Cannabinoid Receptor Activation Prevents the Effects of Chronic Mild Stress on Emotional Learning and LTP in a Rat Model of Depression

Most psychiatric disorders are characterized by emotional memory or learning disturbances. Chronic mild stress (CMS) is a common animal model for stress-induced depression. Here we examined whether 3 days of treatment using the CB1/2 receptor agonist WIN55,212-2 could ameliorate the effects of CMS on emotional learning (ie, conditioned avoidance and extinction), long-term potentiation (LTP) in the hippocampal-accumbens pathway, and depression-like symptoms (ie, coping with stress behavior, anhedonia, and weight changes). We also examined whether the ameliorating effects of WIN55,212-2 on behavior and physiology after CMS are mediated by CB1 and glucocorticoid receptors (GRs). Rats were exposed to CMS or handled on days 1–21. The agonist WIN55,212-2 or vehicle were administered on days 19–21 (IP; 0.5 mg/kg) and behavioral and electrophysiological measures were taken on days 23 and 28. The CB1 receptor antagonist AM251 (IP; 0.3 mg/kg) or the GR antagonist RU-38486 (IP; 10 mg/kg) were co-administered with WIN55,212-2. Our results show that CMS significantly modified physiological and behavioral reactions, as observed by the impairment in avoidance extinction and LTP in the hippocampal-accumbens pathway, and the alterations in depression-like symptoms, such as coping with stress behavior, weight gain, and sucrose consumption. The most significant effect observed in this study was that 3 days of WIN55,212-2 administration prevented the CMS-induced alterations in emotional memory (ie, extinction) and plasticity. This effect was mediated by CB1 receptors as the CB1 receptor antagonist AM251 prevented the ameliorating effects of WIN55,212-2 on extinction and LTP. The GR antagonist RU-38486 also prevented the CMS-induced alterations in extinction and plasticity, and when co-administered with WIN55,212-2, the preventive effects after CMS were maintained. The findings suggest that enhancing cannabinoid signaling could represent a novel approach to the treatment of cognitive deficits that accompany stress-related depression.     

Cannabinoids and Glucocorticoids in the Basolateral Amygdala Modulate Hippocampal–Accumbens Plasticity After Stress

Acute stress results in release of glucocorticoids, which are potent modulators of learning and plasticity. This process is presumably mediated by the basolateral amygdala (BLA) where cannabinoids CB1 receptors have a key role in regulating the hypothalamic–pituitary–adrenal (HPA) axis. Growing attention has been focused on nucleus accumbens (NAc) plasticity, which regulates mood and motivation. The NAc integrates affective and context-dependent input from the BLA and ventral subiculum (vSub), respectively. As our previous data suggest that the CB1/2 receptor agonist WIN55,212-2 (WIN) and glucocorticoid receptor (GR) antagonist RU-38486 (RU) can prevent the effects of stress on emotional memory, we examined whether intra-BLA WIN and RU can reverse the effects of acute stress on NAc plasticity. Bilateral, ipsilateral, and contralateral BLA administration of RU or WIN reversed the stress-induced impairment in vSub–NAc long-term potentiation (LTP) and the decrease in cAMP response element-binding protein (CREB) activity in the NAc. BLA CB1 receptors were found to mediate the preventing effects of WIN on plasticity, but not the preventing effects of RU, after stress. Inactivating the ipsilateral BLA, but not the contralateral BLA, impaired LTP. The possible mechanisms underlying the effects of BLA on NAc plasticity are discussed; the data suggest that BLA-induced changes in the NAc may be mediated through neural pathways in the brain''s stress circuit rather than peripheral pathways. The results suggest that glucocorticoid and cannabinoid systems in the BLA can restore normal function of the NAc and hence may have a central role in the treatment of a variety of stress-related disorders.     

Cannabinoids Prevent the Effects of a Footshock Followed by Situational Reminders on Emotional Processing

Posttraumatic stress disorder (PTSD) can develop following exposure to a traumatic event. Hence, what we do in the first few hours after trauma exposure may alter the trajectory of PTSD. We examined whether cannabinoids can prevent the effects of a single footshock followed by situational reminders (SRs) on emotional processing. Rats were exposed to a footshock (1.5 mA, 10 s) on day 1 followed by exposure to SRs of the shock on days 3 and 5. The CB1/2 receptor agonist WIN55,212-2 or vehicle were injected intraperitoneally 2 h after the shock. After 1 week, PTSD-like symptoms were examined. Exposure to SRs exacerbated the effects of the shock as rats exposed to shock and SRs, but not shock alone, showed impaired extinction of the traumatic event, impaired plasticity in the hippocmapal-accumbens pathway, enhanced latency to startle, and altered expression of CB1 receptors (CB1r) and glucocorticoid receptors (GRs) in the CA1, basolateral amygdala (BLA) and prefrontal cortex (PFC). WIN55,212-2 prevented the effects of the shock and SRs on extinction, plasticity, and startle response. WIN55,212-2 normalized the shock/SR-induced upregulation in CB1r in the PFC, and CA1 and GRs in the CA1, with no effect on BLA downregulation of CB1r and GRs. Shock and SRs caused lasting (1 week) alterations in emotional processing associated with changes in GR and CB1r expression in brain areas related to PTSD. WIN55,212-2 administered after trauma exposure prevented these alterations via PFC- and CA1-CB1r and CA1-GRs.     

Cross-sensitization and cross-tolerance between exogenous cannabinoid antinociception and endocannabinoid-mediated stress-induced analgesia

Footshock stress induces both endocannabinoid mobilization and antinociception. The present studies investigated behavioral plasticity in cannabinoid antinociceptive mechanisms following repeated activation using the tail-flick test. A secondary objective was to ascertain whether blockade of stress antinociception by the CB(1) antagonist rimonabant could be attributed to changes in locomotor activity. The cannabinoid agonist WIN55,212-2 induced hypoactivity in the open field relative to vehicle-treated controls. By contrast, rimonabant, administered at a dose that virtually eliminated endocannabinoid-mediated stress antinociception, failed to alter locomotor behavior (i.e. time resting, ambulatory counts, distance traveled) in rats subjected to the same stressor. Rats exposed acutely to footshock were hypersensitive to the antinociceptive effects of WIN55,212-2 and Delta(9)-tetrahydrocannabinol (Delta(9)-THC). The converse was also true; acute Delta(9)-THC and WIN55,212-2 administration potentiated stress antinociception, suggesting a bidirectional sensitization between endocannabinoid-mediated stress antinociception and exogenous cannabinoid antinociception. Stress antinociception was also attenuated following chronic relative to acute treatment with WIN55,212-2 or Delta(9)-THC. Repeated exposure to footshock (3 min/day for 15 days), however, failed to attenuate antinociception induced by either footshock stress or WIN55,212-2. Our results demonstrate that endocannabinoid-mediated stress antinociception cannot be attributed to motor suppression. Our results further identify a functional plasticity of the cannabinoid system in response to repeated activation. The existence of cross-sensitization between endocannabinoid-mediated stress antinociception and exogenous cannabinoid antinociception suggests that these phenomena are mediated by a common mechanism. The observation of stress-induced hypersensitivity to effects of exogenous cannabinoids may have clinical implications for understanding marijuana abuse liability in humans.     

Cannabinoid CB1-mediated inhibition of stress-induced gastric ulcers in rats

The effect of cannabinoid drugs (i.p.) on cold/restraint stress-induced gastric ulcers was studied in rats. The cannabinoid receptor agonist (WIN 55,212-2, 0.1-1 mg/kg), but not the less active isomer WIN 55,212-3 (1 mg/kg), reduced gastric ulceration. The protective effect of WIN 55,212-2 (1 mg/kg) was counteracted by the cannabinoid CB1 receptor antagonist SR141716A, but not by the cannabinoid CB2 receptor antagonist SR144528. These results indicate that the antiulcer effect of the cannabinoid receptor agonist WIN 55,212-2 is mediated by cannabinoid CB1 receptors.     

Effects of acute systemic and intra-cerebral stimulation of cannabinoid receptors on sensorimotor gating,locomotion and spatial memory in rats

RATIONALE: Cannabinoid CB(1) receptors in the brain are targets of both endocannabinoid signalling and the psychoactive compounds of the hemp plant. They mediate neuronal effects of their ligands in various corticolimbic and striatal circuits by presynaptic regulation of transmitter release. OBJECTIVES/METHODS: This study investigates acute systemic effects of the full CB(1) receptor agonist WIN 55,212-2 (WIN) on prepulse inhibition (PPI) of the acoustic startle response (ASR), locomotor activity and spatial memory retrieval in an eight-arm radial-maze task. Furthermore, we tested the effect of local intra-cerebral micro-infusions of WIN into the nucleus accumbens (NAc), ventral tegmental area (VTA), dorsal (dHIP) and ventral (vHIP) hippocampus and medial prefrontal cortex (mPFC). RESULTS: Systemic WIN (1.2 mg/kg) reduced PPI without affecting ASR, had no effect on locomotion in the open field, but impaired retrieval of spatial memory. Infusions of 5 microg/0.3 microl WIN into either NAc (core or shell), dHIP or VTA did not affect PPI and locomotion immediately afterwards. However, PPI was significantly reduced after intra-mPFC and intra-vHIP infusion of WIN. Furthermore, WIN infusion into dHIP increased the number of reference memory errors in the maze, suggesting impairment of memory retrieval. CONCLUSIONS: Our data support the notion that CB(1) receptor stimulation impairs sensorimotor gating most likely by modulation of neurotransmitter release in mPFC and vHIP. The lack of effects of local WIN infusions in NAc and VTA might be due to low receptor abundance in these regions. Additionally, CB(1) receptor activation in dHIP impairs spatial memory retrieval. Taken together, cortico-hippocampal cannabinoid receptors play an essential role in the regulation of cognitive and behavioural processes.     

Cannabinoids Prevent the Development of Behavioral and Endocrine Alterations in a Rat Model of Intense Stress

Cannabinoids have recently emerged as a possible treatment of stress- and anxiety-related disorders such as post-traumatic stress disorder (PTSD). Here, we examined whether cannabinoid receptor activation could prevent the effects of traumatic stress on the development of behavioral and neuroendocrine measures in a rat model of PTSD, the single-prolonged stress (SPS) model. Rats were injected with the CB1/CB2 receptor agonist WIN55,212-2 (WIN) systemically or into the basolateral amygdala (BLA) at different time points following SPS exposure and were tested 1 week later for inhibitory avoidance (IA) conditioning and extinction, acoustic startle response (ASR), hypothalamic-pituitary-adrenal (HPA) axis function, and anxiety levels. Exposure to SPS enhanced conditioned avoidance and impaired extinction while enhancing ASR, negative feedback on the HPA axis, and anxiety. WIN (0.5 mg/kg) administered intraperitoneally 2 or 24 h (but not 48 h) after SPS prevented the trauma-induced alterations in IA conditioning and extinction, ASR potentiation, and HPA axis inhibition. WIN microinjected into the BLA (5 μg/side) prevented SPS-induced alterations in IA and ASR. These effects were blocked by intra-BLA co-administration of the CB1 receptor antagonist AM251 (0.3 ng/side), suggesting the involvement of CB1 receptors. These findings suggest that (i) there may be an optimal time window for intervention treatment with cannabinoids after exposure to a highly stressful event, (ii) some of the preventive effects induced by WIN are mediated by an activation of CB1 receptors in the BLA, and (iii) cannabinoids could serve as a pharmacological treatment of stress- and trauma-related disorders.     

Influence of intracerebroventricular administration of cannabinergic drugs on morphine state-dependent memory in the step-down passive avoidance test

The effects of cannabinergic drugs on morphine state-dependent memory of passive avoidance task were examined in mice. Pre-training (0.25, 0.5 and 5 mg/kg) and post-training (5 mg/kg) administration of morphine impaired memory retrieval on the test day. Impairment of memory retrieval by morphine (5 mg/kg) on the test day was reversed by pre-test administration of the same dose of the opioid. The pre-test intracerebroventricular administration of the cannabinoid CB1/CB2 receptor agonist (WIN55,212-2) (0.75 and 1 microg/mouse) not only mimicked the effect of pre-test morphine treatment, but also increased this action of the opioid. Furthermore, the pre-test intracerebroventricular administration of CB1 receptor antagonist (AM251) (20 and 100 ng/mouse) prevented the restoration of memory by morphine. Pre-training administration of WIN55,212-2 (1 microg/mouse) led to state-dependent learning with impaired memory retrieval on the test day as well, which was reversed by pre-test administration of the drug (0.5, 0.75 and 1 microg/mouse) or morphine (1 and 5 mg/kg). Restoration of impairment induced by WIN55,212-2 was decreased by both the opioid receptor antagonists, naloxone (0.01 microg/mouse) and AM251 (20 and 100 ng/mouse). In conclusion, the improvement of memory retrieval by morphine treatment on the test day seems to be induced, at least in part, by the cannabinoid CB1 receptors.     

The cannabinoid receptor agonist WIN 55,212-2 facilitates the extinction of contextual fear memory and spatial memory in rats

Rationale Previous studies demonstrated that pharmacological blockade of CB1 cannabinoid receptors decreases the extinction of conditioned fear and spatial memory in rodents. However, the effects of CB1 cannabinoid receptor activation in this response remain unclear.Objectives To evaluate the effects of the cannabinoid agonist WIN 55,212-2 (WIN) and the cannabinoid antagonist SR 147778 (SR) on the extinction of contextual fear memory in rats 24 h or 30 days after fear conditioning.Methods For fear conditioning, rats were placed in the conditioning chamber for 3 min and received a 1-s electric foot shock (1.5 mA). Retrieval testing consisted of a 3-min exposure to the conditioning chamber and extinction training consisted of successive 9-min exposures at 24-h intervals. Rats were also evaluated in the open field and water maze reversal task.Results The administration of SR (1.0 mg/kg, i.p.) and WIN (0.25 mg/kg, i.p.) before extinction training disrupted and facilitated, respectively, the extinction of 24 h contextual fear memory. These effects were not related to any disturbance in memory retrieval, unconditioned freezing expression, or locomotor activity. WIN (0.25 mg/kg, i.p.) also facilitated the extinction of 30-day-old contextual fear memory, while the prior administration of SR (0.2 mg/kg, i.p.) antagonized this response. The facilitative effect of WIN on memory extinction does not seem to be specific for contextual fear memory because it was also observed in the water maze reversal task.Conclusions These results suggest cannabinoid receptor agonists as potential drugs to treat anxiety disorders related to the retrieval of aversive memories.Part of this study was presented at the 18th European College of Neuropsychopharmacology Congress, Amsterdam, The Netherlands, 22–26 October 2005.     

Chronic exposure to cannabinoids before an emotional trauma may have negative effects on emotional function

Chronic direct activation of cannabinoid CB1 receptors (CB1r) may lead to downregulation of CB1r which may in turn result in a depression-like phenotype in certain individuals.We examined the effects of chronic cannabinoid receptor activation before exposure to an emotional traumatic event on CB1r expression in the basolateral amygdala (BLA) and CA1 and on protracted anxiety- and depression-like behaviors. We used exposure to severe shock and situational reminders (SRs) in an inhibitory apparatus as a model for emotional trauma.Chronic treatment with the CB1/2 receptor agonist WIN55,212-2 (1.2?mg/kg, i.p.) before shock exposure had differential effects on depression- and anxiety-like behavioral measures depending on withdrawal periods. In the 24?hrs withdrawal condition, WIN55,212–2 enhanced fear retrieval and impaired extinction, increased anhedonia and despair, but had a therapeutic effect in the startle test. In the 10 days withdrawal condition, WIN55,212–2 enhanced fear retrieval and impaired extinction without preventing the shock/SR-induced negative effects on anhedonia or startle response, but had a therapeutic effect in the despair test.Chronic treatment with WIN55,212–2 was found to down regulate CB1r protein levels in the BLA in the 10 days withdrawal condition, and to upregulate CB1r protein levels in the 24?hrs condition. In the CA1, rats chronically injected with vehicle or WIN55,212–2 demonstrated downregulation of CB1r protein levels.Chronic exposure to cannabinoids prior to an emotional trauma may have deleterious effects on emotional function suggesting that direct CB1/2 receptor activation may not be an optimal way to manipulate the endocannabinoid system in stressful individuals.     

Monitoring extracellular dopamine in the rat nucleus accumbens shell and core during acquisition and maintenance of intravenous WIN 55,212-2 self-administration

Rationale WIN 55,212-2, a potent cannabinoid receptor 1 agonist, is self-administered by animals to evaluate abuse liability of cannabinoids, but to date no information is yet available about its effects on dopaminergic transmission during active response-contingent administration.Objectives This study monitored the changes of extracellular dopamine (DA) in the nucleus accumbens (NAc) shell and core during active intravenous WIN 55,212-2 self-administration (SA).Methods Rats, implanted with a jugular catheter and bilateral intracerebral chronic cannulae, were trained for 3 weeks to self-administer WIN 55,212-2 (12.5 μg/kg) in single daily 1-h sessions under a fixed ratio 1 (FR 1) schedule, than switched to FR 2 for a further week. During SA sessions, microdialysis assays were performed every 3rd day, and then daily starting from the 13th session. Dialysate DA from the NAc shell and core was monitored before, during, and for 30 min after SA.Results Dialysate DA increased during WIN 55,212-2 SA starting from the 1st week in the NAc shell and on the 2nd week in the core. The increase of dialysate DA in the NAc shell was larger than that in the core on all weeks. Dialysate DA did not change during extinction sessions in spite of active nose poking.Conclusions Response-contingent WIN 55,212-2 SA preferentially increases the NAc shell DA output as compared to that of the core independently from the duration of the WIN 55,212-2 exposure. Increase in NAc DA is strictly related to WIN 55,212-2 actions because it is not observed during extinction despite active responding.     

Deficiency in Endocannabinoid Signaling in the Nucleus Accumbens Induced by Chronic Unpredictable Stress

The nucleus accumbens (NAc) is a critical component of the reward circuitry, and dysfunction of the NAc may account for anhedonia and other symptoms of depression. Here, we investigated whether alterations in endocannabinoid (eCB) signaling in the NAc contribute to depression-like behaviors induced by chronic unpredictable stress (CUS) in mice. We compared three types of eCB/CB1 receptor-mediated synaptic plasticity in slices prepared from the NAc core of control and stress-exposed mice: depolarization-induced suppression of excitation, long-term depression, and the depression of field excitatory postsynaptic potentials (fEPSPs) induced by group I metabotropic glutamate receptor agonist DHPG. CUS (5–6-week exposure to stressors), but not sub-CUS (1 week exposure to stressors), induces depression-like behaviors and impairs these forms of eCB/CB1 receptor-mediated plasticity examined in the NAc core. Neither sub-CUS nor CUS altered the tissue contents of the eCBs, anandamide and 2-arachidonoylglycerol in the striatum. However, exposure to CUS, but not to sub-CUS, attenuated the depression of fEPSPs induced by the CB1 receptor agonist WIN 55 212-2. CUS exposure reduced the maximal effect without affecting the EC₅₀ of WIN 55 212-2 to induce fEPSP depression. Thus, impaired CB1 receptor function could account for CUS-induced deficiency in eCB signaling in the NAc. Both CUS-induced deficiency in eCB signaling and depression-like behaviors were reversed by in vivo administration of antidepressant fluoxetine. These results suggest that downregulation of eCB signaling in the NAc occurs after CUS and contributes to the pathophysiology of depression.     

Effect of the cannabinoid receptor ligand, WIN 55,212-2, on superoxide anion and TNF-alpha production by human mononuclear cells

Cannabinoids are known to downregulate immune response but the role for cannabinoid receptors in cannabinoid-induced immunosuppression is still unclear. To address this question, the interference of CB1 and CB2 receptor antagonists with the inhibition of TNF-alpha production by synthetic cannabinoid WIN 55,212-2 was studied using human peripheral blood mononuclear cells (PBMC) in vitro. CB2 (SR 144528) but not CB1 (SR 141716A) receptor antagonist dose dependently interfered with WIN 55,212-2-induced inhibition of TNF-alpha synthesis. Also, WIN 55,212-2 decreased fMLP-induced reactive oxygen species generation in lipopolysaccharide (LPS)-primed PBMC. However, the high concentrations of cannabinoid receptor ligands needed to achieve significant effects suggest that the observed effects may be in part cannabinoid receptor independent.     

Effects of pharmacological manipulations of cannabinoid receptors on severity of dystonia in a genetic model of paroxysmal dyskinesia

Previous studies have shown beneficial effects of the cannabinoid CB(1)/CB(2) receptor agonist (R)-4,5-dihydro-2-methyl-4-(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo [3,2,1-ij]quinolin-6-one mesylate (WIN 55,212-2) in dt(sz) mutant hamsters, a model of idiopathic paroxysmal dystonia (dyskinesia). To examine the pathophysiological significance of the cannabinergic system in the dystonic syndrome, the effect of the cannabinoid CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716A) on severity of dystonia was investigated in dt(sz) mutants which exhibit episodes of dystonic and choreoathetotic disturbances in response to mild stress. SR 141716A (5 and 10 mg/kg i.p.) failed to exert any effects on the severity of dystonia. While the antidystonic efficacy of WIN 55,212-2 (5 mg/kg i.p.) was confirmed, cannabidiol (which has low affinity to cannabinoid receptors) tended to delay the progression of dystonia only at a high dose (150 mg/kg i.p.). The antidystonic and cataleptic effects of WIN 55,212-2 (5 mg/kg i.p.) were completely antagonized by pretreatment with SR 141716A at doses of 2.5 mg/kg (catalepsy) and 10 mg/kg (antidystonic efficacy). These data indicate that the antidystonic efficacy of WIN 55,212-2 is selectively mediated via CB(1) receptors. The lack of prodystonic effects of SR 141716A together with only moderate antidystonic effects of WIN 55,212-2 suggests that reduced activation of cannabinoid CB(1) receptors by endocannabinoids is not critically involved in the dystonic syndrome. In view of previous pathophysiological findings in mutant hamsters, the antidystonic efficacy of WIN 55,212-2 can be explained by modulation of different neurotransmitter systems within the basal ganglia.     

Inhibitory effect of the cannabinoid receptor agonist WIN 55,212-2 on pentagastrin-induced gastric acid secretion in the anaesthetized rat

The effect of the cannabinoid (CB) receptor agonist WIN 55,212-2 on gastric acid secretion was studied in the anaesthetized rat after stimulation with pentagastrin. WIN 55,212-2 (0.5-2 mg/kg, i.v.) was inactive on basal secretion but caused a marked inhibition (80%) of the acid secretion stimulated by pentagastrin (10 microg/kg, i.v.). The enantiomer WIN 55,212-3 (1-3 mg/kg, i.v.) did not significantly modify basal or pentagastrin-induced acid secretion. The inhibitory effect of WIN 55,212-2 against pentagastrin was prevented by the administration of the selective cannabinoid CB1 receptor antagonists SR141716A (1 mg/kg, i.v.) and LY320135 (1 mg/kg, i.v.); by contrast, the CB2 receptor antagonist SR144528 (0.3-1 mg/kg, i.v.) was without effect. The selective CB2 receptor agonist JWH-015 (0.1-10 mg/kg, i.v.) was inactive on the increase of acid output stimulated by pentagastrin. These results suggest that the inhibitory effect of WIN 55,212-2 on pentagastrin-stimulated acid secretion in the anaesthetized rat is mediated by specific cannabinoid receptors. Moreover, the antagonism of WIN 55,212-2-induced effects by the selective CB1 receptor antagonists SR141716A and LY320135 together with the ineffectiveness of both the CB2 receptor agonist JWH-015 and the CB2 receptor antagonist SR144528 indicate that CB1 receptor subtypes are predominantly involved in the antisecretory effect of WIN 55,212-2.     

Activation of cannabinoid CB1 and CB2 receptors suppresses neuropathic nociception evoked by the chemotherapeutic agent vincristine in rats

BACKGROUND AND PURPOSE: The ability of cannabinoids to suppress mechanical hypersensitivity (mechanical allodynia) induced by treatment with the chemotherapeutic agent vincristine was evaluated in rats. Sites of action were subsequently identified. EXPERIMENTAL APPROACH: Mechanical hypersensitivity developed over the course of ten daily injections of vincristine relative to groups receiving saline at the same times. Effects of the CB1/CB2 receptor agonist WIN55,212-2, the receptor-inactive enantiomer WIN55,212-3, the CB2-selective agonist (R,S)-AM1241, the opiate agonist morphine and vehicle on chemotherapy-induced neuropathy were evaluated. WIN55,212-2 was administered intrathecally (i.t.) or locally in the hindpaw to identify sites of action. Pharmacological specificity was established using competitive antagonists for CB1 (SR141716) or CB2 receptors (SR144528). KEY RESULTS: Systemic administration of WIN55,212-2, but not WIN55,212-3, suppressed vincristine-evoked mechanical allodynia. A leftward shift in the dose-response curve was observed following WIN55,212-2 relative to morphine treatment. The CB1 (SR141716) and CB2 (SR144528) antagonists blocked the anti-allodynic effects of WIN55,212-2. (R,S)-AM1241 suppressed vincristine-induced mechanical hypersensitivity through a CB2 mechanism. Both cannabinoid agonists suppressed vincristine-induced mechanical hypersensitivity without inducing catalepsy. Spinal sites of action are implicated in cannabinoid modulation of chemotherapy-induced neuropathy. WIN55,212-2, but not WIN55,212-3, administered i.t. suppressed vincristine-evoked mechanical hypersensitivity at doses that were inactive following local hindpaw administration. Spinal coadministration of both the CB1 and CB2 antagonists blocked the anti-allodynic effects of WIN55,212-2. CONCLUSIONS AND IMPLICATIONS: Cannabinoids suppress the maintenance of vincristine-induced mechanical allodynia through activation of CB1 and CB2 receptors. These anti-allodynic effects are mediated, at least in part, at the level of the spinal cord.     

CB1 receptor-dependent and -independent inhibition of excitatory postsynaptic currents in the hippocampus by WIN 55,212-2

We investigated the effect of a synthetic cannabinoid, WIN 55,212-2 on excitatory postsynaptic currents (EPSCs) evoked by stimulation of Schaffer collaterals in CA1 pyramidal cells. Bath application of WIN 55,212-2 reduced the amplitude of EPSCs in dose-dependent manner tested between 0.01 nM and 30 microM. In rats and mice, this cannabinoid ligand inhibited excitatory synapses in two steps at the nM and muM concentrations. When the function of CB(1) cannabinoid receptors (CB(1)R) was impaired, either by the application of a CB(1)R antagonist AM251, or by using CB(1)R knockout mice, WIN 55,212-2 in microM concentrations could still significantly reduced the amplitude of EPSCs. WIN 55,212-2 likely affected the efficacy of excitatory transmission only at presynaptic sites, since both at low and high doses the paired pulse ratio of EPSC amplitude was significantly increased. The inactive enantiomer, WIN 55,212-3, mimicked the effect of WIN 55,212-2 applied in high doses. In further experiments we found that the CB(1)R-independent effect of 10 microM WIN 55,212-2 at glutamatergic synapses was fully abolished, when slices were pre-treated with omega-conotoxin GVIA, but not with omega-agatoxin IVA. These data suggest that, in the hippocampus, WIN 55,212-2 reduces glutamate release from Schaffer collaterals solely via CB(1)Rs in the nM concentration range, whereas in microM concentrations, WIN 55,212-2 suppresses excitatory transmission, in addition to activation of CB(1)Rs, by directly blocking N-type voltage-gated Ca(2+) channels independent of CB(1)Rs.     

Behavioral effects in adult rats of chronic prepubertal treatment with the cannabinoid receptor agonist WIN 55,212-2

Human and animal studies provide evidence for vulnerable periods of brain development for deleterious effects of cannabinoids. We have recently shown that pubertal chronic cannabinoid treatment leads to long-lasting behavioral deficits, whereas a comparable treatment in adult rats did not affect the animals' behavior. In the present study we examined the effects of an identical chronic cannabinoid treatment in juvenile rats, just before the onset of puberty. Treatment with the synthetic cannabinoid agonist WIN 55,212-2 (WIN) (1.2 mg/kg) or vehicle was extended over 25 days throughout the prepubertal period (postnatal days 15-40) in juvenile rats. The rats received a total of 20 injections intraperitoneally. Adult rats were tested for object recognition memory, performance in a progressive ratio (PR) operant behavior task, locomotor activity and prepulse inhibition (PPI) of the acoustic startle response. Juvenile chronic WIN administration had no effect on object recognition memory, PR performance and locomotor activity in adulthood. However, a PPI deficit was observed in WIN-treated rats when tested as adults that could be reversed by the acute administration of the dopamine receptor antagonist haloperidol (0.1 mg/kg). Additionally, juvenile cannabinoid treatment reduced the number of rearings, as well as the time spent in the center of the open field in adult rats, suggesting increased anxiety. Juvenile chronic cannabinoid treatment induced behavioral disturbances in adult rats that are less severe than those observed after pubertal cannabinoid administration. However, based on the observations of sensorimotor gating deficits and increased anxiety, we conclude that the prepubertal developmental phase, in addition to puberty, also represents a vulnerable time period for persistent adverse effects of cannabinoids.     

Pre-emptive antinociceptive effects of a synthetic cannabinoid in a model of neuropathic pain

The antinociceptive effects of WIN55,212-2, a synthetic cannabinoid, were evaluated in the model of partial sciatic nerve ligation after daily subcutaneous administration of 0.1 mg/kg a week before and two weeks after surgery. Mechanical allodynia and thermal hyperalgesia were evaluated in 46 rats allocated to receive: (1) Vehicle (before surgery)-Vehicle (after surgery); (2) Vehicle-WIN55,212-2; (3) WIN55,212-2-Vehicle; (4) WIN55,212-2-WIN55,212-2; (5) AM251+vehicle; (6) AM251+WIN55,212-2; (7) AM630+vehicle; (8) AM630+WIN55,212-2; (9) Sham receiving vehicle; and (10) Sham receiving WIN55,212-2. The decreased in mechanical allodynia and thermal hyperalgesia by WIN55,212-2 was significantly greater when it was administered during one week before surgery. In conclusion, pre-emptive use of cannabinoids produced greater antinociceptive effects in a model of neuropathic pain and this effect is mediated by cannabinoid CB(1) and CB(2) receptors.     

Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site

Excised outside-out patches from HEK293 cells stably transfected with the human (h) 5-HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5-HT-induced current using the patch clamp technique. In addition, binding studies with radioligands for 5-HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5-HT-induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): 9-THC, WIN55,212-2, anandamide, JWH-015 and CP55940. The WIN55,212-2-induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212-3, an enantiomer of WIN55,212-2, did not affect the 5-HT-induced current. WIN55,212-2 did not change the EC50 value of 5-HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]-SR141716A and the CB1/CB2 receptor ligand [3H]-CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5-HT3A receptor cDNA, WIN55,212-2, CP55940, anandamide and SR141716A did not affect [3H]-GR65630 binding, but 5-HT caused a concentration dependent-inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5-HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5-HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.