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The effect of chronic L-DOPA administration on supersensitive pre- and postsynaptic dopaminergic receptors in rat brain
Life Sciences, Vol. 31, Printed in the U.S.A.
pp.
Pergamon Press
37-44
TRE EFFECT OF CRRONICL-DOPA ADMINISTRATION ON SUPERSENSITIVe PBE- AND POSTSYNAPTICDOPAMINERGIC RECEPTORSIN RAT BRAIN Avinoam Reches, H. Ryan Wagner, De-hua Jiang, Vernice Jackson and Stanley Fahn
Columbia
Department of Neurology University College of Physicians 710 West 168th Street New York, New York 10032
(Received
in final
form April
19,
and Surgeons
1982)
Summary Chronic administration of haloperidol induced supersensitivity dopaminergic receptors in rat brain. of the pre- and postsynaptic The response of the presynaptic receptors was determined by an enhanced inhibitory effect of apomorphine on dopamine synthesis after gamma-butyrolactone injection. This change in the receptor function was detected both in the nigrostriatal and mesolimbic Baloperidol also increased the 3R-spiperone binding pathways. sites in striatal membranes, indicating supersensitivity of the postsynaptic receptors. Subsequent prolonged treatment with high doses of L-DOPA/carbidopa resulted in a decrease in 3H-spiperone binding sites, but had no effect on the supersensitive presynaptic receptors. It is suggested that tardive dyskinesia may be a state of both pre- and postsynaptic dopamine receptor supersensitivity and that chronic L-DOPA treatment may have a differential effect on these sites. Tardive dyskinesia (TD) is an extrapyramidal syndrome which is induced by chronic administration of neuroleptic drugs (1, 2). The pathogenesis of TD is probably related to chronic blockade of the postsynaptic dopaminergic receptors by these drugs and to the ensuing receptor supersensitivity (3). This phenomenon has been established by biochemical (4-S), behavioral (9-13) and electrophysiological (14) studies. If indeed TD results from chronic blockage of the dopaminergic receptors by the neuroleptic drugs then a rational therapeutic approach would be a direct reversal of the neuroleptic induced pathology. This can presumably be achieved by a subsequent chronic administration of direct dopaminergic agonists. In agreement with such an approach are recent reports which demonstrate that both increased receptor binding (15, 16) and behavioral supersensitivity (17, 18) produced by chronic treatment with neuroleptic drugs could be reversed by prolonged administration of high doses of
0024-3205/82/010037-08$03.00/O Copyright (c) 1982 Pergamon Press
Ltd.
38
Chronic DOPA and Dopamine Supersensitivity
Vol. 31, No. 1, 1982
dopaminergic agonists. Presynaptic dopaminergic receptors have an important role in the modulation of dopamine (DA) synthesis and release (19). Thus, any change in their function induced by neuroleptic drugs may have, in turn, a marked effect on the availability of synaptic DA (20). It has been suggested that dopaminergic receptor supersensitivity following chronic exposure to neuroleptic drugs could be a presynaptic, as well as a postsynaptic phenomenon (21). Since chronic treatment with dopaminergic agonists have been recommended in the treatment of TD (22), we studied the effect of prolonged L-DOPA administration on neuroleptic-induced supersensitive preand postsynaptic dopaminergic receptors in both the nigroetriatal and mesolimbic pathways of rat brain. Materials and Methods Drugs and Chemicals: Analytical grade chemicals were purchased from Sigma Chemcial Company (St. Louis, MO), Fisher Scientific Company (Springfield, NJ) and Bioanalytical Systems (West Lafayette, IN). Gamma-butyrolactone (GBL), 3-hydroxybenzylhydrazine-HCl (NSD-1015), and apomorphine were purchased from Sigma Chemical Company. Haloperidol was purchased from McNeil Company (Ft. Washington, PA), L-dihydroxyphenylalanine (L-DOPA) from Nutritional Biochemical Corp. (Cleveland, OH) and carbidopa from Merck, Sharp & Dohme (West Point, PA). 3H-Spiperone (31.3 Ci/mmol) was from New England Nuclear (Boston, MA). Spiperone was a gift from the Janssen Pharmaceutic Co., Inc. (New Brunswick, NJ). Animals 170-200 g at water. Rats combinations according to
and Drug Administration: Male Sprague-Dawley rats weighing the beginning of the study were given free access to food and were divided into five drug treatment groups and given various of chronic saline, haloperidol and L-DOPA plus carbidopa the schedule shown in Table 1.
In Vivo Study of Presynaptic DA Receptors: Sensitivity of the presy=DA receptors was studied as described by Walters and Roth (23). In brief, the administration of GBL blocks the nerve impulse flow of the dopaminergic neurons, following which the accumulation of DA is enhanced. This accumulation of DA can be prevented by inhibition of tyrosine hydroxylase which is accomplished by treatment with apomorphine (24). Since this inhibitor effect of apomorphine occurs in the absence of nerve impulse, it is considered to be mediated via stimulation of presynaptic DA receptors. The responsiveness of the presynaptic receptors to apomorphine can thus be estimated by its effect on the accumulation of DOPA following the administration of GBL in the presence of a DOPA decarboxylase inhibitor. At the end of the treatment schedule (Table 1) rats were injected intraperitoneally (i.p.) with apomorphine (0.5 mg*kg-1), 5 minutes laterwith GBL (750 mg*kg-1 i.p.) and 5 minutes after GBL injection with the DOPA decarboxylase inhibitor NSD-1015 (100 mg*kg'1 i.p.). Group A rats were injected with NSD-1015 only and group B rats were injected with GBL plus NSD-1015, but they did not receive apomorphine injection. All rats were sacrificed by decapitation 30 minutes after the NSD-1015 injection and the brain was rapidly removed. Each brain was split longitudinally. One half was used for DOPA assay and the other half for receptor binding studies. The olfactory tuberclee and nucleus accumbens (medial part) were dissected by exposing the anterior commissure in its entire course. A cut was made from the anterior commissure to the lateral border of the olfactory tubercle. Thus, a brain part was obtained located rostrally and medially to the anterior coma&sure, rostrally to the optic chiasm and including the olfactory tubercles. Thereafter, the corpora striata were removed (25). For
Vol. 31, No. 1, 1982
Chronic DOPA and Dopamine Supersensitivity
39
the sake of simplicity we will refer in the text to these brain regions as "accumbens" and "etriatum". Tissues were dissected on an ice-cold glass plate and immediately frozen on dry ice. Assay for DOPA: Frozen tissue was weighed and sonicated (Kontes MicroUltrasonic cell disrupter) in approximately 20 volumes of ice-cold 0.1 M perchloric acid containing 2 mM Na2EDTA and 10 ul/ml of NaHS03 (0.1 M). After centrifugation (15,600 x g for 15 min) 180 ul supernatant was taken for DOPA extraction (26). 3,4_Dihydroxybenzylamine (DHBA, 25 up) was added as internal standard to quantitate recovery. For the separation and quantification of the catechols, extracts were subjected to reverse phase (Cl6, ODS column, DuPont) high performance liquid chromatography and eluted with 0.1 M monochloroacetate buffer (pH 3.05) containing 1 mM Na2EDTA and 70 The flow rate mg/L of sodium octyl-sulfate as an ion pairing agent (27). was 1.0 ml/min at ambient temperature. The catechols were quantitated using an LC-4A glassy carbon electrode (Bioanalytical Systems) with an applied potential of 0.7 V. Typical retention times were 5.2 min for DOPA and 6.37 min for DHBA 7.5 min for 3,4_dihydroxyphenylacetic acid (DOPAC) and 10.1 min for dopamine. A Spectra-Physics graphic integrator was used for calculations (27). Table l.Drug Treatment Schedule Treatment Group
n
Phase 1 (28 days)
Phase 2 (10 days)
A B c D
5 5 7
no drug no drug saline B.C. saline B.C.
E
8
no drug no no drug no saline p.0. no DOPA/carbidopa no (200/2Omg*kg-1 p.o.) saline p.0. no
F
7
5
Haloperidol (2 mgakg-1s.c.) Haloperidol (2 mg.kg'1s.c.)
Phase 3 (3 days) drug drug drug drug drug
DOPA/carbidopa no drug (200/20 mgakg'1 p.o.)
Legend to Table 1: L-DOPA/carbidopa was given in two divided oral (p.0.) doses as a suspension in saline which was made fresh daily. Haloperidol was used in the form of HaldolH injectable solution (McNeil Lab, Ft. Washington, PA) and was diluted with 0.9% NaCl before subcutaneous (8.c.) injection. 3H-Spiperone binding were performed by a modification of the method described by Seeman et al. (28). Fresh striata were homogenized by Polytron (Brinkman Instruments, Westbury, NY) in approximately 1:lOO w/v ice-cold Tris-EC1 buffer (50 mM, pEl 7.5). To obtain a crude membrane preparation, homogenates were centrifuged (40,000 x g for 10 minutes) and the resulting pellet was washed twice by resuspension and recentrifugation to remove any residual apomorphine. The final pellet was resuspended in 1:150 w/v of ice-cold 'Iris-EC1 (15 mM)-Na2EDTA (5 mM) buffer (pH 7.4) containing 0.01% ascorbate, and preincubated for 10 minutes at 370C. Aliquots of tissue were combined with various concentrations of 3H-spiperone and drugs to a final volume of 1 ml. Protein concentrations as estimated by Lowry assay (29) was approximately 250-400 ng/ml. Incubations were for 12 minutes at 370 C. Bound 3H-spiperone was separated by vacuum filtration over glass fiber filters (Whatman GF/B) followed by washing with 10 ml of the incubation buffer. Filters were placed on Aquasol (New England Nuclear) and counted using liquid scintillation spectrometry. Specific binding was defined as the difference in total binding in the presence and absence of 10 uM unlabelled spiperone. Membranes from each rat were assayed at a single concentration of
40
Chronic
%I-spiperone (0.3 Scatchard analysis
DOPA and Dopamine Supersensitivity
nM) .
(30).
Vol.
31, No.
Membranes from each group were then pooled
Results are expressed as mean f S.B.M. data was determined by two-tailed Student’s
Statistical t-test.
analysis
1, 1982
for of
the
Results In the presence of NSD-1015, DOPA accumulates in striatum and accumbens GBL injected to naive rats 35 minutes before the animals were (Figure 1A). sacrificed increases DOPA concentration in the presence of the DOPA decarboxylase inhibitor NSD-1015 in both the etriatum and accumbene (Figure 1B). This increase was significantly inhibited by apomorphine given prior to GBL injection (Figure lC>. The oral administration of high doses of L-DOPA/carbidopa for 10 days did not change the responsiveness of the presynaptic dopaminergic receptors to apomorphine compared with saline injected controls (Figure 1D). In contrast , pretreatment with haloperidol (2 mg*kg-1 i.p.> for 28 days induced an enhanced response of the presynaptic dopaminergic receptors to apomorphine. The accumulation of DOPA was thus significantly reduced compared to the control rats (Group C) both in the striatum (~(0.005) and accumbens (pCO.005) (Figure 1E). The administration of L-DOPA/carbidopa, for 10 days following the chronic injection of 200/20 mg*kg’l daily, haloperidol did not reverse the enhanced response to apomorphine induced by the neuroleptic. DOPA levels in the latter group (Group F) were not significantly different from those found in rats treated with haloperidol only and were still significantly below control levels in the striatum (p
vol. 31, No. 1, 1982
Chronic DOPA and Dopamine Supersensitivity
ACCUMBENS
S TRIA TUM
APO GEL NSD-1015
--++tt -+++++ t + +
+
+
41
--t++t -+++++ +++++t
+
Figure 1. The Effect of Chronic Treatment With Raloperidol and L-DOPA on Apomorphine-Induced Depression of DOPA Accumulation After GBL Treatment
Rats were given no drugs (A, B), saline only (C), saline followed by L-DOPA/ carbidopa (D), haloperidol followed by saline (E) or haloperidol followed by L-DOPA/carbidopa (F) as described in Table 1. At the end of the treatment schedule the effects of these drugs on the presynaptic dopaminergic receptors was measured as described in Methods. Rats were challenged with either NSD-1015 alone, gamma butyrolactone (GBL) plus NSD-1015, or with apomorphine (APO) plus GBL plus NSD-1015 as outlined above. Groups E and F were significantly different from control rats (Group C> by *p
Table 2.
Treatment Group
n
c D E F
7 5 8 7
Specific 3Wspiperone Binding (fmols/mg protein) Group mean f S.E.M. Bmax 324 363 385 304
f f f f
22 29 ll* 14**
520 581 733 578
Kd(nM) 0.2 0.1 0.1 0.2
Group means represent specific binding at 0.3 nM. 3H-spiperone was averaged from individual animals. Scatchard analysis was conducted from 0.8 to 0.5 nM 3Wspiperone on pooled membranes from animals in each group. *, significantly different from group C by p
42
Chronic
DOPA and Dopamine Supersensitivity
Vol.
31, No.
1, 1982
Discussion The experiments reported here provide evidence that prolonged exposure to haloperidol produced supersensitivity in both presynaptic and postsynaptic dopaminergic receptors. This phenomenon in presynaptic receptors was described earlier by Nowycky and Roth (21) and was recently confirmed also by electrophysiological evidence (31). Supersensitivity of the presynaptic receptors is expressed in our experiments in the enhanced effectiveness of apomorphine to depress the elevated DOPA accumulation which occur in the dopaminergic nerve terminals following impulse flow inhibition by GSL. The neuroleptic-induced change in presynaptic receptor function was detected both in the nigrostriatal and mesolimbic pathways of rat brain (Figure 1). Tardive dyskinesia could be viewed, therefore, as a state of combined pre- and postsynaptic DA receptor supersensitivity. It is worth discussing whether any therapeutic approach should include both types of receptors in consideration. Supersensitivity of the presynaptic receptors may be compensatorally useful in tardive dyskinesia as a means by which synaptic release of DA can be minimized. It may be best therefore, to attempt to “down-regulate” only the postsynaptic DA receptor and leave unaltered the supersensitivity of the presynaptic receptors. Several animal studies have demonstrated that chronic treatment with dopaminergic agonists may down-regulate dopaminergic receptors from an established supersensitive state. These reports include behavioral studies (17, 18, 32, 33), 3H-neuroleptic binding (15, 16), and adenylate cyclase activity measurements (15). That such regulation in 3H-neuroleptic binding sites may also exist in humans has been shown by Lee et al. Since (34). 3Wneuroleptics bind preferentially to postsynaptic receptors (35) and adenylate cyclase is also thought to be coupled to postsynaptic receptors (36, 37), it is possible that these studies reflect the down-regulation that while changes in presynaptic takes place mainly in postsynaptic receptors, receptors are less understood. The data presented in this study suggest that prolonged administration of L-DOPA did not reverse the supersensitivity of the presynaptic dopaminergic receptors induced by chronic pretreatment with haloperidol. The responsiveness of the presynaptic receptors to apomorphine in the haloperidol plus L-DOPA/carbidopa treated rats was similar to that in the haloperidol treated rats and both groups had an enhanced response to the dopaminergic agonist compared with the control group (Figure 1). However, chronic L-DOPA treatment had a significant effect on postsynaptic receptors as measured by 3H-spiperone binding studies. Pretreatment of rats with haloperidol produced an expected increase in the specific binding of 3H-epiperone (7), and subsequent treatment of haloperidol-treated rats with L-DOPA/carbidopa produced an apparent reversal of this effect (Table 2) in agreement with previous reports (15, 16). The data presented here suggest that chronic dopaminergic agonism may have a differential effect on the two types of dopaminergic receptors. Such an effect was found by List and Seeman (16) who reported a significant increase in both 3H-epiperone and 3H-apomorphine binding after chronic Subsequent prolonged treatment with bromocriptine exposure to haloperidol. binding but not the increase in reversed only the increase in 3H-spiperone 3H-apomorphine binding which is thought to label preferentially presynaptic receptors (35). It is possible that supersensitive presynaptic receptors are more resistant to chronic agonist treatment than the postsynaptic receptors. Of special
interest
is
the finding
that
L-DOPA/carbidopa
treatment
alone
Vol. 31, No. 1, 1982
Chronic DOPA and Dopamine Supersensitivity
43
did not down-regulate 3H-spiperone binding. In fact, binding appeared to be slightly elevated in L-DOPA/carbidopa treated rats (Table 2). Inspection of our data suggested that the high values in this group may have reflected unusually low protein levels. It has been shown that the acute administration of L-DOPA induced disaggregation of brain polysomes (38) and transient inhibition of protein synthesis (39). Such an apparent increase in 3H-spiperone binding after chronic L-DOPA treatment was reported earlier by Wilner et al. (40) although the specific question of a possible effect of L-DOPA on protein values in this study was not mentioned. We are currently investigating a cumulative decrease in protein synthesis during chronic L-DOPA treatment. Acknowledgements This work was supported in part by NIH grant NSl5959-02. Dr. Reches is a recipient of a Peggy Engl Fellowship from the Parkinson's Disease Foundation and of a Fogarty Public Health Service International Research Fellowship (NIH TW02884). Dr. Jiang is a recipient of an H. Houston Merritt Fellowship from the Parkinson's Disease Foundation and a fellowship from the People's Republic of China. References 1. 2. 3. 4. 5. 6. 7. 8.
?i. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
L Uhrbrand, A Faurbye, Psychopharmacologia 1:408-418, (1960). GE Crane, Brit J Psychiat 122:395-405, (1973). HL Klawans, Am J Psychiat 130:82-86, (1973). G Gianutsos, MD Hynes, H Lal, Biochem Pharmacol 24:581-582, (1975). K Iwatsubo, DH Clouet, Biochem Pharmacol 24:1499-1503, (1975). P Muller, P Seeman, Life Sci 21:1751-1758, (1977). DR Burke, I Creese, SH Snyder, Science 196:326-328, (1977). JC Schwartz, M Baudry, MP Martres, J Costentin, P Protais, Life Sci 23: 1758-1790, (1978). U Ungerstedt, Acta Physiol Stand Suppl 367:1-122 (1971) HL Klawans, R Rubovits, J Neural Trans 33:235-24: (1972)' D Tarsy, RJ Baldessarini, Neuropharmacol 13:927-910, (1971). RC Smith, JM Davis, Life Sci 19:725-732, (1976). G Gianutsos, KE Moore, Life Sci 20:1585-1592, (1977). G Yarbrough, Eur J Pharmacol 31:367-369, (1975). AJ Friedhoff, K Bonnet, II Rosengarten, Res Comm Chem Path01 Pharmacol 16:411-423, (1977). SJ List, P Seeman, Life Sci 24:1447-1452, (1979). C Ezrin-Waters, P Seeman, Life Sci 22:1027-1032, (1978). WJ Weiner, P Carvey, PA Nausieda, CG Goetz, HL Klawans, Life Sci 28: 2173-2178, (1981). MC Nowycky, RH Roth, Prog Neurol Psychopharmacol 2:139-158, (1978). L Iversen, M Rogowski, R Miller, Mol Pharmacoal 12:251-263, (1976). M Nowycky, R Roth, Naunyn Schmiedeberg's Arch Pharmacol 300:247-254, (1977). M Alpert, AJ Friedhoff, Clin Pharmcol Ther 19:103, (1976). JR Walters, RH Roth, Naunyn-Schmiedeberg's Arch Pharmacol 296:5-14, (1976). W Kehr, A Carlsson, M Lindquist, J Pharm Pharmacol 24~744-747, (1972). A Carlsson, M Lindquist, J Pharm Pharmacol 25:437-440, (1973). R Keller, A Oke, I Mefford, RN Adames, Life Sci 19:995-1004, (1976). GC Davis, PT Kissinger, R Shoup, Anal Chem 53:156-159, (1981). P Seeman, M Chau-Wong, J Tedesco, K Wong, Proc Nat1 Acad Sci (USA) 72:4376-4380, (1975). OH Lowry, NI Rosebrough, A Farr, RJ Randall, J Biol Chem 193:265-275, (1951). G Scatchard: Ann NY Acad Sci 51:660-672, (1949).
44
31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
Chronic DGPA and Dopamine Supersensitivity
Vol. 31, No. 1, 1982
D Gallager, A Pert, W Bunne, Nature 273:309-312, (1978). AV Christensen, I Moller Nielsen, Pscyhopharmacologia 62:111-116, (1979). TF Seeger, EL Gardner, WF Bridger, Brain Res 215:404-409, (1981). T Lee, P Seeman, A Rajput, IJ Farley, 0 Kornykiewicz, Nature 273:59-61, (1978). JI Nagy, T Lee, P Seeman, EC Fibiger, Nature 274~278-280, (1978). R Schwarz, JT Coyle, Brain Res 127:235-249, (1977). IW Kebabian, DB Calne, Nature 277:93-96, (1979). BF Weiss, EN Munro, LA Ordonez, RJ Wurtman, Science 177:613-616, (1972). LE Roel, P Levine, D Rubin, D Markovitz, RN Munro, RJ Wurtman, Life Sci 22:1887-1892, (1978). KD Wilner, IJ Butler, WE Seifert, Y Clement-Cormier, Biochem Pharm 29: 710-716. (1980).
pp.
Pergamon Press
37-44
TRE EFFECT OF CRRONICL-DOPA ADMINISTRATION ON SUPERSENSITIVe PBE- AND POSTSYNAPTICDOPAMINERGIC RECEPTORSIN RAT BRAIN Avinoam Reches, H. Ryan Wagner, De-hua Jiang, Vernice Jackson and Stanley Fahn
Columbia
Department of Neurology University College of Physicians 710 West 168th Street New York, New York 10032
(Received
in final
form April
19,
and Surgeons
1982)
Summary Chronic administration of haloperidol induced supersensitivity dopaminergic receptors in rat brain. of the pre- and postsynaptic The response of the presynaptic receptors was determined by an enhanced inhibitory effect of apomorphine on dopamine synthesis after gamma-butyrolactone injection. This change in the receptor function was detected both in the nigrostriatal and mesolimbic Baloperidol also increased the 3R-spiperone binding pathways. sites in striatal membranes, indicating supersensitivity of the postsynaptic receptors. Subsequent prolonged treatment with high doses of L-DOPA/carbidopa resulted in a decrease in 3H-spiperone binding sites, but had no effect on the supersensitive presynaptic receptors. It is suggested that tardive dyskinesia may be a state of both pre- and postsynaptic dopamine receptor supersensitivity and that chronic L-DOPA treatment may have a differential effect on these sites. Tardive dyskinesia (TD) is an extrapyramidal syndrome which is induced by chronic administration of neuroleptic drugs (1, 2). The pathogenesis of TD is probably related to chronic blockade of the postsynaptic dopaminergic receptors by these drugs and to the ensuing receptor supersensitivity (3). This phenomenon has been established by biochemical (4-S), behavioral (9-13) and electrophysiological (14) studies. If indeed TD results from chronic blockage of the dopaminergic receptors by the neuroleptic drugs then a rational therapeutic approach would be a direct reversal of the neuroleptic induced pathology. This can presumably be achieved by a subsequent chronic administration of direct dopaminergic agonists. In agreement with such an approach are recent reports which demonstrate that both increased receptor binding (15, 16) and behavioral supersensitivity (17, 18) produced by chronic treatment with neuroleptic drugs could be reversed by prolonged administration of high doses of
0024-3205/82/010037-08$03.00/O Copyright (c) 1982 Pergamon Press
Ltd.
38
Chronic DOPA and Dopamine Supersensitivity
Vol. 31, No. 1, 1982
dopaminergic agonists. Presynaptic dopaminergic receptors have an important role in the modulation of dopamine (DA) synthesis and release (19). Thus, any change in their function induced by neuroleptic drugs may have, in turn, a marked effect on the availability of synaptic DA (20). It has been suggested that dopaminergic receptor supersensitivity following chronic exposure to neuroleptic drugs could be a presynaptic, as well as a postsynaptic phenomenon (21). Since chronic treatment with dopaminergic agonists have been recommended in the treatment of TD (22), we studied the effect of prolonged L-DOPA administration on neuroleptic-induced supersensitive preand postsynaptic dopaminergic receptors in both the nigroetriatal and mesolimbic pathways of rat brain. Materials and Methods Drugs and Chemicals: Analytical grade chemicals were purchased from Sigma Chemcial Company (St. Louis, MO), Fisher Scientific Company (Springfield, NJ) and Bioanalytical Systems (West Lafayette, IN). Gamma-butyrolactone (GBL), 3-hydroxybenzylhydrazine-HCl (NSD-1015), and apomorphine were purchased from Sigma Chemical Company. Haloperidol was purchased from McNeil Company (Ft. Washington, PA), L-dihydroxyphenylalanine (L-DOPA) from Nutritional Biochemical Corp. (Cleveland, OH) and carbidopa from Merck, Sharp & Dohme (West Point, PA). 3H-Spiperone (31.3 Ci/mmol) was from New England Nuclear (Boston, MA). Spiperone was a gift from the Janssen Pharmaceutic Co., Inc. (New Brunswick, NJ). Animals 170-200 g at water. Rats combinations according to
and Drug Administration: Male Sprague-Dawley rats weighing the beginning of the study were given free access to food and were divided into five drug treatment groups and given various of chronic saline, haloperidol and L-DOPA plus carbidopa the schedule shown in Table 1.
In Vivo Study of Presynaptic DA Receptors: Sensitivity of the presy=DA receptors was studied as described by Walters and Roth (23). In brief, the administration of GBL blocks the nerve impulse flow of the dopaminergic neurons, following which the accumulation of DA is enhanced. This accumulation of DA can be prevented by inhibition of tyrosine hydroxylase which is accomplished by treatment with apomorphine (24). Since this inhibitor effect of apomorphine occurs in the absence of nerve impulse, it is considered to be mediated via stimulation of presynaptic DA receptors. The responsiveness of the presynaptic receptors to apomorphine can thus be estimated by its effect on the accumulation of DOPA following the administration of GBL in the presence of a DOPA decarboxylase inhibitor. At the end of the treatment schedule (Table 1) rats were injected intraperitoneally (i.p.) with apomorphine (0.5 mg*kg-1), 5 minutes laterwith GBL (750 mg*kg-1 i.p.) and 5 minutes after GBL injection with the DOPA decarboxylase inhibitor NSD-1015 (100 mg*kg'1 i.p.). Group A rats were injected with NSD-1015 only and group B rats were injected with GBL plus NSD-1015, but they did not receive apomorphine injection. All rats were sacrificed by decapitation 30 minutes after the NSD-1015 injection and the brain was rapidly removed. Each brain was split longitudinally. One half was used for DOPA assay and the other half for receptor binding studies. The olfactory tuberclee and nucleus accumbens (medial part) were dissected by exposing the anterior commissure in its entire course. A cut was made from the anterior commissure to the lateral border of the olfactory tubercle. Thus, a brain part was obtained located rostrally and medially to the anterior coma&sure, rostrally to the optic chiasm and including the olfactory tubercles. Thereafter, the corpora striata were removed (25). For
Vol. 31, No. 1, 1982
Chronic DOPA and Dopamine Supersensitivity
39
the sake of simplicity we will refer in the text to these brain regions as "accumbens" and "etriatum". Tissues were dissected on an ice-cold glass plate and immediately frozen on dry ice. Assay for DOPA: Frozen tissue was weighed and sonicated (Kontes MicroUltrasonic cell disrupter) in approximately 20 volumes of ice-cold 0.1 M perchloric acid containing 2 mM Na2EDTA and 10 ul/ml of NaHS03 (0.1 M). After centrifugation (15,600 x g for 15 min) 180 ul supernatant was taken for DOPA extraction (26). 3,4_Dihydroxybenzylamine (DHBA, 25 up) was added as internal standard to quantitate recovery. For the separation and quantification of the catechols, extracts were subjected to reverse phase (Cl6, ODS column, DuPont) high performance liquid chromatography and eluted with 0.1 M monochloroacetate buffer (pH 3.05) containing 1 mM Na2EDTA and 70 The flow rate mg/L of sodium octyl-sulfate as an ion pairing agent (27). was 1.0 ml/min at ambient temperature. The catechols were quantitated using an LC-4A glassy carbon electrode (Bioanalytical Systems) with an applied potential of 0.7 V. Typical retention times were 5.2 min for DOPA and 6.37 min for DHBA 7.5 min for 3,4_dihydroxyphenylacetic acid (DOPAC) and 10.1 min for dopamine. A Spectra-Physics graphic integrator was used for calculations (27). Table l.Drug Treatment Schedule Treatment Group
n
Phase 1 (28 days)
Phase 2 (10 days)
A B c D
5 5 7
no drug no drug saline B.C. saline B.C.
E
8
no drug no no drug no saline p.0. no DOPA/carbidopa no (200/2Omg*kg-1 p.o.) saline p.0. no
F
7
5
Haloperidol (2 mgakg-1s.c.) Haloperidol (2 mg.kg'1s.c.)
Phase 3 (3 days) drug drug drug drug drug
DOPA/carbidopa no drug (200/20 mgakg'1 p.o.)
Legend to Table 1: L-DOPA/carbidopa was given in two divided oral (p.0.) doses as a suspension in saline which was made fresh daily. Haloperidol was used in the form of HaldolH injectable solution (McNeil Lab, Ft. Washington, PA) and was diluted with 0.9% NaCl before subcutaneous (8.c.) injection. 3H-Spiperone binding were performed by a modification of the method described by Seeman et al. (28). Fresh striata were homogenized by Polytron (Brinkman Instruments, Westbury, NY) in approximately 1:lOO w/v ice-cold Tris-EC1 buffer (50 mM, pEl 7.5). To obtain a crude membrane preparation, homogenates were centrifuged (40,000 x g for 10 minutes) and the resulting pellet was washed twice by resuspension and recentrifugation to remove any residual apomorphine. The final pellet was resuspended in 1:150 w/v of ice-cold 'Iris-EC1 (15 mM)-Na2EDTA (5 mM) buffer (pH 7.4) containing 0.01% ascorbate, and preincubated for 10 minutes at 370C. Aliquots of tissue were combined with various concentrations of 3H-spiperone and drugs to a final volume of 1 ml. Protein concentrations as estimated by Lowry assay (29) was approximately 250-400 ng/ml. Incubations were for 12 minutes at 370 C. Bound 3H-spiperone was separated by vacuum filtration over glass fiber filters (Whatman GF/B) followed by washing with 10 ml of the incubation buffer. Filters were placed on Aquasol (New England Nuclear) and counted using liquid scintillation spectrometry. Specific binding was defined as the difference in total binding in the presence and absence of 10 uM unlabelled spiperone. Membranes from each rat were assayed at a single concentration of
40
Chronic
%I-spiperone (0.3 Scatchard analysis
DOPA and Dopamine Supersensitivity
nM) .
(30).
Vol.
31, No.
Membranes from each group were then pooled
Results are expressed as mean f S.B.M. data was determined by two-tailed Student’s
Statistical t-test.
analysis
1, 1982
for of
the
Results In the presence of NSD-1015, DOPA accumulates in striatum and accumbens GBL injected to naive rats 35 minutes before the animals were (Figure 1A). sacrificed increases DOPA concentration in the presence of the DOPA decarboxylase inhibitor NSD-1015 in both the etriatum and accumbene (Figure 1B). This increase was significantly inhibited by apomorphine given prior to GBL injection (Figure lC>. The oral administration of high doses of L-DOPA/carbidopa for 10 days did not change the responsiveness of the presynaptic dopaminergic receptors to apomorphine compared with saline injected controls (Figure 1D). In contrast , pretreatment with haloperidol (2 mg*kg-1 i.p.> for 28 days induced an enhanced response of the presynaptic dopaminergic receptors to apomorphine. The accumulation of DOPA was thus significantly reduced compared to the control rats (Group C) both in the striatum (~(0.005) and accumbens (pCO.005) (Figure 1E). The administration of L-DOPA/carbidopa, for 10 days following the chronic injection of 200/20 mg*kg’l daily, haloperidol did not reverse the enhanced response to apomorphine induced by the neuroleptic. DOPA levels in the latter group (Group F) were not significantly different from those found in rats treated with haloperidol only and were still significantly below control levels in the striatum (p
vol. 31, No. 1, 1982
Chronic DOPA and Dopamine Supersensitivity
ACCUMBENS
S TRIA TUM
APO GEL NSD-1015
--++tt -+++++ t + +
+
+
41
--t++t -+++++ +++++t
+
Figure 1. The Effect of Chronic Treatment With Raloperidol and L-DOPA on Apomorphine-Induced Depression of DOPA Accumulation After GBL Treatment
Rats were given no drugs (A, B), saline only (C), saline followed by L-DOPA/ carbidopa (D), haloperidol followed by saline (E) or haloperidol followed by L-DOPA/carbidopa (F) as described in Table 1. At the end of the treatment schedule the effects of these drugs on the presynaptic dopaminergic receptors was measured as described in Methods. Rats were challenged with either NSD-1015 alone, gamma butyrolactone (GBL) plus NSD-1015, or with apomorphine (APO) plus GBL plus NSD-1015 as outlined above. Groups E and F were significantly different from control rats (Group C> by *p
Table 2.
Treatment Group
n
c D E F
7 5 8 7
Specific 3Wspiperone Binding (fmols/mg protein) Group mean f S.E.M. Bmax 324 363 385 304
f f f f
22 29 ll* 14**
520 581 733 578
Kd(nM) 0.2 0.1 0.1 0.2
Group means represent specific binding at 0.3 nM. 3H-spiperone was averaged from individual animals. Scatchard analysis was conducted from 0.8 to 0.5 nM 3Wspiperone on pooled membranes from animals in each group. *, significantly different from group C by p
42
Chronic
DOPA and Dopamine Supersensitivity
Vol.
31, No.
1, 1982
Discussion The experiments reported here provide evidence that prolonged exposure to haloperidol produced supersensitivity in both presynaptic and postsynaptic dopaminergic receptors. This phenomenon in presynaptic receptors was described earlier by Nowycky and Roth (21) and was recently confirmed also by electrophysiological evidence (31). Supersensitivity of the presynaptic receptors is expressed in our experiments in the enhanced effectiveness of apomorphine to depress the elevated DOPA accumulation which occur in the dopaminergic nerve terminals following impulse flow inhibition by GSL. The neuroleptic-induced change in presynaptic receptor function was detected both in the nigrostriatal and mesolimbic pathways of rat brain (Figure 1). Tardive dyskinesia could be viewed, therefore, as a state of combined pre- and postsynaptic DA receptor supersensitivity. It is worth discussing whether any therapeutic approach should include both types of receptors in consideration. Supersensitivity of the presynaptic receptors may be compensatorally useful in tardive dyskinesia as a means by which synaptic release of DA can be minimized. It may be best therefore, to attempt to “down-regulate” only the postsynaptic DA receptor and leave unaltered the supersensitivity of the presynaptic receptors. Several animal studies have demonstrated that chronic treatment with dopaminergic agonists may down-regulate dopaminergic receptors from an established supersensitive state. These reports include behavioral studies (17, 18, 32, 33), 3H-neuroleptic binding (15, 16), and adenylate cyclase activity measurements (15). That such regulation in 3H-neuroleptic binding sites may also exist in humans has been shown by Lee et al. Since (34). 3Wneuroleptics bind preferentially to postsynaptic receptors (35) and adenylate cyclase is also thought to be coupled to postsynaptic receptors (36, 37), it is possible that these studies reflect the down-regulation that while changes in presynaptic takes place mainly in postsynaptic receptors, receptors are less understood. The data presented in this study suggest that prolonged administration of L-DOPA did not reverse the supersensitivity of the presynaptic dopaminergic receptors induced by chronic pretreatment with haloperidol. The responsiveness of the presynaptic receptors to apomorphine in the haloperidol plus L-DOPA/carbidopa treated rats was similar to that in the haloperidol treated rats and both groups had an enhanced response to the dopaminergic agonist compared with the control group (Figure 1). However, chronic L-DOPA treatment had a significant effect on postsynaptic receptors as measured by 3H-spiperone binding studies. Pretreatment of rats with haloperidol produced an expected increase in the specific binding of 3H-epiperone (7), and subsequent treatment of haloperidol-treated rats with L-DOPA/carbidopa produced an apparent reversal of this effect (Table 2) in agreement with previous reports (15, 16). The data presented here suggest that chronic dopaminergic agonism may have a differential effect on the two types of dopaminergic receptors. Such an effect was found by List and Seeman (16) who reported a significant increase in both 3H-epiperone and 3H-apomorphine binding after chronic Subsequent prolonged treatment with bromocriptine exposure to haloperidol. binding but not the increase in reversed only the increase in 3H-spiperone 3H-apomorphine binding which is thought to label preferentially presynaptic receptors (35). It is possible that supersensitive presynaptic receptors are more resistant to chronic agonist treatment than the postsynaptic receptors. Of special
interest
is
the finding
that
L-DOPA/carbidopa
treatment
alone
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Chronic DOPA and Dopamine Supersensitivity
43
did not down-regulate 3H-spiperone binding. In fact, binding appeared to be slightly elevated in L-DOPA/carbidopa treated rats (Table 2). Inspection of our data suggested that the high values in this group may have reflected unusually low protein levels. It has been shown that the acute administration of L-DOPA induced disaggregation of brain polysomes (38) and transient inhibition of protein synthesis (39). Such an apparent increase in 3H-spiperone binding after chronic L-DOPA treatment was reported earlier by Wilner et al. (40) although the specific question of a possible effect of L-DOPA on protein values in this study was not mentioned. We are currently investigating a cumulative decrease in protein synthesis during chronic L-DOPA treatment. Acknowledgements This work was supported in part by NIH grant NSl5959-02. Dr. Reches is a recipient of a Peggy Engl Fellowship from the Parkinson's Disease Foundation and of a Fogarty Public Health Service International Research Fellowship (NIH TW02884). Dr. Jiang is a recipient of an H. Houston Merritt Fellowship from the Parkinson's Disease Foundation and a fellowship from the People's Republic of China. References 1. 2. 3. 4. 5. 6. 7. 8.
?i. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
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