Cannabinoid Agonist WIN Research Articles

Potentiation by WIN 55,212‐2 of GABA‐activated currents in rat trigeminal ganglion neurones

Although both natural and synthetic cannabinoid compounds have been shown to exert an antinociceptive effect on acute and persistent pain, the anatomical locus of the target of cannabinoid-induced analgesia has not been fully elucidated. Here, we investigated the effects of the cannabinoid agonist WIN 55,212-2 on GABA-activated currents (I(GABA)) in rat primary sensory neurones. In the present study, experiments were performed on neurones freshly isolated from rat trigeminal ganglion (TG) by using whole-cell patch clamp and repatch techniques. GABA-evoked inward currents were potentiated by pretreatment with WIN 55,212-2 in a concentration-dependent manner (10(-10)-10(-8) M). WIN 55,212-2 shifted the GABA concentration-response curve upwards, with an increase of 30.3 +/- 3.7% in the maximal current response but with no significant change in the EC(50) (agonist concentration producing a half-maximal response) value. WIN 55,212-2 potentiated the responses to GABA in a manner independent of holding potential and in the absence of any change in the reversal potential of the current. This potentiation of I(GABA) induced by WIN 55,212-2 was almost completely blocked by AM 251 (3 x 10(-8) M), a CB(1) receptor antagonist, and, using the repatch technique, was found to be abolished after intracellular dialysis with the protein kinase A (PKA) activator cAMP or the PKA inhibitor H89. The potentiation by WIN 55,212-2 of I(GABA) in primary sensory neurones may help to elucidate the mechanism underlying the modulation of analgesia by cannabinoids in the spinal dorsal horn.

Effect of adolescent exposure to WIN 55212-2 on the acquisition and reinstatement of MDMA-induced conditioned place preference

The present study employs a conditioned place preference procedure (CPP) to examine the effects of exposure to the cannabinoid agonist WIN 55212-2 (WIN) (0.1 and 0.5mg/kg) during adolescence on the reinforcing properties of +/-3,4-methylenedioxymetamphetamine hydrochloride (MDMA) (1.25 and 2.5mg/kg) in mice. On postnatal day (PD) 27, animals received a daily injection of the assigned treatment on 5 consecutive days, and three days later the place conditioning procedure was initiated (PD 35). The results suggest that pre-exposure to cannabinoids strengthens the properties of MDMA and favors reinstatement of the craving for the drug, which endorses the gateway hypothesis.

Postnatal maturation of the gastrointestinal tract: A functional and immunohistochemical study in the guinea-pig ileum at weaning

Gastrointestinal motility is mainly controlled by the myenteric plexus. The longitudinal muscle-myenteric plexus (LMMP) preparation from the guinea-pig ileum is the best characterised adult gastrointestinal preparation; it has also been studied in old and neonatal animals, but not at weaning, when milk is substituted with the food typical of adult animals. We used LMMP preparations from weanling and adult guinea-pigs to study different functional parameters and immunohistochemically identified subpopulations of myenteric neurones, including the excitatory motor neurones to the longitudinal muscle (LM-EMN). Excitatory stimuli (low-frequency electrical stimulation, acetylcholine, substance P, and naloxone in morphine-tolerant preparations) produced similar responses in weanling and adult guinea-pigs. The endogenous cannabinoid anandamide, but not the synthetic cannabinoid agonist WIN 55,212-2 or the opioid morphine, inhibited the electrically stimulated twitches less efficaciously, and in vitro tolerance to morphine was also lower in weanling compared to adult animals. The packing densities of the calbindin-immunoreactive neurones (sensory neurones) and of neurones immunoreactive to both calretinin (CR) and neurofilament triplet protein (NFT; ascending interneurones) were slightly but significantly lower in weanling animals, whereas those of the neurones immunoreactive to CR but not NFT (LM-EMN) or immunoreactive to nitric oxide synthase (mainly inhibitory motor neurones) were comparable to the adult. Although guinea-pigs are relatively mature and can even ingest solid food at birth, their myenteric plexus is still not fully mature at the standard time of weaning. The nutritional, behavioural and environmental changes associated with weaning may be essential to attain full maturation of the myenteric plexus and gastrointestinal motility.

Agonism of the endocannabinoid system modulates binge-like alcohol intake in male C57BL/6J mice: involvement of the posterior ventral tegmental area

Recent studies have indicated a role for the endocannabinoid system in the behavioral and physiological effects of alcohol (ethanol), particularly ethanol seeking behaviors. However, its role in modulating binge-like intake and/or the mechanism by which it may exert these effects remain poorly understood. The current study used a newly developed strain-specific animal model of binge drinking, dubbed ‘Drinking In the Dark’ (DID), to determine if facilitation of the endocannabinoid system with the synthetic cannabinoid agonist WIN 55-212,2 (WIN) modulates binge-like ethanol intake in male C57BL/6J (B6) mice. Based on the results of these systemic (i.p.) manipulations, and evidence in support of the involvement of subregions of the Ventral Tegmental Area (VTA) in governing self-administration of ethanol ( Rodd-Henricks et al., (2000) Psychopharmacology (Berl) 149(3):217–224) as well as binge-like intake using the DID model (Moore & Boehm, (2009 Behav Neurosci 123(3):555–563), we extended these findings to evaluate the role of the endocannabinoid system within the anterior and posterior sub regions of the VTA using site-specific microinjections. Consistent with previous research, the lowest systemic dose of WIN (0.5 mg/kg) significantly increased ethanol intake in the first 30 minutes of access whereas the two highest doses (1 and 2 mg/kg) decreased ethanol intake within this time interval. Intra-posterior ventral tegmental area (pVTA) (but not aVTA (anterior ventral tegmental area) microinjections elicited time-dependent and dose-dependent increases (0.25 and 0.5 μg/side) and decreases (2.5 μg/side) in ethanol intake. Importantly, follow-up studies revealed that in some cases alterations in fluid consumption may have been influenced by competing locomotor activity (or inactivity). The present data are consistent with previous research in that agonism of the endocannabinoid system increases ethanol intake in rodents and implicate the pVTA in the modulation of drinking to intoxication. Moreover, the dose-dependent alterations in locomotor activity emphasize the importance of directly assessing multiple (possibly competing) behaviors when evaluating drug effects on voluntary consumption.

Selective lack of tolerance to delayed gastric emptying after daily administration of WIN 55,212‐2 in the rat

The use of cannabinoids to treat gastrointestinal (GI) motor disorders has considerable potential. However, it is not clear if tolerance to their actions develops peripherally, as it does centrally. The aim of this study was to examine the chronic effects of the cannabinoid agonist WIN 55,212-2 (WIN) on GI motility, as well as those in the central nervous and cardiovascular systems. WIN was administered for 14 days, at either non-psychoactive or psychoactive doses. Cardiovascular parameters were measured in anaesthetized rats, whereas central effects and alterations in GI motor function were assessed in conscious animals using the cannabinoid tetrad and non-invasive radiographic methods, respectively. Tests were performed after first (acute effects) and last (chronic effects) administration of WIN, and 1 week after discontinuing treatment (residual effects). Food intake and body weight were also recorded throughout treatment. Blood pressure and heart rate remained unchanged after acute or chronic administration of WIN. Central activity and GI motility were acutely depressed at psychoactive doses, whereas non-psychoactive doses only slightly reduced intestinal transit. Most effects were reduced after the last administration. However, delayed gastric emptying was not and could, at least partially, account for a concomitant reduction in food intake and body weight gain. The remaining effects of WIN administration in GI motility were blocked by the CB1 antagonist AM 251, which slightly accelerated motility when administered alone. No residual effects were found 1 week after discontinuing cannabinoid treatment. The different systems show differential sensitivity to cannabinoids and tolerance developed at different rates, with delayed gastric emptying being particularly resistant to attenuation upon chronic treatment.

Cannabinoid modulation of cortical adrenergic receptors and transporters.

We previously reported that administration of the synthetic cannabinoid agonist WIN 55,212-2 causes an increase in norepinephrine (NE) efflux in the frontal cortex (FC). The present study examined the expression levels of alpha2- and beta1-adrenergic receptors (ARs) as well as the norepinephrine transporter (NET) in the FC of rats following exposure to WIN 55,212-2. Rats received systemic injection of WIN 55,212-2 (3 mg/kg) acutely or for 7 days. Another group of rats received repeated WIN 55,212-2 treatment followed by a period of abstinence. Control rats received vehicle injections. Rats were euthanized 30 min after the last WIN 55,212-2 injection, the FC was microdissected, and protein extracts were probed for alpha2-AR, beta1-AR, and NET. Results showed that beta1-AR expression was significantly decreased following repeated WIN 55,212-2 treatment but significantly increased following a period of abstinence. alpha2-AR expression showed no significant change in all groups examined. NET expression was significantly decreased following acute WIN 55,212-2 treatment, with no changes following chronic administration or a period of abstinence. Alterations in NET may arise from modulation of cannabinoid receptors (CB1) that are localized to noradrenergic axon terminals as we demonstrate colocalization of CB1 receptor and NET in the same cortical axonal processes. The present findings support significant alterations in adrenergic receptor and NET expression in the FC after WIN 55,212 exposure that may underlie the reported changes in attention, cognition, and anxiety commonly observed after cannabinoid exposure.

Peripheral and central sites of action for the non‐selective cannabinoid agonist WIN 55,212‐2 in a rat model of post‐operative pain

Activation of cannabinoid (CB) receptors decreases nociceptive transmission in inflammatory or neuropathic pain states. However, the effects of CB receptor agonists in post-operative pain remain to be investigated. Here, we characterized the anti-allodynic effects of WIN 55,212-2 (WIN) in a rat model of post-operative pain. WIN 55,212-2 was characterized in radioligand binding and in vitro functional assays at rat and human CB(1) and CB(2) receptors. Analgesic activity and site(s) of action of WIN were assessed in the skin incision-induced post-operative pain model in rats; receptor specificity was investigated using selective CB(1) and CB(2) receptor antagonists. WIN 55,212-2 exhibited non-selective affinity and agonist efficacy at human and rat CB(1) versus CB(2) receptors. Systemic administration of WIN decreased injury-induced mechanical allodynia and these effects were reversed by pretreatment with a CB(1) receptor antagonist, but not with a CB(2) receptor antagonist, given by systemic, intrathecal and supraspinal routes. In addition, peripheral administration of both CB(1) and CB(2) antagonists blocked systemic WIN-induced analgesic activity. Both CB(1) and CB(2) receptors were involved in the peripheral anti-allodynic effect of systemic WIN in a pre-clinical model of post-operative pain. In contrast, the centrally mediated anti-allodynic activity of systemic WIN is mostly due to the activation of CB(1) but not CB(2) receptors at both the spinal cord and brain levels. However, the increased potency of WIN following i.c.v. administration suggests that its main site of action is at CB(1) receptors in the brain.

Altered CB1 receptor‐signaling in prefrontal cortex from an animal model of depression is reversed by chronic fluoxetine

Bilateral olfactory bulbectomy in the rat (OBX) induces behavioral, neurochemical, and structural abnormalities similar to those observed in human depression that are normalized after chronic, but not acute, treatment with antidepressants. In our study, OBX animals exhibited significant increases in both CB(1) receptor density ([(3)H]CP55490 binding) and functionality (stimulation of [(35)S]GTPgammaS binding by the cannabinoid (CB) agonist WIN 55212-2) at the prefrontal cortex (PFC). After chronic treatment with fluoxetine (10 mg/kg/day, 14 days, s.c.), OBX-induced hyperactivity in the open-field test was fully abolished. Interestingly, chronic fluoxetine fully reversed the enhanced CB(1)-receptor signaling in PFC observed following OBX. The CB agonist Delta(9)-tetrahydrocannabinol (5 mg/kg, i.p., 1 day) did not produce any behavioral effect in sham-operated animals but returned locomotor activity to control values in OBX rats. As both acute administration of Delta(9)-tetrahydrocannabinol and chronic fluoxetine elicited a similar behavioral effect in the OBX rat, it is not unlikely that the regionally selective enhancement of CB(1) receptor-signaling in the PFC could be related with the altered OBX behavior. Our findings reinforce the utility of this animal model to further investigating the implication of the endocannabinoid system in the modulation of emotional processes and its potential role in the adaptive responses to chronic antidepressants.

Cannabinoid CB1 receptor can mediate gastric mucosal protection in the rat

Event Abstract Back to Event Cannabinoid CB1 receptor can mediate gastric mucosal protection in the rat Nashwan Shujaa1*, Zoltan S. Zadori1 and Klara Gyires1 1 Department of Pharmacology and Pharmacotherapy, Semmelweis University, Hungary Cannabinoid CB1 receptor agonists can modulate several functions of the gastrointestinal tract: they inhibit the gastrointestinal motor function, the stimulated gastric acid secretion and the formation of gastric mucosal ulcerations in acid dependent ulcer models. We aimed to investigate the role of CB1 receptor agonists in an acid independent (ethanol-induced) ulcer model in the rat and to explore any possible interaction between the opioid and cannabinoid systems in this field. Our results showed that the endocannabinoid anandamide (5.75 µmol/kg i.v., 2.8-28.7 nmol/rat i.c.v.), its synthetic analogue methanandamide (1.38-5.53 µmol/kg i.v., 2.27-69 nmol/rat i.c.v.) and the synthetic cannabinoid agonist WIN 55,212-2 (0.047-0.19 µmol/kg i.v., 18.8-75.24 nmol/rat i.c.v.) inhibited the ethanol-induced gastric mucosal damage after both peripheral and central administration. The selective CB1 receptor antagonist SR 141716A (21.6 nmol/rat i.c.v.) reversed the effect of centrally administered anandamide and methanandamide. Moroever, the non-selective opioid receptor antagonist naloxone (27.5 nmol/rat i.c.v.) antagonized the gastroprotective effect of intravenously administered anandamide, methanandamide and WIN 55,212-2. The gastroprotective effect of WIN 55,212-2 (0.19 µmol/kg i.v.) was also reversed by the δ-opioid receptor selective antagonist naltrindole (4.88 nmol/rat i.c.v.) but not by the κ-opioid receptor selective antagonist norBNI (13.6 nmol/rat i.c.v.). In conclusion, the cannabinoid CB1 receptors are likely to be involved in the gastric mucosal defense, this effect seems to be central and correlation between the cannabinoid and opioid system in gastric mucosal protection may be raised. Acknowledgements: Funded by ETT grant 389. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Pathophysiology and neurology - non-degenerative disorders Citation: Shujaa N, Zadori ZS and Gyires K (2009). Cannabinoid CB1 receptor can mediate gastric mucosal protection in the rat. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.041 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Mar 2009; Published Online: 02 Mar 2009. * Correspondence: Nashwan Shujaa, Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary, Nashwan@freemail.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nashwan Shujaa Zoltan S Zadori Klara Gyires Google Nashwan Shujaa Zoltan S Zadori Klara Gyires Google Scholar Nashwan Shujaa Zoltan S Zadori Klara Gyires PubMed Nashwan Shujaa Zoltan S Zadori Klara Gyires Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Residues Accessible in the Binding-Site Crevice of Transmembrane Helix 6 of the CB2 Cannabinoid Receptor

We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the sixth membrane-spanning segment of the CB2 cannabinoid receptor that contribute to the surface of the water-accessible binding-site crevice. Using a background of the mutant C2.59S which is relatively insensitive to the methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 34 consecutive residues in TMH6 of the CB2 receptor. These mutant receptors were then expressed in HEK293 cells. By incubating HEK293 cells stably transfected with CB2 receptors with the small, charged, hydrophilic, thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA), [(3)H]CP55940 binding was significantly inhibited for six mutant receptors. All six of the mutants that reacted with MTSEA were protected from the reaction when pretreated with the cannabinoid agonist WIN55212-2, suggesting that MTSEA modification occurred within the binding crevice. Therefore, the side chains of the residues at these reactive loci (V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62) are on the water-accessible surface of the binding-site crevice. These residues are extracellular to the TMH6 CWXP hinge motif. The pattern of accessibility is consistent with a alpha-helical conformation for this segment of TMH6. Molecular modeling studies performed in the context of the CB2 model show that V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62 face into the CB2 binding pocket, further confirming our SCAM results. These results are similar to the accessibility patterns determined by SCAM studies of TMH6 in the opioid and dopamine D2 receptors.

Behavioural disturbances and altered Fos protein expression in adult rats after chronic pubertal cannabinoid treatment

Cannabis is one of the world's most popular recreational drugs. However, little is known about long-lasting cellular and neurobehavioural effects of chronic cannabinoid intake, especially during puberty where cannabis use among humans is commonly initiated. This study in rats investigates the long-term effect of pubertal cannabinoid treatment on prepulse inhibition (PPI), locomotor activity and on anxiety in the elevated-plus maze during adulthood. Furthermore, changes in adult basic neuronal activity, assessed by c-Fos immunoreactivity (Fos IR), and a potentially altered Fos expression after acute treatment with dopaminergic drugs was evaluated. Chronic treatment with the synthetic cannabinoid full agonist WIN 55,212-2 (WIN; 1.2 mg/kg) was carried out over 25 days of the rats' puberty and subsequent behavioural testing was conducted in adult animals. Finally, Fos IR was evaluated in several brain regions under basal conditions and after acute administration of haloperidol (0.1 mg/kg) and apomorphine (2 mg/kg). Chronic WIN treated animals exhibited a lasting disruption of PPI. These rats were also more active in the open field and less anxious in the elevated-plus maze than their vehicle treated controls. Additionally, when comparing Fos IR in selected brain regions, these animals displayed altered basal neuronal activity and responded differently to acute application of haloperidol or apomorphine. Taken together, these results indicate that chronic stimulation of the cannabinoid receptor CB 1 during the rats' puberty not only leads to persistent behavioural changes but also to cellular long-term adaptations within brain regions critical for drug of abuse or neuropsychiatric diseases.

Non-opioid antinociception produced by brain stem injections of improgan: Significance of local, but not cross-regional, cannabinoid mechanisms

Improgan, a cimetidine derivative which lacks activity at known histamine, opioid or cannabinoid receptors, acts by an unknown mechanism in the periaqueductal gray (PAG) and raphe magnus (RM) to stimulate descending, analgesic circuits. These circuits may utilize cannabinoid mechanisms. To characterize further the nature of these circuits, the effects of intracerebral (i.c.) microinjections of rimonabant (a CB1 receptor inverse agonist) were studied on antinociceptive responses following i.c. microinjections of improgan and the cannabinoid agonist WIN 55,212 (WIN) in rats. Separate intra-RM injections of improgan (30 μg) and WIN (8 μg) produced near-maximal antinociception on both the hot plate (HP) and tail flick (TF) nociceptive tests. Pretreatment with intra-RM rimonabant (20 μg) antagonized the antinociception produced by both intra-RM improgan and intra-RM WIN, but had no effects when given alone. Similar studies with improgan demonstrated rimonabant-sensitive sites within the dorsal and ventrolateral PAG. However, intra-RM pretreatment with rimonabant had no effect on antinociceptive responses following intra-PAG improgan. These studies show that improgan activates pain-relieving mechanisms in the PAG and the RM, both of which may utilize local cannabinoid mechanisms.

Chronic treatment and withdrawal of the cannabinoid agonist WIN 55,212-2 modulate the sensitivity of presynaptic receptors involved in the regulation of monoamine syntheses in rat brain

Brain monoamines are involved in many neurochemical and behavioral effects of cannabinoids, but little is known on the regulation of noradrenaline, dopamine, and serotonin (5-HT) synthesis in cannabinoid addiction. This study investigated in rat brain the chronic effects of the potent cannabinoid agonist WIN 55,212-2 and of rimonabant-precipitated withdrawal, as well as the sensitivity of synthesis-modulating inhibitory receptors, on the accumulation of L-3,4-dihydroxyphenylalanine (DOPA) and 5-HTP after decarboxylase inhibition. Acute WIN (8 mg/kg; 1 h) increased DOPA synthesis in cortex (52%), hippocampus (51%), and cerebellum (56%) and decreased DOPA accumulation in striatum (31%). Acute WIN also decreased the synthesis of 5-HTP in all brain regions (40-53%). Chronic WIN (2-8 mg/kg; 5 days) and/or antagonist-precipitated withdrawal induced tolerance to the acute effects of WIN on the accumulation of DOPA (cortex and striatum) and 5-HTP (all brain regions). The inhibitory effect of clonidine (alpha2-agonist; 1 mg/kg) on the accumulation of DOPA (15-41%) and 5-HTP (22-41%) was markedly decreased or abolished after chronic WIN and precipitated withdrawal, mainly in noradrenergic and serotonergic brain regions, which indicated desensitization of alpha2-autoreceptors and alpha2-heteroreceptors regulating the synthesis of noradrenaline and 5-HT. In WIN-dependent rats (chronic and withdrawal states), the effect of a low dose of (+/-)-8-hydroxy-2-(di-n-propylamino)-tetralin (5-HT1A agonist; 0.1 mg/kg) on the accumulation of precursor amino acids was markedly potentiated in cerebellum and striatum, indicating the induction of supersensitivity of 5-HT1A-autoreceptors and 5-HT1A-heteroreceptors that regulate the synthesis of 5-HT, noradrenaline, and dopamine in these brain regions. These chronic adaptations in presynaptic receptor function could play a relevant role in cannabinoid addiction.

PRECLINICAL STUDY: Acute and chronic cannabinoid treatment differentially affects recognition memory and social behavior in pubertal and adult rats

Although cannabis belongs to the most widely used drugs among adolescents, little is known about its acute and lasting neurobehavioral effects during critical developmental periods. In the present study we investigated acute and long-term behavioral effects of the cannabinoid agonist WIN 55,212-2 (WIN) in pubertal and adult rats. Chronic WIN (1.2 mg/kg)/vehicle treatment was extended over 25 days throughout puberty, from postnatal day (pd) 40 to pd 65, or for a similar time period in adult rats (> pd 80). All animals were tested at three time points for object/social recognition memory, social interaction and spontaneous social behavior. First, acute cannabinoid effects were investigated directly after the first injection. Additionally, behavioral performance was retested 24 hours and 15 days after cessation of WIN treatment. Chronic pubertal WIN treatment induced persistent object/social recognition deficits, indicating a general impairment in short-term information processing. Lasting disturbances in social behavior, social play and self-grooming were also found. Furthermore, behavioral deficits seen after acute WIN administration were more pronounced in pubertal than in adult rats. These results confirm our recent findings that chronic pubertal cannabinoid treatment leads to lasting behavioral alterations in adulthood, and they show that acute cannabinoid administration induces more severe behavioral deficits in pubertal rats than in mature animals. We therefore conclude that an immature brain is more susceptible to the acute and chronic effects of exogenous cannabinoids than an adult organism, which might be explained by an overactive endocannabinoid system and concomittant maturational disturbances in further neurotransmitter systems during pubertal development.

Differential effects of repeated low dose treatment with the cannabinoid agonist WIN 55,212-2 in experimental models of bone cancer pain and neuropathic pain

Pain due to bone malignancies is one of the most difficult types of cancer pain to fully control and may further decrease the patients' quality of life. Animal models of chronic pain conditions resulting from peripheral inflammatory reactions or nerve injuries are responsive to treatment with cannabinoid agonists. However, the use of cannabinoid agonists in humans may be hampered by CNS related side effects and development of tolerance. In the present study, we investigated the effect of repeated low dose administration of the synthetic cannabinoid agonist WIN 55,212-2 on bone cancer pain and neuropathic pain in mice. In addition, we investigated the development of CNS related side effects and tolerance. We found that 0.5 mg/kg/day for 18 days reduced pain related behavior and expression of spinal glial fibrillary acidic protein in the bone cancer pain model but not in the neuropathic pain model. Furthermore, this treatment strategy was not found to induce measurable CNS related side effects or tolerance. Cancer cell viability assays and bone volume fraction assessed by micro computed tomography (μCT) demonstrated that these effects were not due to changes in cancer progression. The difference in WIN 55,212-2 efficacy between the bone cancer and neuropathic pain models may reflect the different pain generating mechanisms, which may be utilized in designing new therapeutic drugs.

The Role of Prefrontal Cortex CB1 Receptors in the Modulation of Fear Memory

Understanding the mechanism of how fear memory can be extinguished could provide potential therapeutic strategies for the treatment of posttraumatic stress disorders. Here we show that infusion of CB1 receptor antagonist into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) retarded cue-alone-induced reduction of fear-potentiated startle. Conversely, cannabinoid agonist WIN55212-2 (WIN) facilitated the extinction. Unexpectedly, administration of WIN without cue-alone trials reduced startle potentiation in a dose-dependent manner. The effect of cannabinoid agonists was mimicked by endocannabinoid uptake or fatty acid amide hydrolase inhibitors. Rats were trained with 10 conditioned stimulus (CS(+)) (yellow light)-shock pairings. Extinction training with CS(+) (yellow light)-alone but not CS(-) (blue light)-alone trials decreased fear-potentiated startle. Intra-IL infusion of WIN before CS(-)-alone trials decreased startle potentiation, suggesting that the cannabinoid agonist decreased conditioned fear irrespective of whether the rats underwent CS(+)- or CS(-)-alone trials. Cannabinoid agonists activated extracellular signal-regulated kinases (ERKs) in mPFC slices, and ERK inhibitor blocked the effect of cannabinoid agonists on fear-potentiated startle. These results suggest that CB1 receptors acting through the phosphorylation of ERK are involved not only in the extinction of conditioned fear but also in the adaptation to aversive situations in general.

Constitutive Activity of the Cannabinoid CB1 Receptor Regulates the Function of Co-expressed Mu Opioid Receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.

Inhibition of spontaneous neurotransmission in the nucleus of solitary tract of the rat by the cannabinoid agonist WIN 55212-2 is not via CB1 or CB2 receptors

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure. Both CB1 and CB2 cannabinoid receptors have been described in the nucleus tractus solitarius (NTS), which receives direct afferent projections of cardiovascular reflexes. In the present study we evaluated the effects of WIN 55212-2 (WIN), a cannabinoid agonist, on fast neurotransmission in the NTS. We recorded spontaneous post-synaptic currents using the whole-cell configuration in NTS cells in brainstem slices from young rats (25–30 days old). Application of 5 μM WIN inhibited the frequency of both glutamatergic and GABAergic sPSCs, without affecting their amplitudes. Effects of WIN were not blocked by application of the CB1 antagonist AM251, the CB2 antagonist AM630 or the vanniloid receptor TRPV1 antagonist AMG9810, suggesting that the effect of WIN is via a non-CB1 non-CB2 receptor. Neither the CB1/CB2 agonist HU210 nor the CB1 agonist ACPA affected the frequency of sPSCs. We conclude WIN inhibits the neurotransmission in the NTS of young rats via a receptor distinct from CB1 or CB2.

The Influence of Mast Cell Mediators on Migration of SW756 Cervical Carcinoma Cells

The role of mast cell mediators on cervical cancer cell migration was assessed using an in vitro assay of scratch wound healing onto monolayers of HPV18-positive cervical carcinoma cells (SW756). Migration of SW756 cells was accelerated by co-culture with the mast cell line LAD2. This effect was inhibited by the H1R antagonist pyrilamine and the cannabinoid agonists 2-arachidonylglycerol (2AG) and Win 55,212-2. Therefore, the specific effects of histamine and cannabinoids on SW756 migration and LAD2 activation were analyzed. Histamine added to the in vitro assay of scratch wound healing either increased or inhibited SW756 migration rate by acting either on H1R or H4R, respectively. Cannabinoids acted on CB1 receptors to inhibit SW756 migration. Supernatants from SW756 cells stimulated LAD2 cell degranulation, which in turn was inhibited by cannabinoids acting via CB2 receptors. RT-PCR showed that SW756 expressed mRNA for CB1, CB2, H1R, H2R, and H4R. On the other hand, LAD2 expressed mRNA for all four HRs and CB2. The results suggest that mast cells could be contributing to cervical cancer cell invasion and spreading by the release of histamine and cannabinoids. Therefore, therapeutic modulation of specific mast cell mediators may be beneficial for cervical cancer treatment.

Changed accumbal responsiveness to alcohol in rats pre-treated with nicotine or the cannabinoid receptor agonist WIN 55,212-2

Alcohol, nicotine, and cannabinoid acutely increase the activity of the mesolimbic dopamine (DA) pathway. Although polysubstance consumption is a common pattern of abuse in humans, little is known about dopamine release following pre-exposure to these drugs. The purpose of this study was to test whether alcohol-induced dopamine release into the nucleus accumbens (NAc) shell is modified by different pre-treatments: water (i.g.), alcohol (1 g/kg, i.g.), nicotine (0.4 mg/kg, s.c.), and WIN 55,212-2 (1 mg/kg, s.c.). Male Wistar rats were treated (i.g.) for 14 days with either water or alcohol. In the following 5 days rats were injected (s.c.) with vehicle, nicotine, or WIN 55,212-2. Finally, a cannula was surgically implanted into the NAc shell and alcohol-induced extracellular dopamine release was monitored in freely moving rats. Alcohol (1 g/kg; i.g.) only increased the release of dopamine when animals were previously treated with water. This DA increase was markedly inhibited by (subchronic) treatment (5 days) with nicotine or WIN 55-212-2 as well as by previous (chronic) exposure to alcohol (14 days). These data demonstrate that pre-treatment with nicotine and the cannabinoid agonist WIN 55,212-2 is able to change the sensitivity of the NAc shell in response to a moderate dose of alcohol. Therefore, cannabinoid and nicotine exposure may have important implications on the rewarding effects of alcohol, because these drugs lead to long-lasting changes in accumbal dopamine transmission.