[3H]5-hydroxytryptamine binding sites in postmortem human brain

Neuro~ha~~~u~~~~~~ Vol.

28, No. IO, pp. 1055-1060.1989

~28-3~8/89

$3.00 + 0.00

Copyright Q 1989Pergamon Press pIc

Printed in Great Britain. All rights reserved

[3H]5-HYDROXYTRYPTAMINE BINDING SITES IN POSTMORTEM HUMAN BRAIN S. C. CHEETHAM*,Y. YAMAGUCHI and R. W. HORTON** Department of Pharmacology and Clinical Pharmacology, St George’s Hospital Medical School, London SW17 ORE, U.K. (Accepted

15 May I989)

sites for [3H]5-hydroxytryptamine (5-HT) in ~stmortem human frontal cortex, hipp~mpus and amygdala were studied by displa~ment with S-HT selective drugs. The results demonstrated the selective labelhng of S-Hf,-like sites by 13H]5-HT in the cortex, with little or no labelling of S-HTz or S-HT, sites. Self-displacement of the binding of [‘HIS-HT is consistent with the presence of a single population of sites, indicating that S-HT is non-selective for the S-HT, subtypes. Around 40% of the 5-HI’, sites in the frontal cortex and amygdala were of the S-HT,, subtype, in contrast to 60% in the hippocampus. The drug RU 24969 consistently displaced with, a high affinity, a greater proportion of [‘HIS-HT sites than did 8-OH-DPAT in all three regions of the brain. The nature of these additional sites was not established. A small proportion (<: 10%) of [‘HIS-HT sites in the frontal cortex appeared to be of the 5-HT,, subtype, as these sites were displaced with high affinity by mianserin. Summary-Binding

Key wor&-+‘H]S-hydroxytryptamine

(5-HT) binding, S-HT,-like binding sites, human postmortem brain.

The division of serotonin (5_hydroxytryptamine, 5-HT) receptors into three major subclasses (S-HT, , 5-HT, and 5-HT,), within the mammalian central nervous system (CNS), is now generally accepted Fozard, Humphrey, (Bradley, Engel, Feniuk, Middlemiss, Mylecharane, Richardson and Saxena, 1986; Kilpatrick, Jones and Tyers, 1987). The 5-HT, class of binding sites have been further subdivided on the basis of distribution and pharmacological selectivity into IA, lB, SC, ID and 1E subtypes (Pedigo, Yamamura and Nelson, 1981; Pazos and Palacios, 1985; Heuring and Peroutka, 1987; Titeler and Herrick-Davis, 1988). The distribution and detailed characterisation of the 5-HT, subtypes has been most extensively studied in the rat and bovine brain. In view of the possible involvement of altered function of 5-HT receptors in several disorders of the CNS (e.g. anxiety, depression), similar detailed characterisation in human brain is desirable. Selective radioligands have been developed for the three major subclasses of S-HT receptors (Peroutka and Snyder, 1979; Leysen, Niemegeers, Van Nueten and Laduron, 1982; Kilpatrick et al., 1987) and for 5-HT,,, 5-HT,, and 5-HT,, sites (Gozlan, El Mestikawy, Pichat, Giowinski and Hamon, 1983; Hoyer, Engel and Kalkman, 1985). As all 5-HT,-like binding sites appear to be labelted by nanomolar concentrations of [3H]5-HT, the sites labelled by this ligand were character&d in postmortem human brain. *Present address: Research Department, The Boots Company PLC., Nottingham NG2 3AA, U.K. **To whom correspondence should be addressed.

METHODS Materials

Hydroxytryptamine creatinine sulphate 5-[ 1,2-3H(N)]-(specific activity 20-29 Ci/mmol) was obtained from New England Nuclear, Boston. Drugs were obtained from the following sources: 5-HT, 5-hydroxytryptamine (Sigma Chemical Co., Poole, Dorset); 8-OH-DPAT, (f)-&hydroxy-Z(di-npropylamino)tetraiin hydrobromide and TFMPP, l[3-(t~fluoromethyl)phenyl]-pi~r~ne hydrochlo~de (Semat Technical (U.K.) Ltd, St Albans, Hertfordshire); buspirone HCl (Bristol-Myers Company, Evansville, Indiana); ipsapirone HCl (Troponwerke, Cologne, F.D.R.); mianserin HCl (Organon Laboratories Ltd, Cambridge); spiperone and ketanserin (Janssen Pharmaceutical Ltd, Wantage, Oxon); S-CT, S-carboxamidotryptamine maleate, GR 38032F, 1,2,3,9-tetrahydro-9-methyl-3-[2-methyllH-imidazol1-yl)methyl]-4H-carbazol-4-one hydrochloride dihydrate (Glaxo Group Research, Ware, Hertfordshire); RU 24969, 5-methoxy-3-( 1,2,3,6-tetrahydro-4-pyridinyl) lH-indole (Centre de Recherche Roussel UCLAF, Romainville, France); MDL 72222, laH, 3a,Sa H-tropan3-yl 3,5-dichloro~nzoate (Merrell Dow Research Institute, Strasbourg, France). Selection of subjects

Tissue (frontal cortex [Brodmann area lo], hippocampus and amygdala) was obtained from 10 male subjects (mean age + SEM, 48 f 2 years) dying suddenly after myocardial infarction. The mean time between death and storage of the tissue (postmortem 1055

deIay) was 38 F 5 hr. Tissue storage was in plastic screw-topped tubes at -80°C until assay.

Rosebrough, Farr and Randall, 1951), using bovine serum albumin as the standard.

Membranes for f3H]5-HIT binding were prepared as described by Cheetham and Horton (1989). Briefly, frozen brain was homogenis~d in ice-cold 0.25 M sucrose (1:20 w/v) with a motor-driven t&on pestle (120 rpm). Myelin and cell debris were removed by centrifngation at 1OOOgfor 10 min. The supernatant was stored on ice and the pellet rehomogenised in sucrose (1: 10 w/v) and centrifuged at 75Og for 10 min. Combined supernatants were diluted with 50 mM Tris-HCI buffer, pH 7.4 (at 2YC) to 1: 100 w/v and centrifuged at 35,OOOgfor 10 min. The pellet was resuspended in Tris buffer (1: 100 w/v) and recentrif~g~ at 35,ooOg for IOmin. To remove endogenous 5-HT, the pellet was resuspended in Tris buffer (1: 50 w/v), incubated at 37°C for 10 min and ~ntrifuged as above. The pellet was washed by resuspension in Tris buffer (1: 100 W/Y) and centrifuged as above. The final pellet was resuspended in Tris buffer (equivalent to 14mg wet weight of tissue per ml for frontal cortex and 7mg wet weight of tissue per ml for hippocampus and amygdala) and imm~~ately used in the binding assay. All centrifugations were carried out at 4°C.

Binding parameters were estimated using weighted least-squares nonlinear regression analysis, fitting a one-component model in elf-displacement of [“HI5-HT or two component mode1 in ~splacement of rH]S-HT by various drugs. The computer programme was an adaptation of the method described by De Lean, Hancock and Lefkowitz (1982). The &, values for @IJS-HT, used in the analysis of competition experiments, were obtained from saturation binding of [sH]S-HT in the same membrane preparation.

Binding assays for {3H]5-HT were carried out in 5 ml plastic tubes. Solutions of ligand and drug were prepared in 50mM Tris-HCl, pN 7.4 (at 25°C). ~u~~r~r~on ~j~d~ng s~~dies. 700 ~1 of freshly prepared membranes, 100 ~1 50 mM Tris-HCI, pH 7.4 containing 40 mM calcium chloride (4mM final), 100 ~1 [3H]5-IIT (3 c~n~ntratio~s, O&32 nM), 100 pl 50mM Tris-I-ICI, pH 7.4 (total binding) or nnla~lled 5-HT (10 PM final, to define non-s~cific binding) were incubated for 10min at 37°C. Ihug d~~~a~e~e~~ studies. For membranes from the frontal cortex the binding of f3HfS-NT at a single concentration of ligand of 3.0 nM was determined as above and in the presence of varying concentrations (5 x 10-‘“-10-3 M) of the following drugs: 5-HT, 8OH-SPAT, ipsapirone, spiperone, 5-CT, buspirone, RU 24969, TFMPP, mianserin, ketan~~n, GR 38032F and MDL 72222. Similarly, in the hippocampus and amygdafa the binding of 3 nM [3H]S-HT was determined as above and in the presence of varying con~n~rations (lO-iO10Tw3 M) of S-HT, I-OH-DPAT and RU 24969. Membrane bound radioactivity was recovered on Whatman CF/B filters by rapid ~it~a~on under vacuum, using a Brandel cell harvester. Filters were washed with 16 ml ice-cold Tris buffer and radioactivity was determined by liquid scintillation counting (6 ml Packard ~intillator 299) at an efficiency of 38--450/o. Ahquots of membrane were stored at -20°C for subsequent determination of protein (Lowry,

REWL'ES

~ispla~ment of the binding of (3H]S-HT in the frontal cortex, hip~campus and amygdala by S-HT and 8-OH-DPAT is shown in Figure I. Kinetic parameters for all drugs studied are summarized in Tables 1-3. In all three regions of the brain, 5-HT displaced the binding of f3H]5-HT from a single population of sites with high a~nity (Fig. 1; Tables l-3). However, some of the drugs studied were able to distinguish high and low affinity binding components, In the frontal cortex, 8-OH-DPAT, ipsapirone, spiperone, 5-CT and buspirone displaced the binding of i3H]5-HT in a biphasic manner, with Hill slopes signifi~ntly less than unity {Table 1). The displa~ment of the binding of [sH]5-HT by these drugs fitted a two-com~nent model, with all five drugs displacing a similar proportion of sites (3S-53%) with high affinity f&t = 7-200 n&i) and the remaining sites with low affinity (KDz= 4-69 FM; Table 1). These drugs displaced from a total num~r of binding sites (936-1184 fmol/mg protein), comparable to the number of sites displaced by S-HT (1160 fmol~mg protein). The binding of E3H]5-HT was also inhibited by 8-OH-DPAT in a biphasic manner in the hippocampus and amygdala (Fig. If, with Hill slopes significantly less than unity (Tables 2 and 3). Again inhibition of the binding of [3H]5-HT by S-OHDPAT fitted a two component model. ~ispla~me~t with high affinity was from a similar proportion of sites in the amygdala (43%) as in the frontal cortex (42%) but from a greater proportion of sites in the hippocampus (63%), In frontal cortex, TFMPP was only a weak dispiacer, with no inhibition of the binding of 13H]S-HT apparent in the nanomolar range and only 32% inhibition of specific binding of [311]5-HT at a eoncentration of 1 PM. The drug RU 24969 displaced the binding of t’HJS.HT in a biphasic manner, in all three regions of the brain, with Hill slopes significantly less than unity (Tables l-3). Also RU 24969 disptaced with a high affinity (1y,, 39-140 nM) from a greater proportion of sites than did 8-OH-DPAT in

Human brain [‘HIS-HT binding sites

1057

go-

P p iE

s E

i?

80-

‘2

0Q-

70-

;

70.

60-

E

60-

b 50k% 9 405 g 30b 20-

*a 50tA g 401

011 II

’

IO

3

9

3

0

’

8 7 6 -log CDrugl M

’

5

30-

’

4

’

3

Fig. 1. Inhibition by S-HT (0) and 8-OH-DPAT (0) of the binding of [3H]S-HT(3.0 nM) in membranes from human frontal cortex (A), hippocampus (B) and amygdala (C). Values are means of 3-6 determinations. the SEM have been omitted for clarity.

S-HT in the frontal cortex. Ketanserin displaced from an apparent single population of sites with low affinity (K,, = 1.6 PM) and a Hiti slope close to unity. Mianserin displaced with high affinity (I;c, = 22 nM)

the frontal cortex (61% vs 42%), hippocampus (90% vs 63%) and amygdala (70% vs 43%). Ketanserin and mianserin displaced only 60% and 55%, respectively, of the [3H]S-HT sites displaced by Table

I. Binding parameters and Hill numbers of various drugs for the displacement of the binding

of 3.0 nM [W]5-HT to membranes from

frontal cortex

t&m.,+ &,xJ

Hill slope

ll60_+ 170 llO2+ 176

0.93 2 0.02 0.49 rf.0.03

I151 * 190

0.50 & 0.04

1184+ 119

0.51 * 0.04

975 * 109

0.38 i: 0.02

Total

KD2

KD! 12.6 + 2.1 7.5 f 1.8

4050 + 800

Ipsapirone

6.9 + 1.3

9500 f 3700

Spipcrone

206+42

5-HT 8.OH-DPAT

3OGOO i 3850

S-CT

55i23

56500 f 15500

Buspirone

701t8

69000 + 15000

RU 24969

139+41

59000 * 13OOu

Mianserin

22+ IO

1180*360

nM; B,,,,,.fmol/mg protein; determinations.

1160& 170 439 * I2 (42.2 + 6.8) 369 + IO (34.8 + 5.2) 622*64 (52.5 & 3.6) 413 f 30 (42.9 + 4.2) 458 f 35 (43.5 it 2.6) 572 + 84 (60.5 + 5.5) 85 f 43 (12.4 k 5.2)

ISSO* 100

Ketanrrin &,,

Bmm,

B,,,,,

as percentage of total [B,,,,,, +

Bmu2

663 It 164 (57.8 It 6.8) 782 + 180 (65.2 i 5.3) 562 F 55 (47.5 +i 3.6) 562 + 79 (57.1 + 4.2) 594+44 (56.5 +- 2.6) 364*46 (39.5 f 5.5) 549 f 35 (87.6 f 5.2) 703 f 29

1052 t 79

0.39 +_0.04

936 + 130

0.41 + 0.03

634 f 78

1.00*0.04

703 f 29

1.13+0.08

B,,,,,* ] is shown in parentheses. Values are means + SEM for 3-6

S. C. CHEETHAM ef al.

1058

Table 2. Binding parameters and Hill numbers of various drugs for the displacement of the binding of 3.0 nM (3H]5-HT to membranes from the hipp~~pus

S-HT 8-OH-DPAT RU 24969

&J, 2.3 f 0.2 3.2 + I.1 39 It. II

KD2

3 ma”,

Bmnx2

1136+22 6150 + 250 19000 + 12000

518 k 104

(63.3 * 1.2)

336 f 40 (36.7 + 1.2)

968 + 24 (89.5 f 4.4)

114+48 (IO.5 rt: 4.4)

Total [%,x, + Yn& I 1136k22 926 + 144

nil1

0.28 f 0.01

1082 k 72

0.74 f 0.02

SlOpe

0.97 * 0.04

KD, nM; BmSi, fmol/mg protein; B,,

as percentage of total I&,,, + B,,,,] is shown in parentheses. Values are means k SEM for 3-6 determinations.

Table 3. Binding parameters and Hill numbers of various drugs for the displacement of the binding of 3.0 nM [‘HIS-HT to membranes from the amygdala ir,, 5-HT 8-OH-DPAT RU 24969

KDl

3 in&

830 f 270

344k1.5

0.89 + 0.10

I .69 i 0.69 49+ 16

53000 * 20000

(42.8 + 3.~1

(57.2 + 3.5)

NO+46 (70.3 * 4.5)

232 rt 24 (29.7 & 4.5)

Total IBma.l+ Lx* 644+9 602 i 48 772 + 90

I

Hill slope 0.88 j, 0.04

0.46 + 0.05 0.39 i: 0.03

K,,. nM: B,,,, fmol/mg protein: B,,, as percentage of total [B,,,, + L?,,,,,~]is shown in parentheses. Values are means i SEM for 3-6 determinations.

from 12% of the mianserin-displaceable sites (equivalent to 7% of sites displaced by S-HT) and with low affinity (&, = 1.2 pM) from the remaining sites (~uivalent to 47% of sites displaced by 5-HT). A Hill slope of unity was however obtained, probably as a result of the small proportion of high affinity sites. The 5-HT, selective compounds, GR 38032F and MDL 72222, did not displace the binding of [3H]5-HT in the frontal cortex, in the nanomolar range and displaced only 31% and IS%, respectively, of specific [3H]S-HT binding at 10m4M. DISCUSSION

Under the assay conditions described, [‘HIS-HT labelled 5-HT,-like binding sites but not 5-HTz or 5-HT, sites. Ketanserin displaced from a single population of sites in the frontal cortex, with an affinity comparable to its affinity for 5-HT,, sites, and much less potently than its affinity for 5-HT, sites (Hoyer, Pazos, Probst and Palacios, 1986a; Hoyer, Pazos, Probst and Palacios, 1986b). Mianserin also displaced the majority of sites (47% of S-HTdisplaceable sites), with an affinity comparable to its affinity for 5-HT,, sites (Hoyer et al., 1986a). The 5-HT, selective antagonists, GR 38032F (Brittain, Butler, Coates, Fortune, Hagan, Hill, Humber, Humphrey, Ireland, Jack, Jordan, Oxford, Straughan and Tyers, 1987) and MDL 72222 (Fozard, 1984), were weak displacers of the binding of [3H]5-HT. These results indicate that [3H]5-HT did not label significant numbers of 5-HT, or 5-HT, sites in the human cortex. In contrast, [3H]5-HT clearly labelled sites with a pharmacological specificity of the 5-HT,, subtype. Drugs with high selectivity for 5-HT,, binding sites (8.OH-DPAT, ipsapirone and buspirone) displaced between 3544% of the binding of [‘HIS-HT in the

frontal cortex with nanomolar affinity. A similar proportion of sites was displaced with high affinity by spiperone (53%) and 5-CT (43%). The proportion of [)H]S-HT sites displaced with high affinity by 5-HT,A-selective drugs in the frontal cortex was comparable to previous reports (26%, Waeber, Dietl, Hoyer, Probst and Palacios, 1988a; 38%, Peroutka, Switzer and Hamik, 1989). Variations between studies may arise from differences in dissection technique, as the proportion of 5-HT,, sites has been reported to vary considerably within the layers of the human frontal cortex; in layer II 65-70% of 5-HT, sites are of the 5-HT,, subtype, whereas there are few in layer III (Hoyer et al., 1986a). The proportion of 5-HT,, sites in the amygdala (43%) was comparable with the frontal cortex, whereas the proportion in the hip~campus was greater (63%). This is compatible with the autoradiographic study of Pazos, Probst and Palacios (1987), with the hippocampus being reported to contain the greatest density of 5-HTIA sites in the human brain. Binding sites of the 5-HT,, subtype have been characterised in the brain of the rat by their high affinity for certain P-adrenoceptor antagonists and for RU 24969 and TFMPP (Asarch, Ransom and Shih, 1985; Hoyer er al., 1985; Peroutka, 1986). The weak displacement of the binding of 13H]5-HT by 1 p M TFMPP in the human frontal cortex, contrasts with almost total displacement by the same concentration in the frontal cortex of the rat (Peroutka, 1986) and clearly illustrates species differences. However, in all three regions of the brain, RU 24969 consistently displaced [3H]5-HT with a high affinity, from a greater proportion of sites than did 8-OHDPAT. Since there is now strong evidence for the absence of 5-HT,, sites in human brain (Hoyer et al., 1986a; Pazos e; al., 1987). alternative explanations

Human brain [3H]5-HT binding sites for the differences between the proportion of sites displaced by 8-OH-DPAT and RU 24969 need to be considered, One possibility is that these additional sites are of the MIT,, subtype, which have been characterised in bovine caudate and human frontal cortex, caudate and globus paflidus (Heuring and Peroutka, 1987; Waeber et al., lP88a; Peroutka et al., 1989) and their distribution determined autoradi~grap~j~ally in human brain (Waeber, SchoefTter, Prtlacios and Hoyer, 1988b). The drug RU 24969 has a 30-40-fold

greater affinity than 8-OH-DPAT for 5-HT,n sites in human frontal cortex (Peroutka et al., 1989), compared to a 4-fold lower affinity than 8-OH-DPAT for 5-WT,A sites (Hoyer et al., 1986a). Thus, high affinity displacement from both SHT,, and S-HT,n sites by RU 24969 might explain these differences. However, two pieces of evidence suggest that this interpretation is incorrect. The SHT,, sites have been reported to represent 43-75X of sites labelled by [3H]5-HT in the human frontal cortex (Waeber et at., 1988b: Herrick-Davis, Titeter, Leonhardt, StrubIe and Price, 1988; Peroutka et al., 1989). Thus, the proportion of SHT,, sites is clearly greater than the difference observed here between 8-OH-DPATand RU 24969high affinity-displaceable sites in the frontal cortex. Secondly, S-CT exhibits nanomolar affinity for both 5-HTiA and 5-HT,n sites in the human cortex (Hoyer ef d, 1986a; Herrick-Davis et al., 1988). Thus, displacement of rH]S-HT from z%-IT,~ and MIT,, sites by 5-C-f would be predicted within a similar concentration range, resulting in a much greater proportion of sites being displaced with high affinity by 5-CT than by I-OH-DPAT. Clearly, this was not the case. Both 5-CT and &OH-DPAT displaced from an identical proportion of sites in the frontal cortex. A further possible explanation for the differences observed between I-OH-DPAT and RW 24969 relates to 5-WT,e binding sites. These sites have a distinct pharmacological specificity and are highly concentrated in the choroid plexus, but are also present in low density in human cortex and hippocampus (Hoyer et nl., 1986bj. Although RU 24969 has a higher affinity than 8-OH-DPAT for 5-HTrc sites in human choroid plexus, the affinity of both drugs is low, with no significant displacement from 5-HTIc sites within the nanomolar range. Thus, the nature of the additional sites, displaced by RU 24969, was not established. Of the drugs studied, only mianserin had nanomolar affinity for 5-HTic sites. The small proportion of [3W]5-I-IT sites displaced with high affinity by mianserin in the frontal cortex was compatible with the reported low density of 5-HT,, sites in human cortex (Hoyer et al., 1986b; Her&k-Davis et a[., f988). It is tempting to speculate that those binding sites displaced with low affinity by the 5-HTiA-selective drugs were 5-HT,n sites. The high density of these

1059

sites, and their micromolar affinity for 5-HT,,selective drugs, is compatible with binding data for [3H]5-HT, obtained in the presence of selective blockade of other 5-HT, subtypes (Herrick-Davis ef al., 1988). However, the high affinity of S-CT for S-HT,, sites would seem to preclude this inte~reta~on. On the basis of the available evidence, the pharmacological specificity of these sites appeared to be more compatible with the recently described 5-HT,, sites (low affinity for 5-ID-t,-selective drugs and for 5-CT, Titeler and Herrick-Davis, 1988). In all three regions of the brain, displacement by 5-HT of [3H]S-HT was well fitted to a single binding site model, suggesting that 5-HT has a similar affinity for all 5-HT,-like sites. However, differences in affinity of 5-HT were observed between the various regions of the brain, suggesting that small differences in affinity may exist. In this paper, analysis of the displacement of [jH]S-H’F was limited to one or two-site binding models. Although the binding parameters, generated by these methods, fitted the experimentai data well, the interpretation was limited by this approach, For example, apparent high affinity sites may consist of unresolved sites of high and intermediate affinity. The proliferation in the variety of 5-HT, sites that have been proposed, now exceeds the methods available to analyse them in a statistically meaningful way, ~~k~o~~e~~e~e~~~-We thank the Weflcome Trust and the Humane Research Trust for financial support and Dr M. R. Crompton for his heip during this study.

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Peroutka S. J. (1986) Pharmacological differentiation and characterization of 5-HT,,.‘. , S-HT,,, and S-HT,, binding sites in rat frontal cortex. J. Neu&hem. 47: X%540. Peroutka S. J. and Snyder S. H. (1979) Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [)H]lysergic acid diethylamide and [‘HIspiroperidol. Molec. Pharmac. 16: 687-699. Peroutka S. J., Switzer J. A. and Hamik A. (1989) Identification of 5-hydroxytryptamine,,, binding sites in human brain membranes. Synapse 3: 61-66. Titeler M. and Herrick-Davis K. (1988) Detection and characterization of the novel 5-HT,, receptor in human and other mammalian brain tissues. Society for Neuroscience Abstr. 221.13. Waeber C.. Diet1 M. M., Hoyer D.. Probst A. and Palacios J. M. (1988a) Visualization of a novel serotonin recognition site (5-HT,,) in the human brain byautoradiography. Neurosci. Left. 88: 11-16. Waeber C., Schoeffter P., Palacios J. M. and Hoyer D. (1988b) Molecular aharmacoloev of S-HT,, recoenition sites: Radioligand binding stud& in human: pig aid calf brain membranes. Naunyn-Schmiedebergs Arch. Pharmac. 337: 595-60

I.