Behavioral Battery for Testing Candidate Analgesics in Mice. II. Effects of Endocannabinoid Catabolic Enzyme Inhibitors and ∆9-Tetrahydrocannabinol.

Enhanced signaling of the endocannabinoid (eCB) system through inhibition of the catabolic enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) has received increasing interest for development of candidate analgesics. This study compared effects of MAGL and FAAH inhibitors with effects of ∆9-tetrahydrocannabinol (THC) using a battery of pain-stimulated, pain-depressed, and pain-independent behaviors in male and female mice. Intraperitoneal injection of dilute lactic acid (IP acid) served as an acute visceral noxious stimulus to stimulate two behaviors (stretching, facial grimace) and depress two behaviors (rearing, nesting). Nesting and locomotion were also assessed in the absence of IP acid as pain-independent behaviors. THC and a spectrum of six eCB catabolic enzyme inhibitors ranging from MAGL- to FAAH-selective were assessed for effectiveness to alleviate pain-related behaviors at doses that did not alter pain-independent behaviors. The MAGL-selective inhibitor MJN110 produced the most effective antinociceptive profile, with 1.0 mg/kg alleviating IP acid effects on stretching, grimace, and nesting without altering pain-independent behaviors. MJN110 effects on IP acid-depressed nesting had a slow onset and long duration (40 minutes to 6 hours), were blocked by rimonabant, and tended to be greater in females. As inhibitors increased in FAAH selectivity, antinociceptive effectiveness decreased. PF3845, the most FAAH-selective inhibitor, produced no antinociception up to doses that disrupted locomotion. THC decreased IP acid-stimulated stretching and grimace at doses that did not alter pain-independent behaviors; however, it did not alleviate IP acid-induced depression of rearing or nesting. These results support further consideration of MAGL-selective inhibitors as candidate analgesics for acute inflammatory pain. SIGNIFICANCE STATEMENT: This study characterized a spectrum of endocannabinoid catabolic enzyme inhibitors ranging in selectivity from monoacylglycerol lipase-selective to fatty acid amide hydrolase-selective in a battery of pain-stimulated, pain-depressed, and pain-independent behaviors previously pharmacologically characterized in a companion paper. This battery provides a method for prioritizing candidate analgesics by effectiveness to alleviate pain-related behaviors at doses that do not alter pain-independent behaviors, with inclusion of pain-depressed behaviors increasing translational validity and decreasing susceptibility to motor-depressant false positives.

Cerebrospinal-fluid drain-related complications in patients undergoing open and endovascular repairs of thoracic and thoraco-abdominal aortic pathologies: a systematic review and meta-analysis.

BACKGROUND: Cerebrospinal-fluid (CSF) drainage is recommended by current guidelines for spinal protection during open and endovascular repairs of thoracic and thoraco-abdominal aortic aneurysms. In the published literature, great variability exists in the rate of CSF-related complications and morbidity. Herein, we perform a systematic review and meta-analysis on the incidence of CSF drainage-related complications, and compare the complication rates between open and endovascular repairs. METHODS: The systematic review was conducted according to the Meta-Analysis of Observational Studies in Epidemiology guidelines. Thirty-four studies (4714 patients) were included in the quantitative analysis. The CSF drainage-related complications were categorised as mild, moderate, and severe. Pooled event rates for each complication category were estimated using a random-effect model. Random-effect uni- and multivariable meta-regression analyses were used to assess the effect of aortic-repair approach (open vs endovascular) and the CSF drainage criteria on CSF drainage-related complications. RESULTS: The pooled event rates were 6.5% [95% confidence interval (CI): 4.3-9.8%] for overall complications, 2% (95% CI: 1.1-3.4%) for minor complications, 3.7% (95% CI: 2.5-5.6%) for moderate complications, and 2.5% (95% CI: 1.6-3.8%) for severe complications. The drainage-related-mortality pooled event rate was 0.9% (95% CI: 0.6-1.4%). The uni- and multivariable meta-regression analyses showed no difference in complication rates between the open and endovascular approaches, or between the different CSF drainage protocols. CONCLUSION: The complication rate for CSF drainage is not negligible. Our results help define a more accurate risk-benefit ratio for CSF drain placement at the time of repair of thoracic and thoraco-abdominal aneurysms.

Apparent CB1 Receptor Rimonabant Affinity Estimates: Combination with THC and Synthetic Cannabinoids in the Mouse In Vivo Triad Model.

Synthetic cannabinoids (SCs) represent an emerging class of abused drugs associated with psychiatric complications and other substantial health risks. These ligands are largely sold over the internet for human consumption, presumably because of their high cannabinoid 1 receptor (CB1R) affinity and their potency in eliciting pharmacological effects similar to Δ9-tetrahydrocannabinol (THC), as well as circumventing laws illegalizing this plant. Factors potentially contributing to the increased prevalence of SC abuse and related hospitalizations, such as increased CB1R efficacy and non-CB1R targets, highlight the need for quantitative pharmacological analyses to determine receptor mediation of the pharmacological effects of cannabinoids. Accordingly, the present study used pA2 and pKB analyses for quantitative determination of CB1R mediation in which we utilized the CB1R-selective inverse agonist/antagonist rimonabant to elicit rightward shifts in the dose-response curves of five SCs (i.e., A-834,735D; WIN55,212-2; CP55,950; JWH-073; and CP47,497) and THC in producing common cannabimimetic effects (i.e., catalepsy, antinociception, and hypothermia). The results revealed overall similarity of pA2 and pKB values for these compounds and suggest that CB1Rs, and not other pharmacological targets, largely mediated the central pharmacological effects of SCs. More generally, affinity estimation offers a powerful pharmacological approach to assess potential receptor heterogeneity subserving in vivo pharmacological effects of SCs.

Stratification of Cannabinoid 1 Receptor (CB1R) Agonist Efficacy: Manipulation of CB1R Density through Use of Transgenic Mice Reveals Congruence between In Vivo and In Vitro Assays.

Synthetic cannabinoids (SCs) are an emerging class of abused drugs that differ from each other and the phytocannabinoid ∆9-tetrahydrocannabinol (THC) in their safety and cannabinoid-1 receptor (CB1R) pharmacology. As efficacy represents a critical parameter to understanding drug action, the present study investigated this metric by assessing in vivo and in vitro actions of THC, two well-characterized SCs (WIN55,212-2 and CP55,940), and three abused SCs (JWH-073, CP47,497, and A-834,735-D) in CB1 (+/+), (+/-), and (-/-) mice. All drugs produced maximal cannabimimetic in vivo effects (catalepsy, hypothermia, antinociception) in CB1 (+/+) mice, but these actions were essentially eliminated in CB1 (-/-) mice, indicating a CB1R mechanism of action. CB1R efficacy was inferred by comparing potencies between CB1 (+/+) and (+/-) mice [+/+ ED50 /+/- ED50], the latter of which has a 50% reduction of CB1Rs (i.e., decreased receptor reserve). Notably, CB1 (+/-) mice displayed profound rightward and downward shifts in the antinociception and hypothermia dose-response curves of low-efficacy compared with high-efficacy cannabinoids. In vitro efficacy, quantified using agonist-stimulated [35S]GTPγS binding in spinal cord tissue, significantly correlated with the relative efficacies of antinociception (r = 0.87) and hypothermia (r = 0.94) in CB1 (+/-) mice relative to CB1 (+/+) mice. Conversely, drug potencies for cataleptic effects did not differ between these genotypes and did not correlate with the in vitro efficacy measure. These results suggest that evaluation of antinociception and hypothermia in CB1 transgenic mice offers a useful in vivo approach to determine CB1R selectivity and efficacy of emerging SCs, which shows strong congruence with in vitro efficacy.

Diacylglycerol lipase ß inhibition reverses nociceptive behaviour in mouse models of inflammatory and neuropathic pain.

BACKGROUND AND PURPOSE: Inhibition of diacylglycerol lipase (DGL)ß prevents LPS-induced pro-inflammatory responses in mouse peritoneal macrophages. Thus, the present study tested whether DGLß inhibition reverses allodynic responses of mice in the LPS model of inflammatory pain, as well as in neuropathic pain models. EXPERIMENTAL APPROACH: Initial experiments examined the cellular expression of DGLß and inflammatory mediators within the LPS-injected paw pad. DAGL-ß (-/-) mice or wild-type mice treated with the DGLß inhibitor KT109 were assessed in the LPS model of inflammatory pain. Additional studies examined the locus of action for KT109-induced antinociception, its efficacy in chronic constrictive injury (CCI) of sciatic nerve and chemotherapy-induced neuropathic pain (CINP) models. KEY RESULTS: Intraplantar LPS evoked mechanical allodynia that was associated with increased expression of DGLß, which was co-localized with increased TNF-α and prostaglandins in paws. DAGL-ß (-/-) mice or KT109-treated wild-type mice displayed reductions in LPS-induced allodynia. Repeated KT109 administration prevented the expression of LPS-induced allodynia, without evidence of tolerance. Intraplantar injection of KT109 into the LPS-treated paw, but not the contralateral paw, reversed the allodynic responses. However, i.c.v. or i.t. administration of KT109 did not alter LPS-induced allodynia. Finally, KT109 also reversed allodynia in the CCI and CINP models and lacked discernible side effects (e.g. gross motor deficits, anxiogenic behaviour or gastric ulcers). CONCLUSIONS AND IMPLICATIONS: These findings suggest that local inhibition of DGLß at the site of inflammation represents a novel avenue to treat pathological pain, with no apparent untoward side effects.

Atheroprotective immunity and cardiovascular disease: therapeutic opportunities and challenges.

Emerging knowledge of the role of atheroprotective immune responses in modulating inflammation and tissue repair in atherosclerotic lesions has provided promising opportunities to develop novel therapies directly targeting the disease process in the artery wall. Regulatory T (Treg) cells have a protective role through release of anti-inflammatory cytokines and suppression of autoreactive effector T cells. Studies in experimental animals have shown that blocking the generation or action of Treg cells is associated with more aggressive development of atherosclerosis. Conversely, cell transfer and other approaches to expand Treg cell populations in vivo result in reduced atherosclerosis. There have been relatively few clinical studies of Treg cells and cardiovascular disease, but the available evidence also supports a protective function. These observations have raised hope that it may be possible to develop therapies that act by enforcing the suppressive activities of Treg cells in atherosclerotic lesions. One approach to achieve this goal has been through development of vaccines that stimulate immunological tolerance for plaque antigens. Several pilot vaccines based on LDL-derived antigens have demonstrated promising results in preclinical testing. If such therapies can be shown to be effective also in clinical trials, this could have an important impact on cardiovascular prevention and treatment. Here, we review the current knowledge of the mode of action of atheroprotective immunity and of the ways to stimulate such pathways in experimental settings. The challenges in translating this knowledge into the clinical setting are also discussed within the perspective of the experience of introducing immune-based therapies for other chronic noninfectious diseases.

Inhibition of monoacylglycerol lipase reduces nicotine withdrawal.

BACKGROUND AND PURPOSE: Abrupt discontinuation of nicotine, the main psychoactive component in tobacco, induces a withdrawal syndrome in nicotine-dependent animals, consisting of somatic and affective signs, avoidance of which contributes to drug maintenance. While blockade of fatty acid amide hydrolase, the primary catabolic enzyme of the endocannabinoid arachidonoylethanolamine (anandamide), exacerbates withdrawal responses in nicotine-dependent mice, the role of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of a second endocannabinoid 2-arachidonylglycerol (2-AG), in nicotine withdrawal remains unexplored. EXPERIMENTAL APPROACH: To evaluate the role of MAGL enzyme inhibition in nicotine withdrawal, we initially performed a genetic correlation approach using the BXD recombinant inbred mouse panel. We then assessed nicotine withdrawal intensity in the mouse after treatment with the selective MAGL inhibitor, JZL184, and after genetic deletion of the enzyme. Lastly, we assessed the association between genotypes and smoking withdrawal phenotypes in two human data sets. KEY RESULTS: BXD mice displayed significant positive correlations between basal MAGL mRNA expression and nicotine withdrawal responses, consistent with the idea that increased 2-AG brain levels may attenuate withdrawal responses. Strikingly, the MAGL inhibitor, JZL184, dose-dependently reduced somatic and aversive withdrawal signs, which was blocked by rimonabant, indicating a CB1 receptor-dependent mechanism. MAGL-knockout mice also showed attenuated nicotine withdrawal. Lastly, genetic analyses in humans revealed associations of the MAGL gene with smoking withdrawal in humans. CONCLUSIONS AND IMPLICATIONS: Overall, our findings suggest that MAGL inhibition maybe a promising target for treatment of nicotine dependence.

In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: antinociceptive activity without cannabimimetic side effects.

BACKGROUND AND PURPOSE: Since monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compound, KML29 (1,1,1,3,3,3-hexafluoropropan-2-yl 4-(bis(benzo[d][1,3]dioxol-5-yl)(hydroxy)methyl)piperidine-1-carboxylate). EXPERIMENTAL APPROACH: In the present study, we tested KML29 in murine inflammatory (i.e. carrageenan) and sciatic nerve injury pain models, as well as the diclofenac-induced gastric haemorrhage model. KML29 was also evaluated for cannabimimetic effects, including measurements of locomotor activity, body temperature, catalepsy, and cannabinoid interoceptive effects in the drug discrimination paradigm. KEY RESULTS: KML29 attenuated carrageenan-induced paw oedema and completely reversed carrageenan-induced mechanical allodynia. These effects underwent tolerance after repeated administration of high-dose KML29, which were accompanied by cannabinoid receptor 1 (CB1 ) receptor desensitization. Acute or repeated KML29 administration increased 2-AG levels and concomitantly reduced arachidonic acid levels, but without elevating anandamide (AEA) levels in the whole brain. Furthermore, KML29 partially reversed allodynia in the sciatic nerve injury model and completely prevented diclofenac-induced gastric haemorrhages. CB1 and CB2 receptors played differential roles in these pharmacological effects of KML29. In contrast, KML29 did not elicit cannabimimetic effects, including catalepsy, hypothermia and hypomotility. Although KML29 did not substitute for Δ(9) -tetrahydrocannabinol (THC) in C57BL/6J mice, it fully and dose-dependantly substituted for AEA in fatty acid amide hydrolase (FAAH) (-/-) mice, consistent with previous work showing that dual FAAH and MAGL inhibition produces THC-like subjective effects. CONCLUSIONS AND IMPLICATIONS: These results indicate that KML29, a highly selective MAGL inhibitor, reduces inflammatory and neuropathic nociceptive behaviour without occurrence of cannabimimetic side effects. LINKED ARTICLES: This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6.

Mouse models of atherosclerosis.

Genetically altered mice carrying mutations of genes encoding crucial components of the immune system and lipid metabolism have been widely used to study the role of immune responses and inflammation in atherosclerosis. These mice are often fed a diet, with a high content of cholesterol and saturated fat in order to induce hypercholesterolemia and arterial lesions. We review the different mouse models of atherosclerosis, type of diets, and techniques to measure lipid deposition and lesion size in the arterial walls. Moreover, the methods used to determine the presence of the immune cells in atherosclerotic lesions are also described here.

Blockade of endocannabinoid-degrading enzymes attenuates neuropathic pain.

Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB(1) or CB(2) receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB(1), but not a CB(2), receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.

The case for the development of novel analgesic agents targeting both fatty acid amide hydrolase and either cyclooxygenase or TRPV1.

Although the dominant approach to drug development is the design of compounds selective for a given target, compounds targeting more than one biological process may have superior efficacy, or alternatively a better safety profile than standard selective compounds. Here, this possibility has been explored with respect to the endocannabinoid system and pain. Compounds inhibiting the enzyme fatty acid amide hydrolase (FAAH), by increasing local endocannabinoid tone, produce potentially useful effects in models of inflammatory and possibly neuropathic pain. Local increases in levels of the endocannabinoid anandamide potentiate the actions of cyclooxygenase inhibitors, raising the possibility that compounds inhibiting both FAAH and cyclooxygenase can be as effective as non-steroidal anti-inflammatory drugs but with a reduced cyclooxygenase inhibitory 'load'. An ibuprofen analogue active in models of visceral pain and with FAAH and cyclooxygenase inhibitory properties has been identified. Another approach, built in to the experimental analgesic compound N-arachidonoylserotonin, is the combination of FAAH inhibitory and transient receptor potential vanilloid type 1 antagonist properties. Although finding the right balance of actions upon the two targets is a key to success, it is hoped that dual-action compounds of the types illustrated in this review will prove to be useful analgesic drugs.

The endogenous cannabinoid system modulates nicotine reward and dependence.

A growing body of evidence suggests that the endogenous cannabinoid system modulates the addictive properties of nicotine, the main component of tobacco that produces rewarding effects. In our study, complementary transgenic and pharmacological approaches were used to test the hypothesis that the endocannabinoid system modulates nicotine reward and dependence. An acute injection of nicotine elicited normal analgesic and hypothermic effects in cannabinoid receptor (CB)(1) knockout (KO) mice and mice treated with the CB(1) antagonist rimonabant. However, disruption of CB(1) receptor signaling blocked nicotine reward, as assessed in the conditioned place preference (CPP) paradigm. In contrast, genetic deletion, or pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for catabolism of the endocannabinoid anandamide, enhanced the expression of nicotine CPP. Although the expression of spontaneous nicotine withdrawal (14 days, 24 mg/kg/day nicotine) was unaffected in CB(1) KO mice, acute administration of rimonabant (3 mg/kg) ameliorated somatic withdrawal signs in wild-type mice. Increasing endogenous levels of anandamide through genetic or pharmacological approaches exacerbated the physical somatic signs of spontaneous nicotine withdrawal in a milder withdrawal model (7 days, 24 mg/kg/day nicotine). Moreover, FAAH-compromised mice displayed increased conditioned place aversion in a mecamylamine-precipitated model of nicotine withdrawal. These findings indicate that endocannabinoids play a role in the rewarding properties of nicotine as well as nicotine dependence liability. Specifically, increasing endogenous cannabinoid levels magnifies, although disrupting CB(1) receptor signaling, attenuates nicotine reward and withdrawal. Taken together, these results support the hypothesis that cannabinoid receptor antagonists may offer therapeutic advantages to treat tobacco dependence.

The psychoactive plant cannabinoid, Delta9-tetrahydrocannabinol, is antagonized by Delta8- and Delta9-tetrahydrocannabivarin in mice in vivo.

BACKGROUND AND PURPOSE: To follow up in vitro evidence that Delta(9)-tetrahydrocannabivarin extracted from cannabis (eDelta(9)-THCV) is a CB(1) receptor antagonist by establishing whether synthetic Delta(9)-tetrahydrocannabivarin (O-4394) and Delta(8)-tetrahydrocannabivarin (O-4395) behave as CB(1) antagonists in vivo. EXPERIMENTAL APPROACH: O-4394 and O-4395 were compared with eDelta(9)-THCV as displacers of [(3)H]-CP55940 from specific CB(1) binding sites on mouse brain membranes and as antagonists of CP55940 in [(35)S]GTPgammaS binding assays performed with mouse brain membranes and of R-(+)-WIN55212 in mouse isolated vasa deferentia. Their ability to antagonize in vivo effects of 3 or 10 mg kg(-1) (i.v.) Delta(9)-tetrahydrocannabinol in mice was then investigated. KEY RESULTS: O-4394 and O-4395 exhibited similar potencies to eDelta(9)-THCV as displacers of [(3)H]-CP55940 (K (i)=46.6 and 64.4 nM, respectively) and as antagonists of CP55940 in the [(35)S]GTPgammaS binding assay (apparent K (B)=82.1 and 125.9 nM, respectively) and R-(+)-WIN55212 in the vas deferens (apparent K (B)=4.8 and 3.9 nM respectively). At i.v. doses of 0.1, 0.3, 1.0 and/or 3 mg kg(-1) O-4394 and O-4395 attenuated Delta(9)-tetrahydrocannabinol-induced anti-nociception (tail-flick test) and hypothermia (rectal temperature). O-4395 but not O-4394 also antagonized Delta(9)-tetrahydrocannabinol-induced ring immobility. By themselves, O-4395 and O-4394 induced ring immobility at 3 or 10 mg kg(-1) (i.v.) and antinociception at doses above 10 mg kg(-1) (i.v.). O-4395 also induced hypothermia at 3 mg kg(-1) (i.v.) and above. CONCLUSIONS AND IMPLICATIONS: O-4394 and O-4395 exhibit similar in vitro potencies to eDelta(9)-THCV as CB(1) receptor ligands and as antagonists of cannabinoid receptor agonists and can antagonize Delta(9)-tetrahydrocannabinol in vivo.

Interactions between THC and cannabidiol in mouse models of cannabinoid activity.

RATIONALE: Interest persists in characterizing potential interactions between Delta(9)-tetrahydocannabinol (THC) and other marijuana constituents such as cannabidiol (CBD). Such interactions may have important implications for understanding the long-term health consequences of chronic marijuana use as well as for attempts to develop therapeutic uses for THC and other CB(1) agonists. OBJECTIVES: We investigated whether CBD may modulate the pharmacological effects of intravenously administered THC or inhaled marijuana smoke on hypoactivity, antinociception, catalepsy, and hypothermia, the well characterized models of cannabinoid activity. RESULTS: Intravenously administered CBD possessed very little activity on its own and, at a dose equal to a maximally effective dose of THC (3 mg/kg), failed to alter THC's effects on any measure. However, higher doses of CBD (ED(50)=7.4 mg/kg) dose-dependently potentiated the antinociceptive effects of a low dose of THC (0.3 mg/kg). Pretreatment with 30 mg/kg CBD, but not 3 mg/kg, significantly elevated THC blood and brain levels. No interactions between THC and CBD were observed in several variations of a marijuana smoke exposure model. Either quantities of CBD were applied directly to marijuana, CBD and THC were both applied to placebo plant material, or mice were pretreated intravenously with 30 mg/kg CBD before being exposed to marijuana smoke. CONCLUSIONS: As the amount of CBD found in most marijuana strains in the US is considerably less than that of THC, these results suggest that CBD concentrations relevant to what is normally found in marijuana exert very little, if any, modulatory effects on CB(1)-receptor-mediated pharmacological effects of marijuana smoke.

Delta9-tetrahydrocannbinol accounts for the antinociceptive, hypothermic, and cataleptic effects of marijuana in mice.

Although it is widely accepted that delta9-tetrahydrocannabinol (delta9-THC) is the primary psychoactive constituent of marijuana, questions persist as to whether other components contribute to marijuana's pharmacological activity. The present experiments assessed the cannabinoid activity of marijuana smoke exposure in mice and tested the hypothesis that delta9-THC mediates these effects through a CB1 receptor mechanism of action. First, the effects of delta9-THC on analgesia, hypothermia, and catalepsy were compared with those of a marijuana extract with equated delta9-THC content after either i.v. administration or inhalation exposure. Second, mice were exposed to smoke of an ethanol-extracted placebo plant material or low-grade marijuana (with minimal delta9-THC but similar levels of other cannabinoids) that were impregnated with varying quantities of delta9-THC. To assess doses, delta9-THC levels in the blood and brains of drug-exposed mice were determined following both i.v. and inhalation routes of administration. Both marijuana and delta9-THC produced comparable levels of antinociception, hypothermia, and catalepsy regardless of the route of administration, and these effects were blocked by pretreatment with the CB1 antagonist SR141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl]. Importantly, the blood and brain levels of delta9-THC were similar in mice exhibiting similar pharmacological effects, regardless of the presence of non-delta9-THC marijuana constituents. The present experiments provide evidence that the acute cannabinoid effects of marijuana smoke exposure on analgesia, hypothermia, and catalepsy in mice result from delta9-THC content acting at CB1 receptors and that the non-delta9-THC constituents of marijuana (at concentrations relevant to those typically consumed) influence these effects only minimally, if at all.

Cannabinoid tolerance and dependence.

The use of marijuana for recreational and medicinal purposes has resulted in a large prevalence of chronic marijuana users. Consequences of chronic cannabinoid administration include profound behavioral tolerance and withdrawal symptoms upon drug cessation. A marijuana withdrawal syndrome is only recently gaining acceptance as being clinically significant. Similarly, laboratory animals exhibit both tolerance and dependence following chronic administration of cannabinoids. These animal models are being used to evaluate the high degree of plasticity that occurs at the molecular level in various brain regions following chronic cannabinoid exposure. In this review, we describe recent advances that have increased our understanding of the impact of chronic cannabinoid administration on cannabinoid receptors and their signal transduction pathways. Additionally, we discuss several potential pharmacotherapies that have been examined to treat marijuana dependence.

Delta(9)-THC-induced cognitive deficits in mice are reversed by the GABA(A) antagonist bicuculline.

RATIONALE: The results of recent in vitro studies have underscored the important role that activation of CB(1) receptors has on GABAergic activity in brain areas associated with memory. OBJECTIVES: The primary purpose of this study was to test the hypothesis that the memory disruptive effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in vivo are mediated through GABAergic systems. Conversely, we also evaluated whether blocking CB(1) receptor signaling would alter memory deficits elicited by GABA agonists. METHODS: The GABA(A) antagonist bicuculline and GABA(B) antagonist CGP 36742 were evaluated for their ability to ameliorate Delta(9)-THC-induced deficits in a mouse working memory Morris water maze task. Mice were also assessed in a T-maze task, as well as non-cognitive behavioral assays. Additionally, the effects of GABA(A) and GABA(B) agonists were assessed in either CB(1) (-/-) mice or wild type mice treated with the CB(1) antagonist SR 141716. RESULTS: Memory deficits resulting from 10 mg/kg Delta(9)-THC in the Morris water maze were completely reversed by bicuculline, though unaffected by CGP 36742. Bicuculline also blocked the disruptive effects of Delta(9)-THC in the T-maze, but failed to alter non-mnemonic effects of Delta(9)-THC. Although CB(1) (-/-) mice exhibited supersensitivity to muscimol-induced water maze deficits compared with wild type control mice, muscimol elicited virtually identical effects in SR 141716-treated and vehicle-treated wild type mice. CONCLUSIONS: This is the first demonstration of which we are aware showing that GABA(A) receptors may play a necessary role in Delta(9)-THC-induced memory impairment in whole animals.

Disruption of CB(1) receptor signaling impairs extinction of spatial memory in mice.

RATIONALE: A growing body of in vitro and in vivo evidence indicates that a central endocannabinoid system, consisting of CB(1) receptors and endogenous cannabinoids, modulates specific aspects of mnemonic processes. Previous research has demonstrated that either permanent or drug-induced disruption of CB(1) receptor signaling interferes with the extinction of a conditioned fear response. OBJECTIVES: In the present study, we evaluated whether the endocannabinoid system also plays a role in extinguishing learned escape behavior in a Morris water maze task. METHODS: CB(1) (-/-) mice and mice repeatedly treated with 3 mg/kg of the CB(1) receptor antagonist SR 141716 (Rimonabant) were trained to locate a hidden platform in the Morris water maze. Following acquisition, the platform was removed and subjects were assigned to either a massed (i.e., five consecutive sessions consisting of four 2-min trials/session) or a spaced (a single, 1-min trial every 2-4 weeks) extinction protocol. RESULTS: Strikingly, both 3 mg/kg SR 141716-treated mice and CB(1) (-/-) mice continued to return to the target location across all five trials in the spaced extinction procedure, while the control mice underwent extinction by the third or fourth trial. In contrast, both the 3-mg/kg SR 141716-treated and CB(1) (-/-) mice exhibited extinction in the massed extinction trial procedure. CONCLUSIONS: These findings indicate that disruption of CB(1) receptor signaling impairs extinction processes in the Morris water maze, thus lending further support to the hypothesis that the endocannabinoid system plays an integral role in the suppression of non-reinforced learned behaviors.

Neuroscience. Stout guards of the central nervous system.

Endocannabinoids have paradoxical effects on the mammalian nervous system: Sometimes they block neuronal excitability and other times they augment it. In their Perspective, Mechoulam and Lichtman discuss new work (Marsicano et al.) showing that activation of the cannabinoid receptor CB1 by the endocannabinoid anandamide protects against excitotoxic damage in a mouse model of kainic acid-induced epilepsy.

Endocannabinoids in cognition and dependence.

Cannabis use is associated with a wide range of pharmacological effects, some of which have potential therapeutic benefit while others result in negative outcomes. Acute cannabinoid intoxication has been well documented to produce deficits in cognitive functioning with concomitant changes in glutamatergic, GABAergic, and cholinergic neurochemical systems in the hippocampus, each of which has been implicated in memory. Additionally, cannabis-dependent individuals abstaining from this drug can undergo a constellation of mild withdrawal effects. The use of the CB(1) cannabinoid receptor antagonist SR141716A and transgenic mice lacking the CB(1) receptor are critical tools for investigating the role of the endocannabinoid system in cognition, drug dependence, and other physiological processes. Converging evidence in which performance in a variety of memory tasks is enhanced following either SR141716A treatment or in CB(1) receptor knockout mice indicates that this system may play an important role in modulating cognition. There are also indications that this system may function to modulate opioid dependence. The purpose of this review is to describe recent advances that have furthered our understanding of the roles that the endocannabinoid system play on both cognition and drug dependence.