And D2-Dopaminergic Receptors in the Primate Cerebral Cortex

Home , Remoxipride

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4353-4356, May 1994 Neurobiology A common action of clozapine, haloperidol, and remoxipride on D1- and D2-dopaminergic receptors in the primate cerebral cortex (neuroleptic drugs/schizophrenia/asociatlon cortex/receptor autoradiography/receptor regulation) MICHAEL S. LIDOW AND PATRICIA S. GOLDMAN-RAKIC Section of Neurobiology, Yale University School of Medicine, New Haven, CT 06510 Contributed by Patricia S. Goldman-Rakic, January 24, 1994

ABSTRACT The potencies of the major neuroleptics used for 6 months to approximate the maintenance of medication in the treatment of schizophrenia, including haloperidol and in clinical practice (14). A control group (group CO) received remoxipride, correlate with their ability to bind D2-dopa- only fruit treats. Five days after final administration ofdrugs, minergic receptors in subcortical structures. On the other the animals were sacrificed, and cortical dopamine receptors hand, the neuroleptic clozapine has a low affinity for these sites, were assayed by quantitative receptor autoradiography. The and the pharmacological basis of its beneficial action is less cortical regions examined are shown in Fig. 1A. They include clear. We have found that chronic treatment with clozapine, prefrontal association [cytoarchitectonic areas 46 and 9 of haloperidol, and remoxipride up-regulates D2 receptors in Walker (15)], temporal association [area 21 of Brodmann specific cortical areas of the rhesus monkey frontal, parietal, (16)], primary motor [area 4 of Brodmann (16)], somatosen- temporal, and occipital lobes. Of particular interest, all three sory [areas 1, 2, and 3 of Brodmann (16)], and primary visual neuroleptics down-regulated D1 receptors in prefrontal and [area 17 of Brodmann (16)] cortex. temporal association regions-the two areas most often asso- Dj-dopaminergic sites were labeled with the antagonist ciated with schizophrenia. This latter finding raises the possi- [3H]SCH23390 (17, 18). An example of [3H]SCH23390 bind- bility that down-regulation of D1 receptors in prefrontal and ing in the prefrontal cortex is shown in Fig. 1B. Tissue temporal cortex may be an important component of the ther- sections were first preincubated for 20 min at room temper- apeutic response to neuroleptic drugs. Further, the common ature in 50 mM Tris'HCl buffer, pH 7.4. They were then effects of three neuroleptics with different pharmacological incubated with 0.1-10 nM [3H]SCH23390 for 90 min at room profiles in the cerebral cortex is consistent with the idea that temperature in 50 mM Tris HCl buffer, pH 7.4, containing 120 this structure is a major therapeutic target in the treatment of mM NaCl, 5 mM KC1, 2 mM CaCl2, 1 mM MgCl2, and 1 pM schizophrenia. mianserin. The latter was added to block binding to 5-HT2, 5-HTic, and a2 sites. After incubation, the tissue was rinsed twice (10 min each) in ice-cold 50 mM Tris HCl buffer, pH The therapeutic effect of neuroleptics has generally been 7.4. Nonspecific binding was determined in the presence of associated with their binding to the D2 class of dopaminergic 1 jmM cis-flupentixol. receptors in the brain (1). However, the beneficial actions of D2-dopaminergic receptors, illustrated in the prefrontal clozapine have not been satisfactorily explained, given that cortex (Fig. 1C), were labeled with the antagonist [1251]epi- this drug does not interact with D2 sites in most regions depride (18). Sections were preincubated as described for examined (2, 3). Numerous previous studies of the effects of [3H]SCH23390 binding and then incubated for 45 min at room prolonged neuroleptic treatment on dopaminergic receptors temperature with 0.1-3.0 nM [1251]-epidepride in 50 mM have focused on subcortical structures (3-8), while the Tris HCl buffer, pH 7.4, containing 120 mM NaCl, 5 mM regulation of cerebral cortical dopaminergic sites by similar KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid, and 0.2 treatment has received little attention. Such data are partic- ,uM idazoxan to prevent labeling of a2 sites. After incuba- ularly important in view of the mounting evidence for the tion, the sections were rinsed as described for [3H]SCH23390 involvement of the cortical dopaminergic system in schizo- binding. Nonspecific labeling was determined in the presence phrenia (for reviews see refs. 9 and 10). To gain further insight of 10 AuM (+)-butaclamol. into the mechanism of action of clozapine, we compared its At the end of the labeling assays, all tissue sections were effect on dopamine receptors in the cerebral cortex ofrhesus dipped in distilled water and apposed to 3H-sensitive Ultro- monkeys with those of two representative neuroleptics, film (Amersham) for 6 weeks ([3H]SCH23390) or 1 week haloperidol and remoxipride. Both of these drugs display a ([125I]epidepride). After development for autoradiography, high affinity for the D2-dopaminergic receptor sites in the the tissue sections were stained with cresyl violet to allow neostriatum and nucleus accumbens, though remoxipride is analysis of the cytoarchitecture. For all animals, binding more selective (11). assays were repeated twice. At least five tissue sections were processed for every concentration of ligand, three for total AND binding and two for nonspecific binding. MATERIALS METHODS The autoradiograms were analyzed with a BDS computer Sixteen rhesus monkeys (Macaca mulatta), 5-7 years ofage, system (Biological Detection Systems, Pittsburgh; ref. 17) and were divided into four groups ofequal size. Group H received the statistical analysis of saturation binding was performed haloperidol (0.2 mg/kg per day), group C1 received clozapine with the nonlinear curve-fitting computer programs KINETIC/ (5.2 mg/kg per day), and group R received remoxipride (3.7 EDBA/LIGAND/LOWRY from Elsevier-Biosoft (Cambridge, mg/kg per day). These doses fall within the recommended U.K.). The analysis is based on specific radiolabeling in tissue range for therapeutic effects in schizophrenic patients (12, exposed to seven different concentrations of radioactive li- 13). The drugs were given orally (in fruit treats) twice a day gand in incubating solutions. This number of data points is sufficient to allow a relatively accurate estimation of B.. The publication costs of this article were defrayed in part by page charge (maximal binding) and Kd (steady-state dissociation constant) payment. This article must therefore be hereby marked "advertisement" values for a one-site receptor model (17). Examples of satu- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ration and Scatchard plots of [3H]SCH23390 and [125I]_ 4353 Downloaded by guest on September 26, 2021 4354 Neurobiology: Lidow and Goldman-Rakic Proc. Natl. Acad Sci. USA 91 (1994) A 0 total nonspecifc A X0 48 °

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Bound (fmol/mg prot.) 0 3 6 9 I3HISCH23390 free(nM)

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FIG. 1. (A) Diagram of the lateral surface of the rhesus monkey 0 00 cerebral cortex. Different patterns indicate the cortical areas exam-

ined in the present study. The plane of section for the slices shown w0 0.9 1.8 2.7 in B and C is indicated by the line. (B) Color-coded autoradiogram %. s~~~~~~ound (final/mg prot.) showing the laminar distribution of [3H]SCH23390 binding in the Ur presence of mianserin (Dj-dopaminergic receptors) in a coronal section from the prefrontal cortex of an untreated rhesus monkey. o~~~.2 0.4 (C) Color-coded autoradiogram of [125I]epidepride labeling in the [1251epidepride free (nM) presence of idazoxan (D2-dopaminergic sites) in cortical section similar to that shown in B. Note that the [3H]SCH23390 binding is densest in layers I, II, MIla, V, and VI, while [125I]epidepride binding FIG. 2. Examples of saturation binding of [3H]SCH23390 (A) and is prominent in layer V. Red coloring indicates the highest density of [125I]epidepride (B) in the area 46 of neuroleptic naive monkey. In binding; black indicates the lowest density of binding. PS, principal each assay, consecutive brain sections were labeled with seven sulcus. concentrations of the radioligand as described in the text. Sections were then apposed to 3H-sensitive Ultrofilm and autoradiograms were generated. Nonspecific labeling for [3H]SCH23390 was defined epidepride are presented in Fig. 2. values for control and Bm.. in the presence of 1 ,uM cis-flupentixol and for [125I]epidepride in the groups were with two-tailed each of the treated compared presence of 10 pM (+)-butaclamol. For each concentration of the Student t tests. The effects of neuroleptic treatments on Kd radioligand, the total binding was obtained from three consecutive values were evaluated with a one-way ANOVA. brain sections, with the fourth and fifth sections providing nonspecific binding. Insets represent the corresponding Scatchard plots. These plots yielded Kd = 0.14 nM andB. = 38.6 fmol/mg ofprotein RESULTS AND DISCUSSION for [3H]SCH23390 and Kd = 0.029 nM and Bm..c = 2.94 fmol/mg of Saturation analysis of[1251]epidepride binding showed that all protein for [125I]epidepride. three neuroleptics produced statistically significant increases density were accompanied by changes in the affinity for (47-52%) in the density of D2-dopaminergic receptor sites in [1251]epidepride (Table 1). Our findings with haloperidol and all layers of the temporal association, primary motor, sOma- remoxipride are in line with the widely reported D2 up- tosensory, and primary visual cortical regions (Fig. 3A). D2 regulation by these drugs in subcortical structures (3-7). receptor sites were also increased in the prefrontal cortex but However, the up-regulation of cortical D2 receptors by by a smaller percentage (11-18%) which did not reach sta- clozapine in widespread areas of the cerebral cortex differs tistical significance (Fig. 3A). None ofthe changes in receptor from the apparent lack of such effect of clozapine on these Table 1. Changes in apparent affinity values for binding of radioligands in the cortex of monkeys chronically treated with neuroleptics Affinity, -log Kd P by one-way Radioligand Cortical area Control Haloperidol Clozepine Remoxipride ANOVA [1251]Epidepride Prefrontal 10.17 + 0.62 10.12 + 0.43 10.05 + 0.67 10.15 ± 0.83 <0.05 Temporal 10.21 ± 0.43 10.08 + 0.99 9.91 ± 0.97 10.32 ± 0.76 <0.05 Motor 10.33 ± 1.01 9.97 ± 0.96 10.02 ± 0.60 10.37 ± 0.83 <0.05 Somatosensory 10.25 ± 0.31 9.86 + 0.81 9.77 ± 0.99 10.17 ± 0.69 <0.05 Visual 10.18 + 0.54 10.06 ± 1.11 9.86 ± 0.88 9.99 ± 0.71 <0.05 [3H]SCH23390 Prefrontal 9.31 ± 0.75 9.97 ± 0.82 9.78 ± 0.93 8.03 ± 1.20 <0.05 Temporal 9.86 + 0.88 8.13 ± 1.13 9.88 ± 1.00 9.74 ± 0.78 <0.05 Motor 9.93 ± 0.89 9.85 ± 0.81 9.99 ± 0.70 8.05 ± 0.97 <0.05 Somatosensory 8.02 ± 1.10 8.05 ± 0.96 9.85 ± 0.62 9.91 ± 1.11 <0.05 Visual 9.65 ± 0.77 8.12 ± 1.10 9.51 ± 0.83 9.33 ± 1.26 <0.05 Since apparent affinities of the ligands have log-normal distributions, the data are presented and analyzed as -log Kd. Downloaded by guest on September 26, 2021 Neurobiology: Lidow and Goldman-Rakic Proc. Natl. Acad. Sci. USA 91 (1994) 4355

[ negligible due to its low affinity for these sites (21). Indeed, A 12251]EpIdepride (D2 receptors) our preliminary binding studies in cell cultures expressing D4-dopaminergic receptors indicate that under the conditions ofthe present experiment, [125I]epidepride may label D4 sites (M.S.L., unpublished results). However, it remains to be 0) determined whether the general increase in the density ofthe 0._0 D2 sites in cortical areas described here is related in any way to the unique composition of D2 dopaminergic receptor subtypes in the cortex. A major finding that could not have been predicted is that E - all three neuroleptics produced decreases in the density of m-- D1-dopaminergic receptor sites. These reductions were substantial (30-34%) and were found exclusively in the prefrontal and temporal association cortices (Fig. 3B) and affected all layers of these cortical areas (Fig. 4). No significant effects were observed in the somatosensory, visual, or motor areas Prefrontal Temporal Motor Somato- Visual cortex cortex cortex sensory cortex (Fig. 3B), nor were alterations in the affinity of D1 receptors (area46) (area2l) (area4) cortex (area 17) to [3H]SCH23390 observed in any of the cortical areas (area 1) studied (Table 1). It is unlikely that the down-regulation of B these receptors results from competitive binding by residual [ 3H]SCH23390 (Dl receptors) neuroleptics, as the animals were not sacrificed until 5 days after the last administration of drug. Indeed, gas chromato- graphic detection analysis of haloperidol and clozapine in cortical tissue, conducted for us by National Medical Serv- 0)0 ices (Willow Grove, PA), failed to detect concentrations of these neuroleptics in the prefrontal and temporal cortices of -6- treated animals above 0.01 nmol/g of tissue, values not E sufficient to interfere with [3H]SCH23390 binding (11). More- 0 over, any significant degree of competition with residual E neuroleptics would have affected the Kd values recorded for x [3H]SCH23390 binding in the tissue of the treated animals. m This, however, was not the case (Table 1). The mechanisms for decrease in the density of cortical D1-dopaminergic receptor sites by chronic neuroleptic treat- Prefrontal Temporal Motor Somato- Visual ment are now open for investigation. For example, down- cortex cortex cortex sensory cortex regulation of D1 sites may be a compensatory reaction to a (area 46) (area 21) (area 4) cortex (area 1 7) chronic increase in cortical dopamine release resulting from (area 1) the blockade of D2 pre- and postsynaptic sites by neuroleptic drugs (23, 24). It should be mentioned, however, that the FIG. 3. Bar graphs representing changes in the density of D2- specific [1251]epidepride (A) and Di-specific [3H]SCH23390 (B) binding in response to chronic haloperidol, clozapine, and remoxipride Control 0 Clozapine treatment in different cortical regions ofrhesus monkey. The data for 03 prefrontal areas 9 and 12 were similar to those for area 46, and the data for somatosensory areas 2 and 3 were similar to those for area 50 1. The B.,, value for each column is an average of four animals. c Error bars are ± SEM. Statistically significant differences between 40 control and treated groups (two-tailed Student t test; P < 0.05) are 0. marked by asterisks. Note that all three neuroleptics up-regulate the O 40 [125I]epidepride binding in most cortical areas. PH]SCH23390 binding is down-regulated only in the prefrontal and temporal regions. 30 sites in striatum and the nucleus accumbens (3-5). An E 20 increase in the density of D2 receptors in response to clozapine treatment has also been observed in the medial pre- 210 frontal cortex ofthe rat (19). It is ofinterest that the prefrontal m cortex was the only area in our study where the D2 receptor was least affected by neuroleptics, including clozapine, per- 0 haps indicating a difference between primates and rodents. I II IIla mb IV V VI The similarity in the effects of clozapine, haloperidol, and Layers remoxipride on cortical D2-dopaminergic receptors may be related to the unique pharmacological and electrophysiolog- FIG. 4. Bar graph representing changes in the density of D1- ical properties of the dopamine neurons that project to the specific [3H]SCH23390 binding in response to chronic clozapine cerebral cortex as compared with those of the nigrostriatal treatment in individual layers of area 46 in the prefrontal cortex. In and mesolimbic dopamine systems (20). Also, the cortex has all cases, B, values are averaged for four animals. Error bars are a substantial proportion ofthe D4 subtype ofthe family of D2 + SEM. Receptor densities significantly different from control values (two-tailed Student t test; P < 0.05) are marked by asterisks. receptors (21), whereas the striatum and nucleus accumbens Note that clozapine significantly down-regulates [3H]SCH23390 contain mainly D2A sites (22). Clozapine has a high affinity for binding in all layers of the prefrontal cortex. Haloperidol and the D4 receptor subtype (21) and could easily up-regulate it, remoxipride have similar effects on [3H]SCH23390 receptors in all while the regulation of D2A receptors by clozapine would be layers of the same cortical area. Downloaded by guest on September 26, 2021 4356 Neurobiology: Lidow and Goldman-Rakic Proc. Natl. Acad. Sci. USA 91 (1994) effect of chronic neuroleptic treatment on cortical dopami- 6. Memo, M., Pizzi, M., Missale, C., Carruba, M. 0. & Spano, nergic release after treatments as prolonged as that used in P. F. (1987) Neuropharmacology 26, 477-480. the present study is not presently known. It is possible, 7. Hall, H. & Sallemark, M. (1987) Pharmacol. Toxicol. 60, 359-363. therefore, that very prolonged exposure to neuroleptics may 8. Laruelle, M., Jaskiw, G. E., Lipska, B. K., Kolachana, J. E. produce depolarization block of the cortical dopaminergic & Weinberger, D. R. (1992) Brain Res. 575, 47-56. innervation (25). Depolarization block can also down- 9. Weinberger, D. R. (1987) Arch. Gen. Psychiatr. 44, 660-669. regulate D1 receptors (26), although this down-regulation 10. Davis, K. L., Kahn, R. S., Grank, K. 0. & Davidson, M. would be related to the elimination of phasic dopamine (1991) Am. J. Psychiatr. 148, 1474-1486. 11. Seeman, P. (1990) Acta Psychiatr. Scand. 82 (Suppl. 358), release (27). Alternatively, the decrease in D, receptor den- 14-20. sity may be secondary to a neuroleptic-induced increase in 12. Pflug, B., Bartels, M., Baner, H., Bunse, J., Gallhofer, B., the level ofresponsiveness ofD2-mediated adenylate cyclase Haas, S., Konzow, W. T. H., Lkieser, E., Kufferle, B., Stein, to dopamine (6). As a strong interaction between D1 and D2 D., Steinberger, K. A. & Weiselmann, G. (1990) Acta Psychi- second messenger systems is well established (28, 29), it is atr. Scand. 82 (Suppl. 358), 142-146. conceivable that an increase in the sensitivity of D2- 13. Dowd, A. L., ed. (1993) Physicians Desk Reference (Medical Economics, Montvall, NJ), 47th Ed. associated adenylate cyclase would be accompanied by an 14. Hyman, S. E. & Arana, G. W. (1987) Handbook ofPsychiatric increase in the sensitivity of the D1-coupled enzyme which, Drug Therapy (Little-Brown, Boston). in turn, would downregulate D1 receptors (8, 30). 15. Walker, A. E. (1940) J. Comp. Neurol. 73, 59-86. Our findings that haloperidol, remoxipride, and clozapine 16. Brodmann, K. (1905) J. Psychol. Neurol. (Leipzig) 9, 177-226. have virtually identical effects in all cortical areas examined 17. Lidow, M. S., Goldman-Rakic, P. S., Gallager, D. W. & may provide the missing link in understanding the therapeutic Rakic, P. (1991) Neuroscience 40, 657-671. 18. Lidow, M. S. (1993) in Receptor Autoradiography: Principles actions of these very different neuroleptic drugs. It seems and Practice, eds. Wharton, J. & Polak, J. M. (Oxford Univ. particularly relevant that the D1 down-regulation was found Press, Oxford, U.K.), pp. 217-236. selectively in the prefrontal and temporal cortical regions 19. Janowsky, A., Neve, K. A., Kinzie, J. M., Taylor, B., De- which, in rhesus monkeys, have especially high concentra- Paulis, T. & Belknar, J. K. (1992) J. Pharmacol. Exp. Ther. tions of dopamine receptors among cortical areas studied 261, 1282-1290. (31). In humans, these areas are also critically involved in 20. Bannon, M. J. & Roth, R. H. (1983) Pharmacol. Rev. 35, 53-67. cognitive and affective functions (for review see refs. 32 and 21. VanTol, H. H. M., Bunzow, J. R., Guan, H. C., Sunahara, 33) and are the areas most often compromised in schizophre- R. K., Seeman, P., Niznik, H. B. & Civelli, 0. (1991) Nature nia (9, 34, 35). All ofthese considerations suggest that the D, (London) 350, 610-614. receptors in these regions may be one of the major sites to 22. Meador-Woodruff, J. H., Mansour, A., Healy, D. J., Kuehn, which neuroleptic treatment is and/or should be directed. R., Zhou, Q. Y., Bunzow, J. R., Akil, H., Civelli, 0. & Watson, S. J. (1991) Neuropsychopharmacology 5, 231-242. Note Added in Proof. Since this paper was submitted for publication, 23. Scatton, B., Glowinski, J. & Julou, L. (1987) Brain Res. 109, an article by et al. came to our attention. It shows that 184-189. Malmberg (36) 24. Bacopoulos, N. C., Spokes, E. G., Bird, E. & Roth, R. H. clozapine binds to the short isoform ofthe D2A-dopaminergic recep- (1979) Science 205, 1405-1407. tor with an affinity comparable to that for D4 receptors. It has also 25. Essig, E. C. & Kilpatrick, I. C. (1991) Psychopharmacology been shown (37) that while the majority of the striatal D2A receptors 104, 194-200. belong to the long type, the short isoform is predominant among D2A 26. Gerfen, C. R., Engler, T. M., Mahan, L. C., Susel, Z., Chase, receptors in the cerebral cortex (particularly in those areas where we T. N., Monsma, F. J. & Sibley, D. R. (1990) Science 250, detected neuroleptic-induced increase in D2A receptors). Thus, the 1429-1431. clozapine-induced up-regulation of D2 sites, detected in the present 27. Grace, A. A. (1991) Neuroscience 41, 1-24. study, may reflect an interaction of this drug with the D4 receptor, 28. Seeman, P., Niznik, H. B., Guan, H. C., Booth, G. & Ulpian, the short isoform of the D2A receptor, or both. C. (1989) Proc. Natl. Acad. Sci. USA 86, 10156-10160. 29. Strange, P. G. (1991) Trends Pharmacol. Sci. 12, 48-49. This work was supported by National Institute of Mental Health 30. Creese, I. & Hess, E. S. (1986) Clin. Neuropharmacol. 9 Center Grant P50-MH44866-O5. Haloperidol was provided by Mc- (Suppl. 4), 14-16. Neil Pharmaceutical (Spring House, PA), clozapine was provided by 31. Lidow, M. S., Goldman-Rakic, P. S., Rakic, P. & Innis, R. B. Sandoz Pharmaceutical (East Hanover, NJ), and remoxipride was (1989) Proc. Natl. Acad. Sci. USA 86, 6412-6416. received from Astra Pharmaceutical (Sodertalje, Sweden). 32. Fuster, J. M. (1980) The Prefrontal Cortex (Raven, New York). 33. Goldman-Rakic, P. S. (1987) in Handbook ofPhysiology, ed. 1. Creese, I., Burt, D. R. & Snyder, S. H. (1976) Science 192, Plum, F. (Am. Physiol. Soc., Bethesda, MD), Section 1, Vol. 481-483. 5, pp. 373-417. 2. Seeman, P., Chau-Wong, M., Tedesco, J. & Wong, K. (1975) 34. Weinberger, D. R. (1988) Trends Neurosci. 11, 367-370. 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