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Home , Levonantradol, Nabilone

Localization in Brain: Relationship to Motor and Reward Systems MILES HERKENHAM Sectrim on Fututional Neuraana~y National InstitUtzr of Mend Hmlth Bui~iiiqg36, Room 20-15 &the&, Matyhnd 20892

INTRODUCI'ION

Marijuana (Omnabri satipa) is one of the oldest and most widely used drugs in the world, with a history of use dating back over 4,000 years.l.2 It was not until about twenty years ago that the principal psychoactive ingredient of the marijuana plant was isolated and found to be A9- (A9-THC).3-5 A9-THC and other natural and synthetic produce characteristic behavioral and cognitive effect^,^.^ most of which can be attributed to actions on the central nervous system8 Marijuana use in this country is widespread, with approximately 40 million Americans having at least tried the drug9 Compulsive use is associated with social and psycho- logical problems in individuals.lo There is little evidence of adverse health side effects or t~xicity.~Jl-lj Until recently, very little was known about the cellular mechanisms through which cannabinoids act. The unique spectrum of cannabinoid effects and the stereoselectivity (enantioselectivity) of action of cannabinoid in behavioral studies (see below) strongly suggested the existence of a specific in blain, but early attempts to identify and characterize such a recognition site were not successful (dis- cussed in re&. 14-16). Without evidence that cannabinoids act through a specific receptor coupled to a functional effector system, researchers were prone to study the effects of cannabinoids on membrane properties, membrane-bound , production, metab- olism, and other neurotransmitter systems in ~im.8J7-1~As pointed out before,20 most of the biochemical studies employed concentrations of A9-THC that were in ex- cess of physiologically meaninfl concentrations that mlght be found in brain (for re- view, see re&. 8, 18). In addition, the criterion of structure-activity relationship was not met-that is, the potencies of various cannabinoids in the in pim assays did not correlate with their relative potencies in eliciting characteristic behavioral effects.8.20 Particularly damapg to the relevance of these in vim studies was the absence of enantioselectivity.20 However, several groups have reported enantioselectivity ofTHC isomers in various behavioral tests in vim. Martin's group bund that the potencies of (-) and (+) forms of each of the crj and tnzm isomers of A9-THC dSer by 10- to 100-bld in producing

19 20 ANNALS NEW YORK ACADEMY OF SCIENCES static in dogs, depressing schedule-controlled responding in monkeys, and in producing hypothermia and inhibiting spontaneous activity in mice.21 Hollister ct ul.22 showed cannabinoid enantioselectivityin human studies using indices of the sub- jective experience, or “high.” May‘s group fbund enantiosclectivity of a series of sp- thetic cannabinoids in tests of motor depression and analge~ia.~~-~~ One of May‘s compounds, (-)-9-nor-9~-hydroxyhexahydmcannabinol@-HHC), was used as a lead compound by Johnson and Melvinx fbr the synthesis of a rather 1- 1- series of structurally novel, dassical and nondassical, cannabinoids fbr studies of their potential use as (FIG. 1). The share physico- chemical properties with the natural cannabinoids and produce many behaviod and physiological effects characteristic of A9-THC, but are 5-1000 times more potent and show high enantioselectivity.

I HCI N-Methyllevonantradol (-)-CP47,407

on (-)-CP55,940 (-)-CP55,244

FIGURE 1. Chemical StNCNCSof A9-tenahydrocannabinol(A9-THC) and three synthetic can- nabinoids Accodng to the nomenclature of Johnson and Melvin,% A9--THC and 9-Nor-98- hydmxyhexahydnxannabinol(8-HHC)are defined as members of the ABC-tricyclic cannabinoid class. CP 55,940 is a hydmxypmpyl analog of a 2-(3-hydmxycydohuryl)phenol,defined as an AC- bicycllc cannabinoid.CP 55,244 is an ACD-mcydic cannabinoid with a rigidly positioned hydmxy- pmpyl moiety. (%produced from Herlcenham ct al.16) HERKENHAM: BRAIN CANNABINOID RECEPTORS 21

The availability of the nonclassical compounds revolutionized the study of the bio- chemical basis of cannabinoid activity. Howlett’s group used them in neuroblastoma cell lines to show inhibition of adenylate cykactivity.27 Such inhibition is enantio- selective, and the pharmacological profile correlates well with that observed by Martin’s group in tests of mouse spontaneous activity, catalepsy, body temperature, and analgesia .28 One of the nonclassical compounds, CP 55,940, was tritiated and used by Howlett’s group to identify and Mycharacterize a unique cannabinoid receptor in membranes from rat brain.l’The results from the centrifugation assay showed that [3H]CP 55,940 receptor binding is saturable, has high afEnity and enantioselectivity, and exhibits char- acteristics expected fbr a neuromodulator receptor associated with a guanine nucleo- tide regulatory (G) protein. Recently, we characterized and validated the binding of [3H]CP 55,940 in slide- mounted brain sections and described assay conditions to autoradiographically visu- alize the CNS distribution of cannabinoid receptors in a number of mammals, in- cluding humans.29 Autoradiography revealed a unique distribution that is similar in all mammalian species examined; binding is most dense in outflow nuclei of the basal gangha-the substantia nigra pars rcticulata and globus pallidus-and in the hippo- campus and cerebellum. The localization of dense receptors in the outflow nuclei of the basal gangha may account fbr some of the actions of cannabinoids. Dense binding localized in the globus pahdus, entopeduncular nudeus, and substantia nigra pars reticulata suggests an as- sociation of cannabinoid receptors with striatal efferent projections to these nuclei and, therefbre, a role fbr cannabinoids in motor control. In addition, binding may be local- ized on mesostriatal dopaminergic neurons, which would implicate a role fbr canna- binoids in direct control of dopamine release and, therefore, brain reward mechanisms. This report summarizes several key htures of our cannabinoid receptor localization studies: 1) validation that the in binding in brain sections is the same binding that mediates the e&cts of cannabinoids in pipg; 2) general htures of brain distribution in several species, including human; and 3) neuronal localization of cannabinoid re- ceptors to motor and/or limbic components of the basal gangha, assessed by making selective chemical lesions of either the striatal GABAergic efferent or dopaminergic afferent pathways interconnecting the caudate-putamen (CPu) and the substantia qra.

MATERIALS AND METHODS BkdingAswrys

Cannabinoid kzptw Bind&

The procedures fbr obtaining cryostat-cut sections of fiesh, frozen brain mounted on “subbed” microscope slides were described previ~usly.’~,~Assay conditions yield- ing 8690% specific binding are: incubation of slide-mounted sections at 37OC fbr 2 h in 50 mMTris-HCl buflkr, pH 7.4,with 5% bovine serum albumin (BSA) and 1-10 nM [3H]CP55,940 (sp. act. 76 Ci/mmole). Slides are washed at O°C fbr 4 h in the same buffer with 1%BSA and then dried. For use in competition studies to characterize and validate binding, natural and synthetic cannabinoid hgands were obtained from the 22 ANNALS NEW YORK ACADEMY OF SCIENCES

National Institute ofDrug Abuse and Pfizer, Inc. Names and stereochemical configura- tions of some of the cannabmoids are shown in FIGURE1. Autoradiography was pertbrmed on 15-25 pm-thick brain sections of rat (Sprague- Dawley), guinea pig (Hartley), dog (beagle), rhesus monkey and human (dying of non- neurological disorders). Sections were incubated in 10 nM [3H]CP 55,940 using op timizcd conditions, then washed, dried, and exposed to tritium-sensitive film (LKB or Amersham) for 3 to 4 weeks before developing. Developed films were digitized with a solid-state video camera and Macintosh I1 computer-based system for densitometry. Receptor densities were quantified using IMAGEe sohare (Wayne Rasband, Re- search Services Branch, NIMH).

Both the D1 and Dz receptor assays were carried out as previously described.30-32 For DI receptor binding, slides were warmed to mom temperature and incubated at 25OC for 2.5 h in 25 mM Tris-HC1 bufir, pH 7.5, with 100 mM NaCI, 1 mM MgCIz, 0.001% ascorbate, and 0.55 nM [3H]SCH 23390 (sp. act. 74.8 Ci/mmole). Slides were washed fbr 10 min in the same bufir at 4OC, dipped in deionized water, and dried. Nonspecific binding was determined by addition of 2 pM cis-flupenthixol and was typically <5% of total binding. Sections were exposed to film for 2 weeks. For Dz receptor binding, sections were incubated at 25OC fbr 1.5 h in 25 mM liis-HC1 buffer, pH 7.5, with 200 mM NaCI, 1 mM MgC12, 0.001% ascorbate, and 1.5 nM [3H]raclopride (sp. act. 64 Ci/mmole). They were washed for 3 min in the same bufir at 4OC with 100 mM NaCI. Nonspecific binding, determined by addition of 5 pM sulpiride, was typically <5% of total binding. Sections were exposed to film for 3 weeks.

Dopamine uptakc sin! Assay conditions were described previ~usly.~~~~Slides were incubated at 2OC for 30 h in 50 mM sodium phosphate buffer, pH 7.5, with 120 mM NaCI, 0.01% BSA, 0.001% ascorbate, 500 nM zmns-flupenthixol, and 0.25 nM [JHIGBR 12935 (sp. act. 53.1 Ci/mmole). They were washed br2 h in same buffer at 2OC. Nonspecific binding, determined by addition of 20 pM mazindol, was typically <15% of total binding. Sec- tions were exposed to film for 8 weeks.

Male rats (Sprague Dawley) were anesthetized and placed in a stereotaxic frame. A cannula was lowered into the caudate-putamen. Via an infusion pump and tubing, 1.5 pl(7.5 Icg) of ibotenate dissolved in normal saline was infused over 8 min. Animals survived for 2 or 4 weeks befbre sacrifice by decapitation. HERKENHAM: BRAIN CANNABINOID RECEmRS 23

6-OHDA Lesbu

Rats were prepared as above but were injected i.p. with desmethylimipramine (15 mg/kg) 30 min befbre infusion. The cannula was lowered into the medial fbrebrain bundle (mfb). Four pl(8 pg) of 6-OHDA dissolved in normal saline with 0.1% ascor- bate added was infused over 8 min. Animals survived fbr 4 weeks befbre sacrifice; at 2 weeks post-lesion they were tested fbr rotational behavior 40 min after adminism- tion of 5 mgkg of & sulfate (Sigma). Only those rats showing greater than 10 rotations per min during a 5-min test were used in the binding experiment.

RESULTS

A large series of cannabinoid and non-cannabinoid drugs was assayed to test fbr com- petitive displacement of [3H]CP 55,940 (TABLE 1). The competition curves and de- rived inhibition constants (Ki's) fbr the natural and synthetic cannabinoids provided a test for validation of binding. We found that highly significant (p < 0.OOOl) corn- lations exist between the Ki's and potencies of the drugs in tests of dog ataxia and human subjective experience, the two most reliable markers of cannabinoid acdv- ity.6,' The K:s also correlate very closely with relative potencies in tests of motor function (ataxia, hypokinesia, catalepsy), analgesia, and inhibition of contractions of guinea pig ileum and adenylate cyclase in neuroblastoma cell lines in piho.29 Enantio- selectivity is striking; the (-) and (+) forms of CP 55,244 differ by more than 10,000-fold in vim, a separation predicted by the rigid structure of the molecule (FIG. 1)26and by potencies in h.Natural cannabinoids lacking psychoactive proper- ties, such as , show extremely low potency at the receptor, and all tested non-cannabinoid drugs have no potency (TABLE 1). Autoradiography showed that in all species very dense binding is fbund in the globus pallidus, substantia nippars reticulata (SNR.), and the molecular layers of the cere- bellum and hippocampal dentate gyrus (FIGS. 2 and 3). Dense binding is also found in the cerebral cortex, other parts of the hippocampal formation, and striatum. In rat, rhesus monkey and human, the SNR contains the highest level of binding (FIG. 3). In dog, the cerebellar molecular layer is most dense (FIG. 2H). In guinea pig and dog, the hippocampal fbrmation has selectively dense binding (FIG. 2E, F). Neocortex in all species has moderate binding across fields, with peaks in superficial and deep layers. Very low and homogeneous binding characterizes the thalamus and most of the brain- stem, including all of the monoamine-containing cell groups, reticular fbrmation, pri- mary sensory, visceromotor and cranial motor nuclei, and the area postrema. The exceptions- hypothalamus, basal amygdala, central gray, nucleus of the solitary tract, and laminae 1-111 and X ofthe spinal cord-show slightly higher but still sparse binding (FIGS. 2 and 3). Quantitative autoradiography reveals very high numbers of receptors, exceeding 1 pmole/mg protein in densely labeled areas. Thus, cannabinoid receptor density is fir in excess of densities of neuropeptide receptors and is similar to levels of cortical benzodiazcpinq35 striatal dopamine,30*J6and whole-brain glutamate recept0rs.3~ 24 ANNALS NEW YORK ACADEMY OF ScIBNCBs

TABLB1, Potencies of Some Cannabmoids in the Section Bind~ngand Other Assays Catalepsy/ Mouse Cyckse Human Ataxia Analgesia Inhibition ''HI@" Compound Ki (nM) (mrn (mglkg) (nM) (m@ CP 55,940 (-AC) 15 (W 0.35 0.7 25 CP 56,667 (+AC) 470 >10 >15 >5,000 CP 55,244 (-ACD) 1.4 0.09 0.09 5 CP 55,243 (+ACD) 18000 >10 >10 >lO,OOo CP 50,556 14 1.5 0.4 100 0.5 CP 53,870 26000 >10 6.5 >5,OOO CP 54,939 14 0.05 0.7 7 120 0.03 100 1 P-HHC 124 0.1 1.6 a-HHC 2590 0.5 >so ( -)A9-THC 420 0.5 10 100 1 ( +)A9-THC 7700 >2.0 >100 A~-THC 498 0.5 8.8 2 1l-OHd9-THC 210 0.05 1.9 1 TMA-AS-THC 2300 8P-OH-A9-THC 4200 10 8a-OH-A9-THC 8700 10 11-OH- 800 Cannabinol 3200 >15 Cannabidiol 53000 inactive >lo0 >30 Cannabigem1 275000 >7 9-COOH-1 1-nor-A9-THC 75000 >40 10 9-COOH-1 1-nor-A8-THC Inactive CP analogs were synthesized at Pfizer Central Research; their structures are given in Johnson and Melvin.26The first 6 analogs are enantiomeric pairs, as are a- and B-HHC and (-) and (+)As- THC. CP 50,556 is Ievonantradol; CP 53,870 is dextronanaadol; CP 54,939 k desacetyl ; TMA-AS-THC is trimethylammonium-A~-THC.The last two compounds arc A9-THC metabolites. Ki's f standard deviations are derived from binding surfice analysis. Dmp which at 10 pM concentration show no inhibition of [3H]CP 55,940 bindug are: amphetamine, P-estradiol, cis-flupenthixol (dopamine receptor hgand), , comcosterone, cydohexyl- adenosine, , etorphine ( meptor &and), y-amino butyric acid (GABA), gluta- mate, leukotriene B4 and D4 (both at 1 pM),lysergic acid diethylamide (LSD), (PCP), prostaglandin E2, h15-1788 ( recc tor Wd), and thujone (the active ingredient of absinthe). (Modified from Herkenham ct af.4 9

The single injection of ibotenate into the caudate-putamen resulted in a small cen- tral site of nonspecific destruction marked by gliosis and a much laqp (appmximately 3 x 4 mm) surrounding area of selective neuronal degeneration, in accordance with previous descriptions of toxicity in the dose mge and location used.% In the &cted striatal territory, the losses on the lesion relative to conrsponding territory on the con- trol side were the most profound fbr the dopamine D1 receptors, showing a 96% re- duction in binding at 4 weeks WLE2). Cannabinoid and D2 receptors were reduced FIGURE 2. Autoradiography of 10 nM [JHICP 55,940 binding in brain. Tritium-sensitive film exposed for 4 weeks, developed and computer digtized. Images were photographed directly from the computer monitor. Gray levels represent relative levels of meptor densities. Sapttal section of rat brain in A. Comnal brain sections of human in B, D, and G; rhesus monkey in C and I; dog in P and H;and rat in J. Horizontal section of guinea pig brain in E. Inatr show non- specific binding in adjacent sections (miniaturized images are shown). A&- :Am, amyg- dala; Br St, brain stem; Cer, cerebellum; CG,centnl py;C, caudate; Col, colliculi; CP, caudate- putamen; Cx, cerebral cortex; DG, dentate gyrus; DH, dorsal horn of spinal cord; Ent Cx, en- torhinal cortex; Ep, entopeduncular nucleus (homolog of GPi); GP, globus pallidus (e, external; i, internal); Hi, hippocampus; Hy, hypothalamus; NTS, nucleus of solitary tract; P, putamen; SNr, substantia nigra pars xticulata; Th, thalamus; VH,ventral horn ofspinal cord. (Reproduced from Herkenham ct id.? 26 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 3. Rclativc densities of cannabinoid reccpton acm brain structures in rat, rhesus monkey, and human. Autoradiographic images were dqytized by a solid-state camera and Macin- tosh I1 computer-based system for quantitaavc densitomcay using ImqS soh(Wayne Ras- band, Rcsmrh Services Branch, NIMH). ?iansmittance lcvels were converted to holcs/mg tissue using tritium standards, then normalized to the densest st~~ctu~in each animal (SNr hr all three). For every section incubated for total binding, an adjacent section was incubated in the presence ofCP 55,244 to permit subtraction of nonspeafic binding on a regional basis. Structure abbreviations not given in FIGURE2 lepd arc: Cing Cx, cingulate cortex; Hipp CA1, hippo- campal field CAl; Med Hypothal, medial hypothalamus; Sp Cd SG, substantia gelatinma of spinal cord ('only rat measured); Rct Form, reticular formation; WM (cc), white matter of corpus callaurn. (Reproduced hmHerkenham ct af.9

by 78-8096 in the a&cted temtory, and the dopaminc uptake site, which mides on afferent dopaminergic axons, was slightly reduced at 4 weeks (not shown). Both cannabinoid and D1 dopamine receptors am lost in similar patterns (FIG. 4) and amounts (GUILE 2) in projection zones of lesioncd striad eflkrent neurons. In agreement with known medial-lateral topography of striad projections,39 receptor losses in both GP and SNR were ptest medially, with sparing of binding in lateral parts receiving projections from unltsioned parts of the lateral and posterior caudate- putamen (FIG.4). For cannabinoid receptor binding, losses in the labeled striato@ pathway were also evident (not shown).

Mfb 6-OHDA Lcrionr

Nissl-stained sections of the substantia nigra showed unilateral loss of neumns in the SNC (FIG. 4e). Autoradiography showed no change in cannabinoid receptor HERKENHAM: BRAIN CANNABINOID RECEmRS 27

TABLE2. Effects of Unilateral Saiatal Ibotenate Lesions at 4 Week Survival (n = 5) Cannabinoid D- 1 D-2 Structure Receptor L % R Receptor L % R Receptor L%R CPU (L) 536 f 37 20 69 f 18 4 54 f 25 18 (R) 2843 f 849 1558 f 111 299 f 22 GP (L) 719 f 182 15 13 f 6 12 (R) 4763 f 969 106 f 28 EP (L) 905 f 340 22 57 f 23 24 (R) 4159 f 834 240 f 39 SNR (L) 1023 f 174 16 164 f 16 13 (R) 6421 f 635 1254 f 97 Values are in fmoles/mg protein and are the means and standard deviations of specific binding to approximately 50% of the total number (&) of receptors and 10% of uptake sites in each region, since concentrations were near the & or below, in the case of [3H]GBR-12935, for each drug. Corresponding locations on the control (R)side were outlined and measured. All left-right differences are significant at the p < 0.005 level of confidence except for the dopamine uptake site, which is significant at thep< 0.05 level (Student‘s paired t-test). Abbreviations: CPu, caudate putamen; EP, entopeduncular nudeus; GP, globus pallidus; SNR, substantia nigra pars compacta. (Data are from Herkenham ct al.32) binding in either the striatum (FIG.4d) or the nigra (FIG.4e), whereas dopamine up take sites were lost throughout the striatum and nipon the lesioned side (FIG. 4f). Quantitative densitometry showed major losses of dopamine uptake sites in the caudate-putamen, accumbens nucleus (ACb), and substantia nip pars compacta (SNC), but no loss of cannabinoid receptors (TABLE3).

DISCUSSION

The section binding assay is easy to perfbrm, is reliable, and shows high sensitivity to manipulations of binding conditions, such as the addition of guanine nucleo-

TABLE3. Effect of Unilateral 6-OHDA Lesions at 4 Week Survival (n = 4) Cannabinoid Dopamine Structure Receptor L%R Uptake Site L%R

~ _____ ~_____ CPU (L) 4395 f 266 99 62 f 57 20 (R) 4422 f 282 311 f 60 ACb (L) 2533 f 384 102 38 f 30 39 (R) 2491 f 463 98 f 13 SNC (L) 1709 f 320 95 If1 7 (R) 1807 f 264 17 f 7 Values are in holeslmg protein and are the means and standard deviations of specific binding to approximately 50% and 10% of the total number (B-) of receptors and uptake sites in each eon,respectively. Densitomehic measures of caudate-putamen (CPu), nudeus accumbens (Acb), and substantia nigra pars compacta (SNC) were each taken from the entire structure at the levels shown in FIGURE3. Left-nght cannabinoid receptor dif€erences were not significant (Student’s paired t-test); dopamine uptake site left-nght di&rences were significant in the CPu (p < 0.01), ACb (p = 0.05),and SNC (p < 0.03). (Data are from Herkenham ct d.32) 28 ANNALS NEW YORK ACADEMY OF SCIENCES

PIGURE 4. Lesion data showing localization of cannabinoid receptors to striaton@ neurons (a+) and not to dopaminecgic nigrostciatal neurons (d-g). As shown in a-c, a unilateral deposit of ibotenate was placed into the caudate-putamen. Nissl-stained section in a shows the area of selective neuronal loss and the enlwd lateral Ventricle (LV). At 4 weeks survival, the losses of cannabinoid receptors in the caudate-putamen (b) and substantia nip pars retidata (SNR) (anmp in c) are shown autoradiographically. The losses are topographic; note sparing of laterally situated striatal neurons and their projections. As shown in d-g, a unilateral lesion of the mesencephalic ascending dopamine system was made by depositing 6-OHDA into the medial forebrain bundle. At 4 weeks suMval, degenerationof dopamine neurons in the substantia nigra pars compacta (SNC) is evident in the Nkl stain (min g) and by the losses of dopamine uptake sites in the SNC (f),whem cannabinoid receptor binding is una&cted in the striatum (d) and nip (c). AbW: ACb, nucleus accumbens; ml, medial lemniscus; lb, offictory tubercle. Magnification bar measw 2 mm. (Modified from Herkenham ct d.32)

tidesz9BSA appears to act as a carrier to keep cannabinoids in solution without ap preciably aflkcting binding kinetics. The low nonspecific binding and absence of binding in white matter indicates that the autoradiographic patterns are not affected by &and lipophilia. The inclusion of BSA in the incubation medium may actually mimic the disposition of cannabinoids administered in piw, as they would quickly com- plex with serum albumin or other carriers in the blood. HERKENHAM: BRAIN CANNABINOID RECEPTORS 29

The structure-activity profile suggests that the receptor defined by the binding of [3H]CP 55,940 is the same receptor that mediates many of the behavioral and phar- macological effects of cannabinoids (TABLE l), including the subjective experience termed the human “high.” All other tested psychoactive drugs, neurotransmitters, ste- roids, and at 10 pM concentrations Med to bind to this receptor. There was no compelling evidence fbr receptor subtypes from that analysis. Autoradiographyof cannabinoid receptors rev& a heterogeneous distribution pat- tern that conforms to cytoarchitectural and hnctional subdivisions in the brain. The distribution is unique-no other pattern of receptors is similar-and it is similar across several mammalian species, including human, suggesting that cannabinoid receptors are phylogenetically stable and conserved in evolution. The distribution appears to be similar to the distribution of the mRNA probe hybridized to a rat brain cannabinoid receptor gene.40 The locations of cannabinoid receptors help to understand cannabinoid pharma- cology. High densities in the hippocampus and cerebral neocortex implicate roles hr cannabinoids in cognitive functions. High densities in axons and terminals of the GABAergic striatal neurons of the basal pgha and of glutamatergic granule cells of the cerebellum suggest a modulatory role in movement systems. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of A9-THC are not lethal. The results of the 6-OHDA lesions indicate that cannabinoid receptors do not re- side on mesencephalic dopamine neurons projecting to either the caudate-putamen or the ACb. Systemically administered A9-THC has been shown to elevate extracel- Mar levels of dopamine in the caudate-putamen41 and ACb.42 The mechanism of ac- tion appears to be indirect, as the effects are attenuated by nal~xone.~~Nevertheless, it has been proposed that drugs which elevate dopamine levels in the striaturn are those that are known to have abuse liability in humans.43~~In humans, cannabinoids can produce a fkeling of as part of the subjective experience known as the mari- juana “high,” but , dizziness, thought disturbances, and sleepiness are also rep~rted.~.~.~~Animals generally will not self-administer A9-THC.4s*MCannabinoids did not lower the threshold fbr electrical self-stimulation in one study.” In another study they did,a but apparently both this phenomenon and the enhancement of basal dopamine efflux from the ACb by A9-THC are strain-specific, occuning only in Lewis rats.49 Thus, the effects of cannabinoids on dopamine circuits thought to be common mediators of reward are indirect and different from those of drugs such as cocaine and which directly affect extracellular dopamine levels and produce craving and powem drugseeking behavior. Accounts of use in humans stlrss the loosening of associations, fiagmen- tation of thought, and confusion on attempting to remember recent accurrences.7.50 These cognitive effects may be mediated by receptors in the cerebral cortex, especially the receptordense hippocampal cortex. The hippocampus “gates” infbrmation during memory consolidation and codes spatial and temporal relations among stimuli and re- sponse~.~~.~~A9-THC causes memory “intrusions,”53 impairs temporal aspects of per- formance,” and suppresses hippocampal electrical activity.5s The localization of cannabinoid receptors in motor areas suggests therapeutic ap plications. Cannabinoids exacerbate hypokinesia in Parkinson’s disease but are ben- eficial for some forms of , txtmor, and spasticity.6.7~~~The association of cannabinoid receptors with GABAergic striatal projection neurons suggests roles fbr cannabinoids in control of movement, perhaps therapeutic roles in hyperkinesis and dystonia. Cannabinoids have been shown to be beneficial hrsome hrms of dystonia, 30 ANNALS NEW YORK ACADEMY OF SCIENCES mmor, and spasticity.6J~~S9Lack of association of cannabinoid receptors with dopa- mine neurons indicates that cannabmoids do not directly a&ct dopamine release asso- ciated with rrward and *-seeking behavior. Further work may show the basis for the reported usefulness in conmlling and stimulating appetite in patients re- ceiving chemothenpy hrcancer or AIDS. Finally, the development of an antagonist could lead to additional therapeutic applications. The section binding assay can be used to screen the potencies of novel drugs and serve to idenrify cannabinoid receptor subtypes, which could lead to renewed intemt in developing cannabinoid drugs without unwanted side &cts.

REFERENCES

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