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BEHAVIORAL PHARMACOLOGY
9-Tetrahydrocannabinol Discrimination Involves Endogenous AnandamideLaboratoire de Biologie et Physiologie Cellulaires, Centre National de la Recherche Scientifique-VMR6187, Université de Poitiers, Poitiers, France (M.So.); Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch (M.So., M.Sc., C.E.W., S.R.G.) and Psychobiology Section, Medications Discovery Research Branch (G.T.), Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland; and B.B. Brodie Department of Neuroscience, University of Cagliari, Cagliari, Italy (M.Sc., W.F.)
Received November 12, 2006; accepted March 6, 2007.
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9-tetrahydrocannabinol (THC), alters extracellular levels of acetylcholine in several brain areas, suggesting an involvement of the cholinergic system in the psychotropic effects of cannabis. Here, we investigated whether drugs acting at either nicotinic or muscarinic receptors can modulate the discriminative effects of THC. In rats that had learned to discriminate effects of 3 mg/kg i.p. injections of THC from injections of vehicle, the nicotinic agonist nicotine (0.1-0.56 mg/kg subcutaneous) and the muscarinic agonist pilocarpine (0.3-3 mg/kg i.p.) did not produce THC-like effects, but they both potentiated the discriminative effects of low doses of THC (0.3-1 mg/kg). Neither the nicotinic antagonist mecamylamine (1-5.6 mg/kg i.p.) nor the muscarinic antagonist scopolamine (0.01-0.1 mg/kg i.p.) altered the discriminative effects of THC, but they blocked the potentiation of discriminative effects of THC by nicotine and pilocarpine, respectively. The cannabinoid CB1 antagonist rimonabant (1 mg/kg i.p.) reversed nicotine- but not pilocarpine-induced potentiation of THC discrimination, suggesting that nicotine potentiation is, at least in part, mediated by release of endogenous cannabinoids in the brain. In addition, when metabolic degradation of the endogenous cannabinoid anandamide was blocked by the fatty acid amide hydrolase inhibitor cyclohexyl carbamic acid 3'-carbamoylbiphenil-3-yl-ester (URB-597; 0.3 mg/kg i.p.) nicotine, but not pilocarpine, produced significant THC-like discriminative effects that were antagonized by rimonabant. Our results suggest that nicotinic and muscarinic cholinergic receptors modulate the discriminative effects of THC by fundamentally different mechanisms. Nicotinic, but not muscarinic, modulation of THC discrimination involves elevations in endogenous levels of anandamide.
; Gold, 2003). In addition, acetylcholine, especially by interacting with dopamine, seems to participate in reward processes (Mansvelder et al., 2003; Cragg, 2006). In the brain, acetylcholine activates two main types of cholinergic receptors, nicotinic receptors and muscarinic receptors. Nicotinic receptors are ion-gated channels (Wonnacott et al., 2005), whereas muscarinic receptors are G protein-coupled receptors (van der Zee and Luiten, 1999). Although their localizations, modes of action, and signaling cascades may differ, nicotinic and muscarinic receptors seem to be similarly implicated in the central effects of acetylcholine.
Cannabis is the most commonly used illegal drug of abuse in western countries. Acute intoxication with cannabis produces euphoria, which is accompanied by impairment of cognitive functions (Goodman and Gilman, 2006). Although these effects of cannabis are thought to be, at least in part, mediated by the cholinergic system, cannabinoid receptor agonists may disrupt performance in a working memory task through noncholinergic mechanisms (Lichtman and Martin, 1996). Cannabinoid and cholinergic systems may interact not only in brain systems involved in memory, such as the hippocampus (Kesner and Hopkins, 2006), but also in brain systems involved in reward, such as the striatum (Di Chiara, 1999), where both nicotinic and cannabinoid CB1 receptors are localized (Martin and Aceto, 1981; Herkenham et al., 1991).
The in vitro effects of cannabinoid CB1 receptor agonists on cholinergic neurotransmission have been well characterized in hippocampal slices, where they decrease long-term potentiation and depression and inhibit the release of acetylcholine (Schlicker and Kathmann, 2001). However, conflicting reports of the effects of cannabinoid CB1 receptor agonists on hippocampal acetylcholine have been reported using in vivo procedures (Gessa et al., 1998; Acquas et al., 2001; Tzavara et al., 2003; Pisanu et al., 2006). For example, Gessa et al. (1998) reported that administration of the natural cannabinoid CB1 receptor agonist 9-tetrahydrocannabinol (THC) or the synthetic CB1 receptor agonist WIN55,212-2 attenuated hippocampal efflux of acetylcholine, whereas Di Chiara and colleagues (Acquas et al., 2001; Pisanu et al., 2006) reported stimulation of hippocampal efflux of acetylcholine with the same compounds. In a recent article, it was suggested that such conflicting findings could be dose-related, with high depressant doses of cannabinoid CB1 receptor agonists acting predominantly in the hippocampus to decrease cholinergic neurotransmission and with low activating doses of CB1 receptor agonists acting predominantly in the septum to increase cholinergic neurotransmission (Tzavara et al., 2003).
Given these contradictory results, it is important to investigate whether behavioral effects of THC related to its abuse can be mediated or modulated by the cholinergic system and whether activation of the cholinergic system facilitates or antagonizes these effects of THC. Drug-discrimination procedures allow for the study of the mechanisms through which drugs of abuse produce central effects that may contribute to the maintenance of drug taking and serve as preclinical animal models for subjective reports of drug effects by humans (Solinas et al., 2006). Although drug-discrimination techniques have been used for decades to investigate the effects of THC (for review, see Wiley, 1999), no study has systematically investigated the involvement of the cholinergic system in the discriminative effects of THC.
In this study, rats learned to discriminate injections of THC from injections of vehicle, and they were then tested with drugs acting at nicotinic and muscarinic receptors to determine whether there was significant cholinergic modulation of the discriminative-stimulus effects of THC. We also tested whether release of the endogenous cannabinoid anandamide could be involved in any modulation observed.
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Drug-Discrimination Apparatus and Procedure. Standard operant-conditioning chambers (Coulbourn Instruments, Allentown, PA) were used. Each chamber contained two levers, separated by a recessed tray into which a pellet dispenser could deliver 45-mg food pellets (F0021; Bioserv, Frenchtown, NJ). Each press of a lever with a force of 0.4 N through 1 mm was recorded as a response and was accompanied by an audible click. The operant-conditioning chambers were controlled by computers using the MED-PC software package (MED Associates, St. Albans, VT). Rats were trained, as described previously (Solinas et al., 2003), under a discrete-trial schedule of food-pellet delivery to respond on one lever after an injection of a training dose of 3 mg/kg THC and on the other lever after an injection of 1 ml/kg THC vehicle. Injections of THC or vehicle were given i.p. 30 min before the start of the session. At the start of the session, a white house light was turned on, and in its presence, the rats were required to make 10 consecutive responses (fixed ratio 10 schedule of food delivery; FR10) on the lever appropriate to the presession treatment. The completion of 10 consecutive responses on the correct lever produced delivery of a 45-mg food pellet and initiated a 45-s time-out during which lever-press responses had no programmed consequences and the chamber was dark. Responses on the incorrect lever had no programmed consequences other than to reset the FR requirement on the correct lever. After each time-out, the white house light was again turned on, and the next trial began. Each session ended after completion of 20 fixed ratio trials or after 30 min elapsed, whichever occurred first.
Discrimination-training sessions were conducted 5 days per week under a double alternation schedule (i.e., DDVVDDVV, and so on, where D is the drug THC and V is the vehicle). Training continued until there were eight consecutive sessions during which rats completed at least 90% of their responses during the session on the correct lever, and no more than four responses occurred on the incorrect lever during the first trial. Test sessions were then initiated. Test sessions were identical to training sessions with the exception that 10 consecutive responses on either one of the two levers ended the trial. Switching responding from one lever to the other lever reset the ratio requirement. In a test phase, a single alternation schedule was introduced, and test sessions were usually conducted on Tuesdays and Fridays. Thus, a 2-week sequence starting on Monday was DTVDTVTDVT (where T is test). In this way, test sessions occurred with equal probability after saline and drug sessions. Test sessions were conducted only if the criterion of 90% accuracy and not more than four incorrect responses during the first trial was maintained in the two preceding training sessions. The first test sessions consisted of different doses of the training drug to establish a THC dose-response curve. Afterward, tests with other compounds alone or in combination with THC began.
Two measures were analyzed: 1) percentage of total lever-presses made on the THC lever, which gives a quantitative indication of whether the drug or combination of drugs tested produces discriminative effects similar to those of the 3-mg/kg training dose of THC; and 2) overall rate of lever-press responding, which gives an indication of any disruption of motor responses produced by the drug or combination of drugs tested. When rates of responding were significantly reduced compared with basal levels, administrations of higher doses of that specific drug or combination of drugs were normally avoided.
Drugs. THC (National Institute on Drug Abuse, National Institutes of Health, Bethesda, MD), 50 mg/ml in ethanol, was dissolved in a solution 40% (w/v) 
-hydroxy-cyclodextrin (Sigma-Aldrich, St. Louis, MO). Rimonabant (National Institute on Drug Abuse) was dissolved in a vehicle containing 2% Tween 80, 2% ethanol, and 96% saline. Nicotine [(-)-nicotine hydrogen tartrate], pilocarpine, mecamylamine, and scopolamine were purchased from Sigma-Aldrich, and they were dissolved in saline solution. URB-597 was a gift of Drs. A. Duranti, A. Tontini, and G. Tarzia (University of Urbino, Urbino, Italy), and dissolved in a vehicle containing 20% dimethyl sulfoxide in saline. Doses were calculated as the salt, with the exception of nicotine, which was calculated as the base. All drugs were injected in a volume of 1.0 ml/kg. THC and vehicle were administered 30 min before the session; nicotine and pilocarpine were administered 15 min before the session; mecamylamine, scopolamine, and URB-597 were administered 40 min before the session; and rimonabant was administered 60 min before the session. The range of doses and pretreatment times for each compound were selected based on published studies showing behavioral effects (when possible discriminative effects) in rats, devoid of toxicity. The doses of nicotine and pilocarpine used in combination with different doses of THC (THC dose-response experiments) were chosen based on the results obtained in this study. The highest dose that did not produce significant depression of rates of responding by itself was used. It should be noted that, with nicotine, a dose of 0.3 mg/kg was initially used for combination studies. However, because 0.3 mg/kg nicotine in combination with THC produced significant disruption of responding, a lower dose, 0.1 mg/kg nicotine, was used for the remainder of the study.
Data Analysis. Discriminative-stimulus data were expressed as the percentage of the total responses on the two levers that were made during the test session on the THC-appropriate lever. Response-rate data were expressed as responses per second averaged over the session, with responding during time-out periods not included in calculations. The data from sessions during which rats did not complete at least one fixed ratio trial were excluded from analysis of drug-lever selection. All results are presented as group means (±S.E.M.). Statistical analysis of the ability of compounds to produce generalization to the discriminative effects of the training dose of THC was done using one-way ANOVA for repeated measures in comparison with vehicle treatments followed, when appropriate, by the Dunnett's post hoc test. A probability value of p < 0.05 was considered significant. ED50 values for each compound or combination were obtained by nonlinear regression analysis with a sigmoidal dose-response (variable slope) equation, using GraphPad Prism 3 software (GraphPad Software, San Diego, CA). The equation was Y = bottom + (top - bottom)/(1 + 10^[(log ED50 - X) x Hill slope)] with the bottom and top values kept constant at 0 and 100%, respectively. Curves were considered parallel when their slopes did not differ significantly, and dose-response curves were considered significantly different when 95% confidence intervals of ED50 values did not overlap. In addition, shifts in dose-response curves were also analyzed by two-way ANOVA for repeated measures with dose and pretreatment as factors followed, when appropriate, by Student-Newman-Keuls post hoc test. Statistical analysis of the effect of any treatment on rates of responding was done using one-way ANOVA for repeated measures in comparison with vehicle treatment followed, when appropriate, by the Dunnett's post hoc test. A probability value of p < 0.05 was considered significant.
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Neither the nicotinic agonist, nicotine (0.1-1 mg/kg s.c.) nor the muscarinic agonist pilocarpine produced significant THC-like discriminative effects (Fig. 1A, top), even at doses that produced significant decreases in rates of responding [F(3,30) = 13.827; p < 0.0001 and F(3,30) = 19.476; p < 0.0001, respectively] (Fig. 1A, bottom).
Nicotine and Pilocarpine Potentiate the Discriminative Effects of THC. Nicotine, at a dose of 0.1 mg/kg, potentiated the discriminative effects of low doses of THC (ED50 = 0.46, 95% confidence intervals = 0.18 to 0.75; Table 1) [treatment effect: F(1,10) = 16.03; p < 0.01] (Fig. 1B, top), without significantly altering rates of responding (Fig. 1B, bottom). Pilocarpine, at a dose of 1.0 mg/kg, also potentiated the discriminative effects of low doses of THC (ED50 = 0.50, 95% confidence intervals = 0.22-0.78; Table 1) [treatment effect: F(1, 10) = 14.70; p < 0.01) = 16.03; p < 0.01] (Fig. 1B, top), and combinations of 1.0 mg/kg pilocarpine with low doses of THC produced only small nonsignificant decreases in rates of responding (Fig. 1B, bottom).
Mecamylamine and Scopolamine Do Not Antagonize the Discriminative Effects of THC. Neither the nicotinic antagonist mecamylamine (1-5.6 mg/kg i.p.) nor the muscarinic antagonist scopolamine reduced the effects of the 3 mg/kg training dose of THC (Fig. 2A, top), even at doses that markedly and significantly decreased rates of responding [F(3,24) = 3.374; p < 0.0001 for mecamylamine and F(3,24) = 12.010; p < 0.0001 for scopolamine] (Fig. 2A, bottom). In addition, neither mecamylamine (3 mg/kg) nor scopolamine (0.03 mg/kg) shifted the dose-response curve for THC discrimination (Fig. 2B, top). Combinations of mecamylamine (3 mg/kg) or scopolamine (0.03 mg/kg) with THC resulted in small but significant decreases in rates of responding compared with basal vehicle rates of responding [F(4,40) = 2.933; p < 0.0001 for mecamylamine and F(4,40) = 4.331; p < 0.01 for scopolamine] (Fig. 2B, bottom).
Potentiation of the Discriminative Effects of THC by Nicotine Is Mediated by Nicotinic Receptors and Potentiation by Pilocarpine Is Mediated by Muscarinic Receptors. A dose of 3 mg/kg mecamylamine, which was ineffective by itself, completely blocked nicotine-induced potentiation of the discriminative effects of THC (ED50 = 0.93, 95% confidence intervals = 0.56-1.31; Table 1) [treatment effect: F(1,9) = 6.80; p < 0.05] (Fig. 3, top) without significantly altering rates of responding (Fig. 3, bottom). Conversely, a dose of 0.03 mg/kg scopolamine, which was ineffective by itself, completely blocked pilocarpine-induced potentiation of the discriminative effects of THC (ED50 = 0.85, 95% confidence intervals = 0.58-1.12; Table 1) [treatment effect: F(1,7) = 7.51; p < 0.05] (Fig. 3, top) without significantly altering rates of responding (Fig. 3, bottom).
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Potentiation of the Discriminative Effects of THC by Nicotine, but Not by Pilocarpine, Is Mediated by Cannabinoid CB1 Receptors. That cholinergic agonists potentiate the effects of THC but cholinergic antagonists do not antagonize them led us to hypothesize that cholinergic potentiation is due to the release of endogenous cannabinoids induced by stimulation of nicotine and muscarinic receptors. To test this possibility, we tried to antagonize nicotine- and pilocarpine-induced potentiation of THC discrimination by blocking CB1 receptors. Rimonabant, at a dose of 1 mg/kg, which we have previously found effective in blocking the discriminative (Solinas et al., 2003, 2004) and reinforcing effects of THC in rats (Zangen et al., 2006), completely reversed nicotine-induced potentiation without affecting rates of responding (ED50 = 1.01, 95% confidence intervals = 0.6436-1.368; Table 1) [treatment effect: F(1,8) = 18.40; p < 0.01] (Fig. 4). In contrast, 1 mg/kg rimonabant did not reverse pilocarpine-induced potentiation of the discriminative effects of THC and did not affect rates of responding (ED50 = 0.53, 95% confidence intervals = 0.29-0.76; Table 1) (Fig. 4).
Nicotine, but Not Pilocarpine, Produces THC-Like Effects When Degradation of the Endocannabinoid Anandamide Is Inhibited by URB-597. To further investigate the possibility that release of endocannabinoids could mediate nicotine- and pilocarpine-induced potentiation of the effects of THC, we investigated whether blockade of metabolic cleavage of the endogenous cannabinoid anandamide would result in nicotine or pilocarpine producing significant THC-like discriminative effects. URB-597 is a selective inhibitor of fatty acid amide hydrolase (FAAH) (Kathuria et al., 2003), the main enzyme responsible for anandamide inactivation, and it is a drug that has been proposed for clinical use as a new anxiolytic and antidepressant (Kathuria et al., 2003; Gobbi et al., 2005). Administration of a 0.3 mg/kg dose of URB-597, which had no THC-like discriminative effects by itself, significantly increased the ability of nicotine to produce THC-like discriminative effects [F(1,7) = 23.85; p < 0.01] (Fig. 5A, top). In addition, URB-597 potentiated the rate-depressant effects of the intermediate 0.3 mg/kg dose of nicotine [F(1,7) = 14.65; p < 0.01] (Fig. 5A, bottom). THC-like effects produced by combinations of URB-597 and nicotine were significantly reduced by administration of 1 mg/kg rimonabant [F(1,7) = 14.65; p < 0.001]. In contrast, URB-597 did not increase the ability of pilocarpine to produce THC-like discriminative effects and did not alter the effects of pilocarpine on rates of responding (Fig. 5B).
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). In that study, the acetylcholinesterase inhibitor physostigmine also did not produce THC-like discriminative effects, and it did not antagonize the discriminative effects of THC (Browne and Weissman, 1981), but only a single dose of each cholinergic compound was studied, and the ability of these single doses to antagonize the discriminative effects of THC were only studied with the training dose of THC. In the present experiments, we replicated the lack of THC-like discriminative effects of muscarinic antagonists and the failure of muscarinic antagonists to reduce the discriminative effects of the training dose of THC and extended these findings to a wide range of THC doses and to the nicotinic agonist nicotine and the nicotinic antagonist mecamylamine. Our results demonstrate that neither nicotinic nor muscarinic receptors mediate the discriminative effects of THC and confirm that the discriminative effects of THC are selectively mediated by cannabinoid CB1 receptors (Wiley, 1999; Jarbe et al., 2001: Solinas et al., 2003). However, when we investigated whether nicotinic or muscarinic agonists could alter the dose-response curve for THC discrimination, we found that both nicotinic and muscarinic agonists produce a clear potentiation of the discriminative effects of THC. This suggests that the cholinergic system is involved in the modulation of the discriminative effects of THC, with nicotinic and muscarinic activation having facilitating effects on THC discrimination.
The present findings are in agreement with previous findings that the reinforcing and anxiolytic effects of nicotine and THC, their ability to produce physical dependence, and their effects on expression of immediate early genes such as c-FOS, are synergistic (Valjent et al., 2002; Balerio et al., 2004, 2006). For example, when ineffective doses of nicotine and THC are given in combination in mice, they produce clear anxiolytic effects in the elevated plus maze (Balerio et al., 2006) and the light-dark box (Valjent et al., 2002), and they produced significant place preferences in an unbiased place conditioning procedure (Valjent et al., 2002). In addition, pharmacological or genetic ablation of cannabinoid CB1 receptors results in decreased behavioral effects of nicotine, including elimination of nicotine-induced conditioned place preferences in mice (Castañé et al., 2002) and rats (Le Foll and Goldberg, 2004) and suppression of intravenous nicotine self-administration behavior in rats (Cohen et al., 2005). Finally, the present results are in agreement with other in vivo findings that low doses of cannabinoid agonists increase cholinergic neurotransmission in the hippocampus (Acquas et al., 2001; Pisanu et al., 2006).
The present and previous findings of facilitation between cannabinoid and cholinergic systems are in contrast with data showing that cannabinoids inhibit acetylcholine release in vitro (Schlicker and Kathmann, 2001) and in vivo (Gessa et al., 1998). It has recently been proposed that such discrepancies could be because high depressant doses of CB1 receptor agonists act predominantly in the hippocampus and decrease cholinergic neurotransmission, whereas low activating doses of cannabinoid CB1 receptor agonists act predominantly in the septum and increase cholinergic neurotransmission (Tzavara et al., 2003). Thus, it seems that our drug-discrimination paradigm is more suited for studying the effects of low activating doses of THC that produce behavioral activation and reward than for studying effects of high doses of THC that produce behavioral depression and aversion (Sañudo-Peña et al., 2000; Ghozland et al., 2002). This would be consistent with our previous findings (Solinas and Goldberg, 2005) that the discriminative effects of THC depend on activation of µ-opioid receptors, which are involved in the rewarding effects of low doses of THC, but not of 
-opioid receptors, which are involved in the depressant and aversive effects of high doses of THC (Ghozland et al., 2002).
Based on our previous work, where opioid modulation of THC discrimination involved release of endogenous -endorphin (Solinas et al., 2004), we initially hypothesized that the present cholinergic potentiation of the effects of THC was related to the ability of THC to release acetylcholine. If this were true, release of acetylcholine by CB1 receptor activation and the consequent stimulation of nicotinic and muscarinic cholinergic receptors would be part of the complex discriminative-stimulus effects of THC, and low doses of THC together with agonists at cholinergic receptors would result in discriminative effects similar to those of higher doses of THC. However, such a mechanism predicts that blockade of cholinergic receptors should block part of the complex discriminative-stimulus effects of THC and result in a rightward shift of the dose-response curve for THC discrimination. In contrast, in the present study, neither nicotinic nor muscarinic antagonists shifted the dose-response curve for THC discrimination to the right, indicating that other mechanisms were responsible for cholinergic modulation of the discriminative effects of THC.
One mechanism that could account for the shift to the left of the dose-response curve for THC discrimination produced by nicotine and pilocarpine and for the lack of a shift to the right with the nicotinic and muscarinic antagonists would be release of endogenous cannabinoids, such as anandamide, by nicotine and pilocarpine. That is, activation of nicotinic or muscarinic receptors would trigger the formation and release of endogenous cannabinoids, and the elevated levels of newly formed endogenous cannabinoids, although too low to produce THC discriminative effects themselves, would facilitate the discriminative effects of low doses of THC.
We have recently demonstrated that intravenous anandamide produces clear THC-like discriminative effects at nondepressant doses when its metabolic inactivation by FAAH enzymes is blocked by URB-597 (Solinas et al., 2007). Our results demonstrate that release of endocannabinoids, especially anandamide, could mediate the nicotine-induced potentiation of THC discrimination in this study. Moreover, the cannabinoid CB1 receptor antagonist rimonabant reversed nicotine-induced potentiation of THC discriminative effects, suggesting that CB1 receptors were responsible for nicotine-induced potentiation of THC discrimination. In addition, blockade of the metabolic inactivation of the endogenous cannabinoid anandamide by URB-597 resulted in significant THC-like discriminative effects of nicotine that were reversed by rimonabant, further suggesting that nicotine releases anandamide. Consistent with our results, it has been recently reported that rats chronically treated with nicotine show increased levels of anandamide (González et al., 2002). However, because chronic treatment with nicotine could be associated with tolerance or sensitization to the effects of subsequent nicotine administrations, further studies are needed to investigate direct effects of acute nicotine on brain levels of endogenous cannabinoids.
In contrast to the results with nicotine, release of anandamide did not seem to be involved in the present pilocarpine-induced facilitation of THC discrimination. A dose of rimonabant that completely antagonized the discriminative and reinforcing effects of THC in previous studies (Solinas et al., 2003, 2004; Zangen et al., 2006) and reversed nicotine-induced potentiation of THC discrimination in the present study did not antagonize pilocarpine-induced potentiation of THC discrimination. In addition, even when FAAH enzymes were blocked by URB-597, pilocarpine did not produce THC-like effects, suggesting again that facilitation of THC discrimination by pilocarpine does not involve endogenous anandamide. Although it is clear that rimonabant did not antagonize the effects of pilocarpine, it is not clear why rimonabant in combination with pilocarpine and THC did not block the effects of THC directly. It is possible that cholinergic and cannabinoid receptors interact with each other at the membrane level (protein-protein interaction mechanisms) or at the level of second messengers. If so, stimulation of cholinergic receptors could lead to changes in the configuration of CB1 receptors that, in turn, could result in an increased affinity of CB1 receptors for CB1 agonists or in a decreased affinity of CB1 receptors for CB1 antagonists. In addition, interactions between cholinergic and cannabinoid receptors could result in the facilitation or counteraction of the effects of CB1 receptor stimulation by CB1 agonists. Similar mechanisms have already been proposed for interactions between opioid and cannabinoid receptors (Berrendero et al., 2003; Kathmann et al., 2006), and it has been suggested that such mechanisms may also underlie in vivo interactions between opioid and cannabinoid systems at the behavioral level (Solinas et al., 2003, 2004; Solinas and Goldberg, 2005).
In conclusion, we found that both nicotinic and muscarinic cholinergic agonists can facilitate but do not mediate the discriminative-stimulus effects of THC. The asymmetric modulation of THC discrimination by cholinergic agonists and antagonists indicates that release of acetylcholine does not play a pivotal role in THC discrimination and that other mechanisms may be responsible for the observed facilitation with nicotine and pilocarpine. Release of the endogenous cannabinoid anandamide could explain nicotine-induced but not pilocarpine-induced facilitation of THC discrimination. Thus, stimulation of nicotinic and muscarinic receptors facilitates THC discrimination by inherently different mechanisms. The THC-like discriminative effects of nicotine after treatment with URB-597 suggest that inhibition of FAAH enzyme activity can unmask or facilitate behavioral effects of nicotine that are similar to low doses of THC, such as rewarding or anxiolytic effects.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CB, cannabinoid; THC, 9-tetrahydrocannabinol; FR, fixed ratio; D, drug, V, vehicle; T, test; rimonabant, SR-141716, N-piperidino-5-(4-chlorophenyl)-4-methylpyrazole-3-carboxamide; FAAH, fatty acid amide hydrolase; URB-597, cyclohexyl carbamic acid 3'-carbamoyl-biphenil-3-yl-ester; ANOVA, analysis of variance; FAAH, fatty acid amide hydrolase; Nic, nicotine; Piloc, pilocarpine; Mecam, mecamylamine; Scopol; scopolamine; Rim, rimonabant; WIN55212-2, (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate salt.
Address correspondence to: Dr. Marcello Solinas, Laboratoire de Biologie et Physiologie Cellulaires, Centre National de la Recherche Scientifique-6187, University of Poitiers, 40 Avenue du Recteur Pineau, 86022, Poitiers, France. E-mail: marcello.solinas{at}univ-poitiers.fr
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