Effects of blockade of metabotropic glutamate receptors in the subthalamic nucleus on haloperidol-induced Parkinsonism in rats

Neuroscience Letters 282 (2000) 21±24 www.elsevier.com/locate/neulet

Effects of blockade of metabotropic glutamate receptors in the subthalamic nucleus on haloperidol-induced Parkinsonism in rats Hideto Miwa a, b,*, Katsunori Nishi a, Tatsu Fuwa a, Yoshikuni Mizuno c a

Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-city, Tokyo 183, Japan Department of Neurology, Juntendo University Urayasu Hospital, 2-1-1 Tomioka, Urayasu-City, Chiba 279-0021, Japan c Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

b

Received 10 December 1999; accepted 15 January 2000

Abstract The present study examined the postural effects of unilaterally local injection of metabotropic glutamate receptor (mGluR) antagonists into the subthalamic nucleus (STN), in rats with haloperidol-induced parkinsonism. In rats which received unilateral microinjections of (1)-a-methyl-4-carboxyphenylglycine (MCPG), a selective, subtype-non-speci®c antagonist of mGluR, but not the vehicle, into the STN, systemic administration of haloperidol induced ipsiversive dystonic posturing. The severity of the dystonic posturing was dose-dependent. However, subtype-speci®c antagonists of group I, II, or III mGluRs induced no dystonic posturing. The present ®ndings suggest that the activity of the STN under conditions of dopamine blockade is facilitated by blockade of mGluRs in the STN, suggesting that mGluRs exert inhibitory in¯uence on glutamate release in the STN. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Subthalamic nucleus; Parkinson's disease; Haloperidol; Circling; Metabotropic glutamate receptor; (1)-a-Methyl-4-carboxyphenylglycine

Based on the current understanding of the neural framework of the basal ganglia, the subthalamic nucleus (STN) and its efferents to the basal ganglia output nuclei, such as the entopeduncular nucleus (EP) and the substantia nigra pars reticulata (SNr), have a key role in the pathophysiological mechanisms underlying parkinsonism [2,6]. It has been proposed that dopamine depletion in the striatum induces overactivity of the STN-EP/SNr axis, as a result of the disinhibition of the STN due to its release from the inhibitory pallido-subthalamic projections, and in turn reduce thalamocortical activity, thereby inducing bradykinesia and rigidity [6]. However, there is a contention that this concept is oversimpli®ed [3,9,15]. There are lines of evidence suggesting that overactivity of the STN under dopamine depletion is not solely a result of disinhibition due to its release from the inhibitory pallido-subthalamic GABAergic projections. First, Hassani et al. [8] showed that the neuronal activity of the STN increases more markedly following striatal dopamine depletion than following globus pallidus (GP) lesions, and that the increase in the discharge rate of STN neurons following GP lesions is * Corresponding author. Tel.: 181-047-353-3111; fax: 181-047381-5054. E-mail address: [email protected] (H. Miwa)

only slight. Next, we reported that the administration of a D2 receptor antagonist in rats with a unilateral GP lesion caused contraversive dystonic posturing or circling [10], suggesting a marked difference between the effects of GP ablation and D2 receptor blockade on basal ganglia output activity. In addition, there are experimental ®ndings suggesting that the neurons in the GP are simply underactive in the dopamine-depleted state; a subset of neurons in the GP has been shown to exhibit a burst-®ring pattern in the dopamine-depleted state [7,14], possibly because of an increase in the excitatory drive exerted by the STN. Moreover, recent reports have revealed that the mRNA level of glutamic acid decarboxylase (GAD67), the rate-limiting enzyme for GABA synthesis, is increased in GP neurons under dopamine-depleted conditions [4,5]. The possibility of excitatory inputs actually contributing to the overactivity of the STN under dopamine depletion has been suggested. It has been proposed that the disinhibition from the pallidal GABAergic efferents under dopamine depletion is also accompanied by an excitatory drive exerted from somewhere in the brain, which induces overactivity of the STN neurons [8,9,11]. As is well known, glutamate is the principal excitatory transmitter in the CNS. If the increase in subthalamic neuron activity under dopamine

0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 84 3- 0

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H. Miwa et al. / Neuroscience Letters 282 (2000) 21±24

depletion is caused by glutamatergic inputs to the STN, it is expected that unilateral blockade of these glutamatergic inputs would result in a certain asymmetrical behavioral response to systemic dopamine blockade. It has been reported that the glutamate blockade in the STN, both of NMDA (N-methyl-d-aspartate) and AMPA (a-amino-3hydroxy-5-methyl-isoxazole-4-proprionate) receptors, prevents the overactivity of the STN under dopamine depletion [11]. Therefore, it is possible to consider that the glutamatergic input to the STN possibly contributes to the overactivity of the STN in parkinsonism. There is also another type of glutamate receptors. Glutamate activates not only ionotropic glutamate receptors such as NMDA and AMPA receptors but also metabotropic glutamate receptors, which couple to G-proteins and control the activity of membrane enzymes and ion channels [19]. Metabotropic glutamate receptors (mGluRs) modulate excitatory synaptic transmission through various transduction pathways [1,13,17]. The aim of the present study is to examine the postural effects of unilateral blockade of mGluRs within the STN under conditions of dopamine blockade. Male Wistar rats (n ˆ 16), weighing 260±290 g at the time of surgery, were used. The animals were housed in cages and kept under a 12/12-h-light/dark cycle in a colony room maintained at 22±238C. Behavioral experiments were performed during the light phase. Food and tap water were made available ad libitum except during experimental sessions. The rats were anesthetized with sodium pentobarbital (50 mg/kg) and mounted on a stereotaxic apparatus with ear bars. After drilling a small burr hole in the skull, a stainless steel guide cannula was implanted 2 mm above the STN, the stereotaxic coordinates of which, according to Paxinos and Watson's atlas [16], were as follows: 3.8 mm posterior to the bregma, 2.4 mm lateral to the sagittal suture, and 7.8 mm from the dura. The implanted guide cannula was normally occluded with a stainless steel dummy cannula. Behavioral experiments were performed following a recovery period of at least one week. The experiments were performed in an observation cage (30 £ 25 £ 50 cm) to which the animals had already become habituated. Animal behavior was recorded by a video camera placed 1.2 m above the observation cage. A vehicle (0.25 ml of saline) or the following drugs were then injected intracerebrally: (1)-a-methyl-4-carboxyphenylglycine (MCPG) (RBI), a non-selective antagonist of mGluRs; l-a-4-carboxyphenylglycine (4-CPG)(SIGMA, USA), a selective antagonist of group I mGluRs; (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG)(RBI, USA), a selective antagonist of group II mGluRs; (^)-a-methyl-4-(phosphonophenyl) glycine (MPPG)(SIGMA, USA), a selective antagonist of group III mGluRs; the drugs were dissolved in 0.1 M PB and the pH was adjusted immediately prior to the injection. The injection cannulae were connected to 10-ml Hamilton microsyringes mounted on an infusion pump (Harvard). All intracerebral injections were delivered in a volume of 0.25 ml. MCPG (2.5 mg, 0.1 mg), 4-CPG (0.25

mg), MCCG (0.25 mg), MPPG (0.25 mg), or the vehicle was infused into the STN via a 30G stainless steel hypodermic cannula at the rate of 0.5 ml/min, and the cannula was kept in situ for at least another minute before removal. During the infusion procedures, the animals were held gently by the experimenter. Fifty minutes after the intracerebral injections, haloperidol (5 mg/kg)(Dainippon, Japan), a potent D2 receptor antagonist, was administered intraperitoneally, and the elicited postural deviation, a static, ®xed postural asymmetry (the term `posturing' can be used to represent this phenomenon) [18], was quantitatively analyzed. Three markings were made on the midline of the animal's longitudinal axis: nose, midpoint of the back line, and tail (Fig. 1). Subsequently, the angle between the nose-back line and the back-tail line were measured, and scored as follows: 0, no ®xed postural alteration; 1, less than 308; 2, 30±598; 3, 60± 898; 4, 908 and greater. Following the experiments, the animals were anesthetized deeply, and transcardially perfused with 4% paraformaldehyde. The brains were removed and frozen. Frozen coronal sections were prepared and stained with Cresyl violet to stain the injection sites. Data were represented as median values. Comparisons between different groups were made using non-parametric ANOVA (Kruskal±Wallis), followed, if indicated, by the Mann±Whitney U-test for a comparison between two groups. A P-value of , 0.05 was considered to denote statistical signi®cance. In rats pretreated by local injection of MCPG unilaterally into the STN, intraperitoneal administration of haloperidol caused dystonic posturing towards the side ipsilateral to the intracerebral injection side (Fig. 2A). This effect of MCPG was dose-dependent. In rats that received intrasubthalamic injections of 4-CPG, MCCG, MPPG or vehicle, no signi®cant dystonic posturing was induced by haloperidol (Fig. 2B). Histological studies con®rmed that the injection sites were located in the STN (Fig. 3). The present study revealed that in rats receiving intracerebral injections of MCPG, a selective, subtype-non-speci®c mGluR antagonist, unilaterally into the STN, systemic

Fig. 1. Quantitative evaluation of dystonic posturing. Three markings, indicating the nose, midpoint of the back, and tail are shown. The angle ù was measured, and scored as follows: 0, no ®xed postural alteration; 1, less than 308; 2, 30±598; 3, 60± 898; 4, 90 and greater.

H. Miwa et al. / Neuroscience Letters 282 (2000) 21±24

Fig. 2. Scores of deviated posturing in response to drug administration. Data represent median values. (A) In animals receiving intrasubthalamic injections of MCPG (n ˆ 5), haloperidol induced signi®cant ipsiversive dystonic posturing (vs. vehicletreated animals). *P , 0:05, 0.25 mg (n ˆ 5) vs. 0.025 mg (n ˆ 5) of MCPG. As a control, an intrasubthalamic injection of vehicle (0.25 ml of saline) was administered (n ˆ 5). There was no statistically signi®cant difference between 0.005 mg MCPG- vs. vehicle-treated animals. The data of vehicle-treated animals were omitted, because no ®xed deviated posturing was elicited in these animals. (B) In animals receiving intrasubthalamic injections of MPPG (n ˆ 4), MCCG (n ˆ 4) or 4-CPG (n ˆ 4), haloperidol induced no signi®cant dystonic posturing. The data of vehicle-treated animals were omitted, because no ®xed deviated posturing was elicited in these animals. Positivity and negativity of the dystonic score represent ipsiversive and contraversive directions, respectively.

administration of haloperidol, a D2 receptor antagonist, induced ipsiversive dystonic posturing. It has been suggested that the pathophysiological mechanism underlying rotational behavior, such as turning or circling, is based on asymmetrically altered basal ganglia output activity [18]. It has also been demonstrated that asymmetrical changes in the activity of the STN neurons contribute to the generation of circling behavior [12]. The direction of the dystonic posturing induced by MCPG is opposite to that hitherto reported to be induced by the AMPA- or NMDA-receptor antagonists. It was reported that unilateral local injections of both AMPA and NMDA receptor antagonists to the STN prevent the overactivity of the STN and induce contraversive posturing under conditions of dopamine blockade. Therefore, the ipsiversive deviated posturing induced by intrasubthalamic injections of MCPG indicate that MCPG has a falicitatory effect on the STN activity. It is possible to speculate that the intrasubthalamic application of MCPG

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facilitated the overactivity of the STN induced by haloperidol, and induced a signi®cant inter-side difference in the neuronal activity of the STN between the intact side and the mGluR antagonists-injected side, thereby producing ipsiversive dystonic posturing. The mGluRs modulate excitatory synaptic transmission, and the receptor family comprises eight subtypes, subdivided into three groups on the basis of sequence similarity and transduction pathways [1,13]. Generally, it has been assumed that activation of group I (mGlu1, mGlu5) mGluRs increases neuronal excitation and excitability with enhanced glutamate release, whereas activation of group II (mGlu2, mGlu3) and group III (mGlu4, mGlu6, mGlu7, mGlu8) mGluRs reduces synaptic excitation with inhibiting glutamate release [1,13]. It was reported that antagonists of mGlu2Rs in particular amplify glutamate release [13], thus suggesting that mGlu2Rs may function as inhibitory autoreceptors. Therefore, it is speculated that MCPG enhances glutamate release in the STN by blocking such inhibitory autoreceptors. Unexpectedly, however, injection of a selective antagonist of group II mGluRs did not induce any dystonic posturing in animals with haloperidol-induced parkinsonism. Since antagonists of group II mGluRs function as inhibitory autoreceptors [1,13], it was expected that the effect of MCPG would be reproduced following injection of a group II mGluR antagonist such as MPPG. Moreover, neither antagonists to group I (4-CPG) and group III (MPPG) receptors also did not induce any dustonic posturing. It remains uncertain why the selective antagonists of speci®c subtypes of mGluRs did not induce any dystonic posturing. Metabotropic GluRs have been found to have heterogeneous functions, and the detailed mechanisms of their actions remain undetermined. Previous studies have suggested that excitatory glutama-

Fig. 3. A schematic representation of the location of the tips of the injection cannulae. (IC, internal capsule; LH, lateral hypothalamus; AMY, amygdala).

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tergic drive to the STN contributes to the overactivity of the STN in parkinsonism [11]. Although further studies will be required to elucidate the actual role of mGluRs in the STN, the present results may imply that mGluRs may contribute to the modulation of the glutamatergic neurotransmission in the STN, presumably by exerting an inhibitory in¯uence. We hope that the present study will contribute to a better understanding of the pathophysiological mechanism underlying parkinsonism. [1] Anwyl, R., Metabotropic glutamate receptors: electrophysilogical properties and role in plasticity. Brain Res. Rev., 29 (1999) 83±120. [2] Alexander, G.E. and Crutcher, M.D., Functional architecture of basal ganglia circuits: neural substrates of parallel processing. Trends Neurosci., 13 (1990) 266±271. [3] Chesselet, M.-F. and Del®s, J.M., Basal ganglia and movement disorders: an update. Trends Neurosci., 19 (1996) 417±422. [4] Del®s, J.M., Ciaramitar, V.M., Parry, T.J. and Chesselet, M.F., Subthalamic nucleus lesions: widespread effects on changes in gene expression induced by nigrostriatal dopamine depletion in rats. J. Nerosci., 15 (1995) 6562±6575. [5] Del®s, J.M., Anegawa, N.J. and Chesselet, M.-F., Glutamate decarboxylase messenger RNA in rat pallidum: comparison of the effects of haloperidol, clozapine and combined haloperidol-scopolamine treatments. Neuroscience, 66 (1995) 67±80. [6] DeLong, M.R., Primate models of movement disorders of basal ganglia origin. Trends Neurosci., 13 (1990) 281±285. [7] Filion, M. and Tremblay, L., Abnormal spontaneous activity of globus pallidus neurons in monkeys with MPTP-induced parkinsonism. Brain Res., 547 (1991) 142±151. [8] Hassani, O.-K., Mouroux, M. and Feger, J., Increased subthalamic neuronal activity after nigral dopaminergic lesion independent of disinhibition via the globus pallidus. Neuroscience, 72 (1996) 105±115.

[9] Levy, R., Hazrati, L.-M., Herrero, M.-T., Vila, M., Hassani, O.K., Mouroux, M., Ruberg, M., Aseni, H., Agid, Y., FeÂger, J., Obeso, J.A., Parent, A. and Hirsch, E.C., Re-evaluation of the functional anatomy of the basal ganglia in normal and parkinsonian states. Neuroscience, 76 (1997) 335±343. [10] Miwa, H., Nishi, K., Fuwa, T. and Mizuno, Y., Dystonic posturing and circling behaviors induced by dopaminergic agents in rats with unilateral globus pallidus lesions. Brain Res., 781 (1998) 268±274. [11] Miwa, H., Nishi, K., Fuwa, T. and Mizuno, Y., Postural effects of unilateral blockade of glutamatergic neurotransmission in the subthalamic nucleus on haloperidol-induced akinesia in rats. Neurosci. Lett., 252 (1998) 167±170. [12] Murer, M.G. and Pazo, J.H., Circling behavior induced by activation of GABAA receptor in the subthalamic nucleus. NeuroReport, 4 (1993) 1219±1222. [13] Nicoletti, F., Bruno, V., Copani, A., Casabona, G. and KnoÈpfel, T., Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders. Trends Neurosci., 19 (1996) 267±271. [14] Pan, H.S. and Walters, J.R., Unilateral lesion of the nigrostriatal pathway decreases the ®ring rate and alters the ®ring pattern of globus pallidus neurons in the rat. Synapse, 2 (1988) 650±656. [15] Parent, A. and Hazrati, L.N., Functional anatomy of the basal ganglia-2. the place of subthalamic nucleus and external pallidum in basal ganglia circuitry. Brain Res. Rev., 20 (1995) 128±154. [16] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. [17] Pin, J.P. and Duvoisin, R., Review: Neurotranmitter receptors I. The metabotropic glutamate receptors: structure and functions. Neuropharmacology, 34 (1995) 1±26. [18] Pycock, C., Turning behavior in animals. Neuroscience, 5 (1980) 461±514. [19] Sugiyama, H., Ito, I. and Hirono, H., A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature, 325 (1987) 531±533.