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Striatal Cannabinoid CB1 Receptor mRNA Expression Is Decreased in the Reserpine-Treated Rat Model of Parkinson's Disease
Experimental Neurology 169, 400 – 406 (2001) doi:10.1006/exnr.2001.7649, available online at http://www.idealibrary.com on
Striatal Cannabinoid CB1 Receptor mRNA Expression Is Decreased in the Reserpine-Treated Rat Model of Parkinson’s Disease M. A. Silverdale, S. McGuire, A. McInnes, A. R. Crossman, and J. M. Brotchie Division of Neuroscience, University of Manchester, Manchester M13 9PT, United Kingdom Received May 22, 2000; accepted January 19, 2001
High levels of both endocannabinoids and endocannabinoid receptors are present in the basal ganglia. Attention has recently focused on the role of endocannabinoids in the control of movement and in movement disorders of basal ganglia origin such as Parkinson’s disease. We investigated CB1 cannabinoid receptor mRNA expression in the reserpine-treated rat model of Parkinson’s disease using in situ hybridization. Reserpine treatment caused a topographically organized reduction in CB1 receptor mRNA expression in the striatum (ranging from 11.6% medially to 53.6% laterally and dorsally). No change in CB1 receptor mRNA expression was observed in the cerebral cortex or septum. This reduction in CB1 receptor mRNA expression may be secondary to increased endocannabinoid stimulation of the receptor as increased basal ganglia endocannabinoid levels have been shown to occur in this model of Parkinson’s disease. The data support the idea that cannabinoid receptor antagonists may provide a useful treatment for the symptoms of Parkinson’s disease. © 2001 Academic Press Key Words: Parkinson’s disease; cannabinoids; reserpine; rat; in situ hybridization; striatum; CB1 receptor.
INTRODUCTION
A cannabinoid receptor was first cloned from a rat cerebral cortex cDNA library (30). This cannabinoid receptor was found to be neuronally located, and its activation was shown to inhibit cAMP production through a pertussis toxin-sensitive G protein (Gi) (30). Subsequently, a peripheral cannabinoid receptor was found (31). The neuronal receptor was termed CB1, and the peripheral receptor CB2. Two endogenous cannabinoid ligands (endocannabinoids) have been identified, 2-arachidonylglycerol (2-AG) and anandamide (8, 12, 38). Recently there has been much progress in understanding the physiological role of endocannabinoids, and the application of pharmacological modulation of cannabinoid receptor-mediated transmission in the treatment of neurological diseases. In particular, 0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
the role of endocannabinoids in the control of movement has been highlighted (18) as the basal ganglia contain the highest concentrations of cannabinoid receptors (24) and endocannabinoids (11) anywhere in the brain. In the rat basal ganglia, cannabinoid receptors are predominantly located on the presynaptic terminals of GABAergic projections from the striatum to the globus pallidus and substantia nigra pars reticulata (the indirect and direct striatal output pathways respectively) (20). Endocannabinoids are released from neurons in response to depolarization and calcium influx (9, 17). Within the globus pallidus, cannabinoids have been shown to enhance GABAergic effects in animal behavioral models (35) and stimulation of cannabinoid receptors in the globus pallidus by exogenous cannabinoid agonists has been shown to modulate cyclic AMP levels (26) and to reduce the uptake of the inhibitory neurotransmitter GABA (2, 29). Striatal neurons of the direct pathway express the D1 receptor and are thought to be activated by dopamine, whereas neurons of the indirect striatopallidal pathway express the D2 receptor and are thought to be inhibited by dopamine (16, 33). In dopamine deficient states, the indirect pathway is overactive (32, 33). We have recently shown that, in the reserpine-treated rat, there are increased levels of endocannabinoids in the globus pallidus (11). We have therefore hypothesized that increased activity of the indirect pathway leads to increased endocannabinoids in the globus pallidus. Enhanced CB1 receptor stimulation would be expected to reduce GABA uptake and enhance GABA transmission. In terms of our current understanding of basal ganglia functional anatomy, enhanced GABA transmission in the globus pallidus would be expected to contribute to the generation of parkinsonian symptoms (1, 3, 4, 7, 28, 32). The fact that the overstimulation of cannabinoid receptors on the indirect pathway is responsible for the generation of parkinsonian symptoms in the reserpine-treated rat is consistent with findings that exogenous cannabinoids reduce the alleviation of akinesia produced by D2 agonists but not D1 agonists
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(27), while CB1 antagonists enhance D2 agonist effects (10, 11). The aim of this study was to investigate the level of CB1 receptor mRNA in the reserpine-treated rat model of Parkinson’s disease. We hypothesized that, if enhanced endocannabinoid transmission in the indirect striatopallidal pathway underlies parkinsonian symptoms, CB1 receptor mRNA expression would be downregulated in a compensatory manner in parkinsonism. MATERIALS AND METHODS
Male Sprague–Dawley rats (250 –320 g, Manchester University BSU) were housed under controlled conditions: temperature (19 –21°C), humidity 55%, and lights on 0800 –2000. Food and water were available ad libitum. Animals were injected at 1700 with either reserpine (3 mg/kg, sc) or vehicle under light halothane anesthesia. Behavioral assessment was performed between 1000 and 1300. The locomotion of animals was measured using an automated movement detection system (Benwick data logger, Linton Instrumentation, UK) as detailed previously (27). All studies were performed under a project license, granted by the Home Office of the UK Government, under the Animals (Scientific Procedures) Act 1986. After assessment of mobility, the animals were killed and brains were removed and rapidly frozen in isopentane cooled to ⫺45°C. Brains were cryostat sectioned (⫺19°C) at 15 m, thaw-mounted onto gelatin/chrome-alum-coated slides, and stored, desiccated at ⫺70°C, until further processing. The method for in situ hybridization was essentially that previously described (13). Briefly, sections were warmed to room temperature, fixed in 4% paraformaldehyde solution for 10 min, rinsed, and then incubated in a fresh solution of 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. Sections were dehydrated in a series of ascending concentrations of ethanol, defatted for 5 min in chloroform, air-dried, and then subsequently hybridized with 35S-tailed oligonucleotides. For experiments using RNase, after incubation in acetic anhydride, and prior to dehydration with ethanol, tissue sections were incubated in RNase A solution (ribonuclease A, Sigma, 20 g/ml) at 37°C for 30 min. Oligonucleotide probes were designed with reference to sequences obtained from GenBank. The probe used was a 48-bp oligonucleotide (GGT GAT GGT ACG GAA GGT GGT GTC TGC AAG GCC ATC TAG GAT CGA CTT), complementary to nucleotides 156 –203 on the CB1 mRNA sequence (30). The probe (50 nM in 1 ml) was 3⬘-end tailed with [ 35S]dATP by incubation for 60 min at 37°C in a 40-l reaction mixture containing 4 pM probe, 4 l terminal transferase buffer, 2 l terminal transferase, and 3.2 l [ 35S]dATP (terminal transferase kit, Promega Cat. No. U2000, [ 35S]dATP, NEN sp act 1250 Ci/mmol). The reaction was terminated by
heating the reaction mixture to 70°C for 10 min and the labeled probe was purified by column chromatography (Biospin 6 columns, Bio-Rad). Hybridization buffer consisted of the 35S-labeled oligonucleotide probe (3 ⫻ 10 6cpm/ml), in 50% formamide, 4⫻ standard saline citrate (4⫻ SSC) (1⫻ SSC ⫽ 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 10% dextran sulfate, 5⫻ Denhardt’s, 200 g/ml salmon testes DNA, 100 g/ml Poly(A), and 2% DTT. For experiments using excess unlabeled probe, in situ hybridization was carried in the presence of a 25-fold excess of unlabeled probe. Aliquots of hybridization buffer were applied to each of the sections on any one slide. Sections were covered with Parafilm coverslips and incubated for 18 h at 42°C in a humid chamber. After incubation, the Parafilm coverslips were floated off in 2⫻ SSC, and the sections were rinsed in a series of washes: 1⫻ SSC at room temperature for 30 min; 1⫻ SSC at 58°C for 30 min; 0.1⫻ SSC at room temperature for 10 min. Sections were then dehydrated for 2 min in 70% and then 95% ethanol then air-dried. The localization of bound probe was revealed by autoradiography by exposure of sections to -Max hyperfilm (Amersham) for 7 days at 4°C. Films were developed using Kodak D-19 and fixed using Kodak Unifix. Densitometric analysis of the autoradiograms was performed using Image-Pro Plus Version 3.0.1 (Media Cybernetics, Silver Spring, MD). Film optical density (OD) was evaluated in the rostral striatum (1.6 mm anterior to bregma) and the caudal striatum (0.8 mm posterior to bregma) (34) (Fig. 1). OD was also evaluated in the deep and superficial layers of the cerebral cortex and in the septum. All images were corrected for nonspecific background OD using an algorithm incorporating division by OD of a blank area of exposed film (Image Pro Plus). The OD of 14C standards (microscale autoradiography standards, Amersham, UK) was also measured on all films. The relationship between the OD and the radioactivity (nCi/g) of the standards was linear within the range of the values measured. The OD of the images was then converted into a 14C radioactivity equivalent (nCi/g). Statistical analysis was performed using three-way ANOVA (Stata 6, Stata Corporation, College Station, TX). The variables were level (rostral/caudal), treatment (reserpine/vehicle), and region (dorsolateral striatum, ventrolateral striatum, dorsomedial striatum, ventromedial striatum, deep cortex, superficial cortex, and septum). If significant, ANOVA was followed by post hoc t test comparisons with Bonferroni corrections. A probability level of P ⬍ 0.05 was considered significant. RESULTS
Eighteen hours following reserpine treatment, rats displayed a parkinsonian state characterized by rigidity, hunched posture, and akinesia. Locomotor activity
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FIG. 1. Diagrammatic representation of the two rostrocaudal levels of the striatum at which the in situ hybridization was performed. The rostral striatum was defined as 1.6 ⫾ 1 mm anterior to bregma (34). The caudal striatum was defined as 0.8 ⫾ 1 mm posterior to bregma (34). The regions of optical density measurement are indicated. The rostral striatum was divided into four regions (dorsolateral (DL), dorsomedial (DM), ventrolateral (VL), and ventromedial (VM)). The caudal striatum was divided into two regions (dorsal (D) and ventral (V)).
in nonreserpinized rats was 1251 ⫾ 228 mobile counts per hour. Following reserpine administration, locomotor activity was less than 1% of that recorded in nonreserpinized rats (9 ⫾ 2 mobile counts per hour, P ⬍ 0.001) (t test). The CB1 receptor probe hybridized to brain sections in a heterogeneous manner. Within the basal ganglia the probe hybridization signal was high in the rostral striatum, the densest signal being seen laterally in contrast to only moderate signal medially. In the caudal striatum, the densest hybridization was seen ventrally, with only moderate labeling dorsally. Very little hybridization signal was detected in the nucleus accumbens. Moderate hybridization was detected in the cerebral cortex and the septal area. No significant hybridization signal was detected in the globus pallidus or in any white matter region (Fig. 2). In situ hybridization was performed on sections previously treated with RNase and on sections in the presence of excess unlabeled probe. No hybridization signal was detected under either of these conditions (data not shown). Reserpine treatment significantly affected the level of CB1 probe hybridization (F(1, 90) ⫽ 103.2, P ⬍ 0.0001). The magnitude of this effect was regulated by both region (F(6, 90) ⫽ 53.4, P ⬍ 0.0001) and level (F(1, 90) ⫽ 136.2, P ⬍ 0.0001). Reserpine treatment caused a topographically organized decrease in the level of CB1 hybridization in the rostral striatum. The decrease in hybridization signal was greater laterally (dorsolateral ⫺53.6%, ventrolateral ⫺48%, both P ⬍ 0.0001 compared to vehicletreated animals) than medially (dorsomedial ⫺11.6%, ventromedial ⫾15.9%, both P ⬍ 0.05 compared to ve-
hicle, both P ⬍ 0.05 compared to dorsolateral and ventrolateral respectively) (Fig. 3). In the caudal striatum, reserpine caused a reduction in CB1 hybridization signal ventrally (⫺40.9%, P ⬍ 0.005 compared to vehicle) but not dorsally (P ⬎ 0.05 compared to vehicle) (Fig. 3). Reserpine treatment did not affect CB1 hybridization in the superficial layers of the cerebral cortex (vehicle 38 ⫾ 2 nCi/g (SEM), reserpine 44 ⫾ 2 nCi/g (SEM), P ⬎ 0.05), deep layers of the cerebral cortex (vehicle 55 ⫾ 1.2 nCi/g (SEM), reserpine 56 ⫾ 2 nCi/g (SEM), P ⬎ 0.05) or the septum (vehicle 37 ⫾ 6 nCi/g (SEM), reserpine 35 ⫾ 3 nCi/g (SEM), P ⬎ 0.05). DISCUSSION
Striatal CB1 receptor mRNA expression was reduced in a topographically organized manner in the reserpine-treated rat. Reserpine treatment depletes noradrenaline, 5-hydroxytryptamine, and dopamine (6). This model is similar to parkinsonian patients who have a depletion not just of dopamine, but of noradrenaline and 5-hydroxytryptamine as well (37). Reserpine treatment elicits a transient syndrome characterized by tremor, rigidity, hypokinesia, and postural flexion (6). The symptoms bear many similarities to those seen in Parkinson’s disease (5) and this model has been widely used to investigate the mechanisms underlying Parkinson’s disease and for the development of new anti-parkinsonian drugs (14, 21, 22, 27, 39). It was in fact in the reserpine-treated rat that the therapeutic potential of dopamine replacement therapy in Parkinson’s disease was first highlighted (5).
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FIG. 2. Typical autoradiographs from the rostral striatum (a, b) and caudal striatum (c, d) from vehicle-treated (a and c) and reserpine-treated (b and d) animals.
The topographical organization of CB1 cannabinoid receptor mRNA expression in this study was essentially identical to that seen in previous studies (20, 24) and no hybridization signal was seen in the presence of excess probe or after RNase pretreatment. Furthermore, the probe sequence used would not hybridize with any other sequence available on GenBank. It is therefore likely that the hybridization signals detected in this study reflect CB1 cannabinoid receptor mRNA expression. CB1 hybridization was observed in the septum. The role of these CB1 receptors is unclear but it is interesting that both septal lesions and exogenous cannabinoids have anxiolytic effects (19, 36). The expression of septal CB1 receptors was not affected by monoaminergic depletion, suggesting that these receptors, unlike the striatal CB1 receptors, are not under monoaminergic control. In addition, no change in CB1 expression was seen in either the superficial or the deep layers of the cerebral cortex. The lack of change in CB1 mRNA expression in the cortex and septum provides evidence that the changes are restricted to particular brain regions and that the changes observed in the basal ganglia are not resulting from a nonspecific down-regulation of CB1 receptor mRNA expression. In previous studies of CB1 mRNA expression in the basal ganglia, the highest levels of expression were found in the medium spiny neurons of the striatum with very little expression in the regions receiving af-
ferents from the striatum, i.e., the globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata (24). Receptor binding studies have, however, shown that the highest density of receptor binding is found in regions receiving striatal output (20, 24). CB1 cannabinoid receptors are therefore likely to be located on the presynaptic terminals of the striatal output neurons. Further evidence for such a localization was provided by studies demonstrating that lesioning the striatal output neurons decreases CB1 receptor binding in the striatal output regions (20). The striatal CB1 probe hybridization signal observed in the present study thus probably reflects synthesis by striatal neurons projecting to the globus pallidus and substantia nigra pars reticulata. The data presented demonstrating down-regulation of striatal CB1 receptor mRNA expression support the hypothesis that increased CB1 receptor stimulation by endogenous endocannabinoids may occur in Parkinson’s disease. This idea is in keeping with findings that the level of the endocannabinoid 2AG is increased in the globus pallidus of the reserpine-treated rat (11), that CB1 antagonists enhance the anti-parkinsonian actions of D2 dopamine receptor agonists (10, 11), and that the anti-parkinsonian actions of D2 dopamine receptor agonists are accompanied by a reduction in pallidal 2AG levels (11). The largest reductions in CB1 mRNA expression found in this study were in the dorsolateral striatum. This area of the basal ganglia has
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FIG. 3. Effect of reserpine treatment on CB1 cannabinoid receptor expression. Data are expressed as means ⫾ SEM (nCi/g) radioactivity equivalent. CB1 mRNA expression was assessed in the rostral striatum (a– d) and caudal striatum (e, f). ***P ⬍ 0.0001, **P ⬍ 0.005, *P ⬍ 0.05.
connections with the motor and somatosensory areas of the cerebral cortex (15, 23) and is thought to have a sensorimotor function as opposed to more medial and ventral areas of the striatum which are thought to have associative and limbic functions respectively (15). The fact that the largest changes in CB1 mRNA expression were seen in sensorimotor areas suggests that increased endocannabinoid levels might be associated with the motor symptoms of Parkinson’s disease. However, it must be noted that previous studies in the 6-hydroxydopamine-lesioned rat model of Parkinson’s disease did not demonstrate a reduction in CB1 receptor mRNA expression (40) or binding (20). Indeed, one study reported an increase in CB1 receptor mRNA expression after 6-hydoxydopamine treatment (25). It is possible that an attempted compensatory decrease in
CB1 receptor synthesis following dopamine depletion may be short-lived and though observed in the reserpine-treated rat may not persist in the 6-hydroxydopamine model where the dopamine depletion is maintained over weeks. Alternatively, it is possible that these previous studies in the 6-hydroxydopamine-lesioned rat were influenced by the fact that apomorphine treatment to assess lesion extent formed part of the study protocol. Indeed, dopamine receptor stimulation has been shown to increase CB1 mRNA expression (40). Another possible reason for the difference between the decrease in CB1 receptor mRNA expression shown in this study and the lack of change in the 6-hydroxydopamine model may be that reductions in 5-hydroxytryptamine or noradrenaline levels, both of which are reduced by reserpine treatment, but not by
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6-hydroxydopamine lesions (6), are responsible for regulating CB1 receptor expression. If increased endocannabinoid stimulation of the CB1 receptor is a component of the neural mechanism underlying Parkinson’s disease, one might expect CB1 antagonists to be useful in the treatment of Parkinson’s disease. Indeed, the cannabinoid antagonist SR141716A markedly potentiates the alleviation of akinesia produced by the D2 agonist quinpirole in the reserpine-treated rat model of Parkinson’s disease (10, 11) and does have mild antiparkinsonian actions in its own right in the MPTP-lesioned monkey (M. Hill, personal communication). In summary, this study provides further evidence for abnormalities of endocannabinoid transmission in a rat model of Parkinson’s disease. We propose that cannabinoid receptor antagonists may provide a useful treatment for Parkinson’s disease. REFERENCES 1.
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Striatal Cannabinoid CB1 Receptor mRNA Expression Is Decreased in the Reserpine-Treated Rat Model of Parkinson’s Disease M. A. Silverdale, S. McGuire, A. McInnes, A. R. Crossman, and J. M. Brotchie Division of Neuroscience, University of Manchester, Manchester M13 9PT, United Kingdom Received May 22, 2000; accepted January 19, 2001
High levels of both endocannabinoids and endocannabinoid receptors are present in the basal ganglia. Attention has recently focused on the role of endocannabinoids in the control of movement and in movement disorders of basal ganglia origin such as Parkinson’s disease. We investigated CB1 cannabinoid receptor mRNA expression in the reserpine-treated rat model of Parkinson’s disease using in situ hybridization. Reserpine treatment caused a topographically organized reduction in CB1 receptor mRNA expression in the striatum (ranging from 11.6% medially to 53.6% laterally and dorsally). No change in CB1 receptor mRNA expression was observed in the cerebral cortex or septum. This reduction in CB1 receptor mRNA expression may be secondary to increased endocannabinoid stimulation of the receptor as increased basal ganglia endocannabinoid levels have been shown to occur in this model of Parkinson’s disease. The data support the idea that cannabinoid receptor antagonists may provide a useful treatment for the symptoms of Parkinson’s disease. © 2001 Academic Press Key Words: Parkinson’s disease; cannabinoids; reserpine; rat; in situ hybridization; striatum; CB1 receptor.
INTRODUCTION
A cannabinoid receptor was first cloned from a rat cerebral cortex cDNA library (30). This cannabinoid receptor was found to be neuronally located, and its activation was shown to inhibit cAMP production through a pertussis toxin-sensitive G protein (Gi) (30). Subsequently, a peripheral cannabinoid receptor was found (31). The neuronal receptor was termed CB1, and the peripheral receptor CB2. Two endogenous cannabinoid ligands (endocannabinoids) have been identified, 2-arachidonylglycerol (2-AG) and anandamide (8, 12, 38). Recently there has been much progress in understanding the physiological role of endocannabinoids, and the application of pharmacological modulation of cannabinoid receptor-mediated transmission in the treatment of neurological diseases. In particular, 0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
the role of endocannabinoids in the control of movement has been highlighted (18) as the basal ganglia contain the highest concentrations of cannabinoid receptors (24) and endocannabinoids (11) anywhere in the brain. In the rat basal ganglia, cannabinoid receptors are predominantly located on the presynaptic terminals of GABAergic projections from the striatum to the globus pallidus and substantia nigra pars reticulata (the indirect and direct striatal output pathways respectively) (20). Endocannabinoids are released from neurons in response to depolarization and calcium influx (9, 17). Within the globus pallidus, cannabinoids have been shown to enhance GABAergic effects in animal behavioral models (35) and stimulation of cannabinoid receptors in the globus pallidus by exogenous cannabinoid agonists has been shown to modulate cyclic AMP levels (26) and to reduce the uptake of the inhibitory neurotransmitter GABA (2, 29). Striatal neurons of the direct pathway express the D1 receptor and are thought to be activated by dopamine, whereas neurons of the indirect striatopallidal pathway express the D2 receptor and are thought to be inhibited by dopamine (16, 33). In dopamine deficient states, the indirect pathway is overactive (32, 33). We have recently shown that, in the reserpine-treated rat, there are increased levels of endocannabinoids in the globus pallidus (11). We have therefore hypothesized that increased activity of the indirect pathway leads to increased endocannabinoids in the globus pallidus. Enhanced CB1 receptor stimulation would be expected to reduce GABA uptake and enhance GABA transmission. In terms of our current understanding of basal ganglia functional anatomy, enhanced GABA transmission in the globus pallidus would be expected to contribute to the generation of parkinsonian symptoms (1, 3, 4, 7, 28, 32). The fact that the overstimulation of cannabinoid receptors on the indirect pathway is responsible for the generation of parkinsonian symptoms in the reserpine-treated rat is consistent with findings that exogenous cannabinoids reduce the alleviation of akinesia produced by D2 agonists but not D1 agonists
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(27), while CB1 antagonists enhance D2 agonist effects (10, 11). The aim of this study was to investigate the level of CB1 receptor mRNA in the reserpine-treated rat model of Parkinson’s disease. We hypothesized that, if enhanced endocannabinoid transmission in the indirect striatopallidal pathway underlies parkinsonian symptoms, CB1 receptor mRNA expression would be downregulated in a compensatory manner in parkinsonism. MATERIALS AND METHODS
Male Sprague–Dawley rats (250 –320 g, Manchester University BSU) were housed under controlled conditions: temperature (19 –21°C), humidity 55%, and lights on 0800 –2000. Food and water were available ad libitum. Animals were injected at 1700 with either reserpine (3 mg/kg, sc) or vehicle under light halothane anesthesia. Behavioral assessment was performed between 1000 and 1300. The locomotion of animals was measured using an automated movement detection system (Benwick data logger, Linton Instrumentation, UK) as detailed previously (27). All studies were performed under a project license, granted by the Home Office of the UK Government, under the Animals (Scientific Procedures) Act 1986. After assessment of mobility, the animals were killed and brains were removed and rapidly frozen in isopentane cooled to ⫺45°C. Brains were cryostat sectioned (⫺19°C) at 15 m, thaw-mounted onto gelatin/chrome-alum-coated slides, and stored, desiccated at ⫺70°C, until further processing. The method for in situ hybridization was essentially that previously described (13). Briefly, sections were warmed to room temperature, fixed in 4% paraformaldehyde solution for 10 min, rinsed, and then incubated in a fresh solution of 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. Sections were dehydrated in a series of ascending concentrations of ethanol, defatted for 5 min in chloroform, air-dried, and then subsequently hybridized with 35S-tailed oligonucleotides. For experiments using RNase, after incubation in acetic anhydride, and prior to dehydration with ethanol, tissue sections were incubated in RNase A solution (ribonuclease A, Sigma, 20 g/ml) at 37°C for 30 min. Oligonucleotide probes were designed with reference to sequences obtained from GenBank. The probe used was a 48-bp oligonucleotide (GGT GAT GGT ACG GAA GGT GGT GTC TGC AAG GCC ATC TAG GAT CGA CTT), complementary to nucleotides 156 –203 on the CB1 mRNA sequence (30). The probe (50 nM in 1 ml) was 3⬘-end tailed with [ 35S]dATP by incubation for 60 min at 37°C in a 40-l reaction mixture containing 4 pM probe, 4 l terminal transferase buffer, 2 l terminal transferase, and 3.2 l [ 35S]dATP (terminal transferase kit, Promega Cat. No. U2000, [ 35S]dATP, NEN sp act 1250 Ci/mmol). The reaction was terminated by
heating the reaction mixture to 70°C for 10 min and the labeled probe was purified by column chromatography (Biospin 6 columns, Bio-Rad). Hybridization buffer consisted of the 35S-labeled oligonucleotide probe (3 ⫻ 10 6cpm/ml), in 50% formamide, 4⫻ standard saline citrate (4⫻ SSC) (1⫻ SSC ⫽ 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 10% dextran sulfate, 5⫻ Denhardt’s, 200 g/ml salmon testes DNA, 100 g/ml Poly(A), and 2% DTT. For experiments using excess unlabeled probe, in situ hybridization was carried in the presence of a 25-fold excess of unlabeled probe. Aliquots of hybridization buffer were applied to each of the sections on any one slide. Sections were covered with Parafilm coverslips and incubated for 18 h at 42°C in a humid chamber. After incubation, the Parafilm coverslips were floated off in 2⫻ SSC, and the sections were rinsed in a series of washes: 1⫻ SSC at room temperature for 30 min; 1⫻ SSC at 58°C for 30 min; 0.1⫻ SSC at room temperature for 10 min. Sections were then dehydrated for 2 min in 70% and then 95% ethanol then air-dried. The localization of bound probe was revealed by autoradiography by exposure of sections to -Max hyperfilm (Amersham) for 7 days at 4°C. Films were developed using Kodak D-19 and fixed using Kodak Unifix. Densitometric analysis of the autoradiograms was performed using Image-Pro Plus Version 3.0.1 (Media Cybernetics, Silver Spring, MD). Film optical density (OD) was evaluated in the rostral striatum (1.6 mm anterior to bregma) and the caudal striatum (0.8 mm posterior to bregma) (34) (Fig. 1). OD was also evaluated in the deep and superficial layers of the cerebral cortex and in the septum. All images were corrected for nonspecific background OD using an algorithm incorporating division by OD of a blank area of exposed film (Image Pro Plus). The OD of 14C standards (microscale autoradiography standards, Amersham, UK) was also measured on all films. The relationship between the OD and the radioactivity (nCi/g) of the standards was linear within the range of the values measured. The OD of the images was then converted into a 14C radioactivity equivalent (nCi/g). Statistical analysis was performed using three-way ANOVA (Stata 6, Stata Corporation, College Station, TX). The variables were level (rostral/caudal), treatment (reserpine/vehicle), and region (dorsolateral striatum, ventrolateral striatum, dorsomedial striatum, ventromedial striatum, deep cortex, superficial cortex, and septum). If significant, ANOVA was followed by post hoc t test comparisons with Bonferroni corrections. A probability level of P ⬍ 0.05 was considered significant. RESULTS
Eighteen hours following reserpine treatment, rats displayed a parkinsonian state characterized by rigidity, hunched posture, and akinesia. Locomotor activity
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FIG. 1. Diagrammatic representation of the two rostrocaudal levels of the striatum at which the in situ hybridization was performed. The rostral striatum was defined as 1.6 ⫾ 1 mm anterior to bregma (34). The caudal striatum was defined as 0.8 ⫾ 1 mm posterior to bregma (34). The regions of optical density measurement are indicated. The rostral striatum was divided into four regions (dorsolateral (DL), dorsomedial (DM), ventrolateral (VL), and ventromedial (VM)). The caudal striatum was divided into two regions (dorsal (D) and ventral (V)).
in nonreserpinized rats was 1251 ⫾ 228 mobile counts per hour. Following reserpine administration, locomotor activity was less than 1% of that recorded in nonreserpinized rats (9 ⫾ 2 mobile counts per hour, P ⬍ 0.001) (t test). The CB1 receptor probe hybridized to brain sections in a heterogeneous manner. Within the basal ganglia the probe hybridization signal was high in the rostral striatum, the densest signal being seen laterally in contrast to only moderate signal medially. In the caudal striatum, the densest hybridization was seen ventrally, with only moderate labeling dorsally. Very little hybridization signal was detected in the nucleus accumbens. Moderate hybridization was detected in the cerebral cortex and the septal area. No significant hybridization signal was detected in the globus pallidus or in any white matter region (Fig. 2). In situ hybridization was performed on sections previously treated with RNase and on sections in the presence of excess unlabeled probe. No hybridization signal was detected under either of these conditions (data not shown). Reserpine treatment significantly affected the level of CB1 probe hybridization (F(1, 90) ⫽ 103.2, P ⬍ 0.0001). The magnitude of this effect was regulated by both region (F(6, 90) ⫽ 53.4, P ⬍ 0.0001) and level (F(1, 90) ⫽ 136.2, P ⬍ 0.0001). Reserpine treatment caused a topographically organized decrease in the level of CB1 hybridization in the rostral striatum. The decrease in hybridization signal was greater laterally (dorsolateral ⫺53.6%, ventrolateral ⫺48%, both P ⬍ 0.0001 compared to vehicletreated animals) than medially (dorsomedial ⫺11.6%, ventromedial ⫾15.9%, both P ⬍ 0.05 compared to ve-
hicle, both P ⬍ 0.05 compared to dorsolateral and ventrolateral respectively) (Fig. 3). In the caudal striatum, reserpine caused a reduction in CB1 hybridization signal ventrally (⫺40.9%, P ⬍ 0.005 compared to vehicle) but not dorsally (P ⬎ 0.05 compared to vehicle) (Fig. 3). Reserpine treatment did not affect CB1 hybridization in the superficial layers of the cerebral cortex (vehicle 38 ⫾ 2 nCi/g (SEM), reserpine 44 ⫾ 2 nCi/g (SEM), P ⬎ 0.05), deep layers of the cerebral cortex (vehicle 55 ⫾ 1.2 nCi/g (SEM), reserpine 56 ⫾ 2 nCi/g (SEM), P ⬎ 0.05) or the septum (vehicle 37 ⫾ 6 nCi/g (SEM), reserpine 35 ⫾ 3 nCi/g (SEM), P ⬎ 0.05). DISCUSSION
Striatal CB1 receptor mRNA expression was reduced in a topographically organized manner in the reserpine-treated rat. Reserpine treatment depletes noradrenaline, 5-hydroxytryptamine, and dopamine (6). This model is similar to parkinsonian patients who have a depletion not just of dopamine, but of noradrenaline and 5-hydroxytryptamine as well (37). Reserpine treatment elicits a transient syndrome characterized by tremor, rigidity, hypokinesia, and postural flexion (6). The symptoms bear many similarities to those seen in Parkinson’s disease (5) and this model has been widely used to investigate the mechanisms underlying Parkinson’s disease and for the development of new anti-parkinsonian drugs (14, 21, 22, 27, 39). It was in fact in the reserpine-treated rat that the therapeutic potential of dopamine replacement therapy in Parkinson’s disease was first highlighted (5).
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FIG. 2. Typical autoradiographs from the rostral striatum (a, b) and caudal striatum (c, d) from vehicle-treated (a and c) and reserpine-treated (b and d) animals.
The topographical organization of CB1 cannabinoid receptor mRNA expression in this study was essentially identical to that seen in previous studies (20, 24) and no hybridization signal was seen in the presence of excess probe or after RNase pretreatment. Furthermore, the probe sequence used would not hybridize with any other sequence available on GenBank. It is therefore likely that the hybridization signals detected in this study reflect CB1 cannabinoid receptor mRNA expression. CB1 hybridization was observed in the septum. The role of these CB1 receptors is unclear but it is interesting that both septal lesions and exogenous cannabinoids have anxiolytic effects (19, 36). The expression of septal CB1 receptors was not affected by monoaminergic depletion, suggesting that these receptors, unlike the striatal CB1 receptors, are not under monoaminergic control. In addition, no change in CB1 expression was seen in either the superficial or the deep layers of the cerebral cortex. The lack of change in CB1 mRNA expression in the cortex and septum provides evidence that the changes are restricted to particular brain regions and that the changes observed in the basal ganglia are not resulting from a nonspecific down-regulation of CB1 receptor mRNA expression. In previous studies of CB1 mRNA expression in the basal ganglia, the highest levels of expression were found in the medium spiny neurons of the striatum with very little expression in the regions receiving af-
ferents from the striatum, i.e., the globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata (24). Receptor binding studies have, however, shown that the highest density of receptor binding is found in regions receiving striatal output (20, 24). CB1 cannabinoid receptors are therefore likely to be located on the presynaptic terminals of the striatal output neurons. Further evidence for such a localization was provided by studies demonstrating that lesioning the striatal output neurons decreases CB1 receptor binding in the striatal output regions (20). The striatal CB1 probe hybridization signal observed in the present study thus probably reflects synthesis by striatal neurons projecting to the globus pallidus and substantia nigra pars reticulata. The data presented demonstrating down-regulation of striatal CB1 receptor mRNA expression support the hypothesis that increased CB1 receptor stimulation by endogenous endocannabinoids may occur in Parkinson’s disease. This idea is in keeping with findings that the level of the endocannabinoid 2AG is increased in the globus pallidus of the reserpine-treated rat (11), that CB1 antagonists enhance the anti-parkinsonian actions of D2 dopamine receptor agonists (10, 11), and that the anti-parkinsonian actions of D2 dopamine receptor agonists are accompanied by a reduction in pallidal 2AG levels (11). The largest reductions in CB1 mRNA expression found in this study were in the dorsolateral striatum. This area of the basal ganglia has
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FIG. 3. Effect of reserpine treatment on CB1 cannabinoid receptor expression. Data are expressed as means ⫾ SEM (nCi/g) radioactivity equivalent. CB1 mRNA expression was assessed in the rostral striatum (a– d) and caudal striatum (e, f). ***P ⬍ 0.0001, **P ⬍ 0.005, *P ⬍ 0.05.
connections with the motor and somatosensory areas of the cerebral cortex (15, 23) and is thought to have a sensorimotor function as opposed to more medial and ventral areas of the striatum which are thought to have associative and limbic functions respectively (15). The fact that the largest changes in CB1 mRNA expression were seen in sensorimotor areas suggests that increased endocannabinoid levels might be associated with the motor symptoms of Parkinson’s disease. However, it must be noted that previous studies in the 6-hydroxydopamine-lesioned rat model of Parkinson’s disease did not demonstrate a reduction in CB1 receptor mRNA expression (40) or binding (20). Indeed, one study reported an increase in CB1 receptor mRNA expression after 6-hydoxydopamine treatment (25). It is possible that an attempted compensatory decrease in
CB1 receptor synthesis following dopamine depletion may be short-lived and though observed in the reserpine-treated rat may not persist in the 6-hydroxydopamine model where the dopamine depletion is maintained over weeks. Alternatively, it is possible that these previous studies in the 6-hydroxydopamine-lesioned rat were influenced by the fact that apomorphine treatment to assess lesion extent formed part of the study protocol. Indeed, dopamine receptor stimulation has been shown to increase CB1 mRNA expression (40). Another possible reason for the difference between the decrease in CB1 receptor mRNA expression shown in this study and the lack of change in the 6-hydroxydopamine model may be that reductions in 5-hydroxytryptamine or noradrenaline levels, both of which are reduced by reserpine treatment, but not by
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6-hydroxydopamine lesions (6), are responsible for regulating CB1 receptor expression. If increased endocannabinoid stimulation of the CB1 receptor is a component of the neural mechanism underlying Parkinson’s disease, one might expect CB1 antagonists to be useful in the treatment of Parkinson’s disease. Indeed, the cannabinoid antagonist SR141716A markedly potentiates the alleviation of akinesia produced by the D2 agonist quinpirole in the reserpine-treated rat model of Parkinson’s disease (10, 11) and does have mild antiparkinsonian actions in its own right in the MPTP-lesioned monkey (M. Hill, personal communication). In summary, this study provides further evidence for abnormalities of endocannabinoid transmission in a rat model of Parkinson’s disease. We propose that cannabinoid receptor antagonists may provide a useful treatment for Parkinson’s disease. REFERENCES 1.
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