WIN 55212-2 Research Articles

Dual Role of PPAR-γ in Induction and Expression of Behavioral Sensitization to Cannabinoid Receptor Agonist WIN55,212-2

Behavioral sensitization (B.S.) is a pathophysiological animal model for stimulant-induced psychosis and addiction. Accumulated evidence indicates that inflammatory processes are involved in psychostimulants effects in the CNS. Cannabinoids like WIN55,212-2 act as potential activators of PPAR-γ and affects the inflammatory status of the CNS. The purpose of this study is to determine PPAR-γ role in induction and expression of B.S. and the coincident inflammatory responses developed by WIN55,212-2 (WIN). Using open-field test, locomotor activity was monitored in animals treated with intraperitoneal low-dose WIN single or repeated injections. Concurrent striatal COX-2 and TNF-α levels and PPAR-γ activity were determined by immunoblotting assay. Effects of concomitant chronic or acute PPAR-γ pharmacological inhibition (with GW9662) were then investigated on behavioral and biochemical variables. WIN enhanced locomotor activity and while administered chronically augmented cytosolic COX-2 and TNF-α and also PPAR-γ nuclear levels. GW9662 co-administration completely prevented the induction of sensitizing effects of chronic WIN and altered the inflammatory responses. However, the expression of B.S. was intensified with GW9662 as assessed by increased locomotion after WIN challenge following 48 h withdrawal. Neuroinflammation and locomotor excitability in animals received just a single-dose WIN were also escalated with GW9662. Our findings conclude that PPAR-γ could play different key roles during B.S. development by WIN. Although PPAR-γ is mostly known for neuroprotective and anti-inflammatory effects, our data indicate that it mediates the B.S. induction by chronic WIN. However, while the B.S. was induced, PPAR-γ could play a homeostatic role opposing the expressed B.S. escalation.

Chronic co-administration of the cannabinoid receptor agonist WIN55,212-2 during puberty or adulthood reverses 3,4 methylenedioxymetamphetamine (MDMA)-induced deficits in recognition memory but not in effort-based decision making

Cannabis and 3,4 methylenedioxymetamphetamine (MDMA, “ecstasy”) are the most frequently combined illegal drugs among young adults in western societies. This study examined the effects of chronic co-administration of the cannabinoid receptor agonist WIN55,212-2 (WIN) and MDMA on working memory and effort-based decision making in rats. Treatment consisted of MDMA (7.5mg/kg), WIN (1.2mg/kg), a combination of these substances (MDMA+WIN) or vehicle over a period of 25days during puberty (PD40-65) or adulthood (PD80-105). Ten days after the last treatment, WIN reversed MDMA-induced working memory deficits in the object recognition test in animals treated during adulthood or puberty, but had no influence on impairment of adult rats in the effort-based T-maze task. No differences were observed between groups of pubertally treated rats in the decision making task. During a subsequent acute drug challenge MDMA and MDMA+WIN decreased high reward choices in both age groups, indicating MDMA-induced cost-aversive choice. Differential long-term interactions on the neuronal level in the hippocampus and MDMA-induced disturbances in cortico-limbic connections are suggested.

Cannabinoids and traumatic stress modulation of contextual fear extinction and GR expression in the amygdala-hippocampal-prefrontal circuit

Considerable evidence suggests that cannabinoids modulate the behavioral and physiological response to stressful events. We have recently shown that activating the cannabinoid system using the CB1/CB2 receptor agonist WIN55,212-2 (WIN) in proximity to exposure to single-prolonged stress (SPS), a rat model of emotional trauma, prevented the stress-induced enhancement of acoustic startle response, the impairment in avoidance extinction and the enhanced negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis (Ganon-Elazar and Akirav, 2012). Some of the effects were found to be mediated by CB1 receptors in the basolateral amygdala (BLA). Here we examined whether cannabinoid receptor activation in a putative brain circuit that includes the BLA, hippocampus and prefrontal cortex (PFC), could prevent the effects of traumatic stress on contextual fear extinction and alterations in glucocorticoid receptor (GR) protein levels. We found that: (i) SPS impaired contextual fear extinction tested one week after trauma exposure and that WIN prevented the stress-induced impairment of extinction when microinjected immediately after trauma exposure into the BLA or hippocampus (5 μg), but not when microinjected into the PFC, (ii) the ameliorating effects of WIN on contextual extinction were prevented by blocking GRs in the BLA and hippocampus, and (iii) SPS up regulated GRs in the BLA, PFC and hippocampus and systemic WIN administration (0.5 mg/kg) after trauma exposure normalized GR levels in the BLA and hippocampus, but not in the PFC. Cannabinoid receptor activation in the aftermath of trauma exposure may regulate the emotional response to the trauma and prevent stress-induced impairment of extinction and GR up regulation through the mediation of CB1 receptors in the BLA and hippocampus. Taken together, the findings suggest that the interaction between the cannabinoid and glucocorticoid systems is crucial in the modulation of emotional trauma.

Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury

Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1−/− mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC–MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein–protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1–JNK1 signaling. WIN reduced TRPV1-induced Ca2+ transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein–protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, Gi/o contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.

Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

The cannabinoid system is known to be involved in the regulation of inflammatory processes. Therefore, drugs targeting cannabinoid receptors are considered as candidates for anti-inflammatory and tissue protective therapy. We demonstrated that the prototypical cannabinoid agonist R(+)WIN55,212-2 (WIN) reduced the secretion of matrix metalloproteinase-9 (MMP-9) in a murine model of cigarette-smoke induced lung inflammation. In experiments using primary cells and cell lines of the monocyte-macrophage-system we found that binding of the cannabinoid-receptor agonist WIN to a stereo-selective, specific binding site in cells of the monocyte-macrophage-system induced a significant down-regulation of MMP-9 secretion and disturbance of intracellular processing, which subsequently down-regulated MMP-9 mRNA expression via a ERK1/2-phosphorylation-dependent pathway. Surprisingly, the anti-inflammatory effect was independent from classical cannabinoid receptors. Our experiments supposed an involvement of TRPV1, but other yet unidentified sites are also possible. We conclude that cannabinoid-induced control of MMP-9 in the monocyte-macrophage system via a cannabinoid-receptor independent pathway represents a general option for tissue protection during inflammation, such as during lung inflammation and other diseases associated with inflammatory tissue damage.

Statistical parametric mapping reveals regional alterations in cannabinoid CB1 receptor distribution and G‐protein activation in the 3D reconstructed epileptic rat brain

The endocannabinoid system is known to modulate seizure activity in several in vivo and in vitro models, and CB(1) -receptor activation is anticonvulsant in the rat pilocarpine model of acquired epilepsy (AE). In these epileptic rats, a unique redistribution of the CB(1) receptor occurs within the hippocampus; however, an anatomically inclusive analysis of the effect of status epilepticus (SE)-induced AE on CB(1) receptors has not been thoroughly evaluated. Therefore, statistical parametric mapping (SPM), a whole-brain unbiased approach, was used to study the long-term effect of pilocarpine-induced SE on CB(1) -receptor binding and G-protein activation in rats with AE. Serial coronal sections from control and epileptic rats were cut at equal intervals throughout the neuraxis and processed for [(3) H]WIN55,212-2 (WIN) autoradiography, WIN-stimulated [(35) S]GTPγS autoradiography, and CB(1) -receptor immunohistochemistry (IHC). The autoradiographic techniques were evaluated with both region of interest (ROI) and SPM analyses. In rats with AE, regionally specific increases in CB(1) -receptor binding and activity were detected in cortex, discrete thalamic nuclei, and other regions including caudate-putamen and septum, and confirmed by IHC. However, CB(1) receptors were unaltered in several brain regions, including substantia nigra and cerebellum, and did not exhibit regional decreases in rats with AE. This study provides the first comprehensive evaluation of the regional distribution of changes in CB(1) -receptor expression, binding, and G-protein activation in the rat pilocarpine model of AE. These regions may ultimately serve as targets for cannabinomimetic compounds or manipulation of the endocannabinoid system in epileptic brain.

Epileptiform activity in the CA1 region of the hippocampus becomes refractory to attenuation by cannabinoids in part because of endogenous γ‐aminobutyric acid type B receptor activity

The anticonvulsant properties of marijuana have been known for centuries. The recently characterized endogenous cannabinoid system thus represents a promising target for novel anticonvulsant agents; however, administration of exogenous cannabinoids has shown mixed results in both human epilepsy and animal models. The ability of cannabinoids to attenuate release of both excitatory and inhibitory neurotransmitters may explain the variable effects of cannabinoids in different models of epilepsy, but this has not been well explored. Using acute mouse brain slices, we monitored field potentials in the CA1 region of the hippocampus to characterize systematically the effects of the cannabinoid agonist WIN55212-2 (WIN) on evoked basal and epileptiform activity. WIN, acting presynaptically, significantly reduced the amplitude and slope of basal field excitatory postsynaptic potentials as well as stimulus-evoked epileptiform responses induced by omission of magnesium from the extracellular solution. In contrast, the combination of omission of magnesium plus elevation of potassium induced an epileptiform response that was refractory to attenuation by WIN. The effect of WIN in this model was partially restored by blocking γ-aminobutyric acid type B (GABA(B) ), but not GABA(A) , receptors. Subtle differences in models of epileptiform activity can profoundly alter the efficacy of cannabinoids. Endogenous GABA(B) receptor activation played a role in the decreased cannabinoid sensitivity observed for epileptiform activity induced by omission of magnesium plus elevation of potassium. These results suggest that interplay between presynaptic G protein-coupled receptors with overlapping downstream targets may underlie the variable efficacy of cannabinoids in different models of epilepsy.

Inhibitory Inputs from Rostromedial Tegmental Neurons Regulate Spontaneous Activity of Midbrain Dopamine Cells and Their Responses to Drugs of Abuse

The rostromedial tegmental nucleus (RMTg), a structure located just posterior to the ventral tegmental area (VTA), is an important site involved in aversion processes. The RMTg contains γ-aminobutyric acid neurons responding to noxious stimuli, densely innervated by the lateral habenula and providing a major inhibitory projection to reward-encoding dopamine (DA) neurons in the VTA. Here, we studied how RMTg neurons regulate both spontaneous firing of DA cells and their response to the cannabinoid agonist WIN55212-2 (WIN), morphine, cocaine, and nicotine. We utilized single-unit extracellular recordings in anesthetized rats and whole-cell patch clamp recordings in brain slices to study RMTg-induced inhibition of DA cells and inhibitory postsynaptic currents (IPSCs) evoked by stimulation of caudal afferents, respectively. The electrical stimulation of the RMTg elicited a complete suppression of spontaneous activity in approximately half of the DA neurons examined. RMTg-induced inhibition correlated with firing rate and pattern of DA neurons and with their response to a noxious stimulus, highlighting that inhibitory inputs from the RMTg strongly control spontaneous activity of DA cells. Both morphine and WIN depressed RMTg-induced inhibition of DA neurons in vivo and IPSCs evoked by RMTg stimulation in brain slices with presynaptic mechanisms. Conversely, neither cocaine nor nicotine modulated DA neuron responses to RMTg stimulation. Our results further support the role of the RMTg as one of the main inhibitory afferents to DA cells and suggest that cannabinoids and opioids might disinhibit DA neurons by profoundly influencing synaptic responses evoked by RMTg activation.

Influence of N-methyl D-aspartate receptor mechanism on WIN55,212-2-induced amnesia in rat dorsal hippocampus

In this study, we investigated the effects of both N-methyl D-aspartate (NMDA) and MK-801 on WIN55,212-2(WIN)-induced amnesia in rats. Step-through inhibitory avoidance of memory was used to examine the retrieval of memory, 24 h after training. All drugs were injected bilaterally into the dorsal hippocampus (intra-CA1) of rats. Pretraining and posttraining or pretesting administration of the nonselective CB1/CB2 receptor agonist, WIN (0.5 µg/rat), decreased the step-through latency. However, amnesia induced by pretraining or posttraining injections of WIN was reversed by a pretest administration of WIN (0.25 and 0.5 µg/rat). Pretest microinjections of different doses of NMDA (0.1, 0.5, and 1 µg/rat) elicited no response, but NMDA (0.5 and 1 µg/rat) did induce full recovery from amnesia induced by WIN (0.5 µg/rat). The posttraining and pretest injection of a higher dose of the NMDA receptor antagonist, MK801 (MK; 4 µg/rat), caused an impairment in the memory retrieval. However, amnesia induced by posttraining injections of MK (4 µg/rat) was reversed by a pretest administration of MK (4 µg/rat). In addition, pretest administration of different doses of the antagonist (2 and 4 µg/rat) induced full recovery of WIN-induced amnesia, but did not influence memory recovery in the subjects, which had received posttraining (0.5 µg/rat) and pretest WIN (0.25 and 0.5 µg/rat). Pretesting coadministration of ineffective doses of WIN (0.1 µg/rat) with NMDA (0.1 µg/rat), but not with MK (1 µg/rat), restored WIN-induced (0.5 µg/rat) amnesia. It can be concluded that the NMDA receptor mechanism located in the dorsal hippocampus may be involved in WIN-induced amnesia.

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.

Pharmacological elevation of anandamide impairs short-term memory by altering the neurophysiology in the hippocampus

In rodents, many exogenous cannabinoid agonists including Δ 9-THC and WIN55,212-2 (WIN-2) have been shown to impair short-term memory (STM) by inhibition of hippocampal neuronal assemblies. However, the mechanisms by which endocannabinoids such as anandamide and 2-arachidonyl glycerol (2-AG) modulate STM processes are not well understood. Here the effects of anandamide on performance of a Delayed-Non-Match-to-Sample (DNMS) task (i.e. STM task) and concomitant hippocampal ensemble activity were assessed following administration of either URB597 (0.3, 3.0 mg/kg), an inhibitor of the Fatty Acid Amide Hydrolase (FAAH), AM404 (1.5, 10.0 mg/kg), a putative anandamide uptake/FAAH inhibitor, or R-methanandamide (3.0, 10.0 mg/kg), a stable analog of anandamide. Principal cells from hippocampal CA3/CA1 were recorded extracellularly by multi-electrode arrays in Long-Evans rats during DNMS task (1–30 s delays) performance and tracked throughout drug administration and recovery. Both R-methanandamide and URB597 caused dose- and delay-dependent deficits in DNMS performance with suppression of hippocampal ensemble activity during the encoding (sample) phase. R-methanandamide-induced effects were not reversed by capsaicin excluding a contribution of TRPV-1 receptors. AM404 produced subtle deficits at longer delay intervals but did not alter hippocampal neuronal activity during task-specific events. Collectively, these data indicate that endocannabinoid levels affect performance in a STM task and their pharmacological elevation beyond normal concentrations is detrimental also for the underlying physiological responses. They also highlight a specific window of memory processing, i.e. encoding, which is sensitive to cannabinoid modulation.

Cannabinoid receptor antagonists AM251 and AM630 activate TRPA1 in sensory neurons

Cannabinoid receptor antagonists have been utilized extensively in vivo as well as in vitro, but their selectivity has not been fully examined. We investigated activation of sensory neurons by two cannabinoid antagonists – AM251 and AM630. AM251 and AM630 activated trigeminal (TG) sensory neurons in a concentration-dependent fashion (threshold 1 μM). AM251 and AM630 responses are mediated by the TRPA1 channel in a majority (90–95%) of small-to-medium TG sensory neurons. AM630 (1–100 μM), but not AM251, was a significantly more potent agonist in cells co-expressing both TRPA1 and TRPV1 channels. We next evaluated AM630 and AM251 effects on TRPV1- and TRPA1-mediated responses in TG neurons. Capsaicin (CAP) effects were inhibited by pre-treatment with AM630, but not AM251. Mustard oil (MO) and WIN55,212-2 (WIN) TRPA1 mediated responses were also inhibited by pre-treatment with AM630, but not AM251 (25 uM each). Co-treatment of neurons with WIN and either AM630 or AM251 had opposite effects: AM630 sensitized WIN responses, whereas AM251 inhibited WIN responses. WIN-induced inhibition of CAP responses in sensory neurons was reversed by AM630 pre-treatment and AM251 co-treatment (25 μM each), as these conditions inhibit WIN responses. Hindpaw injections of AM630 and AM251 did not produce nocifensive behaviors. However, both compounds modulated CAP-induced thermal hyperalgesia in wild-type mice and rats, but not TRPA1 null-mutant mice. AMs also partially regulate WIN inhibition of CAP-induced thermal hyperalgesia in a TRPA1-dependent fashion. In summary, these findings demonstrate alternative targets for the cannabinoid antagonists, AM251 and AM630, in peripheral antihyperalgesia which involve certain TRP channels.

Central Endocannabinoid Signaling Regulates Hepatic Glucose Production and Systemic Lipolysis

OBJECTIVEThe endocannabinoid (EC) system has been implicated as an important regulator of energy homeostasis. In obesity and type 2 diabetes, EC tone is elevated in peripheral tissues including liver, muscle, fat, and also centrally, particularly in the hypothalamus. Cannabinoid receptor type 1 (CB1) blockade with the centrally and peripherally acting rimonabant induces weight loss and improves glucose homeostasis while also causing psychiatric adverse effects. The relative contributions of peripheral versus central EC signaling on glucose homeostasis remain to be elucidated. The aim of this study was to test whether the central EC system regulates systemic glucose fluxes.RESEARCH DESIGN AND METHODSWe determined glucose and lipid fluxes in male Sprague-Dawley rats during intracerebroventricular infusions of either WIN55,212-2 (WIN) or arachidonoyl-2'-chloroethylamide (ACEA) while controlling circulating insulin and glucose levels through hyperinsulinemic, euglycemic clamp studies. Conversely, we fed rats a high-fat diet for 3 days and then blocked central EC signaling with an intracerebroventricular infusion of rimonabant while assessing glucose fluxes during a clamp.RESULTSCentral CB1 activation is sufficient to impair glucose homeostasis. Either WIN or ACEA infusions acutely impaired insulin action in both liver and adipose tissue. Conversely, in a model of overfeeding-induced insulin resistance, CB1 antagonism restored hepatic insulin sensitivity.CONCLUSIONSThus central EC tone plays an important role in regulating hepatic and adipose tissue insulin action. These results indicate that peripherally restricted CB1 antagonists, which may lack psychiatric side effects, are also likely to be less effective than brain-permeable CB1 antagonists in ameliorating insulin resistance.

Cannabinoid‐induced delayed gastric emptying is selectively increased upon intermittent administration in the rat: role of CB1 receptors

Cannabinoids acutely administered depress central, cardiovascular and gastrointestinal functions. These effects might be modified upon repeated administration. Compared to the effects induced by daily administration, those induced by intermittent administration are less known. The effect of intermittent treatment with the CB1/CB2 cannabinoid agonist WIN55,212-2 (WIN) was studied in the rat. Male rats received saline, vehicle or WIN at 0.5 (low-WIN) or 5 (high-WIN) mg kg(-1) week(-1) for 4 weeks. WIN effects on the central nervous system (cannabinoid tetrad tests), cardiovascular function and gastrointestinal motor function were evaluated after the first and last doses, and, where appropriate, 1 week after the last dose. To determine the involvement of CB1 receptors in the chronic effect of WIN, the CB1 receptor antagonist/inverse agonist AM251 (1 mg kg(-1)) was used. High- (but not low-) WIN induced the four signs of the cannabinoid tetrad, and reduced gastrointestinal motility, but did not alter cardiovascular parameters. Upon chronic intermittent administration, tolerance did not clearly develop to WIN effects. Quite the opposite, depression of gastric emptying was intensified. No effect was long-lasting. Repeated administration of AM251 was more efficacious than single administration to block WIN chronic central effects, but the opposite occurred regarding lower intestinal motility. Upon intermittent administration, hypersensitization may develop to some effects (particularly delayed gastric emptying) induced by cannabinoid agonists. CB1 antagonists/inverse agonists may show different efficacy upon repeated or single administration to block cannabinoid-induced central and gastrointestinal effects. Thus, cannabinoid effects are dependent on the pattern of drug administration.

Cannabinoid exposure during zebra finch sensorimotor vocal learning persistently alters expression of endocannabinoid signaling elements and acute agonist responsiveness

BackgroundPreviously we have found that cannabinoid treatment of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. Such persistently altered behavior must be attributable to changes in physiological substrates responsible for song. We are currently working to identify the nature of such physiological changes, and to understand how they contribute to altered vocal learning. One possibility is that developmental agonist exposure results in altered expression of elements of endocannabinoid signaling systems. To test this hypothesis we have studied effects of the potent cannabinoid receptor agonist WIN55212-2 (WIN) on endocannabinoid levels and densities of CB1 immunostaining in zebra finch brain.ResultsWe found that late postnatal WIN treatment caused a long-term global disregulation of both levels of the endocannabinoid, 2-arachidonyl glycerol (2-AG) and densities of CB1 immunostaining across brain regions, while repeated cannabinoid treatment in adults produced few long-term changes in the endogenous cannabinoid system.ConclusionsOur findings indicate that the zebra finch endocannabinoid system is particularly sensitive to exogenous agonist exposure during the critical period of song learning and provide insight into susceptible brain areas.

Effects of the Allosteric Antagonist 1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea (PSNCBAM-1) on CB1 Receptor Modulation in the Cerebellum

1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl] urea (PSNCBAM-1) has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB(1) ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB(1) receptor-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding in cerebellar membranes and on CB(1) ligand modulation of presynaptic CB(1) receptors at inhibitory interneuron-Purkinje cell synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused noncompetitive antagonism in [(35)S]GTPγS binding studies, with higher potency against the CB receptor agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethyl heptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) than for R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3,-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate] [WIN55,212-2 (WIN55)]. In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pretreatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency but having no clear effect on WIN55 actions. The CB(1) antagonist/inverse agonist N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide (AM251) increased mIPSC frequency beyond control; this effect was reversed by PSNCBAM-1. PSNCBAM-1 pretreatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB(1) receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [(35)S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependence associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian central nervous system (CNS). PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB(1) antagonists/inverse agonists in the treatment of CNS disease.

Involvement of dopaminergic and glutamatergic systems of the basolateral amygdala in amnesia induced by the stimulation of dorsal hippocampal cannabinoid receptors

The present study intended to investigate the involvement of dopaminergic and glutamatergic systems of the basolateral amygdala in amnesia induced by the stimulation of dorsal hippocampal cannabinoid receptors in male Wistar rats. The animals were stereotaxically implanted with guide cannulas in the CA1 region of the dorsal hippocampus and basolateral amygdala (BLA), trained in a step-through type passive avoidance task, and tested 24 h after training to measure memory retrieval. Post-training intra-CA1 microinjection of the nonselective CB1/CB2 receptor agonist WIN55,212-2 (WIN) (0.1–0.5 μg/rat) dose-dependently induced amnesia. Post-training intra-BLA administration of the D1/D2 dopamine receptor agonist apomorphine (0.3 and 0.5 μg/rat) plus intra-CA1 administration of 0.1 μg/rat of WIN, which alone did not induce amnesia, inhibited memory formation. The inhibitory effect of 0.5 μg/rat of WIN (intra-CA1) on memory formation was significantly decreased by the D1 dopamine receptor antagonist SCH23390 (0.1–0.5 μg/rat, intra-BLA) or the D2 dopamine receptor antagonist sulpiride (0.02–0.5 μg/rat, intra-BLA) given 5 min before post-training intra-CA1 microinjection of WIN. It is important to note that single intra-BLA microinjection of the same doses of apomorphine, SCH23390 or sulpiride had no effect on memory retrieval in passive avoidance task. On the other hand, post-training co-administration of N-methyl-d-aspartate (NMDA; 0.03 and 0.05 μg/rat, intra-BLA) plus an ineffective dose of WIN (0.1 μg/rat, intra-CA1) induced amnesia. Furthermore, the inhibitory effect of 0.5 μg/rat of intra-CA1 microinjection of WIN on memory formation was significantly decreased by pre-treatment with intra-BLA microinjection of the NMDA receptor antagonist d-2-amino-5-phosphonopentanoic acid (d-AP5; 0.1 and 0.5 μg/rat, intra-BLA). Intra-BLA microinjection of the same doses of NMDA or d-AP5 by itself did not induce any response on memory retrieval. Taken together, these findings support the existence of a functional interaction between dorsal hippocampal and basolateral amygdaloid neural circuits during processing cannabinoid-induced amnesia.

Effects of Drugs of Abuse on Putative Rostromedial Tegmental Neurons, Inhibitory Afferents to Midbrain Dopamine Cells

Recent findings have underlined the rostromedial tegmental nucleus (RMTg), a structure located caudally to the ventral tegmental area, as an important site involved in the mechanisms of aversion. RMTg contains γ-aminobutyric acid neurons responding to noxious stimuli, densely innervated by the lateral habenula and providing a major inhibitory projection to reward-encoding midbrain dopamine (DA) neurons. One of the key features of drug addiction is the perseverance of drug seeking in spite of negative and unpleasant consequences, likely mediated by response suppression within neural pathways mediating aversion. To investigate whether the RMTg has a function in the mechanisms of addicting drugs, we studied acute effects of morphine, cocaine, the cannabinoid agonist WIN55212-2 (WIN), and nicotine on putative RMTg neurons. We utilized single unit extracellular recordings in anesthetized rats and whole-cell patch-clamp recordings in brain slices to identify and characterize putative RMTg neurons and their responses to drugs of abuse. Morphine and WIN inhibited both firing rate in vivo and excitatory postsynaptic currents (EPSCs) evoked by stimulation of rostral afferents in vitro, whereas cocaine inhibited discharge activity without affecting EPSC amplitude. Conversely, nicotine robustly excited putative RMTg neurons and enhanced EPSCs, an effect mediated by α7-containing nicotinic acetylcholine receptors. Our results suggest that activity of RMTg neurons is profoundly influenced by drugs of abuse and, as important inhibitory afferents to midbrain DA neurons, they might take place in the complex interplay between the neural circuits mediating aversion and reward.

Cannabinoid and cholinergic systems interact during performance of a short-term memory task in the rat

It is now well established that cannabinoid agonists such as Δ(9)-tetrahydrocannabinol (THC), anandamide, and WIN 55,212-2 (WIN-2) produce potent and specific deficits in working memory (WM)/short-term memory (STM) tasks in rodents. Although mediated through activation of CB1 receptors located in memory-related brain regions such as the hippocampus and prefrontal cortex, these may, in part, be due to a reduction in acetylcholine release (i.e., cholinergic hypofunction). To determine the interaction between cannabinoid and cholinergic systems, we exposed rats treated with WIN-2 or cholinergic drugs to a hippocampal-dependent delayed nonmatch to sample (DNMS) task to study STM, and recorded hippocampal single-unit activity in vivo. WIN-2 induced significant deficits in DNMS performance and reduced the average firing and bursting rates of hippocampal principal cells through a CB1 receptor-mediated mechanism. Rivastigmine, an acetylcholinesterase inhibitor, reversed these STM deficits and normalized hippocampal discharge rates. Effects were specific to 1 mg/kg WIN-2 as rivastigmine failed to reverse the behavioral and physiological deficits that were observed in the presence of MK-801, an NMDA receptor antagonist. This supports the notion that cannabinoid-modulated cholinergic activity is a mechanism underlying the performance deficits in DNMS. Whether deficits are due to reduced nicotinic or muscarinic receptor activation, or both, awaits further analysis.

A synthetic cannabinoid agonist promotes oligodendrogliogenesis during viral encephalitis in rats

Chronic CNS infection by several families of viruses can produce deficits in prefrontal cortex (PFC) and striatal function. Cannabinoid drugs have been long known for their anti-inflammatory properties and their ability to modulate adult neuro and gliogenesis. Therefore, we explored the effects of systemic administration of the cannabinoid agonist WIN55,212-2(WIN) on prefrontal cortex (PFC) and striatal cytogenesis in a viral model of CNS injury and inflammation based on Borna Disease (BD) virus encephalitis. Active BrdU + progenitor populations were significantly decreased 1 week after BrdU labeling in BD rats [p < 0.001 compared to uninfected (NL) controls] while less than 5% of BrdU + cells colabeled for BDV protein. Systemic WIN (1 mg/kg i.p. twice daily × 7 days) increased the survival of BrdU + cells in striatum (p < 0.001) and PFC of BD rats, with differential regulation of labeled oligodendroglia precursors vs microglia/macrophages. WIN increased the percentage of BrdU + oligodendrocyte precursor cells and decreased BrdU + ED-1-labeled phagocytic cells, without producing pro- or antiviral effects. BDV infection decreased the levels of the endocannabinoid anandamide (AEA) in striatum (p < 0.05 compared to NL rats), whereas 2-AG levels were unchanged. Our findings indicate that: 1) viral infection is accompanied by alterations of AEA transmission in the striatum, but new cell protection by WIN appears independent of its effect on endocannabinoid levels; and 2) chronic WIN treatment alters the gliogenic cascades associated with CNS injury, promoting oligodendrocyte survival. Limiting reactive gliogenesis and macrophage activity in favor of oliogodendroglia development has significance for demyelinating diseases. Moreover, the ability of cannabinoids to promote the development of biologically supportive or symbiotic oligodendroglia may generalize to other microglia-driven neurodegenerative syndromes including NeuroAIDS and diseases of aging.