Jason M. Wiebelhaus

Δ9-Tetrahydrocannabinol and Endocannabinoid Degradative Enzyme Inhibitors Attenuate Intracranial Self-Stimulation in Mice

A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on ICSS. Thus, THC, MAGL inhibition, and dual FAAH-MAGL inhibition not only reduce ICSS, but also decrease other reinforced and nonreinforced behaviors.

Marijuana Dependence: Not Just Smoke and Mirrors

Marijuana (Cannabis sativa) is the most commonly used illicit drug worldwide as well as in the Unites States. Prolonged use of marijuana or repeated administration of its primary psychoactive constituent, Δ9-tetrahydrocannabinol (THC), can lead to physical dependence in humans and laboratory animals. The changes that occur with repeated cannabis use include alterations in behavioral, physiological, and biochemical responses. A variety of withdrawal responses occur in cannabis-dependent individuals: anger, aggression, irritability, anxiety and nervousness, decreased appetite or weight loss, restlessness, and sleep difficulties with strange dreams. But the long half-life and other pharmacokinetic properties of THC result in delayed expression of withdrawal symptoms, and because of the lack of contiguity between drug cessation and withdrawal responses the latter are not readily recognized as a clinically relevant syndrome. Over the past 30 years, a substantial body of clinical and laboratory animal research has emerged supporting the assertion that chronic exposure to cannabinoids produces physical dependence and may contribute to drug maintenance in cannabis-dependent individuals. However, no medications are approved to treat cannabis dependence and withdrawal. In this review, we describe preclinical and clinical research that supports the existence of a cannabinoid withdrawal syndrome. In addition, we review research evaluating potential pharmacotherapies (e.g., THC, a variety of antidepressant drugs, and lithium) to reduce cannabis withdrawal responses and examine how expanded knowledge about the regulatory mechanisms in the endocannabinoid system may lead to promising new therapeutic targets.

Effects of Acute and Repeated Administration of Oxycodone and Naloxone-Precipitated Withdrawal on Intracranial Self-Stimulation in Rats

Incidence of prescription opioid abuse and overdose, often led by oxycodone, continues to increase, producing twice as many overdose deaths as heroin. Surprisingly, preclinical reports relevant to oxycodone’s abuse-related effects are relatively sparse considering its history and patient usage. The goal of this study was to characterize dose- and time-dependent effects of acute and repeated oxycodone administration in a frequency-rate intracranial self-stimulation (ICSS) procedure, an assay often predictive of drug-related reinforcing effects, in male Sprague-Dawley rats. We hypothesized that oxycodone would produce a biphasic profile of rate-increasing and rate-decreasing effects maintained by ICSS similar to μ-opioid receptor agonists. Oxycodone (0.03, 0.3, 1, and 3 mg/kg, s.c.) produced dose- and time-dependent alterations on ICSS, with the predicted biphasic profile of rate-increasing effects at lower stimulation frequencies followed by rate-decreasing effects at higher frequencies. Peak effects were observed between 30 and 60 minutes, which were reversed by naloxone pretreatment (30 minutes). Tolerance to rate-decreasing effects was observed over a 5-day period when rats were treated with 1 mg/kg oxycodone twice a day. Subsequently, the dosing regimen was increased to 3 mg/kg twice a day over 10 days, although further marked tolerance did not develop. When then challenged with 10 mg/kg naloxone, a significant suppression below baseline levels of ICSS-maintained responding occurred indicative of dependence that recovered to baseline within 5 hours. The results of this study provide the first report of acute and chronic effects of oxycodone on responding maintained by ICSS presentation and the use of ICSS-maintained responding to characterize its tolerance and dependence effects.

Discriminative stimulus properties of N-desmethylclozapine, the major active metabolite of the atypical antipsychotic clozapine, in C57BL/6 mice.

N-desmethylclozapine (NDMC) is the major active metabolite of the atypical antipsychotic drug clozapine and may contribute to the therapeutic efficacy of clozapine. Although they share many pharmacological features, it is noteworthy that NDMC is a partial dopamine D2 and cholinergic muscarinic M1/M4 agonist, whereas clozapine is a weak dopamine D2 receptor inverse agonist/antagonist and a nonselective muscarinic antagonist. To better understand the in-vivo pharmacological mechanisms of these drugs, male C57BL/6NHsd-wild-type mice were trained to discriminate 10.0 mg/kg NDMC from vehicle in a two-lever drug discrimination procedure for food reward. It was found that the parent drug clozapine fully substituted for NDMC, whereas the typical antipsychotic drug haloperidol (dopamine D2 antagonist) and the atypical antipsychotic drug aripiprazole (D2 partial agonist) did not substitute for NDMC. These results demonstrated that clozapine and its major metabolite NDMC share in-vivo behavioral properties (i.e. discriminative stimulus properties) that are likely due to shared pharmacological mechanisms that differ from other antipsychotic drugs. The discriminative stimulus properties of NDMC probably reflect a compound cue similar to that of its parent drug clozapine due to its diverse binding profile.

∆9-Tetrahydrocannabinol-dependent mice undergoing withdrawal display impaired spatial memory

RationaleCannabis users display a constellation of withdrawal symptoms upon drug discontinuation, including sleep disturbances, irritability, and possibly memory deficits. In cannabinoid-dependent rodents, the CB1 antagonist rimonabant precipitates somatic withdrawal and enhances forskolin-stimulated adenylyl cyclase activity in cerebellum, an effect opposite that of acutely administered ∆9-tetrahydrocannabinol (THC), the primary constituent in cannabis.ObjectivesHere, we tested whether THC-dependent mice undergoing rimonabant-precipitated withdrawal display short-term spatial memory deficits, as assessed in the Morris water maze. We also evaluated whether rimonabant would precipitate adenylyl cyclase superactivation in hippocampal and cerebellar tissue from THC-dependent mice.ResultsRimonabant significantly impaired spatial memory of THC-dependent mice at lower doses than those necessary to precipitate somatic withdrawal behavior. In contrast, maze performance was near perfect in the cued task, suggesting sensorimotor function and motivational factors were unperturbed by the withdrawal state. Finally, rimonabant increased adenylyl cyclase activity in cerebellar, but not in hippocampal, membranes.ConclusionsThe memory disruptive effects of THC undergo tolerance following repeated dosing, while the withdrawal state leads to a rebound deficit in memory. These results establish spatial memory impairment as a particularly sensitive component of cannabinoid withdrawal, an effect that may be mediated through compensatory changes in the cerebellum.

The metabolites N-desmethylclozapine and N-desmethylolanzapine produce cross-tolerance to the discriminative stimulus of the atypical antipsychotic clozapine in C57BL/6 mice.

It has been previously shown that cross-tolerance to the discriminative stimulus properties of clozapine can be demonstrated with the drug discrimination paradigm. This study examined the ability of N-desmethylclozapine and N-desmethylolanzapine (metabolites of the atypical antipsychotic drugs clozapine and olanzapine, respectively) to induce cross-tolerance to the discriminative stimulus effects of clozapine. After C57BL/6 mice were trained to reliably discriminate 2.5 mg/kg clozapine from vehicle, a clozapine generalization curve was generated. Next, training was suspended and the mice received a maintenance dosing regimen in which they were injected twice daily with 10 mg/kg N-desmethylclozapine for 10 days. Then a second clozapine generalization curve was generated. This was followed by a 10-day washout period during which the mice did not receive drug injections or discrimination training. Finally, a third clozapine generalization curve was generated. These same procedures were followed for N-desmethylolanzapine (10 mg/kg twice daily during maintenance dosing). Both N-desmethylclozapine and N-desmethylolanzapine produced significant rightward shifts in the clozapine generalization curve indicating cross-tolerance between N-desmethylclozapine and clozapine and between N-desmethylolanzapine and clozapine. After a washout period with no training or drug administration this cross-tolerance effect was lost for both metabolites. This cross-tolerance drug discrimination procedure demonstrated in-vivo similarities between these two metabolites and clozapine and suggests that common underlying pharmacological mechanisms were responsible for the cross-tolerance that was observed. These findings also demonstrated that this procedure may be useful for identifying drugs with therapeutic efficacy similar to the atypical antipsychotic clozapine under repeated dosing conditions.

Clozapine and N-Methyl-d-Aspartate have positive modulatory actions on their respective discriminative stimulus properties in C57BL/6 mice

The impairment of N-Methyl-D-Aspartate receptors is thought to contribute to negative symptoms and cognitive deficits. In vitro studies suggest that atypical antipsychotic drugs like clozapine may help to alleviate these deficits by enhancing glutamatergic function. The present study examined the in vivo interaction of clozapine with N-Methyl D-aspartate by training one group of C57BL/6 mice to discrimination 2.5 mg/kg clozapine from vehicle and another group to discriminate 30 mg/kg N-Methyl D-aspartate from vehicle in a two-lever drug discrimination task. Cross-generalization testing revealed that N-Methyl D-aspartate (3-56 mg/kg) failed to substitute for clozapine in the clozapine-trained mice, while clozapine (0.625 mg/kg) produced partial substitution in the N-Methyl D-aspartate-trained mice. Interestingly, administration of a low, non-generalizing dose of each training drug in combination with the full range of doses of the alternate training drug produced full and dose-dependent substitution in both clozapine- and N-Methyl D-aspartate-trained mice. The α(1) antagonist prazosin fully and dose-dependently substituted for both clozapine and N-Methyl D-aspartate. These results suggest that the shared discriminative stimulus properties between clozapine and N-Methyl D-aspartate may be mediated through indirect mechanisms, possibly in part through α(1) adrenergic antagonism.

Nociceptin/orphanin FQ receptors modulate the discriminative stimulus effects of oxycodone in C57BL/6 mice

BACKGROUND Nociceptin/orphanin FQ (NOP) receptor ligands have shown efficacy as putative analgesics and can modulate the abuse-related effects of opioids, suggesting therapeutic applications. The discriminative stimulus effects of a drug are related to their subjective effects, a predictor of abuse potential. To determine whether activation of NOP receptors could alter the subjective effects of an abused opioid analgesic, a novel oxycodone discrimination was established in mice, characterized with positive and negative controls, and its expression evaluated with a NOP receptor agonist. METHODS Adult male C57BL/6 mice were trained to discriminate 1.3 mg/kg oxycodone from vehicle in a two-lever operant procedure. The discrimination was characterized with naloxone challenge, and generalization tests with the μ-opioid receptor agonists, heroin and morphine, and the κ-opioid receptor selective agonist, U50488. Subsequently, effects of the NOP agonist Ro64-6198 were evaluated with and without oxycodone. RESULTS Oxycodone generalization occurred in a dose-dependent manner and was reversed by naloxone pretreatment. Heroin and morphine, but not U50488, substituted for oxycodone. Co-treatment of 1 mg/kg Ro64-6198 with the oxycodone training dose reduced % oxycodone lever responding (%OLR) and restored response rates to vehicle control levels. J-113397, a NOP antagonist, reversed these effects. Co-administration of 1 mg/kg Ro64-6198 with a range of oxycodone doses resulted in rightward dose-effect curve shifts in %OLR and response rates compared to oxycodone alone. CONCLUSIONS These results provide additional evidence that NOP receptor activation can modulate the subjective effects of opioid analgesics and represent the first characterization of oxycodones discriminative stimulus effects in mice.