Lei Ij, Tjkch2

Corrections Proc. Natd Acad. Sci. USA 78 (1981) 7235

Correction. In the article "Purification of guinea pig pH3 mi- Correction. In the article "Resolution of dopamine and sero- gration inhibitory factor" by Heinz G. Remold, Philip L. tonin receptor components of [3H]spiperone binding to rat McCarthy, Jr., andAlma D. Mednis, which appeared in theJuly brain regions" by Stephen J. List and Philip Seeman, which 1981 issue of Proc. NatL Acad. Sci. USA (78, 4088-4091), an appeared in the April 1981 issue of Proc. NatL Acad. Sci. USA undetected printer's error resulted in reversing the placement (78, 2620-2624), the authors request that the following be ofFigs. 1 and 3; the legends of these figures, however, are cor- noted. The correct chemical structure of R43448, which was rectly placed. used by Leysen et aL (1) to distinguish between dopaminergic and serotonergic components of[3H]spiperone binding, may be found in their erratum note (2). The structure is: 1-(4-fluoro- phenylmethyl)- N-{ 1-[2-(2-pyridinyl)ethyl] -4-piperidinyl}-1H- Correction. In the article "Platelet-derived growth factor-mod- benzimidazol-2-amine. ulated proteins: Constitutive synthesis by a transformed cell line" by W. J. Pledger, C. A. Hart, K. L. Locatell, and C. D. Scher, which appeared in the July 1981 issue ofProc. NatL Acad. Sci. USA (78, 4358-4362), an undetected printer's error oc- NlCH2 / F curred in Fig. 6. The figure and the omitted arrows are printed Di below. tjKCH2 - CH2 -N

ST3T3 3T3 1. Leysen, J. E., Gommeren, W. & Laduron, P. M. (1979) Biochem. PPP PDGF PPP PDGF PharmacoL 28, 447-448. 2. Leysen, J. E. Gommeren, W. & Laduron, P. M. (1980) Biochem. 30% 5% 30% 5% PharmacoL 29, 1211. I.. X

I Lei I j,

A, I

* *

FIG. 6. Constitutive synthesis of PDGF-modulated proteins by ST3T3 cells. BALB/c-3T3 cells and ST3T3 cells were grown to conflu- ence in medium supplemented with 10% serum. Three days after the last medium change, the density-arrested cultures were transferred to incorporation medium supplemented with either 30% or 5% PPP or highly purified PDGF at 14 mg/ml for3 hr. [wSIMethionine was added and 20 min later the cultures were harvested. Equal amounts of tri- chloroacetic acid-insoluble contents (20,000 cpm.per sample) were ap- plied to a gel. The arrows (from top to bottom) indicate the positions of pII (Mr 35,000) and pI (Mr 29,000). Downloaded by guest on September 29, 2021 Proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp. 2620-2624, April 1981 Neurobiology

Resolution of dopamine and serotonin receptor components of [3H]spiperone binding to rat brain regions (neuroleptic/neurotransmitter/sulpiride) STEPHEN J. LIST AND PHILIP SEEMAN Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada Communicated by Philip Siekevitz, December 1, 1980 ABSTRACT A procedure was developed to identify receptors inhibition of[3H]spiperone by ADTN has a low Hill coefficient, for dopamine and serotonin separately and selectively by means suggesting that ADTN may compete for multiple sites. Third, of [3H]spiperone and to measure the density of each receptor in there is nothing to prevent [3H]spiperone from labeling either different regions of the rat brain. In the striatum, the binding of serotonin-type or dopamine-type sites in vitro, and it becomes [3H]spiperone to dopamine receptors was inhibited by sulpiride essential, therefore, to coincubate with a drug that selectively but not by quinazolinedione R43448(R43448); in the frontal cortex, precludes the binding of[3H]spiperone to one ofthese two sites. however, the binding of [3H]spiperone to serotonin receptors was Using these important considerations, and using sulpiride and inhibited by R43448 but not by sulpiride. Thus, the density of do- quinazolinedione R43448 (R43448; Janssen, Beerse, Belgium) pamine receptors (D2 sites) was measured by [3H]spiperone binding in the presence of0.1jIM R43448 (to preclude the attachment (11) as selective dopamine and serotonin receptor blockers, re- of the 3H-labeled ligand to serotonin sites), while the density of spectively, we describe here the resolution of [3H]spiperone serotonin receptors (S2 sites) was measured by [3H]spiperone binding into its dopaminergic and serotoninergic components binding in the presence of 10 jIM sulpiride (to preclude the at- for different regions of the rat brain. tachment of the 3H-labeled ligand to dopamine sites). The density of D2 sites was highest in the striatum, followed by the olfactory METHODS tubercle, hypothalamus, substantia nigra, and the lower Rat Brain Homogenates. Freshly dissected brain tissues pons-medulla region. All five regions had similar dissociation con- (male Wistar rats, 200-250 g) were placed in 15 vol ofcold TEAN stants (Kd values) of0.05-0.15 nM. The density ofS2 sites was high- buffer [15 mM Tris'HCl, pH 7.4/5 mM Na2EDTA/0.02% (1.1 est in the frontal cortex, followed by the posterior cortex, olfactory mM) ascorbate/12.5 ,M nialamide], homogenized with a Poly- tubercle, striatum, hypothalamus, and thalamus, and all regions tron (Brinkmann PT-10; setting of7, 10 sec), centrifuged (44,000 had Ka values in the range 0.6-2.3 nM. Thus, because the Kd val- x g) and resuspended (15 vol) four times, and finally stored fro- ues were similar for all regions, and because Scatchard analyses zen at 10 of wet ml for revealed a single set of sites for either D2 or S2 (where detected), (5-ml samples mg original weight per the main criteria for resolving the dopamine and serotonin com- striatum, 20 mg/ml for olfactory tubercle and frontal and pos- of terior cortex, and 50 mg/ml for all other areas). (Posterior cortex ponents [3H]spiperone binding were considered fulfilled. tissue was the posterior 7 mm of cortex; lower pons-medulla Although dopamine receptors in the brain can be directly la- contained all pons and medulla tissue caudal to the brachium beled by either [3H]haloperidol (1-3) or [3H]spiperone (4, 5), pontus.) the latter also labels serotoninergic sites (6, 7) and spirodeca- Binding Assays. From Eppendorf polypropylene pipette none sites (8). In the striatum, for example, [3H]spiperone binds tips, quadruplicate glass test tubes received, in order, 200 ,ul primarily to dopamine receptors, but in the cerebral cortex this ofbuffer (with or without other drugs), 200 ,ul of [3H]spiperone (24 Ci/mmol; New England Nuclear; 1 Ci = 3.7 x 10' becque- H-labeled ligand primarily detects serotoninergic sites (5, 6). rels), and 200 ,ul of homogenate. Final tissue protein per tube Hence, in order to identify dopamine receptors selectively was: approximately 0.08-0.1 mg per tube for olfactory tubercle, and reliably by means of [3H]spiperone, it has now become es- 0.2 mg per tube for frontal and posterior cortex, 0.3-0.35 mg sential to use a procedure that resolves and separates these do- per tube for hippocampus, hypothalamus, thalamus, lower paminergic and serotoninergic components of [3H]spiperone pons-medulla, 0.45 mg per tube for cerebellum, and 0.5 mg binding. Although it has been suggested that dopamine recep- per tube for substantia nigra. After incubation for 60 min at tors be defined as the (±)-6,7-dihydroxy-2-aminotetralin (ADTN)- 20-220C, 0.5-ml samples were vacuum-filtered through What- displaceable component of [3H]spiperone binding, and that se- man GF/B filters and washed with 10 ml of buffer. All results rotonin receptors be defined as the cinanserin-displaceable were averaged from three to five experiments. Unless specified component of [3H]spiperone binding (9, 10), this approach is otherwise, the inhibition of [3H]spiperone binding by various not adequately selective for several reasons. First, excess cin- drugs was tested by using 0.25 nM [3H]spiperone (final con- anserin not only inhibits [3H]spiperone binding to the frontal centration). The definitions for the components of [3H]spiperone cortex (serotoninergic sites) but also inhibits approximately 50% in the of 3. ofthe [3H]spiperone binding to dopaminergic sites in the stria- binding are given legend Fig. tum (10). Second, we have found (unpublished) that excess ADTN is not sufficiently selective, because in addition to in- RESULTS hibiting the binding of[3H]spiperone to the striatum (dopamine It was essential to identify a drug that would inhibit [3H]spiperone component) it considerably inhibits the binding of [3H]spiperone binding in the frontal cortex (mainly serotoninergic in nature) to the frontal cortex (serotonin component). Furthermore, the at concentrations not effective in inhibiting [3H]spiperone binding in the striatum (mainly dopaminergic in nature) and a drug The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: ADTN, (+)-6,7-dihydroxy-2-aminotetralin; R43448, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. quinazolinedione R43448; LSD, lysergic acid diethylamide. 2620 Neurobiology: List and Seeman Proc. Natl. Acad. Sci. USA 78 (1981) 2621 100F

80H bjD ._ 60H- .0n:D SC0 C4- 40H

20k

,,,, ,,,,11 ,,,I ,,,,1 10-8 0-7 10-6 10-5 lo-4 Sulpiride, M

bjD ._ FIG. 1. Binding of [3H]spiperone (0.25 ._- a nM) to receptors. The binding to dopamine receptors was selectively inhibited in the -0 striatum by sulpiride and not by R43448; C4. 0 in the frontal cortex, however, the binding of [3H]spiperone to serotonin receptors was selectively inhibited by R43448 but not by sulpiride. The absolute amounts of total binding (cpm) per filter were: striatum, 1000; olfactory tubercle, 1000; sub- WLJ stantia nigra, 500; frontal cortex, 850; hip- lo-10 10-8 lo-, 10-6 -5 pocampus, 550. Values are means ± SEM 10-9 for three to five independent experiments R43448, M in quadruplicate. that would compete for striatal binding at concentrations not inhibition was 570 ± 11 cpm per filter), indicating that the drugs affecting frontal cortex binding. Sulpiride and R43448 had the were competing for different sites. Furthermore, the sulpiride highest selectivities for the dopaminergic and serotoninergic IC50 values in the olfactory tubercle were the same in the pres- components of [3H]spiperone binding, respectively, out of a ence and absence of 0.1 AM R43448 (Table 1); the IC50 values wide variety of dopaminergic and serotoninergic compounds ef R43448 were also the same in the presence and absence of tested, including methiothepin, cinanserin, mianserin, met- 10 ,M sulpiride. ergoline, methysergide, ADTN, N-propylnorapomorphine, As shown in Fig. 2 Top, the majority of the binding in the apomorphine, pipamperone, and other neuroleptics. striatum, at low [3H]spiperone concentrations, was to the do- As shown in Fig. 1, sulpiride inhibition of [3H]spiperone paminergic site (sulpiride-displaceable binding). Because (+)- binding in the striatum reached a plateau at concentrations between 1 and 10 ,M, whereas no significant inhibition in the Table 1. IC50 values of sulpiride and R43448 against frontal cortex occurred at these concentrations. R43448 was 0.25 nM [3H]spiperone potent in inhibiting the binding of [3H]spiperone to frontal cortex, with the plateau effect occurring at 0. 1 ,uM; concentrations IC50, nM of R43448 up to 0.1 AM, however, produced little inhibition Brain region Sulpiride R43448 of[3H]spiperone binding to the striatum (see also Table 1). Sim- Striatum 150 ilar selectivity was found by using higher concentrations of Frontal cortex - - [3H]spiperone (up to 2.0 nM). Thus, 10 ,uM sulpiride was cho- Olfactory tubercle 120 2.5 sen to block dopaminergic [3H]spiperone binding sites and 0.1 Olfactory tubercle in presence of ,uM R43448 was chosen to block the serotoninergic [3H]spiperone 0.1 gM R43448 110 - binding sites. Olfactory tubercle in presence of The fact that these two drugs selectively inhibited two dis- 10 tLM sulpiride - 1.5 tinct [3H]spiperone binding sites was demonstrated by further Hippocampus - 10 experiments on the olfactory tubercle. Ofthe 1200 cpm per fil- Substantia nigra 150 ter of spiperone-displaceable [3H]spiperone binding in the ol- IC50 value is defined as that drug concentration required to inhibit factory tubercle, sulpiride (at 10 ,uM) inhibited 377 ± 26 cpm 50% of specific binding. Sulpiride-specific binding is defined by the per filter and R43448 (at 0.1 utM) inhibited 177 ± 23 cpm per competition plateau occurring at 5-10 AM sulpiride (see Fig. 1). filter (mean ± SEM). When the two drugs were added together, R43448-specific binding is defined by the competition plateau occur- the inhibition of [3H]spiperone binding was additive (i.e., the ring at 0.1 ,tM R43448 (see Fig. 2). 2622 Neurobiology: List and Seeman Proc. Natl. Acad. Sci. USA 78 (1981)

Striatum butaclamol inhibited 12% more [3H]spiperone binding (com- 1000 pared to sulpiride), this extra 12% may represent a small population of serotoninergic-type sites in the caudate, because (+)-butaclamol is a potent inhibitor of both dopaminergic and 0 600 serotoninergic binding (6, 9). The presence of serotonin sites in the striatum may account for the small but significant inhi- 0 400 O bition ofstriatal binding by low concentrations ofR43448. Non- C' radioactive spiperone inhibited even more binding than did (+)-butaclamol. This latter 14% of[3H]spiperone binding likely represents a specific spirodecanone binding site previously observed by other workers (8, 12). In order to measure the densities and Kd values (dissociation constants) for the components of [3H]spiperone binding, many [3H]spiperone binding isotherms and Scatchard analyses were done on a variety oftissues, using conditions that permitted the selective labeling of D2 or S2 sites by [3H]spiperone (see definitions in legend of Fig. 3; see Figs. 4 and 5 and Table 2). The D2 dopamine receptors were highest in density in the striatum (434 fmol/mg ofprotein), whereas the highest density ofS2 serotonin sites was found in the frontal cortex (666 fmol/mg). The D2 site had a Kd value (0.06-0.15 nM) considerably lower than that for the S2 site (0.57-2.3 nM). The remaining sites for spiperone-displaceable [3H]spiperone binding had lower affinities (kd values up to 10 nM; Table 2). The density ofthese sites was highest in the olfactory tubercle (3527 fmol/mg). The hippo- tor. campus, while displaying a large amount ofspiperone-displace- .0 able [3H]spiperone binding, did not reveal any detectable D2 or S2 components. DISCUSSION Using R43448 to block selectively [3H]spiperone binding to se- [3H]Spiperone, M rotoninergic sites, and using sulpiride to block selectively [3H]spiperone binding to dopaminergic sites, the present study FIG. 2. Components of [3H]spiperone binding to different regions provides the quantitative regional brain distribution of specific of rat brain. In the striatum (Top), 82% of the binding of 0.25 nM serotoninergic, dopaminergic, and other [3H]spiperone binding [3H]spiperone was displaced by nonradioactive spiperone, leaving 8% sites. Furthermore, the regional brain analysis of[3H]spiperone as nondisplaceable (nonspecific) binding. The 82% was composed mostly ofD2 dopamine receptors (as displaced by sulpiride; 66% oftotal binding indicates that there are dopamine receptors in the hy- binding) but also included some S2 serotonin sites, as suggested by the pothalamus and substantia nigra, as has been previously sug- action ofR43448 (at 0.1 uM) or the effect of(+)-butaclamol (which in- gested (10, 13, 14), and also in the lower pons-medulla (see refs. hibited [3H]spiperone binding by 12% more than did sulpiride). The 15 and 16), where dopamine receptors have not previously been frontal cortex (Middle) was mainly composed of S2 sites (R43448-dis- directly detected. placeable), while the hippocampus (Bottom) revealed few D2 or S2 sites. The selectivity of sulpiride for [3H]spiperone/dopamine re- Values are means + SEM for three to five experiments. ceptors was indicated by the inhibition of 10 ,M sulpiride of

Table 2. Components of [3H]spiperone binding Dopaminergic sites (D2) Serotoninergic sites (S2) Other sites Total number Bmax Kd, Kd, Bma, Kd, of sites, Brain region fmol/mg nM Bmax) nM fmol/mg nM fmol/mg Striatum 434 ± 28 0.063 ± 0.02 134 ± 15 0.57 ± 0.1 1470 ± 600 7.0 ± 3.8 2038 ± 600 Olfactory tubercle 107 ± 7 0.082 + 0.008 174 ± 12 0.75 ± 0.1 3246 ± 830 10.1 ± 3.0 3527 ± 830 Hypothalamus 36 ± 3.5 0.15 ± 0.02 128 ± 20 1.4 ± 0.4 530 ± 80 6.5 ± 1.3 694 ± 82 Substantia nigra 31 ± 9.0 0.11 ± 0.05 ND ND 966 ± 270 8.6 ± 2.9 997 ± 270 Lower pons-medulla 25 ± 2.0 0.11 ± 0.02 ND ND 172 ± 23 2.8 ± 0.6 197 ± 23 Frontal cortex ND ND 666 ± 50 1.2 ± 0.2 991 ± 120 2.6 ± 0.5 1657 ± 130 Posterior cortex ND ND 258 ± 41 2.3 ± 0.7 1577 ± 104 5.0 ± 0.4 1835 ± 112 Thalamus ND ND 98 ± 32 0.7 ± 0.3 310 ± 12 1.5 ± 0.3 408 ± 34 Hippocampus ND ND ND ND 120 ± 17 0.6 ± 0.3 Cerebellum ND ND ND ND ND ND ND All values are means ± SEM for three to five independent experiments. Binding is expressed per mg ofprotein. ND, not detectable. D2: selective binding of [3H]spiperone to dopaminergic sites was defined as the binding that occurred in the presence of0.1 AuM R43448 and was inhibited by 10 MM sulpiride. S2: selective binding of[3H]spiperone to serotoninergic sites was defined as the binding that occurred in the presence of10 /M sulpiride and was inhibited by 0.1 ,uM R43448. Binding to "other" sites was defined as the amount of [3H]spiperone binding inhibited by ,M spiperone that remained after the computer accounted for the presence of the D2 and S2 components (see Fig. 5). Thus the total number of [ H]spiperone sites = (Bmax of D2) + (Bm. of S2) + (Bmax of "other"). Neurobiology: List and Seeman Proc. Natl. Acad. Sci. USA 78 (1981) 2623

7000

6000

5000

4000

3000

- 2000 FIG. 3. Representative Scat- chard analyses ([3H]spiperone K1000 binding; range was 0.05-3.0 nM) 0 for each of four brain regions. B, ._ bound; F, free; ND, not detectable. I- 000 .J The total spiperone-displaceable

-& binding of [3H]spiperone was that 60 400 inhibitedby 1 jxM spiperone (larger graphs). The D2 component (seeIn- to 350400 sets) was separately measured and S-4 was the binding of [3H]spiperone that occurred in the presence of0.1 L ,uAM R43448 (to occlude serotonin 250 sites) but that was displaceable by 10 AiM sulpiride. The S2 component 200 (see Insets) was also separately measured and defined as binding of 150 _[3H]spiperone that occurred in the presence of 10 AuM sulpiride (to oc- 100 clude dopamine sites) but that was 50 displaceable by 0.1 jiM R43448. The values for Kd and Bmax of D2 iJ and S2 components are among those 50 100 150 200 250 500 1000 1500 used to calculate the averages given B, fmol/mg protein in Table 2. a large population of[3H]spiperone binding sites in the striatum (6, 9)] was little affected. The selectivity of R43448 (at 0.1 JIM) [dopaminergic binding (5, 6)] while [3H]spiperone binding in for serotoninergic [3H]spiperone sites was demonstrated by the the frontal cortex [mainly serotoninergic [3H]spiperone binding ability ofthis drug to block out a large population of[3H]spiperone sites in the frontal cortex at drug concentrations that had little Hypothalamus 700 effect on [3H]spiperone binding in the striatum. 200 D 2002 In many brain regions, Scatchard analysis of spiperone-dis- 600 150 \ 150 placeable [3H]spiperone binding yielded biphasic or multi- Hippocampus 500- 500 * 100 - 100 phasic curves, one possible interpretation ofwhich is multiple F D2 2 binding sites. In the D2 binding system, which appeared to have ND ND 400 lsz 400 50 50 a dopaminergic distribution in tissue areas (i.e., sites detectable -6 300-t~0+ .300 only in regions with dopaminergic innervation), binding iso- * \tK 50 100 50 100 150 O 200- therms and Scatchard plots became monophasic and revealed t * * Total 200 similar low Kd values (0.05-0.1 nM) in areas where the D2 site 100 100 was present. Although it may seem surprising that no D2 sites

0.03 500 1000 50 100 150 200 250 300 were detected in the frontal cortex, the present results are con- 0 300 ' Pons-medulla sistent with those of other workers, who have failed to detect 150n D2 S2 Thalamus dopaminergic [3H]spiperone binding in whole frontal cortex (6, 250 5500[ 150 100 ND | D2 S2 9, 10); when dopaminergic areas of the frontal cortex are spe- _ 0 400k ND 100 cifically dissected out, however, D2-type binding is detected 5 200 50 (17). 50 300 4., [3H]Spiperone binding isotherms and Scatchard analysis of 10 20 30 40 50 10.0 the S2 system revealed a binding distribution and apparent Kd lOO1 200h values (0.57-2.3 nM) significantly different from those seen for a Total Total the D2 system. 50 100 It has been demonstrated that in the frontal cortex [3H]lysergic 1 acid diethylamide (LSD) labels the [3H]spiperone S2 site as well 20 40 60 80 100 120 100 200 300 400 500 as the [3H]serotonin site (18, 19). The high number of B, fmol/mg protein ~~~~~Total [3H]spiperone S2 sites in the frontal cortex and the low number FIG. 4. Representative Scatchard analyses of [3H]spiperone bind- in the hippocampus observed in the present study are consistent ing to each offour different regions ofrat brain. See Fig. 3 for additional with the previous findings that [3H]LSD in the hippocampus details. appears to label only a single population ofserotoninergic sites, 2624 Neurobiology: List and Seeman Proc. Natl. Acad. Sci - USA 78 (1981) tolamine (a high-affinity a-receptor ligand) of [3H]spiperone binding in any brain areas tested, including frontal cortex, hippocampus, and striata (unpublished results). The present results support the observations ofothers, that [3H]spiperone binds to multiple sites. Our results indicate that multiple sites appear to be present regardless ofthe brain region examined, including brain areas where binding appears to be primarily dopaminergic (e.g., rat striatum) or serotoninergic (e.g., frontal cortex) in nature. By using the D2 and S2 [3H]spiperone binding systems described in this paper, single 180 125 populations of serotoninergic and dopaminergic [ H]spiperone 90.go 0 binding sites can be selectively labeled. Future work on this 0 0.6 1.2 1.8 2.4 3.0 0.7 1.4 2.1 2.8 3.5 bD problem will require a full characterization of the sensitivities g 250 1250 (i.e., IC50 values) ofvarious drugs and neurotransmitters for the 225 1125 Substantia nigra Frontal cortex D2 and S2 sites. 200 1000 j

175 -875 We thank Carla Ulpian for her excellent technical assistance.

150 -750- 125 - 25 1. Seeman, P., Chau-Wong, M., Tedesco, J. & Wong, K. (1975) 100 500- Proc. Nati. Acad. Sci. USA 72, 4376-4380. 2. Seeman, P., Lee, T., Chau-Wong, M., Tedesco, J. & Wong, K. 75 375- (1976) Proc. Natl. Acad. Sci. USA 73, 4354-4358. 50 250- 3. Burt, D. R., Creese, I. & Snyder, S. H. (1976) Mol. Pharmacol. 25 125 12, 800-812. 4. Fields, J. Z., Reisine, T. D. & Yamamura, H. I. (1977) Brain Res. 0.5 1.0 1.5 2.0 2.5 0.6 1.2 1.8 2.4 3.0 136, 578-584. [3H]Spiperone, nM 5. Leysen, J. E., Gommeren, W. & Laduron, P. M. (1978) Biochem. Pharmacol. 27, 307-316. FIG. 5. Examples of computer-fitted lines for spiperone-displace- 6. Leysen, J. E., Niemegeers, C. J. E., Tollenaere, J. P. & Ladu- able binding of [3Hlspiperone (saturation curves, isotherms). The re- ron, P. M. (1978) Nature (London) 272, 168-171. ceptor densities (B,,,.) and dissociation constants (Kd) were first in- 7. Creese, I. & Snyder, S. H. (1978) Eur. J. Pharmacol. 49, dependently obtained by means of the selective labeling procedure 201-202. described for Figs. 3 and 4 and Table 2. Using these values, the com- 8. Howlett, D. R., Morris, H' & Nahorski, S. R. (1979) Mol. Phar- puter then fitted a line as follows: Amount bound (ordinate) = macol. 15, 506-514. In C3BC*-B /(C* + Kd), in which C* is the concentration of 9. Quik, M., Iversen, L. L., Larder, A. & Mackay, A. V. P. (1978) [3H]spiperone and n = 1, 2, and 3 represent the D2 receptors, the.S2 Nature (London) 274, 513-514. sites, and the "other" sites. The computer used a BMDPAR nonlinear 10. Quik, M. & Iversen, L. L. (1979) Eur. J. Pharmacol. 56, regression program (University of California, Los Angeles, 1979) for 323-330. the line-fitting procedure. 11. Leysen, J. E., Gommeren, W. & Laduron, P. M. (1979) Biochem. Pharmacol. 28, 447448. 12. Gorissen, H. & Laduron, P. (1979) Nature (London) 279, 72-74. 13. Quik, M., Emson, P. C. & Joyce, E. (1979) Brain Res. 167, the [3H]serotonin-site (19). The present study also detected a 355-365._ significant number ofS2 [3H]spiperone binding sites in the stria- 14. Reisine, T. D., Nagy, J. I., Fibiger, H. C. & Yamamura, H. I. tum, which again is consistent.with earlier work, which had (1979) Brain Res. 169, 209-214. suggested that in the caudate nucleus [3H]LSD possibly binds 15. Lee, H. K., Chung, P. M. & Wang, S. C. (1979) Eur. J. Phar- to multiple sites for serotonin (20). macol. 53, 29-38. The remaining [3H]spiperone binding sites (after D2 and S2 16. Commissiong, J. W., Gentlemen, S. & Neff, N. H. (1979) Neu- ropharmacology 18, 565-8. sites had been accounted for) detected in spiperone-displace- 17. Marchais, D., Tassin, J. P. & Bockaert, J. (1980) Brain Res. 183, able [3H]spiperone binding isotherms and Scatchard plots had 235-240. apparent Kd values ranging from 1.5 to 10 nM, with the highest 18. Peroutka, S. J. & Snyder, S. H. (1979) Mol. Pharmacol. 16, density ofsites in the olfactory tubercle. These other sites likely 687-699. include those that nonspecifically bind butyrophenones in gen- 19. Seeman, P., Westman, K., Coscina, D. & Warsh, J. J. (1980) Eur. J. Pharmacol. 66, 179-191. eral (8). It is possible that some ofthe binding is to yet-nnklnown 20. Whitaker, P. M. & Seeman, P. (1978) Proc. Nati. Acad. Sci. USA neurotransmitter receptors. Although spiperone has been 75, 5783-5787. shown to have a relatively high affinity for a-adrenergic recep- 21. Peroutka, S. J., U'Prichard, D. C., Greenberg, D. A. & Snyder, tors (21), we have not observed any potent inhibition by phen- S. H. (1977) Neuropharmacology 16, 549-556.