Cannabinoids In Mice Research Articles

Tolerance to cannabinoids in mice

The Cannabis sativa plant, for its analgesic, anti-inflammatory, antiemetic, and anticonvulsant properties, has been used for thousands of years in Chinese, Indian, and Greek cultures and was introduced into Western medicine in the 19th century. There has been a rise in interest in cannabinoids since the main substances of cannabis were identified, as well as the recognition that the endocannabinoid system (ECS) controls multiple processes in pain treatment and neurologic and mental illnesses. However, the ECS has also been associated with negative effects, including harmful effects on emotional and cognitive functions, the development of tolerance and dependence, and withdrawal symptoms after drug cessation in humans. We recently found that repeated intraperitoneal administration of the two main components of cannabis, delta-9-tetrahydrocannabinol (THC) and cannabinolic acid (CBNA), led to the development of tolerance in male mice. In this review, we focus on the evidence demonstrating cannabinoid tolerance in animals. The common mechanisms and main signaling pathways for cannabinoid tolerance, including neuroadaptations primarily at cannabinoid 1 (CB1) receptors, such as desensitization and downregulation, which are mediated by several signaling pathways, are discussed.

Optogenetic brain-stimulation reward: A new procedure to re-evaluate the rewarding versus aversive effects of cannabinoids in dopamine transporter-Cre mice.

Despite extensive research, the rewarding effects of cannabinoids are still debated. Here, we used a newly established animal procedure called optogenetic intracranial self-stimulation (ICSS) (oICSS) to re-examine the abuse potential of cannabinoids in mice. A specific adeno-associated viral vector carrying a channelrhodopsin gene was microinjected into the ventral tegmental area (VTA) to express light-sensitive channelrhodopsin in dopamine (DA) neurons of transgenic dopamine transporter (DAT)-Cre mice. Optogenetic stimulation of VTA DA neurons was highly reinforcing and produced a classical "sigmoidal"-shaped stimulation-response curve dependent upon the laser pulse frequency. Systemic administration of cocaine dose-dependently enhanced oICSS and shifted stimulation-response curves upward, in a way similar to previously observed effects of cocaine on electrical ICSS. In contrast, Δ9 -tetrahydrocannabinol (Δ9 -THC), but not cannabidiol, dose-dependently decreased oICSS responding and shifted oICSS curves downward. WIN55,212-2 and ACEA, two synthetic cannabinoids often used in laboratory settings, also produced dose-dependent reductions in oICSS. We then examined several new synthetic cannabinoids, which are used recreationally. XLR-11 produced a cocaine-like increase, AM-2201 produced a Δ9 -THC-like reduction, while 5F-AMB had no effect on oICSS responding. Immunohistochemistry and RNAscope in situ hybridization assays indicated that CB1 Rs are expressed mainly in VTA GABA and glutamate neurons, while CB2 Rs are expressed mainly in VTA DA neurons. Together, these findings suggest that most cannabinoids are not reward enhancing, but rather reward attenuating or aversive in mice. Activation of CB1 R and/or CB2 R in different populations of neurons in the brain may underlie the observed actions.

Do you feel it now? Route of administration and Δ9-tetrahydrocannabinol-like discriminative stimulus effects of synthetic cannabinoids in mice

A recent push to provide more translationally relevant preclinical models for examination of pharmacological mechanisms underlying inhaled substances of abuse has resulted in the development of equipment and methods that allows exposure of freely moving rodents to aerosolized psychoactive drugs. In the present study, synthetic cannabinoids (CP55,940, AB-CHMINACA, and AMB-FUBINACA) were administered intraperitoneally (i.p.) or aerosolized via a modified electronic cigarette device. Subsequently, the compounds were evaluated in adult male and female C57/Bl6 mice trained to discriminate i.p. 5.6 mg/kg Δ9-tetrahydrocannabinol (THC) for food reinforcement. When administered i.p., THC and AB-CHMINACA were equally potent at producing THC-like effects in both sexes, but CP55,940 and AMB-FUBINACA were more potent in males. Upon aerosol exposure, all compounds continued to produce THC-like effects in both sexes, with AMB-FUBINACA remaining the most potent. In contrast, aerosolized CP55,940 showed substantial decreases in potency in both sexes. Aerosolized nicotine did not substitute for THC in either sex. In females, aerosolized cumyl-4CN-BINACA produced concentration-dependent increases in responding on the THC-associated nosepoke. In addition, the effects of an active concentration of AMB-FUBINACA were reversed by rimonabant, suggesting CB1 receptor mediation. These results show that synthetic cannabinoids produce THC-like effects when injected i.p. or after aerosolization. This study adds to a growing literature suggesting that evaluation of abuse liability of substances via aerosol exposure is feasible and may provide a translationally relevant method that allows for investigation of factors important to the abuse of drugs which humans typically smoke or vape.

Δ(9)-Tetrahydrocannabinol-like effects of novel synthetic cannabinoids in mice and rats.

Novel cannabinoid compounds continue to be marketed as "legal" marijuana substitutes, even though little is known about their molecular and behavioral effects. Six of these compounds (ADBICA, ADB-PINACA, THJ-2201, RCS-4, JWH-122, JWH-210) were tested for in vitro and in vivo cannabinoid-like effects to determine their abuse liability. Binding to and functional activity at CB1 cannabinoid receptors was tested. Locomotor activity in mice was tested to screen for behavioral activity and to identify behaviorally active dose ranges and times of peak effect. Discriminative stimulus effects of the six compounds were tested in rats trained to discriminate Δ(9)-tetrahydrocannabinol (Δ(9)-THC). ADBICA, ADB-PINACA, THJ-2201, RCS-4, JWH-122, and JWH-210 showed high affinity binding at the CB1 receptor at nanomolar affinities (0.59 to 22.5nM), and all acted as full agonists with nanomolar potencies (0.024 to 111nM) when compared to the CB1 receptor full agonist CP 55940. All compounds depressed locomotor activity below 50% of vehicle responding, with depressant effects lasting 1.5 to nearly 4h. All compounds fully substituted (<80% Δ(9)-THC-appropriate responding) for the discriminative stimulus effects of Δ(9)-THC. 3,4-Methylenedioxy-methamphetamine (MDMA) was tested as a negative control and did not substitute for Δ(9)-THC (11% Δ(9)-THC-appropriate responding). All six of the compounds acted at the CB1 receptor and produced behavioral effects common to abused cannabinoid compounds, which suggest that these compounds have substantial abuse liability common to controlled synthetic cannabinoid compounds.

Novel halogenated derivates of JWH-018: Behavioral and binding studies in mice

JWH-018 is a synthetic CB1 and CB2 agonist illegally marketed as products named “Spice” or “herbal blend” for its psychoactive effects which are much higher than those produced by cannabis. In the last year, the European Monitoring Centre for Drugs and Drug Addiction reported to the Italian National Early Warning System the seizure of plant material containing new halogenated derivatives of JWH-018 (JWH-018 Cl and JWH-018 Br). The present study aimed to investigate the in vitro and in vivo activity of these two new synthetic cannabinoids in mice. In vitro competition binding experiments performed on mouse and human CB1 receptors revealed a high affinity and potency of the halogenated compounds. Synthetic cannabinoids (0.01–6 mg/kg i.p.) impaired motor activity and induced catalepsy in mice and their effects were more severe with respect to those evoked by Δ9-THC. Moreover, they increased the mechanical and thermal pain threshold and induced a marked hypothermia. It is interesting to note that whereas high doses of JWH-018 cause seizures, myoclonia and hyperreflexia, the halogenated compounds, in particular JWH-018Br, were less effective. Behavioral and neurological changes were prevented by the selective CB1 receptor antagonist AM 251. These data demonstrate for the first time that JWH-018 Cl and JWH-018 Br act similarly to JWH-018 while inducing less convulsive episodes and myoclonias. These data support the hypothesis that the halogenated compounds may have been introduced onto market to produce similar intoxicating effects as JWH-018 while causing less side effects.

The effects of beta-arrestin1 deletion on acute cannabinoid activity, brain cannabinoid receptors and tolerance to cannabinoids in mice

Context: Previous studies have indicated a role for beta-arrestin2 in the regulation of brain cannabinoid effects and cannabinoid CB1 receptors, but whether beta-arrestin1 has a role has not been investigated. Objective: To determine the role of beta-arrestin1 in cannabinoid activity. Materials and methods: Beta-arrestin1 −/− mice and their wild-type (+/+) counterparts were assayed for antinociceptive and temperature-decreasing effects of two ligands, Δ9-tetrahydrocannabinol (THC) and CP55940, after both single and repeated administration. In vitro assays examined the effects of deletion on CB1 receptor density, agonist-binding and G-protein activation. Results: Deletion of beta-arrestin1 diminished the effects of CP55940 in both antinociception (latency to tail withdrawal) and temperature-depression assays in mice. However, deleting beta-arrestin1 had no effect on the actions of THC in either assay. Antagonist radioligand ([3H]SR141716A) saturation binding indicated no difference between beta-arrestin1 +/+ and −/− mice in the density or affinity for cannabinoid CB1 receptors in brain membranes. CP55940 agonist binding in brain membranes from beta-arrestin1 +/+ mice exhibited high- and intermediate-affinity sites, but beta-arrestin1 −/− membranes exhibited an additional site with low affinity. CP55940 produced greater stimulation of [35S]GTPγS binding to membranes from whole brain of beta-arrestin1 −/− than +/+ mice. The rates of the development of tolerance to chronic THC or CP55940 administration did not appear to be affected by genotype. Discussion: Beta-arrestin1 appeared to mediate the actions of CP55940, but did not affect the activity of THC. Conclusion: Beta-arrestin1 regulates cannabinoid CB1 receptor sensitivity in an agonist-selective manner, but may not be the primary mediator of tolerance to cannabinoid agonists.

Mutation of Putative GRK Phosphorylation Sites in the Cannabinoid Receptor 1 (CB1R) Confers Resistance to Cannabinoid Tolerance and Hypersensitivity to Cannabinoids in Mice

For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ(9)-THC), have delayed tolerance to Δ(9)-THC, and showed increased dependence for Δ(9)-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ(9)-THC was absent in S426A/S430A mutants. Δ(9)-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

Tolerance and cross-tolerance to cannabinoids in mice: schedule-controlled responding and hypothermia

Cannabinoid CB(1) receptor agonists vary in efficacy in vitro; however, relationships between efficacy and behavioral effects are unclear. This study examined the relationship between apparent CB(1) agonist efficacy and in vivo effects. Male C57BL/6J mice responded for food under a fixed ratio 30 schedule; rectal temperature was measured. Sensitivity of the mice to cannabinoid agonists (rank order efficacy in vitro reported to be CP 55940 > anandamide > Δ(9)-tetrahydrocannabinol; Δ(9)-THC) and a non-cannabinoid (the benzodiazepine midazolam) was determined before, during, and after discontinuation of daily Δ(9)-THC treatment (32mg/kg/day, i.p.). Rimonabant was combined with cannabinoids to examine whether CB(1) receptors mediated effects on response rate. Δ(9)-THC, CP 55940, anandamide, and midazolam decreased responding at doses smaller than those producing hypothermia. Rimonabant antagonized the rate-decreasing effects of Δ(9)-THC and CP 55940, but not those of anandamide. Δ(9)-THC treatment produced tolerance for both rate-decreasing and hypothermic effects. Δ(9)-THC treatment did not change sensitivity to the rate-decreasing effects of CP 55940, but produced cross-tolerance to CP 55940 for hypothermic effects. Δ(9)-THC treatment did not modify sensitivity to anandamide and midazolam. CB(1) receptors mediate the operant rate-decreasing effects of Δ(9)-THC and CP 55940, but not anandamide, in mice. CB(1) agonist efficacy is an important determinant of in vivo effects, especially with regard to the magnitude of tolerance and cross-tolerance resulting from daily Δ(9)-THC treatment. This applies not only to different cannabinoids when measuring the same effect but also to the same cannabinoid when measuring different effects.

The schizophrenia susceptibility gene neuregulin 1 modulates tolerance to the effects of cannabinoids

Cannabis increases the risk of schizophrenia in genetically vulnerable individuals. In this study we aim to show that the schizophrenia susceptibility gene neuregulin 1 (Nrg1) modulates the development of tolerance to cannabinoids in mice. Nrg1 heterozygous (HET) and wild-type (WT) mice were treated daily for 15 d with the synthetic analogue of Δ9-tetrahydrocannabinol, CP55,940 (0.4 mg/kg). We measured the impact of this exposure on locomotor activity, anxiety, prepulse inhibition (PPI), body temperature and FosB/ΔFosB immunohistochemistry. Tolerance to CP55,940-induced hypothermia and locomotor suppression developed more rapidly in Nrg1 HET mice than WT mice. Conversely in the light-dark test, while tolerance to the anxiogenic effect of CP55,940 developed in WT mice over days of testing, Nrg1 hypomorphs maintained marked anxiety even after 15 d of treatment. Repeated cannabinoid exposure selectively increased FosB/ΔFosB expression in the lateral septum, ventral part (LSV) of Nrg1 HET but not WT mice. On day 1 of exposure opposite effects of CP55,940 treatment were observed on PPI, i.e. it was facilitated in Nrg1 hypomorphs and impaired in WT mice, despite the drug significantly impairing the acoustic startle reflex equally in both genotypes. These effects of CP55,940 on PPI were not maintained as both genotypes became tolerant to cannabinoid action with repeated exposure. Our results highlight that Nrg1 modulates the development of cannabinoid tolerance dependent on the parameter being measured. Furthermore, these data reinforce the notion that the VLS is an important brain region involved in Nrg1-cannabinoid interactions.

N-Arachidonyl Maleimide Potentiates the Pharmacological and Biochemical Effects of the Endocannabinoid 2-Arachidonylglycerol through Inhibition of Monoacylglycerol Lipase

Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Delta(9)-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB(1) receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB(1)(-/-) mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB(1), non-CB(2) cannabinoid receptors.

Lack of cross-tolerance to the antinociceptive effects of systemic and topical cannabinoids in morphine-tolerant mice

Opioids and cannabinoids produce antinociception through activity at spinal, supraspinal and peripheral sites. Tolerance to the antinociceptive effects of both the opioids and the cannabinoids develop when these agents are administered chronically. Although mutual potentiation of antinociceptive effects have been reported between opioids and cannabinoids, the development of antinociceptive cross-tolerance between these systems has not been demonstrated consistently. In the present investigation, we explored the possibility of antinociceptive cross-tolerance between systemic or topical morphine and systemic or topical cannabinoids in mice. Mice were made tolerant to morphine either by the subcutaneous (s.c.) implantation of a morphine pellet or repeated topical administration and then challenged with the mixed CB 1 and CB 2 receptor agonist WIN55, 212-2 given s.c. or topically. Antinociception was indicated by increased tail-flick latencies to noxious radiant heat. Implantation with morphine pellets did not attenuate the antinociceptive potency of systemic or topical WIN 55,212-2. Moreover, twice-daily topical administration of morphine did not attenuate the antinociceptive potency of WIN 55,212-2 applied topically. These observations suggest that opioids and cannabinoids produce antinociception through mechanisms that are independent of each other at either the systemic or peripheral levels.

Pharmacological evaluation of aerosolized cannabinoids in mice

The reemergence on the debate of the use of marijuana for medicinal purposes has been the impetus for developing an acceptable delivery form of aerosolized cannabinoids. The goals of the present study were to: (1) develop and characterize the physical properties of an aerosolized form of Δ 9-tetrahydrocannabinol (Δ 9-THC), the major psychoactive constituent present in marijuana; and (2) assess the pharmacological effects of cannabinoid inhalation in mice. A Small Particle Aerosol Generator (SPAG) nebulizer, used to generate the aerosol, had an output of approximately 0.154 mg/l of aerosolized Δ 9-THC with a 2.0 μm mass median aerodynamic diameter and a 2.2 geometric standard deviation (GSD). Virtually all the particles were less than 5.0 μm in diameter suggesting that they were sufficiently small to penetrate deeply into the lungs. Inhalation exposure to aerosolized Δ 9-THC in mice elicited antinociceptive effects that were dependent on concentration and exposure time with an estimated Δ 9-THC dose of 1.8 mg/kg. On the other hand, inhalation exposure to Δ 9-THC failed to produce two other indices indicative of cannabinoid activity, hypothermia and decreases in spontaneous locomotor activity. The antinociceptive effects occurred within 5 min of exposure and lasted approximately 40 min in duration. The cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide HCl (SR 141716A), but not naloxone, blocked these antinociceptive effects (AD 50=0.09 mg/kg) indicating a cannabinoid receptor mechanism of action. Similarly, inhalation exposure to a water soluble cannabinoid analog, 3-(5′-cyano-1′,1′dimethylheptyl)-1-(4- N-morpholinobutyrloxy)-Δ 8-tetrahydrocannabinol (O-1057), produced antinociception that was blocked by SR 141716A. These results demonstrate that the development of an aerosolized form of cannabinoids for human medicinal use is feasible.

Anticonvulsant effects of cannabinoids in mice: Drug interactions within cannabinoids and cannabinoid interactions with phenytoin

The anticonvulsant activity of orally administered δ9-tetrahydrocannabinol (δ9-THC), δ8-THC, cannabidiol (CBD) and cannabinol (CBN) was tested in mice utilizing electroshock and chemoshock methods. In doses tested δ9-THC afforded no protection to mice from chemoshock seizures and was effective against electroshock only in high doses (160–200 mg/kg). CBD and CBN (150–200 mg/kg) were without effect in both tests.