Dopamine D2 Receptors in the Cerebral Cortex

Home , Idazoxan, Raclopride

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6412-6416, August 1989 Neurobiology Dopamine D2 receptors in the cerebral cortex: Distribution and pharmacological characterization with [3Hlraclopride (raclopride binding/neostriatum/rat/monkey) MICHAEL S. LIDOW*t, PATRICIA S. GOLDMAN-RAKIC*, PASKO RAKIC*, AND ROBERT B. INNIS*f Section of *Neuroanatomy and tDepartment of Psychiatry, Yale University, School of Medicine, New Haven, CT 06510 Contributed by Pasko Rakic, May 17, 1989

ABSTRACT An apparent involvement of dopamine in the MATERIALS AND METHODS regulation of cognitive functions and the recognition of a widespread dopaminergic innervation of the cortex have fo- Tissue was obtained from three adult rhesus monkeys cused attention on the identity of cortical dopamine receptors. (Macaca mulatta): one male and two females. The animals However, only the presence and distribution of dopamine DI were anesthesized with sodium pentobarbital (40 mg/kg) and receptors in the cortex have been well documented. Compa- perfused with ice-cold phosphate-buffered saline followed by rable information on cortical D2 sites is lacking. We report here 0.1% paraformaldehyde (9; 15). The cerebral cortex and the results of binding studies in the cortex and neostriatum of neostriatum were rapidly removed and immersed in isopen- rat and monkey using the D2 selective antagonist [3H]raclo- tane at -700C for 5 min before storing at -800C until use. For pride. In both structures [3H]raclopride bound in a sodium- competition studies tissue was pooled from several neocor- dependent and saturable manner to a single population of sites tical areas. However, saturation studies were conducted on with pharmacological profiles of dopamine D2 receptors. D2 different cortical areas separately. Rodent brains were ob- sites were present in all regions of the cortex, although their tained from male Sprague-Dawley rats (200-400 g; Charles density was much lower than in the neostriatum. The density River Breeding Laboratories). Animals were decapitated and of these sites in both monkey and, to a lesser extent, rat cortex the cerebral cortex and neostriatum were removed for im- displayed a rostral-caudal gradient with highest concentra- mediate assay. All competition studies using rat cortex came tions in the prefrontal and lowest concentrations in the occipital from the prefrontal cortex. Different cortical areas were cortex, corresponding to dopamine levels in these areas. Thus, assayed separately for the saturation analysis. the present study establishes the presence and widespread The tissue was homogenized in ice-cold 50 mM Tris'HCl distribution of dopamine D2 receptors in the cortex. buffer (pH 7.4) with a Brinkman Polytron PT10. Homoge- nates were centrifuged twice at 20,000 x g for 10 min with an intermediate rehomogenization in fresh buffer. The final The dopaminergic innervation ofthe cerebral cortex has been pellets were resuspended in incubation buffer containing 50 well characterized (1-4), and dopamine receptor-mediated mM Tris-HCI (pH 7.4 at 230C), 120 mM NaCl, 5 mM KCI, 2 effects have also been demonstrated in the cortex (5, 6). mM CaCI2, 1 mM MgCl2, and 0.1% ascorbic acid. The effect However, much less is known about the receptors that of Na' was tested in 50 mM Tris HCl buffer (pH 7.4 at 230C) mediate these effects. It has been only recently that the containing 0.1% ascorbic acid to which different concentra- availability of a Dl-specific antagonist, SCH 23390, has made tions of NaCl were added; effects of K+, Ca2+, and Mg2+ it possible to describe the pharmacology and distribution of were tested in 50 mM Tris'HCl buffer (pH 7.4 at 230C) dopamine D1 sites in both rodents (7, 8) and primate cortex containing 120 mM NaCl and 0.1% ascorbic acid to which (9, 10). On the other hand, dopamine D2 sites in the cortex different concentrations ofKCI, CaCl2, or MgCl2 were added. have not been fully documented, and, indeed, their very Two hundred microliters oftissue homogenate was incubated existence in this structure is controversial. Although D2 with 25 ml of [3H]raclopride and 25 ml of displacer or buffer. receptors in the cortex have been reported in several studies The final dilution of cortical tissue was 1:25 and ofneostriatal (7-9, 11-15), an equal number of studies have obtained tissue was 1:125. Standard incubation was for 30 min at 230C. negative results (10, 16-21). Most studies on cortical D2 sites The effect of incubation temperature was studied at 00, 230, have used either [3H]spiperone, which binds multiple recep- and 370C with incubation time ranging from 15 to 120 min. tor sites (16, 22, 23) or [3H]sulpiride, which has very high The incubation was terminated by rapid filtration and three nonspecific binding in the cortex (11). The few studies that washings with 5 ml ofice-cold 50mM Tris HCl buffer (pH 7.4) used [125I]iodosulpiride, a highly selective D2 antagonist, did through Whatman GF/B filters by using a Brandel M-242 cell not perform saturation analyses of the binding, thus leaving harvester. The radioactivity trapped in the filters was mea- density of the receptors unspecified (24-26). sured in a Packard 3320 liquid scintillation counter. Satura- In light ofthe hypothesis that the D2 receptor is a major site tion studies used 0.1-6 nM [3H]raclopride, and displacement of therapeutic action for neuroleptics (27), it is of interest to studies used 1.0 nM [3H]raclopride. Specific binding was obtain quantitative data on D2 binding sites in various regions defined as the difference in binding with and without 1.0 AmM of the cortex as well as to extend this study to the primate. (+)-butaclomol. Labeling of tissue with 1.0 nM [3H]sulpiride Within the past year the availability of a new substituted was conducted as described for [3H]raclopride. Binding with benzamide, [3H]raclopride, another highly selective D2 an- 1.0 nM [3H]spiperone was performed in the presence of 0.3 tagonist (28), has provided the opportunity to reexamine D2 gM ketanserin. The Pierce BCA protein assay was used to binding sites in the cerebral cortex and to compare these sites determine protein concentration in the tissue. Data were in primates and rodents. We also have compared the cortical analyzed by a nonlinear curve-fitting computer program D2 sites with those in the neostriatum. Abbreviation: 5-HT, 5-hydroxytryptamine. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Yale University, payment. This article must therefore be hereby marked "advertisement" School of Medicine, Section of Neuroanatomy, C303 Sterling Hall in accordance with 18 U.S.C. §1734 solely to indicate this fact. of Medicine, 333 Cedar Street, New Haven, CT 06510.

6412 Downloaded by guest on September 26, 2021 Neurobiology: Lidow et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6413

Z. 200 o200 150 10 3 - 100 0 100- in0 s arsEi0 a Eff100 o7 Naoo ec Cdof a+ and Cdg C C 0 3 E 0I.2. Efc f &,CIaCz +adM20MEon the~spcfic[H]C Effect of Na+ Effect of Ca++ and Mg++ TIME (min) FIG. 2. Effect of Na', Ca2' and Mg2+ on the specific [3HI- raclopride binding in monkey cortex. The effect of Na+ was tested FIG. 1. Effect of temperature on the time course of specific in 50 mM Tris-HCl buffer (pH 7.4 at 230C) -containing 0.1% ascorbic [3H]raclopride binding in monkey cortex. Error bars represent ± acid to which different concentrations of NaCl were added. The SEM of three experiments. effects of Ca2+ and Mg2+ were tested in 50 mM Tris HCl buffer (pH 7.4 at 23°C) containing 120 mM NaCI and 0.1% ascorbic acid to which different concentrations of CaCl2 or MgC12 were added. The column (EDBA/LIGAND, Elsevier-Biosoft, Cambridge, U.K.) and re- labeled 120 mM NaCl in the first data set is the same as the baseline ported as mean ± SEM. for the second data set and is labeled No CaCI2 and MgCl2. Error bars Radioligands were obtained from New England Nuclear. represent + SEM determined from three replications. Drugs were either purchased from Research Biochemicals (Natick, MA) or were gifts from Astra Lakemedel AB, neostriatum had a very low proportion of nonspecific binding Sodertalje, Sweden (raclopride), Smith Kline & French over a wide range of radioligand concentrations (Fig. 3a). The (chlorpromazine), Sandoz (clozapine), Lilly (LY 171555), or specific binding in this structure was saturable with Bma, = 220 Miles (ipsapirone). ± 19 fmol/mg of protein (rat) and 180 ± 9 fmol/mg of protein (monkey). Furthermore, Scatchard plots of [3H]raclopride RESULTS binding were consistent with a single binding site with Kd = 2.5 Effect of Temperature and Buffer Composition on [3H]- ± 0.2 nM and 1.3 ± 0.2 nM in rat and monkey, respectively. Raclopride Binding. Maximal specific binding was reached As in the neostriatum, the specific binding of [3H]raclopride in within 30-min incubation at 230C and was greater in magni- rat and monkey cerebral cortex was saturable and of high tude and more stable over 2 hr than incubation at 00C or 370C affinity (Fig. 3b), and Scatchard plots similarly indicated the (Fig. 1). NaCl in the Tris HCl incubation buffer tended to presence of a single binding site. However, the number of increase specific binding, with a maximal effect at 120 mM [3Hlraclopride-binding sites in the cortex was considerably NaCI (Fig. 2). KCI at 0-15 mM had no influence on total lower than that in the neostriatum: 50-75 times lower in rat and specific binding, measured in the presence of 120 mM NaCl 75-300 times lower in monkey (Fig. 3). Also, saturation studies (data not shown). The divalent cations Mg2' and Ca2+ in the cortex, in contrast to the neostriatum, showed that showed a small depressant effect on specific binding, with [3H]raclopride had a substantial proportion of nonspecific -25% decrease at 8 mM CaCl2 or MgCl2. Nevertheless, 2 mM binding at most concentrations (Fig. 3b). In fact, specific CaCl2 and 1 mM MgCl2 were retained in the incubation buffer binding exceeded nonspecific binding only within a limited for the displacement of[3H]raclopnde binding by D2 agonists, concentration range of the ligand (-0.1-2.5 nM). a process that requires divalent cations (29). Competition Experiments. [3H]Raclopride-binding sites in Saturation Analysis of [311]Raclopride Binding. Saturation neostriatum and cortex showed virtually identical pharma- studies showed that [3H]raclopride binding to rat and monkey cological profiles in both species (Table 1). In all tissues only

a BOUND z ] sv 0 w 0 D $ BOUND (fmol/mg prot) 0 ; (fmol/mg prot) C 150- 6-

Ro n nonspecific uL E 100 _ | °40

~~~~~~~50~~~~~~~~~~~~~~~~

0 1 2 3 4 5 6 0 1 2 3 4 5 6 [3H]RACLOPRIDE FREE (nM) [3H]RACLOPRIDE FREE (nM) FIG. 3. Saturation binding of [3Hlraclopride in monkey neostriatum (a) and cortex (temporal lobe) (b). [3H]Raclopride binding was measured at equilibrium conditions (30 min at 23°C), with specific binding defined as excess over blanks containing 1 AM (+)-butaclamol. (Insets) Corresponding Scatchard plots of the saturation binding data. Each value represents the average of three experiments. These Scatchard plots yielded Kd = 1.3 ± 0.2 nM and Bmax = 180 ± 9 fmol/mg of protein (neostriatum) and Kd = 1.0 + 0.2 and Bmax = 2.3 - 0.1 fmol/mg of protein (cortex). Downloaded by guest on September 26, 2021 ~oo 6414 Neurobiology: Lidow et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 1. Drug displacement of [3H]raclopride binding sites in neostriatum and cortex of monkey and rat Ki, nM Monkey Rat Compounds Striatum Cortex Striatum Cortex D2 antagonists Spiperone 0.010 0.007 0.002 0.002 Haloperidol 1.0 1.4 1.4 1.7 (+)-Butaclomol 2.3 1.9 3.2 1.1 Raclopride 1.3 1.1 2.5 2.3 Fluphenazine 3.4 7.2 1.2 1.0 Chlorpromazine 4.8 4.0 1.9 5.1 Pimozide 6.1 10.1 1.1 1.0 (-)-Sulpiride 38 13 31 27 Clozapine 190 400 130 400 (+)-Sulpiride 440 610 700 540 (-)-Butaclomol >10000 >10000 >10000 >10000 D2 agonists (-)-N-1-propyl- 0.4 (h) 0.6 (h) 0.6 (h) 0.5 (h) norapomorphine 30.2 (1) 26.4 (1) 23.3 (1) 22.5 (1) LY 171555 3.6 (h) 4.7 (h) 3.7 (h) 4.1 (h) 3700 (1) 2700 (1) 3500 (1) 4200 (1) Dopamine 3.5 (h) 4.7 (h) 3.8 (h) 4.5 (h) 4300 (1) 3800 (1) 5000 (1) 3500 (1) (+)-N-1-propylnorapomorphine 620 570 780 650 Other receptors SCH 23390 (D1) 350 400 430 530 SKF 83566 (D1) 480 420 1000 1200 Serotonin (S1) 5900 7600 >10000 >10000 Ipsapirone (SlA) 390 480 820 940 Ketanserin (S2) 230 240 250 310 Mepyramine (histamine) 6800 6500 >10000 >10000 (-)-Propranolol ((3) 7100 6400 >10000 >10000 Prazosin (al) >10000 >10000 >10000 >10000 Clonidine (a2) >10000 >10000 >10000 >10000 Atropine (M) >10000 >10000 >10000 >10000 Bicuculine (GABAA) >10000 >10000 >10000 >10000 The data represent the mean Ki values of three experiments. The pseudo-Hill coefficients of all displacing agents were close to 1.0 except for D2 agonists, which had values of 0.6-0.8. For these ligands K; values are given for both the high (h)- and low (1)-affinity sites. Also in parentheses are the concerned receptor types: D, dopamine; S, serotonin; f3, j3-adrenergic; a, a-adrenergic; M, muscarinic; GABA, y-aminobutyric acid. D2-selective ligands showed high affinity for [3Hlraclopride low rostral-caudal gradient was present, with -50% more binding. Cortical and striatal binding sites demonstrated sites in prefrontal compared with occipital cortex (Fig. 4 similar stereoselectivity, with active isomers [(-)-sulpiride, Inset). [3H]Raclopride binding had similar Kd values in all (+)-butaclomol, and (-)-N-1-propylnorapomorphine] show- cortical regions of both species (Fig. 4). ing much higher affinity for these sites than their inactive Displacement of [3H]Sulpfride and [3H]Spiperone by Raclo- enantiomers [(+)-sulpiride, (-)-butaclomol, and (+)-N-1- pride. To determine whether differences exist between pop- propylnorapomorphine]. Drugs selective for other receptors ulations of D2 receptors labeled by [3H]raclopride and other showed relatively low affinity for the [3H]raclopride binding neuroleptics, we studied the ability of raclopride to compete sites. All ligands used in this study displaced [3H]raclopride for D2 binding sites with [3H]sulpiride (a substituted benza- binding with pseudo-Hill coefficients close to 1.0, with the mide) and [3H]spiperone (a spirodecanone neuroleptic). The exception of D2 agonists, which demonstrated pseudo-Hill binding with [3H]spiperone duplicated common practice, coefficients ranging from 0.6 to 0.8. Such low pseudo-Hill using 0.3 AM ketanserin in the incubation buffer to prevent coefficients are characteristic for inhibition of D2 antagonist binding to 5-hydroxytryptamine type 2 (5-HT2) sites (7-10, binding by agonists (30). 16). The raclopride displacement of [3H]sulpiride binding in Areal Distribution of [3H]Raclopride Binding in the Cortex. neostriatum and cortex of both species showed simple com- The distribution of [3H]raclopride binding sites in monkey petition curves with pseudo-Hill coefficients close to 1.0 (Fig. cortex showed a general rostral-caudal gradient with nearly 5). Raclopride displacement of [3H]spiperone binding in the five times higher density of sites in prefrontal and temporal neostriatum also resulted in a simple competition curve (Fig. regions than in the occipital region (Fig. 4). Motor, soma- 5). In contrast, raclopride displacement of [3H]spiperone tosensory, and posterior parietal cortical regions contained binding in the cortex was shallow, with a pseudo-Hill coef- intermediate concentrations. The rat cortex showed more ficient of -0.6. Computer analysis ofthe displacement curve even distribution of [3H]raclopride binding, although a shal- is consistent with two binding sites: high affinity (Ki = 1.0 Downloaded by guest on September 26, 2021 Neurobiology: Lidow et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6415 _t6 Table 2. Pseudo-Hill coefficients, Ki values, and percentages for CRR and MONKEY high- low-affinity sites obtained by computer analysis of the 04 displacement of [3H]spiperone binding (in the presence of 0.3 A&M ketanserin) to monkey cortex with D2 dopaminergic-, C a-adrenergic-, and 5-HT1A serotonergic-specific ligands Kd (nM)1.30.31.00.21.10.2O.90...2 . High- 0 2 Low-affinity FI. 4 C. E Ligands Pseudo-Hill affinity sites sites (receptor sites) coefficient K;, nM % K1, nM % ~~ ~ ~ ~ ~ ~ ~ ~ At N piN Raclopride (D2) 0.6 1 37 6000 63 E Idazohan (a2) 0.7 10 39 5000 61 ~~~~~~~~~~~~4V Ipsapirone (5-HT1A) 0.6 2 38 3000 62 0 Data represent mean values determined in three experiments. binding of [3H]raclopride in the neostriatum is very low. However, in the cortex the range of [3H]raclopride concen- E ~ ~ 0W trations at which the specific binding was higher than nonspecific binding was extremely limited. Thus, the use of low E~ concentrations (<3 of this is essential for frm moke0 nM) radioligand (mi6itga)adrt(Ist.B n dvle successful labeling of D2 sites in the cortex. This finding may Kd (nM) 1.3±0.3 2 1.1±0.2 0.9±0.2 1.0±0.2 1.0±0.2 explain the failure of previous attempts to use relatively high concentrations of for the FIG. 4. Regional in the [3H]raclopride autoradiographic [3H]raclopride binding neocortical areas of cortical from monkey (main histogram) and rat (Inset). and values study D2 receptors (21). Our results indicate that, Bma, Kd like other are expressed as mean ± SEM determined in three experiments. substituted benzamides, [3H]raclopride shows sodium-dependent binding (11, 31, 32), whereas the divalent cations and nM) accounting for -37% of binding and low affinity (Ki = Ca2' Mg2' have inhibitory effect (32). Contrary 6 /zM) representing the remaining 63% of the sites. To to the recent suggestion that substituted benzamides should determine whether these low-affinity sites represent a2- have very low nonspecific binding when incubation is done at adrenergic and/or 5-HT1A serotonergic receptors, binding of 0°C (12), we like others (33) found that incubation at 23°C for [3H]spiperone to cortical membranes (in the presence of 0.3 30 min resulted in the maximal stable [3H]raclopride binding tkM ketanserin) was displaced with idazoxan (a2-adrenergic to both striatal and cortical tissue. antagonist) and ipsapirone (5-HT1A agonist). The results Cortical Dopamine D2 Receptors. The major finding of this show that displacement of [3H]spiperone binding by both study is that both rodent and primate cortices contain D2 ligands contain high- and low-affinity components (Table 2). dopamine receptor binding sites, which appear virtually The high-affinity sites for idazoxan represented 39% and identical to those in the neostriatum, although the former those for ipsapirone represented 38% of the specific [3H]- sites are present in considerably lower concentration. The spiperone binding in the cortex. low density of D2 binding sites is consistent with the low concentration of dopamine in the cortex as compared with that in the neostriatum. Thus, dopamine concentration in the DISCUSSION rat cortex is 130- to 220-fold lower than in rat neostriatum (2, Specificity of [3lHlRaclopride Binding. Our results support 34) and 130- to 1700-fold lower in monkey cortex than in earlier findings (21, 27) that [3H]raclopride is a potent and monkey neostriatum (1). The D2 receptors in monkey cortex selective D2 ligand, which can be displaced from its binding show a strong rostral-caudal density gradient that closely sites only by D2-selective drugs. We found that nonspecific parallels the concentration gradient of dopamine itself (1). A similar, but less dramatic, gradient was seen in the rat 100 cortex-a finding consistent with the shallow gradient of 1 striatum dopamine in the rodent cortex (2). Apparent Single Population of Dopamine D2 Receptors. Recent electrophysiological studies in the rat have suggested that cortical and striatal D2 receptors have different pharmacological properties (35, 36). However, homogenate binding studies with [3H]sulpiride (16) and [1251]iodosulpiride (25) and the present results with [3H]raclopride have not detected such differences. The reasons for these disparities between Fo electrophysiological and binding studies are unclear but could be due to a unique subpopulation of cortical D2 sites ST _ X F that binding studies with substituted benzamides failed to detect. Indeed, it has been suggested that the substituted 11 10 9 8 7 6 5 11 10 9 8 7 6 benzamides bind only to a subpopulation ofD2 receptors and 5 that other radioligands label the entire population of D2 sites -log [RACLOPRIDE] (M) (29, 37, 38). In our study, we found that, while both [3H]- the FIG. spiperone (in presence of ketanserin) and raclopride S. Raclopride displacement of [3H]sulpiride and [3H]- for the same spiperone binding (in the presence of 0.3 compete population of receptor sites in the ,uM ketanserin) to monkey shows a neostriatum and cortex. Data represent the average of three exper- neostriatum, raclopride high affinity for only a iments. Raclopride displaced [3H]sulpiride with pseudo-Hill coeffi- subpopulation of receptors labeled by [3H]spiperone in the cients 0.98 (neostriatum) and 0.99 (cortex). Ki in the neostriatum cortex. However, [3H]spiperone is known to bind multiple equals 1.3 nM and K. in the cortex equals 1.2 nM. In the- neosetriatum receptor sites (16, 22, 23), and while our assay conditions raclopride displaced [3H]spiperone with pseudo-Hill coefficient 0.85; prevented binding to 5-HT2 sites (16), the low-affinity sites Ki equals 3.0 nM. In the cortex, raclopride displaced [3Hlspiperone detected with raclopride may represent a2-adrenergic and/or with pseudo-Hill coefficient 0.6. Ki values can be seen in Table 2. 5-HTIA receptors (22, 23). Indeed, in competition studies Downloaded by guest on September 26, 2021 M"16 Neurobiology: Lidow et al. Proc. Natl. Acad. Sci. USA 86 (1989) with idazoxan and ipsapirone, we found that more than 12. Stefanini, E., Ortu, A. M., Vernaleone, F. & Gessa, G. L. two-thirds ofthe [3H]spiperone binding was to these receptor (1987) Pharmacol. Res. Commun. 19, 777-791. 13. Liskowsky, D. R. & Potter, L. T. (1985) Life Sci. 36, 1551- sites. Thus, in the neostriatum, the density of D2 receptors is 1559. high enough to make nondopaminergic binding of [3H]- 14. Camus, A., Agid, F. J., Dubois, A. & Asatton, B. (1986) Brain spiperone comparatively insignificant. In contrast, the low Res. 375, 135-149. density of D2 receptors in the cortex together with a sub- 15. Rakic, P., Goldman-Rakic, P. S. & Gallager, D. W. (1988) J. stantial concentration of other high-affinity sites for [3H]- Neurosci. 8, 3670-3690. 16. Altar, C. A., Kim, H. & Marshall, J. F. (1985) J. Pharmacol. spiperone significantly increases the proportion of nondopam- Exp. Ther. 233, 527-538. inergic binding and makes [3H]spiperone a poor label for 17. Bouthenet, M. L., Sales, N. & Schwartz, J. C. (1985) Naunyn- cortical D2 receptors. Schmiedebergs Arch. Pharmacol. 330, 1-8. Functional Significance. A widespread distribution of do- 18. Gehlert, D. R. & Wamsley, J. K. (1985) Neurochem. Int. 7, pamine D2 receptors in the cerebral cortex is of considerable 717-723. this be the site for antipsy- 19. Charuchinda, C., Supavilai, P., Korobath, M. & Palacios, J. M. clinical significance because may (1987) J. Neurosci. 7, 1352-1360. chotic effects of neuroleptics (27). Thus, our findings should 20. Dubois, A., Savata, M., Guret, 0. & Scatton, B. (1986) bring attention to the cortex as a possible site of dysfunction Neuroscience 19, 125-137. in diseases like schizophrenia. Recently, dopamine afferents 21. Kohler, C. & Radesater, A. C. (1986) Neurosci. Lett. 66, have been seen to form synapses with dendritic spines of 85-90. cortical pyramidal cells, placing dopaminergic receptors in a 22. Peroutka, S. J., U'Prichard, D. C., Greenberg, D. A. & Sny- der, S. H. (1977) Neuropharmacology 16, 549-556. strategic position to directly affect the activity of efferents to 23. 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(1986) Psychopharmacology 90, 287-294. Roels, F. & Vanguelin, G. (1988) Neurosci. Lett. 91, 142-147. 38. Magnusson, O., Fowler, C. J., Mohringe, B., Wijkstrom, A. & 11. Carboni, E., Memo, M., Tanda, G. L., Carruba, M. 0. & Ogren, S. 0. (1988) Naunyn-Schmiedeberg's Arch. Pharma- Spasvo, P. F. (1985) Neurochem. Int. 7, 279-284. col. 337, 379-384. Downloaded by guest on September 26, 2021