Cannabinoid Agonist WIN Research Articles

In vitro and non-invasive invivo effects of the cannabinoid-1 receptor agonist AM841 on gastrointestinal motor function in the rat.

Cannabinoids have been traditionally used for the treatment of gastrointestinal (GI) symptoms, but the associated central effects, through cannabinoid-1 receptors (CB1R), constitute an important drawback. Our aims were to characterize the effects of the recently developed highly potent long-acting megagonist AM841 on GI motor function and to determine its central effects in rats. Male Wistar rats were used for invitro and invivo studies. The effect of AM841 was tested on electrically induced twitch contractions of GI preparations (invitro) and on GI motility measured radiographically after contrast administration (invivo). Central effects of AM841 were evaluated using the cannabinoid tetrad. The non-selective cannabinoid agonist WIN 55,212-2 (WIN) was used for comparison. The CB1R (AM251) and CB2R (AM630) antagonists were used to characterize cannabinoid receptor-mediated effects of AM841. AM841 dose-dependently reduced invitro contractile activity of rat GI preparations via CB1R, but not CB2R or opioid receptors. In vivo, AM841 acutely and potently reduced gastric emptying and intestinal transit in a dose-dependent and AM251-sensitive manner. The invivo GI effects of AM841 at 0.1mg/kg were comparable to those induced by WIN at 5mg/kg. However, at this dose, AM841 did not induce any sign of the cannabinoid tetrad, whereas WIN induced significant central effects. The CB1R megagonist AM841 may potently depress GI motor function in the absence of central effects. This effect may be mediated peripherally and may be useful in the treatment of GI motility disorders.

Cannabinoid CB1 receptors mediate the effects of dipyrone.

Dipyrone is a non-steroidal anti-inflammatory drug used primarily as an analgesic and antipyretic. Some hypothesize that dipyrone activity can modulate other pathways, including endocannabinoid signalling. Thus, the aim of the present study was to evaluate the possible role of endocannabinoids in mediating dipyrone activity. This study is based on the tetrad effects of cannabinoids, namely an antinociceptive and cataleptic state, hypolocomotion and hypothermia. Dipyrone (500mg/kg, i.p.) treatment decreased locomotor activity, increased the latency to a thermal analgesic response and induced a cataleptic and hypothermic state. These reactions are similar to the tetrad effects caused by the cannabinoid agonist WIN55,212-2 (3mg/kg, i.p.). The cannabinoid CB1 receptor antagonist AM251 (10mg/kg, i.p.) reversed the effects of dipyrone on locomotor activity, the cataleptic response and thermal analgesia. Both AM251 (10mg/kg, i.p.) and the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (10mg/kg, i.p.) accentuated the reduction in body temperature caused by dipyrone. However, the CB2 receptor antagonist AM630 did not alter the hypothermic response to dipyrone. These results indicate involvement of the endocannabinoid system, especially CB1 receptors, in the analgesic and cataleptic effects of dipyrone, as well as hypolocomotion. However, cannabinoid receptors and TRPV1 were not involved in the hypothermic effects of dipyrone. We hypothesize that the mechanism of action of dipyrone may involve inhibition of cyclo-oxygenase and fatty acid amide hydrolase, which together provide additional arachidonic acid as substrate for endocannabinoid synthesis or other related molecules. This increase in endocannabinoid availability enhances CB1 receptor stimulation, contributing to the observed effects.

Effects of pubertal cannabinoid administration on attentional set-shifting and dopaminergic hyper-responsivity in a developmental disruption model of schizophrenia.

Background:Adolescent exposure to cannabinoids in vulnerable individuals is proposed to be a risk factor for psychiatric conditions later in life, particularly schizophrenia. Evidence from studies in animals has indicated that a combination of repeated pubertal cannabinoid administration with either neonatal prefrontocortical lesion, isolation rearing, or chronic NMDA receptor antagonism administration induces enhanced schizophrenia-like behavioral disruptions. The effects of adolescent exposure to CB1 receptor agonists, however, have not been tested in a developmental disruption model of schizophrenia.Methods:This was tested in the methylazoxymethanol (MAM) model, in which repeated treatment with the synthetic cannabinoid agonist WIN 55,212-2 (WIN; 1.2mg/kg) was extended over 25 days throughout puberty (postnatal days 40–65) in control and MAM rats. The rats received 20 injections, which were delivered irregularly to mimic the human condition. Adult rats were tested for attentional set-shifting task and locomotor response to amphetamine, which was compared with in vivo recording from ventral tegmental area (VTA) dopamine (DA) neurons.Results:MAM-treated rats showed impairment in the attentional set-shifting task, augmented locomotor response to amphetamine administration, and an increased number of spontaneously active DA neurons in the VTA. Interestingly, pubertal WIN treatment in normal animals induced similar changes at adulthood as those observed in MAM-treated rats, supporting the notion that adolescence exposure to cannabinoids may represent a risk factor for developing schizophrenia-like signs at adulthood. However, contrary to expectations, pubertal WIN administration did not exacerbate the behavioral and electrophysiological changes in MAM-treated rats beyond that observed in WIN-treated saline rats (Sal). Indeed, WIN treatment actually attenuated the locomotor response to amphetamine in MAM rats without impacting DA neuron activity states.Conclusions:Taken together, the present results indicate that the impact of cannabinoids during puberty/adolescence on schizophrenia models is more complex than may be predicted.

Post-status epilepticus treatment with the cannabinoid agonist WIN 55,212-2 prevents chronic epileptic hippocampal damage in rats

Repeated seizures are often associated with development of refractory chronic epilepsy, the most common form of which is temporal lobe epilepsy. G-protein-coupled cannabinoid receptors (CB1 and CB2 receptors) regulate neuronal excitability and have been shown to mediate acute anticonvulsant effects of cannabinoids in animal models. However, the potential of cannabinoids to prevent chronic neuronal damage and development of epilepsy remains unexplored. We hypothesized that treatment with a CB receptor agonist after an episode of status epilepticus – but before development of spontaneous recurrent seizures – might prevent the development of functional changes that lead to chronic epilepsy. Using the rat pilocarpine model, a therapeutic approach was simulated by administering the CB agonist, WIN 55,212-2 after an episode of status epilepticus. Epileptic behavior was monitored during development of spontaneous recurrent seizures for up to 6months. Histology, neurochemistry, redox status and NMDA receptor subunit expression were assessed at 6months after pilocarpine-induced seizures. Sub-acute treatment with WIN 55,212-2 (for 15days starting 24h after PILO injection) dramatically attenuated the severity, duration and frequency of spontaneous recurrent seizures. Further, in contrast to vehicle-treated animals, hippocampi from WIN 55,212-2-treated animals showed: normal thiol redox state, normal NR2A and NR2B subunit expression, preservation of GABAergic neurons and prevention of abnormal proliferation of GABAergic progenitors. This study shows for the first time that, after a known inciting event, treatment with a compound targeting CB receptors has the potential to prevent the epileptogenic events that result in chronic epileptic damage.

The CB1 Receptor as an Important Mediator of Hedonic Reward Processing

The endocannabinoid (ECB) system has emerged recently as a key mediator for reward processing. It is well known that cannabinoids affect appetitive learning processes and can induce reinforcing and rewarding effects. However, the involvement of the ECB system in hedonic aspects of reward-related behavior is not completely understood. With the present study, we investigated the modulatory role of the ECB system on hedonic perception, measured by the pleasure attenuated startle (PAS) paradigm for a palatable food reward. Here, a conditioned odor is thought to induce a pleasant affective state that attenuates an aversive reflex-the acoustic startle response. Modulatory effects of the CB1 receptor antagonist/inverse agonist SR1411716 and the cannabinoid agonist WIN 55 212-2 on PAS were examined in rats. PAS was also measured in CB1 receptor knockout (KO) and wild-type (WT) mice. Pharmacological inhibition as well as the absence of CB1 receptors was found to reduce PAS, whereas WIN 55 212-2 administration increased PAS. Finally, presentation of a conditioned reward cue was found to induce striatal FosB/ΔFosB expression in WT mice, but not in KO mice, indicating a reduced stimulation of reward-related brain regions in conditioned KO mice by odor presentation. We here show that in addition to our previous studies in rats, PAS may also serve as a valuable and suitable measure to assess hedonic processing in mice. Our data further indicate that the ECB system, and in particular CB1 receptor signaling, appears to be highly important for the mediation of hedonic aspects of reward processing.

Juvenile cannabinoid treatment induces frontostriatal gliogenesis in Lewis rats

Cannabis abuse in adolescence is associated with a broad array of phenotypical consequences, including a higher risk for schizophrenia and other mental disturbances related to dopamine (DA) imbalances. The great variability of these sequelae likely depends on the key influence of diverse genetic vulnerability factors. Inbred rodent strains afford a highly informative tool to study the contribution of genetic determinants to the long-term effects of juvenile cannabinoid exposure. In this study, we analyzed the phenotypical impact of the synthetic cannabinoid agonist WIN 55,212-2 (WIN; 2mg/kg/day from postnatal day 35–48) in adolescent Lewis rats, an inbred strain exhibiting resistance to psychotomimetic effects of environmental manipulations. At the end of this treatment, WIN-injected animals displayed increased survival of new cells (mainly oligodendroglia precursors) in the striatum and prefrontal cortex (PFC), two key terminal fields of DAergic pathways. To test whether these changes may be associated with enduring behavioral alterations, we examined the consequences of adolescent WIN treatment in adulthood (postnatal days 60–70), with respect to DA levels and metabolism as well as multiple behavioral paradigms. Rats injected with WIN exhibited increased turnover, but not levels, of striatal DA. In addition, cannabinoid-treated animals displayed increases in acoustic startle latency and novel-object exploration; however, WIN treatment failed to induce overt deficits of sensorimotor gating and social interaction. These results indicate that, in Lewis rats, juvenile cannabinoid exposure leads to alterations in frontostriatal gliogenesis, as well as select behavioral alterations time-locked to high DAergic metabolism, but not overt schizophrenia-related deficits.

Cannabinoid Receptor Activation Shifts Temporally Engendered Patterns of Dopamine Release

The ability to discern temporally pertinent environmental events is essential for the generation of adaptive behavior in conventional tasks, and our overall survival. Cannabinoids are thought to disrupt temporally controlled behaviors by interfering with dedicated brain timing networks. Cannabinoids also increase dopamine release within the mesolimbic system, a neural pathway generally implicated in timing behavior. Timing can be assessed using fixed-interval (FI) schedules, which reinforce behavior on the basis of time. To date, it remains unknown how cannabinoids modulate dopamine release when responding under FI conditions, and for that matter, how subsecond dopamine release is related to time in these tasks. In the present study, we hypothesized that cannabinoids would accelerate timing behavior in an FI task while concurrently augmenting a temporally relevant pattern of dopamine release. To assess this possibility, we measured subsecond dopamine concentrations in the nucleus accumbens while mice responded for food under the influence of the cannabinoid agonist WIN 55,212-2 in an FI task. Our data reveal that accumbal dopamine concentrations decrease proportionally to interval duration--suggesting that dopamine encodes time in FI tasks. We further demonstrate that WIN 55,212-2 dose-dependently increases dopamine release and accelerates a temporal behavioral response pattern in a CB1 receptor-dependent manner--suggesting that cannabinoid receptor activation modifies timing behavior, in part, by augmenting time-engendered patterns of dopamine release. Additional investigation uncovered a specific role for endogenous cannabinoid tone in timing behavior, as elevations in 2-arachidonoylglycerol, but not anandamide, significantly accelerated the temporal response pattern in a manner akin to WIN 55,212-2.

Alterations in the Intrinsic Burst Activity of Purkinje Neurons in Offspring Maternally Exposed to the CB1 Cannabinoid Agonist WIN 55212-2

Burst firing plays an important role in normal neuronal function and dysfunction. In Purkinje neurons, where the firing rate and discharge pattern encode the timing signals necessary for motor function, any alteration in firing properties, including burst activity, may affect the motor output. Therefore, we examined whether maternal exposure to the cannabinoid receptor agonist WIN 55212-2 (WIN) may affect the burst firing properties of cerebellar Purkinje cells in offspring. Whole-cell somatic patch-clamp recordings were made from cerebellar slices of adult male rats that were exposed to WIN prenatally. WIN exposure during pregnancy induced long-term alterations in the burst firing behavior of Purkinje neurons in rat offspring as evidenced by a significant increase in the mean number of spikes per burst (p<0.05) and the prolongation of burst firing activity (p<0.01). The postburst afterhyperpolarization potential (p<0.001), the mean intraburst interspike intervals (p<0.001) and the mean intraburst firing frequency (p<0.001) were also significantly increased in the WIN-treated group. Prenatal exposure to WIN enhanced the firing irregularity as reflected by a significant decrease in the coefficient of variation of the intraburst interspike interval (p<0.05). Furthermore, whole-cell voltage-clamp recordings revealed that prenatal WIN exposure significantly enhanced Ca(2+) channel current amplitude in offspring Purkinje neurons compared to control cells. Overall, the data presented here strongly suggest that maternal exposure to cannabinoids can induce long-term changes in complex spike burst activity, which in turn may lead to alterations in neuronal output.

Novel song-stimulated dendritic spine formation and Arc/Arg3.1 expression in zebra finch auditory telencephalon are disrupted by cannabinoid agonism

Cannabinoids are well-established to alter processes of sensory perception; however neurophysiological mechanisms responsible remain unclear. Arc, an immediate-early gene (IEG) product involved in dendritic spine dynamics and necessary for plasticity changes such as long-term potentiation, is rapidly induced within zebra finch caudal medial nidopallium (NCM) following novel song exposure, a response that habituates after repeated stimuli. Arc appears unique in its rapid postsynaptic dendritic expression following excitatory input. Previously, we found that vocal development-altering cannabinoid treatments are associated with elevated dendritic spine densities in motor-(HVC) and learning-related (Area X) song regions of zebra finch telencephalon. Given Arc's dendritic morphological role, we hypothesized that cannabinoid-altered spine densities may involve Arc-related signaling. To test this, we examined the ability of the cannabinoid agonist WIN55212-2 (WIN) to (1) acutely disrupt song-induced Arc expression, (2) interfere with habituation to auditory stimuli, and (3) alter dendritic spine densities in auditory regions. We found that WIN (3mg/kg) acutely reduced Arc expression within both NCM and Field L2 in an antagonist-reversible manner. WIN did not alter Arc expression in thalamic auditory relay nucleus ovoidalis (Ov), suggesting that cannabinoid signaling selectively alters responses to auditory stimulation. Novel song stimulation rapidly increased dendritic spine densities within auditory telencephalon, an effect blocked by WIN pretreatments. Taken together, cannabinoid inhibition of both Arc induction and its habituation to repeated stimuli, combined with prevention of rapid increases in dendritic spine densities, implicates cannabinoid signaling in modulation of physiological processes important to auditory responsiveness and memory.

Diuretic effects of cannabinoid agonists in mice

Cannabinoids both increase urine output and decrease urinary frequency in human subjects. However, these effects have not been systematically evaluated in intact mice, a species commonly used to evaluate the effects of novel cannabinoids. The present studies investigated whether cannabinoid agonists reliably produce diuresis in mice at doses comparable to those that produce other cannabinoid effects and, further, identified the receptors that may mediate these effects. Diuretic effects were measured in male mice over 6h. In some studies, urine was collected and analyzed for electrolyte measurements. In other studies, agonist injections were preceded by pretreatment with cannabinoid CB1 or CB2 selective antagonists, including a peripherally constrained CB1 antagonist. Companion studies evaluated the antinociceptive effects of the cannabinoid agonists in a warm-water tail-withdrawal assay. Direct-acting cannabinoid CB1 agonists Δ9-tetrahydrocannabinol (THC), WIN 55,212, AM7418 and AM4054, had biphasic effects on diuresis, with peak diuretic effects occurring at lower doses than peak antinociceptive effects. Cannabinoid diuresis was similar to κ-opioid agonist-induced diuresis in terms of maximum effects with only moderate loss of Na+. Antagonism studies indicate that the diuretic effects of cannabinoids are CB1-receptor mediated, with both central and peripheral components. These findings suggest that mice may provide a model for understanding the mixed effects of marijuana on urine output, as described in clinical studies, and aid in the development of targeted cannabinoid based therapies for bladder dysfunction.

Interactions between mu opioid receptor agonists and cannabinoid receptor agonists CP55940 and WIN55212–2 in rhesus monkeys: evaluation of treatment‐ and abuse‐related effects

Mu opioid receptor agonists and cannabinoid receptor agonists can be combined to possibly enhance therapeutic effectiveness (e.g., pain treatment); however, it is unclear whether combinations of these drugs enhance abuse liability. These studies examined the effects of cannabinoid receptor agonists CP55940 and WIN55212‐2 on the antinociceptive, discriminative stimulus, and positive reinforcing effects of mu opioid receptor agonists in rhesus monkeys. In one group (n=3), CP55940 (0.001–0.056 mg/kg), and WIN55212‐2 (0.1–1.78 mg/kg) shifted morphine dose‐effect curves leftward up to 8 fold. In monkeys discriminating 3.2 mg/kg morphine from saline (n=3), CP55940 and WIN55212‐2 shifted the morphine discriminative stimulus dose‐effect curves rightward up to 7 fold. In monkeys (n=4) self‐administering heroin (0.32–32.0 μg/kg/infusion), CP55940 and WIN55212‐2 shifted the heroin dose‐effect curves rightward (up to 15 fold) and downward. Cannabinoid receptor agonists CP55940 and WIN55212‐2 enhanced the antinociceptive effects but not the discriminative stimulus or positive reinforcing effects of mu opioid receptor agonists. These data support the view that combining mu opioid receptor agonists and cannabinoid receptor agonists might enhance treatment effectiveness without enhancing abuse liability. Supported by USPHS Grants R01DA05018, K05DA017918 (CPF) and T32DA031115 (DRM).

Cannabinoids may worsen gastric dysmotility induced by chronic cisplatin in the rat

Although cannabinoids have traditionally been used for the treatment and/or prevention of nausea and/or emesis, anorexia and weight loss induced by clinical use of antineoplastic drugs, their efficacy and safety in long-term treatments are still controversial. Our aim was to analyze the effects of the non-selective cannabinoid agonist WIN 55 212-2 (WIN) on gastrointestinal (GI) dysmotility and other adverse effects induced by repeated cisplatin administration in the rat. Male Wistar rats received two intraperitoneal injections once a week for 4 weeks: the first one was WIN, at non-psychoactive doses (0.5 or 1 mg kg(-1)), its vehicle or saline; the second one was cisplatin (2 mg kg(-1)) or saline. Radiographic techniques were used to determine the acute (after first dose), chronic (after last dose), and residual (1 week after treatment finalization) effects of cisplatin and/or WIN on GI motility. Bodyweight gain, food ingestion, and mechanical sensitivity were also tested. Weekly cisplatin induced mechanical allodynia, which WIN prevented, as well as weight gain reduction and anorexia, which WIN did not. Gastric emptying was dose-dependently delayed by cisplatin and this effect was enhanced upon chronic treatment. WIN aggravated cisplatin-induced gastric dysmotility. One week after treatment finalization, only minor alterations of GI motor function were found in rats treated with cisplatin, WIN or both. WIN weekly administered at low doses prevents neuropathy, but does not prevent anorexia or weight loss and aggravates gastric dysmotility induced by cisplatin. Cannabinoids should be handled with caution if chronically administered during chemotherapy.

Characterization of cannabinoid-induced relief of neuropathic pain in rat models of type 1 and type 2 diabetes

Diabetic neuropathy is a frequent complication of diabetes mellitus with a tremendous impact on patients' quality of life, and it remains poorly treated. Cannabinoids relieve the signs of diabetic neuropathy in different experimental models, including streptozotocin- (STZ-) induced type 1 diabetic rodents, and they may also relieve neuropathic signs in type 2 diabetic animals. This study compares the effect of the non-selective cannabinoid agonist WIN 55,212-2 (WIN) in Zucker Diabetic Fatty (ZDF) rats (type 2 diabetes) and in STZ-injected Wistar rats (type 1 diabetes).WIN (or its vehicle) was either systemically administered at a non-psychoactive dose or locally injected. Selective CB1 and CB2 cannabinoid antagonists were used to characterize WIN antineuropathic effects.Both type 1 and type 2 diabetic rats showed mechanical allodynia but not thermal hyperalgesia. WIN alleviated mechanical allodynia in both models of diabetes. In STZ-treated rats, both cannabinoid receptors were involved, whereas in ZDF rats, WIN effects seemed to mainly involve the activation of CB1 receptors. Higher doses of WIN were needed to significantly relieve mechanical allodynia upon intraplantar administration in ZDF vs. STZ-injected rats.Cannabinoids, acting on systemic and/or peripheral receptors, may serve as a new therapeutic alternative for symptom management in painful neuropathy associated with both type 1 and type 2 diabetes. Additionally, our results highlight the need for appropriate selection of diabetic experimental models because the results from studies in STZ-induced diabetic rodents might not be applicable in all diabetic situations.

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.

Cannabinoid agonist WIN 55,212-2 prevents the development of paclitaxel-induced peripheral neuropathy in rats. Possible involvement of spinal glial cells

Spinal glial activation contributes to the development and maintenance of chronic pain states, including neuropathic pain of diverse etiologies. Cannabinoid compounds have shown antinociceptive properties in a variety of neuropathic pain models and are emerging as a promising class of drugs to treat neuropathic pain. Thus, the effects of repeated treatment with WIN 55,212-2, a synthetic cannabinoid agonist, were examined throughout the development of paclitaxel-induced peripheral neuropathy. Painful neuropathy was induced in male Wistar rats by intraperitoneal (i.p.) administration of paclitaxel (1mg/kg) on four alternate days. Paclitaxel-treated animals received WIN 55,212-2 (1mg/kg, i.p.) or minocycline (15mg/kg, i.p.), a microglial inhibitor, daily for 14days, simultaneous with the antineoplastic. The development of hypersensitive behaviors was assessed on days 1, 7, 14, 21 and 28 following the initial administration of drugs. Both the activation of glial cells (microglia and astrocytes) at day 29 and the time course of proinflammatory cytokine release within the spinal cord were also determined. Similar to minocycline, repeated administration of WIN 55,212-2 prevented the development of thermal hyperalgesia and mechanical allodynia in paclitaxel-treated rats. WIN 55,212-2 treatment also prevented spinal microglial and astrocytic activation evoked by paclitaxel at day 29 and attenuated the early production of spinal proinflammatory cytokines (interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α). Our results confirm changes in the reactivity of glial cells during the development of peripheral neuropathy induced by paclitaxel and support a preventive effect of WIN 55,212-2, probably via glial cells reactivity inactivation, on the development of this neuropathy.

Decreased Reward Sensitivity in Rats from the Fischer344 Strain Compared to Wistar Rats Is Paralleled by Differences in Endocannabinoid Signaling

BackgroundThe aim of the present study was to examine if differences in the endocannabinoid (ECB) system might be linked to strain specific variations in reward-related behavior in Fischer344 (Fischer) and Wistar rats.Methodology/Principal FindingsTwo rat strains, the Fischer and the Wistar strain, were tested for different aspects of reward sensitivity for a palatable food reward (sweetened condensed milk, SCM) in a limited-access intake test, a progressive ratio (PR) schedule and the pleasure-attenuated startle (PAS) paradigm. Additionally, basic differences in the ECB system and cannabinoid pharmacology were examined in both rat strains. Fischer rats were found to express lower reward sensitivity towards SCM compared to Wistar rats. These differences were observed for consummatory, motivational and hedonic aspects of the palatable food reward. Western blot analysis for the CB1 receptor and the ECB degrading enzyme fatty acid amide hydrolase (FAAH) revealed a lower expression of both proteins in the hippocampus (HPC) of Fischer rats compared to the Wistar strain. Furthermore, increased cannabinoid-stimulated extracellular-regulated kinase (ERK) phosphorylation was detected in Wistar rats compared to the Fischer strain, indicating alterations in ECB signaling. These findings were further supported by the pharmacological results, where Fischer rats were found to be less sensitive towards the effects of the CB1 receptor antagonist/inverse agonist SR141716 and the cannabinoid agonist WIN 55,212-2.Conclusions/SignificanceOur present findings indicate differences in the expression of the CB1 receptor and FAAH, as well as the activation of ECB signaling pathways between Fischer and Wistar rats. These basic differences in the ECB system might contribute to the pronounced differences observed in reward sensitivity between both rat strains.

Reduced infarct size and accumulation of microglia in rats treated with WIN 55,212-2 after neonatal stroke

Cannabinoids have emerged as brain protective agents under neurodegenerative conditions. Many neuroprotective actions of cannabinoids depend on the activation of specific receptors, cannabinoid receptor type 1 (CB1R) and type 2 (CB2R). The aim of the present study was to determine whether the CB2R and CB1R agonist WIN 55,212-2 (WIN) protects neonatal brain against focal cerebral ischemia-reperfusion and whether anti-inflammatory mechanisms play a role in protection. Seven-day-old rats were subjected to 90-min middle cerebral artery occlusion (MCAO), and injured rats were identified by diffusion-weighted MRI during the occlusion. After reperfusion, rats were subcutaneously administered 1 mg/kg of WIN or vehicle twice daily until sacrifice. MCAO led to increased mRNA expression of CB2R (but not CB1R), chemokine receptors (CCR2 and CX3CR1), and cytokines (IL-1β and TNFα), as well as increased protein expression of chemokines MCP-1 and MIP-1α and microglial activation 24 h after MCAO. WIN administration significantly reduced microglial activation at this point and attenuated infarct volume and microglial accumulation and proliferation in the injured cortex 72 h after MCAO. Cumulatively, our results show that the cannabinoid agonist WIN protects against neonatal focal stroke in part due to inhibitory effects on microglia.

The cannabinoid agonist WIN55212-2 decreases l-DOPA-induced PKA activation and dyskinetic behavior in 6-OHDA-treated rats

Chronic Levodopa (l-DOPA), the gold standard therapy for Parkinson's disease (PD), causes disabling motor complications (dyskinesias) that are associated with changes in the activity of striatal protein kinase A (PKA) and cAMP-regulated phosphoprotein of 32kDa (DARPP-32). In this study, we showed that systemic administration of the cannabinoid agonist WIN55212-2 ameliorated l-DOPA-induced abnormal involuntary movements (AIMs) in the 6-OHDA rat model of PD and reversed l-DOPA-induced PKA hyperactivity via a CB1-mediated mechanism. This effect was accompanied by increased phosphorylation of DARPP-32 at threonine 34, which was partially blocked by CB1 antagonism. Striatal PKA activity was positively correlated with the severity of l-DOPA-induced axial and limb dyskinesias, suggesting a role for the cAMP/PKA signaling pathway in the expression of these motor disturbances.Our results indicate that activation of CB1 receptors, as well as reduction of striatal PKA hyperactivity, might be an effective strategy for the treatment of l-DOPA-induced dyskinesias.

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.

The cannabinoid receptors agonist WIN55212-2 inhibits macrophageal differentiation and alters expression and phosphorylation of cell cycle control proteins

In this study we investigated if and how cannabinoid receptor stimulation regulates macrophageal differentiation, which is one of the key steps in the immune effector reaction. For that reason, we used a well established differentiation model system of human U937 myelocytic leukemia cells that differentiate along the monocyte/macrophage lineage upon stimulation with the phorbol ester PMA. Constant cannabinoid receptor (CB) stimulation was performed using WIN55212-2, a potent synthetic CB agonist. We found that WIN55212-2 inhibited CB1/2-receptor-dependent PMA-induced differentiation of human myelocytic U937 cells into the macrophageal phenotype, which was associated with impaired vimentin, ICAM-1 and CD11b expression. In the presence of WIN55212-2, cdc2 protein and mRNA expression was progressively enhanced and Tyr-15-phosporylation of cdc2 was reduced in differentiating U937 cells. Additionally, p21Waf1/Cip1 expression was up-regulated. PMA-induced apoptosis was not enhanced by WIN55212-2 and differentiation-associated c-jun expression was not altered. In conclusion, we suppose that WIN55212-2-induced signals interferes with cell-cycle-arrest-signaling in differentiating myelocytic cells and thus inhibits macrophageal differentiation. Thus, it is possible that the cannabinoid system is able to influence one of the key steps in the immune effector function, the monocytic-macrophageal differentiation by alteration of cell cycle control proteins cdc2 and p21, and is therefore representing a promising option for therapeutic intervention in exacerbated immune reactions.