- Email: [email protected]
Effects of intranigral cannabinoids on rotational behavior in rats: interactions with the dopaminergic system
ELSEVIER
Neuroscience Letters 206 (1996) 21-24
NHHDSCIENCE LEITfKS
Effects of intranigral cannabinoids on rotational behavior in rats: interactions with the dopaminergic system M . C l a r a S a f i u d o - P e f i a , S a u n d r a L. P a t r i c k , R o b e r t L. P a t r i c k , J. M i c h a e l W a l k e r * Schrier Research Laboratory, Departmentof Psychology, Brown University, P.O. Box 1853, 89 Waterman Street, Providence, R102912, USA
Received 17 October 1995; revised version received2 January 1996; accepted29 January 1996
Abstract
The effect of unilateral intranigral cannabinoid receptor stimulation on rotational behavior was explored. The potent cannabinoid agonist CP 55,940 (5 and 10 ktg/0.5 ~1) induced contralateral turning when microinjected unilaterally into the substantia nigra pars reticulata. In addition, the cannabinoid agonist markedly attenuated the contralateral rotation induced by the dopamine D 1 agonist SKF 82958 and completely reversed the ipsilateral rotation induced by the dopamine D2 agonist quinpirole. In both cases, the coadministration of the cannabinoid agonist together with the D 1 or D2 agonist induced contralateral rotation. It appears that cannabinoids may exert different effects depending on the state of the ongoing chemical activation of this brain circuitry. Keywords: Cannabinoids; Circling; Substantia nigra; Dopamine
Employed since ancient times for both therapeutic and recreational purposes [1], marijuana (Cannabis sativa) remains one of the most widely used drugs in the world. Accordingly, modern research has focused on its potential medical uses and dangers. Recent work has shown that exogenously administered cannabinoids produce their effects by actions on the brain's own cannabinoid neural system. Evidence for this novel neurochemical system includes the cloning of a G-protein coupled cannabinoid receptor, the identification of second messengers and ion channels that mediate the effects of cannabinoids and the identification of putative endogenous ligands for the receptor along with the pathways for their synthesis and degradation [1,3,9,14]. These landmark studies demonstrating the existence of an endogenous cannabinergic neurotransmitter system [3,4,8,9,15} have instigated new lines of research aimed at understanding the functional role of endogenous cannabinoids. A remarkable aspect of the distribution of cannabinoid receptors is their high density in brain areas that participate in motor control [8,15]. The basal ganglia, cerebellum and related structures are enriched in cannabinoid * Corresponding author. Tel.: +I 401 8632727; fax: +1 401 8631300; e-mail: [email protected].
receptors and could therefore mediate the powerful motor effects of cannabinoids [ 1,9,17]. The highest densities of cannabinoid receptors occur in the outflow nuclei of the basal ganglia: the substantia nigra pars reticulata (SNr), the globus pallidus, and the entopeduncular nucleus [8, 15]. A better understanding of the role of cannabinoids in the basal ganglia may be important because these nuclei are involved in many neurodegenerative disorders (for review see [6]). Histochemical and lesion studies have localized cannabinoid receptors to axon terminals innervating the output structures of the basal ganglia [8,15]. Cannabinoid receptors are colocalized with dopamine D l receptors on striatonigral GABA/substance P/dynorphin neurons. However, agonists for the cannabinoid and D1 receptors produce opposite effects on cyclic AMP [6,9] and because of their inhibitory action on N-type calcium channels [14], the effect of cannabinoids on neurotransmitter release would be opposite to that generally accepted for D~ receptors. This suggests that they would likewise exert opposite effects on motor function by their actions at this common neural substrate. In contrast to dopamine D] receptors, the D2 family of receptors in the substantia nigra are mainly located on the dopaminergic neurons, which apparently lack cannabi-
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S0304-3940(96) 12436-6
22
M.C. Sa~udo-Pe~a et al. / Neuroscience Letters 206 (1996) 2 1 - 2 4
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Fig. 1. Effect of unilateral microinjections of various doses of the cannabinoid agonist CP 55,940 into the substantia nigra pars reticulata on turning behavior (*significantly different from vehicle group, P < 0.05). noid receptors [6,8,13,15]. However, cannabinoid receptors are colocalized with D2 receptors on the striatopallidal GABA/enkephalin neurons [6,8,13,15]. The density of cannabinoid receptors in the substantia nigra is comparable to that of dopamine receptors [8], yet very little is known about the functional properties of cannabinoid receptors in the substantia nigra. The present experiments were thus designed (1) to examine the effect of activation of cannabinoid receptors in the SNr on turning behavior and (2) to examine the relationship between cannabinoids and the dopaminergic system. Male Sprague-Dawley rats (Charles River Laboratories, 250-280 g), served as subjects. Animals were anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic frame. In order to avoid excessive tissue damage, the 24-gauge stainless guide cannula was implanted so that its tip was 4.0 mm above the left SNr, 3 . 2 m m anterior, 2 . 2 m m lateral, 4.7 mm ventral to lambda and the skull surface. Stainless steel stylets were used to seal the cannulae. Following at least 3 days recovery, animals were tested for turning behavior after intranigral injection of a drug or combination of drugs. Drugs (dissolved in 60% dimethylsulfoxide) were injected (0.5 ~1 over a 2 min period) using a 31-gauge stainless steel microneedle that extended 4 mm beyond the guide cannula, thus reaching the SNr. After injection, it was left in place for an additional 30 s to allow the drug to diffuse. The animal was then placed in a computerized rotometer for 30 min. Following this single behavioral test, each rat
was perfused transcardially under deep anesthesia with a 10% formalin solution, frozen coronal sections (40/,m) were obtained, stained with cresyl violet and examined under a microscope to localize injection sites. Only the data from those animals with injection sites in the substantia nigra pars reticulata were included in the analyses. Net contralateral half turns (contralateral minus ipsilateral) were used in all data analyses. Three experiments were performed. In the first experiment, we examined the effect of intranigral administration of two doses of the cannabinoid agonist CP 55,940 (5 or 10big). In a second experiment, we examined the effect of coadministration of CP 55,940 with the D1 agonist SKF 82958. Finally, we examined the effect of CP 55,940 on the ipsilateral turning induced by intranigral administration of the D2 agonist quinpirole. Due to heterogeneity of variance, data were analyzed using a non-parametric one-way Kruskal-Wallis test. Post hoc comparisons were carried out using the Mann-Whitney test. The potent cannabinoid agonist CP 55,940 was generously provided by Pfizer (Groton, CT). The DI dopamine receptor agonist SKF 82958 and the D 2 dopamine agonist quinpirole were obtained from Research Biochemicals International (Natick, MA). All other drugs and chemicals were obtained from Sigma (St. Louis, MO).
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Fig. 2. Effect of unilateral microinjections of the D1 dopamine receptor agonist SKF 82958 alone or together with the cannabinoid agonist CP 55,940 into the substantia nigra pars reticulata on turning behavior (*significantly different from vehicle group, **significantly different from the rest of the groups, P < 0.05). The cannabinoid significantly reduced the contralateral turning induced by the dopamine D l agonist.
23
M.C. Sahudo-Pegta et al. I Neuroscience Letters 206 (1996) 2 1 - 2 4
An overall analysis of variance revealed that various drug combinations administered into the SNr produced markedly different effects on rotational behavior (Kruskal-Wallis test, H = 37.3, P < 0.00001). At both doses tested, CP 55,940 induced significant contralateral rotation when injected into the SNr compared to the vehicle group (Fig. 1; Mann-Whitney test, P < 0.03). However, no significant differences in rotation were found between the doses of the cannabinoid. Injections of SKF 82958 (10ktg) in the SNr induced significant contralateral rotational behavior (Mann-Whitney test, P < 0.0002; Fig. 2). Coadministration of the D1 agonist with the cannabinoid agonist at either of the two doses tested significantly reduced the contralateral turning produced by administration of SKF 82958 (P < 0.03). However the rotational behavior was significantly greater than that found in vehicle controls (P < 0.003). In contrast to the D] agonist, the D 2 agonist produced significant ipsilateral circling upon microinjection in the SNr (P < 0.003, Fig. 3). Administration of the cannabinoid with the dopamine D 2 agonist not only reversed the ipsilateral rotation induced by the D2 agonist ( P < 0.0006), it induced significant contralateral rotation (P < 0.02) approximately equal to that produced by the same dose of the cannabinoid alone. The experiments described above yielded two major findings: (1) a cannabinoid agonist produced contralateral circling when unilaterally microinjected into the SNr; and (2) the cannabinoid counteracted the effects of both dopamine D1 and D 2 agonists on rotational behavior. This was surprising, because dopamine D 1 and D~ agonists produced opposite effects. These findings, together with other recent reports on the effects of cannabinoids on nigral function [7,16], suggest that endogenous cannabinoids serve naturally to modulate the activity of the basal ganglia and exert significant influences on dopaminergic neurotransmission. Our data are in agreement with previous reports that activation of dopamine D l receptors in the SNr causes contralateral rotation [2,11]. This effect appears to take place at striatonigral terminals [8,11-13], because stimulation of Dl receptors in the SNr increases the extracellular levels of GABA and dynorphin, and decreases SNr firing with a consequent increase in motor output [6,20]. The decrease in the contralateral rotation induced by the Dl agonist when it is coadministered with the cannabinoid is likely to result from an inhibitory action of the cannabinoid at striatonigral terminals. Cannabinoid and D l receptors are colocalized on striatonigral terminals where they appear to mediate functionally opposite effects, which may account for the suppression of D] receptor-mediated motor effects by the cannabinoid agonist [6, 8,9]. This conclusion is consistent with our previous demonstration that cannabinoids block the inhibition of SNr neurons produced by electrical stimulation of the striatum [ 16]. The residual contralateral rotation observed
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Fig. 3. Effect of unilateral microinjections of the dopamine D 2 agonist quinpirole alone or together with the cannabinoid agonist CP 55,940 into the substantia nigra pars reticulata on turning behavior (*significantly different from vehicle group, **significantly different from the rest of the group). The cannabinoid reversed the ipsilateral tuming induced by the dopamine D 2 agonist.
in these animals supports the idea of a parallel action of cannabinoids at other sites within the substantia nigra. Our results confirm a previous demonstration of ipsilateral turning following unilateral intranigral microinjection of D 2 agonists [2]. This effect appears to result from stimulation of somatodendritic autoreceptors located on the dopaminergic cells of the substantia nigra pars compacta, whose processes invade the pars reticulata [6,8,18]. Thus, D 2 activation inhibits the firing of substantia nigra pars compacta dopaminergic neurons [ 10,18], which may also decrease dopamine release in the substantia nigra and the activation of D 1 receptors, thereby reducing the influence of the striatonigral pathway [5,18]. These factors would account for the ipsilateral circling produced by unilateral administration of the D 2 agonist [2,11 ]. CP 55,940 blocked the ipsilateral turning produced by intranigral administration of a D 2 agonist and induced contralateral rotation of similar magnitude to that produced by the cannabinoid alone. Since D z receptors are concentrated on dopaminergic neurons, which apparently lack cannabinoid receptors, it appears likely that the cannabinoid agonist and the D 2 agonist acted on different neuronal populations. If this is the case, the antagonistic effect would be mainly due to different actions of these agonists on different parts of the circuit rather than to opposing effects on the same neurons. However, a low
24
M.C. Sa~udo-Pe~a et al. / Neuroscience Letters 206 (1996) 2 1 - 2 4
density o f d o p a m i n e D2 receptors m a y occur on striatonigral terminals [12] suggesting other plausible explanations o f the effect. T h e s e e x p e r i m e n t s r e v e a l e d that intranigral administration o f the c a n n a b i n o i d agonist increases m o t o r output and produces contralateral turning. In v i e w of the inhibition o f D1 r e c e p t o r - m e d i a t e d contralateral turning by the cannabinoid, this m a y s e e m surprising at first glance. H o w e v e r , one w o u l d not e x p e c t the effects o f the cannabinoid on striatonigral terminals to account for the effects o f cannabinoids administered alone, because the striatonigral pathway exhibits low tonic activity [21]. By contrast, the main excitatory input to the SNr, which arises f r o m the subthalamic nucleus, is tonically active [19]. T h e subthalamic nucleus has been reported to have m o d e r a t e levels o f m R N A for the cannabinoid receptor [15] and c o u l d therefore contribute to the pool of presynaptic c a n n a b i n o i d receptors found in the SNr. This possibility is consistent with the presence o f residual nigral cannabinoid receptors (>20%) f o l l o w i n g striatal lesions that p r o d u c e a c o m p l e t e loss o f nigral D1 receptors [8], suggesting the p r e s e n c e o f another significant source of cannabinoid receptors in the substantia nigra. Therefore, C P 55,940 m a y act by inhibiting neurotransmitter release from the excitatory terminals derived f r o m subthalamic neurons. This w o u l d r e d u c e the firing rate o f SNr neurons, which w o u l d account for the contralateral rotation observed. O f course, other possibilities cannot be excluded. In conclusion, our data point to a c o m p l e x action o f cannabinoids in the substantia nigra. T h e interaction with the d o p a m i n e r g i c system, a major neurotransmitter system in m o t o r control, and the ability o f the cannabinoid agonist to exert opposite actions d e p e n d i n g on the c h e m i cal activity within this circuitry suggest that e n d o g e n o u s cannabinoids m a y exert important m o d u l a t o r y actions on motor function through actions in the substantia nigra. [1] Abood, M.E. and Martin, B.R., Neurobiology of marijuana abuse, Trends Neurosci., 13 (1992) 201-206. [2] Asin, K.E. and Montana, W.E., Rotation following intranigral injections of a selective D1 or a selective D2 dopamine receptor agonist in rats, Pharmacol. Biochem. Behav., 29 (1988) 89-92. [3l Desarnaud, F., Cadas, H. and Piomelli, D., Anandamide amidohydrolase activity in rat brain microsomes, J. Biol. Chem., 270 (1995) 6030--6035. [4] Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. and Mechoulam, R., Isolation and structure of a brain constituent that binds to the cannabinoid receptor, Nature, 258 (1992) 1946-1949.
[5] Gerfen, C.R., Dopamine receptor function in the basal ganglia, Clin. Neuropharmacol., 18 (1995) S 162-S 177. [6] Graybiel, A.M., Neurotransmitters and neuromodulators in the basal ganglia, Trends Neurosci., 13 (1990) 244-253. [7] Gueudet, C., Santucci, V., Rinaldi-Carmona, M., Soubrie, P. and Le Fur, G., The CB 1 cannabinoid receptor antagonist SR 141716A affects A9 dopamine neuronal activity in the rat, NeuroReport, 6 (1995) 1293-1297. [8] Herkenham, M., Lynn, A.B., de Costa, B.R. and Richfield, E.K., Neuronal localization of cannabinoid receptors in the basal ganglia of the rat, Brain Res., 547 (1991) 267-274. [9] Howlett, A.C., Pharmacology of cannabinoid receptors, Ann. Rev. Pharmacol. Toxicol., 35 (1995) 607~534 [10] Kalivas, P.W. and Duffy, P.A., A comparison of axonal and somatodendritic dopamine release using in vivo dialysis, J. Neurochem., 56 (1991) 961-967. [11] LaHoste, G.J. and Marshall, J.F., Nigral DI and striatal D2 receptors mediate the behavioral effects of dopamine agonists, Behav. Brain Res., 38 (1990) 233-242. [12] Larson, E.R. and Ariano, M.A., Dopamine receptor binding on identified striatonigral neurons, Neurosci. Lett., 172 (1994) 101106. [13] Le Moine, C. and Bloch, B., D1 and D2 dopamine receptor gene expression in the rat striatum: sensitive cRNA probes demonstrate prominent segregation of D1 and D2 mRNAs in distinct neuronal populations of the dorsal and ventral striatum, J. Comp. Neurol., 35 (1995) 418-426. [14] Mackie, K. and Hille, B., Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells, Proc. Natl. Acad. Sci. USA, 89 (1992) 3825-3829. [15] Mailleux, P. and Vanderhaeghen, J.J., Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry, Neuroscience, 48 (1992) 655~588. [16] Miller, A. and Walker, J.M., Effects of a cannabinoid on spontaneous and evoked neuronal activity in the substantia nigra pars reticulata, Eur. J. Pharmacol., 279 (1995) 179-185. [17] Navarro, M., Fernandez-Ruiz, J.J., De Miguel, R., Hernandez, M.L., Cebeira, M. and Ramos, J.A., Motor disturbances induced by an acute dose of D9-tetrahydrocannabinol: possible involvement of nigrostriatal dopaminergic alterations, Pharmacol. Biochem. Behav., 45 (1993) 291-298. [18] Pucak, M.L. and Grace, A.A., Evidence that systemically administered dopamine antagonists activate dopamine neuron firing primarily by blockade of somatodendritic autoreceptors, J. Pharmacol. Exp. Ther., 271 (1994) 1181-1192. [19] Robledo, P. and Feber, J., Excitatory influence of rat subthalamic nucleus to substantia nigra pars reticulata and the pallidal complex: electrophysiological data, Brain Res., 518 (1990) 47-54. [20] You, Z.B., Herrera-Marschitz, M., Nylander, I., Goiny, M., O'Connor, W.T., Ungerstedt, U. and Terenius, L., The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis, II: effects of dopamine D 1 and D2 receptor agonists, Neuroscience, 63 (1994) 427-434. [21] Wilson, C.J., The generation of natural firing patterns in neostriatal neurons. In G.W. Arbuthnott and P.C. Empson (Eds.), Chemical Signalling in the Basal Ganglia, Progress in Brain Research, Vol. 99, Elsevier, Amsterdam, 1993, pp. 277-298.
Neuroscience Letters 206 (1996) 21-24
NHHDSCIENCE LEITfKS
Effects of intranigral cannabinoids on rotational behavior in rats: interactions with the dopaminergic system M . C l a r a S a f i u d o - P e f i a , S a u n d r a L. P a t r i c k , R o b e r t L. P a t r i c k , J. M i c h a e l W a l k e r * Schrier Research Laboratory, Departmentof Psychology, Brown University, P.O. Box 1853, 89 Waterman Street, Providence, R102912, USA
Received 17 October 1995; revised version received2 January 1996; accepted29 January 1996
Abstract
The effect of unilateral intranigral cannabinoid receptor stimulation on rotational behavior was explored. The potent cannabinoid agonist CP 55,940 (5 and 10 ktg/0.5 ~1) induced contralateral turning when microinjected unilaterally into the substantia nigra pars reticulata. In addition, the cannabinoid agonist markedly attenuated the contralateral rotation induced by the dopamine D 1 agonist SKF 82958 and completely reversed the ipsilateral rotation induced by the dopamine D2 agonist quinpirole. In both cases, the coadministration of the cannabinoid agonist together with the D 1 or D2 agonist induced contralateral rotation. It appears that cannabinoids may exert different effects depending on the state of the ongoing chemical activation of this brain circuitry. Keywords: Cannabinoids; Circling; Substantia nigra; Dopamine
Employed since ancient times for both therapeutic and recreational purposes [1], marijuana (Cannabis sativa) remains one of the most widely used drugs in the world. Accordingly, modern research has focused on its potential medical uses and dangers. Recent work has shown that exogenously administered cannabinoids produce their effects by actions on the brain's own cannabinoid neural system. Evidence for this novel neurochemical system includes the cloning of a G-protein coupled cannabinoid receptor, the identification of second messengers and ion channels that mediate the effects of cannabinoids and the identification of putative endogenous ligands for the receptor along with the pathways for their synthesis and degradation [1,3,9,14]. These landmark studies demonstrating the existence of an endogenous cannabinergic neurotransmitter system [3,4,8,9,15} have instigated new lines of research aimed at understanding the functional role of endogenous cannabinoids. A remarkable aspect of the distribution of cannabinoid receptors is their high density in brain areas that participate in motor control [8,15]. The basal ganglia, cerebellum and related structures are enriched in cannabinoid * Corresponding author. Tel.: +I 401 8632727; fax: +1 401 8631300; e-mail: [email protected].
receptors and could therefore mediate the powerful motor effects of cannabinoids [ 1,9,17]. The highest densities of cannabinoid receptors occur in the outflow nuclei of the basal ganglia: the substantia nigra pars reticulata (SNr), the globus pallidus, and the entopeduncular nucleus [8, 15]. A better understanding of the role of cannabinoids in the basal ganglia may be important because these nuclei are involved in many neurodegenerative disorders (for review see [6]). Histochemical and lesion studies have localized cannabinoid receptors to axon terminals innervating the output structures of the basal ganglia [8,15]. Cannabinoid receptors are colocalized with dopamine D l receptors on striatonigral GABA/substance P/dynorphin neurons. However, agonists for the cannabinoid and D1 receptors produce opposite effects on cyclic AMP [6,9] and because of their inhibitory action on N-type calcium channels [14], the effect of cannabinoids on neurotransmitter release would be opposite to that generally accepted for D~ receptors. This suggests that they would likewise exert opposite effects on motor function by their actions at this common neural substrate. In contrast to dopamine D] receptors, the D2 family of receptors in the substantia nigra are mainly located on the dopaminergic neurons, which apparently lack cannabi-
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S0304-3940(96) 12436-6
22
M.C. Sa~udo-Pe~a et al. / Neuroscience Letters 206 (1996) 2 1 - 2 4
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Fig. 1. Effect of unilateral microinjections of various doses of the cannabinoid agonist CP 55,940 into the substantia nigra pars reticulata on turning behavior (*significantly different from vehicle group, P < 0.05). noid receptors [6,8,13,15]. However, cannabinoid receptors are colocalized with D2 receptors on the striatopallidal GABA/enkephalin neurons [6,8,13,15]. The density of cannabinoid receptors in the substantia nigra is comparable to that of dopamine receptors [8], yet very little is known about the functional properties of cannabinoid receptors in the substantia nigra. The present experiments were thus designed (1) to examine the effect of activation of cannabinoid receptors in the SNr on turning behavior and (2) to examine the relationship between cannabinoids and the dopaminergic system. Male Sprague-Dawley rats (Charles River Laboratories, 250-280 g), served as subjects. Animals were anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic frame. In order to avoid excessive tissue damage, the 24-gauge stainless guide cannula was implanted so that its tip was 4.0 mm above the left SNr, 3 . 2 m m anterior, 2 . 2 m m lateral, 4.7 mm ventral to lambda and the skull surface. Stainless steel stylets were used to seal the cannulae. Following at least 3 days recovery, animals were tested for turning behavior after intranigral injection of a drug or combination of drugs. Drugs (dissolved in 60% dimethylsulfoxide) were injected (0.5 ~1 over a 2 min period) using a 31-gauge stainless steel microneedle that extended 4 mm beyond the guide cannula, thus reaching the SNr. After injection, it was left in place for an additional 30 s to allow the drug to diffuse. The animal was then placed in a computerized rotometer for 30 min. Following this single behavioral test, each rat
was perfused transcardially under deep anesthesia with a 10% formalin solution, frozen coronal sections (40/,m) were obtained, stained with cresyl violet and examined under a microscope to localize injection sites. Only the data from those animals with injection sites in the substantia nigra pars reticulata were included in the analyses. Net contralateral half turns (contralateral minus ipsilateral) were used in all data analyses. Three experiments were performed. In the first experiment, we examined the effect of intranigral administration of two doses of the cannabinoid agonist CP 55,940 (5 or 10big). In a second experiment, we examined the effect of coadministration of CP 55,940 with the D1 agonist SKF 82958. Finally, we examined the effect of CP 55,940 on the ipsilateral turning induced by intranigral administration of the D2 agonist quinpirole. Due to heterogeneity of variance, data were analyzed using a non-parametric one-way Kruskal-Wallis test. Post hoc comparisons were carried out using the Mann-Whitney test. The potent cannabinoid agonist CP 55,940 was generously provided by Pfizer (Groton, CT). The DI dopamine receptor agonist SKF 82958 and the D 2 dopamine agonist quinpirole were obtained from Research Biochemicals International (Natick, MA). All other drugs and chemicals were obtained from Sigma (St. Louis, MO).
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Fig. 2. Effect of unilateral microinjections of the D1 dopamine receptor agonist SKF 82958 alone or together with the cannabinoid agonist CP 55,940 into the substantia nigra pars reticulata on turning behavior (*significantly different from vehicle group, **significantly different from the rest of the groups, P < 0.05). The cannabinoid significantly reduced the contralateral turning induced by the dopamine D l agonist.
23
M.C. Sahudo-Pegta et al. I Neuroscience Letters 206 (1996) 2 1 - 2 4
An overall analysis of variance revealed that various drug combinations administered into the SNr produced markedly different effects on rotational behavior (Kruskal-Wallis test, H = 37.3, P < 0.00001). At both doses tested, CP 55,940 induced significant contralateral rotation when injected into the SNr compared to the vehicle group (Fig. 1; Mann-Whitney test, P < 0.03). However, no significant differences in rotation were found between the doses of the cannabinoid. Injections of SKF 82958 (10ktg) in the SNr induced significant contralateral rotational behavior (Mann-Whitney test, P < 0.0002; Fig. 2). Coadministration of the D1 agonist with the cannabinoid agonist at either of the two doses tested significantly reduced the contralateral turning produced by administration of SKF 82958 (P < 0.03). However the rotational behavior was significantly greater than that found in vehicle controls (P < 0.003). In contrast to the D] agonist, the D 2 agonist produced significant ipsilateral circling upon microinjection in the SNr (P < 0.003, Fig. 3). Administration of the cannabinoid with the dopamine D 2 agonist not only reversed the ipsilateral rotation induced by the D2 agonist ( P < 0.0006), it induced significant contralateral rotation (P < 0.02) approximately equal to that produced by the same dose of the cannabinoid alone. The experiments described above yielded two major findings: (1) a cannabinoid agonist produced contralateral circling when unilaterally microinjected into the SNr; and (2) the cannabinoid counteracted the effects of both dopamine D1 and D 2 agonists on rotational behavior. This was surprising, because dopamine D 1 and D~ agonists produced opposite effects. These findings, together with other recent reports on the effects of cannabinoids on nigral function [7,16], suggest that endogenous cannabinoids serve naturally to modulate the activity of the basal ganglia and exert significant influences on dopaminergic neurotransmission. Our data are in agreement with previous reports that activation of dopamine D l receptors in the SNr causes contralateral rotation [2,11]. This effect appears to take place at striatonigral terminals [8,11-13], because stimulation of Dl receptors in the SNr increases the extracellular levels of GABA and dynorphin, and decreases SNr firing with a consequent increase in motor output [6,20]. The decrease in the contralateral rotation induced by the Dl agonist when it is coadministered with the cannabinoid is likely to result from an inhibitory action of the cannabinoid at striatonigral terminals. Cannabinoid and D l receptors are colocalized on striatonigral terminals where they appear to mediate functionally opposite effects, which may account for the suppression of D] receptor-mediated motor effects by the cannabinoid agonist [6, 8,9]. This conclusion is consistent with our previous demonstration that cannabinoids block the inhibition of SNr neurons produced by electrical stimulation of the striatum [ 16]. The residual contralateral rotation observed
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Fig. 3. Effect of unilateral microinjections of the dopamine D 2 agonist quinpirole alone or together with the cannabinoid agonist CP 55,940 into the substantia nigra pars reticulata on turning behavior (*significantly different from vehicle group, **significantly different from the rest of the group). The cannabinoid reversed the ipsilateral tuming induced by the dopamine D 2 agonist.
in these animals supports the idea of a parallel action of cannabinoids at other sites within the substantia nigra. Our results confirm a previous demonstration of ipsilateral turning following unilateral intranigral microinjection of D 2 agonists [2]. This effect appears to result from stimulation of somatodendritic autoreceptors located on the dopaminergic cells of the substantia nigra pars compacta, whose processes invade the pars reticulata [6,8,18]. Thus, D 2 activation inhibits the firing of substantia nigra pars compacta dopaminergic neurons [ 10,18], which may also decrease dopamine release in the substantia nigra and the activation of D 1 receptors, thereby reducing the influence of the striatonigral pathway [5,18]. These factors would account for the ipsilateral circling produced by unilateral administration of the D 2 agonist [2,11 ]. CP 55,940 blocked the ipsilateral turning produced by intranigral administration of a D 2 agonist and induced contralateral rotation of similar magnitude to that produced by the cannabinoid alone. Since D z receptors are concentrated on dopaminergic neurons, which apparently lack cannabinoid receptors, it appears likely that the cannabinoid agonist and the D 2 agonist acted on different neuronal populations. If this is the case, the antagonistic effect would be mainly due to different actions of these agonists on different parts of the circuit rather than to opposing effects on the same neurons. However, a low
24
M.C. Sa~udo-Pe~a et al. / Neuroscience Letters 206 (1996) 2 1 - 2 4
density o f d o p a m i n e D2 receptors m a y occur on striatonigral terminals [12] suggesting other plausible explanations o f the effect. T h e s e e x p e r i m e n t s r e v e a l e d that intranigral administration o f the c a n n a b i n o i d agonist increases m o t o r output and produces contralateral turning. In v i e w of the inhibition o f D1 r e c e p t o r - m e d i a t e d contralateral turning by the cannabinoid, this m a y s e e m surprising at first glance. H o w e v e r , one w o u l d not e x p e c t the effects o f the cannabinoid on striatonigral terminals to account for the effects o f cannabinoids administered alone, because the striatonigral pathway exhibits low tonic activity [21]. By contrast, the main excitatory input to the SNr, which arises f r o m the subthalamic nucleus, is tonically active [19]. T h e subthalamic nucleus has been reported to have m o d e r a t e levels o f m R N A for the cannabinoid receptor [15] and c o u l d therefore contribute to the pool of presynaptic c a n n a b i n o i d receptors found in the SNr. This possibility is consistent with the presence o f residual nigral cannabinoid receptors (>20%) f o l l o w i n g striatal lesions that p r o d u c e a c o m p l e t e loss o f nigral D1 receptors [8], suggesting the p r e s e n c e o f another significant source of cannabinoid receptors in the substantia nigra. Therefore, C P 55,940 m a y act by inhibiting neurotransmitter release from the excitatory terminals derived f r o m subthalamic neurons. This w o u l d r e d u c e the firing rate o f SNr neurons, which w o u l d account for the contralateral rotation observed. O f course, other possibilities cannot be excluded. In conclusion, our data point to a c o m p l e x action o f cannabinoids in the substantia nigra. T h e interaction with the d o p a m i n e r g i c system, a major neurotransmitter system in m o t o r control, and the ability o f the cannabinoid agonist to exert opposite actions d e p e n d i n g on the c h e m i cal activity within this circuitry suggest that e n d o g e n o u s cannabinoids m a y exert important m o d u l a t o r y actions on motor function through actions in the substantia nigra. [1] Abood, M.E. and Martin, B.R., Neurobiology of marijuana abuse, Trends Neurosci., 13 (1992) 201-206. [2] Asin, K.E. and Montana, W.E., Rotation following intranigral injections of a selective D1 or a selective D2 dopamine receptor agonist in rats, Pharmacol. Biochem. Behav., 29 (1988) 89-92. [3l Desarnaud, F., Cadas, H. and Piomelli, D., Anandamide amidohydrolase activity in rat brain microsomes, J. Biol. Chem., 270 (1995) 6030--6035. [4] Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. and Mechoulam, R., Isolation and structure of a brain constituent that binds to the cannabinoid receptor, Nature, 258 (1992) 1946-1949.
[5] Gerfen, C.R., Dopamine receptor function in the basal ganglia, Clin. Neuropharmacol., 18 (1995) S 162-S 177. [6] Graybiel, A.M., Neurotransmitters and neuromodulators in the basal ganglia, Trends Neurosci., 13 (1990) 244-253. [7] Gueudet, C., Santucci, V., Rinaldi-Carmona, M., Soubrie, P. and Le Fur, G., The CB 1 cannabinoid receptor antagonist SR 141716A affects A9 dopamine neuronal activity in the rat, NeuroReport, 6 (1995) 1293-1297. [8] Herkenham, M., Lynn, A.B., de Costa, B.R. and Richfield, E.K., Neuronal localization of cannabinoid receptors in the basal ganglia of the rat, Brain Res., 547 (1991) 267-274. [9] Howlett, A.C., Pharmacology of cannabinoid receptors, Ann. Rev. Pharmacol. Toxicol., 35 (1995) 607~534 [10] Kalivas, P.W. and Duffy, P.A., A comparison of axonal and somatodendritic dopamine release using in vivo dialysis, J. Neurochem., 56 (1991) 961-967. [11] LaHoste, G.J. and Marshall, J.F., Nigral DI and striatal D2 receptors mediate the behavioral effects of dopamine agonists, Behav. Brain Res., 38 (1990) 233-242. [12] Larson, E.R. and Ariano, M.A., Dopamine receptor binding on identified striatonigral neurons, Neurosci. Lett., 172 (1994) 101106. [13] Le Moine, C. and Bloch, B., D1 and D2 dopamine receptor gene expression in the rat striatum: sensitive cRNA probes demonstrate prominent segregation of D1 and D2 mRNAs in distinct neuronal populations of the dorsal and ventral striatum, J. Comp. Neurol., 35 (1995) 418-426. [14] Mackie, K. and Hille, B., Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells, Proc. Natl. Acad. Sci. USA, 89 (1992) 3825-3829. [15] Mailleux, P. and Vanderhaeghen, J.J., Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry, Neuroscience, 48 (1992) 655~588. [16] Miller, A. and Walker, J.M., Effects of a cannabinoid on spontaneous and evoked neuronal activity in the substantia nigra pars reticulata, Eur. J. Pharmacol., 279 (1995) 179-185. [17] Navarro, M., Fernandez-Ruiz, J.J., De Miguel, R., Hernandez, M.L., Cebeira, M. and Ramos, J.A., Motor disturbances induced by an acute dose of D9-tetrahydrocannabinol: possible involvement of nigrostriatal dopaminergic alterations, Pharmacol. Biochem. Behav., 45 (1993) 291-298. [18] Pucak, M.L. and Grace, A.A., Evidence that systemically administered dopamine antagonists activate dopamine neuron firing primarily by blockade of somatodendritic autoreceptors, J. Pharmacol. Exp. Ther., 271 (1994) 1181-1192. [19] Robledo, P. and Feber, J., Excitatory influence of rat subthalamic nucleus to substantia nigra pars reticulata and the pallidal complex: electrophysiological data, Brain Res., 518 (1990) 47-54. [20] You, Z.B., Herrera-Marschitz, M., Nylander, I., Goiny, M., O'Connor, W.T., Ungerstedt, U. and Terenius, L., The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis, II: effects of dopamine D 1 and D2 receptor agonists, Neuroscience, 63 (1994) 427-434. [21] Wilson, C.J., The generation of natural firing patterns in neostriatal neurons. In G.W. Arbuthnott and P.C. Empson (Eds.), Chemical Signalling in the Basal Ganglia, Progress in Brain Research, Vol. 99, Elsevier, Amsterdam, 1993, pp. 277-298.