Brain Cannabinoid CB₂ Receptors Modulate Cocaine’s Actions in Mice

Abstract

The presence and function of cannabinoid CB₂ receptors in the brain have been subject to debate. We report here that systemic, intranasal or intra-accumbens local administration of JWH133, a selective CB₂ receptor agonist, dose-dependently inhibits intravenous cocaine self-administration, cocaine-enhanced locomotion, and cocaine-enhanced accumbens dopamine (DA) in wild-type (WT) and CB₁ receptor-knockout (CB₁^−/−), but not CB₂^−/−, mice. This inhibition is mimicked by GW405833, another CB₂ receptor agonist with a different chemical structure, and is blocked by AM630, a selective CB₂ receptor antagonist. Intra-accumbens JWH133 alone dose-dependently decreases, while intra-accumbens AM630 elevates, extracellular DA and locomotion in WT and CB₁^−/− mice, but not in CB₂^−/− mice. Intra-accumbens AM630 also blocks the reduction in cocaine self-administration and extracellular DA produced by systemic administration of JWH133. These findings, for the first time, suggest that brain CB₂ receptors modulate cocaine’s rewarding and locomotor-stimulating effects, likely by a DA-dependent mechanism.

Behavioral and psychoactive effects of cannabinoids are mediated by activation of brain cannabinoid receptors^{1, 2}. Two major cannabinoid receptors (CB₁ and CB₂) have been identified. Since CB₁ receptors are highly expressed in the brain^{2, 3} and CB₂ receptors are found primarily in the periphery^{4, 5}, it has been heretofore generally believed that the behavioral and psychotropic effects of cannabinoids are CB₁-mediated^{1, 2} and that CB₂ receptor ligands have no psychoactive effects⁶. However, the purported lack of brain CB₂ receptors has been challenged by recent reports of low densities of CB₂ receptors on microglia⁷ and neuronal^8–11 cells in several brain regions - including the anterior olfactory nucleus, cerebral cortex, cerebellum, hippocampus, striatum and brainstem. Further, activation of CB₂ receptors by 2-arachidonoylglycerol, JWH015 or JWH133 inhibits locomotion^{10, 11}, morphine-6-glucuronide-induced emesis¹¹ and neuropathic pain^{12, 13}, while stimulating neural progenitor proliferation¹⁴ and producing neuroprotective effects^{15, 16}. More recent studies suggest that CB₂ receptor activation inhibits neuronal firing in dorsal-root ganglia and spinal cord^{17, 18} and GABAergic transmission in rat cerebral cortex¹⁹. These data suggest that functional CB₂ receptors may be expressed on central nervous system neuronal cells, prompting us to re-examine the role of CB₂ receptors in drug reward and addiction. To this end, we here used highly selective CB₂ receptor agonists and antagonists, combined with specific CB₁ receptor-knockout (CB₁^−/−) and CB₂ receptor-knockout (CB₂^−/−) mice, to investigate possible involvement of brain CB₂ receptors in cocaine’s behavioral and neurochemical effects.

RESULTS

JWH133 inhibits intravenous cocaine self-administration

To determine whether CB₂ receptor activation alters intravenous cocaine self-administration, we used JWH133, a highly selective CB₂ receptor agonist (200-fold selectivity for CB₂ versus CB₁)^{20, 21}, and AM630, a highly selective CB₂ receptor antagonist (160-fold selectivity for CB₂ versus CB₁)^{20, 21}, as pharmacological tools. We found that over 50% of wild-type (WT) (20 of 34) and CB₂^−/− (22 of 36) mice, while only about 30% of CB₁^−/− (10 of 36) mice acquired stable intravenous cocaine self-administration, defined as 20 or more infusions per 3-h session, with a regular pattern of self-administration achieved after 10 days of training (Supplementary Fig. 1). Strikingly, CB₁^−/− mice displayed a significant reduction in both total number and rate (infusions per h) of cocaine infusions on days 1–5, compared to WT or CB₂^−/− mice (Supplementary Fig. 1a, b). In addition, the majority of CB₁^−/− mice (7 of 10) displayed a distinct “burst-like” drug-taking pattern with long inter-burst intervals, while WT and CB₂^−/− mice displayed evenly-paced drug-taking without significant difference between the two strains (Supplementary Fig. 1c). These findings suggest that deletion of CB₁ receptors may lower cocaine’s rewarding efficacy, leading to a compensatory increase in drug intake during each individual drug-taking episode. This is further supported by the finding that CB₁^−/− mice displayed a significant reduction in break-point level for cocaine self-administration under progressive-ratio (PR) reinforcement, compared to WT mice (Supplementary Fig. 1d). Since PR break-point, defined as maximal work performed by the animal to get a cocaine infusion, is cocaine dose-dependent and positively correlated to reward strength²², the reduction in PR break-point observed in CB₁^−/− mice suggests a reduction in cocaine’s reward strength and/or motivation for cocaine-taking behavior. This is consistent with previous findings that CB₁ receptor deletion impairs cocaine’s rewarding, locomotor-stimulating, and DA-elevating effects^{23, 24}.

Intraperitoneal (i.p.) administration of JWH133 (10, 20 mg/kg) produced a significant and dose-dependent reduction in cocaine self-administration and cocaine intake in both WT and CB₁^−/− mice, but not in CB₂^−/− mice (Fig. 1a). This inhibition lasted for no longer than 24 hrs after 20 mg/kg JWH133 (Fig. 1b, c). Pretreatment with AM630, a selective CB₂ receptor antagonist, but not with AM251, a selective CB₁ receptor antagonist²⁵, significantly attenuated JWH133-induced inhibition of cocaine self-administration (Fig. 1d). This suggests that JWH133’s attenuating effect is mediated by activation of CB₂, not CB₁, receptors. This conclusion is further supported by the additional finding that systemic administration of GW405833 (3, 10 mg/kg, i.p.), another highly selective but structurally distinct CB₂ receptor agonist²⁶, also inhibited cocaine self-administration in WT mice (Fig. 2a).

Figure 1.

Effects of JWH133 on cocaine self-administration. (a) Systemic administration of JWH133 (10, 20 mg/kg, i.p., 30 min prior to testing) inhibits cocaine self-administration under FR1 reinforcement in WT (one-way ANOVA, F_2,16 = 13.09, P < 0.001) and CB₁^−/−(F_2,10 = 5.01, P < 0.05), but not CB₂^−/− (F_2,14=0.56, P = 0.58), mice. (b) Time course of JWH133’s attenuation of cocaine self-administration in WT mice on the test day. (c) Time course of recovery of cocaine self-administration in WT mice after JWH133 administration. (d) In WT mice, JWH133-induced attenuation of cocaine self-administration is prevented by pretreatment with the CB₂ receptor antagonist AM630 (10 mg/kg, i.p., 30 min prior to JWH133), but not by pretreatment with the CB₁ receptor antagonist AM251 (3 mg/kg, i.p.) (F_5,40 = 6.31, P < 0.001). Neither AM630 nor AM251 altered cocaine self-administration in WT mice. Data are means ± s.e.m. * P < 0.05, ** P < 0.01, compared to vehicle (Veh) control groups. ^### P < 0.001, compared to pre-JWH133 (−24 h) condition.

Figure 2.

Effects of GW405833 or JWH133 on cocaine self-administration. (a) GW405833 (3, 10 mg/kg, i.p.) dose-dependently inhibited cocaine self-administration under FR1 reinforcement in WT mice (one-way ANOVA, F_2,6 = 20.03, P < 0.01). (b) JWH133 (10, 20 mg/kg) or AM251 (3 mg/kg, i.p.) significantly lowered the cocaine self-administration break-point under PR reinforcement in WT mice (F_3,37 = 13.83, P < 0.001). (c) Intranasal microinjections of JWH133 (50, 100 µg/nostril) dose-dependently inhibited cocaine self-administration under FR1 reinforcement (F_2,18 = 14.34, P < 0.001). (d) Intravenous injection of the same micro-quantity (100, 200 µg) of JWH133 as used intranasally had no effect on cocaine self-administration (F_2,16 = 1.59, P = 0.23). (e) Intra-NAc microinjections of JWH133 (0.3, 1, 3 µg/side) dose-dependently inhibited cocaine self-administration under FR1 reinforcement in WT mice. This inhibition was blocked by intra-NAc co-administration of AM630 (3 µg/side) (F_3,24 = 4.49, P < 0.05). (f) Intra-NAc administration of JWH133 (3 µg/side) had no effect on cocaine self-administration in CB₂^−/− mice (F_1,10 = 2.37, P = 0.15). Data are means ± s.e.m. * P < 0.05, *** P < 0.001, compared to vehicle control group.

To determine whether JWH133-induced attenuation of cocaine self-administration was due to a reduction in cocaine’s rewarding efficacy, we studied JWH133’s effect on i.v. cocaine self-administration under PR reinforcement. We found that systemic administration of JWH133 (10, 20 mg/kg, i.p.) significantly lowered the PR break-point for cocaine self-administration in WT mice (Fig. 2b), suggesting a reduction in cocaine’s reward strength and/or motivation for drug-taking behavior after JWH133 administration. We previously showed that CB₁ receptor blockade by AM251 significantly lowered the PR break-point for cocaine self-administration in rats²⁷. We therefore also tested AM251 in the present study, and found that AM251 (3 mg/kg) lowered the PR break-point for cocaine self-administration in WT mice (Fig. 2b). These data suggest that the JWH133-induced reduction in cocaine self-administration resulted from a reduction in cocaine’s rewarding efficacy.

JWH133 inhibits cocaine self-administration by activation of brain CB₂ receptors

To further determine whether JWH133’s action was mediated by activation of brain or peripheral CB₂ receptors, we first studied the effects of intranasal microinjections of JWH133 on i.v. cocaine self-administration. Extensive studies have shown that a wide variety of compounds that cannot penetrate the blood-brain barrier can be delivered directly from nose into brain²⁸. We found that intranasal microinjections of JWH133 (50, 100 µg/10 µl/side) dose-dependently inhibited i.v. cocaine self-administration (Fig. 2c). To explore the possibility that effects of intranasal JWH133 might be mediated by drug absorption into the nasal vasculature with subsequent venous delivery of drug to pharmacological site(s) of action, we observed the effects of i.v. injection of the same micro-quantities of JWH133 as used intranasally on cocaine self-administration. We found that i.v. microinjections of JWH133 (100, 200 µg) had no effect on cocaine self-administration (Fig. 2d). These data suggest that intranasal JWH133-induced pharmacological effects are mediated by activating brain rather than peripheral CB₂ receptors. To further explore this issue, we observed the effects of local administration of JWH133 into the nucleus accumbens (NAc) on cocaine self-administration. We found that intra-NAc microinjections of JWH133 (0.3, 1, 3 µg/side) significantly and dose-dependently inhibited cocaine self-administration in WT mice (Fig. 2e), but not in CB₂^−/− mice (Fig. 2f). This inhibition was blocked by intra-NAc co-administration of AM630 (3 µg/side).

JWH133 itself has no reinforcing or aversive effects

We further examined whether JWH133 itself has cocaine-like rewarding effects. To address this issue, we first trained mice to acquire stable cocaine self-administration, and then cocaine was replaced by JWH133 (1 mg/kg/infusion) or vehicle. We found that neither JWH133 nor vehicle sustained stable self-administration in mice previously trained for cocaine self-administration (Supplementary Fig. 2a). In fact, the self-administration behavior underwent gradual extinction over the 5 days of substitution testing. This extinction pattern was essentially identical to that seen when vehicle was substituted for cocaine. However, when JWH133 or vehicle was replaced by cocaine, self-administration behavior returned to levels previously observed during stable cocaine self-administration. In addition, we also found that cocaine (10, 20 mg/kg, i.p.) produced a significant conditioned place preference, while JWH133, at the same doses, produced neither conditioned place preference nor place aversion in WT mice (Supplementary Fig. 2b). These findings suggest that JWH133 has no cocaine-like reinforcing nor aversive effects in mice.

JWH133 inhibits cocaine-enhanced locomotion

To determine whether JWH133’s effect on cocaine self-administration generalizes to other cocaine actions, we investigated the effects of JWH133 on cocaine-enhanced locomotion. Systemic administration of 10 mg/kg cocaine produced a significant increase in locomotion in all 3 mouse strains (Fig. 3). Pretreatment with JWH133 (10, 20 mg/kg, 30 min prior to cocaine) dose-dependently attenuated cocaine-enhanced locomotion in WT (Fig. 3a) and CB₁^−/− (Fig. 3b) mice, but not in CB₂^−/− (Fig. 3c) mice. Systemic administration of the same doses of JWH133 alone also significantly inhibited locomotion in a dose-dependent manner in WT and CB₁^−/− mice, but not in CB₂^−/− mice (Fig. 4a), suggesting an effect mediated by activation of CB₂ receptors. Since the same doses of JWH133 alone failed to alter locomotor performance on a fast-running rotarod device in all 3 mouse strains (Supplementary Fig. 3), we infer that JWH133’s inhibition of cocaine self-administration or locomotion is not produced by nonspecific impairment of locomotor capacity.

Figure 3.

Systemic administration of JWH133 (10, 20 mg/kg, i.p., 30 min prior to cocaine) dose-dependently inhibited cocaine-enhanced locomotion in WT (a, two-way ANOVA for repeated measures over time, F_2,16 = 14.45, P < 0.001) and CB₁^−/− (b, F_2,18=12.57, p<0.001), but not in CB₂^−/− (c, F_2,12 = 0.17, P = 0.85), mice. Data are means ± s.e.m. ** P < 0.01, *** P < 0.001, compared to vehicle treatment group.

Figure 4.

Effects of systemic or local intra-NAc administration of JWH133 or AM630 on locomotor activity. (a) Systemic administration of JWH133 (10, 20 mg/kg, i.p.) dose-dependently inhibited locomotion in WT (one-way ANOVA, F_2,24 = 8.03, P = 0.002) and CB₁^−/− (F_2,25 = 13.44, P < 0.001) mice, but not in CB₂^−/− (F_2,14 = 3.36, P > 0.05) mice. (b) Intra-NAc microinjections of JWH133 (1, 3 µg/side) significantly inhibited locomotion in WT (F_2,14=4.17, p<0.05) and CB₁^−/− (F_2,12 = 4.91, P < 0.05), but not in CB₂^−/− (F_2,14 = 0.04, P > 0.05), mice (c) Systemic administration of AM630 failed to alter locomotion in any strain of mice. (d) Intra-NAc administration of AM630 (1, 3, 10 µg/side) significantly augmented locomotion in WT (F_3,21 = 4.62, P < 0.05) and CB₁^−/− (F_2,12 = 10.57, P < 0.01), but not in CB₂^−/−(F_2,14 = 0.05, P > 0.05), mice. Data are means ± s.e.m. * P < 0.05, ** P < 0.01, compared to vehicle control group.

To further determine whether such locomotor inhibition is mediated by activation of brain CB₂ receptors, we observed the effects of intra-NAc JWH133 and/or AM630 on locomotion. We found that intra-NAc microinjections of JWH133 (1, 3 µg/side) significantly inhibited locomotion in WT or CB₁^−/− mice, but not in CB₂^−/− mice (Fig. 4b), in a dose-dependent manner similar to systemic administration (Fig. 4a). We note that systemic administration of AM630 failed to alter locomotion in any mouse strain tested (Fig. 4c). However, when locally administered into the NAc, AM630 (1, 3, 10 µg/side) significantly increased locomotor activity (Fig. 4d) in WT and CB₁^−/− mice, but not in CB₂^−/− mice. These data suggest that CB₂ receptors may tonically modulate locomotion. A higher brain AM630 level, achieved by local rather than by systemic administration, appears to be required to block endocannabinoid action on brain CB₂ receptors.

JWH133 inhibits cocaine-enhanced extracellular DA in the nucleus accumbens

Given the crucial role of the mesolimbic DA system in cocaine self-administration and modulation of locomotion²⁹, we further investigated the effects of JWH133 on basal and cocaine-enhanced DA in the NAc by in vivo microdialysis. We did not see significant differences in basal levels of extracellular NAc DA between WT and CB₂^−/− mice (Supplementary Fig. 4). However, CB₁^−/− mice displayed a significant basal reduction, compared to WT mice (Supplementary Fig. 4). Consistent with the findings in cocaine self-administration and locomotion, systemic administration of JWH133 (3, 10, 20 mg/kg, i.p.) also significantly and dose-dependently lowered extracellular NAc DA in WT (Fig. 5a) and CB₁^−/− (Fig. 5b) mice, but not in CB₂^−/− (Fig. 5c) mice. This reduction in NAc DA was blocked by AM630 (10 mg/kg, i.p.) in CB₁^−/− mice (Fig. 5b), suggesting that JWH133’s DA-inhibiting effect is mediated by activation of CB₂ receptors. Moreover, pretreatment with the same doses of JWH133 also significantly attenuated cocaine-enhanced NAc DA in WT (Fig. 5d), CB₁^−/− (Fig. 5e), but not in CB₂^−/− (Fig. 5f) mice.

Figure 5.

Systemic administration of JWH133 (3, 10, 20 mg/kg, i.p.) dose-dependently inhibited basal (a, b, c) or cocaine-enhanced (d, e, f) extracellular NAc DA in WT (a, two-way ANOVA for repeated measures over time, F_3,29 = 25.97, P < 0.001; d, F_2,19 = 4.47, P < 0.05) and CB₁^−/− (b, F_3,28 = 10.07, P < 0.001; e, F_2,16 = 4.78, P < 0.05) mice, but not in CB₂^−/− (c, F_2,23 = 0.10, P > 0.05; f, F_2,22 = 1.53, P > 0.05) mice. AM630 alone (10 mg/kg, i.p.) failed to alter NAc DA in CB₁^−/− mice, while AM630 pretreatment (10 mg/kg, i.p.) prevented JWH133-induced inhibition of NAc DA in CB₁^−/− mice (b). Data are means ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, compared to pre-drug baseline.^# P < 0.05,^## P < 0.01, compared to vehicle treatment group.

To determine whether this inhibition is mediated by activation of brain or peripheral CB₂ receptors, we also observed the effects of intranasal or intra-NAc local administration of JWH133 on extracellular DA. We found that intranasal administration of JWH133 (100 µg/nostril) produced a significant reduction in extracellular NAc DA in WT and CB₁^−/− mice, but not in CB₂^−/− mice (Fig. 6a). Similarly, intra-NAc local perfusion of JWH133 (1–1000 µM) significantly lowered extracellular DA in both WT and CB₁^−/− mice, but not in CB₂^−/− mice (Fig. 6b). In fact, an unexpected increase in extracellular DA was observed in CB₂^−/− mice after local administration of JWH133. The underlying mechanisms are unclear. One possibility is that JWH133 may bind to other (non-CB₂) receptors in CB₂^−/− mice, producing an increase in extracellular DA. Congruent with this finding, intra-NAc local perfusion of AM630 (1, 10, 100 µM) elevated extracellular DA in a concentration-dependent manner in both WT and CB₁^−/− mice, but not in CB₂^−/− mice (Fig. 6c), suggesting that endocannabinoids tonically modulate NAc DA release by activation of brain CB₂ receptors. Further, AM630-enhanced extracellular DA appears more robust in CB₁^−/− mice than in WT mice (Fig. 6c), suggesting higher endocannabinoid tone on brain CB₂ receptors in CB₁^−/− mice. Moreover, intra-NAc local perfusion of AM630 also blocked the reduction in extracellular NAc DA produced by systemic administration of JWH133 seen in WT and CB₁^−/− mice (Fig. 6c, d), suggesting that JWH133-induced inhibition of DA release is mediated by activation of NAc CB₂ receptors. The locations of microdialysis probes or microinjection cannulae were within the NAc (Supplementary Fig. 5).

Figure 6.

Effects of intranasal or intra-NAc local perfusion of JWH133 or AM630 on extracellular NAc DA. (a) Intranasal administration of JWH133 (50 µg/nostril) significantly lowered extracellular DA in WT and CB₁^−/− mice, but not in CB₂^−/− mice (two-way ANOVA for repeated measures over time, F_2,15 = 10.81, P = 0.001). (b) Intra-NAc local perfusion of JWH133 lowered extracellular DA in WT and CB₁^−/− mice in a dose-dependent manner, while elevating extracellular DA in CB₂^−/− mice (F_2,18=47.00, P < 0.001). (c) Intra-NAc local perfusion of AM630 elevated extracellular DA in WT and CB₁^−/− mice, but not in CB₂^−/− mice (F_2,18 = 12.13, P < 0.001). Further, AM630-enhanced extracellular DA appears more robust in CB₁^−/− mice than in WT mice (F_1,12 = 7.50, P < 0.05). (d) Renormalized data over new baselines 1 h before JWH133 administration from the data in Panel c, illustrating that intra-NAc local perfusion of AM630 blocked JWH133’s action on extracellular DA in WT and CB₁^−/− mice. Data are means ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, compared to pre-drug baseline.

DISCUSSION

Here we report that systemic administration of the CB₂ receptor agonist JWH133 significantly and dose-dependently inhibits intravenous cocaine self-administration under both FR1 and PR reinforcement and inhibits cocaine-enhanced locomotion and extracellular NAc DA in WT and CB₁^−/− mice, but not in CB₂^−/− mice. This effect was mimicked by GW405833 (another selective CB₂ receptor agonist), and blocked by AM630, a selective CB₂ receptor antagonist, but not by AM251, a selective CB₁ receptor antagonist, suggesting an effect mediated by activation of CB₂ receptors. Further, intranasal microinjections of JWH133, but not intravenous injections of the same micro-quantities of JWH133 as injected intranasally, also significantly and dose-dependently inhibited intravenous cocaine self-administration, suggesting an effect mediated by activation of brain, not peripheral, CB₂ receptors. This is further supported by the finding that local intra-NAc administration of JWH133 also significantly inhibited cocaine self-administration in a dose-dependent manner, an effect that was blocked by intra-NAc co-administration of AM630. In addition, intra-NAc local administration of JWH133 dose-dependently lowered, while AM630 elevated, basal levels of locomotion and extracellular NAc DA. Intra-NAc local perfusion of AM630 blocked the reduction in cocaine self-administration and NAc DA produced by systemic administration of JWH133. These data suggest that both the behavioral and neurochemical effects of JWH133 are mediated by activation of brain CB₂ receptors.

We note that systemic administration of AM630 failed to alter, while intra-NAc local administration of AM630 elevated, extracellular DA and locomotion, suggesting that local AM630 administration is more effective than systemic administration. This may be related to AM630’s relatively poor pharmacokinetic properties and/or blood-brain barrier passage. In addition, we also note that intra-NAc AM630 significantly elevated extracellular DA and locomotion, but failed to alter cocaine self-administration. This may be related to previous findings that locomotion is largely DA-dependent³⁰, while cocaine self-administration is dependent on both DA and non-DA mechanisms³¹. We also note that pharmacological blockade of NAc CB₂ receptors elevated, while genetic deletion of CB₂ receptors did not alter, basal extracellular DA in the NAc. The reasons are unclear. One possibility is that CB₂ receptor deletion-induced disinhibition of NAc DA release may be compromised by actions in other brain loci that modulate the mesolimbic DA system. Another possibility is that neuroadaptative processes may antagonize CB₂ receptor inactivation-induced DA neuronal disinhibition after CB₂ receptor deletion. Whatever the exact mechanism(s), the present data strongly suggest that brain CB₂ receptors functionally modulate the mesolimbic DA system and DA-related functions. Activation of brain CB₂ receptors by JWH133 inhibits both the behavioral and neurochemical effects of cocaine. Since JWH133 neither alters locomotor performance as assessed by the rotarod test, nor produces drug rewarding or aversive effects as assessed by i.v. self-administration and conditioned place preference, JWH133-induced inhibition of cocaine self-administration is most likely mediated by attenuation of cocaine’s rewarding efficacy secondary to the reduction in cocaine-enhanced NAc DA rather than by nonspecific locomotor impairment or malaise.

We fully recognize that these findings challenge the currently accepted opinion that selective CB₂ receptor agonists have no CNS effects. This opinion is largely based upon previous reports that the selective CB₂ receptor agonist AM1241 neither inhibits locomotion or rotarod performance, nor produces catalepsy or hypothermia in rats or mice³². In addition, AM1241 also failed to alter brain functional activity as assessed by pharmacological MRI³³. The ineffectiveness of AM1241 may be related to the relatively lower doses (30 µg-3.3 mg/kg) used in those studies, relatively poor selectivity, and species differences in CB₂ receptor response to AM1241^34–36. For example, AM1241 is reported to act as a full or partial agonist at human CB₂ receptors³⁵, while acting as an inverse agonist at rodent CB₂ receptors³⁶. Further, the analgesic effects produced by AM1241 are reported to be blocked by the opioid receptor antagonist naloxone³⁷, suggesting that AM1241 may interact with other, non-cannabinoid, receptors. However, the CB₂ receptor agonist GW405833, at high doses (30–100 mg/kg), produces significant CNS effects such as analgesia, sedation and catalepsy²⁶, consistent with our finding that GW405833 (3–10 mg/kg) significantly inhibits cocaine self-administration in mice.

The presence of CB₂ receptors in the CNS, in particular on neurons, has been subject to debate^{10, 38}. Previous studies using in situ hybridization and Northern blot assays failed to detect CB₂ receptor mRNA in brain^{5, 39, 40}. However, recent studies with more sensitive RT-PCR and immunolabeling techniques have claimed to find significant CB₂ receptor expression in microglia and subpopulations of neuronal cells in brain^7–11. By using highly sensitive and specific Taqman probes, we have recently identified two CB₂ receptor isoforms (CB_2A, CB_2B) in both brain and peripheral tissues, which display significant species differences in both structure and expression between humans, rats and mice⁴¹. It is now well accepted that CB₂ receptors are expressed on microglia and a subset of neurons with levels increasing under certain pathological conditions such as neuroinflammation and brain injury³⁸. There are two possibilities to explain the present findings. First, a low density of CB₂ receptors may be expressed on mesolimbic DA neurons. Since CB₂ receptors are G_i/o coupled⁴², activation of CB₂ receptors on DA neurons in the midbrain ventral tegmental area (VTA) may directly inhibit VTA DA neurons and decrease NAc DA release, and therefore inhibit intravenous cocaine self-administration and cocaine-enhanced locomotion as observed in the present study. Although direct evidence of CB₂ receptor expression in the mesolimbic DA neurons is lacking at present, functional CB₂ receptors are found in other neurons. For example, CB₂ receptor mRNA is expressed on striatal GABAergic neurons in non-human primates⁴³, and activation of CB₂ receptors inhibits GABAergic neurotransmission in the medial entorhinal cortex of the rat¹⁹. In addition, CB₂ receptors are also found on neurons in the dorsal-root ganglion (DRG) and spinal cord (SC)^{44, 45}, and activation of CB₂ receptors on DRG-SC neurons inhibits neuronal response to noxious stimuli^{45, 46}, thereby contributing to the antinociceptive effects of CB₂ receptor agonists⁴⁷. The second possibility is that activation of CB₂ receptors located on microglial cells or astrocytes in the VTA and/or NAc may indirectly inhibit NAc DA release by releasing cytokines and inflammatory factors⁴⁸, thereby inhibiting cocaine self-administration and cocaine-enhanced locomotion as observed in the present study.

Whatever the mechanisms, the present findings, for the first time, suggest that activation of brain CB₂ receptors inhibits cocaine’s rewarding and psychomotor-stimulating effects, which is congruent with a rapidly expanding corpus of published reports implicating brain CB₂ receptors in modulating a variety of CNS functions such as locomotion¹⁰, pain^{13, 47}, emesis¹¹, neurogenesis¹⁴, and neuroprotection¹⁵. This finding not only challenges current views that CB₂ receptors are absent from the CNS and that CB₂ receptor ligands lack CNS effects, but also suggests that brain CB₂ receptors may be a novel target for the pharmacotherapy of drug abuse and addiction.