Jacob Bell
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Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson's disease
VINCENZO DI MARZO12, MICHAEL P. HILL" , TIZIANA BISOGNO2, ALAN R. CROSSMAN* and JONATHAN M. BROTCHIE*1
* School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom; Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, 80072, Arco Felice, Naples, Italy; and
" Motac Neuroscience Ltd., Manchester MI3 9XX, U.K.
1Correspondence: J.B., 1.124 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Rd, Manchester, M13 9PT, U.K. E-mail: j.brotchie@man.ac.uk and V.D.M., Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, Via Tolano 6, 80072 Arco Felice Naples, Italy. E-mail: vdimarzo@icmib.na.cnr.it
ABSTRACT
In recent years, cannabinoid receptors and their endogenous ligands (endocannabinoids) have been identified within the brain. The high density of CB1 cannabinoid receptors within the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement and in basal ganglia-related movement disorders such as Parkinson's disease. However, whether endocannabinoids play a role in regulating motor behavior in health and disease is unknown. Here we report the presence in two regions of the basal ganglia, the globus pallidus and substantia nigra, of the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide. The levels of the latter compound are ~threefold higher than those previously reported in any other brain region. In the reserpine-treated rat, an animal model of Parkinson's disease, suppression of locomotion is accompanied by a sevenfold increase in the levels of the 2AG in the globus pallidus, but not in the other five brain regions analyzed. Stimulation of locomotion in the reserpine-treated rat by either of the two selective agonists of D2 and D1 dopamine receptors, quinpirole and R-(±)-3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (Cl-APB), respectively, results in the reduction of both anandamide and 2AG levels in the globus pallidus. Finally, full restoration of locomotion in the reserpine-treated rat is obtained by coadministration of quinpirole and the selective antagonist of the cannabinoid CB1 receptor subtype, SR141716A. These findings indicate a link between endocannabinoid signaling in the globus pallidus and symptoms of Parkinson's disease in the reserpine-treated rat, and suggest that modulation of the endocannabinoid signaling system might prove useful in treating this or other basal ganglia-related movement disorders.–Di Marzo, V., Hill, M. P., Bisogno, T., Crossman, A. R., Brotchie, J. M. Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson's disease.
Key Words: anandamide - 2-arachidonoyl glycerol - cannabinoids - dopamine - receptors
INTRODUCTION
ALTHOUGH THE PSYCHOACTIVE properties of cannabis have been widely exploited and studied, the physiological role of cannabinoid receptor-mediated signaling is unknown. The search for such a role has been heightened by the identification of two endogenous ligands for the cannabinoid receptors, 2-arachidonoylglycerol (2-AG) and anandamide (1 2 3) , named 'endocannabinoids'. These molecules were suggested to have a neuromodulatory action (for a review, see ref 4 ), but to date there has been no clear demonstration of a role for these endocannabinoids in controlling any aspect of behavior.
The high levels of 'central' cannabinoid (CB1) receptors within the basal ganglia (5) suggest a potential role for endocannabinoids in the control of voluntary movement and in basal ganglia-related movement disorders such as Parkinson's disease (6 7 8) . Within the basal ganglia, cannabinoid receptors are particularly prominent on the terminals of GABAergic projections from the striatum to the globus pallidus and substantia nigra pars reticulata (the 'indirect' and 'direct' striatal output pathways, respectively) (5) . The interplay between activity in these pathways is key to the selection and initiation of appropriate voluntary movements and is tightly regulated by the dopaminergic nigrostriatal system (9 , 10) . Dopamine receptors exert differential control of striatal outputs, D2-class dopamine receptors inhibiting the activity of the indirect pathway while the direct pathway is activated by D1-class dopamine receptors (11) .
Stimulation of cannabinoid receptors in the globus pallidus, by exogenous cannabinoid receptor agonists, reduces uptake of the inhibitory neurotransmitter GABA (12 , 13) and reduces voluntary movements, producing Parkinson-like symptoms (14) . Stimulation of cannabinoid receptors also reduces the anti-parkinsonian actions of D2, but not D1, dopamine receptor agonists (15) . It is possible, therefore, to hypothesize a role for endocannabinoids in the modulation of signaling by the indirect striatal output.
In this study, we have measured the levels and distribution of anandamide and 2-AG in rat brain by using a highly sensitive and specific isotope dilution gas chromatography-mass spectrometric (GC-MS) method (16) . Furthermore, we have compared endocannabinoid levels in different brain regions of vehicle-treated or reserpine-treated rats, a widely used animal model of Parkinson's disease, in the presence or absence of dopamine receptor agonists. We provide evidence suggesting that changes in endocannabinoid signaling in the globus pallidus of the reserpine-treated rat correlate with the modulation of voluntary movement in this model.
MATERIALS AND METHODS
Animal treatment
Male Sprague Dawley rats (200—250 g) were injected subcutaneously with 3 mg kg-1 reserpine (dissolved in 1% glacial acetic acid in distilled water) or vehicle (1 ml kg-1) under light inhalation anesthesia (Halothane). Assessment of locomotion was carried out 18 h later. Animals were acclimatized in the experimental room for at least 30 min before drugs were administered and the assessment commenced. Drugs administered were Cl-APB (R-(±)-3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide, 0.1 mg/kg), quinpirole (0.1 mg/kg), and SR141716A (N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride, 1 mg/kg). The locomotion of animals was measured using an automated movement detection system (Benwick Data-logger, Linton Instrumentation, Diss, U.K.), which consisted of a frame (52 cmx36 cm) containing an array of infra-red beams (organized into a grid 19x13) situated along the exterior of a frame. Animals were isolated from the frame by a Perspex box placed inside the frame. The movement of animals was detected when the animal passed from one grid to another and so broke an infrared beam. Locomotor activity was monitored using Amlogger software (Linton Instrumentation), which logged data at 5 min intervals over a period of 60 min. The index of locomotion used in the studies was mobile counts, which are recorded when the central position of the animal changes by more than two grids in any one second period. All studies were performed under a project license granted by the Home Office of the U.K. Government under the Animals (Scientific Procedures) Act 1986.
Tissue dissection, extraction, and endocannabinoid level measurement
Anandamide and 2-AG in lipid extracts from different brain regions were quantitated following the isotope dilution GC-MS procedure described previously (16) . Briefly, brain areas from eight rats belonging to each different group of treatments were dissected, pooled, and frozen in liquid nitrogen in the Manchester laboratory. A maximum of 3 min elapsed between tissue dissection and freezing. The frozen tissue was sent on dry ice to the Naples laboratory, stored at -80°C, and weighed prior to its extraction with 3 volumes of chloroform/methanol/Tris-HCI 50 mM buffer (pH 7.4) (2: 1: 1 by vol). The extraction mixture contained 2 nmol each of d8-anandamide and d8-2-AG synthesized from d8 arachidonic acid and ethanolamine or glycerol, as described previously (1 , 22) . The two phases were separated and the aqueous phase extracted three more times with an equal volume of chloroform. The pooled organic phases were dried down in a rotating evaporator, transferred to siliconized vials, and purified by normal phase-high pressure liquid chromatography (NP-HPLC) carried out as described previously (22) . Monoarachidonoylglycerols and anandamide standards were eluted after 18—23 min and 27—28 min, respectively. The mono-arachidonoylglycerol HPLC fraction contained both 1- (3) and 2-stereoisomers. HPLC fractions were dried down under a flow of nitrogen and derivatized with 15 µl N-methyl-N-tris-methyl-sylyl-trifluoroacetamide containing 1% trimethylchlorosylane for 2 h at room temperature, thus yielding the tris-methyl-sylyl derivatives of anandamide and 2-AG. The two derivatized fractions were analyzed separately by GC-MS carried out as described previously (16 , 22) . The tris-methyl-sylyl ethers of both deuterated and nondeuterated anandamide, 2-AG and 1- (3) arachidonoylglycerol standards were eluted after ~18, 19, and 19.5 min, respectively. MS detection was run in the selected ion monitoring mode to improve sensitivity. Selected ions for anandamide were at m/z = 427 and 419, corresponding to the molecular ions of d8-anandamide and nondeuterated anandamide, and m/z = 412 and 404, corresponding to the loss of a methyl group in both compounds. Selected ions for 2-AG were at m/z = 530 and 522, corresponding to the molecular ions of d8-2-AG, and nondeuterated 2 AG and m/z = 515 and 507, corresponding to the loss of a methyl group in both compounds. The endogenous cannabinoids were identified on the basis of the presence, at the same retention time as the deuterated internal standards, of the corresponding MS signals with the appropriate relative abundance. The amounts of anandamide and 2-AG were calculated from the peak area ratios between the signals at m/z = 404 and 412, and m/z = 507 and 515, respectively. A linear correlation between these area ratios and the amounts of anandamide (25—10000 pmol) or 2-AG (50—10000 pmol) standards was observed in separate studies. In the case of 2-AG, the amounts of the 1- (3) isomers, which are almost exclusively formed during tissue workup and lipid purification (23) and were 25—30% of the total monoarachidonoylglycerols, were added to the amounts of the 2-isomer. Each brain region in each group of animals was analyzed in quadruplicate. Measurements in each monoplicate were carried out in duplicate. Results are expressed in pmol/g wet tissue weight and are means ± SEM of the four separate determinations. Means from different groups were compared by using 1-way ANOVA, followed by Tukey-Kramer analysis. It can be estimated that 96 different samples (six brain regions in four different groups of treatments, each in quadruplicate, each from eight rats) had to be extracted, purified, and analyzed in the present study.
RESULTS AND DISCUSSION
We have used a sensitive and specific assay using isotope dilution GC-MS (16) to measure the levels and distribution of the endocannabinoids anandamide and 2-AG in the brain of vehicle-treated Sprague Dawley rats (Fig. 1 ). Anandamide levels were similar in striatum, cerebral cortex, cerebellum, and hippocampus and the values reported here are similar to those previously described for these regions (16 , 17) . Although cannabinoid receptors are expressed at high levels within the globus pallidus and substantia nigra, the levels of endocannabinoids within these important motor regions have not previously been reported. The levels of 2AG in these two regions were similar to other brain regions and generally lower than those previously observed in Wistar rat brain (16) . However, the levels of anandamide in the globus pallidus and substantia nigra were ~threefold higher than those previously reported in any brain region (Fig. 1B ). In the globus pallidus this difference was statistically significant (P<0.05). These data further support the speculation (12 13 14) that endocannabinoids may play a key role in the functioning of the striatal outputs in the control of movement.
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Figure 1. Levels of the endocannabinoids 2-AG (A) and anandamide (B) in vehicle-treated rats (□) and after induction of hypokinesia by reserpine treatment (▪). Data are presented as mean ± SEM of 4 separate experiments each with samples from 8 individual animals. ***P<0.001 compared to vehicle by 1-way ANOVA with post hoc Tukey-Kramer test. Abbreviations, SNr, substantia nigra pars reticulata; GP, globus pallidus.
To investigate this potential role of endocannabinoids in motor control, we have used the reserpine-treated rat as a model system in which the initiation and execution of movements can easily be assessed and manipulated. In this model, catecholamine stores are depleted and a motor syndrome characterized by decreased initiation and speed of voluntary movements, rigidity, and a hunched posture is observed (18) . These symptoms bear many similarities to those seen in Parkinson's disease (19) and are ameliorated by dopaminergic anti-parkinsonian drugs (18 19 20 21) . Thus, the reserpine-treated rodent provides a useful model of Parkinson's disease; indeed, it was in this model that the key role of decreased dopamine transmission in parkinsonism was identified and the therapeutic potential of dopamine replacement therapy in Parkinson's disease first highlighted (19) . Other currently used animal models of this disorder, e.g., the 6-hydroxy-dopamine (6-OHDA) -lesioned rat or the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -lesioned monkey were not utilized in this study for two different reasons: 1) although more selective than reserpine for the depletion of dopamine stores, 6-OHDA-induced lesions do not lead to overt symptoms of Parkinson's disease, an important point for our study aimed at finding a possible correlation between these symptoms and endocannabinoid levels; 2) it was estimated that at least 72 MPTP-lesioned monkeys would have been necessary to carry out a study with equivalent statistical rigor as the present one.
After treatment of rats with reserpine, locomotion was dramatically reduced (locomotor activity in vehicle-treated rats was 1251±228 mobile counts/h compared to 9±2 in reserpine-treated rats, P<0.001, t test, n=12) as has been previously described. In association with this decreased locomotion, a sevenfold elevation in the levels of 2AG in the globus pallidus was observed (Fig. 1) . Although average 2AG levels increased slightly in all the other brain regions analyzed, this effect was never as marked as in the globus pallidus (e.g., a 2.5-fold stimulation was found in striatum) and was never statistically significant. There was no statistically significant reserpine-induced change in the levels of anandamide in any brain region. These data highlight the fact that catecholamines play an important role in the inhibitory regulation of 2AG synthesis in the globus pallidus. The most likely mechanism underlying such an effect would be an action of dopamine on the striatal neurons projecting to the globus pallidus. In situations of decreased dopamine transmission, the activity of the indirect striatal output pathway is increased (9) . Increased depolarization of striatopallidal terminals after reserpine treatment may enhance 2AG synthesis, as depolarization has been shown to elevate 2AG levels in other cells (22 , 23) . The mechanism underlying this depolarization-induced increase in 2AG levels involves activation of calcium-dependent enzymes such as phospholipase C (23 , 24) , phosphatidic acid phosphohydrolase, and phospholipase A2 (25) . The functional relevance of this enhanced endocannabinoid synthesis in the globus pallidus may be great, since (as described above) activation of cannabinoid receptors in the globus pallidus by exogenous agonists has been shown to elicit parkinsonian symptoms (14) . Thus, enhanced 2AG signaling in the globus pallidus might be part of the neural mechanisms underlying hypokinesia in the reserpine-treated rat.
These data also underscore the complexity by which endocannabinoid levels might be regulated in vivo. Thus, catecholamines appear to regulate 2AG levels differentially in different populations of striatal output neurons (levels in the substantia nigra, the target of the direct striatal output pathway, were not significantly elevated) whereas different endocannabinoids are regulated differentially within a given region (pallidal anandamide levels were not elevated after reserpine treatment). The lack of effect on anandamide levels may be due to the fact that there is high endocannabinoid tone at rest in the globus pallidus and the basal amounts of anandamide actually represent the maximal level of synthesis possible in this structure. As anandamide levels are elevated by neuronal depolarization (26) , this would suggest that basal firing rate in the indirect striatal output pathway is sufficient to maximally stimulate anandamide synthesis. During the early preparation of this paper, Giuffrida et al. (27) reported that anandamide, but not 2AG, is released into microdialysates from the dorsal striatum of freely moving rats. In a previous study (23) the same authors found that 2-AG, but not anandamide, could be produced by electrically stimulated hippocampal slices. These reports point to the possible differential regulation of different endocannabinoids in different brain regions.
Administration of the D2 dopamine receptor agonist quinpirole (0.1 mg/kg) to reserpine-treated rats reduced 2AG and anandamide levels in the globus pallidus (Fig. 2 ). Anandamide levels were reduced by 75% whereas 2AG levels were reduced by 53%. These falls in anandamide and 2AG levels were accompanied by increased locomotion in reserpine-treated rats (Fig. 3 ).Quinpirole did not significantly alter 2AG or anandamide levels in any other region of the brain (data not shown). The reduction in pallidal 2AG and anandamide levels accompanying alleviation of parkinsonian symptoms by the D2 dopamine receptor agonist is further suggestive of a causative role for enhanced endocannabinoid signaling in the indirect pathway in parkinsonism. Indeed, stimulation of D2 receptors alleviates parkinsonism by reducing the activity of the indirect striatal output pathway (28) . The reduction in 2AG and anandamide levels may be due to this reduction in activity and, in the case of 2AG, reflect reversal of the depolarization-induced activation of calcium-dependent enzymes described above; anandamide levels have also been shown to be maintained by depolarization (26) . It is worthwhile mentioning that infusion with quinpirole was recently shown to enhance anandamide release from the dorsal striatum of normal rats (27) . The authors presented no evidence to show that endogenous dopamine tonically increases anandamide release from the striatum, and we found here no decrease in anandamide levels in the striatum of catecholamine-depleted rats (Fig. 1B ). However, this report, taken together with our present data, may suggest that in different regions of the basal ganglia, and under different conditions, stimulation of D2 dopamine receptors may have opposing consequences on endocannabinoid biosynthesis.
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Figure 2. Effect of D1 and D2 dopamine receptor stimulation on the levels of the endocannabinoids 2 AG (A) and anandamide (B) in the globus pallidus in the reserpine-treated rat. Levels of endocannabinoids were measured after alleviation of parkinsonian symptoms by the D2 dopamine receptor agonist quinpirole (0.1 mg/kg) and the dopamine D1 receptor agonist Cl-APB (0.1 mg/kg). Data are expressed as % control (vehicle-treated, reserpinized animals) and presented as the mean ± SEM of 4 independent experiments, each with samples from 8 individual animals. *P<0.05, **P<0.01, ***P<0.001, compared to vehicle, 1-way ANOVA with post hoc Tukey-Kramer test.
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Figure 3. Effect of the CB1 cannabinoid receptor antagonist SR141716A (1 mg/kg) on locomotion induced by administration of either a D2 dopamine receptor agonist (A, B) or a D1 dopamine receptor agonist (C, D) in the reserpine-treated rat. A, C) Data are presented as mean mobile counts per 5 min period (± SEM, n=12) after administration of vehicle (▴), the D2 dopamine receptor agonist quinpirole (0.1 mg/kg) alone (□), quinpirole plus SR141716A (▪), the D1 dopamine receptor agonist Cl-APB (0.1 mg/kg) alone (□), or Cl-APB plus SR141716A (▪). B, D) Data are presented as total mobile counts cumulated over a 1 h assessment period. *P<0.05, ***P<0.01 compared to vehicle, " " P<0.01 compared to agonist alone, 1-way ANOVA with post hoc Tukey-Kramer test.
Administration of the D1 dopamine receptor agonist Cl-APB (0.1 mg/kg) was also accompanied by a reduction in endocannabinoid levels in the globus pallidus (Fig. 2) and an increase in locomotion (Fig. 3) . Whether this reduction in endocannabinoid levels in the globus pallidus could be responsible for the anti-parkinsonian actions of Cl-APB is unclear since the anti-parkinsonian actions of D1 receptor stimulation are mediated through activation of the direct, rather than indirect, striatal output pathway (28) . However, basal ganglia outputs to the thalamus are a vital link in basal ganglia-thalamo-cortical-basal ganglia loops, the cortical components of which impinge directly onto striatal output neurons (10) . Therefore, activation of the direct pathway might indirectly lead to decreased excitation of the indirect striatopallidal pathway that (as described above) would be expected to reduce levels of endocannabinoids in the globus pallidus. In this case, the reversal in abnormalities in endocannabinoid transmission in the globus pallidus induced by Cl-APB might be a consequence of an anti-parkinsonian action resulting from normalization of abnormalities in the direct pathway. In any event, these data, taken together, point to a clear reverse correlation in reserpine-treated rats between locomotion and endocannabinoid levels in the globus pallidus.
To address further the issue of whether the anti-parkinsonian actions of dopamine receptor stimulation are mediated through a reduction in endocannabinoid signaling, we assessed the behavioral effects of treatment of reserpinized rats with quinpirole and the selective antagonist of CB1 receptors, SR141716A (29) . SR141716A markedly potentiated the ability of the D2 dopamine receptor agonist to elicit locomotion in the reserpine-treated rat (Fig. 3) . This finding strongly suggests that endocannabinoids counteract the ability of D2 receptor stimulation to elicit locomotion and might be responsible, in part at least, for the abnormalities in the indirect pathway that underlie the generation of parkinsonian symptoms. SR141716A had no significant effect on locomotion induced by the D1 agonist Cl-APB (Fig. 3) . This suggests that the reduction in 2AG levels brought about by Cl-APB (and the subsequent stimulation of the direct striatal output pathway) is indeed a consequence rather than a cause of its anti-parkinsonian action, as suggested above. The lack of effect of SR141716A on D1 agonist-induced locomotion and the finding that SR141716A has no effect on locomotion in non-parkinsonian animals (data not shown) also suggest that it is unlikely that the effects of SR141716A in enhancing D2 agonist-mediated locomotion are due to a nonspecific increase in locomotion or to the proposed inverse agonist properties of this compound (30) . Furthermore, our findings with SR141716A and quinpirole in the reserpine-treated rat are in agreement with an analogous enhancement by the CB1 antagonist of quinpirole-induced locomotion in normal rats reported early during the preparation of this manuscript (27) .
Our data show that reducing not only the levels, but also the action, of endocannabinoids in the globus pallidus may lead to enhanced locomotion in the reserpine-treated rat. These findings, therefore, suggest that the selection, initiation, and execution of movement by stimulation of the GABAergic indirect striatal output pathway, in the parkinsonian brain at least, may be under a negative control by endocannabinoids. Enhanced pallidal GABAergic transmission is a key component of the neural mechanisms underlying the symptoms of parkinsonism (9) . Stimulation of cannabinoid receptors on terminals of the indirect pathway reduces GABA reuptake (12 , 13) , thereby enhancing GABA transmission in the globus pallidus. Enhanced endocannabinoid signaling in the globus pallidus would thus elicit parkinsonian symptoms.
In conclusion, high levels of endocannabinoids are found in regions of the basal ganglia receiving input from the striatum. Endocannabinoids and dopamine may have a close functional relationship whereby, under physiological conditions, dopamine acts to suppress 2AG signaling in the globus pallidus while endocannabinoids act as a 'brake' on dopamine-receptor-stimulated locomotion in this as well as other areas of the basal ganglia (see also ref 27 ). Overactivation of the endocannabinoid system in the globus pallidus may play a part in the generation of parkinsonian symptoms. We suggest that endocannabinoids provide a neurochemical substrate for the interactions between transmitter systems that are, according to current thinking, fundamental to the appropriate selection and initiation of voluntary movements. Our findings may help to identify novel avenues of therapeutic intervention in Parkinson's disease (31) .
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Figure 3C.
ACKNOWLEDGMENTS
We thank Ms. Gerolmina Ambrosino and Mr. Ralph Davies for valuable technical assistance. This work was supported by the MRC (U.K., to J.M.B. and A.R.C.) and the MURST (3933, to V.D.M.).
FOOTNOTES
2 Affiliated with the National Institute for the Chemistry of Biological Systems, C.N.R.
Received for publication August 20, 1999. Revision received November 5, 1999.
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Source: Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson's disease
VINCENZO DI MARZO12, MICHAEL P. HILL" , TIZIANA BISOGNO2, ALAN R. CROSSMAN* and JONATHAN M. BROTCHIE*1
* School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom; Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, 80072, Arco Felice, Naples, Italy; and
" Motac Neuroscience Ltd., Manchester MI3 9XX, U.K.
1Correspondence: J.B., 1.124 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Rd, Manchester, M13 9PT, U.K. E-mail: j.brotchie@man.ac.uk and V.D.M., Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, Via Tolano 6, 80072 Arco Felice Naples, Italy. E-mail: vdimarzo@icmib.na.cnr.it
ABSTRACT
In recent years, cannabinoid receptors and their endogenous ligands (endocannabinoids) have been identified within the brain. The high density of CB1 cannabinoid receptors within the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement and in basal ganglia-related movement disorders such as Parkinson's disease. However, whether endocannabinoids play a role in regulating motor behavior in health and disease is unknown. Here we report the presence in two regions of the basal ganglia, the globus pallidus and substantia nigra, of the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide. The levels of the latter compound are ~threefold higher than those previously reported in any other brain region. In the reserpine-treated rat, an animal model of Parkinson's disease, suppression of locomotion is accompanied by a sevenfold increase in the levels of the 2AG in the globus pallidus, but not in the other five brain regions analyzed. Stimulation of locomotion in the reserpine-treated rat by either of the two selective agonists of D2 and D1 dopamine receptors, quinpirole and R-(±)-3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (Cl-APB), respectively, results in the reduction of both anandamide and 2AG levels in the globus pallidus. Finally, full restoration of locomotion in the reserpine-treated rat is obtained by coadministration of quinpirole and the selective antagonist of the cannabinoid CB1 receptor subtype, SR141716A. These findings indicate a link between endocannabinoid signaling in the globus pallidus and symptoms of Parkinson's disease in the reserpine-treated rat, and suggest that modulation of the endocannabinoid signaling system might prove useful in treating this or other basal ganglia-related movement disorders.–Di Marzo, V., Hill, M. P., Bisogno, T., Crossman, A. R., Brotchie, J. M. Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson's disease.
Key Words: anandamide - 2-arachidonoyl glycerol - cannabinoids - dopamine - receptors
INTRODUCTION
ALTHOUGH THE PSYCHOACTIVE properties of cannabis have been widely exploited and studied, the physiological role of cannabinoid receptor-mediated signaling is unknown. The search for such a role has been heightened by the identification of two endogenous ligands for the cannabinoid receptors, 2-arachidonoylglycerol (2-AG) and anandamide (1 2 3) , named 'endocannabinoids'. These molecules were suggested to have a neuromodulatory action (for a review, see ref 4 ), but to date there has been no clear demonstration of a role for these endocannabinoids in controlling any aspect of behavior.
The high levels of 'central' cannabinoid (CB1) receptors within the basal ganglia (5) suggest a potential role for endocannabinoids in the control of voluntary movement and in basal ganglia-related movement disorders such as Parkinson's disease (6 7 8) . Within the basal ganglia, cannabinoid receptors are particularly prominent on the terminals of GABAergic projections from the striatum to the globus pallidus and substantia nigra pars reticulata (the 'indirect' and 'direct' striatal output pathways, respectively) (5) . The interplay between activity in these pathways is key to the selection and initiation of appropriate voluntary movements and is tightly regulated by the dopaminergic nigrostriatal system (9 , 10) . Dopamine receptors exert differential control of striatal outputs, D2-class dopamine receptors inhibiting the activity of the indirect pathway while the direct pathway is activated by D1-class dopamine receptors (11) .
Stimulation of cannabinoid receptors in the globus pallidus, by exogenous cannabinoid receptor agonists, reduces uptake of the inhibitory neurotransmitter GABA (12 , 13) and reduces voluntary movements, producing Parkinson-like symptoms (14) . Stimulation of cannabinoid receptors also reduces the anti-parkinsonian actions of D2, but not D1, dopamine receptor agonists (15) . It is possible, therefore, to hypothesize a role for endocannabinoids in the modulation of signaling by the indirect striatal output.
In this study, we have measured the levels and distribution of anandamide and 2-AG in rat brain by using a highly sensitive and specific isotope dilution gas chromatography-mass spectrometric (GC-MS) method (16) . Furthermore, we have compared endocannabinoid levels in different brain regions of vehicle-treated or reserpine-treated rats, a widely used animal model of Parkinson's disease, in the presence or absence of dopamine receptor agonists. We provide evidence suggesting that changes in endocannabinoid signaling in the globus pallidus of the reserpine-treated rat correlate with the modulation of voluntary movement in this model.
MATERIALS AND METHODS
Animal treatment
Male Sprague Dawley rats (200—250 g) were injected subcutaneously with 3 mg kg-1 reserpine (dissolved in 1% glacial acetic acid in distilled water) or vehicle (1 ml kg-1) under light inhalation anesthesia (Halothane). Assessment of locomotion was carried out 18 h later. Animals were acclimatized in the experimental room for at least 30 min before drugs were administered and the assessment commenced. Drugs administered were Cl-APB (R-(±)-3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide, 0.1 mg/kg), quinpirole (0.1 mg/kg), and SR141716A (N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride, 1 mg/kg). The locomotion of animals was measured using an automated movement detection system (Benwick Data-logger, Linton Instrumentation, Diss, U.K.), which consisted of a frame (52 cmx36 cm) containing an array of infra-red beams (organized into a grid 19x13) situated along the exterior of a frame. Animals were isolated from the frame by a Perspex box placed inside the frame. The movement of animals was detected when the animal passed from one grid to another and so broke an infrared beam. Locomotor activity was monitored using Amlogger software (Linton Instrumentation), which logged data at 5 min intervals over a period of 60 min. The index of locomotion used in the studies was mobile counts, which are recorded when the central position of the animal changes by more than two grids in any one second period. All studies were performed under a project license granted by the Home Office of the U.K. Government under the Animals (Scientific Procedures) Act 1986.
Tissue dissection, extraction, and endocannabinoid level measurement
Anandamide and 2-AG in lipid extracts from different brain regions were quantitated following the isotope dilution GC-MS procedure described previously (16) . Briefly, brain areas from eight rats belonging to each different group of treatments were dissected, pooled, and frozen in liquid nitrogen in the Manchester laboratory. A maximum of 3 min elapsed between tissue dissection and freezing. The frozen tissue was sent on dry ice to the Naples laboratory, stored at -80°C, and weighed prior to its extraction with 3 volumes of chloroform/methanol/Tris-HCI 50 mM buffer (pH 7.4) (2: 1: 1 by vol). The extraction mixture contained 2 nmol each of d8-anandamide and d8-2-AG synthesized from d8 arachidonic acid and ethanolamine or glycerol, as described previously (1 , 22) . The two phases were separated and the aqueous phase extracted three more times with an equal volume of chloroform. The pooled organic phases were dried down in a rotating evaporator, transferred to siliconized vials, and purified by normal phase-high pressure liquid chromatography (NP-HPLC) carried out as described previously (22) . Monoarachidonoylglycerols and anandamide standards were eluted after 18—23 min and 27—28 min, respectively. The mono-arachidonoylglycerol HPLC fraction contained both 1- (3) and 2-stereoisomers. HPLC fractions were dried down under a flow of nitrogen and derivatized with 15 µl N-methyl-N-tris-methyl-sylyl-trifluoroacetamide containing 1% trimethylchlorosylane for 2 h at room temperature, thus yielding the tris-methyl-sylyl derivatives of anandamide and 2-AG. The two derivatized fractions were analyzed separately by GC-MS carried out as described previously (16 , 22) . The tris-methyl-sylyl ethers of both deuterated and nondeuterated anandamide, 2-AG and 1- (3) arachidonoylglycerol standards were eluted after ~18, 19, and 19.5 min, respectively. MS detection was run in the selected ion monitoring mode to improve sensitivity. Selected ions for anandamide were at m/z = 427 and 419, corresponding to the molecular ions of d8-anandamide and nondeuterated anandamide, and m/z = 412 and 404, corresponding to the loss of a methyl group in both compounds. Selected ions for 2-AG were at m/z = 530 and 522, corresponding to the molecular ions of d8-2-AG, and nondeuterated 2 AG and m/z = 515 and 507, corresponding to the loss of a methyl group in both compounds. The endogenous cannabinoids were identified on the basis of the presence, at the same retention time as the deuterated internal standards, of the corresponding MS signals with the appropriate relative abundance. The amounts of anandamide and 2-AG were calculated from the peak area ratios between the signals at m/z = 404 and 412, and m/z = 507 and 515, respectively. A linear correlation between these area ratios and the amounts of anandamide (25—10000 pmol) or 2-AG (50—10000 pmol) standards was observed in separate studies. In the case of 2-AG, the amounts of the 1- (3) isomers, which are almost exclusively formed during tissue workup and lipid purification (23) and were 25—30% of the total monoarachidonoylglycerols, were added to the amounts of the 2-isomer. Each brain region in each group of animals was analyzed in quadruplicate. Measurements in each monoplicate were carried out in duplicate. Results are expressed in pmol/g wet tissue weight and are means ± SEM of the four separate determinations. Means from different groups were compared by using 1-way ANOVA, followed by Tukey-Kramer analysis. It can be estimated that 96 different samples (six brain regions in four different groups of treatments, each in quadruplicate, each from eight rats) had to be extracted, purified, and analyzed in the present study.
RESULTS AND DISCUSSION
We have used a sensitive and specific assay using isotope dilution GC-MS (16) to measure the levels and distribution of the endocannabinoids anandamide and 2-AG in the brain of vehicle-treated Sprague Dawley rats (Fig. 1 ). Anandamide levels were similar in striatum, cerebral cortex, cerebellum, and hippocampus and the values reported here are similar to those previously described for these regions (16 , 17) . Although cannabinoid receptors are expressed at high levels within the globus pallidus and substantia nigra, the levels of endocannabinoids within these important motor regions have not previously been reported. The levels of 2AG in these two regions were similar to other brain regions and generally lower than those previously observed in Wistar rat brain (16) . However, the levels of anandamide in the globus pallidus and substantia nigra were ~threefold higher than those previously reported in any brain region (Fig. 1B ). In the globus pallidus this difference was statistically significant (P<0.05). These data further support the speculation (12 13 14) that endocannabinoids may play a key role in the functioning of the striatal outputs in the control of movement.
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Figure 1. Levels of the endocannabinoids 2-AG (A) and anandamide (B) in vehicle-treated rats (□) and after induction of hypokinesia by reserpine treatment (▪). Data are presented as mean ± SEM of 4 separate experiments each with samples from 8 individual animals. ***P<0.001 compared to vehicle by 1-way ANOVA with post hoc Tukey-Kramer test. Abbreviations, SNr, substantia nigra pars reticulata; GP, globus pallidus.
To investigate this potential role of endocannabinoids in motor control, we have used the reserpine-treated rat as a model system in which the initiation and execution of movements can easily be assessed and manipulated. In this model, catecholamine stores are depleted and a motor syndrome characterized by decreased initiation and speed of voluntary movements, rigidity, and a hunched posture is observed (18) . These symptoms bear many similarities to those seen in Parkinson's disease (19) and are ameliorated by dopaminergic anti-parkinsonian drugs (18 19 20 21) . Thus, the reserpine-treated rodent provides a useful model of Parkinson's disease; indeed, it was in this model that the key role of decreased dopamine transmission in parkinsonism was identified and the therapeutic potential of dopamine replacement therapy in Parkinson's disease first highlighted (19) . Other currently used animal models of this disorder, e.g., the 6-hydroxy-dopamine (6-OHDA) -lesioned rat or the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -lesioned monkey were not utilized in this study for two different reasons: 1) although more selective than reserpine for the depletion of dopamine stores, 6-OHDA-induced lesions do not lead to overt symptoms of Parkinson's disease, an important point for our study aimed at finding a possible correlation between these symptoms and endocannabinoid levels; 2) it was estimated that at least 72 MPTP-lesioned monkeys would have been necessary to carry out a study with equivalent statistical rigor as the present one.
After treatment of rats with reserpine, locomotion was dramatically reduced (locomotor activity in vehicle-treated rats was 1251±228 mobile counts/h compared to 9±2 in reserpine-treated rats, P<0.001, t test, n=12) as has been previously described. In association with this decreased locomotion, a sevenfold elevation in the levels of 2AG in the globus pallidus was observed (Fig. 1) . Although average 2AG levels increased slightly in all the other brain regions analyzed, this effect was never as marked as in the globus pallidus (e.g., a 2.5-fold stimulation was found in striatum) and was never statistically significant. There was no statistically significant reserpine-induced change in the levels of anandamide in any brain region. These data highlight the fact that catecholamines play an important role in the inhibitory regulation of 2AG synthesis in the globus pallidus. The most likely mechanism underlying such an effect would be an action of dopamine on the striatal neurons projecting to the globus pallidus. In situations of decreased dopamine transmission, the activity of the indirect striatal output pathway is increased (9) . Increased depolarization of striatopallidal terminals after reserpine treatment may enhance 2AG synthesis, as depolarization has been shown to elevate 2AG levels in other cells (22 , 23) . The mechanism underlying this depolarization-induced increase in 2AG levels involves activation of calcium-dependent enzymes such as phospholipase C (23 , 24) , phosphatidic acid phosphohydrolase, and phospholipase A2 (25) . The functional relevance of this enhanced endocannabinoid synthesis in the globus pallidus may be great, since (as described above) activation of cannabinoid receptors in the globus pallidus by exogenous agonists has been shown to elicit parkinsonian symptoms (14) . Thus, enhanced 2AG signaling in the globus pallidus might be part of the neural mechanisms underlying hypokinesia in the reserpine-treated rat.
These data also underscore the complexity by which endocannabinoid levels might be regulated in vivo. Thus, catecholamines appear to regulate 2AG levels differentially in different populations of striatal output neurons (levels in the substantia nigra, the target of the direct striatal output pathway, were not significantly elevated) whereas different endocannabinoids are regulated differentially within a given region (pallidal anandamide levels were not elevated after reserpine treatment). The lack of effect on anandamide levels may be due to the fact that there is high endocannabinoid tone at rest in the globus pallidus and the basal amounts of anandamide actually represent the maximal level of synthesis possible in this structure. As anandamide levels are elevated by neuronal depolarization (26) , this would suggest that basal firing rate in the indirect striatal output pathway is sufficient to maximally stimulate anandamide synthesis. During the early preparation of this paper, Giuffrida et al. (27) reported that anandamide, but not 2AG, is released into microdialysates from the dorsal striatum of freely moving rats. In a previous study (23) the same authors found that 2-AG, but not anandamide, could be produced by electrically stimulated hippocampal slices. These reports point to the possible differential regulation of different endocannabinoids in different brain regions.
Administration of the D2 dopamine receptor agonist quinpirole (0.1 mg/kg) to reserpine-treated rats reduced 2AG and anandamide levels in the globus pallidus (Fig. 2 ). Anandamide levels were reduced by 75% whereas 2AG levels were reduced by 53%. These falls in anandamide and 2AG levels were accompanied by increased locomotion in reserpine-treated rats (Fig. 3 ).Quinpirole did not significantly alter 2AG or anandamide levels in any other region of the brain (data not shown). The reduction in pallidal 2AG and anandamide levels accompanying alleviation of parkinsonian symptoms by the D2 dopamine receptor agonist is further suggestive of a causative role for enhanced endocannabinoid signaling in the indirect pathway in parkinsonism. Indeed, stimulation of D2 receptors alleviates parkinsonism by reducing the activity of the indirect striatal output pathway (28) . The reduction in 2AG and anandamide levels may be due to this reduction in activity and, in the case of 2AG, reflect reversal of the depolarization-induced activation of calcium-dependent enzymes described above; anandamide levels have also been shown to be maintained by depolarization (26) . It is worthwhile mentioning that infusion with quinpirole was recently shown to enhance anandamide release from the dorsal striatum of normal rats (27) . The authors presented no evidence to show that endogenous dopamine tonically increases anandamide release from the striatum, and we found here no decrease in anandamide levels in the striatum of catecholamine-depleted rats (Fig. 1B ). However, this report, taken together with our present data, may suggest that in different regions of the basal ganglia, and under different conditions, stimulation of D2 dopamine receptors may have opposing consequences on endocannabinoid biosynthesis.
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Figure 2. Effect of D1 and D2 dopamine receptor stimulation on the levels of the endocannabinoids 2 AG (A) and anandamide (B) in the globus pallidus in the reserpine-treated rat. Levels of endocannabinoids were measured after alleviation of parkinsonian symptoms by the D2 dopamine receptor agonist quinpirole (0.1 mg/kg) and the dopamine D1 receptor agonist Cl-APB (0.1 mg/kg). Data are expressed as % control (vehicle-treated, reserpinized animals) and presented as the mean ± SEM of 4 independent experiments, each with samples from 8 individual animals. *P<0.05, **P<0.01, ***P<0.001, compared to vehicle, 1-way ANOVA with post hoc Tukey-Kramer test.
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Figure 3. Effect of the CB1 cannabinoid receptor antagonist SR141716A (1 mg/kg) on locomotion induced by administration of either a D2 dopamine receptor agonist (A, B) or a D1 dopamine receptor agonist (C, D) in the reserpine-treated rat. A, C) Data are presented as mean mobile counts per 5 min period (± SEM, n=12) after administration of vehicle (▴), the D2 dopamine receptor agonist quinpirole (0.1 mg/kg) alone (□), quinpirole plus SR141716A (▪), the D1 dopamine receptor agonist Cl-APB (0.1 mg/kg) alone (□), or Cl-APB plus SR141716A (▪). B, D) Data are presented as total mobile counts cumulated over a 1 h assessment period. *P<0.05, ***P<0.01 compared to vehicle, " " P<0.01 compared to agonist alone, 1-way ANOVA with post hoc Tukey-Kramer test.
Administration of the D1 dopamine receptor agonist Cl-APB (0.1 mg/kg) was also accompanied by a reduction in endocannabinoid levels in the globus pallidus (Fig. 2) and an increase in locomotion (Fig. 3) . Whether this reduction in endocannabinoid levels in the globus pallidus could be responsible for the anti-parkinsonian actions of Cl-APB is unclear since the anti-parkinsonian actions of D1 receptor stimulation are mediated through activation of the direct, rather than indirect, striatal output pathway (28) . However, basal ganglia outputs to the thalamus are a vital link in basal ganglia-thalamo-cortical-basal ganglia loops, the cortical components of which impinge directly onto striatal output neurons (10) . Therefore, activation of the direct pathway might indirectly lead to decreased excitation of the indirect striatopallidal pathway that (as described above) would be expected to reduce levels of endocannabinoids in the globus pallidus. In this case, the reversal in abnormalities in endocannabinoid transmission in the globus pallidus induced by Cl-APB might be a consequence of an anti-parkinsonian action resulting from normalization of abnormalities in the direct pathway. In any event, these data, taken together, point to a clear reverse correlation in reserpine-treated rats between locomotion and endocannabinoid levels in the globus pallidus.
To address further the issue of whether the anti-parkinsonian actions of dopamine receptor stimulation are mediated through a reduction in endocannabinoid signaling, we assessed the behavioral effects of treatment of reserpinized rats with quinpirole and the selective antagonist of CB1 receptors, SR141716A (29) . SR141716A markedly potentiated the ability of the D2 dopamine receptor agonist to elicit locomotion in the reserpine-treated rat (Fig. 3) . This finding strongly suggests that endocannabinoids counteract the ability of D2 receptor stimulation to elicit locomotion and might be responsible, in part at least, for the abnormalities in the indirect pathway that underlie the generation of parkinsonian symptoms. SR141716A had no significant effect on locomotion induced by the D1 agonist Cl-APB (Fig. 3) . This suggests that the reduction in 2AG levels brought about by Cl-APB (and the subsequent stimulation of the direct striatal output pathway) is indeed a consequence rather than a cause of its anti-parkinsonian action, as suggested above. The lack of effect of SR141716A on D1 agonist-induced locomotion and the finding that SR141716A has no effect on locomotion in non-parkinsonian animals (data not shown) also suggest that it is unlikely that the effects of SR141716A in enhancing D2 agonist-mediated locomotion are due to a nonspecific increase in locomotion or to the proposed inverse agonist properties of this compound (30) . Furthermore, our findings with SR141716A and quinpirole in the reserpine-treated rat are in agreement with an analogous enhancement by the CB1 antagonist of quinpirole-induced locomotion in normal rats reported early during the preparation of this manuscript (27) .
Our data show that reducing not only the levels, but also the action, of endocannabinoids in the globus pallidus may lead to enhanced locomotion in the reserpine-treated rat. These findings, therefore, suggest that the selection, initiation, and execution of movement by stimulation of the GABAergic indirect striatal output pathway, in the parkinsonian brain at least, may be under a negative control by endocannabinoids. Enhanced pallidal GABAergic transmission is a key component of the neural mechanisms underlying the symptoms of parkinsonism (9) . Stimulation of cannabinoid receptors on terminals of the indirect pathway reduces GABA reuptake (12 , 13) , thereby enhancing GABA transmission in the globus pallidus. Enhanced endocannabinoid signaling in the globus pallidus would thus elicit parkinsonian symptoms.
In conclusion, high levels of endocannabinoids are found in regions of the basal ganglia receiving input from the striatum. Endocannabinoids and dopamine may have a close functional relationship whereby, under physiological conditions, dopamine acts to suppress 2AG signaling in the globus pallidus while endocannabinoids act as a 'brake' on dopamine-receptor-stimulated locomotion in this as well as other areas of the basal ganglia (see also ref 27 ). Overactivation of the endocannabinoid system in the globus pallidus may play a part in the generation of parkinsonian symptoms. We suggest that endocannabinoids provide a neurochemical substrate for the interactions between transmitter systems that are, according to current thinking, fundamental to the appropriate selection and initiation of voluntary movements. Our findings may help to identify novel avenues of therapeutic intervention in Parkinson's disease (31) .
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Figure 3C.
ACKNOWLEDGMENTS
We thank Ms. Gerolmina Ambrosino and Mr. Ralph Davies for valuable technical assistance. This work was supported by the MRC (U.K., to J.M.B. and A.R.C.) and the MURST (3933, to V.D.M.).
FOOTNOTES
2 Affiliated with the National Institute for the Chemistry of Biological Systems, C.N.R.
Received for publication August 20, 1999. Revision received November 5, 1999.
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Source: Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson's disease
