Facilitation of CB1 receptor-mediated neurotransmission decreases marble burying behavior in mice

Obsessive-compulsive disorder (OCD) is a common psychiatric disorder characterized by the occurrence of obsessions and compulsions. Glutamatergic abnormalities have been related to the pathophysiology of OCD. Cannabinoids inhibit glutamate release in the central nervous system, but the involvement of drugs targeting the endocannabinoid system has not yet been tested in animal models of repetitive behavior. Thus, the aim of the present study was to verify the effects of the CB1 receptor agonist WIN55,212-2, the inhibitor of anandamide uptake AM404 and the anandamide hydrolysis inhibitor URB597, on compulsive-associate behavior in male C57BL/6J mice submitted to the marble burying test (MBT), an animal model used for anti-compulsive drug screening. WIN55,212-2 (1 and 3 mg/kg), AM404 (1 and 3 mg/kg) and URB597 (0.1, 0.3 and 1 mg/kg) induced a significant decrease in the number of buried marbles compared to controls. Pretreatment with the CB1 receptor antagonist, AM251, prevented both WIN55,212-2 and URB597 effects. These results suggest a potential role for drugs acting on the cannabinoid system in modulating compulsive behavior.     

CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus

In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30–300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction–liquid chromatography–tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K⁺, with or without addition of Ca²⁺, or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.     

Cannabinoids modulate hippocampal memory and plasticity

Considerable evidence demonstrates that cannabinoid agonists impair whereas cannabinoid antagonists improve memory and plasticity. However, recent studies suggest that the effects of cannabinoids on learning do not necessarily follow these simple patterns, particularly when emotional memory processes are involved. We investigated the involvement of the cannabinoid system in hippocampal learning and plasticity using the fear‐related inhibitory avoidance (IA) and the non‐fear‐related spatial learning paradigms, and cellular models of learning and memory, i.e., long‐term potentiation (LTP) and long‐term depression (LTD). We found that microinjection into the CA1 of the CB1/CB2 receptor agonist WIN55,212‐2 (5 μg/side) and an inhibitor of endocannabinoid reuptake and breakdown AM404 (200 ng/side) facilitated the extinction of IA, while the CB1 receptor antagonist AM251 (6 ng/side) impaired it. WIN55,212‐2 and AM251 did not affect IA conditioning, while AM404 enhanced it, probably due to a drug‐induced increase in pain sensitivity. However, in the water maze, systemic or local CA1 injections of AM251, WIN55,212‐2, and AM404 all impaired spatial learning. We also found that i.p. administration of WIN55,212‐2 (0.5 mg/kg), AM404 (10 mg/kg), and AM251 (2 mg/kg) impaired LTP in the Schaffer collateral‐CA1 projection, whereas AM404 facilitated LTD. Our findings suggest diverse effects of the cannabinoid system on CA1 memory and plasticity that cannot be categorized simply into an impairing or an enhancing effect of cannabinoid activation and deactivation, respectively. Moreover, they provide preclinical support for the suggestion that targeting the endocannabinoid system may aid in the treatment of disorders associated with impaired extinction‐like processes, such as post‐traumatic stress disorder. © 2009 Wiley‐Liss, Inc.     

Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons

To understand the functional significance and mechanisms of action in the CNS of endogenous and exogenous cannabinoids, it is crucial to identify the neural elements that serve as the structural substrate of these actions. We used a recently developed antibody against the CB1 cannabinoid receptor to study this question in hippocampal networks. Interneurons with features typical of basket cells showed a selective, intense staining for CB1 in all hippocampal subfields and layers. Most of them (85.6%) contained cholecystokinin (CCK), which corresponded to 96.9% of all CCK-positive interneurons, whereas only 4.6% of the parvalbumin (PV)-containing basket cells expressed CB1. Accordingly, electron microscopy revealed that CB1-immunoreactive axon terminals of CCK-containing basket cells surrounded the somata and proximal dendrites of pyramidal neurons, whereas PV-positive basket cell terminals in similar locations were negative for CB1. The synthetic cannabinoid agonist WIN 55,212-2 (0.01-3 microM) reduced dose-dependently the electrical field stimulation-induced [3H]GABA release from superfused hippocampal slices, with an EC50 value of 0. 041 microM. Inhibition of GABA release by WIN 55,212-2 was not mediated by inhibition of glutamatergic transmission because the WIN 55,212-2 effect was not reduced by the glutamate blockers AP5 and CNQX. In contrast, the CB1 cannabinoid receptor antagonist SR 141716A (1 microM) prevented this effect, whereas by itself it did not change the outflow of [3H]GABA. These results suggest that cannabinoid-mediated modulation of hippocampal interneuron networks operate largely via presynaptic receptors on CCK-immunoreactive basket cell terminals. Reduction of GABA release from these terminals is the likely mechanism by which both endogenous and exogenous CB1 ligands interfere with hippocampal network oscillations and associated cognitive functions.     

Hippocampal endocannabinoids play an important role in induction of long-term potentiation and regulation of contextual fear memory formation

Recent studies show contradictory results regarding the contribution of endocannabinoids in fear memory formation and long-term synaptic plasticity. In this study, we investigated the effects of both cannabinoid receptor type 1 (CB1 receptor) antagonist AM281 and anandamide reuptake inhibitor AM404 on the formation of contextual fear memory in adult mice. Both i.p. and intra-hippocampal injections of AM281 promoted contextual fear memory while a high dose of AM404 inhibited it. These findings demonstrate that CB1 receptor-mediated signaling negatively contributes to contextual fear memory formation. We further investigated the induction of long-term potentiation (LTP) in CA1 pyramidal neurons of hippocampal slices and found that AM281 impaired the induction of LTP. Additionally, the blockade of LTP by AM281 was completely prevented by bath application of picrotoxin, a selective antagonist of GABA(A) receptor. Taken together, these results indicate that activation of CB1 receptor contributes to induction of LTP via a GABA(A) receptor-mediated mechanism.     

Differential stimulatory effects of cannabinoids on VIP release and NO synthase activity in synaptosomal fractions from rat ileum

Cannabinoid-1 (CB1) and CB2 receptors are present on neurons of the enteric nervous system. Our aim was to study whether cannabinoid receptor activation is involved in the regulation of VIP release and NO synthesis in isolated fractions of nerve terminals from rat ileum. VIP was measured by RIA and NO synthesis was analyzed using a L-[3H]arginine assay. Anandamide stimulated VIP release (basal: 245.9+/-12.4pg/mg, 10(-6)M: 307.6+/-11.7pg/mg, [n=6, P<0.05], 10(-7)M: 367.0+/-26.1pg/mg, [n=6, P<0.01]). The cannabinoid receptor agonist WIN 55,212-2 had similar effects (basal: 250.5+/-37.4pg/mg, 10(-6)M: 320.9+/-34.7pg/mg; [n=4, P<0.05]). The stimulatory effect of anandamide was blocked by the selective CB2 receptor antagonist, SR144528 (10(-7)M) (anandamide 10(-6)M: 307.6+/-11.7pg/mg; +SR144528: 249.0+/-26.3pg/mg, [n=6, P<0.05]), whereas the selective CB1 receptor antagonist SR141716 A had no effect. NO synthesis was stimulated by anandamide ([fmol/mg/min] basal: 0.08+/-0.01, 10(-6)M: 0.16+/-0.03; 10(-7)M: 0.13+/-0.02, n=4, P<0.05) and WIN 55,212-2 ([fmol/mg/min] basal: 0.05+/-0.01, 10(-6)M: 0.1+/-0.02, n=4, P<0.05). The anandamide reuptake inhibitor, AM 404 increased basal NOS activity ([fmol/mg/min] control: 0.1+/-0.04, 10(-6)M: 0.28+/-0.08, n=7, P<0.05). The stimulatory effect of anandamide on NO synthase was not antagonized by antagonists at the CB1, CB2 or TRPV1 receptor, respectively. In conclusion, in enteric nerves anandamide stimulates VIP release by activation of a CB2 receptor specific pathway, while the stimulation of NO production suggests the existence of an additional type of cannabinoid receptor in the enteric nervous system.     

Differential effects of endocannabinoids on [(3)H]-GABA uptake in the rat globus pallidus

In the globus pallidus, cannabinoid CB(1) receptors are localized pre-synaptically on GABAergic neurons. We assessed the influence of the endocannabinoids, anandamide, 2-arachidonoyl-glycerol (2-AG) and noladin ether, on the uptake of [(3)H]-GABA in pallidal slices from rat. Both 2-AG and noladin ether increased [(3)H]-GABA uptake (by 40.8 +/- 8.0% and 38.4 +/- 12.5%). The effect of 2-AG was blocked by the cannabinoid CB(1) receptor antagonist AM 251. In contrast, neither anandamide nor the agonist WIN 55,212-2 had an effect on [(3)H]-GABA uptake. Different roles might be played by different endocannabinoids, both physiologically and in basal ganglia disorders, such as Parkinson's disease.     

Antinociceptive effect of cannabinoid agonist WIN 55,212-2 in rats with a spinal cord injury

Spinal cord injury (SCI) pain exhibits many symptoms associated with peripheral neuropathic pain, including increased tactile hypersensitivity. One novel approach to ameliorate SCI pain is the use of cannabinoid (CB) ligands. The current study evaluated the efficacy of the nonselective CB receptor agonist WIN 55,212-2 on tactile hypersensitivity in rats following a brief compression to the thoracic spinal cord. The withdrawal thresholds of the hind paws following SCI were significantly decreased, indicating tactile hypersensitivity. Systemic injection of WIN 55,212-2 increased withdrawal thresholds in a dose-dependent manner. Pretreatment with the CB(1) receptor subtype-selective antagonist AM 251 completely abolished the antinociceptive effect of WIN 55,212-2 whereas pretreatment with the CB(2) receptor subtype-selective antagonist AM 630 did not alter the antinociceptive effect of WIN 55,212-2. These data indicate that a CB(1)-selective agonist may be novel therapeutic treatment for clinical SCI pain.     

Cannabinoids ablate release of TNFalpha in rat microglial cells stimulated with lypopolysaccharide

Upon activation, brain microglial cells release proinflammatory mediators, such as TNFalpha, which may play an important role in eliciting neuroinflammatory processes causing brain damage. As cannabinoids have been reported to exert anti-inflammatory and neuroprotective actions in the brain, we here examined the effect of both synthetic and endogenous cannabinoids on TNFalpha release elicited by bacterial endotoxin lypopolysaccharide (LPS) in cultured microglia. Exposure of primary cultures of rat cortical microglial cells to LPS significantly stimulated TNFalpha mRNA expression and release. The endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG), as well as the synthetic cannabinoids (+)WIN 55,212-2, CP 55,940, and HU210, inhibited in a concentration-dependent manner (1-10 microM) the LPS-induced TNFalpha release. Unlike the high-affinity cannabinoid receptor agonist (+)WIN 55,212-2, the low-affinity stereoisomer (-)WIN 55,212-2 did not exert any significant inhibition on TNFalpha release. Given this stereoselectivity, the ability of (+)WIN 55,212-2 to inhibit LPS-induced TNFalpha release from microglia is most likely receptor-mediated. By RT-PCR we found that the two G(i/o) protein-coupled cannabinoid receptors (type 1 and 2) are both expressed in microglial cultures. However, selective antagonists of type 1 (SR141716A and AM251) and type 2 (SR144528) cannabinoid receptors did not affect the effect of (+)WIN 55,212-2. Consistent with this finding is the observation that the ablative effect of (+)WIN 55,212-2 on LPS-evoked release of TNFalpha was not sensitive to the G(i/o) protein inactivator pertussis toxin. In addition, the cAMP elevating agents dibutyryl cAMP and forskolin both abolished LPS-induced TNFalpha release, thus rendering unlikely the possibility that (+)WIN 55,212-2 could ablate TNFalpha release through the inhibition of adenylate cyclase via the G(i)-coupled cannabinoid receptors type 1 and 2. In summary, our data indicate that both synthetic and endogenous cannabinoids inhibit LPS-induced release of TNFalpha from microglial cells. By showing that such effect does not appear to be mediated by either CB receptor type 1 or 2, we provide evidence suggestive of the existence of yet unidentified cannabinoid receptor(s) in brain microglia.     

Cannabinoid receptor agonist WIN 55,212-2 inhibits rat cortical dialysate gamma-aminobutyric acid levels

The effects of the cannabinoid receptor agonist WIN 55,212-2 (0.1-5 mg/kg i.p.) on endogenous extracellular gamma-aminobutyric acid (GABA) levels in the cerebral cortex of the awake rat was investigated by using microdialysis. WIN 55,212-2 (1 and 5 mg/kg i.p.) was associated with a concentration-dependent decrease in dialysate GABA levels (-16% +/- 4% and -26% +/- 4% of basal values, respectively). The WIN 55,212-2 (5 mg/kg i.p.) induced-inhibition was counteracted by a dose (0.1 mg/kg i.p.) of the CB(1) receptor antagonist SR141716A, which by itself was without effect on cortical GABA levels. These findings suggest that cannabinoids decrease cortical GABA levels in vivo, an action that might underlie some of the cognitive and behavioral effects of acute exposure to marijuana.     

Effects of WIN55,212-2 on voltage-gated sodium channels in trigeminal ganglion neurons of rats

OBJECTIVES: This study was carried out to investigate the effects of WIN55,212-2, a potential cannabinoid receptor agonist, on voltage-gated sodium currents I(Na) in cultured trigeminal ganglion neurons of rats, and to investigate whether the anti-nociceptive effects of cannabinoid receptor subtype 1 (CB1) were produced through its modulation on I(Na). METHODS: Whole cell patch clamp techniques were used to record I(Na) before and after WIN55,212-2 was perfused in cultured trigeminal ganglion neurons of rats. RESULTS: WIN55,212-2 (0.01 micromol/l) could enhance I(Na) slightly by 11.5 +/- 4.7% (n=7, p<0.05), and this effect could not be blocked by AM251, the CB1 receptor antagonist. However, WIN55,212-2 could inhibit I(Na) in concentration dependent manner at concentrations from 0.1 to 100 micromol/l. The inhibitory rates were 17.4 +/- 6.0, 22.5 +/- 7.8, 43.9 +/- 9.4 and 73.9 +/- 6.7% respectively by 0.1, 1, 10, 100 micromol/l WIN55,212-2, and the EC(50) was 17.8 micromol/l (n=7, p<0.05 or p<0.01). This inhibitory effect could be blocked partly by 1 micromol/l AM251 (n=7, p<0.05). WIN55,212-2 (0.01 micromol/l) shifted the active curve of I(Na) leftward slightly (n=7, p<0.05), but had no effect on its stable inactive curve (n=7, p>0.05). WIN55,212-2 (10 micromol/l) did not affect the active and stable inactive curves of I(Na) (n=7, p>0.05). CONCLUSION: WIN55,212-2 had bidirectional (two phases) effects on I(Na) in trigeminal ganglion neurons. It might act on different receptors, and the CB1 receptor participated in its modulation on I(Na).     

Uptake blockade of endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition

Previous studies supporting a possible physiological role for an endogenous cannabinoid, arachidonylethanolamide (AEA, anandamide), showed a significant increase in AEA content in the nucleus tractus solitarius (NTS) after an increase in blood pressure (BP) and prolonged baroreflex inhibition of renal sympathetic nerve activity (RSNA) after exogenous AEA microinjections into the NTS. These results, along with other studies, support the hypothesis that endogenous AEA can modulate the baroreflex through cannabinoid CB(1) receptor activation within the NTS. This study was performed to characterize the physiological role of endogenously released cannabinoids (endocannabinoids) in regulating baroreflex control of RSNA through actions in the NTS. Endocannabinoid effects were assessed by measuring the RSNA baroreflex response to increased pressure after bilateral microinjections of AM404, an endocannabinoid transport inhibitor, into the NTS of adult male Sprague Dawley rats. AM404 blocks uptake of endocannabinoids and enhances the effects of any endocannabinoids released [M. Beltramo, et al., Functional role of high-affinity anandamide transport, as revealed by selective inhibition, Science 277 (5329) (1997) 1094-1097.] into the NTS. Therefore, it was hypothesized that microinjections of AM404 should exhibit effects similar to microinjections of exogenous AEA. In this study, AM404 microinjections into the NTS were found to significantly prolong baroreflex inhibition of RSNA compared to control, similar to effects of exogenous AEA. This effect is thought to result from an increased endocannabinoid presence in the NTS, leading to prolonged CB(1) receptor activation. These results indicate that endocannabinoids released in the NTS have the potential to modulate baroreflex control at this site in the central baroreflex pathway.     

Cannabinoids inhibit the release of [3H]glutamate from rodent hippocampal synaptosomes via a novel CB1 receptor-independent action

In this study we investigated the effect of cannabinoids on [3H]glutamate release from hippocampal synaptosomes of rat and CB1-null mutant mouse. In the rat, cannabinoid receptor agonists, i.e. CP55,940 (EC50, 0.84 microm), WIN55,212-2 (EC50, 3.47 microm), ACEA (EC50, 17.8 microm), and R-(+)-methanandamide (EC50, 19.8 microm) concentration-dependently inhibited the 25-mm-K+ depolarization-evoked release of [3H]glutamate and, among them, WIN55,212-2 displayed the greatest efficacy. The CB1 receptor antagonists SR141716A (1-5 microm) and AM251 (1 microm) and the VR1 vanilloid receptor antagonist capsazepine (10 microm) did not antagonize the effect of the agonists. SR141716A by itself attenuated the evoked [3H]glutamate release. WIN55,212-2 inhibited the release of [3H]glutamate in CB1 -/- mice as well. These data demonstrate that the action of cannabinoids on glutamate release in the hippocampus is pharmacologically distinct and independent from the cloned CB1 receptor.     

Locomotor activity and occupancy of brain cannabinoid CB1 receptors by the antagonist/inverse agonist AM281

The goals of this study were to examine the relationship between intravenous doses of the cannabinoid CB1 receptor antagonist AM281 (N-(morpholin-4-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and the degree of occupancy of this receptor, and to relate occupancy to the ability of this compound to antagonize the sedative effects of the cannabinoid receptor agonist WIN 55,212-2. Occupancy was determined by measuring the ability of intravenous doses of AM281 to inhibit in vivo binding of [(131)I]AM281 in brain areas, and locomotor activity was assessed by measuring the rate of beam crossings in a photocell apparatus. As previously documented, WIN 55,212-2 (1 mg/kg, i.v.) significantly reduced locomotor activity at early times after administration. Co-injection of AM281 (0.3 mg/kg i/v) and WIN 55, 212-2 restored the rate of beam crossings to that seen on injection of vehicle. In addition, AM281 (0.3 mg/kg i/v) approximately doubled locomotor activity between 60-120 min when injected alone. The IC(50) value for displacement of [(131)I]AM281 by AM281 was 0.45 mg/kg. These observations confirm earlier indications that AM281 is a CB1 receptor antagonist or inverse agonist and suggest the existence of an endogenous cannabinoid tone that moderates exploratory locomotor activity.     

Differential effects of the sleep-inducing lipid oleamide and cannabinoids on the induction of long-term potentiation in the CA1 neurons of the rat hippocampus in vitro

Cannabinoids have been shown to impair cognition in vivo and block long-term potentiation (LTP), a candidate experimental model of learning and memory in vitro, via cannabinoid receptor (CB1) activation. cis-Oleamide (cOA) is an endogenous sleep-inducing lipid with putative cannabinomimetic properties. We hypothesise that cOA is cannabinomimetic and perform a comparative study with synthetic and endogenous cannabinoids on their effects on synaptic conditioning via two different patterns of stimulation in the hippocampal slice. CB1 agonists, R(+)-WIN55212-2 and anandamide, but not cOA blocked high frequency stimulation (HFS)-LTP. R(+)-WIN55212-2 and cOA (stereoselectively) attenuated responses to theta-burst-LTP, while anandamide did not. The anandamide transport inhibitor, AM404, attenuated HFS-LTP, an effect reversed by the CB1 receptor antagonist SR141716A but not mimicked by the vanilloid receptor agonist capsaicin. TFNO, an inhibitor of fatty acid amide hydrolase (FAAH), the enzyme responsible for degrading anandamide, failed to block HFS-LTP alone or in combination with cOA. On the contrary, this combination was as effective as cOA on its own in attenuating theta-burst-LTP. cOA effects on theta-burst-LTP were prevented in the presence of the GABA(A) receptor blocker picrotoxin, but not by pretreatment with SR141716A. These findings suggest that cOA neither directly activates CB1 receptors nor acts via the proposed "entourage" effect [Nature 389 (1997) 25] to increase titres of anandamide through FAAH inhibition. The selective effects of cOA on theta-burst-conditioning may reflect modulation of GABAergic transmission. Anandamide uptake inhibition, but not blockade of FAAH, effectively increases synaptic concentrations of endocannabinoids.     

Evaluation of the neuroprotective effect of cannabinoids in a rat model of Parkinson's disease: importance of antioxidant and cannabinoid receptor-independent properties

We have recently demonstrated that two plant-derived cannabinoids, Delta9-tetrahydrocannabinol and cannabidiol (CBD), are neuroprotective in an animal model of Parkinson's disease (PD), presumably because of their antioxidant properties. To further explore this issue, we examined the neuroprotective effects of a series of cannabinoid-based compounds, with more selectivity for different elements of the cannabinoid signalling system, in rats with unilateral lesions of nigrostriatal dopaminergic neurons caused by local application of 6-hydroxydopamine. We used the CB1 receptor agonist arachidonyl-2-chloroethylamide (ACEA), the CB2 receptor agonist HU-308, the non-selective agonist WIN55,212-2, and the inhibitors of the endocannabinoid inactivation AM404 and UCM707, all of them administered i.p. Daily administration of ACEA or WIN55,212-2 did not reverse 6-hydroxydopamine-induced dopamine (DA) depletion in the lesioned side, whereas HU-308 produced a small recovery that supports a possible involvement of CB2 but not CB1 receptors. AM404 produced a marked recovery of 6-hydroxydopamine-induced DA depletion and tyrosine hydroxylase deficit in the lesioned side. Possibly, this is caused by the antioxidant properties of AM404, which are derived from the presence of a phenolic group in its structure, rather than by the capability of AM404 to block the endocannabinoid transporter, because UCM707, another transporter inhibitor devoid of antioxidant properties, did not produce the same effect. None of these effects were observed in non-lesioned contralateral structures. We also examined the timing for the effect of CBD to provide neuroprotection in this rat model of PD. We found that CBD, as expected, was able to recover 6-hydroxydopamine-induced DA depletion when it was administered immediately after the lesion, but it failed to do that when the treatment started 1 week later. In addition, the effect of CBD implied an upregulation of mRNA levels for Cu,Zn-superoxide dismutase, a key enzyme in endogenous defenses against oxidative stress. In summary, our results indicate that those cannabinoids having antioxidant cannabinoid receptor-independent properties provide neuroprotection against the progressive degeneration of nigrostriatal dopaminergic neurons occurring in PD. In addition, the activation of CB2 (but not CB1) receptors, or other additional mechanisms, might also contribute to some extent to the potential of cannabinoids in this disease.     

Anandamide suppression of Na+ currents in rat dorsal root ganglion neurons

Anandamide, the ethanolamide of arachidonic acid, is an endogenous cannabinoid. It is an agonist at CB1 and CB2 cannabinoid receptors as well as the vanilloid receptor, VR1. It is analgesic in inflammatory and neuropathic pain. Both central and peripheral mechanisms are considered to participate in its analgesia. Primary sensory neurons express Na+ currents that are involved in the pathogenesis of pain. We examined the effect of anandamide on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ currents in rat dorsal root ganglion neurons. Anandamide inhibited both Na+ currents in a concentration-dependent manner. At a membrane potential of -80 mV, the current inhibition was greater in TTX-S than TTX-R currents (K(d); 5.4 microM vs. 38.4 microM). The activation and inactivation became faster in TTX-R current but not in TTX-S current. Anandamide did not alter the activation voltage in either type of current. It, however, produced a hyperpolarizing shift of the steady-state inactivation voltage in both types of currents. The maximum availability at a large negative potential was not reduced by anandamide. Thus, anandamide seems to affect inactivated Na+ channels rather than resting channels. The inhibition of Na+ currents was not reversed by AM 251 (a CB1 antagonist), AM 630 (a CB2 antagonist) or capsazepine (a VR1 antagonist), suggestive of a direct action of anandamide on Na+ channels. The inhibition of Na+ currents in sensory neurons may contribute to the anandamide analgesia.     

Cannabinoid agonist WIN55,212-2 induces apoptosis in cerebellar granule cells via activation of the CB1 receptor and downregulation of bcl-xL gene expression

The endogenous cannabinoid system is involved in the regulation of a number of physiologic effects in both the central and peripheral nervous systems. Its role in the control of neuronal cell proliferation has attracted major attention because of its potential implications for new therapeutic strategies. In the present study, we demonstrated that treatment of cultured cerebellar granule cells with the synthetic cannabinoid WIN55,212-2, causes cell-body and nuclear shrinkage, which are hallmarks of neuronal apoptosis, as well as concentration-dependent decrease in cell viability. Staining with the fluorescent nuclear dye, Hoechst 33258, revealed apoptosis in 27.1% and 58.5% of cells exposed to 1 and 10 microM of WIN55,212-2, respectively (P < 0.01 and P < 0.001 vs. control respectively) after 36 hr. After 24 hr of exposure to WIN55,212-2, mRNA levels for the anti-apoptotic gene bcl-xL were reduced to 45.6% of those found in control (P < 0.01). These effects were completely reverted when cells were exposed to the synthetic cannabinoid in the presence of the specific CB1-receptor antagonist, SR141716A (1 microM). Moreover, the pro-apoptotic effect of 10 microM WIN55,212-2 could be reduced by the addition to the incubation medium of a cell-permeant inhibitor of caspase-1 (50 nM). Finally, WIN55,212-2 significantly increased caspase-1 activity after 24 hr. These findings show that the activation of CB1 receptors on cerebellar granule cells induces apoptotic cell death, which is associated with downregulation of the anti-apoptotic gene, bcl-xL, and at least in part, activation of caspase-1.     

The anandamide transport inhibitor AM404 reduces ethanol self-administration

The endocannabinoid system mediates in the pharmacological actions of ethanol and genetic studies link endocannabinoid signaling to alcoholism. Drugs activating cannabinoid CB1 receptors have been found to promote alcohol consumption but their effects on self-administration of alcohol are less clear because of the interference with motor performance. To avoid this problem, a novel pharmacological approach to the study of the contribution of the cannabinoid system in alcoholism may be to use drugs that locally amplify the effects of alcohol on endogenous cannabinoids. In the present study we addressed this model by studying the effects of the anandamide transport inhibitor N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404) on both ethanol self-administration and reinstatement of alcohol-seeking behavior in rats. The results show that AM404 significantly reduced ethanol self-administration in a dose-dependent manner but failed to modify reinstatement for lever pressing induced by the stimulus associated with alcohol. This effect was not due to a motor depressant effect and was not related to a decrease in general motivational state, as it was not effective in other reward paradigms such as lever pressing for a saccharin solution. The mechanism of action of AM404 does not involve cannabinoid CB1 receptors as the CB1-selective antagonist SR141716A did not block the reduction of ethanol self-administration induced by the anandamide uptake blocker. Moreover, 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-delta 8-tetrahydrocannabinol (HU-210), a classical cannabinoid receptor agonist, did not affect ethanol self-administration. The effects of AM404 are not mediated by either vanilloid VR1 receptors or cannabinoid CB2 receptors because it is not antagonized by either the VR1 receptor antagonist capsazepine or the CB2 antagonist AM630. These results indicate that AM404 may be considered as an innovative approach to reduce alcohol consumption.     

Species and strain differences in the expression of a novel glutamate-modulating cannabinoid receptor in the rodent hippocampus

A novel, non-CB1 cannabinoid receptor has been defined by the persistence of inhibition of glutamatergic EPSPs by the cannabinoid receptor agonist WIN55,212-2 in mice lacking the cloned CB1 receptor (CB1-/-) (Hajos et al., 2001). This novel receptor was also distinguished from CB1 by its sensitivity to the antagonist SR141716A and its insensitivity to the antagonist AM251 (Hajos & Freund, 2002). We have chosen to refer to this putative receptor as CBsc due to its identification on Schaffer collateral axon terminals in the hippocampus. We examined properties of CBsc receptors in Sprague Dawley (SD) rats and two strains of wild-type (WT) mice (C57BL/6J and CD1) used as backgrounds for two independent lines of CB1-/- mice (Ledent et al., 1999; Zimmer et al., 1999). The inhibition of synaptic glutamate release by WIN55,212-2 was observed in hippocampal slices from WT CD1 mice and SD rats but was absent in WT C57 mice. We also found that AM251 and SR141716A antagonized the effect of WIN55,212-2 in hippocampal slices from CD1 mice and SD rats demonstrating a lack of selectivity of these ligands for CB1 and CBsc receptors in these animals. The results indicate that the glutamate-modulating CBsc cannabinoid receptor is present in the hippocampi of CD1 mice and SD rats but not in C57BL/6J mice. Thus, we have identified animal models that may permit the study of cannabinoids independently of the novel CBsc receptor (C57CB1+/+), the CBsc receptor independently of the cloned CB1 receptor (CD1CB1-/-), or in the absence of both receptors (C57CB1-/-).