chronic ▵9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors...

ChronicD9-Tetrahydrocannabinol Treatment Produces a Time-Dependent Loss of Cannabinoid Receptors and Cannabinoid

Receptor-Activated G Proteins in Rat Brain

*Christopher S. Breivogel, Steven R. Childers, Sam A. Deadwyler, Robert E. Hampson,Leslie J. Vogt, and *Laura J. Sim-Selley

Department of Physiology and Pharmacology, Center for the Neurobiological Investigation of Drug Abuse, and Center forInvestigative Neuroscience, Wake Forest University School of Medicine, Winston-Salem, North Carolina; and*Department of

Pharmacology and Toxicology and the Institute for Drug and Alcohol Studies, Medical College of Virginia of VirginiaCommonwealth University, Richmond, Virginia, U.S.A.

Abstract: Chronic treatment of rats with D9-tetrahydro-cannabinol (D9-THC) results in tolerance to its acute be-havioral effects. In a previous study, 21-day D9-THCtreatment in rats decreased cannabinoid activation of Gproteins in brain, as measured by in vitro autoradiographyof guanosine-59-O-(3-[35S]thiotriphosphate) ([35S]GTPgS)binding. The present study investigated the time courseof changes in cannabinoid-stimulated [35S]GTPgS bind-ing and cannabinoid receptor binding in both brain sec-tions and membranes, following daily D9-THC treatmentsfor 3, 7, 14, and 21 days. Autoradiographic resultsshowed time-dependent decreases in WIN 55212-2-stimulated [35S]GTPgS and [3H]WIN 55212-2 binding incerebellum, hippocampus, caudate-putamen, and glo-bus pallidus, with regional differences in the rate andmagnitude of down-regulation and desensitization. Mem-brane binding assays in these regions showed qualita-tively similar decreases in WIN 55212-2-stimulated[35S]GTPgS binding and cannabinoid receptor binding(using [3H]SR141716A), and demonstrated that de-creases in ligand binding were due to decreases in max-imal binding values, and not ligand affinities. These re-sults demonstrated that chronic exposure to D9-THCproduced time-dependent and region-specific down-regulation and desensitization of brain cannabinoid re-ceptors, which may represent underlying biochemicalmechanisms of tolerance to cannabinoids. Key Words:[35S]GTPgS autoradiography—Guanosine-59-O-(3-thio-triphosphate)—CB1 receptor—Desensitization—Down-reg-ulation.J. Neurochem. 73, 2447–2459 (1999).

Two types of cannabinoid receptors, CB1 (Matsudaet al., 1990) and CB2 (Munro et al., 1993), have beencloned, with CB1 receptors the predominant cannabinoidreceptors in brain (Pertwee, 1997). Cannabinoid com-pounds exert their CNS effects by binding to brain can-nabinoid receptors (Compton et al., 1993) to activate Gi-and Go-type G proteins (Howlett et al., 1986; Devane

et al., 1988). Receptor activation of G proteins can bemeasured by agonist-stimulated binding of the hydroly-sis-resistant GTP analogue, guanosine-59-O-(3-[35S]thio-triphosphate) ([35S]GTPgS), to Ga subunits in mem-branes (Hilf et al., 1989; Lorenzen et al., 1993; Traynorand Nahorski, 1995; Selley et al., 1996) or brain sections(Sim et al., 1995). This technique has been used previ-ously to demonstrate thatD9-tetrahydrocannabinol (D9-THC), the primary active constituent of marijuana(Gaoni and Mechoulam, 1964), is a low-efficacy partialagonist at brain cannabinoid receptors because it stimu-lates only 20% as much [35S]GTPgS binding as thesynthetic ligands WIN 55212-2 or levonantradol (Simet al., 1996a; Burkey et al., 1997; Breivogel et al., 1998).

Chronic administration of cannabinoids results in tol-erance to their acute behavioral effects (Abood et al.,1993; Oviedo et al., 1993; Rodrı´guez de Fonseca et al.,1994; Deadwyler et al., 1995; Fan et al., 1996; Romeroet al., 1997; Rubino et al., 1997), and the degree oftolerance to each effect varies (Fan et al., 1994, 1996).Measurement of the underlying changes in cannabinoidreceptors that accompany tolerance has yielded variableresults. Abood et al. (1993) found no changes in canna-binoid receptor binding or CB1 mRNA in homogenatesof whole mouse brain. However, region-specific changesin rat brain cannabinoid receptors and CB1 mRNA havebeen reported after chronicD9-THC treatment (Oviedoet al., 1993; Rodrı´guez de Fonseca et al., 1994; Romero

Resubmitted manuscript received August 16, 1999; accepted August16, 1999.

Address correspondence and reprint requests to Dr. C. S. Breivogelat Department of Pharmacology and Toxicology, Medical College ofVirginia of Virginia Commonwealth University, Box 98051, 1112 E.Clay Street, Richmond, VA 23298, U.S.A.

Abbreviations used:BSA, bovine serum albumin; GDP, guanosinediphosphate; [35S]GTPgS, guanosine-59-O-(3-[35S]thiotriphosphate);PIA, R(2)N6-(2-phenylisopropyl)adenosine;D9-THC, D9-tetrahydro-cannabinol.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 1999 International Society for Neurochemistry

et al., 1997, 1998a,b; Zhuang et al., 1998; Corchero etal., 1999). Several studies have examined in vitro CB1receptor function following chronic cannabinoid treat-ment. Fan et al. (1996) reported a 50% reduction in[3H]CP55940 binding sites in cerebellar membranes, butno desensitization of cannabinoid inhibition of adenylylcyclase. Sim et al. (1996a) found region-specific de-creases in cannabinoid-stimulated [35S]GTPgS bindingto brain sections, which has been confirmed recently byother laboratories (Romero et al., 1998a; Corchero et al.,1999).

Behavioral studies by Deadwyler et al. (1995) showedtime-dependent tolerance to the memory-disruptive ef-fects of daily D9-THC injections that was completewithin 30 days. Using the same treatment paradigm, ourlaboratory demonstrated decreases in cannabinoid recep-tor activation of G proteins in a number of regions of ratbrain using agonist-stimulated [35S]GTPgS autoradiog-raphy (Sim et al., 1996a). However, none of the previousstudies have determined the rate at which these changesin cannabinoid receptors and cannabinoid receptor acti-vation of G proteins occurred across different brain re-gions. Moreover, it is not known whether these alter-ations in ligand binding occurred as a result of changes inthe number (Bmax) or affinity (KD) of binding sites, or achange in the potency (EC50) or efficacy (Emax) of can-nabinoids to activate G proteins. The present study wasperformed to extend previous work by examining thetime course of the adaptations of brain cannabinoid re-ceptors and receptor activity following 3-, 7-, 14-, and21-day treatments withD9-THC. The same treatmentparadigm has been used by Zhuang et al. (1998) toinvestigate the effects ofD9-THC treatment on CB1receptor mRNA in the same brain regions examined inthis study. The results from these two studies provide acomprehensive, region-specific examination of the adap-tation of cannabinoid receptors in rat brain to chronicD9-THC treatment.

MATERIALS AND METHODS

MaterialsMale Sprague–Dawley rats were purchased from Zivic

Miller Laboratories, Inc. (Zelienople, PA, U.S.A.). [5,7-naph-thyl-3H]WIN 55212-2 ([3H]WIN 55212-2; 45.5 Ci/mmol),[35S]GTPgS (1,250 Ci/mmol), and Reflections film were pur-chased from New England Nuclear Corp. (Boston, MA,U.S.A.). [3H]SR141716A (65 Ci/mmol) and Hyperfilmbmaxwere obtained from Amersham Life Sciences (ArlingtonHeights, IL, U.S.A.).D9-THC was obtained from NIDA/Re-search Triangle Institute. WIN 55212-2 andR(2)N6-(2-phe-nylisopropyl)adenosine (PIA) were purchased from ResearchBiochemicals International (Natick, MA, U.S.A.). SR141716Awas a generous gift from Dr. Francis Barth at Sanofi Recherche(Montpellier, France). Guanosine diphosphate (GDP) for mem-brane binding assays and GTPgS were purchased fromBoehringer Mannheim (Indianapolis, IN, U.S.A.). GDP for[35S]GTPgS autoradiography was purchased from SigmaChemical Co. (St. Louis, MO, U.S.A.). All other reagent-gradechemicals were obtained from Sigma or Fisher Scientific (Pitts-burgh, PA, U.S.A.).

D9-THC treatmentD9-THC was dissolved in ethanol and prepared for injection

as previously described (Heyser et al., 1993). The ethanolsolution was suspended in a 1:4:1 ratio with Pluronic F68detergent in ethanol and saline, and the ethanol was evaporatedunder a stream of nitrogen gas. TheD9-THC was diluted to 10mg/ml in saline for injection. Animals received a single dailyintraperitoneal injection of 10 mg/kgD9-THC or an equalvolume of vehicle (control) for 3, 7, 14, or 21 days. Animalswere killed by decapitation 24 h after the last injection. Brainswere removed and hemisected along the midline; the left halfwas used for autoradiography, and the right was dissected formembrane assays, as described below.

[35S]GTPgS autoradiographyBrains were immersed in isopentane at235°C. Twenty-

micrometer horizontal sections were cut on a cryostat, thaw-mounted onto gelatin-coated slides, and stored at280°C untiluse. Sections were rinsed in assay buffer [50 mM Tris-HCl, 3mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, 0.1% (wt/vol)bovine serum albumin (BSA), pH 7.4] at 25°C for 10 min andthen with 2 mM GDP in assay buffer at 25°C for 15 min. Theywere then incubated with 0.04 nM [35S]GTPgS and 2 mM GDPin the presence or absence of 10mM WIN 55212-2 in assaybuffer at 25°C for 2 h. For all assays, WIN 55212-2 wasdissolved at 10 mM in dimethyl sulfoxide and then diluted inassay buffer before addition to the assay. Basal [35S]GTPgSbinding was assessed in the absence of agonist on adjacentsections. Slides were rinsed twice in cold 50 mM Tris buffer,pH 7.4, and once in deionized water, dried, and exposed to filmfor 48 h. Films were digitized with a Sony XC-77 video cameraand analyzed using the NIH Image program for Macintoshcomputers. Images were quantified by densitometric analysiswith 14C-labeled standards. Values are expressed as femto-moles of radioligand bound per gram of tissue and corrected for35S based upon incorporation of35S into brain paste standards(Sim et al., 1996b).

[3H]WIN 55212-2 receptor autoradiographyBrains were processed as described above. Sections were

preincubated in 20 mM HEPES with 0.5% (wt/vol) BSA and 1mM MgCl2, pH 7.0, for 20 min at 30°C and then incubated in1 nM [3H]WIN 55212-2 in this buffer for 80 min at 30°C.Nonspecific binding was assessed in the presence of 1mMunlabeled WIN 55212-2 on adjacent sections. Slides wererinsed four times for 10 min each in the preincubation buffer at25°C and then twice in deionized water at 4°C. Slides weredried thoroughly and exposed to Hyperfilmbmax for 3 weeks.Films were analyzed as described above.3H-labeled standardswere used for quantification, and values are expressed as fem-tomoles of radioligand bound per milligram of tissue. [3H]WIN55212-2 provided;90% specific binding in the regions ofinterest.

Membrane preparationsThe cerebellum, hippocampus, and striatum plus globus

pallidus (including caudate-putamen and globus pallidus) weredissected from the right half of each brain on ice and stored at280°C. On the day of the assay, each sample was thawed andhomogenized with a Tissumizer (Tekmar, Cincinnati, OH,U.S.A.) in cold assay buffer [50 mM Tris-HCl, 3 mM MgCl2,0.2 mM EGTA, 100 mM NaCl, 0.1% (wt/vol) BSA, pH 7.4]and centrifuged at 48,000g for 10 min at 4°C. Pellets wereresuspended in assay buffer and then centrifuged at 48,000gfor 10 min at 4°C. Pellets were homogenized in assay buffer,

J. Neurochem., Vol. 73, No. 6, 1999

2448 C. S. BREIVOGEL ET AL.

preincubated for 10 min at 30°C in 0.004 U/ml adenosinedeaminase (EC 3.5.4.4; Sigma) to remove endogenous adeno-sine, and then assayed for protein content (Bradford, 1976)before addition to assay tubes. All three membrane assays wereperformed using the same assay conditions simultaneously oneach tissue preparation.

Agonist-stimulated [35S]GTPgS binding assaysFor saturation binding analysis, membranes were incubated

with 0.5–20 nM unlabeled GTPgS in the presence or absence of3 mM WIN 55212-2. WIN 55212-2 concentration–effect anal-ysis was performed by incubating membranes with 1–10,000nM WIN 55212-2; each assay also included a single triplicateof 1 mM PIA to measure adenosine receptor-stimulated[35S]GTPgS binding. All assays were conducted at 30°C for2 h in assay buffer (see above) including 10–15mg of mem-brane protein with 0.1% (wt/vol) BSA, 30mM GDP, and 0.05nM [35S]GTPgS in a final volume of 1 ml. Nonspecific bindingwas determined in the absence of WIN 55212-2 and the pres-ence of 30mM unlabeled GTPgS. Reactions were terminatedby rapid filtration under vacuum through Whatman GF/B glass-fiber filters, followed by three washes with 4°C Tris buffer, pH7.4. Bound radioactivity was determined by liquid scintillationspectrophotometry at 95% efficiency for35S after overnightextraction of the filters in 4 ml of ScintiSafe Econo 1 scintil-lation fluid (Fisher).

[3H]SR141716A receptor binding assaysMembranes were diluted with assay buffer and incubated

under the same conditions as for the [35S]GTPgS bindingassays. Saturation binding analyses were performed by incu-bating 3.5–5 mg of membrane protein with 0.02–1 nM[3H]SR141716A in the presence and absence of 1mM unla-beled SR141716A to determine nonspecific and specific bind-ing, respectively. SR141716A was dissolved at 10 mM in 95%ethanol and then diluted in assay buffer (see above) beforeaddition to the assay. Assays were conducted in assay buffer for2 h at 30°C and included 0.1% (wt/vol) BSA, 30mM GDP, and0.05 nM unlabeled GTPgS. Reactions were terminated by rapidfiltration under vacuum through Whatman GF/B glass-fiberfilters that had been soaked in Tris buffer, pH 7.4, containing0.5% (wt/vol) BSA, followed by three washes with 4°C Trisbuffer, pH 7.4, containing 0.05% (wt/vol) BSA. Bound radio-activity was determined (at 45% efficiency for3H) as above. At0.2 nM, [3H]SR141716A provided 49–65% specific binding,depending on the brain region.

Data analysisNet agonist-stimulated [35S]GTPgS binding values were cal-

culated by subtracting basal binding values (obtained in theabsence of agonist) from agonist-stimulated values (obtained inthe presence of agonist) under otherwise identical conditions.Membrane binding values (including [3H]SR141716AKD andBmax, [35S]GTPgS apparentKD and apparentBmax, and WIN55212-2 EC50 andEmax) were determined by iterative nonlin-ear regression fitting to appropriate equations using JMP forMacintosh (SAS, Cary, NC, U.S.A.).KD andBmax values werefit to B 5 ([L]pBmax)/([L] 1 KD), whereB is the amount ofligand bound at each concentration of ligand [L]. EC50 andEmax values were fit toE 5 ([L]pEmax/([L] 1 EC50), whereEis the amount of net WIN 55212-2-stimulated [35S]GTPgSbinding at each concentration of WIN 55212-2 [L]. Differencesamong values within a brain region were determined byANOVA, and significant differences from control values atp , 0.05 were determined by using JMP to perform Dunnett’s

test. Differences among regions were determined by theTukey–Kramer test for multiple comparisons atp , 0.05.Unless otherwise indicated, all data presented are means6 SEM; autoradiographic values were determined in quadru-plicate sections from five animals per condition, and membranebinding values were determined in triplicate from three to fiveanimals per condition.

RESULTS

Autoradiography of cannabinoid-stimulated[35S]GTPgS binding and cannabinoid receptorbinding

Animals were treated for 3, 7, 14, or 21 days withD9-THC or vehicle to examine the time course ofchanges in brain cannabinoid receptors after chronicD9-THC treatment. Adjacent horizontal sections fromhemisected brains were processed for WIN 55212-2-stimulated [35S]GTPgS binding and [3H]WIN 55212-2receptor binding. Sections were analyzed at the appro-priate two levels to examine the cerebellum and globuspallidus. In addition, the hippocampus and caudate-putamen were analyzed at these same brain levels [indi-cated as dorsal (Fig. 1A) and ventral (Fig. 1B)]. Assimilar results were obtained in the dorsal and ventralportions of these regions, the data are presented aspooled data in the text.

WIN 55212-2-stimulated [35S]GTPgS autoradiogra-phy. WIN 55212-2-stimulated [35S]GTPgS autoradiog-raphy revealed high levels of cannabinoid receptor-acti-vated G proteins in the caudate-putamen, globus palli-dus, hippocampus, cerebellum, and cortex (Fig. 1). Fourregions were analyzed densitometrically to correspond tomembrane binding assays: cerebellum, hippocampus,caudate-putamen, and globus pallidus; autoradiographicresults presented as striatum plus globus pallidus are datapooled from caudate-putamen and globus pallidus. Basal[35S]GTPgS binding in these regions was not affected byD9-THC treatment: the values ranged from 2066 14fmol/g in the globus pallidus to 2386 10 fmol/g in thehippocampus of control animals. In contrast, the levels ofnet WIN 55212-2-stimulated [35S]GTPgS binding (WIN55212-2-stimulated minus basal [35S]GTPgS binding)were decreased significantly by chronicD9-THC treat-ment in the regions examined, with the exception ofglobus pallidus. However, the time course and magni-tude of the decrease in net WIN 55212-2-stimulated[35S]GTPgS binding differed between regions. This canbe seen in the representative autoradiograms shown inFig. 1, as well as in the densitometric data presented inTable 1. The most rapid change was identified in thehippocampus, where net WIN 55212-2-stimulated[35S]GTPgS binding was decreased significantly by day3 to 66% of control (p , 0.05). Net WIN 55212-2-stimulated [35S]GTPgS binding decreased to 35% ofcontrol at day 7, and remained at this level through days14 and 21 (p , 0.001 vs. control andp , 0.05 vs. day 3).

A slower time course of desensitization was found forcannabinoid-stimulated [35S]GTPgS binding in the cer-ebellum, although the magnitude of the decrease corre-


2449CHRONIC THC EFFECTS ON CANNABINOID RECEPTORS

sponded to that measured in the hippocampus. There wasno significant effect at day 3. On day 7, net WIN 55212-2-stimulated [35S]GTPgS binding decreased to 56% ofcontrol (p , 0.005). A further decrease was found onday 14, where net WIN 55212-2-stimulated [35S]GTPgSbinding was 35% of control, and remained at this levelon day 21 (p , 0.001 vs. control andp , 0.05 vs. day 3).

The caudate-putamen and globus pallidus exhibitedthe slowest time course for desensitization of the regions

examined. Furthermore, the magnitude of the decrease inagonist-stimulated [35S]GTPgS binding in these regionswas less than that measured in the hippocampus andcerebellum. In the caudate-putamen, net WIN 55212-2-stimulated [35S]GTPgS binding at day 3 was not signif-icantly different from control. On day 7, values were65% of control (p , 0.05). Further decreases weremeasured on days 14 and 21, where net WIN 55212-2-stimulated [35S]GTPgS binding was 59% and 45% of

FIG. 1. Autoradiograms of cannabinoid-stimulated [35S]GTPgS binding and cannabinoid receptor binding in brain sections from ratstreated for various times with D9-THC. Horizontal sections at dorsal (A) and ventral (B) levels of brains from rats that were treated for3–21 days with 10 mg/kg/day D9-THC or vehicle (control) are shown. The top half of each brain shows cannabinoid-activated G proteins,obtained by incubating slide-mounted sections with 0.04 nM [35S]GTPgS, 2 mM GDP, and 10 mM WIN 55212-2 in Tris buffer, pH 7.4,containing 100 mM NaCl. The bottom half of each brain shows cannabinoid receptor binding, obtained by incubating sections with 1nM [3H]WIN 55212-2 in HEPES buffer, pH 8.0, under high-affinity agonist binding conditions (no Na1 or GDP). Sections shown arerepresentative of five brains for each condition, which showed similar results.



control, respectively (p , 0.005). In the globus pallidus,net WIN 55212-2-stimulated [35S]GTPgS binding wasnot significantly different from control at any time point,including day 21, which was 74% of control (p 5 0.068by Dunnett’s test). A similar pattern of desensitizationwas found in the caudate-putamen and globus pallidus,although the time course was faster and the magnitude ofthe decrease was greater in the caudate-putamen. Pooledautoradiographic data from caudate-putamen and globuspallidus are presented as striatum plus globus pallidus inFig. 2 in order to be able to compare these results moredirectly with those obtained in membrane homogenatesof striatum plus globus pallidus (see below and Fig. 6).Figure 2 provides a summary of the data obtained fromall regions for both WIN 55212-2-stimulated[35S]GTPgS and [3H]WIN 55212-2 autoradiography (seebelow).

[ 3H]WIN 55212-2 receptor autoradiography.Sectionsadjacent to those used for [35S]GTPgS autoradiographywere processed with [3H]WIN 55212-2 to examine thetime course of the effects of chronicD9-THC treatmenton cannabinoid receptor binding. High levels of canna-binoid receptors were found in the same regions de-scribed above: cerebellum, hippocampus, caudate-puta-men, globus pallidus, and cortex (Fig. 1). Qualitatively,the time course of receptor down-regulation was similarto that of desensitization measured in the [35S]GTPgSbinding assay. However, in most regions, the magnitudeof the decrease in receptor binding was less than thedecrease in cannabinoid-activated G proteins. Represen-tative autoradiograms are shown in Fig. 1, and cannabi-noid receptor binding values are presented in Table 2. Inthe hippocampus, no significant decrease in cannabinoidreceptor binding was detected until day 7, at which timeit was 76% of control and remained at this level throughdays 14 and 21 (p , 0.001). A similar time course wasseen in the cerebellum, where cannabinoid receptor bind-ing was reduced significantly from control at days 7, 14,

and 21 (p , 0.05), to;80% of control. No significantchanges were detected in the globus pallidus at any timepoint. In the caudate-putamen, decreased cannabinoidreceptor binding was measured primarily in the ventral

TABLE 1. Net WIN 55212-2-stimulated [35S]GTPgS binding to brain sectionsfrom D9-THC-treated rats

Region

Net WIN 55212-2-stimulated [35S]GTPgS binding (fmol/mg)

Control 3 days 7 days 14 days 21 days

Cblm 1286 8.8 1016 9.6 716 14a 456 4.0a 456 8.0a

Hipp (D) 2116 8.8 1456 11b 836 13a 936 16a 756 15a

Hipp (V) 2106 17 1326 16a 706 12a 666 8.0a 656 13a

C-P (D) 2256 13 1916 8.0 1426 10b 1356 20a 816 20a

C-P (V) 2056 16 1626 18 1386 9.6b 1186 16a 1126 8.8a

GP 5206 21 5596 14 5226 19 4886 37 3836 66

[35S]GTPgS autoradiography was performed by incubating slide-mounted brain sectionswith 0.04 nM [35S]GTPgS and 2 mM GDP in the presence and absence of 10mM WIN55212-2. Net agonist-stimulated binding values (expressed as fmol/g of tissue) were obtainedby subtracting basal binding values (measured in the absence of agonist) from those obtainedin the presence of agonist on adjacent sections. Data shown are means6 SEM determined inquadruplicate for each of five brains for each condition. Cblm, cerebellum; Hipp (D), dorsallevel of the hippocampus; Hipp (V), ventral level of the hippocampus; C-P (D), dorsal levelof the caudate-putamen; C-P (V), ventral level of the caudate-putamen; GP, globus pallidus.

a p , 0.005,bp , 0.05 versus control values.

FIG. 2. Comparison of cannabinoid-stimulated [35S]GTPgSbinding and cannabinoid receptor binding by autoradiographyfollowing various treatment times with D9-THC. Net agonist-stimulated [35S]GTPgS binding values and [3H]WIN 55212-2binding values were each determined by densitometric analysisof autoradiograms such as those shown in Fig. 1. Data shownare means 6 SEM expressed as a percentage of control values(presented in Tables 1 and 2). †p , 0.05, ‡p , 0.005 versuscontrol values.



part of the region. The level of cannabinoid receptorbinding in the caudate-putamen decreased to;76% ofcontrol at day 7 (p , 0.005). The level of receptorbinding increased somewhat at day 14, and then returnedto 79% of control at day 21 (p , 0.005). Analysis ofpooled data from the caudate-putamen and globus palli-dus (designated as striatum plus globus pallidus) re-vealed that cannabinoid receptor binding decreased to;85% of control at days 7 and 21 (p , 0.05). Pooleddata from the striatum plus globus pallidus, hippocam-pus, and cerebellum are presented in Fig. 2.

Analysis of cannabinoid-stimulated [35S]GTPgSbinding and cannabinoid receptor binding inmembrane homogenates

Membrane assays were performed in cerebellum, hip-pocampus, and striatum plus globus pallidus to deter-mine whether changes in receptor and [35S]GTPgS bind-ing observed in sections resulted from changes in bind-ing site number (Bmax) or affinity (KD) or agonist potency(EC50) or efficacy (Emax) in a homogeneous preparationof a given brain region. All samples from each regionwere assayed for cannabinoid activation of G proteins by[35S]GTPgS binding using two assays: concentration–effect curves of WIN 55212-2 to determine whetherchronicD9-THC treatment affected agonist potency andefficacy for G-protein activation (Selley et al., 1997), andsaturation analysis of net WIN 55212-2-stimulated[35S]GTPgS binding to determine whether desensitiza-tion changed the ability of the agonist to increase theaffinity of Ga for [35S]GTPgS (i.e., apparentKD) or tostimulate a maximal number of Ga (i.e., apparentBmax)(Breivogel et al., 1997a). The CB1-selective antagonist,[3H]SR141716A, was used to measure cannabinoid re-ceptor density and affinity under the same assay condi-tions used for [35S]GTPgS binding. All three assays wereconducted simultaneously on the same membrane prep-aration of each tissue sample.

WIN 55212-2 concentration–effect curves.The po-tency (EC50) and efficacy (Emax) values for WIN55212-2 in [35S]GTPgS binding were determined fromWIN 55212-2 concentration–effect curves (Fig. 3).These results showed no significant effect of chronic

FIG. 3. WIN 55212-2 concentration–effect curves for the can-nabinoid receptor-mediated stimulation of [35S]GTPgS bindingto brain membranes from D9-THC-treated rats. Membranes fromcerebellum, hippocampus, and striatum plus globus palliduswere incubated with 0.05 nM [35S]GTPgS and 30 mM GDP in thepresence of 1–10,000 nM WIN 55212-2. Data are means 6 SEMfor control and 21-day-treated membranes.

TABLE 2. [ 3H]WIN 55212-2 binding to brain sections fromD9-THC-treated rats

Region

[3H]WIN 55212-2 binding (fmol/mg)

Control 3 days 7 days 14 days 21 days

Cblm 1036 3.6 926 5.4 846 2.7a 836 1.9a 866 3.1a

Hipp (D) 576 2.8 546 1.4 476 1.0b 466 0.3b 456 1.1b

Hipp (V) 646 4.0 536 2.2b 456 1.0b 496 1.2b 456 1.1b

C-P (D) 446 3.7 466 2.4 396 1.7 446 1.9 386 3.2C-P (V) 726 2.7 616 3.1a 556 1.6b 666 2.0 576 3.6b

GP 1196 9.2 1246 7.2 1096 3.7 1276 3.7 1056 2.0

Cannabinoid receptor binding was determined in sections by incubating sections with 1 nM[3H]WIN 55212-2 in the presence and absence of 1mM WIN 55212-2 to determine nonspe-cific and total binding. Specific binding (expressed as fmol/mg of tissue) was calculated bysubtracting nonspecific from total binding values that were determined on adjacent sections.Data shown are means6 SEM determined in quadruplicate for each of five brains for eachcondition. Cblm, cerebellum; Hipp (D), dorsal level of the hippocampus; Hipp (V), ventrallevel of the hippocampus; C-P (D), dorsal level of the caudate-putamen; C-P (V), ventral levelof the caudate-putamen; GP, globus pallidus.

a p , 0.05,bp , 0.005 versus control values.



D9-THC treatment on the EC50 values of WIN 55212-2(see below). The effects of chronicD9-THC treatment onthe maximal stimulation of [35S]GTPgS binding by WIN55212-2 varied across regions. There was no significanteffect on maximal stimulation in cerebellum at any timepoint, but the maximal stimulation by WIN 55212-2 wasdecreased by 31% from control values (p , 0.05) inhippocampus membranes from 21-day-treated animals,and by 34% from control values (p , 0.05) in striatumplus globus pallidus membranes from 21-day-treated an-imals.

Nonlinear regression analysis of WIN 55212-2 con-centration–effect curves revealed that the effect of ago-nist in all three regions was better fit to two-componentsigmoidal curves than to single-component curves. Thus,both high- and low-potency EC50 andEmax values weredetermined for the stimulation of [35S]GTPgS binding(Fig. 3). The high-potency sites exhibited mean EC50values of 12.46 2.9 nM, 9.3 6 2.8 nM, and 7.46 6.5nM for WIN 55212-2 in control cerebellum, hippocam-pus, and striatum plus globus pallidus, respectively. Thelow-potency sites in control tissue displayed EC50 valuesfor WIN 55212-2 of 4536 75 nM in cerebellum, 4596 37 nM in hippocampus, and 2656 178 nM in striatumplus globus pallidus. Comparison of the basal levels of[35S]GTPgS binding (determined in the absence of WIN55212-2) and WIN 55212-2 EC50 values between tissuefrom control andD9-THC-treated animals from eachbrain region revealed significant differences in neitherbasal binding nor EC50 values, indicating no detectableeffects of residualD9-THC in the membranes. Although

the chronicD9-THC treatment had no effect on the EC50values of WIN 55212-2-stimulated [35S]GTPgS binding,the apparent potency of cannabinoids to activate G pro-teins was decreased because a higher concentration ofdrug would be required to obtain the same effect ob-served in control tissue (see Fig. 3). This apparent de-crease in potency could manifest as tolerance to canna-binoids in chronically treated whole animals.

High- and low-potencyEmax values for all three re-gions are presented in Table 3.D9-THC treatment did notresult in significant changes in theEmax values for cere-bellum, except for a small decrease in the high-potencyEmax value at 7 days, which was not observed at 14 or 21days. The hippocampus showed significant decreases (p, 0.005) in high-potencyEmax values at all treatmenttimes from days 3 to 21 (40–45% decreases), but nosignificant effect of theD9-THC treatment on low-po-tency Emax values (ANOVA, p 5 0.13). Thus, the de-creases in WIN 55212-2 stimulation of [35S]GTPgSbinding in hippocampus were due primarily to decreasesin the component of [35S]GTPgS binding stimulated byWIN 55212-2 with high potency. In striatum plus globuspallidus, despite the statistically significant 34% decrease( p , 0.05 by Dunnett’s test) in the overall stimulation of[35S]GTPgS binding by WIN 55212-2 at day 21 (Fig. 3),none of the decreases in the high-potency (40% at day21,p 5 0.51) or low-potency (31% at day 21,p 5 0.14)Emax values reached statistical significance.

[ 35S]GTPgS binding saturation analysis.The next setof experiments assayed net agonist-stimulated [35S]-GTPgS binding to obtain apparentKD and apparentBmax

TABLE 3. WIN 55212-2-stimulated [35S]GTPgS binding in brain membranesfrom D9-THC-treated rats

Region, treatment

WIN 55212-2Emax (fmol/mg)[35S]GTPgS Bmax

(pmol/mg)High affinity Low affinity

CerebellumControl 906 6 1326 7 7.86 0.73 days 736 6 1246 12 9.06 0.67 days 586 4a 1316 2 7.46 0.314 days 886 3 1216 6 9.86 1.121 days 766 6 1336 7 8.76 0.8

HippocampusControl 906 1 1156 18 10.76 0.53 days 526 8a 1126 12 9.06 0.87 days 546 6a 816 5 7.66 0.9b

14 days 526 6a 866 5 6.96 0.6a

21 days 496 8a 916 8 6.06 0.5a

Striatum plus globus pallidusControl 1086 34 2496 48 10.56 0.73 days 816 37 1746 29 12.76 1.47 days 866 22 1636 20 12.16 0.714 days 696 8 1816 25 10.96 0.921 days 656 21 1716 21 8.36 1.3

Emax values were obtained by incubating membranes with 1–10,000 nM WIN 55212-2 and0.05 nM [35S]GTPgS. Bmax values were determined by net WIN 55212-2-stimulated[35S]GTPgS binding with 0.05 nM [35S]GTPgS plus 0.5–20 nM unlabeled GTPgS with andwithout 3 mM WIN 55212-2.

a p , 0.005,bp , 0.05 compared with control values.



values for cannabinoid-activated G proteins in each re-gion. These values are termed “apparent” because of thepresence of GDP in the assay, which is necessary foragonist stimulation of [35S]GTPgS binding, but com-petes for [35S]GTPgS binding at the guanine nucleotidebinding site of the G protein (Breivogel et al., 1998). Incontrast to results obtained in purified G proteins, how-ever, the binding of [35S]GTPgS to native membranes isreversible (Hilf et al., 1992; Breivogel et al., 1998),allowing the data to be analyzed under the assumptionsof saturable and reversible ligand binding.

Net agonist-stimulated values are obtained by sub-tracting the basal binding values from those obtained inthe presence of agonist at each concentration of GTPgS.Net WIN 55212-2-stimulated [35S]GTPgS binding satu-ration analyses (Fig. 4) showed similar effects ofD9-THC treatment to the WIN 55212-2 concentration–effectof [35S]GTPgS binding.D9-THC treatment had no effecton [35S]GTPgS binding apparentKD values in any re-gion, which had mean values of 1.86 0.3 nM, 3.46 0.5nM, and 2.06 0.2 nM for control cerebellum, hippocam-pus, and striatum plus globus pallidus, respectively. Fur-thermore, there was no effect ofD9-THC treatments onthe levels of basal [35S]GTPgS binding in these assays,as in the concentration–effect assays. ApparentBmax

values in cerebellum were not affected by the chronicD9-THC treatment (ANOVA,p 5 0.20). In striatum plusglobus pallidus, there was no decrease in apparentBmaxvalues until day 21 (21% decrease from control), thesame time point where the significant decrease inEmaxwas observed; however, the decrease in apparentBmaxfailed to reach statistical significance (p 5 0.38 byDunnett’s test). ApparentBmax values decreased by 30–45% in hippocampus from days 7 to 21 (p , 0.05 at day7 andp , 0.005 at days 14 and 21) (Table 3). Theseresults indicated that the chronicD9-THC-induced de-creases in the maximal effect of the agonist (Emax) seenin the WIN 55212-2 concentration–effect curves forhippocampus were due to a decrease in the number of Gproteins being activated by WIN 55212-2 (apparentBmax), and not a change in the apparent affinity of theWIN 55212-2-activated G proteins for [35S]GTPgS (ap-parentKD).

To determine whether the effects of chronicD9-THCtreatment were specific to cannabinoid receptors,[35S]GTPgS assays were conducted in the presence of amaximally effective concentration of the adenosine A1receptor agonist, PIA. In control membranes, PIA stim-ulated [35S]GTPgS binding by 2806 6% in cerebellum,2306 10% in hippocampus, and 1026 11% in striatumplus globus pallidus. In contrast to its effects on canna-binoid-stimulated [35S]GTPgS binding, chronicD9-THChad no effect on PIA-stimulated [35S]GTPgS binding atany treatment time in any region (data not shown). Theseresults are analogous to those obtained previously wherechronic D9-THC treatment had no effect on GABAB-stimulated [35S]GTPgS binding to sections (Sim et al.,1996a).

Cannabinoid receptor binding.The number and affin-ity of cannabinoid receptors were quantified by satura-tion binding analysis of the CB1-selective antagonist[3H]SR141716A. This antagonist radioligand was usedso that receptor binding could be performed under thesame assay conditions (high concentrations of sodiumand GDP) as the [35S]GTPgS binding assays. Figure 5shows representative Scatchard plots obtained in cere-bellum, hippocampus, and striatum plus globus pallidusmembranes from control and 21-day-treated animals.Results showed no change inKD values for[3H]SR141716A in any region at any time point (Table4). MeanKD values in control tissue were not signifi-cantly different among regions with values of 0.256 0.07 nM, 0.12 6 0.02 nM, and 0.146 0.02 nM incerebellum, hippocampus, and striatum plus globus pal-lidus, respectively. Both cerebellum and hippocampusshowed significant decreases in [3H]SR141716ABmax(ANOVA, p 5 0.011 and 0.001, respectively) beginningat 3 days.D9-THC treatments resulted in maximal de-creases in receptor numbers of 50% for cerebellum and55% for hippocampus at 21 days (Table 4). Striatum plusglobus pallidus showed no significant changes (ANOVA,p 5 0.231) in [3H]SR141716ABmax values, which had amean of 2.36 0.2 pmol/mg in control tissue.

FIG. 4. Saturation analysis of net cannabinoid receptor-stimu-lated [35S]GTPgS binding to brain membranes from D9-THC-treated rats. Membranes from cerebellum, hippocampus, andstriatum plus globus pallidus were incubated with 30 mM GDPand 0.05 nM [35S]GTPgS plus 0.5–20 nM unlabeled GTPgS in thepresence and absence of 3 mM WIN 55212-2. Saturation datawere analyzed to obtain KD and Bmax values of agonist-induced[35S]GTPgS binding to activated G proteins. Data are means6 SEM for control and 21-day-treated membranes.



Figure 6 summarizes the time-course data obtained inmembrane homogenate binding assays. The values ofWIN 55212-2 concentration–effectEmax of [35S]GTPgSbinding and theBmax of [3H]SR141716A receptor bind-ing in cerebellum, hippocampus, and striatum plus glo-bus pallidus are shown as a percentage of control valuesand as a function of the time of treatment withD9-THC.These values represent the effects of chronicD9-THC oncannabinoid receptor levels ([3H]SR141716ABmax) andcannabinoid receptor activity for G-protein coupling([35S]GTPgS Emax). Each brain region exhibited funda-

mentally unique effects. In cerebellum, there was a rapidand profound decrease in cannabinoid receptors with nodecrease in receptor-activated G proteins. In striatumplus globus pallidus, there was a slow, but significant,decrease in receptor-activated G proteins with no de-crease in receptor levels. In hippocampus, rapid andsignificant decreases in both receptors and receptor-acti-vated G proteins were observed.

DISCUSSION

The present study extends the findings of Sim et al.(1996a) to show that both desensitization and down-regulation of cannabinoid receptors occur with chronicD9-THC treatment and, further, that these responses aretime-dependent and region-specific. For example, rapidand profound decreases in cannabinoid-stimulated[35S]GTPgS binding and cannabinoid receptor bindingwere found in hippocampus, whereas these alterationswere less pronounced and slower in the striatum andglobus pallidus. Although other studies have examinedcannabinoid receptor and cannabinoid-stimulated[35S]GTPgS autoradiography following chronicD9-THCtreatment (Romero et al., 1997, 1998a,b; Corchero et al.,1999), this is the first study to determine systematicallythe time course of cannabinoid receptor desensitizationand down-regulation in multiple brain regions simulta-neously. Furthermore, this is the first study to correlatethe effects of chronicD9-THC on cannabinoid receptorsand receptor-stimulated [35S]GTPgS binding betweenbrain sections and membranes, to provide a completeassessment of changes in receptor/G-protein coupling.Although these results are generally consistent with theresults of studies by other laboratories, some importantdifferences should be addressed. Romero et al. (1998a)have reported that a 5 day-treatment withD9-THC doesnot significantly affect WIN 55212-2-stimulated[35S]GTPgS binding in the caudate-putamen or globuspallidus, whereas significant decreases in cannabinoidreceptor binding were found in most brain regions ex-amined, including caudate-putamen, but not globus pal-lidus. These findings are consistent with the presentresults, except that a significant decrease in cannabinoidreceptor binding in caudate-putamen reported at 5 daysby Romero et al. (1998a) was not observed until day 21

FIG. 5. Scatchard plots of [3H]SR141716A binding to cannabi-noid receptors in brain membranes from D9-THC-treated rats.Membranes from cerebellum, hippocampus, and striatum plusglobus pallidus were incubated with 30 mM GDP and 0.05 nMGTPgS plus 0.02–1 nM [3H]SR141716A in the presence andabsence of 1 mM unlabeled SR141716A. Saturation data wereused to obtain KD and Bmax values of cannabinoid receptors for[3H]SR141716A. Representative data are shown for control and21-day-treated membranes.

TABLE 4. Parameters of [3H]SR141716A binding in brain membranes fromD9-THC-treated rats

Treatment

Cerebellum Hippocampus Striatum plus globus pallidus

Bmax (pmol/mg) KD (nM) Bmax (pmol/mg) KD (nM) Bmax (pmol/mg) KD (nM)

Control 5.26 1.0 0.256 0.07 4.66 0.2 0.126 0.02 2.36 0.2 0.146 0.023 days 3.26 0.2a 0.176 0.05 3.36 0.5a 0.146 0.03 2.16 0.4 0.146 0.017 days 2.46 0.1b 0.126 0.02 2.56 0.1b 0.146 0.02 2.66 0.3 0.216 0.0414 days 3.56 0.3 0.216 0.01 2.56 0.2b 0.126 0.02 2.16 0.6 0.236 0.1121 days 2.66 0.4a 0.166 0.03 2.06 0.2b 0.156 0.03 2.06 0.4 0.176 0.03

Bmax andKD values were determined under [35S]GTPgS binding conditions using 0.02–1 nM [3H]SR141716A in the absence and presence of 1mM SR141716A, to determine total and nonspecific binding, respectively.

a p , 0.05,bp , 0.005 compared with control values.



in the present study. Corchero et al. (1999) also exam-ined the time course of decreases in cannabinoid receptorbinding and cannabinoid receptor-stimulated [35S]-GTPgS binding, but this analysis was restricted to thecaudate-putamen. Those results differ considerably fromthis study, as well as that of Romero et al. (1998a); WIN55212-2-stimulated [35S]GTPgS binding was decreasedsignificantly in the caudate-putamen from days 1 through14 ofD9-THC treatment. The reason for this discrepancyis not clear, but may result from differences inD9-THCtreatment or in in vitro assay conditions between labo-ratories.

All of the previous studies of the effects of chronicD9-THC administration on brain cannabinoid receptorsused single concentrations of radioligand binding tobrain sections, which did not allow for a distinctionbetween loss of ligand binding sites and loss of ligandbinding affinity (Sim et al., 1996a; Romero et al.,1997,1998a,b; Corchero et al., 1999). The current studyindicates that decreases in cannabinoid-stimulated[35S]GTPgS binding and cannabinoid receptor bindingwere attributable to losses in the number of cannabinoidreceptors and cannabinoid receptor-activated G proteins.These results support and extend previous work on the

effects of chronicD9-THC on cannabinoid receptor/Gprotein coupling. Furthermore, these data support previ-ous results from our laboratory (Breivogel et al., 1997b),which indicate that cannabinoid receptors exhibit quan-titative differences in measurements of signal transduc-tion responses depending upon their regional localiza-tion.

An important consideration in this study was thatresidualD9-THC might remain in the tissue, due to itslipophilic nature. As we have found thatD9-THC is apartial cannabinoid agonist (Sim et al., 1996a), this is animportant concern because residualD9-THC could de-crease WIN 55212-2-stimulated [35S]GTPgS binding. Inthe previous study by Sim et al. (1996a), stringent con-trols were included that excluded this possibility. Theresults of the current study further indicate that residualD9-THC is not present because there were no differencesbetween control andD9-THC-treated samples in (a) basal[35S]GTPgS binding in either membranes or brain sec-tions, (b) WIN 55212-2 EC50 values in membranes, and(c) [3H]SR141716AKD values in membranes. In addi-tion, cannabinoid receptor desensitization was apparentlyhomologous because neither GABAB (Sim et al., 1996a)nor adenosine A1 (present results) activation of G pro-teins was affected by chronicD9-THC treatment.

Qualitatively similar results were obtained in bothisolated membranes and brain sections. In both prepara-tions, the fastest and greatest decrease in cannabinoidreceptor binding and receptor-stimulated [35S]GTPgSbinding occurred in the hippocampus, with a slowerresponse in cerebellum, and the smallest and slowestresponse in striatum plus globus pallidus. However,some important differences were seen using the twomethods; in particular, desensitization was greater inmagnitude using [35S]GTPgS autoradiography comparedwith membrane binding assays, whereas the degree ofapparent receptor down-regulation was greater in mem-branes than in sections. It is possible that these discrep-ancies were due to technical differences. For example,each preparation provides different degrees of anatomi-cal resolution, and membranes are prepared from dis-sected regions in which microheterogeneities may belost. Furthermore, brain sections preserve cellular orga-nization, whereas soluble components and subcellularcompartmentalization are lost during homogenization ofmembranes. Thus, differences in desensitization mayhave been due to dephosphorylation or to loss of com-ponents, such as arrestin, that mediate receptor/G-proteinuncoupling (Lefkowitz et al., 1992), or loss of internal-ized CB1 receptors (Wenzhen et al., 1999).

Finally, quantitative differences in receptor bindingbetween membranes and sections may have been due tothe use of different radioligands, [3H]WIN 55212-2 and[3H]SR141716A. For example, if there is significantexpression of CB2 (Skaper et al., 1996) or undiscoveredcannabinoid receptor types in brain, the fact that receptorbinding was measured in brain sections with a nonselec-tive agonist, and in membranes with a CB1-selectiveantagonist, may explain the lower degree of down-regu-

FIG. 6. Comparison of cannabinoid-stimulated [35S]GTPgSbinding and cannabinoid receptor density in brain membranesfollowing various treatment times with D9-THC. [35S]GTPgS Emaxvalues were determined as the maximal effect of the cannabi-noid receptor agonist for the stimulation of [35S]GTPgS bindingas described in Fig. 3. [3H]SR141716A Bmax values were deter-mined by saturation analysis of cannabinoid receptors as de-scribed in Fig. 5. Data shown are means 6 SEM expressed as apercentage of control values (presented in Tables 3 and 4).†p , 0.05, ‡p , 0.005 versus control values.



lation measured in sections. SR141716A has been shownto be an inverse agonist in CB1 receptor-transfectedChinese hamster ovary cells (Bouaboula et al., 1997),which could complicate the interpretation of the receptorbinding data using [3H]SR141716A because inverse ago-nists may prefer binding to non-precoupled receptorsover G protein-precoupled receptors. However, this li-gand is a neutral antagonist in brain membranes (Breivo-gel et al., 1998), and moreover, only a single binding sitefor [3H]SR141716A was detected. Thus, it appears that[3H]SR141716A binding measures all CB1 receptors inthese membranes.

Membrane binding assays in cerebellum indicated thatD9-THC treatment resulted in a 50% decrease in canna-binoid receptor binding without affecting cannabinoidreceptor activation of G proteins in cerebellum. Theseresults agree with previous findings that similar canna-binoid-induced decreases in cerebellar cannabinoid re-ceptors did not affect CP55940-inhibited cyclic AMPaccumulation in membranes (Fan et al., 1996), and con-firm previous results from our laboratory that indicated asmall degree of cannabinoid receptor reserve forG-protein activation in cerebellar membranes (Breivogelet al., 1997b). In the presence of large receptor reserve,receptor down-regulation should decrease agonist po-tency. The fact that no significant changes in agonistEC50 values were observed following chronicD9-THCtreatment also suggests that the receptor reserve in cer-ebellar membranes is relatively small. In contrast, resultsobtained by autoradiography would not indicate receptorreserve for cannabinoid receptors in any of these regionsbecause small losses in cannabinoid receptors were ac-companied by larger decreases in cannabinoid receptor-activated G proteins. These discrepancies may beexplained by differences between the membrane andautoradiographic assays: compartmentalization or se-questration in whole cells may prevent or limit coupling,thereby reducing receptor reserve for G-protein activa-tion in sections. Alternatively, as lipophilic cannabinoidradioligands do not distinguish between internalized andsurface CB1 receptors (Wenzhen et al., 1999), the recep-tor binding observed in sections may have appearedgreater than the amount of functional receptor remaining.

Saturation analyses in membranes demonstrated thatall decreases in cannabinoid receptor binding were due tochanges in maximal binding quantities, and not affinities.WIN 55212-2 stimulated [35S]GTPgS binding with twoapparent potencies, and the loss of stimulation in hip-pocampus was preferentially from the number of high-potency (low EC50) sites. However, a recent study ofWIN 55212-2-stimulated azidoanilido[32P]GTP bindingto rat cerebellar membranes demonstrated that WIN55212-2 activates different G-protein subtypes withvarying potencies, which may contribute to the multi-component concentration–effect curves observed for thestimulation of [35S]GTPgS binding by WIN 55212-2(P. L. Prather, H. Zhang, C. S. Breivogel, and S. R.Childers, manuscript submitted). Thus, the present studyindicates that the greatest loss of cannabinoid activation

of G proteins was from the pool of G proteins that areactivated by WIN 55212-2 with higher potency.

As the D9-THC treatment used in the present studywas identical to that used to study the time course oftolerance to the memory-disruptive effects ofD9-THC(Deadwyler et al., 1995), it is reasonable to comparethose behavioral results with the hippocampal receptorand G-protein data in the present study. Deadwyler et al.,(1995) reported gradual onset of tolerance at 5–16 days,which increased at days 16–21, before complete adapta-tion occurred at 30 days. Time-course data in the presentstudy revealed that desensitization and down-regulationof cannabinoid receptors in hippocampus occurred rap-idly from 3 to 7 days and remained at that level from 7to 21 days. Thus, the biochemical alterations measured inhippocampus may correspond to the first phase of toler-ance to the memory-disruptive effects ofD9-THC. Thefinding that cannabinoid receptor desensitization variesacross brain regions supports previous findings of differ-ent magnitudes of tolerance to catalepsy, hypothermia,decreased locomotion, and analgesia (Fan et al., 1994,1996), and predicts that tolerance to different CNS ef-fects of cannabinoids may develop at different rates.

The results of CB1 receptor and [35S]GTPgS bindingin membranes and sections contribute to the elucidationof the underlying biochemical mechanisms of toleranceto cannabinoids. It is clear that chronic exposure of braincannabinoid receptors toD9-THC generally resulted inadaptations that decreased the effect of agonist. De-creases in agonist-stimulated [35S]GTPgS binding repre-sented functional uncoupling of cannabinoid receptorsfrom the entire signal transduction pathway because ac-tivation of the G protein is the first step in this cascade.Similar to previous results in other G protein-coupledreceptor systems (Law et al., 1983; Lefkowitz et al.,1990), it has been shown that desensitization and inter-nalization of CB1 cannabinoid receptors are mediated byphosphorylation of the intracellular domain of the recep-tor (Garcia et al., 1998; Wenzhen et al., 1999). Loss ofreceptors may be due to increases in degradation ordecreases in synthesis, processing, or expression of re-ceptor protein. A study by Zhuang et al. (1998) foundtransient, region-specific alterations in CB1 mRNA, inthe same groups ofD9-THC-treated rats and over thesame time course as reported here. Therefore, it is clearthat chronic treatment withD9-THC produces profoundeffects on the brain cannabinoid receptor system at mul-tiple levels, which may vary significantly across differentbrain regions. Such changes could account, at least inpart, for the different time course and degree of behav-ioral tolerance that develops in different systems uponrepeated cannabinoid administration.

Acknowledgment: Doug Byrd, Joanne Konstantopoulos,Shou-Yuan Zhuang, and Ruoyu Xiao provided excellent tech-nical assistance. These studies were supported by U.S. PublicHealth Service grants DA-07625 (S.A.D.), DA-06784 and DA-00634 (S.R.C.), DA-00287 (L.J.S.-S.), and DA-07246 (C.S.B.)from NIDA.



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chronic ▵9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors...

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