). Current available treatments for opioid dependence, such as methadone and buprenorphine, possess their own abuse liability (Cicero and Inciardi, 2005) and are not fully effective at alleviating withdrawal (Dyer et al., 1999, Kuhlman et al., 1998). Thus, new pharmacotherapies that lack abuse potential are needed to alleviate opioid withdrawal.
Extracts from cannabis and the primary constituent of marijuana, Δ9-tetrahydrocannabinol (THC), have long been known to ameliorate somatic morphine withdrawal signs (Birch, 1889, Hine et al., 1975). THC produces the bulk of its pharmacological effects through two known G-protein coupled receptors, cannabinoid type-1 (CB1; Matsuda et al., 1990) and type-2 (CB2; Munro et al., 1993). These receptors, as well as the endogenous cannabinoids (endocannabinoids) 2-arachidonoylglycerol (2-AG; Mechoulam et al., 1995, Sugiura et al., 1995) and N-arachidonoylethanolamine (anandamide, AEA; Devane et al., 1992) comprise the endogenous cannabinoid system. These endocannabinoids are rapidly degraded by the respective enzymes fatty acid amide hydrolase (FAAH; Cravatt et al., 2001) and monoacylglycerol lipase (MAGL; Dinh et al., 2002). Selective inhibitors of these endocannabinoid degradative enzymes reduce somatic signs of opioid withdrawal (e.g., jumping, paw fluttering, head/body shaking, weight loss, diarrhea; Ramesh et al., 2013, Ramesh et al., 2011). However, it is unknown whether the anti-withdrawal effects extend to the affective components of morphine withdrawal.
Opioid-dependent individuals undergoing withdrawal experience aversive subjective effects, a process that is modeled in the Pavlovian conditioned place avoidance (CPA) paradigm. In this assay morphine-dependent rats (Gracy et al., 2001, Hand et al., 1988, Parker and Rennie, 1992, Schnur et al., 1992, Stinus et al., 2000, Stinus et al., 1990, Watanabe et al., 2003) or mice (Broseta et al., 2005, Maldonado et al., 2003, Olson et al., 2006, Sato et al., 2005, Shoblock and Maidment, 2005) undergo conditioning trials in which naloxone precipitates an aversive interoceptive stimulus that is paired with a distinct chamber. Following subsequent placement into the test apparatus, the subjects spend less time in the conditioning chamber than in the control chamber (i.e., CPA). In this assay, lower doses of naloxone produce CPA than those doses necessary to elicit somatic withdrawal signs (Caillé et al., 1999, Frenois et al., 2002). Furthermore, the α2 adrenergic agonist clonidine, which is known to reduce opioid withdrawal in humans (Gold et al., 1978, Gossop, 1988), attenuates opioid withdrawal CPA (Kosten, 1994, Schulteis et al., 1998a). Given the colocalization of CB1 and mu opioid receptors in the locus coeruleus (Scavone et al., 2010), periaqueductal gray (Wilson-Poe et al., 2012) and nucleus accumbens (Pickel et al., 2004), cannabinoid receptors are advantageously positioned to compensate for the hyperactivity in neurons that are key to the expression of both somatic and aversive aspects of opioid withdrawal (Frenois et al., 2002, Lane-Ladd et al., 1997, Nestler and Tallman, 1988, Stinus et al., 1990, Widnell et al., 1994).
The purpose of the present study was to test whether stimulation of CB1 receptors via administration of THC or inhibition of endocannabinoid catabolic enzymes would prevent the acquisition of naloxone-precipitated morphine withdrawal CPA and withdrawal-related jumping behavior in mice. To this end, the MAGL inhibitor JZL184 (Long et al., 2009a) or FAAH inhibitor PF-3845 (Ahn et al., 2009) was administered at the time of conditioning. In addition, we evaluated whether the dual FAAH/MAGL inhibitor SA-57 (Niphakis et al., 2012) would prevent morphine withdrawal CPA. Of note, combined inhibition of these two major endocannabinoid hydrolytic enzymes elicits enhanced cannabimimetic activity compared with single enzyme inhibition (Long et al., 2009b, Seillier et al., 2014, Wise et al., 2012). Moreover, each of these inhibitors reduces naloxone-precipitated and spontaneous somatic withdrawal signs in morphine-dependent mice (Ramesh et al., 2013, Ramesh et al., 2011). Finally, each inhibitor was tested in non-dependent mice to ascertain whether the compounds elicited reward-related or aversive effects in the place conditioning assay.
Section snippets
Subjects
Male ICR mice (6–8 weeks old; Harlan, Indianapolis, IN) with a body mass of 27–32 g were used for all experiments. Mice were group-housed (four per cage) on a 12/12 light/dark cycle (lights on at 0600 h) and given food and water ad libitum. All animal protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee, were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal
Naloxone-precipitated morphine withdrawal produces conditioned place avoidance behavior at a dose that does not elicit this effect in non-dependent mice
Initial experiments were designed to determine the optimal dose of naloxone that produced CPA in morphine dependent mice, but not in placebo-pelleted mice. The dose–response relationship of naloxone (0.03, 0.056, and 0.1 mg/kg) was examined in morphine- and placebo-pelleted mice. Naloxone produced a significant CPA in morphine-pelleted mice [F(3,74) = 4.3; p < 0.01; Fig. 1A] as well as in placebo-pelleted animals [F(3,80) = 4.3; p < 0.01; Fig. 1B]. Post hoc analyses revealed that morphine-pelleted mice
Discussion
The experiments in the present study tested whether inhibition of endocannabinoid catabolic enzymes or THC administration would reduce aversive aspects of morphine withdrawal. This hypothesis was tested using a Pavlovian conditioning procedure in which morphine-dependent mice display a CPA to a compartment associated with naloxone-precipitated withdrawal. Neither THC nor the endocannabinoid catabolic inhibitors significantly reduced morphine withdrawal CPA, though each of these compounds, with
Author disclosures
Role of funding source
Financial support was provided by the National Institutes of Health grants [T32DA007027, R01DA032933, P01DA009789, and P01DA01725] and a Toni Rosenberg Fellowship. The funding source had no involvement in the research reported in this manuscript or the writing of the manuscript.
Contributors
T.F. Gamage conducted the bulk of the studies, data analyses, and writing of the manuscript. B.M. Ignatowska-Jankowska conducted a subset of the experiments as well as contributed to the
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