© Masson, Paris, 1981 J. PhysioL. Paris, 1981, 77, 351-362 Symposium The Serotoninergic Neuron

CNRS, Marseille, July 9-11, 1980 Serotoninergic receptors in brain tissue" properties and identification of various aH-ligand binding sites in vitro

J.E. LE¥SEN

Department of Biochemidal Pharmacology, Janssen Pharmaceutica, B-2340 Beerse.

compounds, known as labelled ligands. This allowed SUMMARY: detection of high affinity binding to membranes in In vitro binding studies to serotoninergic receptors were suspension. In vitro binding studies were much performed using 3H-LSD, 3H-5-HT and 3H-spiperone. An facilitated by the empirical finding that labelled mem- overview is given on findings using these three ligands with branes were retained through adsorption on glass-fibre respect to the following : (1) Localization of specific binding filters after vacuum filtration, thus providing a fast and sites, i.e., in various animal species, the regional distribution in simple technique for separating <tryptamine-induced clonic seizures in rats. position of the assay medium ; binding kinetic prop- There was a very significant correlation between binding erties ; receptor regulation in vivo. affinities for 3H-LSD and 3H-5-HT labelled sites in the hippo- campus. These hippocampal binding sites are different from III. Identity of the binding sites : differences between those for 3H-LSD and 3H-spiperone in the frontal cortex, sites for various 3H-ligands ; pharmacological specificity Binding to 3H-5-HT or _H-LSD sites in the hippocampus could of the membranous binding sites ; chemical composition not be related to a pharmacological effect, of the macromolecular complex constituting the binding site. Key-words : Review. Serotoninergic receptors. In vitro binding. 3H_5_hydroxytryptamine. 3H_LSD. 3H.spiperone. IV. Function of the receptors. Serotoninergic receptors on membranes were first studied by biochemical means using 3H-lysergic acid INTRODUCTION diethylamide (LSD) to label specific binding sites. Using an equilibrium dialysis method, FARROW and VAN The biochemical study of specific binding sites on VUNAKtS (1972, 1973) demonstrated saturable and ste- membranes for neurotransmitters or drugs, and the reospecific d-LSD binding in rat cerebral cortex. The relationship between such sites and pharmacologically or physiologically defined receptors, became possible occurrence of high affinity stereospecific LSD binding through the availability of highly radioactively labelled sites in several rat brain areas was confirmed by BEN- NETT and AGHAJANIAN (1974) using the rapid filtration method. Ever since, membranous serotoninergic recep- • Re_:uenpremi_relecturele10juillet1980, endeuxi_melecturele14aoflt1980, tors have been widely studied using 3H-LSD, 3H-5- 352 J.E. LEYSEN .Journal de Pb.;'sto/osie hydroxytryptamine (5-HT) and 3H-spiperone, mainly in oral cortex and also occurred in high amounts in the brain tissue of various mammals, but also in peripheral striatum. In the latter area, however, 3H-LSD was tissues, lower animal species and cell cultures, shown to label dopaminergic receptors (BURT et al., The present review is focussed on the investigation 1976). Serotoninergic receptors labelled with 3H- of brain serotoninergic receptors. An attempt is made to spiperone were most abundant in rat frontal cortex summarize the relevant literature, to indicate problems (LEYSEN and LADURON, 1977 ; LEYSEN el al., 1978 b). and gaps in our knowledge with respect to the four major Similar binding sites could be detected in the limbic points mentioned above, areas (nucleus accumbens and tuberculum olfactorium) The review is supplemented with some original provided that binding to dopaminergic receptors was work, recently performed in our laboratories, relating prevented by the addition of an excess of a selective binding affinities of drugs for sites labelled by 3H-5-HT, dopamine antagonist (domperidone) or dopamine ago- 3H-LSD and 3H-spiperone in rat frontal cortex and nist (2-(N,N-dipropyl)amino-5,6-dihydroxytetralin or hippocampus with the potencies of the drugs in in vivo ADTN) (LEYSEN et al., 1979 ; QUtK and IVERSEN, and in vitro pharmacological tests. 1979). It thus appeared that the regional distribution of I specific binding sites for the various 3H-ligands used for labelling serotoninergic receptors was different and METHODOLOGY did not correspond to the content of endogenous 5-HT nor to the distribution of 5-HT uptake sites in the In general, a similar technique was used by most brain. researchers. Fresh or frozen brain tissue is homogenized Investigation of the subcellular distribution of the and a particular membrane fraction is prepared by specific binding sites by diffential centrifugation re- centrifugation techniques. The washed, and occasionally vealed the highest enrichment in the microsomal fraction preincubated membrane fraction is suspended in high for all three ligands (BENNETT and SNYDER, 1975, 1976 ; dilution (about 1 mg protein/ml) in a given assay LEYSEN et al., 1978 a). However, by discontinuous medium at physiological pH. Membrane suspensions are gradient centrifugation of lysed mitochot_drial fractions, incubated with nanomolar concentrations of a 3H-ligand 3H-5-HT and 3H-LSD binding were mostly found in the in the absence or the presence of unlabeUed drugs. When synaptosome ghost membrane fraction (BENNETT and maximal binding is reached, the incubation mixture is SNYDER, 1975, 1976 ; FILLION et al., 1978). rapidly filtered under vacuum through glass fibre filters Cellular distribution of binding sites was inves- and rinsed. Radioactivity on the filters is counted in a tigated following various kinds of lesions in rat brain. liquid scintillation counter. Specific 3H-LSD and 3H-5- Lesions of the mid-brain raphe nuclei by 5,6-dihydroxy- HT binding was usually defined by the portion tryptamine did not produce a change in the maximal inhibited using excess unlabelled LSD or 5-HT number of 3H-LSD or 3H-5-I-tT binding sites in the respectively. Specific 3H-spiperone binding was defined cortex (BENNETT and AGHAJANIAN, 1974 ; BENNETT by inhibition using unlabelled (+)-butaclamol or and SNYDER, 1975, 1976) or the forebrain (FILLION et cinanserine. Binding data are analysed according to al., 1978 ; SEGAWA et ai., 1979). However, after 5,7- transformations of the law of mass action or first and dihydroxytryptamine lesions, 3H-5-HT binding sites second order rate equations (see SEGEL, 1975). Besides were reported to be 40 % increased in the hippocampus the binding assay, various other techniques are used in and 25 % in the striatum. However, the latter appeared receptor research such as centrifugation, cell-lesion, not to be a significant change (NELSON et ai., 1978). The enzyme treatment, solubilization and purification. KD-values seemed not to be significantly altered in any of the studies. 5-HT uptake sites were always completely abolished, indicating that 5-HT neurons had been I. -- LOCALIZATION sufficiently lesioned. It was concluded that 3H-LSD and 3H-5-HT binding sites seemed not to occur on presynaptic The regional distribution in the brain of specific membranes of these neurons. Following kainic acid 3H-LSD and 3H-5-HT binding was studied in rats lesions in rat striatum, high affinity _H-5-HT binding (BENNETT and AGHAJANIAN, 1974 ; BENNETT and disappeared and low affinity binding became apparent SNYDER, 1976 ; FILLION el al., 1976 ; NELSON el ai., (FILLION et al., 1979). Therefore, high affinity 3H-5-HT 1978) and in monkeys (BENNETT and SNYDER, 1975), binding sites were assumed to be localized on striatal stereospecific 3H-spiperone binding was studied in rat inter-neurons. Following 6-hydroxydopamine lesions of brain (LADURON el al., 1978). The 3H-5-HT binding the nigrostriatal pathway, 3H-5-HT binding sites in the was most enriched in the hippocampus and the striatum striatum remained unchanged, indicating that the sites and less was found in cerebral cortex areas. In con- were not localized on dopaminergic neurons (REISINE et trast, 3H-LSD binding was most enriched in the cere- ai., 1979). Effects of brain lesions on 3H-spiperone Vo/. 77, n ° 2, 1981 IN VITRO BINDING TO SEROTONINERGIC RECEPTORS 353

binding to brain serotoninergic receptors have not yet washing and short preincubation of the membranes was been reported so far. necessary to remove endogenous 5-HT, resulting in a 4-fold increase in the apparent binding affinity of 3H- 11. m PROPERTIES 5-HT (NELSON et al., 1978). We have recently noticed that KD-values vary considerably amongst several batches A) In vitro binding properties, of 3H-5-HT, even when impurities in the 3H-ligand are Binding properties can be considerably influenced beyond the detection limit of a thin layer chroma- by assay conditions. In particular, 3H-5-HT binding tography analysis. Therefore, variations in Ko-values are seemed to be sensitive to the ionic composition of the difficult to interpret and cannot be taken as an indication medium. The specific binding showed a 20-35 % increase for variations in binding sites. in 4 mM Ca ++ and a 30 % reduction in high Association and dissociation rates at 37 °C ap- concentrations of Na + and K + (BENNETT and SNYDER, peared to be fast (tl/2 --<2 rain) for all 3H-ligands, and 1975, 1976; NELSON et aL, 1978). aH-LSD and 3H- cooperative effects of binding were not apparent in spiperone binding were much less affected. However, we dissociation studies using rat cortex tissue (BENNETT and recently observed that in the presence of physiological SNYDER, 1976 ; LEYSEN and GOMMEREN, 1978). concentrations of Na ÷ (120 mM), K ÷ (5 mM), Mg ÷ ÷ When studying the inhibition of 3H-ligand binding (1 mM) and Ca ÷ ÷ (2 mM) and of 10 _tM pargyline and by unlabelled drugs, various anomalies may be 0.1 o/o ascorbic acid, the non-specific binding of encountered. For the inhibition of 3H-spiperone binding 3H-5-HT, 3H-LSD and 3H-spiperone was considerably by various 5-HT antagonists and agonists, it was reduced and specific binding of 3H-5-HT and 3H-LSD observed that 60 % of the binding was stereospecifically apparently increased with respect to the bindings inhibited by (+)-butaclamol, and that this part of the measured in Tris buffer not containing these ions. This binding apparently involved serotoninergic receptors. was not observed by others (GRIPENBERG and JANSSON, However, when using unlabelled spiperone, an addi- 1978). The effects of nucleotides on the in vitro binding tional 20 0/0 of the binding was inhibited with high is not clear. NELSON reported that 2 mM ATP with and affinity. This part of the binding appeared to be related without 2 mM Mg ÷ ÷ decreased 3H-5-HT binding in rat to the spirodecanone moiety of the molecule and showed forebrain b_/ 50-70 % (NELSON et ai., 1978), whereas chemical specificity, but lacked a relationship with a PEROUTKAdid not find any effect of 1 mM ATP, ADP, pharmacological effect of the compounds (LEYSEN and AMP and GMP on 3H-5-HT, 3H-LSD and 3H-spiperone GOMMEREN, 1978 ; HOWLETT et al., 1979). These binding in rat frontal cortex (PEROUTKA et al., 1979). In findings demonstrate the importance of an accurate contrast, 1 mM GTP and GDP reduced 3H-5-HT definition of specific binding and the danger of using the binding by 40 % and 3H-LSD binding by 20 %, whereas unlabelled ligand. Indeed, membranes may accidentally 3H-spiperone binding was not affected. The guanine contain high affinity and saturable binding sites which nucleotides appeared to decrease the apparent binding specifically bind certain structural moieties, but which affinity of agonists without changing the number of are not related to a pharmacological or physiological binding sites (PEROUTKA et al., 1979). receptor. Even more confusing are the numerous reports on Furthermore, it has been observed that inhibition of the investigation of binding affinities of the 3H-ligands. 3H-LSD bindirig by unlabelled 5-HT and vice versa, For 3H-5-HT, KD-values, derived from Scatchard produced shallow inhibition isotherms or slopes in Hill analysis, varied between 1.4 and 8 nM for high affinity plots of less than unity (BENNETT and SNYDER, 1975 ; binding, and occasionally, a concomitant lower affinity FILLION et ai., 1978). Others noted that the inhibition of site (KD = 30 nM) was observed (FILLION et al., 1978 ; 3H-5-HT binding by quipazine, yielded a slope < 1 in SECAWA et al., 1979). The KD-values of 3H-LSD ranged the Hill plot, whereas inhibition by antagonists such as between 4 and 10 nM. Curved Scatchard plots were metergoline, cinanserine, pizotifen yielded a slope = 1 observed for 3H-spiperone binding in rat frontal cortex (NELSON et al., 1978). One parsimonious interpretation microsomal fraction (Ko-values : 0.6 and 1.4 nM), was that interactions between various compounds were whereas plots were slraight using the mitochondrial not always competitive. However, a systematic study of fraction (Ko = 1.3 nM) (LEYSEN et al., 1978 a). Using a the type of inhibition between various compounds using total membrane fraction, PEROUTKA found a KD-value various 3H-ligands in different assay conditions has not of 0.7 nM (PEROLITKA and SNYDER, 1979). The various been carried out. In the case of the binding of dopamine studies reported in the literature were performed using agonists and antagonists to dopaminergic receptors on tissue from different species, different brain areas and striatal membranes, it has recently been argued that subcellular fractions and variable assay conditions. This physicochemical events at the surface of the membrane renders the comparison and the evaluation of the data micelles may cause anomalous binding patterns (LEYSEN nearly impossible. NELSON claimed that extensive and GOMMEREN, 1980). In particular, when using 354 J.E. LEYSEN Journal de PhysloloRie

compounds of different physicochemical nature, such as 3H-antagonists have to be compared. In addition, the water-soluble agonists and lipophilic antagonists, specificity and binding affinity of a wide variety of different surface effects may be provoked. Prior to the compounds have to be investigated. As mentioned interaction with the binding sites, compounds with before, 3H-ligands thus far used to label serotoninergic different physicochemical properties will differentially receptors are: 3H-5-HT, 3H-LSD and 3H-spiperone. interact with the surface of the membrane micelles and 3H-Methiothepine appeared unsuitable because of its too already be non-competitive at this level. With these high non-specific binding(NELSON et ai., 1979). Binding assumptions, it may be argued that observations of of 3H-5-HT has mainly been studied in forebrain, cortex irregular binding patterns are not necessarily an and hippocampus. Binding of 3H-LSDand3H-spiperone indication of the involvement of multiple sites. Surface to serotoninergic receptors had to be studied in cortical phenomena always occur during interactions between areas, since in brain areas rich in dopaminergic receptors compounds in solution and suspended micelles {either such as the striatum and the mesolimbic areas, these membrane micelles or detergent micelles which contain compounds also label dopaminergic receptors with high _ solubilized >_receptors). This is often overlooked in in affinity. As mentioned above, the latter binding can be vitro binding studies. Since they may considerably prevented by adding, in excess, a selective unlabelled influence the binding, they should be taken into account dopamine receptor blocker, but this has only been when analyzing binding data. occasionally applied (WHITAKER and SEEMAN, 1978; LEYSENet al., 1979). B) In vivo regulation of the binding sites. Sites labelled with 3H-5-HT in the cerebral cortex, The ontogenetic development of 3H-5-HT binding caudate or forebrain exhibited a very high selectivity sites was investigated in rat cerebral cortex areas and towards structural derivatives of 5-HT. The 5-hydroxyl superior collicus (UZSECOV et al., 1979). Area specific group appeared necessary for a binding affinity at decline in the number of receptors from birth to nanomolar concentrations. Besides these, some ergot adulthood was noticed. However, in these studies derivatives also showed high binding affinity, such as optimal binding was measured at pH 8.5-9.0, which was metergoline, ergotamine and d-LSD. The binding sites different from all other studies. The instability of the 3H- 5-HT in these conditions might cause problems for appeared stereoselective since d-LSD was much more measuring specific binding, potent than the l-enantiomer. 5-HT antagonists lacking The effect of various drug treatments on 3H-5-HT an indole nucleus, such as cyproheptadine, mianserin binding in rat brain has been investigated by several and pizotifen, showed moderate binding affinity. authors ; data are summarized in Table 1. Experimental Harmaline derivatives and 5-hydroxyindole acetic acid, protocols differed widely, findings were unclear and although containing an indole nucleus, did not occupy irreproducible either in different brain areas or in the sites at micromolar concentrations and neither did neurotransmitters other than 5-HT (BENNETT and dift'erent laboratories. If anything, drugs which enhance the endogenous 5-HT concentration in the synaptic cleft SNYDER, 1976 ; FILLION et al., 1978 ; NELSON el ai., tend to decrease the number of 5-HT sites, without 1978 ; WHITAKER and SEEMAN, 1978 b). Sites labelled changing the Ko-values. Repeated administration of by 3H-LSD in the cerebral cortex displayed a different LSD decreased both the Bmax and Ko-values. Metergoline drug selectivity. Various 5-HT antagonists were potent seemed to increase Bmax in some brain areas after competitors, whereas 5-HT derivatives were less potent. prolonged treatment. After a single administration of However, 5-HT showed a higher binding affinity than methiothepin, Bmax was increased 3-6 days afterwards, other neurotransmitters (BENNETT and SNYDER, 1976 ; The 5-HT depletor, p-chlorophenylalanine, seemed to FILLION et al., 1978). Neither 3H-5-HT nor 3H-LSD sites increase the Bronx-value considerably after prolonged seemed pharmacologically related to 5-HT uptake sites treatment. Effects of drug treatment on 3H-LSD and {NELSON et aL, 1978) and antidepressants revealed only 3H-spiperone binding to serotoninergic receptors have weak binding affinity {'FANG and SEEMAN, 1980). not yet been reported, except for monoamine oxidase Binding affinities of compounds for sites labelled by 3H- spiperone in rat frontal cortex correlated significantly inhibitors, which do not seem to affect the binding. However, after treatment with monoamine reuptake with binding affinities for 3H-LSD labelled sites in the blockers, variations in adrenergic receptors were same area. Besides 5-HT antagonists, neuroleptics with reported along with the variations in 3H-5-HT binding antiserotoninergic properties bound to these sites at nanomolar concentrations. The binding affinities sites (references in Table 1). correlated significantly with the potencies of the drugs to antagonize tryptamine-induced clonic seizures in rats in 111. -- IDENTITY vivo (LEYSEN and LADURON, 1977; LEYSEN et a]., To identify binding sites with a neurotransmitter 1978 b). Hence, sites labelled by the agonist 3H-5-HT receptor, the sites labelled by various 3H-agonists and and by the antagonist 3H-spiperone, seemed to differ in VoL 77, n ° 2, 1981 IN VITRO BINDING TO SEROTONINERGIC RECEPTORS 355

TABLE !. -- Effect of various in vivo drug treatments of rats on specific J H-5-HT binding in brain areas.

Treatment 3H-5-HTbinding* Dose Duration Drug mpk, daily treatment, drug free Brain area Bma_ KD Reference

5-HT releasing agent d-fenfluramine 2.5 (2 ×) l.p. 14 d 5 d Diencephalon l -- SAMANINet at.. 1980 id. id. Other areas ,x, -- id. 28 d id. Diencephalon 1 -- id. id. id. Cortex 1 -- id. id. id. Brain stem _ -- id. id. id. Striatum ,_, -- id. Mono-amine reuptake id. id. Hippocampus ,x, -- id. blockers nisoxetin + fluoxetin** 10 + 10 (I ×) 21 d 48 h Frontal cortex 1 -- MAGGI et al., 1980 id. id. Hippocampus ,x, -- id. fluoxetin 10 (2 x ) i.e. 4 d 24 h Cortex ,x, ,x, SAVAGEet al., 1979 chlorimipramine 10 (2 x) I.P. 4 d 24 h Cortex ,,,, ,x, id. desmethylimipramine 6 (1 x ) i.e. 1, 4, 6, 24 h Cortex _ % BERGSTROMand KELLAR,1979 12w 2 w id. Cortex 1 ',,, id. various tricyclies 10 (2 x) J.P. 3 w 0.5-3 h Forebrain high affin. 11 ,x, SE(JAWA et al., 1979 low affin. 11 _, 30 (1 x ) l.p. acute 0.5 h Forebrain ,x, ,x, id. MA 0 inhibitors (A) clorgyline I (! x) l.P. 4 d 24 h Cortex 1 * SAVAGEet aL, 1979 (B) deprenyl 1 (1 x ) i.P. 4 d 24 h Cortex ,,,, ',,, id. 5-HT depictor p-chlorophenyl-alanine 300 (1 x ) acute 2 d Forebrain ,x, I l STEIGRADet aL, 1978 3 inj. 6-8 d intervals 2 d Forebrain ! ,x, id. 100 (1 X) 3 d ! d Forebrain "_, It id. 6 d 1 d Forebrain "_, 11 id. 9 d 1 d Forebrain ,x, 1 id. 12 d 1 d Forebrain I 1 id. Receptor agonist/antagonist LSD 0.1 (4 x) ,.P. 4 d 24 h Forebraln 1 1 TRULSONand JACOBS, 1979 Brain stem 1 1 id. bromo LDS O. I (4 × ) LP. 4 d 24 h Forebrain ,,,, ,x, id. metergoline 1 (2 × ) i.P. 14 d 5 d Various areas * -- SAMANIN et al., 1980 28 d id. Cortex I I -- id. id. id. Striatum l -- id. id. id. Hippocampus ! -- id. id. id. Diencephalon ,x, -- id. id. id. Brainstem * _ id. methiothepine 20 (1 x ) I.p. acute 1 h Forebrain i m NELSON et aL, 1979 id. 1 d Forebraln ,_, m id. id. 2 d Forebraln _, -- id. id. 3 d Forebrain ! m id. id. 3 d Hippocampus I ,x, id. id. 6 d Forebrain I -- id.

• - No inlo,nmtion ; - no challge ; I i,_crease ; I decrease ; I : < 50 e/0 ; II :50-100 o/0 ; 111 : > 100 % of controls. • * Separate administration of the drugs caused no change in _lt-5-HT binding in that study.

both distribution in the brain (see above) and in drug sites : 9.2 and 8.5 pmoles/g tissue. The latter apparently specificity. The fact that it concerns two distinct sites was only concerned part of the 3H-spiperone binding sites recently emphasized by PEROUTKA and SNVDER, who since we found a total amount of 21.9 pmoles/g tissue designated 5-HT-I receptors the sites labelled by (LADURON et al., 1978) and curved Scatchard plots 3H-5-HT which could be inhibited by 300 nM unlabelled (LEYSEN et al., 1978 a ; PEDIGO et al., 1978). PEROUTKA 5-HT, and 5-HT-2 receptors the sites labelled by 3H-spi- showed that in rat frontal cortex, 3H-LSD labelled both perone which could be inhibited by 30 nM unlabelled the 3H-spiperone and the 3H-5-HT sites (PEROUTKA and spiperone (PEROUTKA and SNYDER, 1979). In these SNYDER, 1979). studies using rat frontal cortex, they found an While the antagonist sites can be related' to a approximately equal amount of both types of binding pharmacological effect, this has not been the case yet for 356 J.E. LEYSEN Journal de Physiologic

the agonist sites. Because of apparent regional methodological specifications are given in Table I1. As variations, and a lack of sufficient data in the literature, reported before, a significant correlation was found we investigated relative binding affinities of a large series between binding affinities for 3H-spiperone and 3H-LSD of compounds for both 3H-spiperone and 3H-LSD sites labelled sites in the frontal cortex (Fig. 1). However, in in rat frontal cortex and for both 3H-5-HT and 3H-LSD the hippocampus, binding affinities for the 3H-LSD sites sites in rat hippocampus. The ICs0-values and correlated significantly with binding affinities for the3H-

TABLEII. -- Relative binding affinities (plCso, M)* of various compounds for JH-iigand binding sites in rat brain areas.

Frontal cortex Hippoeampus Compound (abbreviation) 3H-spiperone 3H-LSD 3H-5-HT 3H-LSD

Serotonin (5-HT) 5.5 ± 0.1 6.1 + 0.l 8.0 ± 0.6 7.8 ± 0.1 Bufotenin (BUFO) 5.8 ± 0.1 6.2 ± 0.07 7.1 ± 0.2 7.2 ± 0.3 Tryptamine (TRYP) 4.78 ± 0.07 5.3 ± 0.1 6.6 ± 0.2 5.9 ± 0.5 Apomorphine (APO) 5.0 ± 0.1 5.5 ± 0.l 5.2 ± 0,2 5 2(N.N-dipropyl)amino-5.6- dihydroxytetralin (TETRAL) 4.1 ± 0. l 4.4 + 0.1 < 5 < 5 Dopamine(DA) 3.6 + 0.1 3.7 ± 0.1 4.6 + 0.5 < 5 Noradrenaline (NA) _ -- 3.6 ± 0.2 < 5 Acetylcholine (ACh) _ -- < 4 < 3 ".., Ergotamine(ERGOT) 7.4 ± 0.1 7.9 ± 0.1 8.1 ± 0.4 8.7 ± 0.1 • " Lysergic acid diethylamide (LSD) 7.6 + 0.1 8.3 ± 0.1 7.6 + 0,2 8.0 ± 0.1 _'_ Metergoline (METER) 8.4 ± 0.2 8.6 ± 0.1 7.6 ± 0.2 7.8 ± 0.2 "_'i" Bromocryptine (BRCR) 7.4 ± 0.1 -- 7.1 -4-0.2 7.6 ± 0.3 -,, Methysergide (METHYS) 7.45 ± 0.05 8.1 ± 0.05 6.9 ± 0.0l 7.2 ± 0.2 "_ Cyproheptadine (CYPRO) 7.7 ± 0.1 7.55 ± 0.05 6.1 + 0.1 6.2 ± 0.1 NPizotifen (PIZOT) 7.7 ± 0.1 7.7 ± 0.1 5.7 ± 0.2 5.4 + 0.2 '-_ Mianserin (MIAN) 7.4 ± 0.1 7.35 ± 0.05 5.9 ± 0.4 5.8 ± 0.1 "_CinanserinKetotifen (KETOT)(CINAN) 5.06.9 ± 0.20.05 4.76.95 ± 0.20.05 5.05.4 ± 0.20.1 5.05.1 ±± 0.2 Spiperone (SPIP) 8.45 + 0.05 8.2 ± 0.0l 6.7 ± 0.2 7.12 ± 0.2 Methiothepine (METIT) 8.23 ± 0.07 7.95 ± 0.05 7.1 ± 0.4 7.3 ± 0.3 Propericiazine (PROPE) 8.1 ± 0,1 7.2 ± 0.1 5.2 ± 0.2 5.6 ± 0.2 Chlorprothixene (CPTX) 8.0 ± 0.05 7.61 ± 0.09 6.0 ± 0.1 6.3 ± 0.l -,, Pipamperone (PIPAM) 7.79 ± 0.06 7.57 ± 0.07 5.2 ± 0.2 5.6 ± 0.2 \Clothiapine (CLOTHIA) 7.74 ± 0.08 7.4 ± 0.1 -- -- Iknperidol (BENPE) 7.7 + 0.05 7.6 ± 0.1 6.6 ± 0.4 7.0 ± 0.3 • Milenperone OVtILEN) 7.4 ± 0.l 7.6 ± 0.1 6.1 ± 0.2 6.4 ± 0.1 ",,,, Azaperone (AZAP) 7.45 ± 0.06 7.5 ± 0.1 6.9 ± 0.5 6.8 ± 0.5 "x, Flupenthixoi (FPTX) 7.4 ± 0.05 6.75 ± 0.05 5.0 + 0.2 5.6 ± 0.2 "Clozapine (CLOZ) 7.32 ± 0.09 7.08 ± 0.07 5.4 ± 0.2 6.2 ± 0.2 Chlorpromazine (CPZ) 7.21 ± 0.07 6.6 ± 0.1 5.4 + 0.2 5.65 ± 0.2 (+)-butaclamol ((+)-BUT) 7.0 ± 0.1 7.38 ± 0.06 5.5 ± 0.2 6.6 ± 0.2 Haloperidol (HAL) 6.83 ± 0.07 6.15 ± 0.05 5.0 ± 0.2 5.5 ± 0.2 Bromperidol (BROMPE) 6.4 ± 0.2 m 5.1 ± 0.2 5.6 ± 0.2 Domperidone (DOMP) 6.4 ± 0.2 m 5.0 ± 0.2 5.5 ± 0.2 Oxiperomide (OXIP) 6.05 ± 0.05 6.35 ± 0.05 6.9 ± 0.2 7.1 ± 0.2 (--)-butaclamol < 6 < 6 < 5 -- Astimizole (ASTI) 6.8 ± 0.2 -- 5.0 ± 0.2 5.8 ± 0.2 Oxatomide (OXAT) 7.2 ± 0.2 6.0 ± 0.2 5.0 + 0.2 5.8 ± 0.2 Doxepin (DOXE) 7.1 ± 0.1 6.7 ± 0.2 5.2 ± 0.2 5.7 ± 0.2 Phenoxybenzamine (PHBZ) m _ 5.7 ± 0.5 5.8 ± 0.2 Phentolamine (PHENT) < 6 -- 5.0 ± 0.2 5.6 ± 0.2 Alprenolol (ALP) < 6 -- 5.3 ± 0.2 6.5 ± 0.2 Atropine 4.5 -- < 4 < 5

Assay conditions "H-ligand concentration 2 nM 2 nM 3 nM 3 nM non-specific binding 2 taM(+)-BUT 1taM LSD 2 oM LSD 2 laM 5-HT total membrane fraction 25 mg/2.2 ml 25 mg/2.2 ml 25 mg/2.2 ml 25 rag/2.2 ml buffer Tris-HCl Tris-HCl Tris-HCI Tris-HCI . 50 raM, pH 7.6 50 mM, pH 7.4 50 mM, pH 7.6 50 mM, pH 7.6 Salts** _ Salts** Salts** incubation temperature, time 37 °C, 10min 37 °C, 10rain 37 °C, 10rain 37 °C, 10 rain

* plCs0-values are the means of at least 3 independent determinations in duplicate, using 5 drug concentrations per inhibition curve. IC50(nM) = 109plc (M),i.e., the drug concentration inhibited 50 % of the specific binding of the labelled ligand. ** Salts : NaCI 120 raM, KC! 5 raM, CaCI22 raM, MgCI2 1raM, pargyline 101aM, ascorbic acid 0.1%. Voi. 77, n ° 2, 1981 IN VITRO BINDING TO SEROTONINERGIC RECEPTORS 357

RAT FRONTAL CORTEX ]

4' /TETRAL • KETOT

5 _TRYP

z(_ / oAPO bindinFI(;, gI. affini-- Correlatties o,fiondrugsbet{plCween_o, relatM) ivefor _ 6" o OXAT / O 5 tit _H-spiperone and JH-LSD labelled sites _ HAL in rat frontal cortex, c / J eOXlPeBUFO Spearman rank correlation coef- _ oCPZ ,/ ficient : r = .87, n = 32, p < .001. e OOXE Compounds and assay conditions are _ QFPT /. INAN indicated in Table II. z 7. eCL o¢ ilkPROPE MIAN/ "3 CLOTHIAe i_,n O(+)BUT 0 p]p_ILi_i i _" _._ cma C PTXe ----I[--_BENPE.... 0 '_ / PIZOT METITI Br(.SDI IERGOT 8" / eMETHY S / iISPIP OLSD 9 ; i ; i

plCso of 3H SPIPI2nM) BINDING

5-HTsites (Fig. 2). As indicated in Fig. 2, themost active Potencies of drugs to antagonize 5-HT-induced compounds in these tests were compounds with an indole contractions in the rat fundus in vitro (VAN NUETEN and nucleus such as 5-HT and ergot derivatives. There was no VANHOUTTE, 1980) did not correlate with binding correlation between binding affinities for 3H-spiperone affinities either in the frontal cortex or in the sites ill the frontal cortex and 3H-5-HT sites in the hippocampus. According to these observations, the sites hippocampus (Fig. 3). From these results it appears that labelled by 3H-spiperone and 3H-LSD in the frontal in the frontal cortex the sites labelled by 3H-LSD at 2 nM cortex could be related to t>, show the overall binding characteristics of the 3H- since these sites show binding properties which can be spiperone sites, indicating that in these conditions correlated with 5-HT mediated pharmacological effects. _( 5-HT-2 receptors )> should be more involved. In the However, both the'role and the significance of these sites hippocampus, 3H-spiperone sites were hardly measur- in the brain remains to be clarified. On the other hand, able, but _H-5-HT sites were present in high amounts, the 3H-5-HT binding sites were suggested to be coupled Since 3H-LSD is able to label these sites too, the hippo- to a 5-HT sensitive adenylate cyclase (FILLION et al.," campal 3H-LSD binding sites are similar to the 3H-5-HT 1979). This remains, however, to be proven. So far, the sites. As shown in Figs. 4 and 5, binding affinities for binding characteristics of the 3H-5-HT labelled sites 3H-spiperone sites in the frontal cortex correlated cannot be related to a pharmacological or physiological significantly with the drug potencies to antagonize effect of 5-HT and therefore cannot truly be called tryptamine-induced clonic seizures in rats (NIEMEGEERS t_ receptors >>. Extreme care has to be taken in the and JANSSEN, 1979) and with the potencies to antagonize identification of <

.,PPoc,MPosI

/ / / /

5 CINANo • 0_ ETOT ./BROMPE PIZOT e / /-.HAL O P/PA.MtIiS'PHENT FiG. 2. -- Correlation between relative o-- ";il-'Frx binding affinities of drags (plCstu ,44)jar z MIAN• .,T Z 6" • TRYP _ _PHBZ • -"/_OXATTI 3H-5-HTand hiJHppocampus-LSD labelled. sites in rat CYPROI¢ ¢ •CLOZ Spearman rank correlation coef- _" CPTX• ODECLEN ficient : r = .86, n = 36, p < .001. " Compounds and assay conditions are _-_ / OMILEN _ •ALP indicated in Table It. I:::i / _1 +) BUT tn IAZAP .J :I: 7- OXlP_/" •BENP "_ • SPIP BUFOT_ •METHYS O / MERIT m / OBRCR (HO),_"_ o,., S HT• /-0 METER 11"1 8" / • LSD R'"_. R / R'"N _ IBr)

/ • ERGOT °/ t 9,/'

pICs0 of 3H 5HT (3nM) BINDING

effects. Since so far, high binding affinity to the 5-HT constituting the binding site in the membrane has been labelled sites seemed to be restricted to certain indole investigated by treating the membranes with various derivatives, the possibility of the involvement of reagents, enzymes and detergents (BENNETT and SNV- structural recognition sites cannot be disregarded. DER, 1976). A summary of the findings is given in The chemical nature of the macromolecules Table 111. Further attempts to identify the <( binding ))

TABLE111. -- Effect of various membrane treatments on specoric binding in rat cortex (BENNETTand SNYDER, 1976).

I :decrease;l :increase:l < 50 % ;11:50% ; 111 : > 50 %.

3H.5.HT 3H.LS D Important for Nol important for binding binding

Sulfhydryl reagents 1_ 11 SH Carboxyl reagents 111 11 COOH Tryptophane reagents 1 1 "] i 1 Tra-chymypsinotryTpsinPCK 1111 111 j_ Integral protein Phospholipase A 111 111 Fatty acids Phospholipase D I I -- Polar ()of phospholipids Neuraminidase I I -- Sialic acid i:i Triton X 100 111 I l I )

TweenDeoxycholate 11111 11111 j_ Membrane structure Vol. 77, n o 2, 1981 IN VITRO BINDING TO SEROTONINERGIC RECEPTORS 359

El) D_ FPTXOXATHAL eOOMP =E 5 PIPAM • • • • BROMPE • APO _<.) PROPE• • • DOX o CLOZO •CINAN I1.0_ O(+) BUT .__ PIZOT• • PHBZ • MIAN z Fl(i. 3. -- Correlation between relative -- 6' C_l_o .OMILEN binding affinities of drugs (plC_o, M) for 0 --CYPRO JH-spiperone sites in rat frontal cortex _ •BENPE and 3H-$-HT sites in rat hippocampus, cZa Spearman rank correlation coef- -- •TRYP ficien/ : r = .33, n = 35, p > .01. ca •SPIP Compounds and assay conditions are _r-- • METHYSAZAP •OXIP indicated in Table 11, " 7 c-_ •METIT • BRGR • BUFO t- -r m • LSD -r

ulo • 5HT o 8 • ERGOT O.

plC50 3H SPIP (2nM) BINDING IN FRONTAL CORTEX

plCs0(M) OF 3H 5HTIgnM) BINDING IN RAT HIPPOCAMPUS (e)

8 "/ 6 5J IO00-_ ' ' / ' KETOo / KETOe / / / / FIG,4. -- Correlation between potencies I00- / of drugs to antagonize tryptamine- ca / induced clonic seizures in rats in viva -- (Nt6"M6'GL'_Sand JANSSe_, 1979) and / relative binding affinities of drugs _ / (plC_o, M) for _H-spiperone sites in rat ua __- / / frontal cortex (o) or for _H-5-HTsites in z _-- l0 CLOZO O sCl_Jq oOXl P eOXI P CINANe rat hippocampus to). < < / Spearman rank correlation coef- _. '*" ficient for ]H-spiperone : r = -.87, --z . IANCPZ_ _ o•/HAL MIAN n= 22, p< .001; for 3H-5-HT : r = - .51,

O "P_oPAM(_O/CAI_-_A:_HYS •,+)SOT METHYSl_7.AP MILIENeCPI• "X_•PtZO(+_BUTePIPAMFPTX con nd= it22,ionsp are> .0ind1. icCaomtedpoinundsTableand11.assay :_ t ,.._.to 8- o".--rcr_ • c_Ro",.,_s° -_ _.j CYPRO,PIZO/ l_lwl_ L BENP o _ BENP

_ 0_ / 0 METER • METER

plCs0(M) OF 3H $PIP(2nM) BINDING IN RAT FRONTAL CORTEX (O) 360 J.E. LF_YSEN Journal de Physiologic

plC50lM) OF _H 5HT (3nM) BINDING IN RAT HtPPOCAMPU$(el 8 7 6 5 10-6 I /I . I J _z / / FiG. 5. -- Correlation between potencies oz oo_" eoxlp of drugs to anlagonize 5-tiT-induced / contractionsinratcaudalarteryinvitro I0-?, < 0 Asll / #_[_l (VaN NU£TEN and VANItOt.'TrE, 1980) (plCj_ M) for _H-spiperonesites in rat / ]rontal cortex (o), or for 3H-5-HTsites in -- OOXAT/ OXAT• rat hippocampus (e). i io-e.:" _ FI_X./t/3t+)0 HAl.Burr// o KEtO I+) BUT• HALFl_Ot anficidenrelativeSt pefoarmr _aH-bninltingspipraneronk affinecor:relationitiesr =of - drugsco.8ef-2,

. " PIP _IA_Nu_ ClN_J_ _'_I_M Ite_ AN_ Lo_DM_IN:N n-.=41, 24, n=p< 24,.001fOr; >p .0l.3H-5-ComHTpou:nrds= CYPROo/ MErHYS METHY eCYPRO and assay conditions are indicated in METERo o_O sQBENP • CM'Xe • ePROPE _'_"_ METER• ME_ BENP Table It. _ 10.9_ METIT _CFTXPIZO PIZO _ O$pIp,PROPE eSPIP

< ,o-,o/

plC50 (M) OF 3H SPIP(2nM) BINDING IN RAT FRONTAL CORTEX(O)

'macromolecules have not been reported yet. The still using selective agents and careful scoring of phar- elusive goal of the chemical composition of the receptor mac•logical effects might be an interesting tool in can only be achieved after solubilization and purification clarifying the issues. A rewarding goal would be to of the binding macromolecule, extend our knowledge regarding the involvement of receptor alterations in diseased states. Preliminary studies in this area indicate a decrease in LSD binding IV. -- FUNCTION sites, but unchanged 5-HT binding sites in schizophrenia The function of the binding sites on membranes in (BENNETT et ai., 1979) and decreased LSD and spiperone brain tissue, and their relation to the action of 5-HT, is binding sites in Huntington's disease (ENNA et ai., 1976 ; REISINE et al., 1977). However, much more research is unknown and represents a vast and complicated field for. needed to find out the relevance and significance of these future research. To be investigated are e.g. the receptor changes in the etiology of mental diseases. immediate effects on the membrane or cell of receptor triggering. Examples of reactions which may be initiated are : (1) phospholipid turnover, known to play a role in CONCLUSION various receptor systems, e.g., phosphatidyl inositol- phosphatidic acid turnover (MICHELL, 1979), phos- Measurements of in vitro binding to membranes pholipid methylation (STRITTMATTER el t/L, 1979), require little skill and simple technical equipment. The acylation-deacylation (SCHREY and RUBIN, 1979), etc. ; subject is fashi'onable and many isolated experiments (2) receptor coupling with enzymes, e.g., adenylate are being reported. However, the biochemistry and cyclase as described for the B-receptor system physicochemistryofligand interactions with membranes (SIROSBERG, 1980); (3) receptor coupling with ion are complicated and comprise many unknown and channels, etc. therefore uncontrollable phenomena. Assay conditions The in vivo behavioural effects resulting from may considerably influence membrane conformations, triggering serotoninergic receptors in various brain areas and have to be carefully checked and standardized to remain to be explored. In vivo receptor binding make observations reproducible. Binding patterns experiments have shown that 3H-LSD accumulates should be critically interpreted, taking into account specifically in certain brain areas. In the cortex, it was possible anomalies caused by surface events on shown that labelling could be selectively displaced by membrane micelles or by degradation of unstable 3H- serotonin agonists and antagonists (DUCHEMIN et al., ligands. Since < Vol. 77, n ° 2, 1981 IN VITRO BINDING TO SEROTONINERGIC RECEPTORS 361 should only be identified with a given receptor when the GRtPENBERGJ., JANSSONS.E.(1978). Binding of 3H-5-hydroxytryp- tamine to synaptic plasma membranes of rat brain. Acta Physiol. relationship with physiological or pharmacological Scand., 102, 123-125. effects can be made. Finally, receptor events are only the HowLutt D.R., MORRIS H., NAHORSKI S.R. (1979). Anomalous start of a chain of reactions resulting in a response, properties of 3H-spiperone binding sites in various areas of the rat limbic system. MoL PharmacoL, 15, 506-514. When trying to understand receptor functions and LAOURONP.M., JANSSV.n P.F.M., LEVSEN J.E. (1978). Spiperone: regulations, not only the site of attachment, but the a ligand of choicefor neuroleptic receptors.2. Regional distri- whole system has to be studied. This review has shown PbutionharmacoLand ,in27vivo. 317-321.displacement of neuroleptic drugs. Biochem. that the knowledge about serotoninergic receptors is LEVSEN J.E., GOMMEREN W. (1978). Different kinetic properties of rather limited. In order to resolve the intriguing problem neuroleptic receptor binding in the rat striatum and frontal cortex. of what the features and functions of serotoninergic LEYSENLife J.E.,Sci., GOM23, 447-452.MERENW. (1980). Optimal conditions for 3H-apo- receptors are, extensive and carefully designed further morphine binding and anomalous equilibrium binding of 3H-apo- research is necessary, morphine and 3H-spiperone to rat striatal membranes : involve- ment of surface phenomena versus multiple binding sites. J. Neurochem., in press. LEYSEN J.E., GOMMEREN W., LADORON P. (1978a). Kinetic ACKNOWLEDGEMENT investigation of [3H]-spiperone binding sites in rat frontal cortex. Arch. Int. Physiol. Biochim., 86, 874-875. Part of this work was supported by a grant from I.W.O.N.L. The LEYSEN J.E., GOMMEREN W., LADURON P.M. (1979). Distinction author sincerely thanks Drs. C.J.E. NII:MP-GEERS and J.M. VAN between dopaminergic and serotonergic components of neuro- NUETEN (Dept. of Pharmacology, Janssen Pharmaceutica) for leptic binding sites in limbic brain areas. Biochem. Pharmacol., providing data of in vivo and in vitro pharmacological tests. 28, 447-448. Thanks are due to Dr. J.P. TOLLENAERE and D. ASHTON for their LEYSEN J.E., LADLIRON P.M. (1977). A serotonergic component of help in preparing the manuscript, neuroleptic receptors. Arch. Int. Pharmacodyn. Ther., 230, 337- 339. I.'EYSENJ.E., NIEMEGEERSC.J.E., TOLLENAERE J.P., LADLIRONP.M. (1978 b). Serotonergic component of neuroleptic receptors. REFERENCES Nature, 272, 168-171. MAGGI A., U'PRICHARD D.C., t'NNA _.J. (1980). Differential effects of antidepressant treatment on brain monoaminergic BENNETt J.L., AGHAJANIAN G.K. (1974). D-LSD binding to brain receptors. Eur. J. PharmacoL, 61, 91-98. homogenates : possible relationship to serotonin receptors. L(l'e MtCHEt.L R.H. (1979). lnositol phospholipids in membrane function. 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