Cannabinoid interactions with glucocorticoid receptors in rat hippocampus

Brain Research, 534 (1990) 135-141 Elsevier

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Cannabinoid interactions with glucocorticoid receptors in rat hippocampus J. Charles Eldridge and Philip W. Landfield Department of Physiology and Pharmacology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27103 (U.S.A.)

(Accepted 5 June 1990) Key words: Tetrahydrocannabinol; Glucocorticoid receptors; Hippocampus

Previous studies have found that chronic administration of A9-tetrahydrocannabinol (THC), a psychoactive cannabinoid, can induce brain aging-like degenerative changes in hippocampal structures (e.g., pyramidal cell loss, glial reactivity). Normal aging changes in the hippocampus appear to be partly corticosteroid-dependent. Because THC is similar in molecular structure to corticosteroids (CORT), therefore, we have suggested that THC may act to induce pathology in the hippocampus through CORT receptors. The possibility of THC interactions with CORT receptors was tested more directly in the present studies. Binding of [3H]dexamethasone (DEX) to hippocampal cytosol, in vitro, was inhibited partially, but not completely, by 100-fold excess unlabeled THC and cannabidiol (CBD), a non-psychoactive cannabinoid. Even at 10,000-fold molar excess, moreover, THC could displace only 50% of radiolabeled DEX binding and CBD could inhibit only 22% of tracer binding. Scatchard plot analyses also pointed to a possible non-competitive site for cannabinoid interaction with glucocorticoid receptors. In addition, several studies utilizing the synthetic steroid RU-28362 indicated that THC interacts primarily with the type II class of glucocorticoid receptors. In a separate study, adrenalectomized rats were treated daily for 14 days with 5-10 mg/kg THC or vehicle, and examined 24 h later for [3H]CORT binding in hippocampal cytosol. In THC-treated animals, the BmaXfor type II binding was reduced to a degree almost comparable to the down-regulation seen after chronic stress or high corticosteroid administration. Thus, these data are consistent with the hypothesis that THC interacts with the brain's glucocorticoid receptor system, and appear to raise the possibility that this receptor system represents one endogenous target for THC effects on neuropathology and perhaps, on behavioral functions as well. INTRODUCTION Although numerous pharmacologic actions of A 9tetrahydrocannabinol (THC) and other psychoactive cannabinoids have been found on a wide range of hormonal, neuronal and behavioral variables 2'6'17'2°'2a' 32,33,55, the specific cellular and molecular mechanisms in brain that underlie these actions are not well understood (cf. ref. 28). Recent studies on the long-term effects of T H C appear to raise the possibility that one endogenous receptor site may be the hippocampal glucocorticoid receptor system. That is, long-term treatment (9 months) with a reasonably well-tolerated dose of T H C resulted in aging-like degenerative changes in the structure of the hippocampus of young-adult rats (e.g., pyramidal cell loss, astrocyte reactivity) 25. Treatment for 3 months has also been reported to alter hippocampal ultrastructure 44. Similar brain aging changes appear to be partly glucocorticoid-dependent (e.g., they are retarded by 9 months of adrenalectomy 22,24 and are accelerated by 6 months of chronic stress or 3 months of treatment with glucocorticoids21'23"43). Moreover, this same long-term T H C

treatment resulted in elevated pituitary-adrenal activity and adrenal gland hypertrophy 1'25, indicating that the well-established stimulatory effects of T H C on the pituitary-adrenal axis 3'7'9'1s do not diminish over months of treatment. Because cannabinoids and steroids exhibit a number of similarities in molecular structure 6'28'51, therefore, we previously suggested that T H C may exert its neurotoxic effects through direct interactions with hippocampal glucocorticoid receptors 25. This could occur either through an agonist action of T H C at the steroid receptor, which mimics the cytotoxic effects of corticosteroids, or through an antagonist action at the receptor level, which interferes with negative feedback and results in elevation of endogenous C O R T secretion (which, in turn, could mediate neurotoxicity). The search for specific brain binding sites for canna° binoids has proven difficult (cf. reviews in refs. 6, 28). Early studies of T H C effects on endocrine systems 7,9,14,28 raised the possibility of an interaction with peripheral steroid receptors, and one early study of hepatoma cells found dexamethasone-displaceable binding of radiola-

Correspondence: J.C. EIdridge, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, NC 27103, U.S.A.

136 beled A8-THC (ref. 14). In addition, several studies have r e p o r t e d T H C interactions with sex steroid receptors in p e r i p h e r a l tissues 37'39. On the other hand, another study, using Scatchard and sucrose gradient analyses, found contradictory data and little evidence of direct estrogen r e c e p t o r binding by T H C 36. With regard to neuronal steroid r e c e p t o r sites for T H C , several studies have r e p o r t e d direct effects of T H C on brain neurochemistry and neuronal physiology2'6A3'2°'55, but few, if any, data directly link T H C binding to brain steroid receptors. O t h e r studies, using less hydrophobic analogs, have seen cannabinoid-like binding sites in brain m e m b r a n e s (rather than in cytosol where steroid receptors are concentrated). Nye and co-workers 35 r e p o r t e d a membrane site possessing a m o d e s t affinity (K 0 = 89 nM) for a r a d i o l a b e l e d quaternary a m m o n i u m cannabinoid, trim e t h y l - a m m o n i u m AS-THC ( T M A ) . The sites were saturable, but both psychoactive and nonpsychoactive cannabinoids displaced binding of T M A . M o r e recently, a synthetic agent, CP-55,940 (CP), which appears to possess strong cannabinoid-like behavioral activity, has been found to bind to specific m e m b r a n e sites in rat brain 5"15. A cortical m e m b r a n e p r e p a r a t i o n was found to bind [3H]CP with a K d of 0.13 nM and a concentration of 1300 fmol/mg protein 5, and autoradiographic studies showed dense binding sites in basal ganglia and in the h i p p o c a m p u s 15. H o w e v e r , the possibility of a cannabinoid binding site on brain m e m b r a n e s clearly does not preclude the presence of a separate site on intracellular steroid receptors, given the multiplicity of T H C effects 6' 20,28,32

It has long been recognized that the hippocampus contains essentially the highest concentrations of glucocorticoid receptors ( G C R ) of any brain structure 31'47, and that these receptors are highly labile to up-regulation following a d r e n a l e c t o m y ( A D X ) 11"29'53, or to downregulation following several weeks or months of chronic stress of high C O R T administration 12'42"43'53. In addition, recent studies using synthetic steroids 4°'52 have shown that there are multiple classes (types I and II) of G C R in the h i p p o c a m p u s 4'34'41, which exhibit differential topographic distribution 4'48. This has led to the concept that type I receptors (which have a somewhat higher affinity for C O R T ) are essentially occupied fully at baseline C O R T secretion levels, whereas type II receptors (which have a somewhat lower affinity for C O R T but slightly higher affinity for d e x a m e t h a s o n e ) are occupied substantially by e n d o g e n o u s C O R T only during episodes of stress and elevated C O R T secretion 4m. In the present studies, both r e c e p t o r types were investigated to test further the possibility that T H C interacts with G C R . Aspects of these d a t a have been described in abstract form 10.

MATERIALS AND METHODS

Animals Subjects were specific-pathogen-free male Fischer-344 rats obtained from Harlan Industries at 3 months of age. Animals were housed in a barrier facility and were provided food and water ad libitum. To avoid interaction of endogenous steroids with steroid receptors, all animals used in the in vitro studies were adrenalectomized (ADX) 48 h prior to study. Surgery was performed in a small animal surgery facility, via a pair of mid-dorsal incisions, under ketamine/xylazine anesthesia and semi-sterile conditions. Muscle wounds were sutured and animals were treated postoperatively with 5000 IU BiciUin and maintained in heated cages for 24 h. Following surgery, the drinking water was substituted by 1% saline. Animals studied in the in vivo experiment were maintained on drinking saline supplemented with minimal steroid doses (see below).

Reagents The principal receptor assay buffer (TEG) was made with 5 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 10 mM molybdate, 10% glycerol, pH 7.40. Dextran-charcoal suspension was prepared in TEG without glycerol. Gradient buffers contained sucrose in TEG. Radioligands were [1,2,4,6,7-3H]dexamethasone ([3H]DEX, 95.7 Ci/mmol or 212.5 dpm/fmol) and [1,2,6,7-3H]corticosterone ([3H]CORT, 75.0 Ci/mmol or 166.5 dpm/fmol) (Amersham Corp.). Specific type II binding was identified by displacement of a fraction of total binding with RU-28362 (RU), a synthetic steroid that is highly specific for type II receptors16"4°'41. Total receptor binding (type I and type II) was assessed by displacement with excess non-labeled DEX (which binds to both type I and type II receptors with similar affinity), and type I was calculated as the difference between total displacement (DEX) and displacement of type II (RU). THC and cannabidiol (CBD) were supplied by the National Institute of Drug Abuse, as was a radioimmunoassay (RIA) kit for determination of plasma THC. RIAs for plasma corticosterone were performed with standard methods, described elsewhere12'~. All other reagents, including Lowry reagents and dextran-charcoal, were obtained from Sigma Chemical Co.

Cytosol preparation Animals were killed by decapitation and the hippocampi were dissected rapidly, placed in ice-cold TEG, and homogenized by 2 bursts of 5 s each with a Polytron (Brinkman Instruments). Homogenates were centrifuged in a Ti-40 rotor (Beckman Instruments) at 105,000 x g for 60 min, and the resulting supernatant (cytosol) was used for receptor binding studies. Samples were kept on ice or under refrigeration from the time of tissue dissection until the end of the procedure.

Scatchard plot analyses Using a microassay modification that permits Scatchard plot analysis in hippocampi from a single animal~1, the assay was performed on cytosols from paired hippocampi of each animal, in a volume of 3.5 ml, with average cytosol protein of 1.68 mg/ml, and incubated with 5 doses of tracer, plus or minus competitors. Tracer dilutions ranged from 1.25 to 20 nM of [3H]DEX. In each experiment, one set of duplicate tracer tubes contained no unlabeled hormone, one set contained unlabeled RU at 125 nM to 2 k~M (100-fold vs. tracer) and one set contained unlabeled DEX at 750 nM to 10 pM (500-fold). In addition, 4 other sets of tracer tubes contained, as unlabeled competitors: THC or CBD alone (125 nM to 2 pM), THC plus RU, or CBD plus RU (same ranges as each alone). The hydrophobicity of THC is well-recognized6"2~'z4,but, at these concentrations, both cannabinoids and steroids appeared to be evenly suspended in the TEG assay buffer. Duplicate incubations at each dose and condition were made of 50/~1 cytosol in each tube, which were incubated overnight at 4 °C. Receptor-bound tracer was then separated from free tracer with dextran-charcoal (0.1%/1.0%) and centrifugation at 2000 × g. Half the supernatant volume was

137 removed from each tube and counted in an LKB Model 1217 RackBeta counter. Efficiency loss and quench were corrected by external standardization and dpm results were computed by an on-board processor. Receptor binding measures were normalized for protein content (mean content per tube = 1.68 mg/ml cytosol), analyzed by the Lowry method. Estimates of specific receptor content were all based on subtraction of values in tubes containing various unlabeled competitors from the value in tubes containing no competitor (total binding), at comparable doses; type I receptor content was obtained by subtracting the displaced binding in the RU tubes (type II) from the displacement in the DEX tubes (total receptor).

Sucrose gradient analysis Aiiquots of cytosol (3.5 mg/ml protein) were incubated for 4 h with 5 nM [3H]DEX alone or in the presence of 500 nM unlabeled THC, CBD or RU-28362. Excess unbound iigand was removed with dextran-charcoal and the cytosols were layered on 5-20% gradients of sucrose in TEG. Tubes were centrifuged using an SW-55 rotor (Beckman Instruments) for 17 h at 325,000 x g. Fractions were collected dropwise from the bottom and counted. 14C-Methylated bovine serum albumin (BSA) (Amersham Corp.) was layered on simultaneously run gradients to mark the location of 5.5 S.

Glucocorticoid receptor down-regulation by THC ADX rats were injected s.c. once daily with THC in Pluronic F-68 (Wyandotte Chemical Co.) vehicle for 14 days (5 mg/kg for the first 7 days, followed by 10 mg/kg for the second 7 days) (n = 6), or with Pluronic vehicle alone (n = 5) for 14 days. The use of Pluronic F-68 vehicle has been described in detail previously45 and has been shown to be relatively non-toxic. Specific preparations used in our laboratory in long-term studies are described elsewhere24. Animals were maintained on 1% saline drinking water supplemented with 50 /~g/ml corticosterone, 25/zg/ml deoxycorticosterone, 2% glucose and 0.1% ethanol, which represented a minimal steroid replacement dose22'3s, but helped to maintain body weight and health during treatment. Both hormones were removed from the drinking water 48 h prior to killing, to allow clearance of steroids and restoration of unoccupied receptors for optimal binding analysis. Twenty-four h after the final treatment, animals were decapitated and both hippocampi were removed, cytosol was prepared and receptor binding was analyzed in each animal by Scatchard plot analyses, as described above. The only changes were that radiolabeled CORT was used instead of DEX, and that only 4 doses were incubated per cytosol (1.25-10 nM). Trunk blood was also collected at killing and plasma prepared for subsequent measurement of THC and CORT by RIA.

displaced only 11.5 + 5.8% of l a b e l e d D E X (nonsignificant vs. no a d d e d competitor). H o w e v e r , displacement by T H C was significantly less than that found with unlabeled D E X or R U ( T H C vs. R U , P < 0.01). Incubation with unlabeled R U , a specific type II agonist, displaced 80.8 + 3 . 9 % , which defined type II binding under these conditions. A d d i t i o n of either 250 n M T H C or C B D to 250 nM R U failed to displace m o r e b o u n d [3H]DEX than did R U alone, indicating that the displacement of tracer by T H C alone likely occurred at the same sites displaced by R U . Similar results were o b t a i n e d when h i p p o c a m p a l cytosols incubated with [3H]DEX were analyzed in sucrose density gradients (Fig. 2). Co-incubation with 100-fold excess unlabeled T H C r e d u c e d total p e a k binding of tracer by 28.5% ( P < 0.05), whereas 100-fold C B D reduced binding by an average of 9.2% (NS). The presence of cannabinoid did not shift the location of the r e c e p t o r p e a k , but r e d u c e d the magnitude of binding within the same single p e a k in the 8 S region. The kinetics of glucocorticoid r e c e p t o r interaction with T H C were e x a m i n e d by Scatchard analyses (4 experiments) of h i p p o c a m p a l cytosol incubated with various doses of [3H]DEX plus or minus excess unlabeled T H C , and o t h e r unlabeled inhibitors (Fig. 3). Incubation of tracer with 500-fold excess u n l a b e l e d D E X p r o d u c e d a linear plot with an average Bmax of 276.5 + 17.9 fmol/mg protein. This plot represents total specific G C R binding, c o m p o s e d of both types I and II sites. T h e plot for D E X is not curvilinear because, although two r e c e p t o r sites were occupied, both sites exhibit relatively similar affin-

COMPETITION AT 2.5 nMOLAR [3H]DEX DEX

Statistical analyses Comparisons of binding in the presence of various unlabeled compounds were assessed by analysis of variance (ANOVA), and individual condition contrasts were assessed by post-hoc tests (Crunch Software Corp.).

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Fig. 1. Competition of cannabinoids for binding to hippocampal glucocorticoid receptors. Cytosol incubated with 2.5 nM [3H]DEX in the presence of 250 nM unlabeled ligands (DEX, dexamethasone; RU, RU-28362; THC, A9-tetrahydrocannabinol; CBD, cannabidiol). Displacement by unlabeled DEX was designated as 100% inhibition for each experiment (mean = 234.4 _+ 16.1 fmol/mg protein). Histograms represent means _+ S.E.M. (No. of experiments) of percent inhibition of total specific binding by each unlabeled ligand or combination of two ligands. RU displacement represents type II binding; the difference between type II (RU) and total (DEX) represents type I capacity.

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Fig. 2. Representative sucrose density gradient analysis of cannabinoid competition in hippocampai cytosol. Cytosol aliquots were incubated with 5 nM [3H]DEX alone (A) or in the presence of 500 nM THC (O), cannabidioi (CBD) (O) or RU-28362 (&). After removal of unbound steroid, cytosols were layered on 5-20% sucrose and centrifuged for 17 h at 325,000 x g. Fractions of 150 al were collected dropwise from the bottom. Sedimentation of [14C]BSA indicated on plot. Peak binding occurred in the 8-9 S region, as did inhibition by cannabinoids. ities for D E X 27'41. The type I K d for D E X binding in rat h i p p o c a m p u s is a p p r o x i m a t e l y 3 - 4 nM, whereas the type II K d is a p p r o x i m a t e l y 2 - 3 nM (cf. refs. 4, 41). The plot from analyses of tubes containing excess RU-28362 was therefore parallel to the D E X plot and had an average x-intercept of 224.4 _+ 17.1 fmol/mg. The R U plot represents the type II population of G C R ; the

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Fig. 3. Scatchard plot of cannabinoid competition with [3H]DEX binding to hippocampal CORT receptors. Cytosols were incubated with 1.25-20 nM radiolabeled DEX alone or plus 100-fold excess unlabeled DEX, RU-28362, THC or CBD. Points represent means + S.E.M. of 4 experiments each. DEX plot indicates total specific receptor displacement (Ka = 3.22 nM, B,,.~x = 276.9 fmol/mg). RU plot indicates type II displacement (Kd = 3.41 nM, Bmax = 224.4 fmoi/mg). The slope of the THC plot was parallel to those for DEX and RU (Kd = 3.29 nM) but THC displaced fewer sites (Bin,. = 108.7 fmol/mg). Unlabeled cannabidiol produced a slight displacement of bound tracer (estimated B~a, = 46.7 fmol/mg, but precision of the plot was poor (Kd = 17.6 nM, r2 = 0.622; all other plots showed r2 > 0.95).

Fig. 4. Dose-response curves of cannabinoid inhibition of binding by [3H]dexamethasone in hippocampal cytosol. Cytosol aliquots were incubated with 10 nM [3H]DEX plus doses of competitor indicated on the abscissa scale. Plotted points represent the average of duplicate tubes at each dose. The maximum binding of control tubes, containing cytosol and tracer without competitor, was set as 100% binding. Binding that remained after maximum displacement by non-labeled DEX (non-specific binding) was set as 0% binding, and those non-specific counts were subtracted from all other readings before calculation of percent binding. type I population (average: 52.1 fmol/mg) was determined from the difference in binding at each point between the R U and D E X plots. W h e n 100-fold excess unlabeled T H C was co-incub a t e d with tracer and cytosol, the result was also a plot parallel to the others, but with a lower Bmax (average: 108.7 + 12.3 fmol/mg). Thus, in Scatchard analyses also, T H C displaced a significant fraction of, but not all (as defined by the D E X plot), r e c e p t o r binding. T h e slope of the curve for [3H]DEX binding was not changed in the presence of T H C , as indicated by KdS in the range of 3 nM. This could reflect a non-competitive site of action. Combining excess unlabeled R U and T H C in the same tubes produced no greater inhibition than did R U alone. Incubation with 100-fold excess C B D produced slight displacement in some incubations. A Scatchard plot of the combined C B D data produced a mean Bmax of 46.7 + 8.0 fmol/mg; however, the mean K d was 17.6 nM (Fig. 3). To determine whether much higher concentrations of T H C could displace all of the type II population, an inhibition analysis was carried out in 4 e x p e r i m e n t s by incubating increasing doses of unlabeled inhibitor with the same concentration of r a d i o l a b e l e d D E X (10 nM) and cytosol in all tubes (Fig. 4). Because D E X occupied both type I and type II G C R types, the displacement curve of RU-28362, a specific type II iigand, was less than 100% of the unlabeled D E X displacement curve. The R U curve reached a m a x i m u m of 30.5% (69.5% displacement) at 2 p M , a 200-fold excess of tracer concentration. The IC5o of inhibition was similar for both D E X and R U , at 7 nM. H o w e v e r , unlabeled T H C could displace only 50% of D E X tracer at concentrations as

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14.1 fmoi/mg) (P < 0.05). Type I binding capacity was not altered by THC treatment, nor were affinity measures of either receptor type. Plasma from the THCtreated animals contained 19.7 + !.7 ng/ml THC, measured by RIA, while vehicle-treated controls had undetectable levels (<1.0 ng/ml). These levels of THC in the treated animals were in the low range for human psychoactivity. Plasma CORT levels were undetectable (<10 ng/ml) in all vehicle and THC-treated animals, except for one THC-treated rat showing a CORT value of 105.8 ng/ml (presumably because of incomplete adrenalectomy). However, this animal's values for hippocampal GCR binding were close to the mean for the entire THC group. DISCUSSION

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Fig. 5. Down-regulation of glucocorticoid receptor binding in hippocampus following chronic THC treatment. Adrenalectomized rats were treated for 14 days with THC, 10 mg/kg/day (n = 6) or pluronic vehicle alone (n = 5). Cytosols were prepared from each pair of hippocampi and analyzed for both receptor types by Scatchard plots, as described in the Materials and Methods section. Each point represents the mean -+ S.E.M. of group binding data. Upper panel: type I receptor binding showed no change with treatment (K a for THC treatment = 1.88 nM; for vehicle = 1.60 nM; Bma. = 65.9 fmol/mg protein for THC and 67.6 fmol/mg for vehicle treatment). Lower panel: type II receptor binding demonstrated down-regulation in THC-treated animals (THC Bma. = 121.9 fmol/mg protein vs. vehicle = 167.2 fmol/mg, P < 0.05) but little change of receptor affinity (THC K d = 2.18 nM vs. vehicle = 3.04 nM).

high as 100/zM (10,000-fold molar excess) (Fig. 4). At higher concentrations, THC did not remain in solution (0.1% ethanol was present in all cannabinoid and steroid incubation tubes). The IC5o for this degree of inhibition was estimated from the displacement curve as 250 nM (38 times the IC5o for DEX). CBD, at a maximum of 100 /zM, displaced only 22% of total DEX binding. The estimated IC5o for CBD inhibition was 200 nM.

Glucocorticoid receptor down-regulation by THC Following daily administration of 5-10 mg/kg THC to ADX rats for 14 days, a significant reduction of type II binding was observed (Fig. 5). A group-averaged Scatchard plot for type II binding showed a significantly reduced Bma x in the hippocampus of THC-treated rats (121.9 + 8.1 fmol/mg) vs. vehicle-treated rats (167.2 +

These studies suggest that a naturally occurring, highly potent cannabinoid, A9-THC, is able to interact both in vitro and in vivo with the glucocorticoid receptor system of rat hippocampus. THC appeared to interact specifically with the type II class of GCR because no additional displacement of type I occurred when excess T H C and RU were combined. However, the pattern of interaction suggests that THC does not bind in the brain as a high affinity glucocorticoid. Unlabeled THC at 100-fold the [3H]DEX concentration was not able to displace type II sites fully and even 10,000-fold excess of THC (100/zM) displaced only 50% of radiolabeled DEX sites. Although THC might function as a very low affinity competitor for the type II site, 100 #M is well above the usual pharmacological levels. Therefore, the inability of this concentration of THC to displace DEX completely may indicate either that cannabinoids bind only to an as yet unrecognized subclass of type II C O R T receptors, or that they exert inhibitory influences on all type II sites by non-competitive (allosteric) means. Resolving these issues will require further analyses, using radiolabeled cannabinoids and various doses and classes of ligands. Nevertheless, it seems of interest that several investigators 49'5° have found that some steroidal and non-steroidal compounds are able to compete against CORT binding to its receptor with kinetics that appear to be non-competitive, i.e., allosteric, in nature. A second binding site on the glucocorticoid receptor has been identified which is hydrophobic 19, and topographically close to the agonist binding domain 49'5°. Thus, it appears plausible that THC may interact with an allosteric site on the CORT receptor. As noted in the Introduction, recent studies have identified a high-affinity binding site for a cannabinoid-like agent (CP) in brain membranes 5,15. Nevertheless, it seems possible, perhaps probable, that the wide range of THC actions is mediated by more than

140 one receptor type. Alternatively, since corticosteroid receptor binding has also been seen in brain membrane preparations 54, some of the membrane binding could also involve steroid receptors. The results showing THC-induced down-regulation of hippocampal G C R in A D X animals (Fig. 5) provide important indications that T H C interacts directly with G C R in vivo, under physiological conditions, and therefore, that this interaction could conceivably mediate THC-induced neuropathology 25. Because T H C was administered to A D X animals, the down-regulatory effects of T H C cannot be accounted for by the well-established T H C stimulation of endogenous glucocorticoid secretion 6'7"9'18. The extent of down-regulation induced by T H C was almost of the magnitude reported for chronic stress or with high C O R T administration 4'12'44'53. However, future studies will need to utilize additional techniques (e.g., R I A of local brain regions or uptake of radioactive ligands) 8'3° that permit direct analyses of cannabinoid uptake by G C R in adrenal-intact animals, in order to assess interactions under n o n - A D X conditions. The observation that chronic T H C can reduce glucocorticoid binding to hippocampal G C R , which are importantly involved in behavioral plasticity and a range of other functions, suggest that T H C might alter the effects of adrenal steroids on behavior, perhaps by dampening the brain's responsiveness to stressful stimuli. It seems conceivable that this, in turn, could be perceived as 'reinforcing' or could contribute to the pronounced

tolerance that develops for many pharmacological actions of cannabinoids 6. However, considerable additional work will be needed to evaluate the possible role of G C R in mediating the various neuropharmacological actions of THC. Because hippocampal G C R apparently participate in negative feedback control of A C T H 4'3~, the stimulatory effects of T H C on the pituitary-adrenal axis have led to the suggestion that the drug may antagonize the negative feedback actions of C O R T 9. However, the apparent downregulatory action of T H C (Fig. 5) could reflect a partial agonist-like action. Therefore, it appears possible that T H C exerts mixed agonist and antagonist actions on brain GCR. In summary, the present studies tested two predictions of the view 25 that the long-term neurotoxic effects of T H C (and perhaps other neurobehavioral effects) may be mediated via direct interactions with hippocampal G C R : (1) T H C should displace brain tissue corticosteroid binding in vitro; and (2) T H C should modify the G C R population in vivo. The data are consistent with these predictions and therefore appear to lend additional support to the above hypothesis.

REFERENCES

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Acknowledgements. We thank Deidre Fleenor-Heyser and Lisa B. Cadwallader for excellent and extensive technical contributions to this study. This work was supported in part by grants DA-03637 and DA-06218 from the National Institute on Drug Abuse, and by a Biomedical Support Grant S07-RR-05404 from the National Institutes of Health. We also express our appreciation to Centre de Recherches Roussel-Uclaf for providing the compound RU-28362.

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