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Effect of exogenousL -DOPA on behavior in the rat: an in vivo voltammetric study
Brain Research, 49t1 (1989) 332-338
332
Elsevier BRE 14591
Effect of exogenous L-DOPA on behavior in the rat: an in vivo voltammetric study Taizo Nakazato 1 and Akitane A k i y a m a 2 1Department of Neurology, Juntendo University School of Medicine, Tokyo (Japan) and 2Department of Electronic Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama (Japan) (Accepted 6 December 1988)
Key words: L-Dihydroxyphenylalanine; Dopamine; 3-Methoxytyrosine; In vivo vottammetry; Behavior; Dihydroxyphenylalanine-induced dyskinesia; Rat
L-DOPA was administered intraperitoneally (i.p.) or intraventricularly (i.v.t,) to freely moving rats to investigate the effects of exogenous L-DOPA itself on behavior. Striatal dopamine (DA) in the extraceUular fluid was examined with microcomputercontrolled in vivo voltammetry, and behavioral change was observed. When L-DOPA was administered (i.p.) after pretreatment with benserazide, a peripheral DOPA decarboxylase inhibitor, behavioral change was elicited before the elevation in DA and suppressed before its reduction. After pretreatment with NSD-1015, a central DOPA decarboxylase inhibitor, behavioral change was also elicited, although DA was still not increased. When L-DOPA was injected (i.v.t.), the behavioral effect was manifested at once; DA was still unchanged at this time, but it increased after behavioral activity reached the maximum level. L-DOPA was also injected (i.v.t.) into rats with striatal lesions induced by 6-hydroxydopamine (i.v.t.). Behavioral change was manifested promptly after the injection. When the dose-response curves to different dosages of L-DOPA were examined in normal rats without striatal lesions, it was found to exhibit a steeper rise than tha: of DA. Finally, when rats were injected (i.p. or i.v.t.) with 3-O-methyl-DOPA (3-methoxytyrosine), a major metabolite of L-DOPA, no behavioral change was elicited, and no increase in DA was recognized. These experimental results indicated that L-DOPA is related directly to the manifestation of behavioral change.
INTRODUCTION L-DOPA therapy is today's most important strategy for the treatment of Parkinson's disease. There are, however, many problems associated with this therapy: psychiatric symptoms 33, D O P A - i n d u c e d dyskinesia2°, and the on-off p h e n o m e n o n 25. B a r b e a u and Roy 2 pointed out that a high dosage of L-DOPA increased the incidence of dyskinesia and that combination therapy with L-DOPA and benserazide was more hazardous than the use of L-DOPA alone. Lesser et al. 17 also reported that the long-term application of L-DOPA resulted in diminished response to the drug and caused the on-off p h e n o m e n o n and the wearing-off p h e n o m e n o n . Klawans et al. ~4 showed that chronic L-DOPA treatment
in the guinea pig resulted in decreased sensitivity of dopamine ( D A ) receptors, possibly causing dyskinesia. Moreover, the D A receptor binding was demonstrated to decrease in the striatum of Parkinson's disease patients treated with L-DOPA 16. However, it is not known whether these L-DOPAinduced p h e n o m e n a are caused by D A converted from L-DOPA, L-DOPA itself, or the metabolites. Sharma and Fahn 28 pointed out that L-DOPA is an agonist to D A receptors, and Ogawa and Yamamoto 23 also reported that L-DOPA b o u n d directly to the D A binding site. However, Ungerstedt 32 and Melamed et a1.19 found no evidence for the action of L-DOPA. Thus, there is still no definite solution to this problem. The present study was designed to investigate the
Correspondence: T. Nakazato. Present address: Department of Physiology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
333 action of L-DOPA on the central nervous system and clarify the mechanism of L-DOPA-induced dyskinesia. For this purpose, the concentrations of D A and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum of the rat were recorded using microcomputer-controlled in vivo voltammetry after the intraventricular (i.v.t.) administration of L-DOPA. At the same time, behavioral changes induced by these drugs were observed. The concentrations of D A and D O P A C were also examined after the intraperitoneal (i.p.) administration of L-DOPA following pretreatment with the D O P A decarboxylase inhibitors: benserazide and NSD-1015 (ref. 4). Since 3-O-methyl-DOPA (3-OM-DOPA, 3-methoxytyrosine) is a major metabolite of L-DOPA in the brain 3, the drug was also administered (i.p. and i.v.t.) to test its effect on D A release and behavior.
Preparation of freely moving animal model Male Wistar rats (n = 10, 300-400 g) were fixed in a stereotaxic apparatus under pentobarbital anesthesia (50 mg/kg). A carbon fiber electrode of 7 #m in diameter was inserted unilaterally into the striatum, and a stainless-steel cannula was placed contralaterally into the lateral ventricle for the administration of drugs. The details were described in a previous paper z2. Four out of 10 rats were administered (i.v.t.) with 200/~g of 6-hydroxydopamine (6-OHDA) dissolved in 24/d of normal saline for 18 min, after pretreatment with desipramine (25 mg/kg, i.p.) 6. Ten days later, 6 - O H D A was injected again, using the same method. Measurements and drug administrations were done at least 14 days after either the operation or the second 6 - O H D A injection.
MATERIALS AND METHODS
Drug administration Animals were injected (i.p.) with 200 mg/kg of L-DOPA or 3-OM-DOPA, and pretreated with 50 mg/kg of benserazide or 100 mg/kg of NSD-1015 30 min before L-DOPA administration. Intraventricular injection was performed with a microcomputercontrolled autoburette. O n e / A of the solution was injected at the rate of 1/16 ktl/2 s every 45 s. L-DOPA, DA, and 3-OM-DOPA were normally dissolved at the concentration of 5 mg/ml in normal saline deaerated by nitrogen gas, and 40/~1 of the solution was injected in 30 min. L-DOPA concentrations were sometimes changed to clear distinctly the difference of time courses between D A concentration and behavior. If a rat's behavior became
Electrochemical measurement Many studies of the in vivo voltammetric technique have been reported ~°'13"31. The many problems associated with this technique involve its imperfect selectivity and the short lifetime of the electrodes. However, some of the problems have been solved by using the microcomputer-controlled potentiostatic pulse polarization technique described in previous papers a'21. The electrochemical procedures can be summarized as follows. The electrode was pretreated by an anodic-cathodic triangular wave (_+1.5 V, 10 V/s in the slope) before every measurement. Two s after the wave, a triple pulse was applied to determine the concentrations of DA. The first pulse was 50 mV for 660 ms, the second 150 mV for 660 ms and the third 250 mV for 22.5 ms. The currents were measured at the end of every pulse. Changes in D A concentrations were measured every 3 min. A modified differential pulse voitammogram (DPV) ranging from 100 to 500 mV in 25 mV steps was used for qualitative analysis, which was performed every 30 min. The influence of the triangular activation wave and that of the consecutive triple pulse on brain were not determined precisely. However, many of our in vivo studies have shown that these treatments do not cause behavioral change, alteration in the sensitivity of the electrode, or change of the concentration of DA zx.
TABLE I
Behavioral ratingscale Score
Definition Asleep, lying down with eyes closed. Lying down with eyes opened, little movement. Lying down with head up, slow periodic sniffing. Getting up with periodic sniffing. Some rearing with sniffing and slow head swinging, some turning. Frequent rearing with prominent sniffing, head swinging, and turning. Continuous rearing with faster head swinging, or much turning.
334
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Fig. 3. Time course of DA intensity (O) and behavioral changes ( i ) after L-DOPA (440 ktg) injection (i.v.t.). LDOPA was injected between the solid vertical bars.
Histological procedures hyperactive and life-threatening because of the drug, injections were discontinued. To construct d o s e response curves, various concentrations of the drugs were dissolved in 40 /A of normal saline. Since L - D O P A is difficult to dissolve at high concentrations, L - D O P A p r e p a r e d and d o n a t e d by Sankyo (Japan) was used when a high concentration was needed.
A f t e r the experiment, the animals were perfused intracardially with 10% formaline u n d e r deep anesthesia. The brains were sliced into 50-ktm-thick sections by a freezing microtome. The sections were stained with Cresyl violet. The positions of electrodes and cannula were confirmed microscopically.
Behavioral observations
Intraperitoneal administration of L-DOPA
The rats were placed in a box 30 x 20 × 25 cm in size, and behavioral changes were observed, while D A intensities and D P V s were simultaneously measured. Behavior was scored by the behavioral rating scale in Table I (refs. 8, 11).
W h e n 200 mg/kg of L - D O P A was injected (i.p.) 30 rain after p r e t r e a t m e n t with benserazide, behavioral activity increased about 10 min after the start of the injection and reached the m a x i m u m level at about 60
RESULTS
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Fig. 4. Time course of behavioral change after injection (i.v.t.) of L-DOPA (10 mg/ml) into a normal (O) and 6-OHDAlesioned (&) rats. Injections of L-DOPA were continued from 0 min to the time indicated by the solid or dashed vertical bars in the normal or the 6-OHDA-lesioned rats, respectively.
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Fig. 5. Time course of behavioral change after injection (i.v.t.) of D A (5 mg/m1) into normal ( 0 ) and 6 - O H D A lesioned (A) rats. Injections of D A were continued until the time indicated in solid or dashed bars in the normal or the 6-OHDA-lesioned rat, respectively.
min; D A intensity, however, peaked at about 180 min (Fig. 1). L-DOPA was also administered (i.p.) 30 min after the injection of NSD-1015, a central D O P A decarboxylase inhibitor that penetrates the blood-brain barrier (BBB) (Fig. 2). Behavioral activity began to increase at almost the same time as in the case of benserazide pretreatment, but the effect lasted for a longer period. During this period, D A intensity started to increase slightly at 140 min, reached the maximum level at 350 min, then decreased.
Intraventricular administration of L-DOPA and DA When 400/~g (20 kd) of L-DOPA was injected (i.v.t.) for 15 min (Fig. 3), the rats abruptly became
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active at 3-4 min after the injection, but D A intensity in the striatal extracellular fluid (ECF) did not show an increase at this time. After the L-DOPA injection was completed, D A intensity gradually increased and reached the maximum level at about 100 min after injection. The rats had already become less active by this time. L-DOPA (10 mg/ml) was administered (i.v.t.) to 6-OHDA-lesioned rats (Fig. 4). The animals became hyperactive immediately after injection. L-DOPA injection was, therefore, discontinued at about 8 min (90/~g), as shown by the broken line in the figure, but the behavioral effect continued for 150 min. When normal rats without striatal lesions were administered L-DOPA, appearance of the hyperactivity was slightly delayed compared with that in the lesioned rat. This effect, however, was not prolonged. The 6-OHDA-lesioned rat was administered D A (5 mg/ml) (i.v.t.) (Fig. 5). During the injection, almost the same effect on behavior was exhibited as with L-DOPA, but the behavioral changes elicited by DA did not continue after injection was discontinued at 12 min (80/~g). In the normal rat, 200 ktg of D A was administered for 30 min, and the effect was smaller than that in the lesioned rat. Dose-response curves were made by plotting the maximal behavioral effect induced by different dosages of L-DOPA and D A (Figs. 6 and 7). Comparison of the two curves reveals that a marked behavioral change was produced by /ldministration of more than 100 pg of L-DOPA. This large effect on
336 behavior was not observed in the case of DA.
Administration of 3-OM-DOPA When 200 mg/kg of 3-OM-DOPA were intraperitoneally administered to the normal rat, no behavioral change was observed. Intraventricular injection of 200 ~g of the drug also failed to produce behavioral change. During these experiments, DA and D O P A C concentrations in the striatal ECF were unchanged. DISCUSSION
Behavioral effect of L-DOPA It has already been shown that L-DOPA administered is converted to DA, which then acts on the central nervous system. The present experiments suggest that L-DOPA itself also has the ability to elicit behavioral change in rats. The evidence leading to this conclusion is as follows. First, as shown in Fig. 3, when rats were administered L-DOPA (i.v.t.), behavioral change was observed within 3 to 4 min. D A intensity, however, did not increase at this time, but was observed to increase after more than 20 min. The appearance of behavioral change occurred earlier with L-DOPA injection (i.v.t.) than with D A injection, as shown in Fig. 5. Second, when rats were administered L-DOPA (i.p.) after pretreatment with a peripheral D O P A decarboxylase inhibitor, benserazide (Fig. 1), behavioral change was elicited after 5-10 min, and reached a peak 60-90 rain later. Bartholini and Pletscher 3 reported that L-DOPA administered reached its maximal concentration in the rat brain 1 h later. This time span corresponds closely with the time at which maximal behavioral activity appeared in the present experiment. Third, when L-DOPA was administered (i.p.) after pretreatment with NSD-1015, which inhibited its conversion to D A in the brain, as expected, the increase in D A concentration in the ECF was greatly suppressed compared with that following the case of pretreatment with benserazide. The beginning of the behavioral change, however, was not delayed, and the change persisted much longer in the case of NSD-1015. Melamed et al. 19 reported that when rats with
unilateral striatal lesions were administered (i.p.) with 50 mg/kg of L-DOPA after pretreatment of 100 mg/kg of NSD-1015, circling behavior was suppressed. Therefore, it was suggested that exogenous L-DOPA itself was not responsible for behavior. However, in the present experiment, 200 mg/kg of L-DOPA was injected into the normal rats pretreated with NSD-1015, and behavioral change was observed. The conflicting result obtained in the present experiment might have been due to different experimental conditions: normal rats were used and 200 mg/kg of L-DOPA was injected. Since more L-DOPA reached the central nervous system than in the study of Melamed et al., behavioral change may have been elicited. Fourth, when L-DOPA was administered (i.v.t.) to the 6-OHDA-lesioned rat, behavioral change was elicited immediately after injection as shown in Fig. 4. This should be considered partially due to the supersensitivity of the D A receptor 9, as already demonstrated in Fig. 5. However, in spite of the fact that the lesioned rats have reduced D O P A decarboxylase activity in the striatum 3°, behavioral change after L-DOPA injection was observed to appear at almost the same time that it did after D A injection to the lesioned rat, suggesting that L-DOPA also induces behavioral change. It is interesting to discuss the dose-response curves shown in Figs. 6 and 7. With D A injection (i.v.t.), behavioral response increased gradually with increasing doses, whereas with L-DOPA, the increase in the response was apparently larger at doses of more than 100/~g. These results may indicate that exogenous L-DOPA and its partially converted DA act on D A receptors, and consequently the larger behavioral effect was produced by L-DOPA injection. It has been demonstrated that D O P A decarboxylase activity was reduced in the striatum of 6OHDA-lesioned rats 3°. In this state, it is thought that the following conditions are produced when L-DOPA is administered: the conversion from LD O P A to D A is suppressed and the extracellular concentration of L-DOPA in the striatum is higher than normal ~2. These conditions are also thought to occur in patients with Parkinson's disease TM. Therefore, in the on-off phenomenon that appears in long-term L-DOPA therapy 29, the L-DOPA concen-
337
tration in the striatal E C F is thought to be very high just before the a p p e a r a n c e of the off p h e n o m e n o n . A slight decrease in the concentration of L - D O P A m a y elicit a great reduction in the behavioral effect, as shown in Fig. 6. A s a result, the on-off p h e n o m enon m a y develop. In the p e a k dose of dyskinesia 2°, the inverse is thought to occur: a slight increase in L - D O P A in the E C F induces a very large effect on behavior.
striatal E C F after 3 - O M - D O P A administration (i.p. and i.v.t.). No increase in D A or D O P A C concentration was recognized in the present in vivo vol, t a m m e t r i c system. This result is consistent with the result in papers of Chalmers et al. 7 and Reches and Fahn 26, all of whom failed to d e m o n s t r a t e D A formation of D A from 3 - O M - D O P A .
ACKNOWLEDGEMENTS 3-OM-DOPA 3 - O M - D O P A is r e p o r t e d to be a m a j o r metabolite of exogenous L - D O P A in animals 15 and humans 24.
This study e x a m i n e d w h e t h e r 3 - O M - D O P A also elicited behavioral change. Since this drug passes through the B B B 27, 200 mg/kg was intraperitoneally administered, but no behavioral change was observed. T h e n , 200/ag were intraventricularly administered, but again no change was observed. Bartholini et al. 5 r e p o r t e d that 3 - O M - D O P A is a p r e c u r s o r of D A . Therefore, changes in D A and D O P A C concentrations were m e a s u r e d in the REFERENCES 1 Akiyama, A., Kato, T., Ishii, K. and Yasuda, E., In vitro measurement of dopamine concentration with carbon fiber electrode, Anal. Chem., 57 (1985) 1518-1522. 2 Barbeau, A. and Roy, M., Ten-years results of treatment with levodopa plus benserazide in Parkinson's disease. In EC. Rose and R. Capildeo (Eds.), Progress in Parkinson's Disease, Pitman, London, 1981, pp. 241-247. 3 Bartholini, G. and Pletscher, A., Cerebral accumulation and metabolism of ~4C-DOPA after selective inhibition of peripheral decarboxylase, J. Pharmacol. Exp. Therap. , 161 (1968) 14-20. 4 Bartholini, G. and Pletscher, A., Effect of various decarboxylase inhibitors on the cerebral metabolism of dihydroxyphenylalanine, J. Pharm. Pharmacol., 21 (1969) 323-324. 5 Bartholini, G., Kuruma, I. and Pletscher, A., 3-0Methyldopa, a new precursor of dopamine, Nature (Lond.), 230 (1971) 533-534. 6 Breese, G.R. and Traylor, T.D., Depletion of brain noradrenaline and dopamine, Br. J. Pharmacol., 42 (1971) 88-99. 7 Chalmers, J.P., Draffan, G.H., Reid, J.L., Thorgeirsson, S.S. and Davies, D.S., Demethylation of 3-O-methyldopa in the rat, Life Sci., 10 (1971) 1243-1251. 8 Creese, I. and Iversen, S.D., Blockade of amphetamine induced motor stimulation and stereotypy in the adult rat following neonatal treatment with 6-hydroxydopamine, Brain Research, 55 (1973) 369-382. 9 Creese, I., Burt, D.R. and Snyder, S.H., Dopamine receptor binding enhancement accompanies lesioned-induced behavioral supersensitivity, Science, 197 (1977)
The authors wish to thank A k i h i r o Shimizu, MS, of the Mitsubishi Research Institute for his technical support. They would also like to express their appreciation to Emeritus Professor H i r o t a r o Narabayashi and Dr. Hisamasa Imai of the D e p a r t m e n t of Neurology, Professor A k i r a Takeuchi of the D e p a r t m e n t of Physiology, J u n t e n d o University, for their helpful discussions. They extend their gratitude to Sankyo Pharmaceutical Co. and N i p p o n Roche K.K. in Tokyo for their kind donations of L - D O P A and benserazide, respectively. 596--598. 10 Gonon, F., Buda, M., Cespuglio, R., Jouvet, M. and Pujol, J.-F., In vivo electrochemical detection of catechols in the neostriatum of anaesthetized rats: dopamine or DOPAC?, Nature (Lond.), 286 (1980) 902-904. 11 Gonzalez, L.P., Alterations in amphetamine stereotypy following acute lesions of substantia nigra, Life Sci., 40 (1987) 899-908. 12 Hefti, F., Melamed, E. and Wurtman, R.J., The site of dopamine formation in rat striatum after L-DOPA administration, J. Pharmacol. Exp. Therap. , 217 (1981) 189-197. 13 Kissinger, P.T., Hart, J.B. and Adams, R.N., Voltammetry in brain tissue - - a new neurophysiological measurement, Brain Research, 55 (1973) 209-213. 14 Klawans, H.L., Goetz, C., Nausieda, P.A. and Weiner, W.J., Levodopa-induced dopamine receptor hypersensitivity, Ann. Neurol., 2 (1977) 125-129. 15 Kuruma, I., Bartholini, G. and Pletscher, A., L-DOPAinduced accumulation of 3-o-methyldopa in brain and heart, Eur. J. Pharmacol., 10 (1970) 189-192. 16 Lee, T., Seeman, P., Rajput, A., Farley, I.J. and Hornykiewicz, O., Receptor basis for dopaminergic supersensitivity in Parkinson's disease, Nature (Lond.), 273 (1978) 59-60. 17 Lesser, R.P., Fahn, S., Snider, S.R., Cote, L.J., Isgreen, W.P. and Barrett, R.E., Analysis of the clinical problems in parkinsonism and the complications of long-term levodopa therapy, Neurology, 29 (1979) 1253-1260. 18 Lloyd, K. and Hornykiewicz, O., Parkinson's disease: activity of L-DOPA decarboxylase in discrete brain regions, Science, 170 (1970) 1212-1213. 19 Melamed, E., Hefti, F., Bitton, V. and Globus, M., Suppression of L-dopa-induced circling in rats with nigral
338
20
21
22
23
24
25
26
lesions by blockade of central dopa-decarboxylase: implications for mechanism of action of L-dopa in parkinsonism, Neurology, 34 (1984) 1566-1570. Muenter, M.D., Sharpless, N.S., Tyce, G.M. and Darley, F.L., Patterns of dystonia ('I-D-I' and 'D-I-D') in response to L-DOPA therapy for Parkinson's disease, Mayo Clin. Proc., 52 (1977) 163-174. Nakazato, T., Akiyama, A. and Shimizu, A., Microcomputer-controlled in vivo voltammetry, Biogenic Amines, 5 (1988) 339-350. Nakazato, T. and Akiyama, A., In vivo voltammetric study of 6-hydroxydopamine-induced neuronal degradation, J. Neurochem., 51 (1988) 1007-1013. Ogawa, N. and Yamamoto, M., Neurotransmitters and their receptors in parkinsonism. In Y. Kuroiwa and Y. Toyokura (Eds.), Current Problems in Treatment of Parkinson's Disease, DMW Japan, Tokyo, 1985, pp. 23-32. Pletscher, A., Bartholini, G. and Tissot, R., Metabolic fate of L-[14C]DOPA in cerebrospinal fluid and blood plasma of humans, Brain Research, 4 (1967) 106-109. Quinn, N., Parkes, J.D. and Marsden, C.D., Control of on/off phenomenon by continuous intravenous infusion of levodopa, Neurology, 34 (1984) 1131-1136. Reches, A. and Fahn, S., 3-O-Methyldopa blocks dopa metabolism in rat corpus striatum, Ann. Neurol., 12 (1982) 267-271.
27 Rivera-calimlim, L., Absorption, metabolism and distribution of [14C]-o-methyldopa and [14C]-L-DOPA after oral administration to rats, Br. J. Pharmacol., 5(7 (1974) 259-263. 28 Sharma, J.N. and Fahn, S., Microiontophoretic studies with L-dopa and putative neurotransmitters on the neurons of caudate nucleus of rat, Soc. Neurosci. Abstr.. 5 (1979) 599. 29 Shoulson, I., Glaubiger, G.A. and Chase, T.N., On-off response: clinical and biochemical correlations during oral and intravenous levodopa administration in parkinsonian patients, Neurology, 25 (1975) 1144-1148. 30 Sims, K.L. and Bloom, EE., Rat brain L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan decarboxylase activities: differential effect of 6-hydroxydopamine, Brain Research, 49 (1973) 165-175. 31 Stamford, J.A., In vivo voltammetry: promise and perspective, Brain Res. Rev., 10 (1985) 119-135. 32 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, Acta Physiol. Scand., Suppl. 367 (1971) 69-93. 33 Yahr, M.D., Duvoison, R.C., Schear, M.J., Barrette, R.E. and Hoehn, M.M., Treatment of parkinsonism with levodopa, Arch. Neurol., 21 (1969) 343-354.
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Elsevier BRE 14591
Effect of exogenous L-DOPA on behavior in the rat: an in vivo voltammetric study Taizo Nakazato 1 and Akitane A k i y a m a 2 1Department of Neurology, Juntendo University School of Medicine, Tokyo (Japan) and 2Department of Electronic Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama (Japan) (Accepted 6 December 1988)
Key words: L-Dihydroxyphenylalanine; Dopamine; 3-Methoxytyrosine; In vivo vottammetry; Behavior; Dihydroxyphenylalanine-induced dyskinesia; Rat
L-DOPA was administered intraperitoneally (i.p.) or intraventricularly (i.v.t,) to freely moving rats to investigate the effects of exogenous L-DOPA itself on behavior. Striatal dopamine (DA) in the extraceUular fluid was examined with microcomputercontrolled in vivo voltammetry, and behavioral change was observed. When L-DOPA was administered (i.p.) after pretreatment with benserazide, a peripheral DOPA decarboxylase inhibitor, behavioral change was elicited before the elevation in DA and suppressed before its reduction. After pretreatment with NSD-1015, a central DOPA decarboxylase inhibitor, behavioral change was also elicited, although DA was still not increased. When L-DOPA was injected (i.v.t.), the behavioral effect was manifested at once; DA was still unchanged at this time, but it increased after behavioral activity reached the maximum level. L-DOPA was also injected (i.v.t.) into rats with striatal lesions induced by 6-hydroxydopamine (i.v.t.). Behavioral change was manifested promptly after the injection. When the dose-response curves to different dosages of L-DOPA were examined in normal rats without striatal lesions, it was found to exhibit a steeper rise than tha: of DA. Finally, when rats were injected (i.p. or i.v.t.) with 3-O-methyl-DOPA (3-methoxytyrosine), a major metabolite of L-DOPA, no behavioral change was elicited, and no increase in DA was recognized. These experimental results indicated that L-DOPA is related directly to the manifestation of behavioral change.
INTRODUCTION L-DOPA therapy is today's most important strategy for the treatment of Parkinson's disease. There are, however, many problems associated with this therapy: psychiatric symptoms 33, D O P A - i n d u c e d dyskinesia2°, and the on-off p h e n o m e n o n 25. B a r b e a u and Roy 2 pointed out that a high dosage of L-DOPA increased the incidence of dyskinesia and that combination therapy with L-DOPA and benserazide was more hazardous than the use of L-DOPA alone. Lesser et al. 17 also reported that the long-term application of L-DOPA resulted in diminished response to the drug and caused the on-off p h e n o m e n o n and the wearing-off p h e n o m e n o n . Klawans et al. ~4 showed that chronic L-DOPA treatment
in the guinea pig resulted in decreased sensitivity of dopamine ( D A ) receptors, possibly causing dyskinesia. Moreover, the D A receptor binding was demonstrated to decrease in the striatum of Parkinson's disease patients treated with L-DOPA 16. However, it is not known whether these L-DOPAinduced p h e n o m e n a are caused by D A converted from L-DOPA, L-DOPA itself, or the metabolites. Sharma and Fahn 28 pointed out that L-DOPA is an agonist to D A receptors, and Ogawa and Yamamoto 23 also reported that L-DOPA b o u n d directly to the D A binding site. However, Ungerstedt 32 and Melamed et a1.19 found no evidence for the action of L-DOPA. Thus, there is still no definite solution to this problem. The present study was designed to investigate the
Correspondence: T. Nakazato. Present address: Department of Physiology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
333 action of L-DOPA on the central nervous system and clarify the mechanism of L-DOPA-induced dyskinesia. For this purpose, the concentrations of D A and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum of the rat were recorded using microcomputer-controlled in vivo voltammetry after the intraventricular (i.v.t.) administration of L-DOPA. At the same time, behavioral changes induced by these drugs were observed. The concentrations of D A and D O P A C were also examined after the intraperitoneal (i.p.) administration of L-DOPA following pretreatment with the D O P A decarboxylase inhibitors: benserazide and NSD-1015 (ref. 4). Since 3-O-methyl-DOPA (3-OM-DOPA, 3-methoxytyrosine) is a major metabolite of L-DOPA in the brain 3, the drug was also administered (i.p. and i.v.t.) to test its effect on D A release and behavior.
Preparation of freely moving animal model Male Wistar rats (n = 10, 300-400 g) were fixed in a stereotaxic apparatus under pentobarbital anesthesia (50 mg/kg). A carbon fiber electrode of 7 #m in diameter was inserted unilaterally into the striatum, and a stainless-steel cannula was placed contralaterally into the lateral ventricle for the administration of drugs. The details were described in a previous paper z2. Four out of 10 rats were administered (i.v.t.) with 200/~g of 6-hydroxydopamine (6-OHDA) dissolved in 24/d of normal saline for 18 min, after pretreatment with desipramine (25 mg/kg, i.p.) 6. Ten days later, 6 - O H D A was injected again, using the same method. Measurements and drug administrations were done at least 14 days after either the operation or the second 6 - O H D A injection.
MATERIALS AND METHODS
Drug administration Animals were injected (i.p.) with 200 mg/kg of L-DOPA or 3-OM-DOPA, and pretreated with 50 mg/kg of benserazide or 100 mg/kg of NSD-1015 30 min before L-DOPA administration. Intraventricular injection was performed with a microcomputercontrolled autoburette. O n e / A of the solution was injected at the rate of 1/16 ktl/2 s every 45 s. L-DOPA, DA, and 3-OM-DOPA were normally dissolved at the concentration of 5 mg/ml in normal saline deaerated by nitrogen gas, and 40/~1 of the solution was injected in 30 min. L-DOPA concentrations were sometimes changed to clear distinctly the difference of time courses between D A concentration and behavior. If a rat's behavior became
Electrochemical measurement Many studies of the in vivo voltammetric technique have been reported ~°'13"31. The many problems associated with this technique involve its imperfect selectivity and the short lifetime of the electrodes. However, some of the problems have been solved by using the microcomputer-controlled potentiostatic pulse polarization technique described in previous papers a'21. The electrochemical procedures can be summarized as follows. The electrode was pretreated by an anodic-cathodic triangular wave (_+1.5 V, 10 V/s in the slope) before every measurement. Two s after the wave, a triple pulse was applied to determine the concentrations of DA. The first pulse was 50 mV for 660 ms, the second 150 mV for 660 ms and the third 250 mV for 22.5 ms. The currents were measured at the end of every pulse. Changes in D A concentrations were measured every 3 min. A modified differential pulse voitammogram (DPV) ranging from 100 to 500 mV in 25 mV steps was used for qualitative analysis, which was performed every 30 min. The influence of the triangular activation wave and that of the consecutive triple pulse on brain were not determined precisely. However, many of our in vivo studies have shown that these treatments do not cause behavioral change, alteration in the sensitivity of the electrode, or change of the concentration of DA zx.
TABLE I
Behavioral ratingscale Score
Definition Asleep, lying down with eyes closed. Lying down with eyes opened, little movement. Lying down with head up, slow periodic sniffing. Getting up with periodic sniffing. Some rearing with sniffing and slow head swinging, some turning. Frequent rearing with prominent sniffing, head swinging, and turning. Continuous rearing with faster head swinging, or much turning.
334
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Histological procedures hyperactive and life-threatening because of the drug, injections were discontinued. To construct d o s e response curves, various concentrations of the drugs were dissolved in 40 /A of normal saline. Since L - D O P A is difficult to dissolve at high concentrations, L - D O P A p r e p a r e d and d o n a t e d by Sankyo (Japan) was used when a high concentration was needed.
A f t e r the experiment, the animals were perfused intracardially with 10% formaline u n d e r deep anesthesia. The brains were sliced into 50-ktm-thick sections by a freezing microtome. The sections were stained with Cresyl violet. The positions of electrodes and cannula were confirmed microscopically.
Behavioral observations
Intraperitoneal administration of L-DOPA
The rats were placed in a box 30 x 20 × 25 cm in size, and behavioral changes were observed, while D A intensities and D P V s were simultaneously measured. Behavior was scored by the behavioral rating scale in Table I (refs. 8, 11).
W h e n 200 mg/kg of L - D O P A was injected (i.p.) 30 rain after p r e t r e a t m e n t with benserazide, behavioral activity increased about 10 min after the start of the injection and reached the m a x i m u m level at about 60
RESULTS
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Fig. 4. Time course of behavioral change after injection (i.v.t.) of L-DOPA (10 mg/ml) into a normal (O) and 6-OHDAlesioned (&) rats. Injections of L-DOPA were continued from 0 min to the time indicated by the solid or dashed vertical bars in the normal or the 6-OHDA-lesioned rats, respectively.
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min; D A intensity, however, peaked at about 180 min (Fig. 1). L-DOPA was also administered (i.p.) 30 min after the injection of NSD-1015, a central D O P A decarboxylase inhibitor that penetrates the blood-brain barrier (BBB) (Fig. 2). Behavioral activity began to increase at almost the same time as in the case of benserazide pretreatment, but the effect lasted for a longer period. During this period, D A intensity started to increase slightly at 140 min, reached the maximum level at 350 min, then decreased.
Intraventricular administration of L-DOPA and DA When 400/~g (20 kd) of L-DOPA was injected (i.v.t.) for 15 min (Fig. 3), the rats abruptly became
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active at 3-4 min after the injection, but D A intensity in the striatal extracellular fluid (ECF) did not show an increase at this time. After the L-DOPA injection was completed, D A intensity gradually increased and reached the maximum level at about 100 min after injection. The rats had already become less active by this time. L-DOPA (10 mg/ml) was administered (i.v.t.) to 6-OHDA-lesioned rats (Fig. 4). The animals became hyperactive immediately after injection. L-DOPA injection was, therefore, discontinued at about 8 min (90/~g), as shown by the broken line in the figure, but the behavioral effect continued for 150 min. When normal rats without striatal lesions were administered L-DOPA, appearance of the hyperactivity was slightly delayed compared with that in the lesioned rat. This effect, however, was not prolonged. The 6-OHDA-lesioned rat was administered D A (5 mg/ml) (i.v.t.) (Fig. 5). During the injection, almost the same effect on behavior was exhibited as with L-DOPA, but the behavioral changes elicited by DA did not continue after injection was discontinued at 12 min (80/~g). In the normal rat, 200 ktg of D A was administered for 30 min, and the effect was smaller than that in the lesioned rat. Dose-response curves were made by plotting the maximal behavioral effect induced by different dosages of L-DOPA and D A (Figs. 6 and 7). Comparison of the two curves reveals that a marked behavioral change was produced by /ldministration of more than 100 pg of L-DOPA. This large effect on
336 behavior was not observed in the case of DA.
Administration of 3-OM-DOPA When 200 mg/kg of 3-OM-DOPA were intraperitoneally administered to the normal rat, no behavioral change was observed. Intraventricular injection of 200 ~g of the drug also failed to produce behavioral change. During these experiments, DA and D O P A C concentrations in the striatal ECF were unchanged. DISCUSSION
Behavioral effect of L-DOPA It has already been shown that L-DOPA administered is converted to DA, which then acts on the central nervous system. The present experiments suggest that L-DOPA itself also has the ability to elicit behavioral change in rats. The evidence leading to this conclusion is as follows. First, as shown in Fig. 3, when rats were administered L-DOPA (i.v.t.), behavioral change was observed within 3 to 4 min. D A intensity, however, did not increase at this time, but was observed to increase after more than 20 min. The appearance of behavioral change occurred earlier with L-DOPA injection (i.v.t.) than with D A injection, as shown in Fig. 5. Second, when rats were administered L-DOPA (i.p.) after pretreatment with a peripheral D O P A decarboxylase inhibitor, benserazide (Fig. 1), behavioral change was elicited after 5-10 min, and reached a peak 60-90 rain later. Bartholini and Pletscher 3 reported that L-DOPA administered reached its maximal concentration in the rat brain 1 h later. This time span corresponds closely with the time at which maximal behavioral activity appeared in the present experiment. Third, when L-DOPA was administered (i.p.) after pretreatment with NSD-1015, which inhibited its conversion to D A in the brain, as expected, the increase in D A concentration in the ECF was greatly suppressed compared with that following the case of pretreatment with benserazide. The beginning of the behavioral change, however, was not delayed, and the change persisted much longer in the case of NSD-1015. Melamed et al. 19 reported that when rats with
unilateral striatal lesions were administered (i.p.) with 50 mg/kg of L-DOPA after pretreatment of 100 mg/kg of NSD-1015, circling behavior was suppressed. Therefore, it was suggested that exogenous L-DOPA itself was not responsible for behavior. However, in the present experiment, 200 mg/kg of L-DOPA was injected into the normal rats pretreated with NSD-1015, and behavioral change was observed. The conflicting result obtained in the present experiment might have been due to different experimental conditions: normal rats were used and 200 mg/kg of L-DOPA was injected. Since more L-DOPA reached the central nervous system than in the study of Melamed et al., behavioral change may have been elicited. Fourth, when L-DOPA was administered (i.v.t.) to the 6-OHDA-lesioned rat, behavioral change was elicited immediately after injection as shown in Fig. 4. This should be considered partially due to the supersensitivity of the D A receptor 9, as already demonstrated in Fig. 5. However, in spite of the fact that the lesioned rats have reduced D O P A decarboxylase activity in the striatum 3°, behavioral change after L-DOPA injection was observed to appear at almost the same time that it did after D A injection to the lesioned rat, suggesting that L-DOPA also induces behavioral change. It is interesting to discuss the dose-response curves shown in Figs. 6 and 7. With D A injection (i.v.t.), behavioral response increased gradually with increasing doses, whereas with L-DOPA, the increase in the response was apparently larger at doses of more than 100/~g. These results may indicate that exogenous L-DOPA and its partially converted DA act on D A receptors, and consequently the larger behavioral effect was produced by L-DOPA injection. It has been demonstrated that D O P A decarboxylase activity was reduced in the striatum of 6OHDA-lesioned rats 3°. In this state, it is thought that the following conditions are produced when L-DOPA is administered: the conversion from LD O P A to D A is suppressed and the extracellular concentration of L-DOPA in the striatum is higher than normal ~2. These conditions are also thought to occur in patients with Parkinson's disease TM. Therefore, in the on-off phenomenon that appears in long-term L-DOPA therapy 29, the L-DOPA concen-
337
tration in the striatal E C F is thought to be very high just before the a p p e a r a n c e of the off p h e n o m e n o n . A slight decrease in the concentration of L - D O P A m a y elicit a great reduction in the behavioral effect, as shown in Fig. 6. A s a result, the on-off p h e n o m enon m a y develop. In the p e a k dose of dyskinesia 2°, the inverse is thought to occur: a slight increase in L - D O P A in the E C F induces a very large effect on behavior.
striatal E C F after 3 - O M - D O P A administration (i.p. and i.v.t.). No increase in D A or D O P A C concentration was recognized in the present in vivo vol, t a m m e t r i c system. This result is consistent with the result in papers of Chalmers et al. 7 and Reches and Fahn 26, all of whom failed to d e m o n s t r a t e D A formation of D A from 3 - O M - D O P A .
ACKNOWLEDGEMENTS 3-OM-DOPA 3 - O M - D O P A is r e p o r t e d to be a m a j o r metabolite of exogenous L - D O P A in animals 15 and humans 24.
This study e x a m i n e d w h e t h e r 3 - O M - D O P A also elicited behavioral change. Since this drug passes through the B B B 27, 200 mg/kg was intraperitoneally administered, but no behavioral change was observed. T h e n , 200/ag were intraventricularly administered, but again no change was observed. Bartholini et al. 5 r e p o r t e d that 3 - O M - D O P A is a p r e c u r s o r of D A . Therefore, changes in D A and D O P A C concentrations were m e a s u r e d in the REFERENCES 1 Akiyama, A., Kato, T., Ishii, K. and Yasuda, E., In vitro measurement of dopamine concentration with carbon fiber electrode, Anal. Chem., 57 (1985) 1518-1522. 2 Barbeau, A. and Roy, M., Ten-years results of treatment with levodopa plus benserazide in Parkinson's disease. In EC. Rose and R. Capildeo (Eds.), Progress in Parkinson's Disease, Pitman, London, 1981, pp. 241-247. 3 Bartholini, G. and Pletscher, A., Cerebral accumulation and metabolism of ~4C-DOPA after selective inhibition of peripheral decarboxylase, J. Pharmacol. Exp. Therap. , 161 (1968) 14-20. 4 Bartholini, G. and Pletscher, A., Effect of various decarboxylase inhibitors on the cerebral metabolism of dihydroxyphenylalanine, J. Pharm. Pharmacol., 21 (1969) 323-324. 5 Bartholini, G., Kuruma, I. and Pletscher, A., 3-0Methyldopa, a new precursor of dopamine, Nature (Lond.), 230 (1971) 533-534. 6 Breese, G.R. and Traylor, T.D., Depletion of brain noradrenaline and dopamine, Br. J. Pharmacol., 42 (1971) 88-99. 7 Chalmers, J.P., Draffan, G.H., Reid, J.L., Thorgeirsson, S.S. and Davies, D.S., Demethylation of 3-O-methyldopa in the rat, Life Sci., 10 (1971) 1243-1251. 8 Creese, I. and Iversen, S.D., Blockade of amphetamine induced motor stimulation and stereotypy in the adult rat following neonatal treatment with 6-hydroxydopamine, Brain Research, 55 (1973) 369-382. 9 Creese, I., Burt, D.R. and Snyder, S.H., Dopamine receptor binding enhancement accompanies lesioned-induced behavioral supersensitivity, Science, 197 (1977)
The authors wish to thank A k i h i r o Shimizu, MS, of the Mitsubishi Research Institute for his technical support. They would also like to express their appreciation to Emeritus Professor H i r o t a r o Narabayashi and Dr. Hisamasa Imai of the D e p a r t m e n t of Neurology, Professor A k i r a Takeuchi of the D e p a r t m e n t of Physiology, J u n t e n d o University, for their helpful discussions. They extend their gratitude to Sankyo Pharmaceutical Co. and N i p p o n Roche K.K. in Tokyo for their kind donations of L - D O P A and benserazide, respectively. 596--598. 10 Gonon, F., Buda, M., Cespuglio, R., Jouvet, M. and Pujol, J.-F., In vivo electrochemical detection of catechols in the neostriatum of anaesthetized rats: dopamine or DOPAC?, Nature (Lond.), 286 (1980) 902-904. 11 Gonzalez, L.P., Alterations in amphetamine stereotypy following acute lesions of substantia nigra, Life Sci., 40 (1987) 899-908. 12 Hefti, F., Melamed, E. and Wurtman, R.J., The site of dopamine formation in rat striatum after L-DOPA administration, J. Pharmacol. Exp. Therap. , 217 (1981) 189-197. 13 Kissinger, P.T., Hart, J.B. and Adams, R.N., Voltammetry in brain tissue - - a new neurophysiological measurement, Brain Research, 55 (1973) 209-213. 14 Klawans, H.L., Goetz, C., Nausieda, P.A. and Weiner, W.J., Levodopa-induced dopamine receptor hypersensitivity, Ann. Neurol., 2 (1977) 125-129. 15 Kuruma, I., Bartholini, G. and Pletscher, A., L-DOPAinduced accumulation of 3-o-methyldopa in brain and heart, Eur. J. Pharmacol., 10 (1970) 189-192. 16 Lee, T., Seeman, P., Rajput, A., Farley, I.J. and Hornykiewicz, O., Receptor basis for dopaminergic supersensitivity in Parkinson's disease, Nature (Lond.), 273 (1978) 59-60. 17 Lesser, R.P., Fahn, S., Snider, S.R., Cote, L.J., Isgreen, W.P. and Barrett, R.E., Analysis of the clinical problems in parkinsonism and the complications of long-term levodopa therapy, Neurology, 29 (1979) 1253-1260. 18 Lloyd, K. and Hornykiewicz, O., Parkinson's disease: activity of L-DOPA decarboxylase in discrete brain regions, Science, 170 (1970) 1212-1213. 19 Melamed, E., Hefti, F., Bitton, V. and Globus, M., Suppression of L-dopa-induced circling in rats with nigral
338
20
21
22
23
24
25
26
lesions by blockade of central dopa-decarboxylase: implications for mechanism of action of L-dopa in parkinsonism, Neurology, 34 (1984) 1566-1570. Muenter, M.D., Sharpless, N.S., Tyce, G.M. and Darley, F.L., Patterns of dystonia ('I-D-I' and 'D-I-D') in response to L-DOPA therapy for Parkinson's disease, Mayo Clin. Proc., 52 (1977) 163-174. Nakazato, T., Akiyama, A. and Shimizu, A., Microcomputer-controlled in vivo voltammetry, Biogenic Amines, 5 (1988) 339-350. Nakazato, T. and Akiyama, A., In vivo voltammetric study of 6-hydroxydopamine-induced neuronal degradation, J. Neurochem., 51 (1988) 1007-1013. Ogawa, N. and Yamamoto, M., Neurotransmitters and their receptors in parkinsonism. In Y. Kuroiwa and Y. Toyokura (Eds.), Current Problems in Treatment of Parkinson's Disease, DMW Japan, Tokyo, 1985, pp. 23-32. Pletscher, A., Bartholini, G. and Tissot, R., Metabolic fate of L-[14C]DOPA in cerebrospinal fluid and blood plasma of humans, Brain Research, 4 (1967) 106-109. Quinn, N., Parkes, J.D. and Marsden, C.D., Control of on/off phenomenon by continuous intravenous infusion of levodopa, Neurology, 34 (1984) 1131-1136. Reches, A. and Fahn, S., 3-O-Methyldopa blocks dopa metabolism in rat corpus striatum, Ann. Neurol., 12 (1982) 267-271.
27 Rivera-calimlim, L., Absorption, metabolism and distribution of [14C]-o-methyldopa and [14C]-L-DOPA after oral administration to rats, Br. J. Pharmacol., 5(7 (1974) 259-263. 28 Sharma, J.N. and Fahn, S., Microiontophoretic studies with L-dopa and putative neurotransmitters on the neurons of caudate nucleus of rat, Soc. Neurosci. Abstr.. 5 (1979) 599. 29 Shoulson, I., Glaubiger, G.A. and Chase, T.N., On-off response: clinical and biochemical correlations during oral and intravenous levodopa administration in parkinsonian patients, Neurology, 25 (1975) 1144-1148. 30 Sims, K.L. and Bloom, EE., Rat brain L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan decarboxylase activities: differential effect of 6-hydroxydopamine, Brain Research, 49 (1973) 165-175. 31 Stamford, J.A., In vivo voltammetry: promise and perspective, Brain Res. Rev., 10 (1985) 119-135. 32 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, Acta Physiol. Scand., Suppl. 367 (1971) 69-93. 33 Yahr, M.D., Duvoison, R.C., Schear, M.J., Barrette, R.E. and Hoehn, M.M., Treatment of parkinsonism with levodopa, Arch. Neurol., 21 (1969) 343-354.