Activation Of Cannabinoid CB1 Receptors Research Articles

Cannabinoid Receptor 1 associates to different molecular complexes during GABAergic neuron maturation.

CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long-lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot-gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), voltage-dependent anion channel 1 (VDAC1), myosin phosphatase Rho-interacting protein (MPRIP) or usher syndrome type-1C protein-binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic-associated proteins. We describe also interacting partners specifically immunoprecipitated with either N-terminal or C-terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development.

Orally Active Peptide Vector Allows Using Cannabis to Fight Pain While Avoiding Side Effects.

The activation of cannabinoid CB1 receptors (CB1R) by Δ9-tetrahydrocannabinol (THC), the main component of Cannabis sativa, induces analgesia. CB1R activation, however, also causes cognitive impairment via the serotonin 5HT2A receptor (5HT2AR), a component of a CB1R–5HT2AR heteromer, posing a serious drawback for cannabinoid therapeutic use. We have shown that peptides reproducing CB1R transmembrane (TM) helices 5 and 6, fused to a cell-penetrating sequence (CPP), can alter the structure of the CB1R–5HT2AR heteromer and avert THC cognitive impairment while preserving analgesia. Here, we report the optimization of these prototypes into drug-like leads by (i) shortening the TM5, TM6, and CPP sequences, without losing the ability to disturb the CB1R–5HT2AR heteromer, and (ii) extensive sequence remodeling to achieve protease resistance and blood–brain barrier penetration. Our efforts have culminated in the identification of an ideal candidate for cannabis-based pain management, an orally active 16-residue peptide preserving THC-induced analgesia.

Kahweol, a natural diterpene from coffee, induces peripheral antinociception by endocannabinoid system activation.

Kahweol is a compound derived from coffee with reported antinociceptive effects. Based on the few reports that exist in the literature regarding the mechanisms involved in kahweol-induced peripheral antinociceptive action, this study proposed to investigate the contribution of the endocannabinoid system to the peripheral antinociception induced in rats by kahweol. Hyperalgesia was induced by intraplantar injection of prostaglandin E2(PGE2) and was measured with the paw pressure test. Kahweol and the drugs to test the cannabinoid system were administered locally into the right hind paw. The endocannabinoids were purified by open-bed chromatography on silica and measured by LC-MS. Kahweol (80 µg/paw) induced peripheral antinociception against PGE2-induced hyperalgesia. This effect was reversed by the intraplantar injection of the CB1 cannabinoid receptor antagonist AM251 (20, 40, and 80 μg/paw), but not by the CB2 cannabinoid receptor antagonist AM630 (100 μg/paw). Treatment with the endocannabinoid reuptake inhibitor VDM11 (2.5 μg/paw) intensified the peripheral antinociceptive effect induced by low-dose kahweol (40 μg/paw). The monoacylglycerol lipase (MAGL) inhibitor, JZL184 (4 μg/paw), and the dual MAGL/fatty acid amide hydrolase (FAAH) inhibitor, MAFP (0.5 μg/paw), potentiated the peripheral antinociceptive effect of low-dose kahweol. Furthermore, kahweol increased the levels of the endocannabinoid anandamide, but not of the other endocannabinoid 2-arachidonoylglycerol nor of anandamide-related N-acylethanolamines, in the plantar surface of the rat paw. Our results suggested that kahweol induced peripheral antinociception via anandamide release and activation of CB1 cannabinoid receptors and this compound could be used to develop new drugs for pain relief.

The cannabinoid CB2 receptor agonist LY2828360 synergizes with morphine to suppress neuropathic nociception and attenuates morphine reward and physical dependence

The opioid crisis has underscored the urgent need to identify safe and effective therapeutic strategies to overcome opioid-induced liabilities. We recently reported that LY2828360, a slowly signaling G protein-biased cannabinoid CB2 receptor agonist, suppresses neuropathic nociception and attenuates the development of tolerance to the opioid analgesic morphine in paclitaxel-treated mice. Whether beneficial effects of LY2828360 are dependent upon the presence of a pathological pain state are unknown and its impact on unwanted opioid-induced side-effects have never been investigated. Here, we asked whether LY2828360 would produce synergistic anti-allodynic effects with morphine in a paclitaxel model of chemotherapy-induced neuropathic pain and characterized its impact on opioid-induced reward and other unwanted side-effects associated with chronic opioid administration. Isobolographic analysis revealed that combinations of LY2828360 and morphine produced synergistic anti-allodynic effects in suppressing paclitaxel-induced mechanical allodynia. In wildtype (WT) mice, LY2828360 blocked morphine-induced reward in a conditioned place preference assay without producing reward or aversion when administered alone. The LY2828360-induced attenuation of morphine-induced reward was absent in CB2 knockout (CB2KO) mice. In the absence of a neuropathic pain state, LY2828360 partially attenuated naloxone-precipitated opioid withdrawal in morphine-dependent WT mice, and this withdrawal was itself markedly exacerbated in CB2KO mice. Moreover, LY2828360 did not reliably alter morphine-induced slowing of colonic transit or attenuate tolerance to morphine antinociceptive efficacy in the hot plate test of acute nociception. Our results suggest that cannabinoid CB2 receptor activation enhances the therapeutic properties of opioids while attenuating unwanted side-effects such as reward and dependence that occur with sustained opioid treatment.

Genetic Manipulation of sn-1-Diacylglycerol Lipase and CB1 Cannabinoid Receptor Gain-of-Function Uncover Neuronal 2-Linoleoyl Glycerol Signaling in Drosophila melanogaster.

Introduction: In mammals, sn-1-diacylglycerol lipases (DAGL) generate 2-arachidonoylglycerol (2-AG) that, as the major endocannabinoid, modulates synaptic neurotransmission by acting on CB1 cannabinoid receptors (CB1R). Even though the insect genome codes for inaE, which is a DAGL ortholog (dDAGL), its products and their functions remain unknown particularly because insects lack chordate-type cannabinoid receptors. Materials and Methods: Gain-of-function and loss-of-function genetic manipulations were carried out in Drosophila melanogaster, including the generation of both dDAGL-deficient and mammalian CB1R-overexpressing flies. Neuroanatomy, dietary manipulations coupled with targeted mass spectrometry determination of arachidonic acid and 2-linoleoyl glycerol (2-LG) production, behavioral assays, and signal transduction profiling for Akt and Erk kinases were employed. Findings from Drosophilae were validated by a CB1R-binding assay for 2-LG in mammalian cortical homogenates with functionality confirmed in neurons using high-throughput real-time imaging in vitro. Results: In this study, we show that dDAGL is primarily expressed in the brain and nerve cord of Drosophila during larval development and in adult with 2-LG being its chief product as defined by dietary precursor availability. Overexpression of the human CB1R in the ventral nerve cord compromised the mobility of adult Drosophilae. The causality of 2-LG signaling to CB1R-induced behavioral impairments was shown by inaE inactivation normalizing defunct motor coordination. The 2-LG-induced activation of transgenic CB1Rs affected both Akt and Erk kinase cascades by paradoxical signaling. Data from Drosophila models were substantiated by showing 2-LG-mediated displacement of [3H]CP 55,940 in mouse cortical homogenates and reduced neurite extension and growth cone collapsing responses in cultured mouse neurons. Conclusions: Overall, these results suggest that 2-LG is an endocannabinoid-like signal lipid produced by dDAGL in Drosophila.

Cannabinoid CB2 receptors mediate the anxiolytic-like effects of monoacylglycerol lipase inhibition in a rat model of predator-induced fear

The endocannabinoid system is a key regulator of the response to psychological stress. Inhibitors of monoacylglycerol lipase (MGL), the enzyme that deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG), exert anxiolytic-like effects in rodent models via 2-AG-dependent activation of CB1 cannabinoid receptors. In the present study, we examined whether the MGL inhibitor JZL184 might modulate persistent predator-induced fear in rats, a model that captures features of human post-traumatic stress disorder. Exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a volatile chemical that is innately aversive to some rodent species, produced in male rats a long-lasting anxiety-like state that was measured 7 days later in the elevated plus maze test. Systemic administration of JZL184 [4, 8 and 16 mg/kg, intraperitoneal (IP)] 4 h before testing caused dose-dependent inhibition of MGL activity and elevation of 2-AG content in brain tissue. Concomitantly, the inhibitor suppressed TMT-induced fear behaviors with a median effective dose (ED50) of 4 mg/kg. A similar behavioral response was observed with another MGL inhibitor, KML29 (4 and 16 mg/kg, IP). Surprisingly, the effect of JZL184 was prevented by co-administration of the CB2 inverse agonist AM630 (5 mg/kg, IP), but not the CB1 inverse agonist rimonabant (1 mg/kg, IP). Supporting mediation of the response by CB2 receptors, the CB2 agonist JWH133 (0.3, 1 and 3 mg/kg, IP) also produced anxiolytic-like effects in TMT-stressed rats, which were suppressed by AM630. Notably, (i) JWH133 was behaviorally ineffective in animals that had no prior experience with TMT; and (ii) CB2 mRNA levels in rat prefrontal cortex were elevated 7 days after exposure to the aversive odorant. The results suggest that JZL184 attenuates the behavioral consequences of predator stress through a mechanism that requires 2-AG-mediated activation of CB2 receptors, whose transcription may be induced by the stress itself.

Impairment of Synaptic Plasticity by Cannabis, Δ9-THC, and Synthetic Cannabinoids.

The ability of neurons to dynamically and flexibly encode synaptic inputs via short- and long-term plasticity is critical to an organism's ability to learn and adapt to the environment. Whereas synaptic plasticity may be encoded by pre- or postsynaptic mechanisms, current evidence suggests that optimization of learning requires both forms of plasticity. Endogenous cannabinoids (eCBs) play critical roles in modulating synaptic transmission via activation of cannabinoid CB1 receptors (CB1Rs) in many central nervous system (CNS) regions, and the eCB system has been implicated, either directly or indirectly, in several forms of synaptic plasticity. Because of this, perturbations within the eCB signaling system can lead to impairments in a variety of learned behaviors. One agent of altered eCB signaling is exposure to "exogenous cannabinoids" such as the primary psychoactive constituent of cannabis, Δ9-THC, or illicit synthetic cannabinoids that in many cases have higher potency and efficacy than Δ9-THC. Thus, by targeting the eCB system, these agonists can produce widespread impairment of synaptic plasticity by disrupting ongoing eCB function. Here, we review studies in which Δ9-THC and synthetic cannabinoids impair synaptic plasticity in a variety of neuronal circuits and examine evidence that this contributes to their well-documented ability to disrupt cognition and behavior.

Different receptor mechanisms underlying phytocannabinoid- versus synthetic cannabinoid-induced tetrad effects: Opposite roles of CB1 /CB2 versus GPR55 receptors.

Cannabis or cannabinoids produce characteristic tetrad effects-analgesia, hypothermia, catalepsy and suppressed locomotion, which are believed to be mediated by the activation of cannabinoid CB1 receptors. Given recent findings of CB2 and GPR55 receptors in the brain, we examined whether these receptors are also involved in cannabinoid action. We compared Δ9 -tetrahydrocannabinol (Δ9 -THC)-, WIN55212-2-, or XLR11-induced tetrad effects between wild-type (WT) and each genotype of CB1 -, CB2 - or GPR55-knockout (KO) mice and then observed the effects of antagonists of these receptors on these tetrad effects in WT mice. Systemic administration of Δ9 -THC, WIN55212-2 or XLR11 produced dose-dependent tetrad effects in WT mice. Genetic deletion or pharmacological blockade of CB1 receptors abolished the tetrad effects produced by all three cannabinoids. Unexpectedly, genetic deletion of CB2 receptor abolished analgesia and catalepsy produced by Δ9 -THC or WIN55212-2, but not by XLR11. Microinjections of Δ9 -THC into the lateral ventricles also produced tetrad effects in WT, but not in CB1 -KO mice. CB2 -KO mice displayed a reduction in intraventricular Δ9 -THC-induced analgesia and catalepsy. In contrast to CB1 and CB2 receptors, genetic deletion of GPR55 receptors caused enhanced responses to Δ9 -THC or WIN55212-2. Antagonisim of CB1 , CB2 or GPR55 receptors produced alterations similar to those observed in each genotype mouse line. These findings suggest that in addition to CB1 , both CB2 and GPR55 receptors are also involved in some pharmacological effects produced by cannabinoids. CB1 /CB2 , in contrast to GPR55, receptors appears to play opposite roles in cannabinoid action.

α2-Adrenoceptor agonist induces peripheral antinociception via the endocannabinoid system.

Xylazine is an α2 adrenoceptor agonist that is extensively used in veterinary medicine and animal experimentation procedures to produce analgesia, sedation and muscle relaxation without causing general anesthesia. Considering the lack of knowledge of the mechanisms involved in peripheral antinociception induced by xylazine and the potential interactions between the adrenergic and endocannabinoid systems, the present study investigated the contribution of the latter system in the mechanism of xylazine. The rat paw pressure test, in which hyperalgesia was induced by the intraplantar injection of prostaglandin E2, was performed. Xylazine administered via an intraplantar injection (25, 50 and 100μg) induced a peripheral antinociceptive effect against prostaglandin E2 (2μg)-induced hyperalgesia. This effect was blocked by treatment with the selective CB1 cannabinoid antagonist AM251 (20, 40 and 80μg) but not by the selective CB2 cannabinoid antagonist AM630 (100μg). The anandamide reuptake inhibitor VDM11 (2.5μg) intensified the peripheral antinociceptive effect of a submaximal dose of xylazine (25μg), and the inhibitor of endocannabinoid enzymatic hydrolysis, MAFP (0.5μg), showed a tendency towards this same effect. In addition, liquid-chromatography mass spectrometric analysis indicated that xylazine (100μg) treatment was associated with an increase in anandamide levels in the rat paws treated with PGE2. The present results provides evidence that the peripheral antinociceptive effect of the α2 adrenoceptor agonist xylazine probably results from anandamide release and subsequent CB1 cannabinoid receptor activation.

Δ9-THC and related cannabinoids suppress substance P- induced neurokinin NK1-receptor-mediated vomiting via activation of cannabinoid CB1 receptor

Δ9-THC suppresses cisplatin-induced vomiting through activation of cannabinoid CB1 receptors. Cisplatin-evoked emesis is predominantly due to release of serotonin and substance P (SP) in the gut and the brainstem which subsequently stimulate their corresponding 5-HT3-and neurokinin NK1-receptors to induce vomiting. Δ9-THC can inhibit vomiting caused either by the serotonin precursor 5-HTP, or the 5-HT3 receptor selective agonist, 2-methyserotonin. In the current study, we explored whether Δ9-THC and related CB1/CB2 receptor agonists (WIN55,212–2 and CP55,940) inhibit vomiting evoked by SP (50 mg/kg, i.p.) or the NK1 receptor selective agonist GR73632 (5 mg/kg, i.p.). Behavioral methods were employed to determine the antiemetic efficacy of cannabinoids in least shrews. Our results showed that administration of varying doses of Δ9-THC (i.p. or s.c.), WIN55,212–2 (i.p.), or CP55,940 (i.p.) caused significant suppression of SP-evoked vomiting in a dose-dependent manner. When tested against GR73632, Δ9-THC also dose-dependently reduced the evoked emesis. The antiemetic effect of Δ9-THC against SP-induced vomiting was prevented by low non-emetic doses of the CB1 receptor inverse-agonist/antagonist SR141716A (<10 mg/kg). We also found that the NK1 receptor antagonist netupitant can significantly suppress vomiting caused by a large emetic dose of SR141716A (20 mg/kg). In sum, Δ9-THC and related cannabinoids suppress vomiting evoked by the nonselective (SP) and selective (GR73632) neurokinin NK1 receptor agonists via stimulation of cannabinoid CB1 receptors.

Novel object recognition memory in REM sleep-deprived rats: Role of the cannabinoid CB1 receptor

A survey of the literature indicates that both rapid eye movement sleep deprivation (RSD) and activation of cannabinoid CB1 receptor (CB1R) may impair novel object recognition (NOR) memory in rodents. To our knowledge, so far, no previous study has investigated the probable effects of RSD on the different phases of NOR memory. Moreover, far too little attention has been paid to the potential role of the CB1R in the effects of RSD on object memory. Therefore, the major objective of this study was to investigate the probable role of the CB1R in the acquisition, consolidation, retrieval, and reconsolidation of NOR memory in the RSD rats. A 12-h paradigm of RSD using the multiple platform method did not affect acquisition, but it impaired the consolidation, retrieval, and reconsolidation of NOR memory. Administration of the CB1R antagonist rimonabant (1 or 3 mg/kg, i.p.) did not have significant effects on the acquisition and reconsolidation, but it improved RSD-induced impairment of the consolidation and retrieval of object memory, especially at the dose of 3 mg/kg. In addition, the RSD paradigm did not affect the levels of plasma corticosterone as an important marker of stress in rat. The results revealed that RSD may have different effects on the different phases of NOR memory which may not be attributable to the effects of stress. Our findings would seem to suggest that the CB1R can be targeted to, at least partially, modulate the adverse effects of RSD on the process of NOR memory.

Δ9-Tetrahydrocannabinol elicited 22-kHz ultrasonic vocalization changes after air puff stimulus through CB1 receptor in adult rats

Δ9-Tetrahydrocannabinol (THC) is known to have various pharmacological effects mediated through activation of cannabinoid CB1 and CB2 receptors in rodents. In adult rats, 22- and 50-kHz ultrasonic vocalizations (USVs) serve as an effective communication system and as indicators of negative and positive states, respectively. The present study was performed to determine whether THC affects USVs in adult rats, and to determine the roles of cannabinoid receptors in these effects. THC (1, 3 mg/kg) was administered intraperitoneally to adult male Wistar rats 60 min before measurement of USVs. The CB1 antagonist, SR141716 (3, 6 mg/kg), or CB2 antagonist, AM630 (1, 10 mg/kg), was administered intraperitoneally 10 min before THC. USVs were measured during a 5-minute period without air puff stimulus or with air puff stimulus. THC did not affect 22- or 50-kHz USVs without air puff stimulus. On the other hand, THC significantly increased the number of 22-kHz USVs, but not 50-kHz USVs, after air puff stimulus. Moreover, SR141716 at 6 mg/kg, but not AM630 at either dose, inhibited the increase in number of 22-kHz USVs induced by THC after air puff stimulus. These results suggest that THC induced changes in sensitivity to aversive air puff stimuli through CB1 receptors, and as a result increased emission of 22-kHz USVs in rats.

Brain permeant and impermeant inhibitors of fatty-acid amide hydrolase suppress the development and maintenance of paclitaxel-induced neuropathic pain without producing tolerance or physical dependence in vivo and synergize with paclitaxel to reduce tumor cell line viability in vitro

Activation of cannabinoid CB1 receptors suppresses pathological pain but also produces unwanted side effects, including tolerance and physical dependence. Inhibition of fatty-acid amide hydrolase (FAAH), the major enzyme catalyzing the degradation of anandamide (AEA), an endocannabinoid, and other fatty-acid amides, suppresses pain without unwanted side effects typical of direct CB1 agonists. However, FAAH inhibitors have failed to show efficacy in several clinical trials suggesting that the right partnership of FAAH inhibition and pathology has yet to be identified. We compared efficacy of chronic treatments with a centrally penetrant FAAH inhibitor (URB597), a peripherally restricted FAAH inhibitor (URB937) and an orthosteric pan-cannabinoid agonist (WIN55,212-2) in suppressing neuropathic pain induced by the chemotherapeutic agent paclitaxel. Each FAAH inhibitor suppressed the development of paclitaxel-induced neuropathic pain and reduced the maintenance of already established allodynia with sustained efficacy. Tolerance developed to the anti-allodynic efficacy of WIN55,212-2, but not to that of URB597 or URB937, in each dosing paradigm. Challenge with the CB1 antagonist rimonabant precipitated CB1-dependent withdrawal in paclitaxel-treated mice receiving WIN55,212-2 but not URB597 or URB937. When dosing with either URB597 or URB937 was restricted to the development of neuropathy, paclitaxel-induced allodynia emerged following termination of drug delivery. These observations suggest that both FAAH inhibitors were anti-allodynic rather than curative. Moreover, neither URB597 nor URB937 impeded the ability of paclitaxel to reduce breast (4T1) or ovarian (HeyA8) tumor cell line viability. In fact, URB597 and URB937 alone reduced 4T1 tumor cell line viability, albeit with low potency, and the dose matrix of each combination with paclitaxel was synergistic in reducing 4T1 and HeyA8 tumor cell line viability according to Bliss, Highest Single Agent (HSA) and Loewe additivity models. Both FAAH inhibitors synergized with paclitaxel to reduce 4T1 and HeyA8 tumor cell line viability without reducing viability of non-tumor HEK293 cells. Neither FAAH inhibitor reduced viability of non-tumor HEK293 cells in either the presence or absence of paclitaxel, suggesting that nonspecific cytotoxic effects were not produced by the same treatments. Our results suggest that FAAH inhibitors reduce paclitaxel-induced allodynia without the occurrence of CB1-dependence in vivo and may, in fact, enhance the anti-tumor actions of paclitaxel in vitro.

Ketamine induces central antinociception mediated by endogenous cannabinoids and activation of CB1 receptors

The participation of endocannabinoids in central and peripheral antinociception induced by several compounds has been shown by our group. In this study, we investigated the effect of endocannabinoids on the central antinociception induced by ketamine. The nociceptive threshold for thermal stimulation was measured using the tail-flick test in Swiss mice. The drugs were administered intracerebroventricularly. Probabilities less than 5% (p < 0.05) were considered to be statistically significant (Two-way ANOVA/Bonferroni’s test). The CB1-selective cannabinoid receptor antagonist AM251 (2 and 4 μg) completely reversed the central antinociception induced by ketamine (4 μg) in a dose-dependent manner. In contrast, the CB2-selective cannabinoid receptor antagonist AM630 (2 and 4 μg) did not antagonize this effect. Additionally, the administration of the anandamide amidase inhibitor MAFP (0.2 μg) and anandamide uptake inhibitor VDM11 (4 μg) significantly enhanced the antinociception induced by a low dose of ketamine (2 μg). It was concluded that central antinociception induced by ketamine involves the activation of CB1 cannabinoid receptors. Mobilization of cannabinoids might be required for the activation of those receptors, since inhibitors of the endogenous cannabinoids potentiate the effect of Ketamine.

Cannabinoids-induced peripheral analgesia depends on activation of BK channels

The endogenous cannabinoid system is involved in the physiological inhibitory control of pain and is of particular interest for the development of therapeutic approaches for pain management. Selective activation of the peripheral CB1 cannabinoid receptor has been shown to suppress the heightened firing of primary afferents, which is the peripheral mechanism underlying neuropathic pain after nerve injury. However, the mechanism underlying this effect of CB1 receptor remains unclear. The large-conductance calcium-activated potassium (BK) channels have been reported to participate in anticonvulsant and vasorelaxant effects of cannabinoids. We asked whether BK channels participate in cannabinoids-induced analgesia and firing-suppressing effects in primary afferents after nerve injury. Here, using mice with chronic constriction injury (CCI)-induced neuropathic pain, antinociception action and firing-suppressing effect of HU210 were measured before and after BK channel blocker application. We found that local peripheral application of HU210 alleviated CCI-induced pain behavior and suppressed the heightened firing of injured fibers. Co-administration of IBTX with HU210 significantly reversed the analgesia and the firing-suppressing effect of HU210. This result indicated that the peripheral analgesic effects of cannabinoids depends on activation of BK channels.

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

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

Endocannabinoid CB1 receptors are involved in antiepileptogenic effect of low frequency electrical stimulation during perforant path kindling in rats

IntroductionAdministration of low-frequency electrical stimulation (LFS) at the kindling site has an antiepileptogenic effect. In the present study, we investigated the role of cannabinoid receptors type 1 (CB1) in mediating the inhibitory effects of LFS on the development of perforant path kindled seizures. MethodsFor seizure generation, rats were kindled by electrical stimulation of perforant path in semi-rapid kindling manner (12 stimulations per day at 10 min intervals at afterdischarge threshold intensity).To determine the effect of LFS (0.1 ms pulse duration at 1 Hz, 800 pulses) on seizure generation, LFS was applied to the perforant path 5 min after the last kindling stimulation daily. AM281, a CB1 receptor antagonist, was microinjected into the lateral ventricle immediately after the last kindling stimulation (before LFS application) at the doses of 0.5 and 2 μg/μl during kindling procedure. The expression of cannabinoid receptors in the dentate gyrus was also investigated using immunohistochemistry. ResultsApplication of LFS had inhibitory effect on development of kindled seizures (kindling rate). Microinjection of AM281 (0.5 μg/μl) immediately after the last kindling stimulation (before LFS application) reduced the inhibitory effect of LFS on the kindling rate and suppressed the effects of LFS on potentiation (increasing the magnitude) of both population spike amplitude and population excitatory postsynaptic potential slope during kindling acquisition. AM281 pretreatment also prevented the effects of LFS on kindling-induced increase in early and late paired pulse depression. The higher dose of AM281 (2 μg/μl) failed to exert the effects observed with its lower dose (0.5 μg/μl). In addition, there was a decreased CB1 receptors immunostaining in kindled animals compared to control. However, application of LFS following kindling stimulations led to overexpression of CB1 receptors in the dentate gyrus. ConclusionObtained results showed that activation of overexpressed cannabinoid CB1 receptors by endogenous cannabinoids may have a role in mediating the inhibitory effect of LFS on perforant path kindled seizures.

Controlled-Deactivation CB1 Receptor Ligands as a Novel Strategy to Lower Intraocular Pressure.

Nearly half a century has passed since the demonstration that cannabis and its chief psychoactive component Δ9-THC lowers intraocular pressure (IOP). Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a condition that places millions at risk of blindness. It is likely that Δ9-THC exerts much of its IOP-lowering effects via the activation of CB1 cannabinoid receptors. However, the initial promise of CB1 as a target for treating glaucoma has not thus far translated into a credible therapeutic strategy. We have recently shown that blocking monoacylglycerol lipase (MAGL), an enzyme that breaks the endocannabinoid 2-arachidonoyl glycerol (2-AG), substantially lowers IOP. Another strategy is to develop cannabinoid CB1 receptor agonists that are optimized for topical application to the eye. Recently we have reported on a controlled-deactivation approach where the “soft” drug concept of enzymatic deactivation was combined with a “depot effect” that is commonly observed with Δ9-THC and other lipophilic cannabinoids. This approach allowed us to develop novel cannabinoids with a predictable duration of action and is particularly attractive for the design of CB1 activators for ophthalmic use with limited or no psychoactive effects. We have tested a novel class of compounds using a combination of electrophysiology in autaptic hippocampal neurons, a well-characterized model of endogenous cannabinoid signaling, and measurements of IOP in a mouse model. We now report that AM7410 is a reasonably potent and efficacious agonist at CB1 in neurons and that it substantially (30%) lowers IOP for as long as 5 h after a single topical treatment. This effect is absent in CB1 knockout mice. Our results indicate that the direct targeting of CB1 receptors with controlled-deactivation ligands is a viable approach to lower IOP in a murine model and merits further study in other model systems.

Examination of the effects of cannabinoid ligands on decision making in a rat gambling task

Although exposure to delta-9-tetrahydrocannabinol (THC) is perceived to be relatively harmless, mounting evidence has begun to show that it is associated with a variety of cognitive deficits, including poor decision making. THC-induced impairments in decision making are thought to be the result of cannabinoid CB1 receptor activation, and although clinical literature suggests that chronic activation via THC contributes to perturbations in decision making, acute CB1 receptor modulation has yielded mixed results. Using an animal model to examine how CB1-specific ligands impact choice biases would provide significant insight as to how recruitment of the endocannabinoid system may influence decision making. Here, we used the rat gambling task (rGT), a validated analogue of the human Iowa Gambling Task, to assess baseline decision making preferences in male Wistar rats. After acquisition rGT performance was measured. Animals were challenged with the CB1 receptor antagonist rimonabant, the partial agonist THC, and the synthetic agonist WIN55,212-2. Animals were also treated acutely with the fatty acid amide hydrolase (FAAH) inhibitor URB597 to selectively upregulate the endocannabinoid anandamide. Blockade of the CB1 receptor produced a trend improvement in decision making in animals who preferred the advantageous task options, yet left choice unaffected in risk-prone rats. Neither CB1 receptor agonist had strong effects on decision making, but a high dose THC decreased premature responses, whereas WIN55,212-2 did the opposite. URB597 did not affect task performance. These results indicate that although chronic CB1 receptor activation may be associated with impaired decision making, acute modulation has modest effects on choice and instead may play a substantive role in regulating impulsive responding.

Corticosterone-Induced Changes in Inhibitory Neurotransmission in Hippocampal Field CA1 Synapses Depending on the Activity of Inhibitory Synapses Expressing Cannabinoid Receptors

Objectives. To study the effects of corticosterone (100 nM) on spontaneous inhibitory postsynaptic currents(sIPSC) in pyramidal neurons in hippocampal field CA1 and to identify the role of inhibitory synapses expressing cannabinoid receptors in the changes induced by corticosterone. Materials and methods. Experiments were performed on living slices of rat ventral hippocampus using a patch clamp method in the whole cell configuration. Results and conclusions. Addition of corticosterone led to a significant increase in the frequency of sIPSC in the first 10 min, after which the magnitude of this parameter returned to baseline. In addition, the use of corticosterone led to a significant decrease in sIPSC amplitude 40 min after the start of exposure. Activation of cannabinoid CB1 receptors had weak effects on the properties of sIPSC, though addition of corticosterone did not induce the rapid rise in sIPSC frequency occurring at 10 min in controls or the drop in sIPSC amplitude (at 40 min in controls). These data indicate that both the rapid and slow effects of corticosterone developing in the ventral hippocampus are associated with the activity of CB1-expressing inhibitory synapses.