Evidence for spontaneous cannabinoid withdrawal in mice.

Although the behavioral effects of acute and chronic exposure to cannabinoids have been extensively studied in mice, spontaneous withdrawal following exposure to cannabinoids has not been well characterized in this species. To address this issue, different groups of mice were treated for 5 days with saline, 20-36 mg/kg/day of the CB partial agonist Δ9-tetrahydrocannabinol (Δ9-THC), or 0.06-0.1 mg/kg/day of the CB high-efficacy agonist AM2389. Initial studies assessed changes in observable behavior (paw tremors) that were scored from the recordings taken at 4 or 24 h after the last injection. Subsequently, radiotelemetry was used to continuously measure body temperature and locomotor activity before (baseline), during, and after the 5-day dosing regimens. Results show that increases in paw tremors occurred following 5-day exposure to AM2389 or Δ9-THC. In telemetry studies, acute AM2389 or THC decreased both temperature and activity. Rapid tolerance occurred to the hypothermic effects of the cannabinoids, whereas locomotor activity continued to be suppressed following each drug injection. In contrast, increases in locomotor activity were evident 12-72 h after discontinuing daily injections of either 0.06 or 0.1 mg/kg/day AM2389. Increases in locomotor activity were also noted in mice treated daily with 30 or 36, but not 20 mg/kg/day Δ9-THC; these effects were smaller and appeared later than effects seen in AM2389-treated mice. These results indicate that the discontinuation of daily treatment with a CB high-efficacy agonist will yield evidence of spontaneous withdrawal that may reflect prior dependence, and that the degree of cannabinoid dependence may vary in relation to the dose or efficacy of the agonist injected daily.

Altered sleep during spontaneous cannabinoid withdrawal in male mice.

Cessation of cannabinoid use in humans often leads to a withdrawal state that includes sleep disruption. Despite important health implications, little is known about how cannabinoid abstention affects sleep architecture, in part because spontaneous cannabinoid withdrawal is difficult to model in animals. In concurrent work we report that repeated administration of the high-efficacy cannabinoid 1 (CB1) receptor agonist AM2389 to mice for 5 days led to heightened locomotor activity and paw tremor following treatment discontinuation, potentially indicative of spontaneous cannabinoid withdrawal. Here, we performed parallel studies to examine effects on sleep. Using implantable electroencephalography (EEG) and electromyography (EMG) telemetry we examined sleep and neurophysiological measures before, during, and after 5 days of twice-daily AM2389 injections. We report that AM2389 produces decreases in locomotor activity that wane with repeated treatment, whereas discontinuation produces rebound increases in activity that persist for several days. Likewise, AM2389 initially produces profound increases in slow-wave sleep (SWS) and decreases in rapid eye movement (REM) sleep, as well as consolidation of sleep. By the third AM2389 treatment, this pattern transitions to decreases in SWS and total time sleeping. This pattern persists following AM2389 discontinuation and is accompanied by emergence of sleep fragmentation. Double-labeling immunohistochemistry for hypocretin/orexin (a sleep-regulating peptide) and c-Fos (a neuronal activity marker) in lateral hypothalamus revealed decreases in c-Fos/orexin+ cells following acute AM2389 and increases following discontinuation, aligning with the sleep changes. These findings indicate that AM2389 profoundly alters sleep in mice and suggest that sleep disruption following treatment cessation reflects spontaneous cannabinoid withdrawal.

Two Janus Cannabinoids That Are Both CB2 Agonists and CB1 Antagonists.

The cannabinoid signaling system includes two G protein-coupled receptors, CB1 and CB2 These receptors are widely distributed throughout the body and have each been implicated in many physiologically important processes. Although the cannabinoid signaling system has therapeutic potential, the development of receptor-selective ligands remains a persistent hurdle. Because CB1 and CB2 are involved in diverse processes, it would be advantageous to develop ligands that differentially engage CB1 and CB2 We now report that GW405833 [1-(2,3-dichlorobenzoyl)-5-methoxy-2-methyl-3-[2-(4-morpholinyl)ethyl]-1H-indole] and AM1710 [1-hydroxy-9-methoxy-3-(2-methyloctan-2-yl)benzo[c]chromen-6-one], described as selective CB2 agonists, can antagonize CB1 receptor signaling. In autaptic hippocampal neurons, GW405833 and AM1710 both interfered with CB1-mediated depolarization-induced suppression of excitation, with GW405833 being more potent. In addition, in CB1-expressing human embryonic kidney 293 cells, GW405833 noncompetitively antagonized adenylyl cyclase activity, extracellular signal-regulated kinase 1/2 phosphorylation, phosphatidylinositol 4,5-bisphosphate signaling, and CB1 internalization by CP55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol). In contrast, AM1710 behaved as a low-potency competitive antagonist/inverse agonist in these signaling pathways. GW405833 interactions with CB1/arrestin signaling were complex: GW405833 differentially modulated arrestin recruitment in a time-dependent fashion, with an initial modest potentiation at 20 minutes followed by antagonism starting at 1 hour. AM1710 acted as a low-efficacy agonist in arrestin signaling at the CB1 receptor, with no evident time dependence. In summary, we determined that GW405833 and AM1710 are not only CB2 agonists but also CB1 antagonists, with distinctive and complex signaling properties. Thus, experiments using these compounds must take into account their potential activity at CB1 receptors.

Protection from Radiation-Induced Pulmonary Fibrosis by Peripheral Targeting of Cannabinoid Receptor-1.

Radiation-induced pulmonary fibrosis (RIF) is a severe complication of thoracic radiotherapy that limits its dose, intensity, and duration. The contribution of the endocannabinoid signaling system in pulmonary fibrogenesis is not known. Using a well-established mouse model of RIF, we assessed the involvement of cannabinoid receptor-1 (CB1) in the onset and progression of pulmonary fibrosis. Female C57BL/6 mice and CB1 knockout mice generated on C57BL/6 background received 20 Gy (2 Gy/min) single-dose thoracic irradiation that resulted in pulmonary fibrosis and animal death within 15 to 18 weeks. Some C57BL/6 animals received the CB1 peripherally restricted antagonist AM6545 at 1 mg/kg intraperitoneally three times per week. Animal survival and parameters of pulmonary inflammation and fibrosis were evaluated. Thoracic irradiation (20 Gy) was associated with marked pulmonary inflammation and fibrosis in mice and high mortality within 15 to 18 weeks after exposure. Genetic deletion or pharmacological inhibition of CB1 receptors with a peripheral CB1 antagonist AM6545 markedly attenuated or delayed the lung inflammation and fibrosis and increased animal survival. Our results show that CB1 signaling plays a key pathological role in the development of radiation-induced pulmonary inflammation and fibrosis, and peripherally restricted CB1 antagonists may represent a novel therapeutic approach against this devastating complication of radiotherapy/irradiation.

The VMAT-2 inhibitor tetrabenazine affects effort-related decision making in a progressive ratio/chow feeding choice task: reversal with antidepressant drugs.

Behavioral activation is a fundamental feature of motivation, and organisms frequently make effort-related decisions based upon evaluations of reinforcement value and response costs. Furthermore, people with major depression and other disorders often show anergia, psychomotor retardation, fatigue, and alterations in effort-related decision making. Tasks measuring effort-based decision making can be used as animal models of the motivational symptoms of depression, and the present studies characterized the effort-related effects of the vesicular monoamine transport (VMAT-2) inhibitor tetrabenazine. Tetrabenazine induces depressive symptoms in humans, and also preferentially depletes dopamine (DA). Rats were assessed using a concurrent progressive ratio (PROG)/chow feeding task, in which they can either lever press on a PROG schedule for preferred high-carbohydrate food, or approach and consume a less-preferred lab chow that is freely available in the chamber. Previous work has shown that the DA antagonist haloperidol reduced PROG work output on this task, but did not reduce chow intake, effects that differed substantially from those of reinforcer devaluation or appetite suppressant drugs. The present work demonstrated that tetrabenazine produced an effort-related shift in responding on the PROG/chow procedure, reducing lever presses, highest ratio achieved and time spent responding, but not reducing chow intake. Similar effects were produced by administration of the subtype selective DA antagonists ecopipam (D1) and eticlopride (D2), but not by the cannabinoid CB1 receptor neutral antagonist and putative appetite suppressant AM 4413, which suppressed both lever pressing and chow intake. The adenosine A2A antagonist MSX-3, the antidepressant and catecholamine uptake inhibitor bupropion, and the MAO-B inhibitor deprenyl, all reversed the impairments induced by tetrabenazine. This work demonstrates the potential utility of the PROG/chow procedure as a rodent model of the effort-related deficits observed in depressed patients.

Dopaminergic modulation of effort-related choice behavior as assessed by a progressive ratio chow feeding choice task: pharmacological studies and the role of individual differences.

Mesolimbic dopamine (DA) is involved in behavioral activation and effort-related processes. Rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. In the present study, the effects of several drug treatments were assessed using a progressive ratio (PROG)/chow feeding concurrent choice task. With this task, rats can lever press on a PROG schedule reinforced by a preferred high-carbohydrate food pellet, or alternatively approach and consume the less-preferred but concurrently available laboratory chow. Rats pass through each ratio level 15 times, after which the ratio requirement is incremented by one additional response. The DA D(2) antagonist haloperidol (0.025-0.1 mg/kg) reduced number of lever presses and highest ratio achieved but did not reduce chow intake. In contrast, the adenosine A(2A) antagonist MSX-3 increased lever presses and highest ratio achieved, but decreased chow consumption. The cannabinoid CB1 inverse agonist and putative appetite suppressant AM251 decreased lever presses, highest ratio achieved, and chow intake; this effect was similar to that produced by pre-feeding. Furthermore, DA-related signal transduction activity (pDARPP-32(Thr34) expression) was greater in nucleus accumbens core of high responders (rats with high lever pressing output) compared to low responders. Thus, the effects of DA antagonism differed greatly from those produced by pre-feeding or reduced CB1 transmission, and it appears unlikely that haloperidol reduces PROG responding because of a general reduction in primary food motivation or the unconditioned reinforcing properties of food. Furthermore, accumbens core signal transduction activity is related to individual differences in work output.

Effects of cannabinoid receptor antagonists on maintenance and reinstatement of methamphetamine self-administration in rhesus monkeys.

Cannabinoid receptor antagonists have shown some promise as treatments capable of reducing abuse and relapse to a number of abused drugs. In rodents, such effects have been observed with methamphetamine self-administration. However, the effects of cannabinoid receptor antagonists on methamphetamine self-administration and relapse have not been studied in primates. In the present study, rhesus monkeys were trained to respond on a three-component operant schedule. During the first 5-min component, fixed-ratio responses were reinforced by food, during the second 90- or 180-min component fixed-ratio responses were reinforced by i.v. methamphetamine. The third component was identical to the first. There was a 5-min timeout between each component. The effects of the cannabinoid receptor antagonists AM 251 and rimonabant were tested at various doses against self-administration of 3microg/kg/injection methamphetamine, and 1mg/kg AM 251 and 0.3mg/kg rimonabant were tested against the methamphetamine dose-effect function. The 1mg/kg dose of AM 251 was also tested for its ability to alter reinstatement of extinguished self-administration responding. The cannabinoid receptor antagonist AM 251 was found to reduce methamphetamine self-administration at doses that did not affect food-reinforced responding. The cannabinoid receptor antagonist rimonabant had similar, but less robust effects. AM 251 also prevented reinstatement of extinguished methamphetamine seeking that was induced by re-exposure to a combination of methamphetamine and methamphetamine-associated cues. These results indicate that cannabinoid receptor antagonists might have therapeutic effects for the treatment of methamphetamine dependence.

Some effects of CB1 antagonists with inverse agonist and neutral biochemical properties.

The CB1 inverse agonist/antagonist SR141716A recently has been introduced for the management of obesity (rimonabant; Acomplia) and appears to have beneficial effects. However, its utility may be hampered in some individuals by adverse effects including nausea or emesis or by mood depression. The recent development of biochemically 'neutral' antagonists such as AM4113 (Sink et al., 2007) has allowed an initial evaluation of the proposition that adverse effects of SR141716A are associated with its inverse agonist activity. Thus far, data comparing SR141716A and AM4113 across several species indicate that both drugs produce dose-related direct effects on operant behavior within the same range of doses that serve to antagonize the behavioral and hypothermic effects of a CB1 agonist. However, initial observations suggest that AM4113 may not produce preclinical indications of nausea or emesis. Further studies with AM4113 and other novel CB1 antagonists differing in efficacy should amplify our understanding of the relationship between the pharmacological activity of CB1 antagonists and their behavioral effects.

The novel cannabinoid CB1 receptor neutral antagonist AM4113 suppresses food intake and food-reinforced behavior but does not induce signs of nausea in rats.

Drugs that interfere with cannabinoid CB1 transmission suppress various food-motivated behaviors, and it has been suggested that such drugs could be useful as appetite suppressants. Biochemical studies indicate that most of these drugs assessed thus far have been CB1 inverse agonists, and although they have been shown to suppress food intake, they also appear to induce nausea and malaise. The present studies were undertaken to characterize the behavioral effects of AM4113, which is a CB1 neutral antagonist, and to examine whether this drug can reduce food-reinforced behaviors and feeding on diets with varying macronutrient compositions. Biochemical data demonstrated that AM4113 binds to CB1 receptors, but does not show inverse agonist properties (ie no effects on cyclic-AMP production). In tests of spontaneous locomotion and analgesia, AM4113 reversed the effects of the CB1 agonist AM411. AM4113 suppressed food-reinforced operant responding with rats responding on fixed ratio (FR) 1 and 5 schedules of reinforcement in a dose-dependent manner, and also suppressed feeding on high-fat, high-carbohydrate, and lab chow diets. However, in the same dose range that suppressed feeding, AM4113 did not induce conditioned gaping, which is a sign of nausea and food-related malaise in rats. These results suggest that AM4113 may decrease appetite by blocking endogenous cannabinoid tone, and that this drug may be less associated with nausea than CB1 inverse agonists.

Conformational memories and the endocannabinoid binding site at the cannabinoid CB1 receptor.

Endocannabinoid stucture-activity relationships (SAR) indicate that the CB1 receptor recognizes ethanolamides whose fatty acid acyl chains have 20 or 22 carbons, with at least three homoallylic double bonds and saturation in at least the last five carbons of the acyl chain. To probe the molecular basis for these acyl chain requirements, the method of conformational memories (CM) was used to study the conformations available to an n-6 series of ethanolamide fatty acid acyl chain congeners: 22:4, n-6 (K(i) = 34.4 +/- 3.2 nM); 20:4, n-6 (K(i) = 39.2 +/- 5.7 nM); 20:3, n-6 (K(i) = 53.4 +/- 5.5 nM); and 20:2, n-6 (K(i) > 1500 nM). CM studies indicated that each analogue could form both extended and U/J-shaped families of conformers. However, for the low affinity 20:2, n-6 ethanolamide, the higher populated family was the extended conformer family, while for the other analogues in the series, the U/J-shaped family had the higher population. In addition, the 20:2, n-6 ethanolamide U-shaped family was not as tightly curved as were those of the other analogues studied. To quantitate this variation in curvature, the radius of curvature (in the C-3 to C-17 region) of each member of each U/J-shaped family was measured. The average radii of curvature (with their 95% confidence intervals) were found to be 5.8 A (5.3-6.2) for 20:2, n-6; 4.4 A (4.1-4.7) for 20:3, n-6; 4.0 A (3.7-4.2) for 20:4, n-6; and 4.0 A (3.6-4.5) for 22:4, n-6. These results suggest that higher CB1 affinity is associated with endocannabinoids that can form tightly curved structures. Endocannabinoid SAR also indicate that the CB1 receptor does not tolerate large endocannabinoid headgroups; however, it does recognize both polar and nonpolar moieties in the headgroup region. To identify a headgroup orientation that results in high CB1 affinity, a series of dimethyl anandamide analogues (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (K(i) = 7.42 +/- 0.86 nM), (R)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (K(i) = 185 +/- 12 nM), (S)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (K(i) = 389 +/- 72 nM), and (S)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (K(i) = 233 +/- 69 nM) were then studied using CM and computer receptor docking studies in an active state (R) model of CB1. These studies suggested that the high CB1 affinity of the R,R stereoisomer is due to the ability of the headgroup to form an intramolecular hydrogen bond between the carboxamide oxygen and the headgroup hydroxyl that orients the C2 and C1' methyl groups to have hydrophobic interactions with valine 3.32(196), while the carboxamide oxygen forms a hydrogen bond with lysine 3.28(192) at CB1. In this position in the CB1 binding pocket, the acyl chain has hydrophobic and C-H.pi interactions with residues in the transmembrane helix (TMH) 2-3-7 region. Taken together, the studies reported here suggest that anandamide and its congeners adopt tightly curved U/J-shaped conformations at CB1 and suggest that the TMH 2-3-7 region is the endocannabinoid binding region at CB1.