Brain Research, 512 (1990) 125-131 Elsevier

125

BRES 15299

Blockade of excitatory neurotransmission in the globus pallidus induces rigidity and akinesia in the rat" implications for excitatory neurotransmission in pathogenesis of Parkinson's diseases Lechoslaw Turski, Thomas Klockgether*, Waldemar A. Turski**, Michael Schwarz*** and Karl-Heinz Sontag Max-Planck Institute for Experimental Medicine, G6ttingen (F.R. G.) (Accepted 15 August 1989) Key words: (-)-2-Amino-7-phosphonoheptanoic acid; N-Methyl-D-aspartic acid; Globus pallidus; Caudate-putamen; Nucleus accumbens; Muscle tone; Catalepsy; Electromyogram; Parkinson's disease

Bilateral microinjections of the selective N-methyl-o-aspartate (NMDA) antagonist, (-)-2-amino-7-phosphonoheptanoate (AP7), 0.02-0.5 nmol, into the globus paUidus and ventral-posterior portions of the caudate-putamen result in an increase in the muscle tone (rigidity) and catalepsy (akinesia) in rats. NMDA blocked the actions of AP7 on motility in sensitive regions of the globus pallidus and caudate-putamen. Topographical differences in the action of AP7 in the striatum were detected in the dorsal-ventral and rostral-caudal direction. Microinjections of AP7 into the nucleus accumbens induced neither an increase in the muscle tone nor catalepsy in rats, while ventral regions of the caudate-putamen were sensitive to both actions of AP7. Microinjections of AP7 into the dorsal caudate-putamen resulted in a moderate or no increase in the muscle tone. AP7 failed to induce catalepsy from dorsal regions of the caudate-putamen. These data identify the globus pallidus and a defined subregion of the caudate-putamen as crucial sites where excitatory neurotransmission acts to regulate the final set-point of the respective output neurons providing modulation of the passage of motor information through the basal ganglia. INTRODUCTION Rigor, akinesia and tremor are hallmarks of Parkinson's disease 19,2°. Clinical observations in humans and lesion studies in monkeys established that the basal ganglia are essential for the pathogenesis of parkinsonism 8. Destruction of nigrostriatal dopaminergic pathways is believed to cause the major motor deficits in parkinsonian patients 2°. Humans and monkeys subjected to the action of a selective toxin, 1-methyl-4-phenyl-l,2,3, 6-tetrahydropyridine (MPTP), show an immediate development of bradykinesia and rigor 14. Microinjection and lesion studies in rodents also demonstrated increases in muscle tone and akinesia following destruction of dopaminergic nigrostriatal pathways with 6-hydroxydopamine, or as a result of inactivation of this pathway with either local application of a dopamine (DA) antagonist, haloperidol, into the caudate-putamen (CP), or placement of a G A B A A agonist muscimol into the substantia nigra pars compacta 5,28,3°. Experimental analysis of neuronal networks mediating

dopamine-dependent functions in the basal ganglia established that the globus pallidus (GP), the substantia nigra pars reticulata (SNR) and the entopeduncular nucleus (EP) are essential for their transmission and modulation 23. The excitation in the basal ganglia is probably mediated by dicarboxylic amino acids (Lglutamate or L-aspartate) 7,9,26. L-Glutamate or L-aspartate are believed to subserve neurotransmitter function in cortical pathways terminating in the striatum, the substantia nigra and the EP 6'16. Dicarboxylic amino acids are also suggested to be neurotransmitters in the subthalamopallidal and subthalamonigral pathways 9,e6. Excitatory neurotransmission in the basal ganglia may therefore operate in the regulation of muscle tone. On this assumption we have tried to define the involvement of amino acid-mediated excitation in the striatum and GP in motor control using microinjections of a selective N-methyl-D-aspartate ( N M D A ) antagonist, 2-amino-7phosphonoheptanoate (AP7). We have explored the effect of AP7 on the regulation of muscle tone and on the motility of rats subjected to microinjections into different

* Present address: Department of Neurology, Eberhardt-Karls-University, Schnarrenberg Clinics, Hoppe-Seyler-Str. 3, D-7400 Tiibingen, ER.G. ** Present address: Department of Pharmacology, Medical School, Jaczewskiego 8, PL-20--090 Lublin, Poland. *** Present address: Department of Neurology, Alfried-Krupp-Hospital, Alfried-Krupp-Str. 21, D-4300 Essen, ER.G. Correspondence: L. Turski. Present address: Department of Neuropsychopharmacology, Schering AG, Sellerstr. 6-8, D-1000 Berlin 65, F.R.G. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

126 s u b r e g i o n s of the GP, C P and nucleus a c c u m b e n s ( A C B ) . S o m e of t h e s e data h a v e b e e n c o m m u n i c a t e d to the M e e t i n g " E x c i t a t o r y A m i n o A c i d s ' 8 8 " in M a n a u s 33 and to

the

2nd

International

Meeting

of the

European

B e h a v i o u r a l P h a r m a c o l o g y Society in A t h e n s 29. MATERIALS AND METHODS

Animals Male Wistar rats (Winkelmann, Borchen, F.R.G.), 200-240 g in weight, were housed under environmentally controlled conditions (06.00-18.00 h light-dark cycle; 22-24 °C) and permitted free access to food and water for at least 3 days prior to surgery. The assignment of rats to experimental groups was random. The assessment of catalepsy and monitoring of the electromyogram (EMG) took place between 8.00 and 12.00 h.

Stereotaxic surgery The rats were anesthetized with sodium pentobarbital (Nembutal; Ceva, Neuilly-sur-Seine, France), 50 mg/kg i.p., and implanted with guide cannulae directed towards CP, ACB, or GP 13. Coordinates for microinjections are shown in Table I. Following the surgery the rats were housed individually. The microinjections were performed bilaterally in unanesthetized rats 4-7 d after surgery. The drugs were delivered in a volume of 0.5/A at a rate of 0.5/A/min. No rats were used for more than one pair of microinjections.

Electromyography The activity in the electromyogram (EMG) was recorded from the gastrocnemius muscle (GS) of the hind-limb of unanesthetizcd rats by means of pairs of teflon-insulated stainless-steel wire electrodes (Cooner Wire, Chabworth, CA, U.S.A. AS 632 SS) inserted percutaneously into the muscle. The rats were placed separately in ventilated Plexiglas boxes and their hindlimbs, which were gently secured with adhesive tape, were allowed to hang through slots in the bottom of boxes. The electrical signals were amplified, bandpass filtered (5 Hz to 10 kHz) and rectified. The EMG was recorded continuously and the average integrated activity was determined over 5 min periods and expressed in arbitrary units"'. To ensure that only tonic EMG activity was recorded, bursts of phasic activity induced by movements of the rat were discarded. The data from the EMG experiments were analysed by means of Mann-Whitney U-test.

Morphological techniques For morphological examination by light microscopy, the rats were sacrificed upon completion of the experiments and their brains were prepared for verification of cannulae tip placement 3°. RESULTS

A P 7 increases muscle tone following intrapallidal administration Globus pallidus. T h e initial a p p r o a c h was to m i c r o i n ject A P 7 , a selective N M D A a n t a g o n i s t , into the G P and

Drugs

into d i f f e r e n t s u b r e g i o n s of the rat striatum. B i l a t e r a l

(-)-2-Amino-7-phosphonoheptanoic acid (AP7; Tocris, Buckhurst Hill, Essex, U.K.) and N-methyl-o-aspartic acid (NMDA; Tocris) were brought into solution with a minimum quantity of 1 N NaOH, and the final volume was made up with saline. The pH was adjusted to 7.4.

p r o d u c e d a dose- and t i m e - d e p e n d e n t increase of the

Catalepsy

tion of A P 7 , 0.5 (n = 8) and 0.1 (n = 5) n m o l , was

Catalepsy was measured by means of the bar test 10 min after microinjection into the GP, CP or ACB (immediately before recording of the EMG). For the bar test, animals were placed with both front paws on the edge of a wooden block of 9.0 cm height and the time was estimated until the rat left this position (descent latency). An animal was considered to be cataleptic if it maintained a given position for at least 30 s. The time (up to 180 s) during which an animal maintained its position was the estimation of catalepsy.

m i c r o i n j e c t i o n s of A P 7 ,

d e c r e a s e d during the f o l l o w i n g 120 min (Fig. 1). Intrapallidal a d m i n i s t r a t i o n of A P 7 , 0.02 n m o l (n = 8), only m o d e r a t e l y i n c r e a s e d muscle t o n e (Fig. 1). M i c r o i n j e c tions of s o l v e n t (n = 5) into t h e G P did not p r o d u c e

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Fig. 1. Left panel: time course of action and dose relationship of AP7 after bilateral microinjection into the GP on a tonic activity recorded in the EMG from the GS muscle in rats. Abscissa, time (min) after beginning of the microinjection into the GP; ordinate, EMG activity in the GS muscle. Median values. Right panel: catalepsy after microinjection of AP7 into the GP. Ordinate, descent latency (s). Median values.

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Fig. 2. Original EMG recordings 20-25 min after injection of solvent, AP7, or AP7 and NMDA into the GP. The lower tracings (b) represent the rectified EMG activity whereas the upper tracings (a) represent the integrated activity. A: EMG activity following microinjection of solvent into the GP. B: tonic EMG activity after microinjection of AP7, 0.5 nmol, into the GP. C: EMG activity in the GS muscle in a rat subjected to co-administration of AP7 and NMDA in equimolar doses of 0.5 nmol. increases of the muscle tone (Figs. 1 and 2A). N M D A , 0.5 nmol, when co-administered into the G P with AP7, 0.5 n m o l (n = 5), totally blocked the increase in the muscle tone induced by the latter (Fig. 2C). Ventral caudate-putamen. Microinjections of A P 7 into the ventral portions of the CP also induced increases of the muscle tone (Fig. 3). This effect was dose- and t i m e - d e p e n d e n t but was less p r o n o u n c e d than that p r o d u c e d by intrapallidal administration of equimolar dosages (Fig. 1). The maximal E M G activity elicited by microinjections of A P 7 , 0.5 (n = 24) and 0.1 (n = 5) nmol, into the ventral CP was seen i m m e d i a t e l y after the beginning of the recording session and it slowely abated

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Fig. 3. Left panel: time course of action and dose relationship of AP7 after bilateral microinjection into the ventral CP on a tonic activity recorded in the EMG from the GS muscle in rats. Abscissa, time (min) after beginning of the microinjection into the ventral CP; ordinate, EMG activity in the GS muscle. Median values. Right panel: catalepsy after microinjection of AP7 into the ventral CP. Ordinate, descent latency (s). Median values.

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during the following 120 min (Fig. 3). A n increase in muscle tone following microinjections of A P 7 , 0.02 nmol (n = 8), into the CP was seen only during the first 10 min of recording (Fig. 3). N M D A , 0.5 nmol (n = 4), blocked the increase of muscle tone induced by administration of A P 7 , 0.5 nmol, into the CP. Solvent injections into the ventral CP (n = 5) did not trigger increases of muscle tone (Fig. 3). Dorsal caudate-putamen. Placement of A P 7 into the dorsal CP induced a m o d e r a t e increase of muscle tone when a dose of 0.5 nmol was a d m i n i s t e r e d (Fig. 4). The E M G activity r e c o r d e d from rats subjected to such t r e a t m e n t was less than that registered in rats with microinjections of e q u i m o l a r dosages into the G P or ventral CP (Figs. 1 and 3). Nucleus accumbens. Bilateral administration of A P 7 , 0.5 nmol (n = 8), into the A C B did not result in changes in the muscle tone (Fig. 5).

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Fig. 5. The tonic activity in the EMG (left panel) and catalepsy (right panel) following microinjeetion of AP7, 0.5 nmol, into the GP, ventral portions of CP and ACB. Left panel: abscissa, time (min) after beginning of the microinjection into the GP, CP or ACB; ordinate, EMG activity in the GS muscle. Median values. Right panel: ordinate, descent latency (s). Median values.

128

AP7 induces catalepsy from the globus pallidus and ventral caudate-putamen Globus pallidus. Bilateral microinjections of AP7 into the GP produced immediate appearance of catalepsy in rats (Fig. 1). The catalepsy induced by AP7 from the GP was dose-dependent (Fig. 1). AP7, 0.5 nmol (n = 8), elicited pronounced catalepsy (maximal catalepsy score -180 s) in all tested rats, while moderate degree of catalepsy was measured following dosages of 0.1 (n = 5) and 0.02 (n = 8) nmol (Fig. 1). NMDA, 0.5 nmol n = 5), co-administered with AP7, 0.5 nmol, into the GP attenuated the catalepsy response. Microinjections of solvent into the GP did not produce catalepsy in five rats (Fig. 1). Ventral caudate-putamen. Microinjections of AP7 placed in the ventral portions of the CP also produced catalepsy (Fig. 3). The catalepsy was observed immediately after administration of AP7 and had dose-dependent characteristics (Fig. 3). NMDA, 0.5 nmol (n = 4), attenuated catalepsy induced by AP7, 0.5 nmol. Microinjections of solvent did not produce catalepsy from the ventral CP (Fig. 3). Dorsal caudate-putamen. AP7, 0.02-0.5 nmol (n = 34), did not trigger catalepsy from the dorsal CP (Fig. 4). Nucleus accumbens, Microinjections of AP7, 0.5 nmol (n = 8), into the ACB produced no catalepsy (Fig. 5).

Topography of motor effects of AP7 in the striatum and globus pallidus The topography of motor effects of AP7 in the basal ganglia and globus pallidus was assessed by means of bilateral microinjections of the dose of 0.5 nmol (Figs. 5-7). It is apparent that the action of AP7 on muscle tone and catalepsy in the ventral striatum and globus pallidus increases along the rostro-caudal axis, being minimal in the rostral part of the ventral CP (AP 9410) and maximal in the GP (AP 6570)(Figs. 5 and 7). The cataleptogenic

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Fig. 6. The tonic activity in the EMG (left panel) and catalepsy (right panel) following microinjection of AP7, 0.5 nmol, into the dorsal portions of CP. Left panel: abscissa, time (min) after beginning of the microinjection into the dorsal CP; ordinate, EMG activity in the GS muscle. Median values. Right panel: ordinate, descent latency (s). Median values.

action of AP7 generally matches the action of the drug on muscle tone, but the cataleptogenic potential of AP7 in the mid-ventral (AP 7470) and posterior-ventral (At' 6570) portions of the CP is similar to that in the GP (Fig. 5). No differences were seen along the rostro-caudal axis in the dorsal CP (Fig. 6). In the dorsal CP AP7 produced neither pronounced increases in the muscle tone nor triggered catalepsy (Figs. 6 and 7). The analysis of the topography of increases in the muscle tone and catalepsy produced by AP7 shows that the GP and posteriorventral portions of the CP mediate motor effects of AP7. The regions sensitive to increases of the muscle tone are slightly broader than those responding with catalepsy (Fig. 7). DISCUSSION

Blockade of excitation by AP7, an antagonist of the NMDA subtype of glutamate receptors, in the GP or mid- and posterior portions of the CP disturbs motility in rats. Microinjections of AP7 into the GP and CP caused an increase of muscle tone which resembles rigidity and produced catalepsy which represents the maximal degree of akinesia in the rat. The receptor specificity of this action is substantiated by the finding that NMDA blocked the increase in muscle tone and catalepsy induced by AP7. The depressive effect on motility of AP7 was detectable throughout the entire extent of the GP and in the ventral and posterior portions of the CP. This topographical selectivity of action of AP7 matches observations with rats following microinjections of the GABAA agonist muscimoi into the basal ganglia zT. With regard to EMG activity, muscimol produced an increase in the muscle tone from mid- and posterior-ventral subregions of the CP and from the GP 27. Similar topography was reported in the rat for the cataleptogenic action of muscimo123'27. Microinjections of another NMDA-antagonist 2amino-5-phosphonopentanoate (AP5) into the anterodorsal CP were previously reported to induce stereotyped sniffing in rats 24. Others 3 reported a potentiation of morphine and methadone catalepsy in rats by application of AP7 into the anterior CP. Microinjections of AP5 or AP7 into the ACB were found to increase locomotor activity in rats 3"4. Electromyographic monitoring has previously demonstrated that the CP and its GABAergic efferent pathways to the SNR and EP mediate the regulation of muscle tone 3°'32. Inactivation of the striatonigral and striatoentopeduncular pathways with muscimol in the ventral CP or bicuculline in the SNR or EP increases muscle tone 27"2s. A similar mechanism of action may be proposed for AP7 in the CP, since impairment of excitatory

129

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