European Journal of Pharmacology. 178 (1990) 115-120
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Elsevier EJP 20573 Short c o m m u n i c a t i o n
Effect of chronic treatment of MPTP monkeys with dopamine D-I and/or D-2 receptor agonists C6fine G a g n o n 1, Paul J. B6dard 2 a n d Th6r6se Di Paolo 1 I School of Pharmacy, Laval University, (~tebec, Qc, GIK ?P4 Canada. and Laboratory of Molecular Endocrinology. CHUL Research Center, Ste-Foy, Qc, G1V 4G2 Canada, "Neurobiology Laboratory. L'Enfant-Jesus Hospital. Quebec. Qc. GIJ I Z4 Canada and Department of Anatomy, Laval University, Faculty of Medicine, Quebec. Qc. GI K 71'4 Canada
Received 11 December1989, accepted 9 January 1990
A severe parkinsonian syndrome developed in four monkeys after administration of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). One monkeys subsequently remained untreated, and each of the three others were treated daily for at least one month with Sinemet, bromocriptine or SKF 38393. An intact control monkey (not MPTP-treated) was also included in the experiment. Sinemet and bromocriptine relieved the parkinsonian symptoms, whereas SKF 38393 was ineffective. The animal treated with Sinemet developed dyskinesia while those treated with bromocriptine or SKF 38393 did not. MPTP decreased dopamine levels by more than 99~ in the striatum of all monkeys. Striatal D-1 and D-2 dopamine receptor densities as evaluated by autoradiography of [3H]SCH 23390 and [3HI spiperone binding were increased by 66 and 51~, respectively, after MPTP. Sinemet, bromocriptine or SKF 38393 treatment decreased D-2 receptor density, respectively, by 17, 84 and 35~ and D-1 receptor density by 28, 33 and 6~ vs. that in MPTP-treated animals. Our results suggest that dopamine receptor changes could be implicated in the loss of efficacy and in the side-effects of these treatments. Dopamine D-1 receptors; Dopamine D-2 receptors; MPTP (1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine); Parkinson; (Monkey)
1. Introduction The i.v. administration of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) can induce a Parldnsoa-like syndrome in humans and in nonhuman primates (Bums et al., 1983). This neurotoxin causes selective destruction of dopaminergic neurons in the substantia nigra pars compacta (Bums et al., 1983). Treatment with the dopamine agonist, L-DOPA, relieves parldnsonian symptoms, but long-term treatment with this drug induces important side-effects such as dyskinesias in
Correspondence to: T. Di Paolo, Laboratory of Molecular Endocrinology, CHUL Research Center, 2705 Laurier Bouleyard, Sainte-Foy,Quebec, GIV 4G2 Canada.
humans (Mones et al., 1971) and monkeys (B6dard et al., 1986; Burns et al., 1983). L-DOPA-induced dyskinesias are often thought to be related to supersensitivity of striatal dopamine (DA) receptors caused by prolonged denervation (Creese et al., 1977). In contrast, de novo treatment of parkinsonians with the D-2-specific agonist, bromocriptine, improves the parkinsonian syndrome while inducing little or no dyskinesia (Rinne, 1987; Lees and Stem, 1981); the efficacy of this drug may however diminish with time. Bromocriptine can correct motor deficits in MPTP-parkinsonian monkeys without inducing dyskinesia, while it decreases the density of the supersensitive postsynaptic DA receptors (B6dard et al., 1986). Recently, we have investigated the behavioral and biochemical effects of treatment of
0014-2999/90/$03.50 © 1990 ElsevierScience Publishers B.V. (BiomedicalDivision)
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MPTP monkeys with various DA agonists specific for one t~ve of DA receptors (D-I or D-2) (F~ardeau et aL, 1988). Our results showed that chronic treatment with L-DOPA or bromocriptine re~eves parkinsonian symptoms of MPTP mow keys whereas SKI: 38393 was ineffective. As opposed to L-DOPA neither bromocriptine nor SKF 38393 induced dyskinesias. Biochemical study of bra/n tissue homogenates showed that chronic treatment with L-DOPA, bromocriptine or SKF 3.8393 decreased D-2 receptor supersensitivity. The p ~ of the present study was to compare the modulation of D-1 and D-2 DA receptors after chronic treatment of MPTP-treated monkeys with DA agonists (Sinemet (L-DOPA + carbidopa), brom~riptine or SKF 38393). Quantitative autorad/ography of [3H]spiperone (D-2 antagonist) and [3H]SCH 23390 (D-1 antagonist) binding was used to see if the therapeutic action and side-effects induced by chronic dopaminergic treatment could be related to localized D-1 a n d / o r D-2 receptor changes which could have been missed when tissue homogenates were studied.
2. Materials and meflu~ts
Z1. Animals and treatments Five female monkeys (Macaca fascicularis) weighing around 3.0 kg and of approximately the same age were used in this study. The animals were ovariectomized bilaterally under ketamine hydrochloride anesthesia three months before the experiments were started. Four animals received a saline solution of MPTP (Aldrich Chemical) administered i.v. (0.3 mg/kg) repeatedly until a stable parkinsonian syndrome was observed (cumulative dose 0.3-1.5 mg/kg). The fifth animal remained untreated and served as control. One week after the last dose of MPTP, the four akinetic animals were started on daily oral treatment with the various DA agonists or saline for at least one month. Each animal received only one drug. Sinemet (L-DOPA/carbidopa) was administered in doses of 50 mg/kg and the D-2 agonist bromocriptine (Parlodel) and the selective D-1 agonist SKF 38393 were given in doses of 5 mg/kg.
All drugs were suspended in water and introduced directly into the oesophagus with a syringe and plastic tube. Behavior was evaluated as previously described (Falardeau et al., 1988).
2.2. Tissue preparation and autoradiography After one mouth of treatment, three days after the last dose of L-DOPA/carbidopa, bromocriptine or SKF 38393, the animals were killed with an overdose of pentobarbital. The brains were rapidly removed, frozen and stored at - 8 0 ° C . For autoradiography, the brains were cut into coronal sections (20/~m) on a cryostat ( - 1 8 ° C ) thaw-mounted onto gelatin and chrome alumcoated slides. The slide-mounted coronal sections of caudate and putamen were preincubated for 15 min at room temperature in a phosphate buffer (mM: 81 Na2HPO4, 19 KH2PO4 and 2 MgCI 2, pH 7.40-7.45). The sections were then incubated (60 rain at room temperature) with 1 nM of [3H]spiperone (D-2 antagonist) (68-95 Ci/mmol) or 1 nM of [3H]SCH 23390 (D-1 antagonist) (75-85 Ci/mmol). In the D-2 receptor assay, ketanserin (50 nM) was added to the incubation medium to block 5-HT2 serotonin receptors. Non-specific binding was determined in adjacent sections with the addition of 1 pM (+)-butaclatool (D-2) or 1/~M SCH 23390 (D-l). The sections were then rinsed for 15 rain in the appropriate cold (4°C) phosphate buffer then for 10 s in distilled water. The sections were dried overnight at room temperature. The slide-mounted tissue sections were then apposed to 3H-sensitive film (Hyperfilm RPN13, LKB) along with a set of tritium standards (3H-microscale, Amersham) for 15 days at - 2 0 o C. The films were developed with D-19 developer (Kodak) for 4 min, fixed in rapid fixer (Kodak) for 10 min and rinsed in water for 30 min before drying. The autoradiograms of the sections were analyzed with an image analyzer system (RAS-1000, Amersham). Quantitative values were obtained for direct autoradiography of 3H-microscale (Amersham) with brain sections. Data were obtained from four regions: anterior and posterior caudate as well as anterior and posterior putamen. As similar effects of the treatments on DA receptors were observed in these
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regions, we expressed the results of each brain region as • of their respective value for each specific brain area of the unlesioned monkey then pooled the data to "calculate an average change of striatal density as a ~o of density in the unlesioned monkey + S.E.M.
2.3. Catecholamine determination Caudate-putamen tissue from alternate coronal sections was dissected for catecholamine determination. DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assayed by HPLC with electrochemical detection, essentially as described by Di Paolo et al. (1986). Data were obtained from four brain regions: anterior and posterior caudate as well as anterior and posterior putamen. As a similar extent of denervation was obtained after MPTP throughout the striatum, we expressed the results for each brain region as a ~ of the value for the corresponding brain area in the unlesioned monkey. We then pooled the data to calculate an average change of striatal concentration as a ~ of that in the unlesioned monkey + S.E.M.
2. 4. Statistical analysis The multiple-range test of Duncan-Kramer (Kramer, 1956) was used for comparison of the data obtained for receptor and catecholamine levels.
3. Results As we previously reported (B~.,dard et al., 1986; Falardeau et al., 1988), the animals ~ e progressively akinetic following the injection of MPTP and a maximum was reached after 3 days. L-DOPA treatment improved the parkinsonian symptoms, but bucco-lingual dyskinesia soon appeared (fourth day) and increased in the weeks following. MPTP monkeys treated with bromocriptine displayed increased locomotor activity and fewer stereotyped movements than did the L-DOPAtreated monkeys (no dyskinesia was seen). The effect of bromocriptine only became evident after 3 days whereas the effect of L-DOPA was seen after the first dose. The L-DOPA effect began 90 min after drug administration and lasted approximately 6 h. SKF 38393 treatment had no apparent effect on locomotor activity and no dyskinesia or stereotypies were observed. MPTP caused a decrease of 99% or more of DA levels in the striatum and a smaller decrease of DOPAC (>I 75~o) and HVA (>t 90%) (table 1). As DA depletion was similar in the four striatal regions assayed, the catecho!amine results were expressed as % of their respective control values and were pooled as shown in table 1. DA depletion in the striatum of all the MPTP monkeys was also similar. The control values for DA, DOPAC and HVA concentrations, in n g / m l of pr.,tein, were, respectively; anterior caudate: 88.79, 13.41,159.99; posterior caudate: 114.97, 7.13, 178.26; anterior
TABLE 1 Effect of treatment of MPTP-treated monkeys with Sinemet, bromocriptine or SKF 38393 on specific [3H]spiperone (1 nM) and [3H]SCH 23390 (1 nM) binding to D-2 and D-1 receptors, as well as on DA, DOPAC a~d HVA concentrations in the striatum of these monkeys. The data, as calculated from the values obtained from four regions, anterior and posterior caudate as well as anterior and posterior putamen, were expressed as % of those for the unlesioned monkey and were pooled to calculate an average change of density or concentration + S.E.M. Catecholamine levels were determined in duplicate or in triplicate in each region, while the density of DA receptors is she mean of 10-25 slices for each region, n.d.: non-detectable. Values given are ~ of intact. Group
Receptor specific binding D-1
Intact MPTP MPTP+Sinemet MPTP+bromocriptine M P T P + S K F 38393
100.0+4.3 166.4+5.7 119.9+2.9 111.7+4.9 156.3+4.8
DA conc.
DOPAC conc.
100.00+6.30 n.d. a 0.70+0.23 a 0.11 + 0 . 1 i a 0.59+0.30 a
100.00 + 9.65 18.71+ 4.38 20.73+10.99 18.24+ 2.70 74,34+:14.47
HVA conc.
D-2 a a.b b a,d.e
100.0+6.3 151.4+6.5 a 125.1-1-4.0 a,b.e 23.8+2.2 a,b 99.0+45a,d.e
a a a a
100.00+6.27 0.62+0.18 10.605:2.97 0.48+:0.22 0.95+:0.25
a.b a a.b a.b
a p < 0.01 vs. intact; b p < 0.01 vs. MPTP; c p < 0.05 and d p < 0.01 vs. MPTP + Sinemet; e p < 0.01 vs. MPTP + bromocriptine.
putml~en: 117.72, 14.36, 294.21 and posterior putamen: 160.76, 12.79, 528.2. Denervation by means of MPTP caused an increase of D-1 and D-2 DA receptors as compared to the intact control (fig. 1 and table 1). D-]
D-2
~NTACT
+
J
MPTP
MP~'rP 4-
SL~-VEI"
MPTP `4.
BF~OMOC~P~NE
Treatment w.~th DA agonist (L-DOPA, bromocriptine or SKF 38393) decreased this supersensitivity. The changes in [3H] spiperone and [3H]SCH 23390 binding to the D-2 and D-1 receptors, respectively, after MPTP and the various treatments were similar in the four regions studied. The resuits were therefore expressed as % of their respecfive control values and pooled as shown in table 1. Denervation by means of MPTP caused a sh,'ailar up-regulation of 66% for D-1 and 51% for D-2 receptor density. Treatment with each of the three drugs reversed the denervation supersensitivity. Sinemet, bromocriptine or SKF 28393 treatment reduced D-2 receptor density by 17, 84 and 35%, respectively, and D-1 receptor density by 28, 33 and 6%, respectively, as compared to that of MPTP-treated animals. Bromocriptine treatment not only reversed the striatal denervation supersensitivity but reduced the binding to D-2 DA receptors by 76% as compared to that in the intact control striatum. The control values for the densities of D-1 and D-2 receptors in fmol/mg of tissue were, respectively: anterior caudate: 280.4, 176.0; posterior caudate: 251.4, 121.1; anterior p cry+men: 261.3, i52.5 and posterior putamen 309.2, 157.9.
4. Discussion
p" \+
MPTP ÷
SKF 38393
Fig. 1. Autoradiograms of [JH]SCH 23390 (1 nM) and [3H]spiperone (1 nM) binding to D-1 and D-2 DA receptors in the posterior caudate and putamen of one intact, one MPTPtreated monkey and three MPTP-treated monkeys receiving chronicafiy Sinemet, bromocriptine or SKF 38393. Autoradiograms show the total binding. Non-specific binding for D-1 and D-2 receptors was approximately 10 and 40~ of the total binding, respectively.
In the present experiment, as in previous ones (Btdard et al., 1986; Falardeau et al., 1988), monkeys exhibiting parkinsonian symptoms following an MPTP injection responded well to both L-DOPA and bromocriptine. Once again, LDOPA (mixed D-1 and D-2 activity) was shown to induce more stereotyped behavior and dyskinesia than did bromocriptine (D-2 agonist). SKF 38393 (D-1 agonist) had no effect on locomotor activity and did not induce dyskinesia. AH MPTP animals were denervated extensively and to the same extent and showed a decrease of more than 99% of DA in the striatum as compared to the intact control. Thus the receptor changes observed were most likely due to the drug treatment rather than to denervation, since the striata of these monkeys were similarly denervated. The present results are preliminary as only one animal per
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group was used. We nevertheless consider these results representative since in our present evaluation by autoradiography as in our previous study with tissue homogenates from two to three monkeys per group, D-2 receptor density increased in the striatum of the MPTP-treated monkey (2596) and dopaminergic treatments decreased this supersensitivity (Falardeau et al., 1988). Interestingly in the present autoradiographic assay of receptors, the decrease in D-2 receptors observed after treatment of the MPTPtreated monkey with the Do2 agonist, bromocriptine, was even more dramatic than the effect in homogenates (Falardeau et al., 1988). Indeed D-2 receptor density in the striatum of this monkey was 7696 less than that in the intact monkey. It is tempting to speculate that this may be related to the loss of responsiveness to bromocriptine seen in patients. We cannot, however, exclude some degree of residual binding of bromocriptine to D-2 receptors. However, the presence of bromocriptine on D-2 DA receptors should lead to a decrease of affinity in binding experiments. This was not been observed in our previous experiments where bromocriptine left the [3H]spiperone binding affinity to D-2 receptors unchanged (B6dard et al, 1986) or even slightly increased (Falardeau et al., 1988). D-1 receptors such as the D-2 subtype were also increased (6696) in the striatum after the injection of MPTP. Treatment with bromocriptine or L-DOPA decreased this supersensitivity by 33 and 2896, respectively, as compared to MPTP alone. The decrease in binding of D-1 receptors after treatment with L°DOPA was approximately the same as for D-2 receptors (2896 and 1796, respectively), whereas the treatment with bromocriptine induced a greater decrease in density of D-2 receptors (8496) than of D-1 receptors (3396) as compared to that in the MPTP monkey. It is surprising to note that treatment with SKF 38393 did not significantly change (696 vs. MPTP) the density of D-1 receptors. This may be related to the fact that SKF 38393 is not only a D-1 agonist but also a weak D-1 antagonist. Our results suggest that there is an interaction between D-1 and D-2 receptors. Indeed treatment of MPTP-treated monkeys with a specific D-2
agonist had an effect on the density of D-1 receptors while the specific D-1 agonist affected Do2 receptors. The results of several biochemical and behavioral studies also suggest that there are important functional interactions between D-1 and D-2 receptors. For instance, Barone et al. (1987) observed that, in hemiparkinsonian monkeys, the D-2 DA agonist, LY 171555, but not the D-1 agonist, SKF 38393, reduced parkinsonian signs and induced rotation away from the side of the nigral lesion. When administered together, however, SKF 38393 diminished the LY 171555-induced rotation away from the side of the nigral lesion. In summary, the present work extends our previous observations that chronic treatment with L-DOPA is more likely to induce dyskinesia in parkinsor6an monkeys than is treatment with bromocriptine and that this may be due to a different modulation of D-1 and D-2 receptors. MPTP induces denervation supersensitivity of both D-1 and D-2 striatal DA receptors and this is reversed by D-I, D-2 or mixed D - l / D - 2 agonists. The receptor changes are, however, not limited to the receptor with which the selective agonist is supposed to interact.
Acknowledgements This research was supported by the Parkinsonian Foundation of Canada and the Medical Research Council of Canada. We wish to thank M. Michel Daigle for technical assistance.
References Barone P., K.S. Bankiewicz, G.U. Corsini, l.J. Kopin and T.N. Chase, 1987, Dopaminergic mechanisms in hemiparkinsonian monkeys, Neurology 37, 1592. B~dard, P.J., T. Di Paolo, P. Falardeau and R. Boucher, 1986, Chronic treatment with levodopa, but not bromocriptine induces dyskinesia in MPTP-parkinsonian monkeys. Correlation with [3H]-spiperone binding, Brain Res. 379, 294. Bums, R.S., C.C. Chiueh, S.P. Markey0 M.H. Ebert, D.M. Jacobowitz and 13. Kopin, 1983, A primate model for parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-l,2,3,6 tetrahydropyridine, Proc. Natl. Acad. Sci. U.S.A. 80, 4546.
Cre~e~ L, D.IL Bert and S.H. Snvder, 1977, Dopamine receptor bind/rig enhancement accompanies lesion-induced behavioral supersensitivity, Science 197, 596. D~ P ~ , T., PJ. B6dard, M. Daigle and R. Boucher, 1986, Longqerm effect of MPTP on central and peripheral ~ t e c ~ a m i n e and indolamine concentrations in monkeys, Brain Res. 379, 286. Fahrdeau, P., S. Bouchard, PJ. B6dard. IL Boncher and T. Di Pat'qo~ 1988, Behavioral and biochemical effects of chronic treatment with D-1 and/or D-2 dopamine agonists in MPTP monke)~ European J. Pharmacol. 150, 59. Kxamer, C.Y., 1956, Extension of multiple-range tests to group
means with unequal number of replications, Biometrics 12, 307. Lees, A.J. and G.M. Stem, 1981, Sustained bromocriptine therapy in previously untreated patients with Parkinson's disease, J. Neurol. Neurosurg. Psychiat. 43, 1020. Mones, R.J., T. Elizan, GJ. Sand Siegel, 1971, Analysis of L-dopa-induced dyskinesias in 51 patients with parkinsonism, J. Neurol. Neurosurg. Psychiat. 34, 668. Rinne, V.K, 1987, Early combination of bromocriptine and levodopa in the treatment of Parkinson's disease: a 5 year follow-up, Neurology 37, 826.
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Elsevier EJP 20573 Short c o m m u n i c a t i o n
Effect of chronic treatment of MPTP monkeys with dopamine D-I and/or D-2 receptor agonists C6fine G a g n o n 1, Paul J. B6dard 2 a n d Th6r6se Di Paolo 1 I School of Pharmacy, Laval University, (~tebec, Qc, GIK ?P4 Canada. and Laboratory of Molecular Endocrinology. CHUL Research Center, Ste-Foy, Qc, G1V 4G2 Canada, "Neurobiology Laboratory. L'Enfant-Jesus Hospital. Quebec. Qc. GIJ I Z4 Canada and Department of Anatomy, Laval University, Faculty of Medicine, Quebec. Qc. GI K 71'4 Canada
Received 11 December1989, accepted 9 January 1990
A severe parkinsonian syndrome developed in four monkeys after administration of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). One monkeys subsequently remained untreated, and each of the three others were treated daily for at least one month with Sinemet, bromocriptine or SKF 38393. An intact control monkey (not MPTP-treated) was also included in the experiment. Sinemet and bromocriptine relieved the parkinsonian symptoms, whereas SKF 38393 was ineffective. The animal treated with Sinemet developed dyskinesia while those treated with bromocriptine or SKF 38393 did not. MPTP decreased dopamine levels by more than 99~ in the striatum of all monkeys. Striatal D-1 and D-2 dopamine receptor densities as evaluated by autoradiography of [3H]SCH 23390 and [3HI spiperone binding were increased by 66 and 51~, respectively, after MPTP. Sinemet, bromocriptine or SKF 38393 treatment decreased D-2 receptor density, respectively, by 17, 84 and 35~ and D-1 receptor density by 28, 33 and 6~ vs. that in MPTP-treated animals. Our results suggest that dopamine receptor changes could be implicated in the loss of efficacy and in the side-effects of these treatments. Dopamine D-1 receptors; Dopamine D-2 receptors; MPTP (1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine); Parkinson; (Monkey)
1. Introduction The i.v. administration of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) can induce a Parldnsoa-like syndrome in humans and in nonhuman primates (Bums et al., 1983). This neurotoxin causes selective destruction of dopaminergic neurons in the substantia nigra pars compacta (Bums et al., 1983). Treatment with the dopamine agonist, L-DOPA, relieves parldnsonian symptoms, but long-term treatment with this drug induces important side-effects such as dyskinesias in
Correspondence to: T. Di Paolo, Laboratory of Molecular Endocrinology, CHUL Research Center, 2705 Laurier Bouleyard, Sainte-Foy,Quebec, GIV 4G2 Canada.
humans (Mones et al., 1971) and monkeys (B6dard et al., 1986; Burns et al., 1983). L-DOPA-induced dyskinesias are often thought to be related to supersensitivity of striatal dopamine (DA) receptors caused by prolonged denervation (Creese et al., 1977). In contrast, de novo treatment of parkinsonians with the D-2-specific agonist, bromocriptine, improves the parkinsonian syndrome while inducing little or no dyskinesia (Rinne, 1987; Lees and Stem, 1981); the efficacy of this drug may however diminish with time. Bromocriptine can correct motor deficits in MPTP-parkinsonian monkeys without inducing dyskinesia, while it decreases the density of the supersensitive postsynaptic DA receptors (B6dard et al., 1986). Recently, we have investigated the behavioral and biochemical effects of treatment of
0014-2999/90/$03.50 © 1990 ElsevierScience Publishers B.V. (BiomedicalDivision)
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MPTP monkeys with various DA agonists specific for one t~ve of DA receptors (D-I or D-2) (F~ardeau et aL, 1988). Our results showed that chronic treatment with L-DOPA or bromocriptine re~eves parkinsonian symptoms of MPTP mow keys whereas SKI: 38393 was ineffective. As opposed to L-DOPA neither bromocriptine nor SKF 38393 induced dyskinesias. Biochemical study of bra/n tissue homogenates showed that chronic treatment with L-DOPA, bromocriptine or SKF 3.8393 decreased D-2 receptor supersensitivity. The p ~ of the present study was to compare the modulation of D-1 and D-2 DA receptors after chronic treatment of MPTP-treated monkeys with DA agonists (Sinemet (L-DOPA + carbidopa), brom~riptine or SKF 38393). Quantitative autorad/ography of [3H]spiperone (D-2 antagonist) and [3H]SCH 23390 (D-1 antagonist) binding was used to see if the therapeutic action and side-effects induced by chronic dopaminergic treatment could be related to localized D-1 a n d / o r D-2 receptor changes which could have been missed when tissue homogenates were studied.
2. Materials and meflu~ts
Z1. Animals and treatments Five female monkeys (Macaca fascicularis) weighing around 3.0 kg and of approximately the same age were used in this study. The animals were ovariectomized bilaterally under ketamine hydrochloride anesthesia three months before the experiments were started. Four animals received a saline solution of MPTP (Aldrich Chemical) administered i.v. (0.3 mg/kg) repeatedly until a stable parkinsonian syndrome was observed (cumulative dose 0.3-1.5 mg/kg). The fifth animal remained untreated and served as control. One week after the last dose of MPTP, the four akinetic animals were started on daily oral treatment with the various DA agonists or saline for at least one month. Each animal received only one drug. Sinemet (L-DOPA/carbidopa) was administered in doses of 50 mg/kg and the D-2 agonist bromocriptine (Parlodel) and the selective D-1 agonist SKF 38393 were given in doses of 5 mg/kg.
All drugs were suspended in water and introduced directly into the oesophagus with a syringe and plastic tube. Behavior was evaluated as previously described (Falardeau et al., 1988).
2.2. Tissue preparation and autoradiography After one mouth of treatment, three days after the last dose of L-DOPA/carbidopa, bromocriptine or SKF 38393, the animals were killed with an overdose of pentobarbital. The brains were rapidly removed, frozen and stored at - 8 0 ° C . For autoradiography, the brains were cut into coronal sections (20/~m) on a cryostat ( - 1 8 ° C ) thaw-mounted onto gelatin and chrome alumcoated slides. The slide-mounted coronal sections of caudate and putamen were preincubated for 15 min at room temperature in a phosphate buffer (mM: 81 Na2HPO4, 19 KH2PO4 and 2 MgCI 2, pH 7.40-7.45). The sections were then incubated (60 rain at room temperature) with 1 nM of [3H]spiperone (D-2 antagonist) (68-95 Ci/mmol) or 1 nM of [3H]SCH 23390 (D-1 antagonist) (75-85 Ci/mmol). In the D-2 receptor assay, ketanserin (50 nM) was added to the incubation medium to block 5-HT2 serotonin receptors. Non-specific binding was determined in adjacent sections with the addition of 1 pM (+)-butaclatool (D-2) or 1/~M SCH 23390 (D-l). The sections were then rinsed for 15 rain in the appropriate cold (4°C) phosphate buffer then for 10 s in distilled water. The sections were dried overnight at room temperature. The slide-mounted tissue sections were then apposed to 3H-sensitive film (Hyperfilm RPN13, LKB) along with a set of tritium standards (3H-microscale, Amersham) for 15 days at - 2 0 o C. The films were developed with D-19 developer (Kodak) for 4 min, fixed in rapid fixer (Kodak) for 10 min and rinsed in water for 30 min before drying. The autoradiograms of the sections were analyzed with an image analyzer system (RAS-1000, Amersham). Quantitative values were obtained for direct autoradiography of 3H-microscale (Amersham) with brain sections. Data were obtained from four regions: anterior and posterior caudate as well as anterior and posterior putamen. As similar effects of the treatments on DA receptors were observed in these
117
regions, we expressed the results of each brain region as • of their respective value for each specific brain area of the unlesioned monkey then pooled the data to "calculate an average change of striatal density as a ~o of density in the unlesioned monkey + S.E.M.
2.3. Catecholamine determination Caudate-putamen tissue from alternate coronal sections was dissected for catecholamine determination. DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assayed by HPLC with electrochemical detection, essentially as described by Di Paolo et al. (1986). Data were obtained from four brain regions: anterior and posterior caudate as well as anterior and posterior putamen. As a similar extent of denervation was obtained after MPTP throughout the striatum, we expressed the results for each brain region as a ~ of the value for the corresponding brain area in the unlesioned monkey. We then pooled the data to calculate an average change of striatal concentration as a ~ of that in the unlesioned monkey + S.E.M.
2. 4. Statistical analysis The multiple-range test of Duncan-Kramer (Kramer, 1956) was used for comparison of the data obtained for receptor and catecholamine levels.
3. Results As we previously reported (B~.,dard et al., 1986; Falardeau et al., 1988), the animals ~ e progressively akinetic following the injection of MPTP and a maximum was reached after 3 days. L-DOPA treatment improved the parkinsonian symptoms, but bucco-lingual dyskinesia soon appeared (fourth day) and increased in the weeks following. MPTP monkeys treated with bromocriptine displayed increased locomotor activity and fewer stereotyped movements than did the L-DOPAtreated monkeys (no dyskinesia was seen). The effect of bromocriptine only became evident after 3 days whereas the effect of L-DOPA was seen after the first dose. The L-DOPA effect began 90 min after drug administration and lasted approximately 6 h. SKF 38393 treatment had no apparent effect on locomotor activity and no dyskinesia or stereotypies were observed. MPTP caused a decrease of 99% or more of DA levels in the striatum and a smaller decrease of DOPAC (>I 75~o) and HVA (>t 90%) (table 1). As DA depletion was similar in the four striatal regions assayed, the catecho!amine results were expressed as % of their respective control values and were pooled as shown in table 1. DA depletion in the striatum of all the MPTP monkeys was also similar. The control values for DA, DOPAC and HVA concentrations, in n g / m l of pr.,tein, were, respectively; anterior caudate: 88.79, 13.41,159.99; posterior caudate: 114.97, 7.13, 178.26; anterior
TABLE 1 Effect of treatment of MPTP-treated monkeys with Sinemet, bromocriptine or SKF 38393 on specific [3H]spiperone (1 nM) and [3H]SCH 23390 (1 nM) binding to D-2 and D-1 receptors, as well as on DA, DOPAC a~d HVA concentrations in the striatum of these monkeys. The data, as calculated from the values obtained from four regions, anterior and posterior caudate as well as anterior and posterior putamen, were expressed as % of those for the unlesioned monkey and were pooled to calculate an average change of density or concentration + S.E.M. Catecholamine levels were determined in duplicate or in triplicate in each region, while the density of DA receptors is she mean of 10-25 slices for each region, n.d.: non-detectable. Values given are ~ of intact. Group
Receptor specific binding D-1
Intact MPTP MPTP+Sinemet MPTP+bromocriptine M P T P + S K F 38393
100.0+4.3 166.4+5.7 119.9+2.9 111.7+4.9 156.3+4.8
DA conc.
DOPAC conc.
100.00+6.30 n.d. a 0.70+0.23 a 0.11 + 0 . 1 i a 0.59+0.30 a
100.00 + 9.65 18.71+ 4.38 20.73+10.99 18.24+ 2.70 74,34+:14.47
HVA conc.
D-2 a a.b b a,d.e
100.0+6.3 151.4+6.5 a 125.1-1-4.0 a,b.e 23.8+2.2 a,b 99.0+45a,d.e
a a a a
100.00+6.27 0.62+0.18 10.605:2.97 0.48+:0.22 0.95+:0.25
a.b a a.b a.b
a p < 0.01 vs. intact; b p < 0.01 vs. MPTP; c p < 0.05 and d p < 0.01 vs. MPTP + Sinemet; e p < 0.01 vs. MPTP + bromocriptine.
putml~en: 117.72, 14.36, 294.21 and posterior putamen: 160.76, 12.79, 528.2. Denervation by means of MPTP caused an increase of D-1 and D-2 DA receptors as compared to the intact control (fig. 1 and table 1). D-]
D-2
~NTACT
+
J
MPTP
MP~'rP 4-
SL~-VEI"
MPTP `4.
BF~OMOC~P~NE
Treatment w.~th DA agonist (L-DOPA, bromocriptine or SKF 38393) decreased this supersensitivity. The changes in [3H] spiperone and [3H]SCH 23390 binding to the D-2 and D-1 receptors, respectively, after MPTP and the various treatments were similar in the four regions studied. The resuits were therefore expressed as % of their respecfive control values and pooled as shown in table 1. Denervation by means of MPTP caused a sh,'ailar up-regulation of 66% for D-1 and 51% for D-2 receptor density. Treatment with each of the three drugs reversed the denervation supersensitivity. Sinemet, bromocriptine or SKF 28393 treatment reduced D-2 receptor density by 17, 84 and 35%, respectively, and D-1 receptor density by 28, 33 and 6%, respectively, as compared to that of MPTP-treated animals. Bromocriptine treatment not only reversed the striatal denervation supersensitivity but reduced the binding to D-2 DA receptors by 76% as compared to that in the intact control striatum. The control values for the densities of D-1 and D-2 receptors in fmol/mg of tissue were, respectively: anterior caudate: 280.4, 176.0; posterior caudate: 251.4, 121.1; anterior p cry+men: 261.3, i52.5 and posterior putamen 309.2, 157.9.
4. Discussion
p" \+
MPTP ÷
SKF 38393
Fig. 1. Autoradiograms of [JH]SCH 23390 (1 nM) and [3H]spiperone (1 nM) binding to D-1 and D-2 DA receptors in the posterior caudate and putamen of one intact, one MPTPtreated monkey and three MPTP-treated monkeys receiving chronicafiy Sinemet, bromocriptine or SKF 38393. Autoradiograms show the total binding. Non-specific binding for D-1 and D-2 receptors was approximately 10 and 40~ of the total binding, respectively.
In the present experiment, as in previous ones (Btdard et al., 1986; Falardeau et al., 1988), monkeys exhibiting parkinsonian symptoms following an MPTP injection responded well to both L-DOPA and bromocriptine. Once again, LDOPA (mixed D-1 and D-2 activity) was shown to induce more stereotyped behavior and dyskinesia than did bromocriptine (D-2 agonist). SKF 38393 (D-1 agonist) had no effect on locomotor activity and did not induce dyskinesia. AH MPTP animals were denervated extensively and to the same extent and showed a decrease of more than 99% of DA in the striatum as compared to the intact control. Thus the receptor changes observed were most likely due to the drug treatment rather than to denervation, since the striata of these monkeys were similarly denervated. The present results are preliminary as only one animal per
119
group was used. We nevertheless consider these results representative since in our present evaluation by autoradiography as in our previous study with tissue homogenates from two to three monkeys per group, D-2 receptor density increased in the striatum of the MPTP-treated monkey (2596) and dopaminergic treatments decreased this supersensitivity (Falardeau et al., 1988). Interestingly in the present autoradiographic assay of receptors, the decrease in D-2 receptors observed after treatment of the MPTPtreated monkey with the Do2 agonist, bromocriptine, was even more dramatic than the effect in homogenates (Falardeau et al., 1988). Indeed D-2 receptor density in the striatum of this monkey was 7696 less than that in the intact monkey. It is tempting to speculate that this may be related to the loss of responsiveness to bromocriptine seen in patients. We cannot, however, exclude some degree of residual binding of bromocriptine to D-2 receptors. However, the presence of bromocriptine on D-2 DA receptors should lead to a decrease of affinity in binding experiments. This was not been observed in our previous experiments where bromocriptine left the [3H]spiperone binding affinity to D-2 receptors unchanged (B6dard et al, 1986) or even slightly increased (Falardeau et al., 1988). D-1 receptors such as the D-2 subtype were also increased (6696) in the striatum after the injection of MPTP. Treatment with bromocriptine or L-DOPA decreased this supersensitivity by 33 and 2896, respectively, as compared to MPTP alone. The decrease in binding of D-1 receptors after treatment with L°DOPA was approximately the same as for D-2 receptors (2896 and 1796, respectively), whereas the treatment with bromocriptine induced a greater decrease in density of D-2 receptors (8496) than of D-1 receptors (3396) as compared to that in the MPTP monkey. It is surprising to note that treatment with SKF 38393 did not significantly change (696 vs. MPTP) the density of D-1 receptors. This may be related to the fact that SKF 38393 is not only a D-1 agonist but also a weak D-1 antagonist. Our results suggest that there is an interaction between D-1 and D-2 receptors. Indeed treatment of MPTP-treated monkeys with a specific D-2
agonist had an effect on the density of D-1 receptors while the specific D-1 agonist affected Do2 receptors. The results of several biochemical and behavioral studies also suggest that there are important functional interactions between D-1 and D-2 receptors. For instance, Barone et al. (1987) observed that, in hemiparkinsonian monkeys, the D-2 DA agonist, LY 171555, but not the D-1 agonist, SKF 38393, reduced parkinsonian signs and induced rotation away from the side of the nigral lesion. When administered together, however, SKF 38393 diminished the LY 171555-induced rotation away from the side of the nigral lesion. In summary, the present work extends our previous observations that chronic treatment with L-DOPA is more likely to induce dyskinesia in parkinsor6an monkeys than is treatment with bromocriptine and that this may be due to a different modulation of D-1 and D-2 receptors. MPTP induces denervation supersensitivity of both D-1 and D-2 striatal DA receptors and this is reversed by D-I, D-2 or mixed D - l / D - 2 agonists. The receptor changes are, however, not limited to the receptor with which the selective agonist is supposed to interact.
Acknowledgements This research was supported by the Parkinsonian Foundation of Canada and the Medical Research Council of Canada. We wish to thank M. Michel Daigle for technical assistance.
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