Lateral hypothalamus involvement in control of stress response by bed nucleus of the stria terminalis endocannabinoid neurotransmission in male rats.

The endocannabinoid neurotransmission acting via local CB1 receptor in the bed nucleus of the stria terminalis (BNST) has been implicated in behavioral and physiological responses to emotional stress. However, the neural network related to this control is poorly understood. In this sense, the lateral hypothalamus (LH) is involved in stress responses, and BNST GABAergic neurons densely innervate this hypothalamic nucleus. However, a role of BNST projections to the LH in physiological responses to stress is unknown. Therefore, using male rats, we investigated the role of LH GABAergic neurotransmission in the regulation of cardiovascular responses to stress by CB1 receptors within the BNST. We observed that microinjection of the selective CB1 receptor antagonist AM251 into the BNST decreased the number of Fos-immunoreactive cells within the LH of rats submitted to acute restraint stress. Treatment of the BNST with AM251 also enhanced restraint-evoked tachycardia. Nevertheless, arterial pressure increase and sympathetically-mediated cutaneous vasoconstriction to restraint was not affected by CB1 receptor antagonism within the BNST. The effect of AM251 in the BNST on restraint-evoked tachycardia was abolished in animals pretreated with the selective GABAA receptor antagonist SR95531 in the LH. These results indicate that regulation of cardiovascular responses to stress by CB1 receptors in the BNST is mediated by GABAergic neurotransmission in the LH. Present data also provide evidence of the BNST endocannabinoid neurotransmission as a mechanism involved in LH neuronal activation during stressful events.

mGluR5-Mediated eCB Signaling in the Nucleus Accumbens Controls Vulnerability to Depressive-Like Behaviors and Pain After Chronic Social Defeat Stress.

Stress contributes to major depressive disorder (MDD) and chronic pain, which affect a significant portion of the global population, but researchers have not clearly determined how these conditions are initiated or amplified by stress. The chronic social defeat stress (CSDS) model is a mouse model of psychosocial stress that exhibits depressive-like behavior and chronic pain. We hypothesized that metabotropic glutamate receptor 5 (mGluR5) expressed in the nucleus accumbens (NAc) normalizes the depressive-like behaviors and pain following CSDS. Here, we show that CSDS induced both pain and social avoidance and that the level of mGluR5 decreased in susceptible mice. Overexpression of mGluR5 in the NAc shell and core prevented the development of depressive-like behaviors and pain in susceptible mice, respectively. Conversely, depression-like behaviors and pain were exacerbated in mice with mGluR5 knockdown in the NAc shell and core, respectively, compared to control mice subjected to 3 days of social defeat stress. Furthermore, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, reversed the reduction in the level of the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the NAc of susceptible mice, an effect that was blocked by 3-((2-methyl-1, 3-thiazol-4-yl) ethynyl) pyridine hydrochloride (MTEP), an mGluR5 antagonist. In addition, the injection of CHPG into the NAc shell and core normalized depressive-like behaviors and pain, respectively, and these effects were inhibited by AM251, a cannabinoid type 1 receptor (CB1R) antagonist. Based on these results, mGluR5-mediated eCB production in the NAc relieves stress-induced depressive-like behaviors and pain.

GPR55 in the brain and chronic neuropathic pain.

There is a clear need for novel and improved therapeutic strategies for alleviating chronic neuropathic pain, as well as a need for better understanding of brain mechanisms of neuropathic pain, which are less understood than spinal and peripheral mechanisms. The G protein-coupled receptor 55 (GPR55), is a lysophosphatidylinositol (LPI)-sensitive receptor that has also been involved in cannabinoid signaling. It is expressed throughout the central nervous system, including the periaqueductal gray (PAG), a brainstem area and key element of the descending pain control system. Behaviors, pharmacology, biochemistry tools, and stereotaxic microinjections were used to determine if GPR55 plays a role in pain control in a chronic constriction injury (CCI) neuropathic pain model in rats. It was found that the blockade of GPR55 action in the PAG can restore and drive a descending control system to mitigate neuropathic pain. Our data demonstrate that GPR55 play a role in the descending pain control system, and identify GPR55 at supraspinal level as a neuropathic pain brain mechanism.

The interaction effect of sleep deprivation and cannabinoid type 1 receptor in the CA1 hippocampal region on passive avoidance memory, depressive-like behavior and locomotor activity in rats.

Increasing evidence shows the interaction effect of cannabinoids and sleep on cognitive functions. In the present study, we aimed to investigate the interaction effect of cannabinoids type 1 receptor (CB1r) in the CA1 hippocampal region and sleep deprivation (SD) on passive avoidance memory and depressive-like behavior in male Wistar rats. We used water box apparatus to induce total SD (TSD) for 24 h. The shuttle-box was applied to assess passive avoidance memory and locomotion apparatus was applied to assess locomotor activity. Forced swim test (FST) was used to evaluate rat's behavior. ACPA (CB1r agonist) at the doses of 0.01, 0.001 and 0.0001 µg/rat, and AM251 (CB1r antagonist) at the doses of 100, 10 and 1 ng/rat were injected intra-CA1, five minutes after training via stereotaxic surgery. Results showed SD impaired memory. ACPA at the doses of 0.01 and 0.001 µg/rat impaired memory and at all doses did not alter the effect of SD on memory. AM251 by itself did not alter memory, while at lowest dose (1 ng/rat) restored SD-induced memory deficit. Both drugs induced depressive-like behavior in a dose-dependent manner. Furthermore, both drugs decreased swimming at some doses (ACPA at 0.0001 µg/rat, AM251 at 0.001 and 0.01 ng/rat). Also, ACPA at the highest dose increased climbing of SD rats. In conclusion, we suggest CB1r may interact with the effect of SD on memory. Additionally, cannabinoids may show a dose-dependent manner in modulating mood and behavior. Interestingly, CB1r agonists and antagonists may exhibit a similar effect in some behavioral assessments.

The modulation of striatonigral and nigrotectal pathways by CB1 signalling in the substantia nigra pars reticulata regulates panic elicited in mice by urutu-cruzeiro lancehead pit vipers.

Cannabinoid receptor type 1 (CB1R) is widely distributed in the substantia nigra pars reticulata (SNpr). However, the role of CB1R at the SNpr level in threatening situations is poorly understood. We investigated the role of CB1R in the SNpr on the expression of fear responses in mice confronted with urutu-cruzeiro pit vipers. First, a bidirectional neurotracer was injected into the SNpr; then, immunostaining of the vesicular GABA transporter was conducted at the levels of the striatum (CPu) and deep layers of the superior colliculus (dlSC). In addition, CB1R immunostaining and GABA labelling were performed in the SNpr. Using a prey-versus-snake paradigm, mice were pretreated with the CB1R antagonist AM251 (100 pmol) and treated with the endocannabinoid anandamide (AEA, 5 pmol) in the SNpr, followed by bicuculline (40 ng) in the dlSC, and were then confronted with a snake. Bidirectional neural tract tracers associated with immunofluorescence showed the GABAergic striatonigral disinhibitory and nigrotectal inhibitory pathways. Furthermore, we showed that CB1R labelling was restricted to axonal fibres surrounding SNpr GABAergic cells. We also demonstrated a decrease in the defensive behaviours of mice treated with AEA in the SNpr, but this effect was blocked by pre-treatment with AM251 in this structure. Taken together, our results show that the panicolytic consequences of the AEA enhancement in the SNpr are signalled by CB1R, suggesting that CB1R localised in axon terminals of CPu GABAergic neurons in the SNpr modulates the activity of the nigrotectal GABAergic pathway during the expression of defensive behaviours in threatening situations.

URB597 abrogates anxiogenic and depressive behaviors in the methamphetamine-withdrawal mice: Role of the cannabinoid receptor type 1, cannabinoid receptor type 2, and transient receptor potential vanilloid 1 channels.

BACKGROUND: Methamphetamine is an addictive stimulant that possesses toxicity in the brain when taken repeatedly or at higher doses. Methamphetamine neurotoxicity is associated with numerous forms of mental impairment, including depression and anxiety. Evidence has also demonstrated that the endocannabinoid system is involved in the regulation of anxiety and depression. AIMS: This study was designed to determine the involvement of the endocannabinoid system in anxiety- and depression-related behaviors in methamphetamine-withdrawal male NMRI mice. METHODS: The elevated plus maze and forced swim test were used to assess the level of anxiety and depression. RESULTS: We found that methamphetamine (30 mg/kg, intraperitoneal) evoked depressive- and anxiogenic-like effects at 3 days post-administration. Injection of URB597 (5-10 ng/mouse, intracerebroventricular), 10 min before the test, prevented the emotional deficits induced by methamphetamine withdrawal. Moreover, the cannabinoid receptor type 1 antagonist AM251 (1 µg/mouse) or cannabinoid receptor type 2 antagonist AM630 (5 and 10 µg/mouse) suppressed the antidepressant activity in the methamphetamine-withdrawal mice treated with URB597. The transient receptor potential vanilloid 1 antagonist capsazepine (25 µg/mouse) prevented while capsazepine (100 µg/mouse) potentiated the antidepressant efficacy in the methamphetamine-withdrawal mice treated with URB597. The higher dose of AM630 and two higher doses of capsazepine had antidepressant efficacy, by themselves. Furthermore, capsazepine (50 µg/mouse) increased locomotion in the methamphetamine-withdrawal mice treated with URB597. CONCLUSIONS: The results suggest that URB597 has a potential for preventing methamphetamine withdrawal-evoked anxiety and depression. Cannabinoid type 1 receptors, cannabinoid type 2 receptors and transient receptor potential vanilloid 1 differently affect depression-related behaviors in methamphetamine-withdrawal mice treated with URB597.

Early-life stress exacerbates the effects of WIN55,212-2 and modulates the cannabinoid receptor type 1 expression.

Early-life stress induces an abnormal brain development and increases the risk of psychiatric diseases, including depression, anxiety and substance use disorders. We have developed a reliable model for maternal neglect, named maternal separation with early weaning (MSEW) in CD1 mice. In the present study, we evaluated the long-term effects on anxiety-like behaviours, nociception as well as the Iba1-positive microglial cells in this model in comparison to standard nest (SN) mice. Moreover, we investigated whether MSEW alters the cannabinoid agonist WIN55,212-2 effects regarding reward, spatial and emotional memories, tolerance to different cannabinoid responses, and physical dependence. Adult male offspring of MSEW group showed impaired responses on spatial and emotional memories after a repeated WIN55,212-2 treatment. These behavioural impairments were associated with an increase in basolateral amygdala and hippocampal CB1-expressing fibres and higher number of CB1-containing cells in cerebellum. Additionally, MSEW promotes a higher number of Iba1-positive microglial cells in basolateral amygdala and cerebellum. As for the cannabinoid-induced effects, rearing conditions did not influence the rewarding effects of WIN55,212-2 in the conditioned place preference paradigm. However, MSEW mice showed a delay in the development of tolerance to the cannabinoid effects. Moreover, CB1-positive fibres were reduced in limbic areas in MSEW mice after cannabinoid withdrawal precipitated with the CB1 antagonist SR141617A. These findings support that early-life stress promotes behavioural and molecular changes in the sensitivity to cannabinoids, which are mediated by alterations in CB1 signalling in limbic areas and it induces an increased Iba1-microglial marker which could interfere in emotional memories formation.

Precipitated Δ9-THC withdrawal reduces motivation for sucrose reinforcement in mice.

Withdrawal from Δ9-tetrahyrocannibidol (THC) is associated with a host of dysphoric symptoms that increase probability of relapse. To date, many animal models of THC withdrawal rely on withdrawal-induced somatic withdrawal signs leaving withdrawal-suppressed behavior relatively unexplored. As compared with withdrawal-induced behaviors, ongoing behavior that is suppressed by withdrawal is a useful behavioral endpoint because it 1) more effectively models the subjective aspects of withdrawal and 2) identifies pharmacotherapies that restore behavior to baseline levels, rather than eliminate behavior induced by withdrawal. The current study assessed effects of spontaneous and rimonabant-precipitated THC withdrawal in mice responding on a progressive-ratio (PR) schedule of sucrose water reinforcement. Once behavior stabilized, male and female mice were administered THC (10 mg/kg, s.c.) or vehicle for five or six days. THC was either discontinued and behavior monitored for three days during abstinence, or the CB1 antagonist rimonabant (2 mg/kg, i.p.) was used to precipitate withdrawal. Whereas spontaneous THC withdrawal had no effect on PR performance, THC-treated mice were differentially sensitive to rimonabant administration via large decreases in break point, overall response rate, and run rate relative to vehicle-treated mice. Importantly, pretreatment with the CB1 positive allosteric modulator ZCZ011 (10 mg/kg, i.p.) did not prevent precipitated-withdrawal-induced behavioral impairment. These extend findings of earlier studies suggesting operant baselines are useful tools to study subjective effects of cannabinoid withdrawal. Additionally, operant baselines allow withdrawal pharmacotherapies to be tested in a restoration-of-function context, which may be more sensitive, selective, and clinically relevant.

Novel therapeutic and drug development strategies for tobacco use disorder: endocannabinoid modulation.

INTRODUCTION: Tobacco use disorder (TUD) is a chronic relapsing condition. Existing pharmacotherapy can assist smokers to initiate smoking cessation, but relapse rates remain high. Novel therapeutics are required to help people quit and also to prevent relapse. The endocannabinoid system has been increasingly implicated in reward and addiction processes and the cannabinoid CB1 receptor inverse agonist rimonabant has been shown to be effective at promoting smoking cessation but has been associated with adverse psychiatric side effects. AREAS COVERED: Multiple converging factors likely contribute to the maintenance of smoking and cause relapse including nicotine reinforcement, propensity to reinstate drug seeking (induced by nicotine priming, nicotine-associated cues, and stress), the severity of withdrawal signs and executive function status. Studies assessing the impact of endocannabinoid (CB1 receptor, CB2 receptor, anandamide, and 2-arachidonoylglycerol) modulation on these addiction-related factors are reviewed. Future research avenues are also discussed. EXPERT OPINION: Endocannabinoid research in TUD is at a relatively early stage. Based on current evidence, CB1 receptor neutral antagonists and fatty acid amide hydrolase inhibitors demonstrate positive effects in studies assessing several addiction-related factors. This suggests they offer the greatest promise as novel cessation and anti-relapse agents.

Behavioral assessment of rimonabant under acute and chronic conditions.

Cannabinoid subtype 1 receptor (CB1R) antagonists were originally developed as anti-obesity agents. Unfortunately, SR1417116A (rimonabant), the first marketed inverse agonist of CB1R, produced CNS-related adverse effects including depression and suicidal ideation, and thus it was withdrawn from the market. These effects of rimonabant became evident in patients following chronic dosing. Standard preclinical toxicity studies failed to detect these adverse effects. The goal of these studies was to perform an integrated battery of behavioral assays to better understand the behavioral effects of rimonabant following both acute and chronic administration. In the present study, acute dosing with rimonabant in rats significantly decreased food consumption; decreased measures of locomotor activity; increased scratching, grooming and wet-dog shakes; and increased defecation. Subsequently, animals were tested using a chronic dosing regimen but prior to drug administration for that day. The highest dose of rimonabant tested significantly decreased marble burying behavior, presumably anxiolysis. There were also significant effects in social interaction after chronic dosing. Our results did not reveal significant rimonabant-induced anxiogenic behaviors. Future studies to characterize behavioral screens for anxiogenic effects of CB1 antagonists in rodents should further explore social interaction paradigms and potential comorbid factors of rimonabant dosing such as sex, age, and obesity.

Effects of combined 5-HT2A and cannabinoid receptor modulation on a schizophrenia-related prepulse inhibition deficit in mice.

RATIONALE: Prepulse inhibition of the startle reflex (PPI) is disrupted in several psychiatric disorders including schizophrenia. Understanding PPI pharmacology may help elucidate the pathophysiology of these disorders and lead to better treatments. Given the advantages of multi-target approaches for complex mental illnesses treatment, we have investigated the interaction between receptors known to modulate PPI (5-HT1A and 5-HT2A) and the neuromodulatory endocannabinoid system. OBJECTIVES: To investigate serotonin and cannabinoid receptor (CBR) co-modulation in a model of PPI disruption relevant to schizophrenia METHODS: Male Swiss mice were pretreated with WIN 55,212-2 (CBR agonist), rimonabant (CB1R inverse agonist), 8-OH-DPAT (5-HT1A/7 agonist), and volinanserin (5-HT2A antagonist) or with a combination of a cannabinoid and a serotonergic drug. PPI disruption was induced by acute administration of MK-801. RESULTS: WIN 55,212-2 and rimonabant did not change PPI nor block MK-801-induced deficits. 8-OH-DPAT increased PPI in control mice and, in a higher dose, inhibited MK-801-induced impairments. Volinanserin also increased PPI in control and MK-801-treated mice, presenting an inverted U-shaped dose-response curve. Co-administration of either cannabinoid ligand with 8-OH-DPAT did not change PPI; however, the combination of volinanserin with rimonabant increased PPI in both control and MK-801-exposed mice. CONCLUSIONS: WIN 55,212-2 and rimonabant had similar effects in PPI. Moreover, serotonin and cannabinoid receptors interact to modulate PPI. While co-modulation of CBR and 5-HT1A receptors did not change PPI, a beneficial effect of 5-HT2A and CB1R antagonist combination was detected, possibly mediated through potentiation of 5-HT2A blockade effects by concomitant CB1R blockade.

CB1 receptor antagonist rimonabant protects against chronic intermittent hypoxia-induced bone metabolism disorder and destruction in rats.

OBJECTIVE: The endocannabinoid system (ECS) regulates bone turn-over and remodeling. Chronic intermittent hypoxia (CIH) occurring during obstructive sleep apnea (OSA) may lead to disorders of the ECS and bone metabolism abnormalities. This study aimed to investigate whether or not the cannabinoid receptor 1 (CB1R) antagonist rimonabant (Ri) alleviates bone metabolism abnormalities and bone destruction induced by chronic intermittent hypoxia (CIH). METHODS: Healthy male Sprague Dawley (SD) rats (n=48) were randomly divided into 6 groups of 8 rats: 2 normal control (NC) groups, 2 intermittent hypoxia (IH) groups, and 2 IH + Ri groups. Rats in NC groups breathed room air for 4 weeks (4w NC group) and 6 weeks (6w NC group). Rats in IH groups experienced IH environment for 4 weeks (4w IH group) and 6 weeks (6w IH group). In addition to the same IH exposure, rats in IH + Ri group were given daily intraperitoneal injection of Ri at the dosage of 1.5 mg/kg/d for 4 weeks (4w IH + Ri group) and 6 weeks (6w IH + Ri group). Levels of serum tartrate-resistant acid phosphatase (TRAP, a marker of bone resorption) were determined by ELISA. Hematoxylin and eosin (HE) staining was performed on bone sections to observe the changes in bone microstructure. Expression of CB1R in bone tissue was determined by immunohistochemistry. RESULTS: TRAP levels were higher in the 4w IH and 6w IH groups than in the 4w NC and 6w NC groups; TRAP levels were lower in the 4w IH + Ri and 6w IH + Ri groups than in the 4w IH and 6w IH groups. HE staining showed that the morphology of bone cells in the NC group was normal, but the 4w IH group had mild edema of bone cells, reduction in trabecular bone, and destruction of bone microstructure. Changes were more severe in the 6w IH group than 4w IH. The 4w IH + Ri group was slightly improved compared with the 4w IH group. The 6w IH + Ri group was improved compared with the 4w IH + Ri group. The results of immunohistochemistry showed that the expression of CB1R in IH group was significantly higher than that in NC group. The expression of CB1R in the IH + Ri group was lower than that in the IH group. With the prolongation of hypoxia, the expression of CB1R in bone cells of IH group increased. The expression level of CB1R in IH + Ri group decreased with the prolongation of intervention time. Correlation analysis showed that the expression rate of CB1R in bone cells was positively correlated with the level of TRAP in serum. CONCLUSION: CIH increases serum TRAP levels and triggers metabolic bone disorder by activating bone CB1R. Intervention with CB1R antagonist (rimonabant) reduces the bone dysmetabolism in the CIH rat model.

Neuronal nicotinic acetylcholine receptors mediate ∆9 -THC dependence: Mouse and human studies.

Cessation from prolonged use of ∆9 -tetrahydrocannabinol (THC), the primary active compound responsible for the cannabimimetic effects of cannabis, results in a mild to moderate withdrawal syndrome in humans and laboratory animals. Whereas manipulations of the endogenous cannabinoid system (eg, cannabinoid receptors and endocannabinoid regulating enzymes) alter nicotine withdrawal, in this study we asked the reciprocal question. Do nicotinic acetylcholine receptors (nAChRs) modulate THC withdrawal? To assess the role of different nAChR subtypes in THC withdrawal, we used transgenic mouse, preclinical pharmacological, and human genetic correlation approaches. Our findings show that selective α3ß4* nAChR antagonist, AuIB, and α3ß4* nAChR partial agonist, AT-1001, dose-dependently attenuated somatic withdrawal signs in THC-dependent mice that were challenged with the cannabinoid-1 receptor antagonist rimonabant. Additionally, THC-dependent α5 and α6 nAChR knockout (KO) mice displayed decreased rimonabant precipitated somatic withdrawal signs compared with their wild-type counterparts. In contrast, ß2 and α7 nAChR KO mice showed no alterations in THC withdrawal signs. Moreover, deletion of ß2 nAChR did not alter the reduced expression of somatic signs by the preferred α6ß4* antagonist, BulA [T5A;P60]. Finally, the human genetic association studies indicated that variations in the genes that code for the α5, α3, ß4, and α6 nAChRs were associated with cannabis disorder phenotypes. Overall, these findings suggest that α3ß4* and α6ß4* nAChR subtypes represent viable targets for the development of medications to counteract THC dependence.

Investigation of the Involvement of the Endocannabinoid System in TENS-Induced Antinociception.

Transcutaneous electrical nerve stimulation (TENS) promotes antinociception by activating the descending pain modulation pathway and consequently releasing endogenous analgesic substances. In addition, recent studies have shown that the endocannabinoid system controls pain. Thus, the present study investigated the involvement of the endocannabinoid system in TENS-induced antinociception of cancer pain using a cancer pain model induced by intraplantar (i.pl.) injections of Ehrlich tumor cells in male Swiss mice. Low- and high-frequency TENS was applied for 20 minutes to the mice's paws, and to investigate the involvement of the endocannabinoid system were used the N-(peperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pitazole-3-carboixamide (AM251), a cannabinoid CB1 receptor antagonist and (5Z,8Z,11Z,14Z)-5,8,11,14-eicosatetraenyl-methylester phosphonofluoridic acid (MAFP), an inhibitor of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase, injected by via i.pl., intrathecal (i.t.), and intradorsolateral periaqueductal gray matter (i.dl.PAG). Furthermore, liquid chromatography-tandem mass spectrometry, western blot, and immunofluorescence assays were used to evaluate the endocannabinoid anandamide levels, cannabinoid CB1 receptor protein levels, and cannabinoid CB1 receptor immunoreactivity, respectively. Low- and high-frequency TENS reduced the mechanical allodynia induced by Ehrlich tumor cells and this effect was reversed by AM251 and potentiated by MAFP at the peripheral and central levels. In addition, TENS increased the endocannabinoid anandamide levels and the cannabinoid CB1 receptor protein levels and immunoreactivity in the paw, spinal cord, and dorsolateral periaqueductal gray matter. These results suggest that low- and high-frequency TENS is effective in controlling cancer pain, and the endocannabinoid system is involved in this effect at both the peripheral and central levels. PERSPECTIVE: TENS is a nonpharmacological strategy that may be used to control cancer pain. Identification of a new mechanism involved in its analgesic effect could lead to the development of clinical studies as well as an increase in its application, lessening the need for pharmacological treatments.

Stress-induced modulation of endocannabinoid signaling leads to delayed strengthening of synaptic connectivity in the amygdala.

Even a brief exposure to severe stress strengthens synaptic connectivity days later in the amygdala, a brain area implicated in the affective symptoms of stress-related psychiatric disorders. However, little is known about the synaptic signaling mechanisms during stress that eventually culminate in its delayed impact on the amygdala. Hence, we investigated early stress-induced changes in amygdalar synaptic signaling in order to prevent its delayed effects. Whole-cell recordings in basolateral amygdala (BLA) slices from rats revealed higher frequency of miniature excitatory postsynaptic currents (mEPSCs) immediately after 2-h immobilization stress. This was replicated by inhibition of cannabinoid receptors (CB1R), suggesting a role for endocannabinoid (eCB) signaling. Stress also reduced N-arachidonoylethanolamine (AEA), an endogenous ligand of CB1R. Since stress-induced activation of fatty acid amide hydrolase (FAAH) reduces AEA, we confirmed that oral administration of an FAAH inhibitor during stress prevents the increase in synaptic excitation in the BLA soon after stress. Although stress also caused an immediate reduction in synaptic inhibition, this was not prevented by FAAH inhibition. Strikingly, FAAH inhibition during the traumatic stressor was also effective 10 d later on the delayed manifestation of synaptic strengthening in BLA neurons, preventing both enhanced mEPSC frequency and increased dendritic spine-density. Thus, oral administration of an FAAH inhibitor during a brief stress prevents the early synaptic changes that eventually build up to hyperexcitability in the amygdala. This framework is of therapeutic relevance because of growing interest in targeting eCB signaling to prevent the gradual development of emotional symptoms and underlying amygdalar dysfunction triggered by traumatic stress.

Cannabinoid Antagonist Drug Discrimination in Nonhuman Primates.

Despite a growing acceptance that withdrawal symptoms can emerge following discontinuation of cannabis products, especially in high-intake chronic users, there are no Food and Drug Administration (FDA)-approved treatment options. Drug development has been hampered by difficulties studying cannabis withdrawal in laboratory animals. One preclinical approach that has been effective in studying withdrawal from drugs in several pharmacological classes is antagonist drug discrimination. The present studies were designed to examine this paradigm in squirrel monkeys treated daily with the long-acting CB1 agonist AM2389 (0.01 mg/kg) and trained to discriminate the CB1 inverse agonist/antagonist rimonabant (0.3 mg/kg) from saline. The discriminative-stimulus effects of rimonabant were both dose and time dependent and, importantly, could be reproduced by discontinuation of agonist treatment. Antagonist substitution tests with the CB1 neutral antagonists AM4113 (0.03-0.3 mg/kg), AM6527 (0.03-1.0 mg/kg), and AM6545 (0.03-1.0 mg/kg) confirmed that the rimonabant discriminative stimulus also could be reproduced by CB1 antagonists lacking inverse agonist action. Agonist substitution tests with the phytocannabinoid ∆9-tetrahydrocannabinol (0.1-1.0 mg/kg), synthetic CB1 agonists nabilone (0.01-0.1 mg/kg), AM4054 (0.01-0.03 mg/kg), K2/Spice compound JWH-018 (0.03-0.3 mg/kg), FAAH-selective inhibitors AM3506 (0.3-5.6 mg/kg), URB597 (3.0-5.6 mg/kg), and nonselective FAAH/MGL inhibitor AM4302 (3.0-10.0 mg/kg) revealed that only agonists with CB1 affinity were able to reduce the rimonabant-like discriminative stimulus effects of withholding daily agonist treatment. Although the present studies did not document physiologic disturbances associated with withdrawal, the results are consistent with the view that the cannabinoid antagonist drug discrimination paradigm provides a useful screening procedure for examining the ability of candidate medications to attenuate the interoceptive stimuli provoked by cannabis discontinuation. SIGNIFICANCE STATEMENT: Despite a growing acceptance that withdrawal symptoms can emerge following the discontinuation of cannabis products, especially in high-intake chronic users, there are no FDA-approved pharmacotherapies to assist those seeking treatment. The present studies systematically examined cannabinoid antagonist drug discrimination, a preclinical animal model that is designed to appraise the ability of candidate medications to attenuate the interoceptive effects that accompany abrupt cannabis abstinence.

A medium throughput rodent model of relapse from addiction with behavioral and pharmacological specificity.

One of most formidable problems in the treatment of addiction is the high rate of relapse. The discovery of medicines to help mitigate relapse are aided by animal models that currently involve weeks of training and require surgical preparations and drug delivery devices. The present set of experiments was initiated to investigate a rapid 8-day screening method that utilizes food instead of intravenous drug administration. Male Sprague-Dawley rats were trained in a reinstatement paradigm in which every lever press produced a 45 mg food pellet concurrently paired with a light and tone. Behavior was subsequently extinguished with lever responses producing neither food nor food-associated stimuli. Reinstatement of responding was evaluated under conditions in which the first three responses of every 5 min time bin produced a food pellet along with food-associated stimuli. The mGlu5 receptor antagonists MPEP and MTEP produced a significant reduction in reinstatement while failing to alter responding where every response produced food. The cannabinoid CB1 receptor antagonist rimonabant and the mGlu2/3 receptor agonist LY379268 also selectively reduced reinstatement. Other compounds including clozapine, d-amphetamine, chlordiazepoxide, ABT-431, naltrexone and citalopram were without effect. The results suggest that relapse-like behavioral effects can be extended to non-pharmacological reinforcers. Drug effects demonstrated both behavioral and pharmacological specificity. The present experimental design thus allows for efficient and rapid assessment of the effects of drugs that might be useful in the treatment of addiction-associated relapse.

Ketamine-induced antidepressant like effects in mice: A possible involvement of cannabinoid system.

The purpose of this study was to explore the possible interaction between ketamine and cannabinoid system in the modulation of depression-related responses using the forced swimming test (FST), tail suspension test (TST) and open-field test (OFT) in mice. Our results revealed that intra-peritoneal (i.p.) injection of ketamine (5 and 10 mg/kg), a non-competitive NMDA antagonist, dose-dependently produced antidepressant-like effect in the FST. Moreover, i.p. administration of both CB1 and CB2 receptor drugs: ACPA (1 mg/kg; CB1 receptor agonist), AM251 (1 mg/kg; CB1 receptor antagonist), GP1a (2 mg/kg; CB2 receptor agonist) and AM630 (0.5 mg/kg; CB2 receptor antagonist) exhibited antidepressant action. Interestingly, the concomitant administration of ineffective doses of ketamine and cannabinoid receptor antagonists provoked the antidepressant-like effects as compared to control group. It should be considered, all above mentioned doses of drugs could not change locomotor activity in the OFT. It seems that possible interaction between ketamine and cannabinoid system may modulate depression-related behavior.

Role of the endocannabinoid system in the dorsal hippocampus in the cardiovascular changes and delayed anxiety-like effect induced by acute restraint stress in rats.

BACKGROUND: The dorsal hippocampus has a central role in modulating cardiovascular responses and behavioral adaptation to stress. The dorsal hippocampus also plays a key role in stress-associated mental disorders. The endocannabinoid system is widely expressed in the dorsal hippocampus and modulates defensive behaviors under stressful conditions. The endocannabinoid anandamide activates cannabinoid type 1 receptors and is metabolized by the fatty acid amide hydrolase enzyme. AIMS: We sought to verify whether cannabinoid type 1 receptors modulate stress-induced cardiovascular changes, and if pharmacological fatty acid amide hydrolase inhibition in the dorsal hippocampus would prevent the cardiovascular responses and the delayed anxiogenic-like behavior evoked by restraint stress in rats via cannabinoid type 1 receptors. METHODS: Independent groups received intra-dorsal-hippocampal injections of N-(piperidin-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-hpyrazole-3-carboxamide (AM251; cannabinoid type 1 receptor antagonist/inverse agonist, 10-300 pmol) and/or cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597; fatty acid amide hydrolase inhibitor, 10 pmol) before the restraint stress session. Cardiovascular response during restraint stress or later behavioral parameters were evaluated. RESULTS: Acute restraint stress altered the cardiovascular response, characterized by increased heart rate and mean arterial pressure, as well as decreased tail cutaneous temperature. It also induced a delayed anxiogenic-like effect, evidenced by reduced open arm exploration in the elevated plus maze 24 h after stress. AM251 exacerbated the stress-induced cardiovascular responses after injection into the dorsal hippocampus. In contrast, local injection of URB597 prevented the cardiovascular response and the delayed (24 h) behavioral consequences of restraint stress, effects attenuated by pretreatment with AM251. CONCLUSION: Our data corroborate previous results indicating that the hippocampal endocannabinoid system modulates the outcome of stress exposure and suggest that this could involve modulation of the cardiovascular response during stress exposure.

Cannabis effects on lipoproteins.

PURPOSE OF REVIEW: The endocannabinoid system affects several physiological functions. A family of endocannabinoid receptors is susceptible to cannabis constituents. Cannabis is widely used in our society and following its recent legalization in Canada, we focus on how exposure to cannabis and pharmacologic cannabinoid receptor type 1 (CB1) inhibition affect lipoprotein levels. RECENT FINDINGS: Several groups have reported that exposure to cannabis does not increase weight despite the marked increase in caloric intake. In observational studies, the effect of smoked cannabis exposure on plasma lipids is variable. Some studies in specific patient populations with longer exposure to cannabis seemed to identify slightly more favorable lipoprotein profiles in the exposed group. Several larger controlled clinical trials using orally administered rimonabant, a CB1 receptor antagonist, have consistently shown relative improvements in weight and plasma levels of triglyceride and high-density lipoprotein cholesterol among patients receiving the treatment. SUMMARY: The widely variable findings on the relationship of cannabis in various forms with plasma lipids preclude any definitive conclusions. Cannabis has complex effects on the cardiovascular system and its effects on lipid profile must be considered in this overall context. Further properly controlled research is required to better understand this topic.