Dana E. Selley

Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects

2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for anandamide, is mediated by fatty acid amide hydrolase (FAAH) and, for 2-AG, is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, intimating a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL, JZL184, that, upon administration to mice, raises brain 2-AG by 8-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia, and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.

Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system

Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

Essential role for RGS9 in opiate action

Regulators of G protein signaling (RGS) are a family of proteins known to accelerate termination of effector stimulation after G protein receptor activation. RGS9-2, a brain-specific splice variant of the RGS9 gene, is highly enriched in striatum and also expressed at much lower levels in periaqueductal gray and spinal cord, structures known to mediate various actions of morphine and other opiates. Morphine exerts its acute rewarding and analgesic effects by activation of inhibitory guanine nucleotide-binding regulatory protein-coupled opioid receptors, whereas chronic morphine causes addiction, tolerance to its acute analgesic effects, and profound physical dependence by sustained activation of these receptors. We show here that acute morphine administration increases expression of RGS9-2 in NAc and the other CNS regions, whereas chronic exposure decreases RGS9-2 levels. Mice lacking RGS9 show enhanced behavioral responses to acute and chronic morphine, including a dramatic increase in morphine reward, increased morphine analgesia with delayed tolerance, and exacerbated morphine physical dependence and withdrawal. These findings establish RGS9 as a potent negative modulator of opiate action in vivo, and suggest that opiate-induced changes in RGS9 levels contribute to the behavioral and neural plasticity associated with chronic opiate administration.

An essential role for ΔFosB in the nucleus accumbens in morphine action

The transcription factor ΔFosB is induced in the nucleus accumbens (NAc) and dorsal striatum by the repeated administration of drugs of abuse. Here, we investigated the role of ΔFosB in the NAc in behavioral responses to opiates. We achieved overexpression of ΔFosB by using a bitransgenic mouse line that inducibly expresses the protein in the NAc and dorsal striatum and by using viral-mediated gene transfer to specifically express the protein in the NAc. ΔFosB overexpression in the NAc increased the sensitivity of the mice to the rewarding effects of morphine and led to exacerbated physical dependence, but also reduced their sensitivity to the analgesic effects of morphine and led to faster development of analgesic tolerance. The opioid peptide dynorphin seemed to be one target through which ΔFosB produced this behavioral phenotype. Together, these experiments demonstrated that ΔFosB in the NAc, partly through the repression of dynorphin expression, mediates several major features of opiate addiction.

Distinct Patterns of ΔFosB Induction in Brain by Drugs of Abuse

The transcription factor ΔFosB accumulates and persists in brain in response to chronic stimulation. This accumulation after chronic exposure to drugs of abuse has been demonstrated previously by Western blot most dramatically in striatal regions, including dorsal striatum (caudate/putamen) and nucleus accumbens. In the present study, we used immunohistochemistry to define with greater anatomical precision the induction of ΔFosB throughout the rodent brain after chronic drug treatment. We also extended previous research involving cocaine, morphine, and nicotine to two additional drugs of abuse, ethanol and Δ9‐tetrahydrocannabinol (Δ9‐THC, the active ingredient in marijuana). We show here that chronic, but not acute, administration of each of four drugs of abuse, cocaine, morphine, ethanol, and Δ9‐THC, robustly induces ΔFosB in nucleus accumbens, although different patterns in the core vs. shell subregions of this nucleus were apparent for the different drugs. The drugs also differed in their degree of ΔFosB induction in dorsal striatum. In addition, all four drugs induced ΔFosB in prefrontal cortex, with the greatest effects observed with cocaine and ethanol, and all of the drugs induced ΔFosB to a small extent in amygdala. Furthermore, all drugs induced ΔFosB in the hippocampus, and, with the exception of ethanol, most of this induction was seen in the dentate. Lower levels of ΔFosB induction were seen in other brain areas in response to a particular drug treatment. These findings provide further evidence that induction of ΔFosB in nucleus accumbens is a common action of virtually all drugs of abuse and that, beyond nucleus accumbens, each drug induces ΔFosB in a region‐specific manner in brain. Synapse 358–369, 2008.

Repeated low-dose administration of the monoacylglycerol lipase inhibitor JZL184 retains cannabinoid receptor type 1-mediated antinociceptive and gastroprotective effects.

The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug–induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [3H]SR141716A binding and CP55,940 [(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5′-O-(3-[35S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ9-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.

Prolonged recovery rate of CB1 receptor adaptation after cessation of long-term cannabinoid administration.

Long-term cannabinoid administration produces region-dependent CB1 receptor desensitization and down-regulation. This study examined the time course for normalization of CB1 receptors and G-protein activation using 3H-labeled N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A) and guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS binding), respectively, in hippocampus and striatum/globus pallidus (GP). Mice were treated with escalating doses of Δ9-tetrahydrocannabinol (Δ9-THC) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN55,212-2) for 15 days, and tissue was collected 1, 3, 7, or 14 days after final injection. [3H]SR141716A and WIN55,212-2-stimulated [35S]GTPγS binding were decreased in both regions 1 day after treatment. WIN55,212-2-stimulated G-protein activation in striatum/GP returned to control level at 3 days after cessation of treatment with either drug but did not return to control level in hippocampus until 14 days. CB1 receptor binding did not recover to control levels until day 7 or 14 after treatment in striatum/GP and hippocampus, respectively. The mechanism of CB1 binding site down-regulation was investigated after long-term Δ9-THC treatment. Analysis of CB1 receptor mRNA in hippocampus and striatum/GP showed that transcriptional regulation could not explain prolonged recovery rates from CB1 receptor down-regulation. In contrast, CB1 receptor protein, as determined by immunoblot analysis, matched the down-regulation and recovery rates of CB1 receptor binding sites relatively closely. These data demonstrate that cannabinoid-induced decreases in CB1 receptor function persist for relatively long time periods after cessation of long-term drug treatment and that CB1 receptor signaling recovers more quickly in striatum/GP than hippocampus. Moreover, down-regulation of CB1 receptor binding sites does not seem to result mainly from transcriptional regulation, suggesting that adaptive regulation of CB1 receptors in brain primarily occurs at the protein level.

CB1 cannabinoid receptor activity is modulated by the cannabinoid receptor interacting protein CRIP 1a.

The CB1 cannabinoid receptor is a G-protein coupled receptor that has important physiological roles in synaptic plasticity, analgesia, appetite, and neuroprotection. We report the discovery of two structurally related CB1 cannabinoid receptor interacting proteins (CRIP1a and CRIP1b) that bind to the distal C-terminal tail of CB1. CRIP1a and CRIP1b are generated by alternative splicing of a gene located on chromosome 2 in humans, and orthologs of CRIP1a occur throughout the vertebrates, whereas CRIP1b seems to be unique to primates. CRIP1a coimmunoprecipitates with CB1 receptors derived from rat brain homogenates, indicating that CRIP1a and CB1 interact in vivo. Furthermore, in superior cervical ganglion neurons coinjected with CB1 and CRIP1a or CRIP1b cDNA, CRIP1a, but not CRIP1b, suppresses CB1-mediated tonic inhibition of voltage-gated Ca2+ channels. Discovery of CRIP1a provides the basis for a new avenue of research on mechanisms of CB1 regulation in the nervous system and may lead to development of novel drugs to treat disorders where modulation of CB1 activity has therapeutic potential (e.g., chronic pain, obesity, and epilepsy).

Cannabinoid CB1 receptor‐interacting proteins: novel targets for central nervous system drug discovery?

The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G‐protein‐coupled receptors (GPCRs), including CB1 and CB2 receptors. The CB1 receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB1 receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G‐proteins. These proteins, which include the GPCR kinases, β‐arrestins, GPCR‐associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor‐interacting proteins: CRIP1a/b, are thought to play important roles in the regulation of intracellular trafficking, desensitization, down‐regulation, signal transduction and constitutive activity of CB1 receptors. This review examines CB1 receptor‐interacting proteins, including heterotrimeric G‐proteins, but with particular emphasis on non‐G‐protein entities, that might comprise the CB1 receptosomal complex. The evidence for direct interaction with CB1 receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.