β-Arrestin2 Regulates Cannabinoid CB1 Receptor Signaling and Adaptation in a Central Nervous System Region–Dependent Manner

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    00doy Words: Beta-arrestin, cannabinoid receptors, GPCR, statisticalrametric mapping, tolerance

    B1 receptors (CB1Rs) are widely distributed in the centralnervous system (CNS) (1) and mediate the central effects of

    9-tetrahydrocannabinol (THC) and cannabinoids (2). Thedocannabinoid system is implicated in numerous physiologicalcesses and is a potential therapeutic target for disorders includ-neurodegenerative and neuropsychiatric diseases and chronic

    in (3). However, therapeutic use is limited by psychoactive andtor side effects.Moreover, repeated cannabinoid treatment pro-ces tolerance to cannabinoid-mediated in vivo effects (4). Re-n-specific CB1R desensitization and downregulation occur injunction with tolerance (5), but themolecular mechanisms thatderlie CB1R adaptations and tolerance are not well defined.CB1Rs primarily activate Gi/o-proteins, which regulate adenylyllases, ion channels, and kinases (6). Persistent cannabinoid ex-sure induces CB1R uncoupling from G-proteins (desensitization), with subsequent receptor internalization (8) and degradationwnregulation) (9,10). Onemechanism for these adaptations oc-

    phosphorylation of activated receptors and subsequent -arrestinbinding (11). -arrestin2 is one of two arrestin isoforms in the brain(12), and findings in cell models support a role for -arrestin2 inCB1R adaptations. Coexpression of GRK3 and -arrestin2 was re-quired for rapid desensitization of CB1R-mediated potassium cur-rents following exposure to WIN55,212-2 in Xenopus oocytes (8).Similarly, expression of dominant negative -arrestin2 attenuateddesensitization ofWIN55,212-2-mediated inhibition of glutamater-gic neurotransmission in hippocampal neurons (13). Immunohisto-chemical studies show that CB1Rs are codistributed with -arres-tin2 in certain CNS regions (12,14), suggesting that -arrestin2might regulate CB1R signaling in the CNS.

    Because there are no pharmacological -arrestin inhibitors,-arrestin2 knockout (arr2-KO)mice (15) provide amodel to studyits role in regulatingGPCRs in vivo (16). Acute administrationof THCto arr2-KO mice revealed enhanced sensitivity to its antinocicep-tive and hypothermic effects (17). However, direct evidence for therole of -arrestin2 in CB1R adaptations and tolerance followingrepeated THC is lacking.

    We adapted statistical parametric mapping (SPM) to analyze[35S]GTPS autoradiography (18). SPMhas the advantage of assess-ing changes in G-protein activation in an unbiased and anatomi-cally inclusive manner. We applied SPM to examine the role of-arrestin2 in CB1R regulation by comparing cannabinoid-stimu-lated [35S]GTPS binding in brains from vehicle and THC-treatedarr2-KO and wild-type (WT) littermates. Combining this approachwith behavioral assessment following THC administration allowedus to compare CB1R signaling with behavioral responses observedin thearr2-KOmice.Wedemonstrate region-specific regulation of

    m the Department of Pharmacology and Toxicology and Institute forDrug and Alcohol Studies (PTN, DES, LJS-S), Virginia CommonwealthUniversity, Richmond, Virginia; and Departments of Molecular Thera-peutics and Neuroscience (CLS, KMR, LMB), The Scripps Research Insti-tute, Jupiter, Florida.dress correspondence to Laura J. Sim-Selley, Ph.D., Box 980524, 1112 E.Clay Street, Richmond, VA 23298; E-mail: ljsimsel@vcu.edueived Aug 4, 2011; revised Oct 25, 2011; accepted Nov 30, 2011.

    BIOL PSYCHIATRY 2012;71:71472406-3223/$36.00i:10.1016/j.biopsych.2011.11.027 2012 Society of Biological Psychiatry-Arrestin2 Regulates Cannignaling and Adaptation inegionDependent Mannerter T. Nguyen, Cullen L. Schmid, Kirsten M. Raehalura J. Sim-Selley

    ckground: Cannabinoid CB1 receptors (CB1Rs) mediate the effectsrijuana. Repeated THC administration produces tolerance and dduces motor and psychoactive side effects. -arrestin2 mediates rse CB1R effects and receptor regulation is unclear.

    thods: CB1R signaling and behaviors (antinociception, hypothermd-type mice after THC administration. Cannabinoid-stimulated [35Srametric mapping and region-of-interest analysis.

    sults: -arrestin2 deletion increased CB1R-mediated G-protein acicle-treated mice. arr2-KO mice exhibited enhanced acute THCalepsy. After repeated THC administration, arr2-KOmice showeddal periaqueductal gray, and spinal cord and attenuated tolerances found in hypothalamus, cortex, globus pallidus, and substantia nigC-induced catalepsy was observed in arr2-KO mice.

    nclusions: -arrestin2 regulation of CB1R signaling following acugest that multiple, overlapping mechanisms regulate CB1Rs. Theponses toacuteTHCanddecreased tolerance to theantinociceptivevelopment of cannabinoid drugs thatminimize CB1R interactionswuced motor suppression.inoid CB1 ReceptorCentral Nervous System

    na E. Selley, Laura M. Bohn, and

    9-tetrahydrocannabinol (THC), the psychoactive component indence, which limit therapeutic development. Moreover, THCtor desensitization, internalization, and signaling, but its role in

    atalepsy) were assessed in -arrestin2-knockout (arr2-KO) andS and [3H]ligand autoradiography were assessed by statistical

    in subregions of the cortex but did not affect CB1R binding, iniated antinociception and hypothermia, with no difference inced CB1R desensitization and/or downregulation in cerebellum,C-mediated antinociception. In contrast, greater desensitizationarr2-KO comparedwithwild-typemice. Enhanced tolerance to

    d repeated THC administration was region-specific, and resultsrvations that arr2-KO mice display enhanced antinociceptivectsof thedrug, yet enhanced tolerance to catalepsy, suggest that-arrestin2might produce improved cannabinoid analgesicswith

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  • CB1Rs by-arrestin2 that parallel changes in THC-mediated behav-ior












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    P.T. Nguyen et al. BIOL PSYCHIATRY 2012;71:714724 715and tolerance.

    thods andMaterials

    Detailed Methods are provided in Supplement 1.

    ceMale WT and arr2-KO mice (littermates, 4 months) (15) wereected intraperitoneally with THC (10 mg/kg) or vehicle (1:1:18anol:cremaphor: .9% saline) twice daily for 6.5 days (subchronicatment). Twenty-four hours after the final injection, mice wereallengedwith increasing doses of THC (3, 7, 20, 26, and 44mg/kg,raperitoneally) every 40 min, with responses assessed 30 miner each injection. Studies followed the National Institutes ofalthGuidelines for the Care and Use of Laboratory Animals.

    haviorAntinociception was assessed using the warm-water (52C) tail-mersion assay (19). Duplicate measurements determined base-e responses, but mice were assessed only once following eachection to minimize tissue damage. A trained observer assessedmobility by determining the time mice spent motionless on atal ring-stand over 5 min (20). Mice were gently restrained, anddy temperaturewasmeasuredusinga rectal probe thermometer).Micewere sacrificedbydecapitation24hours after testing. Thenal cord and brain were extracted, frozen, and stored at80C.r antinociception and catalepsy, data are presented as the per-tageof themaximumpossible effect (%MPE)100% [(exper-ental response latency basal response latency)/(maximal pos-le response basal response latency)]. Nonlinear regressionalysis was calculated using GraphPad Prism software (La Jolla,lifornia).

    S]GTPS and [3H]SR141716A BindingWhole spinal cord was collected (Supplement 1), tissue wasmogenized, and agonist-stimulated [35S]GTPS binding wasducted as published (10). Concentration-effect curves were

    nerated using .013 mol/L CP55,940 or .0310 mol/LN55,212-2. Percent stimulation [(agonist-stimulated basal)/sal] 100%. Curveswere fit using nonlinear regression inGraph-d Prism. [3H]SR141716A binding was performed as published) using [3H]SR141716A (.12.5 nmol/L), and nonspecific bindingsmeasuredwith 5mol/L SR141716A. Datawere fit to a one-sitedel in GraphPad Prism. Statistical comparisons used Student-wman-Keuls post hoc test.

    S]GTPS and [3H]CP55,940 AutoradiographyAgonist-stimulated [35S]GTPS autoradiography was con-cted on duplicate serial sections as published (7,18). Basal and55,940-stimulated [35S]GTPS binding were conducted in ad-ent sections. CP55,940 is a high-efficacy agonist for G-proteinivation but does not activate non-CB1 sites in brain sections). Net stimulation (nCi/g) (agonist-stimulated basal).]CP55,940autoradiographywasmodified fromHerkenham et al.and Moise et al. (22). Total binding was assessed with 3 nmol/L]CP55,940 and nonspecific binding was measured using 10ol/L CP55,940. Image reconstructions, SPM, and region-of-inter-(ROI) analysis were conducted as published (18,23). The ROIasurements were made on original unprocessed images, aver-ed across hemispheres, and analyzed by two-way analysis ofiance (significance p .05) and Student-Newman-Keuls postc com