zyxwvutsrqponm zyxwvutsrqponm zyxwvutsrqpo zyxwvutsrqpon Movemenf Disorders Vol. 7 , No. 1, 1992, pp. 43-47 0 1992 Movement Disorder Society Effect of LY 171555 and CY 208-243 on Tremor Suppression in the MPTP Monkey Model of Parkinsonism Baltazar Gomez-Mancilla, RenC Boucher, and Paul J. BCdard Centre de Recherche en Neurobiologie, Hbpital de I’Enfant-.Thus, Quebec, Canada zyxwv zyxwvuts Summary: The antitremor effect of the D, agonist LY 171555 and of the D, agonist CY 208-243 alone and in combination was tested in a monkey previously rendered parkinsonian by MPTP and displaying exceptionally a rest tremor in the limbs. The D, agonist suppressed rest tremor in a dose-dependent fashion. The D, agonist by itself had no effect but it potentiated the effect of a small dose of LY 171555. Key Words: LY 171555-D, agonist-CY 208-243D, agonist-Monkey-Parkinsonism-MPTP-Tremor. __ have also observed one monkey with alternating 7 Hz rest tremor in the limbs more evident in the left arm. The tremor recorded on EMG (see Fig. 3) was identified to be a resting tremor because it occurred while the animal was in a resting state. Furthermore, it abated or disappeared in response to action or intentional movements. In order to evaluate the functional interaction of D, and D, agonists on tremor suppression in this animal, we have tested selective dopamine (DA) agonists for the two receptors. Parkinson’s disease (PD) is a clinical syndrome characterized principally by resting tremor, bradyand hypokinesia, rigidity, and loss of postural reflexes. The available evidence indicates that diminished striatal dopamine levels is a sufficient condition for the production of a parkinsonian syndrome (1). Replacement therapy by levodopa was introduced by Birkmayer and Hornykiewicz in 1961 (2). Striatal dopamine receptors on which dopamine exerts its action have been characterized as D, and D, based on whether or not they activate dopaminesensitive adenylate cyclase (3). Recently, it has been reported that, in monkeys with unilateral ventromedial tegmental lesions, D, agonists decreased tremor while D, agonists did not (4). A parkinsonian syndrome can be induced in the monkey by the toxin MPTP. Rest tremor is not usually a prominent feature of the syndrome in the monkey (1). However, a rest tremor has been identified in the African green monkey after injection of MPTP. The tremor has a frequency of 4 to 5 Hz, and is virtually indistinguishable from parkinsonian resting tremor (5). There is one report of an old Macaca fascicularis treated with MPTP showing a resting tremor with a frequency of 3.8 Hz (6). We METHODS Animal Model The experiment was performed in one female Macaca fascicularis weighing 3 kg. The toxin 1methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) was dissolved in warm saline and injected i.v. at a dose of 0.3 mg/kg. The injection was repeated several times at weekly intervals until the parkinsonian signs were stable. The total dose received was 6.0 mg. A severe parkinsonian syndrome developed after the last dose and after several weeks it was noted that the animal displayed an almost constant rest tremor. The tremor had been present for more than a year when the present experiment was performed. The study was carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory zyxwvu zyxwvuts Address correspondence and reprint requests to Dr. P. J . BBdard at Centre de Recherche en Neurobiologie, HBpital de I’Enfant-JCsus, 1401, 18’ Rue, QuBbec, Canada GIJ 124. 43 44 zyxwvutsrqp zyxwvutsr zyxwvu zyxwvutsr zy B . GOMEZ-MANCILLA ET AL. Animals as adopted and promulgated by the National Institutes of Health. Administration of the Drugs and Assessment of Tremor The animal was placed in a chair, to immobilize the trunk and the neck. The upper limbs were restrained with loose tape to facilitate the evaluation of tremor at rest. Tremor at rest was present most of the time but would disappear during short periods. The animal had received several types of antiparkinsonian drugs before this study, including dopamine agonists, levodopa, and anticholinergic agents. However, it had been drug free for 4 months at the beginning of this study. The experiment started with the administration of LY 171555 and CY 208-243 given separately in increasing doses, with an interval of 2 days between each trial. In the second part, a combination of increasing doses of LY 171555 with a constant dose of CY 208-243was given with an interval of 5 days between each assay. One milliliter of saline solution was given as a control. All experiments were repeated at least three times. The effect of the drugs was quantified by using a stopwatch to record the cumulative time during which the animal had stopped trembling during the experimental period. The cumulative time during which the tremor was absent was quantified every 15 min until the effect of the drug was over. The observer was placed across a one-way screen. All drugs were administrated at 10 a.m. Data Analysis A two-way analysis of variance for repeated measures was applied to the serial assays. Differences between control and experimental data were assessed by means of Dunnett's a posteriori test. Tukey's a posteriori test was used for treatment comparison (7). RESULTS The D, agonist CY 208-243 at doses of 0.001, 0.01, and 1.0mglkg injected S.C. had no effect on tremor, and doses over 1.0 mglkg produced agitation and aggressive behavior (data not shown). The D, agonist LY 171555 injected S.C. at doses of 0.001, 0.01, and 0.1 mg/kg stopped tremor in a dose-related fashion as shown in Fig. 1. The effect of LY 171555 at 0.001 mg/kg was not statistically different from control, but at doses of 0.01 and 0.1 mg/kg, the differences were significant in comparison with control. The antitremor effect produced by doses of 0.01 and 0.1 mg/kg lasted approximately 120 min and the percentage of the time during which tremor was stopped was 30 and 69%, respectively, as shown in Fig. 1. Figure 2 shows the time course of the effects on tremor produced by LY 171555 at 0.001, 0.01,and 0.1 mg/kg given alone and in combination with CY 208-243at 0.01 mg/kg. The percentage of time spent without tremor is shown for each period of 15 min of observation. As can be seen, LY 171555 at 0.001 mg/kg injected alone or in combination with CY 208-243 had no effect on tremor (Fig. 2A). Electromyographic (EMG) Recordings Direct recording of tremor was performed using needle intramuscular wires. EMG recordings were made from the biceps and triceps muscles, using an ink-writing polygraph. The EMG recording was carried out before and during pharmacological therapy, under the same conditions as for assessment of tremor. zyxwvutsrq zy Drugs CY 208-243 [( -)-(6aR)(12bR)-4,6,6a,7,8,12bhexahydro-7-methylindolo(4,3-ab)-phenanthridine, Sandoz, Basel, Switzerland] was mixed with an equal weight of tartaric acid and dissolved in 0.9% sterile saline (pH 4.0) and then injected S.C. LY 171555 (Quinpirole, Eli Lilly, U.S.A.) was dissolved in 0.9% sterile saline and then injected S.C. FIG. 1. Histograms of tremor suppression induced by saline (n = 3)andLY 171555at0.001,0.01, andO.l mg/kg(n = 3). Values were calculated in percent of 120 min. **p < 0.01 by comparison with control values: Dunnett's a posteriori test; + p < 0.05 by comparison with LY 0.1 values and ""p < 0.05 by comparison with LY 0.01 values: Tukey's a posteriori test. + zyxwvutsrqpo Movement Disorders, Vol. 7, N o . 1 , 1992 zy zyx zyxwvutsrqponm TREMOR SUPPRESSION IN MONKEY MODEL OF PARKINSONISM A z 0 fn u) zyxwvuts zyxwvut w a n n3 .E 15 - ----t -t- 10 - ZE 0 3 a 45 + 5- lY(.Wl) lY(.Wl)CY zyxwvutsrqponm - - 7 9 0 B Iy(a1) ---c ** ** zyxwvut * . ? . . . . . - i n 1 1 I 30 90 60 120 150 TIME (min) C z 15 - 0 v) In UJ K 10 ** ** ** -1 - 2 :. 0 5- 1 a + 0, _ . 30 IVl.1) A n nK- tistically significant when compared with that of LY 171555 at 0.01 mg/kg. When LY 171555 was injected at 0.1 mg/kg, the antitremor effect lasted 120 min, and when combined with CY 208-243 at 0.01 mg/kg, the effect was reduced to 105 min. The difference was statistically significant in comparison with LY 171555 given alone (Fig. 2C). EMG recordings of biceps and triceps before treatment are shown in Fig. 3. The rest tremor observed in the agonist and antagonist muscles caused an alternating oscillation with a frequency of 7-8 Hz (Fig. 3A). The EMG pattern after the administration of CY 208-243 at 0.01 mg/kg was not different from control (Fig. 3A). By contrast, the administration of LY 171555 at 0.01 mg/kg caused a decrease in the amplitude of the EMG activity without variation of the frequency when compared with control (Fig. 3B). The combination of LY 171555 at 0.01 mg/kg and CY 208-243 at 0.01 mg/kg produced a decrease in the amplitude that is more prominent when compared with either drug given alone (Fig. 3C). . . 60 DISCUSSION The location of DA receptors in the nigrostriatal system has been well characterized. D, receptors appear to be localized, particularly on the cell bodies of noncholinergic neurons, where they are postsynaptic receptors for the nigrostriatal dopaminergic axons, and on the presynaptic terminals of the striatopallidal and striatonigral GABAergic neurons (8). Autoreceptors on both the cell bodies or the presynaptic terminals of nigrostriatal dopaminergic neurons are of the D, type (9,lO). The functional role o€ D, and D, receptors has been described and this interaction can be antagonistic, as in the regulation of cholecystokinin, GABA, and acetylcholine as well as adenylate cyclase (1 1) or synergistic observed in both biochemical and behavioral paradigms (11,12). Our results demonstrate that CY 208-243, a novel D, agonist (13) at doses of 0.01 and 0.1 mglkg that increase locomotion, is ineffective in stopping tremor. These results are in agreement with other studies that showed that SKF-38393 was also ineffective in stopping tremor in monkeys (4). All dopamine receptor agonists presently in clinical use for PD are predominantly D, agonists. In the MPTP model, D2 agonists also relieve the cardinal parkinsonian symptoms (14). It is therefore not surprising that in our study the D, agonist LY 17 1555 at several increasing doses stopped tremor zyxw zyxwvutsrq I 90 I 120 TIME (min) FIG. 2. Tremor suppression by saline or LY 171555 at several increasing doses given alone and in combination with CY 208243. (A) Saline (n = 3), LY 171555 at 0.001 mg/kg given alone (n = 3) and in combination with CY 208-243 at 0.01 mg/kg (n = 3). (B) Saline (n = 3), LY 171555 at 0.01 mgikg given alone (n = 3) and in combination with CY 208-243 at 0.01 mg/kg (n = 3). (C) Saline, LY 171555 at 0.1 mg/kg given alone (n = 3) and in combination with CY 208-243 at 0.01 mglkg (n = 3). Values were expressed by counting the cumulative time during which the tremor was absent, during periods of 15 min, until the effect of the drug disappeared. **p < 0.01 by comparison with control values: Dunnett’s a posteriori test. Figure 2B shows that the effect of LY 171555 at 0.01 mg/kg given alone on tremor lasted 120 min, and when it was combined with CY 208-243 also at 0.01 mg/kg, the antitremor effect lasted 165 min. The increased effect of such a combination was sta- Movement Disorders, Vol. 7, No. I , 1992 zyxw zyxwvutsrqp B . GOMEZ-MANCILLA ET AL. 46 A mom CONTROL I ' l I l zyxwvutsrqponmlk . . ) . ' I , zyxwvutsrqpo zyxwvuts TRICEPS BICEPS 01 0.01 mg/kg TRICEPS B BICEPS CONTROL TRICEPS BICEPS LY 0.01 mgPlrg -1 TRICEPS C r zyxwvut BICEPS CONTROL TRICEPS BICEPS LY+cY TRlCEPS [ 5m" zyxwvutsrqpo Y -. 7 0.6- FIG. 3. EMG recording made from biceps and triceps muscles, using needle intramuscular wire and ink-writing polygraph. EMG was performed before (control) and after (30 min) CY-208243 at 0.01 mgfkg and LY 171555 at 0.01 mg/kg given alone or in combination using the same conditions of assessment of tremor. in a dose-related fashion. Indirect biochemical studies have showed the presence of D, receptors on striatal interneurons of cholinergic type (9,15,16). More recently, it has been described that most of them contain detectable D2 receptor mRNA (17). Since the striatal cholinergic interneurons possess D, receptors that inhibit the release of acetylcholine (9), and D, agonists as well as anticholinergic drugs (18) suppress tremor, we may assume that D, agonists and anticholinergic drugs act on the same chain of neurons for suppressing rest tremor in PD. It has been hypothesized from behavioral experiments that D, receptors gate the level of response to D, receptor activation ( l S 2 1 ) . D, agonists could therefore potentiate the action of D, agonists or allow full expression of the effect of D, agonists (20). Movement Disorders, Vol. 7, N o . I , 1992 However, in our experiments, we observed that when the antitremor effect of LY 171555 is near maximal, CY 208-243 will reduce its efficacy rather than potentiating it, as one would expect of a partial D, agonist. We have described previously that the number of D, and D, receptors increased (upregulation) in monkeys treated with MPTP (22). We (unpublished results) and others (23) have recently observed that CY 208-243 has a clear effect on locomotor activity and this effect is dependent upon the denervation state of the MPTP-treated animal. This also suggests that CY 208-243 acts preferentially on receptors in the high-affinity state. However, CY 208-243 has no beneficial effect by itself on tremor. This could be explained by the fact zy zyx zyxwvutsrq zyx zyxwvut TREMOR SUPPRESSION IN MONKEY MODEL OF PARKINSONISM that D, receptors are not located on striatal cholinergic interneurons, which appear to be important in the pathophysiology of this symptom (9). This may also be the reason for the lack of efficacy of SKF38393, another D, agonist (4,12). The present results will have to be reinterpreted in light of the recent demonstration by methods of molecular biology of a D, receptor localized preferentially in the limbic structures and the ventral striatum as well as A9 and A10 dopaminergic cell bodies (10). However, the identification of D, receptors in nonhuman primates remains to be confirmed. Initial reports (10) describe LY 171555 as having potentially strong D, agonist properties. As for CY 208-243, it was described as a D, agonist in view of the fact that it stimulates adenylate cyclase but has no effect on prolactin (1). However, in ligand and binding studies, CY 208-243 did not appear to be very specific (13). This could suggest that CY 208243 has D, and D, agonist properties that would explain the effect on adenylate cyclase, the locomotor effect, and the apparent nonselectivity in binding studies, but no D, effect, which would explain the lack of action on prolactin. We can therefore conclude that the relief of tremor by L-dopa or DA agonists in MPTP-treated monkeys is mostly related to the D, dopamine receptor, probably via cholinergic interneurons. However, adding to the D, agonist a dose of D, agonist that is ineffective by itself may increase its efficacy in a certain dose range. 47 7. Winer BJ, ed. Statistical principles in experimental design. New York: McGraw-Hill, 1971. 8. Richfield EK, Young AB, Penney JB. Comparative distribution of dopamine D, and D, receptors in the basal ganglia of turtles, rats, cats and monkeys. J Comp Neurol 1987;262: 446463. 9. Dawson VL, Dawson TM, Filloux FM, Wamseley JK. Evidence of dopamine D,-receptors on cholinergic interneurons in the rat caudate-putamen. Life Sci 1988;42:1933-1939. 10. Sokoloff P, Giros B, Martres MP, Bouthenet ML, Schwartz JC. Molecular cloning and characterization of a novel dopamine receptor (D,) as a target for neuroleptics. Nature (Lond) 1990;347:146-151. 11. Baron P, David TA, Braun A, Chase TN. Dopaminergic mechanisms and motor functions: characterization of D, and D, dopamine receptors interaction. Eur J Pharmacol 1986; 123:109-114. 12. Gershanik 0,Heikkila RE, Duvoisin RC. Behavioral correlations of dopamine receptor activation. Neurology 1983;33: 1489. 13. Markstein R, Seilen MP, Vigault JM, Urwyler S, Enz A, Dixons K. Pharmacologicalproperties of CY 208-243a novel D1 agonist. In: Sandler M, Dahlstrom A, Belkmaker R, eds. Progress in catecholamine research. New York: Alan R. Liss, 198859. 14. BCdard PJ, Gomez-Mancilla B, Boucher R, Di Paolo T. Pharmacological strategies in Parkinson’s disease based on work in MPTP-monkeys. In: Schneider JS, Gupta M, eds. Current concepts in Parkinson’s disease (in press). 15. Joyce JN, Marshall JF. Quantitative autoradiography of dopamine D, sites in rat caudate-putamen: localization to intrinsic neurons and not to neocortical afferents. Neuroscience 1987;20:773-795. 16. Lehmann J, Langer SZ. The striatal cholinergic interneuron: synaptic target of dopaminergic terminals? Neuroscience 1983~10:1105-1120. 17. LeMoine Tison F, Bloch B. D, dopamine receptor gene expression by cholinergic neurons in rat striatum. Neurosci Lett 1990;117:24&252. 18. Comella CL, Tanner CM. Anticholinergic drugs in the treatment of Parkinson’s disease. In: Koller CW, Paulson G, eds. Therapy of Parkinson’s disease. New York: Marcel and Dekker Inc., 1990:123-141. 19. Clark D, White FJ. Review: D1 dopamine receptors. The search for a function: a critical evaluation of the D,/D, dopamine receptor classification and its functional implications. Synapse 1987;1:347-388. 20. Carlson JH, Bergstrom DA, Walters JR. Stimulation of both D, and D, dopamine receptors appears necessary for full expression of postsynaptic effects of dopamine agonist. Brain Res 1987;400:205-211. 21. Rouillard C, BCdard PJ. Specific D, and D, agonists and antagonists have synergistic effects in the 6-OHDA circling model in the rat. Neuropharmacology 1988;27:1257-1264. 22. Falardeau P, BCdard PJ, Di Paolo T. Relation between brain dopamine loss and D, dopamine density in MPTP monkeys. Neurosci Lett 1988;86:225-229. 23. Temlett JA, Chong NP, Oertel WH, Jenner P, Marsden CD. The D, dopamine receptor partial agonist, CY 208-243 exhibits antiparkinsonian activity in MPTP-treated marmosets. Eur J Pharmacol 1988;156:197-206. zyxwv zyxwvu REFERENCES 1. Weiner WJ, Lang AE, eds. Movement disorders: A comprehensive study. New York: Mount Kisco, 1989. 2. Birkmayer W, Hornykiewicz 0. Der L-Dioxyphenylalanin (L-DOPA) effekt bei der Parkinson Akinesie. Wien Kfin Wochenschr 1961 ;73:787. 3. Kebabian JK, Calne DB. Multiple receptors for dopamine. Nature (Lond) 1979;277:92. 4. Goldstein M. Monkeys with unilateral ventromedial tegmental lesions of the brainstem. Models for Parkinson’s disease and Lesch Nyhan syndrome. Prog Neuropsychopharmacol Biol Psychiatry 1989;13:311-318. 5 . Tertud JW, Langston JW, Redmond DE Jr, Roth RH, Sladek JR, Angle RW. MPTP-induced tremor in human and non-human primates. Neurology 1986;36:308-312. 6. Degryse AD, Colpaert FC. Symptoms and behavior features induced by I-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in an old Java monkey (Macaca cynomolgus fascicularis (Raffles). Brain Res Bull 1986;16:561-571. Movement Disorders, Vol. 7, N o . 1 , 1992