And D2-Dopaminergic Receptors in the Primate Cerebral Cortex
Total Page：16
File Type：pdf, Size：1020Kb
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 ° ° 36 N E 24_ 12 18 Bound (fmol/mg prot.) 0 3 6 9 I3HISCH23390 free(nM) B 3.2 ° §.z 2.4- 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 nonspe- cific 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.