Opioid and Cannabinoid Modulation of Precipitated Withdrawal in -Tetrahydrocannabinol and Morphine-Dependent Mice

The goal of the present study was to elucidate the relationship between cannabinoid and opioid systems in drug dependence. The CB1 cannabinoid receptor antagonist SR 141716A precipitated both paw tremors and head shakes in four different mouse strains that were treated repeatedly with -tetrahydrocannabinol ( -THC). SR 141716A-precipitated -THC withdrawal was ameliorated in -opioid receptor knockout mice compared with the wild-type control animals and failed to occur in mice devoid of CB1 cannabinoid receptors. An acute injection of morphine in -THC-dependent mice undergoing SR 1417161A-precipitated withdrawal dose dependently decreased both paw tremors, antagonist dose 50 (AD50) (95% CL) 0.035 (0.03–0.04), and head shakes, AD50 (95% CL) 0.07 (0.04–0.12). In morphine-dependent mice, the opioid antagonist naloxone precipitated head shakes, paw tremors, diarrhea, and jumping. As previously reported, naloxone-precipitated morphine withdrawal failed to occur in -opioid knockout mice and was significantly decreased in CB1 cannabinoid receptor knockout mice. Acute treatment of -THC in morphinedependent mice undergoing naloxone-precipitated withdrawal blocked paw tremors, AD50 (95% CL) 0.5 (0.3–1.0), and head shakes AD50 (95% CL) 0.6 (0.57–0.74) in dose-dependent manners, but failed to diminish the occurrence of diarrhea or jumping. Finally, naloxone and SR 141716A failed to elicit any overt effects in -THC-dependent and morphine-dependent mice, respectively. These findings taken together indicate that the -opioid receptor plays a modulatory role in cannabinoid dependence, thus implicating a reciprocal relationship between the cannabinoid and opioid systems in dependence. Cannabis sativa has been the most prevalently used illicit drug in the United States for the last several decades (Johnston et al., 1998). Also contributing to increases in the use of this drug is the growing popular support for its decriminalization for medicinal purposes. The occurrence of a positive correlation between marijuana use and marijuana dependence (Chen et al., 1997) raises concern that physical withdrawal might become an issue when a recreational user or patient abruptly discontinues the drug. Indeed, more than 25 years ago, an abrupt cannabinoid withdrawal syndrome was described in human subjects following discontinuation from chronic oral -THC (Jones and Benowitz, 1976; Jones et al., 1976). More recently, subjects undergoing abrupt withdrawal from repeated administration of either oral -THC (Haney et al., 1999a) or marijuana smoke inhalation (Haney et al., 1999b) exhibited abstinence symptoms that included subjective effects of anxiety, irritability, and stomach pain, as well as decreases in food intake. Before the availability of the CB1 cannabinoid antagonist SR 141716A (Rinaldi-Carmona et al., 1994), studies investigating cannabinoid abstinence withdrawal in laboratory animals yielded contradictory findings. Whereas some evidence supported the occurrence of cannabinoid dependence (Kaymakcalan, 1979; Beardsley et al., 1986), other studies failed to observe any abrupt withdrawal signs (McMillan et al., 1970; Leite and Carlini, 1974). In contrast, administration of SR 141716A precipitated reliable withdrawal signs in several species that had been treated repeatedly with cannabinoids, including mice (Cook et al., 1998; Hutcheson et al., 1998), rats (Aceto et al., 1995; Tsou et al., 1995), and dogs (Lichtman et al., 1998). As in the case of opioids, mice will self-administer the cannabinoid aminoalkylindole WIN 55,212-2 (Martellotta et al., 1998). Through the use of these models, the neurochemical mechanisms underlying cannabinoid dependence can now be systematically investigated. Substantial evidence is mounting that the antinociceptive effects, drug reinforcing actions, and dependence liability of morphine and -THC share common neuroanatomical sites. In particular, converging data suggest that cannabinoids influence opioid withdrawal. Anatomical studies have found that CB1 cannabinoid receptor and -opioid receptor mRNA This work was supported by National Institutes of Health Grants DA 03672 and DA 09789. ABBREVIATIONS: -THC, -tetrahydrocannabinol; SR 141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1Hpyrazole-3-carboxamide HCl; AD50, antagonist dose 50; CL, confidence limits. 0022-3565/01/2983-1007–1014$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 298, No. 3 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3812/923555 JPET 298:1007–1014, 2001 Printed in U.S.A. 1007 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from are colocalized in brain limbic areas associated with dependence (Navarro et al., 1998). It has long been known that -THC produces a moderate amelioration of naloxone-precipitated withdrawal in morphine-dependent mice (Bhargava, 1976a,b, 1978) and rats (Hine et al., 1975). The endogenous cannabinoid anandamide was also shown to decrease naloxone-induced morphine withdrawal (Vela et al., 1995). Other compelling evidence supporting a link between opioid dependence and the cannabinoid system is that CB1 cannabinoid receptor knockout mice exhibited substantial decreases in both morphine self-administration and naloxoneprecipitated morphine withdrawal (Ledent et al., 1999). This amelioration of opioid withdrawal suggests the possibility that endocannabinoids modulate opioid dependence. Alternatively, epistasis in which the affect of gene disruption is modified by the genetic background on which it is placed can also account for amelioration of naloxone-precipitated morphine withdrawal in CB1 cannabinoid receptor knockout mice. Whereas a CD-1 background strain was used for the CB1 cannabinoid receptor knockout mice in the opioid dependence report (Ledent et al., 1999), the present study used a C57BL/6 background strain for the knockouts. A reduction in opioid dependence in both backgrounds would provide further support for the involvement of the cannabinoid system in opioid dependence. In addition, we examined whether -THC would reduce naloxone-precipitated morphine withdrawal reactions and whether SR 141716A would precipitate withdrawal in morphine-dependent mice. In contrast to the growing body of research that is establishing a role for cannabinoid receptors on modulating opioid dependence, relatively few studies have focused on the influence of opioid receptors on cannabinoid dependence. The finding that SR 141716A-precipitated -THC withdrawal was significantly attenuated in preproenkephalin-deficient mice suggests that opioid systems may modulate cannabinoid dependence (Valverde et al., 2000). In the present study, we investigated whether the expression of SR 141716A-precipitated cannabinoid withdrawal would also be altered in -opioid receptor-deficient mice. In addition, the effects of acute morphine administration on cannabinoid withdrawal were examined. Because previous research has yielded mixed results on whether an opioid antagonist can precipitate withdrawal in -THC-dependent animals (McMillan et al., 1971; Hirschhorn and Rosecrans, 1974; Kaymakcalan et al., 1977), we also evaluated whether naloxone would precipitate withdrawal effects in -THC-dependent mice. In all experiments, mice were evaluated for head shakes and paw tremors, two cannabinoid withdrawal behaviors that can be reliably quantitated (Cook et al., 1998), as well as for diarrhea and jumping, indices indicative of opioid withdrawal (Way et al., 1969). Materials and Methods Subjects. Swiss-Webster and ICR mice were purchased from Harlan Laboratories (Dublin, VA). C57BL/6 and DBA/2 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice deficient of the -opioid receptor (Loh et al., 1998) and CB1 cannabinoid receptor (Zimmer et al., 1999) mice were born in the Virginia Commonwealth University vivarium from breeding pairs that were initially provided by Drs. Horace Loh (University of Michigan, Ann Arbor, MI) and Andreas Zimmer (National Institutes of Health, Bethesda, MD), respectively. All subjects were male, weighed 22 to 30 g, and were housed six animals per cage in an American association for the Accreditation of Laboratory Animal Care-approved facility. The study was approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University. Mice were given unlimited access to food and water and were maintained on a 12:12-h light/dark cycle. Drugs. -THC, SR 141716A, morphine sulfate, and morphine sulfate pellets (25 or 75 mg) were provided by the National Institute on Drug Abuse (Bethesda, MD). Naloxone hydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO). -THC and SR 141716A were dissolved in ethanol, followed by addition of Emulphor-620 (Rhone-Poulenc, Princeton, NJ), and diluted with 0.9% saline to form a vehicle mixture of ethanol/Emulphor/saline in a ratio of 1:1:18. Morphine and naloxone were dissolved in 0.9% saline. All drug injection volumes were made based on mouse body weight, with 0.1 ml of dissolved drug volume given for every 10 g of body weight. -THC, morphine, and naloxone were given s.c. and SR 141716A