NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.2 117

The 5-HT3 in Mammalian : A New Target for the Development of Psychotropic ? Jose Antonio Apud, M.D., Ph.D.

3 K�Y �oRJ?s: ; Serotonin receptors; 5-HT3 The 5-HTl site, which can also be labeled by [ H]5- bindmg sItes; 5-HT3 receptor antagonists; -gated HT, was characterized by a dissociation constant for Na+-K+ channel 5-HT in the nanomolar range. The combination of ap­ propriate specifIc ligands and computer-assisted anal­ Serotonin (5-HT) is a in the central 3 ysis of [ H]5-HT binding isotherms in the eNS re­ nervoussystem (eNS) of vertebrates and invertebrates. vealed fIve different subtypes of 5-HTl binding sites: Invert ebrates, 5-HT participates in the regulationof var­ 5-HTIA, 5-HTIB, 5-HTlC, 5-HTm, and 5-HTIE (Pedigo ious physiologic functions, including pain perception, et al. 1981; Pazos et al. 1984; Peroutka 1986; Heuring blood pressure, sleep, homeothermia, and sexual ac­ and Peroutka 1987; Leonhardt et a1. 1989). The recep­ tivity. It is also believed that 5-HT may participate in tor status of the 5-HTIE subtype, however, is still a theexpression of symptoms of certain psychiatric dis­ pending question. In contrast, the 5-HT2 site was orders, such as and anxiety. In this context, characterized as a 5-HT receptor with low (micromo­ most of our knowledge concerning the participation of lar) affinity for 5-HT but high (nanomolar) affinity for S-HTand 5-HT receptors in psychopathology has come a series of compounds (, , cinanse­ fromthe characterization of the mechanisms of action rin, and ) (Bradley et a1. 1985; Glennon 1987) of various drugs that are effectivein relieving the symp­ that antagonize certain behavioral responses elicited by toms of psychiatric disorders. the administration of 5-HT-mimetic drugs (Peroutka et That the 5-HT receptor population in the periph­ al. 1981). erymight be heterogeneous was fIrstsuggested by the A third type of 5-HT receptor, the 5-HT3 recogni­ early pioneering work of two independent groups tion site, has also been identifIed (Fozard et a1. 1979; (Rochae Silva et a1. 1953; Gaddum and Hameed 1954). Fozard 1984; Richardson et a1. 1985; Hoyer and Neijt It was not until the late 1970s, however, that Peroutka 1987; Kilpatrick et al. 1987; Resier and Hamprecht 1989; andSnyder (1979) described, in the eNS, the existence Peters and Lambert 1989; Barnes et a1. 1989a). The of twodiff erent 5-HT recognition sites labeled by lyser­ 5-HT3 receptor, which represents the so-called "M" gic acid diethylamine, the serotonin-1 (5-HT1) and the receptor identifIed by Gaddum and Piccarelli (1957) in serotonin-2 (5-HT2) binding sites. the guinea pig intestine, was initially described in pe­ ripheral in which it mediates depolarization Fromthe Fidia-Georgetown Institutefor the Neurosciences, George­ of neurotransmitter release (Bradley et a1. 1985). More townUniversity Medical School, Washington D.C. (On leave from recently, 5-HT3 binding sites were also identifIed in theCatedra de Farmacologia y Toxicologia, Facultad de Medicina neuronal cell lines (Hoyer and Neijt 1987; Resier and Universidad de Buenos Aires and ININFA,CONICET, Buenos Aires: Argentina. ) Hamprecht 1989; Peters and Lambert 1989), in discrete A�dressreprint requests to: Jose Antonio Apud, Fidia-Georgetown . brain areas (Kilpatrick et a1. 1987; Barnes et al. 1988; Institute for the NeuroSCiences, Georgetown University Medical Peroutka and Hamik 1988; Barnes et al. 1989a; Waeber School, 3900 Reservoir Road NW, Washington, D.C. 20007. Received August 1, 1991; revised January 30, 1992; accepted Febru­ et a1. 1989), and in the spinal cord (Glaum and Ander­ ary 12, 1992. son 1988; Hamon et a1. 1989). Whether these binding

Cll9?3 American College of Pubhshed by Elsevier Science Publishing Co., Inc. 65SAvenue of the Americas, New York, NY 10010 0893-133X/93/$6.00 118 J.A. Apud NEUROPSYCHOPHARMACOLOGY 1993 -VOL. 8, NO.2

sites represent functional receptors and meet the criteria have as competitive antagonists of 5-HT action on pe­ for 5-HT3 receptors (Bradley et al. 1985) has been ad­ ripheral tissues possessing 5-HT3 receptors. dressed by a series of studies showing behavioral, bio­ The broad pharmacologic profIles of compounds chemical, and electro physiologic responses to both with affinity for the neural 5-HT3 receptor hampered 5-HT3 and antagonist drugs (Costall et al. 1987, the defInition of this receptor. By chemically substitut­ 1990; Blandina et al. 1988; Yakel and Jackson 1988; ing compounds that bind with rather low specifIcity to Barnes et al. 1989b; Blandina et al. 1989; Imperato et the 5-HT3 site, it became possible to synthesize selec­ al. 1989; Peters and Lambert 1989; Hagan et al. 1990). tive 5-HT3 receptor and antagonists (Fozard A 5-HT receptor that does not meet the criteria for and Gittos 1983; Richardson et al. 1985; Fake et al. 1987). a 5-HTl, 5-HT2, or 5-HT3 receptor has also been re­ On the basis of the assumption that different confor­ cently described. This receptor, which is found in mations of the 5-HT molecule are required for activa­ mouse embryo colliculi neurons and in the guinea pig tion of the so-called "0" (5-HT2) and "M" (5-HT3) hippocampus, has been classifIed as 5-HT4 (Dumuis et receptors, Richardson et aI. (1985) produced both al. 1988). A series of different putative 5-HT receptors specifIc agonists and antagonists for the 5-HT3 recep­ (5-HTlp, drosophila 5-HT receptor, the ­ tor with 5-HT as the starting compound. By systematic preferring receptors of the invertebrates, the stomach methyl substitutions in the nucleus and ethyl­ fundus receptor) have also been identifIed in periph­ amine side chain, the conformational freedom of the eral tissues of vertebrates and invertebrates. 5-HT molecule was reduced. This strategy provided The 5-HTl, 5-HT2, and 5-HT4 receptors are coupled somewhat specifIc 5-HT2 (a-methyl-5-HT) and highly to second messenger systems. Activation of 5-HTIA, specifIc 5-HT3 (2-methyl-5-HT) receptor agonists. 5-HTIB, and 5-HT1O receptors inhibits, whereas activa­ Moreover, by extending the ethylamine chain of 5-HT tion of the 5-HT4 receptor stimulates, adenylate cyclase and producing rigid analogues in which the terminal (Bradley et al. 1985; Dumuis et al. 1988). On the other is included in a tropine or homeotropine ring hand, activation of both 5-HTIC and 5-HT2 receptors system, the selective 5-HT3 antagonist (3a-tropanyl)­ increases phosphoinositide (PI) metabolism (Conn and IH-indole-3-carboxylic acid ester (ICS 205-930) was ob­ Sanders-Bush 1984; Conn et al. 1986; Nakaki et al. 1984). tained (Richardson et aI. 1985). It was also shown recently that 5-HT3 receptor activa­ The synthesis of a series of compounds structur­ tion stimulates PI hydrolysis in the rat cortex (Edwards ally related to ( - )- led to the discovery of an­ et al. 1991). Whether the increase of PI turnover is the other selective 5-HT3 . Substitution result of a direct 5-HT3 receptor-mediated mechanism by chlorine in the ring of a number of sub­ or else an indirect effect mediated through a still un­ stituted benzoic acid esters of tropine led to the syn­ known pathway is not yet clearly understood. thesis of l-a-H,3-a,5-a- H-tropan-3-yl-3, 5-dichloroben­ In the last several years, the 5-HTIA, 5-HTIC, zoate (MOL 72222), a selective and competitive 5-HT3 5-HT1O, and 5-HT2 receptors have been cloned (Fargin receptor antagonist (Fozard and Gittos 1983; Fozard et aI. 1988; Julius et al. 1988; Pritchett et al. 1988; Ham­ 1984a). blin and Metcalf 1991). More recently, the 5-HT3 recep­ By restricting the conformational freedom of the tor has also been cloned (Maricq et al. 1991). The char­ diethylaminoethylside chain and changing the aromatic acteristics of the cloned 5-HT3 receptor are largely nucleus of the molecule, a series of consistent with the properties of the purifIed receptor 3-indazole carboxamides was obtained, Among these obtained from brain tissue and neural tumor cell lines compounds, endo-N-(9-methyl-9-azabicyclo[3,3,I]non- (McKernan et al. 1990a, 1990b; Miquel et aI. 1990) (see 3-yl)-I-methyl--carboxamide (BRL 43694) was below). found to possess potent and selective 5-HT3 antagonis­ properties (Fake et al. 1987). Other compounds with selective activity at the DEVELOPMENT OF SELECTIVE DRUGS 5-HT3 receptor have also been identifIed. Among them, ACTIVE AT THE 5-HT3 RECEPTOR 1,2,3,9-tetrahydro-9- methyl-3[ (2-methyl-lH-imidazol­ l-yl)methyl]-4H-carbazol-4-1 hydrochloride hydrate ' It has long been known that both morphine and cocaine (GR38032) (Butler et aI. 1988) and 4-amino-N-(1- block the excitatory action of 5-HT on peripheral neu­ azabicyclo[2,2.2.]oct-yl)-5-chloro-2-methoxybenzamide rons (Rocha e Silva et al. 1953; Gaddum and Hameed hydrochloride hydrate () (Smith et al. 1988) 1954). Only recently, however, was evidence presented also proved to be potent 5-HT3 receptor antagonists. indicating that both drugs induce their effects via a possesses high affInity for the 5-HT3 bind­ specifIc competitive antagonistic action on 5-HT3 ing site in nervous tissue (Peroutka and Hamik 1988) receptors (Fozard et al. 1983). Further studies demon­ but also binds with relatively high affInity to other 5-HT strated that metoclopramideand a series of substituted receptors (Glennon et al. 1986). benzamides (Fozard et al. 1978; Fozard 1984) also be- Recently a computer-aided molecular modeling NEUROPSYCHOPHARMACOLOGY 1993- VOL. 8, NO. 2 5-HT3 Receptors in Brain 119

PARENT PRODUCT were consistent with those expected for a 5-HT3 recep­ tor (Bradley et a1. 1985). Moreover, the inability of ade­ nine and guanine nucleotides to affectagonist binding suggests that 5-HT3 recognition sites are not coupled H0'Q)NH' to G-proteins (Hoyer and Neijt 1988). 2-

-(-)oocaine MOL 72222 Watling et al. 1988; Barnes et al. 1988; Barnes et al. 1990; Peroutka and Hamik 1988). The highest affinityfor the 5-HT3 site (Kd = 0. 066 nmollL) was shown by PH]GR67330 when compared to other labeled 5-HT3 receptor ligands (Kd values of 0.24 to 1.2 nmollL). The Bmax values for these ligands ranged between 32 and Hetcx:lcpramidc BIU. 43694 145 fentomole per milligram of protein (Barnes et a1.

Figure1. Synthesis of potent and specifIcdrugs [PRODUCT] 1990; Kilpatrick et a1. 1987, 1989, 1990; Peroutka 1988; with affInity for the 5-HT3 receptor derived from different Barnes et al. 1988; Watling et al. 1988). Equilibrium satu­ compounds [PARENT DRUGS] that bind the 5-HT3 site with ration analysis for the radioligands revealed that bind­ low specifIcity. ing was to a single saturable high-affi.nity site. Binding was also reversible, stereospecific, and unevenly dis­ (CAMM) system has been used to identify an initial tributed throughout the rat CNS (see below) (Kilpatrick three-component pharmacophore for specifIc 5-HT3 et al. 1987, 1990; Barnes et a1. 1988; Watling et al. 1988; receptor ligands such as ICS 205-930, GR38032F, Milburn and Peroutka 1989; Barnes et a1. 1990). The zacopride, and the novel compound 3-[2-(guanidyl­ afflnity of drugs for the 5-HT3 binding site methyl)-4-thiazolyl]indole (Rizzi et a1. 1990). The labeled with any of the 5-HT3 receptor ligands clearly CAMM represents a powerful tool in the drug-design indicates that the various compounds bind to an iden­ fIeld because it helps to explain or predict a variety of tical 5-HT3 binding site. molecular properties. The ability of various 5-HTuptake blockers that pos­ sess action to displace with nanomolar affinity [3H]quipazine from rat cortical membranes 5·RT3 RECOGNITION SITES IN NERVOUS (Schmidt and Peroutka 1989) led to the suggestion that SYSTEM TISSUE: BIOCHEMICAL the 5-HT3 receptor might be involved in the pharmaco­ CHARACTERIZATION AND DISTRIBUTION logic action of . However, in mem­ branes of the rat entorhinal cortex labeled with 3 3 During the last 3 years, various 5·HT3 receptor antag­ [ H]zacopride or of N1E-1l5 cells labeled with [ H]ICS onists have been labeled with radioactive isotopes. The 205-930, 5-HT uptake blockers display very low affi.nity fIrst ra dioligand used to identify 5-HT3 receptors in for the 5-HT3 binding site (Hoyer et al. 1989). These tissue was [3H]ICS 205·930. Scatchard results support the idea that [3H]quipazine might also analysis with cell membranes of either NG108-15 bind to a site unrelated to the 5-HT3 recognition site in neuroblastoma-glioma cells (Hoyer and Neijt 1987) or nervous system tissue (for example, the 5-HT trans­ NIE·115 neuroblastoma cells (Hoyer and Neijt 1988) in­ porter in the presynaptic terminal). In these experi­ dicated that [3H]ICS 205-930 labeled a single popula­ ments, it was also shown that potent atypical antipsy­ tion of binding sites, with a Bmax of 60.4 and 40.5 fen­ chotic drugs (, , and ) show tomole per milligram of protein and a pKd of 8.91 and higher affi.nity (pKd > 6) for the 5-HT3 binding site than 9.2 mollL, respectively. The binding was competitive metoclopramide, 5-HT, and 2-methyl-5-HT, drugs cur­ and stereos elective (Hoyer and Neijt 1988), and the in­ rently used in the characterization of 5-HT3 receptors hibition profIles of various potent 5-HT3 receptor in nervous system tissue. If the action of agonists and antagonists on [3H]ICS 205-930 binding atypical neuroleptics involves an interaction with brain 120 J.A. Apud NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.2

Table 1. 5-HT3 Receptors in the

Transducer Biochemical Agonists Antagonists Radioligands Mechanism Neurophysiology Characteristics

5-HT MDL72222 3H-GR67330 2-methyl-5HT rcs 205 930 3H-GR65630 I-phenyl biguanide GR38032 F 3H-rCS 205-930 Ligand-gated Fast depolarization Heterooligomeric complex GR65630 3H-BRL43694 Na+-K+ channel of 250 kD MW MDL 73,147F 3H-5-HT Metoclopramide 3H-zacopride Zacopride 3H-S-zacopride 3H-quipazine

Distribution Biochemical Molecular Regulation Characteristics Very High High Low Low or Absent

DA release or turnover 1462 Nucleotides encoding Area postrema Cortex Septum (nigrostriatal & 487 amino acids Hippocampus Cerebellum mesocortical systems) 27% Homology with Amygdala Hypothalamus Thalamus ACh release (cortex) a subunit of Nuclei solitary NA release? ACh receptor; 22% tract homology with GABAA Spinal trigeminal receptor and with the nucleus 48 kD subunit of the Substantia gelatinosa Hydrophobicity analysis predict a protein containing four hydro­ phobic transmembrane regions Ligand-gated receptor superfamily characteristics

5-HT3 binding sites, then 5-HT3 receptors may repre­ al. 1989a). The 5-HT3 recognition sites in human brain sent a new target for the development of atypical neu­ meet the criteria for 5-HT3 receptors (Bradley et al. roleptics. This assumption, however, is not supported 1985). However, characterization of 5-HT3 binding by preliminary studies, which showed that zacopride sites with various radioligands will be necessary to elu­ failed to modify positive symptoms in schizophrenic cidate the biochemical processes that mediate the cen· patients (Newcomer et al. 1990). tral actions of 5-HT3 antagonists in human brain. The 5-HT3 binding sites initially described in the In all species studied, the area postrema, which is rat brain and tumor cell lines were also found in the the site of the chemoreceptor trigger zone, possesses of other animal species, including humans the highest concentration of 5-HT3 binding sites (Pratt (Peroutka 1988; Kilpatrick et al. 1989; Barnes et al. et al. 1990). In rat and mouse brain, high densities 1989a). Specificbinding of [3H]quipazine was detected of [3H]GR65630, [3H]GR67330, [3H](S)-zacopride, or in whole brain membranes of pig but not of cow, dog, [3H]quipazine specific binding sites are present in the turtle, mouse, guinea pig, chicken, or rabbit (Peroutka entorhinal, frontal, retrosplenic, cingulate, temporal, 1988). The ability of various serotonergic compounds parietal, and occipital cortex and in the amygdala and to displace [3H]quipazine with a similar affmity pattern hippocampus. In these same species, 5-HT3 specifIc in rat and pig cortical membranes suggests the pres­ binding sites are very low in number or undetectable ence of a similar 5-HT3 receptor in the brains of these in the nucleus accumbens, striatum, thalamus, hypo­ two species (Peroutka 1988). thalamus, and cerebellum (Kilpatrick et al. 1987, 1988, Binding sites of [3H]zacopride (Bameset al. 1989a), 1989, 1990; Milburn and Peroutka 1989; Barnes et al. [3H]GR65630 (Kilpatrick et al. 1989), and [3H]ICS 205- 1990). In rat spinal cord, 5-HT3 binding sites seem to 930 (Waeber et al. 1989) have also been identifiedin hu­ be located presynaptically on capsaicin-sensitive pri· man brain. Binding of [3H]zacopride in hippocampus mary afferent fIbers (Glaum and Anderson 1988; Ha· and amygdala was shown to be saturable, specific, and mon et al. 1989). Interestingly, the ferret possesses high 3 of high affinity. Scatchard transformation of the data concentrations of [ H]GR65630 binding sites in the nu­ revealed Kd values of 2.64 and 2.93 nmol/L and Bmax cleus accumbens, , amygdala, and stri­ values of 55 and 44 fentomole per milligram of protein atum (Kilpatrick et al. 1989). in amygdala and hippocampus, respectively (Barnes et Biochemical and auto radiographic localization of NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO. 2 5-HT3 Receptors in Brain 121

3 5·HT3 binding sites with [ H]GR65630 (Kilpatrick et al. tween the 5-HT3 receptor and these three receptors, it 3 3 1989), [ H]zacopride (Barnes et al. 1989a), or [ H]ICS was predicted that the 5-HT3 receptor belongs to the 205·930 (Waeber et al. 1989) in human brain membranes superfamily of ligand-gated ion channels. The recep­ or slices has revealed a 5-HT3 receptor distribution tors in this family possess a heterooligomeric structure, similar to that of rodent brain. However, in contrast probably comprising four or fIve subunits, which to­ with other mammalian species, human cortical areas gether form the hydrophilic pore of the ion-specific possess low concentrations of 5-HT3 binding sites channel. (Waeber et al. 1989). Relatively high concentrations of 5·HT3 recognition sites are observed particularly in the nuclei of the solitary tract, the spinal trigeminal nucleus, CLONING OF THE 5-HT3 RECEPTOR and the substantia gelatinosa of the spinal cord (Wae­ ber et al. 1989). Molecular biological studies further supported the con­ cept that the 5-HT3 receptor belongs to the superfamily of ligand-gated ion channels. Maricq et al. (1991) iso­ SOLUBILIZATION AND PURIFICATION lated two messenger ribonucleic acid (mRNA) fractions OF THE 5-HT3 RECEPTOR from polyadenylated mRNA of NCB-20 cells that gave rise to functional expression of 5-HT-gated currents af­ Two groups have recently solubilized from rat brain ter injection into Xenopus oocytes. Construction of a (McKernanet al. 1990a) and NG108-15 neuroblastoma­ complementary deoxyribonucleic acid (cDNA) library glioma cells (Miquel et al. 1990) a 5-HT3 receptor with in an RNA expression vector for the two positive mRNA biochemical characteristics similar to those of the 5-HT3 fractions led to the identification of a single positive receptor identifiedin the corresponding membrane frac­ clone (p5HT3R-A). Oocytes injected with transcripts tions(Kilpatrick et al. 1987, 1989; Hoyer and Neijt 1988; from this clone produced a fast inward current in re­ Barnes et al. 1990). Further studies led to the purifica­ sponse to 5-HT that was similar to that observed in tionof the solubilized receptor from the NCB20 neuro­ clonal cell lines and brain tissues possessing native nal·glioma hybrid cell line, in which the number of 5-H3 receptors (Yakel et al. 1988; Neijt et al. 1988a). 5·HT3 recognition sites per milligram of protein is 20 Sequence analysis of the clone for the 5-HT3 recep­ to 30 times the number in rat brain (McKernan 1990b). tor revealed a 2131-bp cDNA, which encodes a protein A 1700-fold purifIcation of the 5-HT3 receptor was of 487 amino acids. Interestingly, the molecular mass of achievedby afunity chromatography, with the selective the predicted protein (56 kD) is similar to that of one 5·HT3 receptor antagonist L-685,603 immobilized in of the fragmentsof the purifIedreceptor (see above) (Mi­ agarose as the ligand. Scatchard analysis and competi­ quel et al. 1990; McKernan et al. 1990a). The hydropa­ 3 tion studies with the radioligand [ H]Q ICS 205-930 thy profIle of the cloned 5-HT3 receptor predicts a confrrmed the presence of a single population of 5-HT3 structure consisting of four hydrophobic transmem­ bindingsites in the purifIed preparation (McKernan et brane regions, a large extracellular amino terminal do­ al. 1990b). main, and a long intracellular loop connecting the third Polyacrylamide gel electrophoresis of the purifIed and fourth transmembrane regions (Maricq et al. 1991). receptor revealed two bands that migrated with appar­ The cloned 5-HT3 receptor shows 27% identity entmolecular masses of 54 and 38 kD (McKernan et al. with the a subunit of the Torpedo californica nicotinic 1990b). Recent radiation inactivation studies have ACh receptor and 22% identity to both the /31 subunit shown that, in rat brain, the binding sites labeled by of the bovine GABAA receptor and the 48-kD subunit [3H]zacopride and by [3H]GR65630 possess molecular of the rat glycine receptor (Maricq et al. 1991). mass es of 35 kD (Bolanos et al. 1990) and of 49 kD Crude membranes of COS-7 cells transfected with (Lummis el al. 1990), respectively. Gel-exclusion chro­ the 5-HT3 receptor cDNA specificallybound the selec­ matography and sucrose-density gradient centrifuga­ tive 5-HT3 receptor antagonist [3H]GR63650; more­ tionof the solubilized receptor and gel fIltration of the over, kinetic studies showed that the affinity and Hill 3 purifiedmaterial suggested that the 5-HT3 receptor has coefficient values for [ H]GR63650 binding to these an apparent molecular mass of 250 kD. membranes (Maricq et al. 1991) were identical to those The apparent molecular mass of the purifIed5-HT 3 observed in NCB-20 cells or rat brain tissue bearing na­ receptor(250 kD) is in the same range as that determined tive 5-HT3 receptors (Kilpatrick et al. 1987; McKernan for three ligand-gated ion channels: the gamma­ et al. 1990b). The expression of specifIc 5-HT3 binding aminobutyric acid type A (GABAA) receptor (Sigel et sites in COS-7 cells and the generation of currents in al.1983; Stephenson 1988; Mamalaki et al. 1989), the oocytes (see below) with a single cDNA clone indicate glycinereceptor (Pfeifferet al. 1982), and the nicotonic that functional homomeric 5-HT3 receptors can be ex­ (ACh) receptor (Whiting and Lindstrom pressed in different biological systems (Maricq et al. 1986). Considering the physicochemical similarities be- 1991). 122 J.A. Apud NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.2

The distribution of the 5-HT3 receptor mRNA was results in a fast excitatory 5-HT3 receptor-mediated re­ determined with the use of polymerase chain reaction sponse (Yakel and Jackson 1988; Derkach et al.1989, (PCR). With oligonucleotide primers corresponding to Yang 1990; Maricq et al. 1991). positions 84-114 and 682-712 of the cloned 5-HT3 Unlike other receptors for biogenic amines, both receptor sequence, a single PCR product was identifIed native and cloned 5-HT3 receptors are directly coupled in the mouse cortex, brainstern, midbrain, spinal cord, to an ion channel that is permeable to the monovalent and heart (Maricq et al. 1991). The observed distribu­ cations Na+ and K+ (Derkach et al.1989; Yakel et al. tion of the mRNA is consistent with ligand-binding 1990; Maricq et al. 1991). Activation of 5-HT3 receptors studies, with the exception that mRNA was not de­ in voltage-clamped membranes results in an opening tected in the intestine.The absence of a PCR product of ligand-gated Na+-K+ channels with an increase in in the intestine, which is rich in 5-HT3 binding sites the inward current that flows through these channels (Kilpatrick et al. 1987), suggests that a distinct 5-HT3 (Peters and Usherwood 1983). The large inward driv­ receptor subtype encoded by a separate gene may ex­ ing force for Na+ results in a greater Na+ influx and ist in this . a reduced K+ efflux through the channel, with the can· sequence that the resting membrane potential ( - 55 mV) is progressively driven toward the Na+ equilibrium ELECTROPHYSIOLOGIC RESPONSES TO 5-HT3 potential (+55 mY); however, when the membrane RECEPTOR ACTIVATION IN THE BRAIN potential reaches around 0 mV (the reversal potenti al for the Na+-K+ ion channel), the inward Na+ current Serotonin has been shown to produce three different is reduced and the outward K+ current is increased types of electrophysiologic response in cultured mouse (Yakel and Jackson 1988; Lambert et al. 1989).The in­ hippocampal and striatal neurons (Yakel et al. 1988), crease in the membrane potential triggers the opening the most frequent of which is an inhibitory response of Ca2+ channels, which leads to an influx of extracel­ that results from receptor-mediated activation of an in­ lular Ca2+ into the cell. The influx of Ca2+ does not wardly rectifying K + conductance. A fast excitatory re­ modify the membrane potential, but Ca2+ functions as sponse (present in 6% to 10% of cells tested), which was an intracellular messenger to couple the activation of apparent after delivery of 5-HT by pressure ejection, the 5-HT3 receptor-linked ion channel to transmitter was evident after 35 msec, peaked at 200 msec, and release. Transmitter release is modulated by Ca2+ lasted approximately 2 to 4 seconds (Yakel et al. 1988). probably through facilitation of a transient fusion of the A similar fast depolarizing response to 5-HT is also ob­ synaptic vesicle membrane with the presynaptic termi· served in clonal cell lines (Yakel and Jackson 1988; N eijt nal membrane. Whether Ca2+ promotes vesicular fu­ et al. 1988a; Yang 1990) and in Xenopus oocytes injected sion directly or through a Ca2+-binding protein that with RNA transcripts obtained from the 5-HT3 recep­ promotes fusion is not yet known. tor cDNA (Maricq et al. 1991). The fast depolarizing cur­ In clonal cell lines, extracellular Ca2+ and Mg2+ ex· rent evoked by 5-HT is reproduced by the 5-HT3 recep­ ert a regulatory influence on 5-HT3 receptor-mediated tor agonists 2-methyl-5-HT, 1- and responses by modulating the amplitude and duration l-(m-chlorophenyl) biguanide, and is antagonized by of 5-HT -induced currents (Peters et al. 1988). Both cat­ selective 5-HT3 receptor antagonists (Neijt et al.1986, ions reduce the conductance of the native 5-HT3 recep­ 1988a; Yakel et al. 1988; Yakel and Jackson 1988; Maricq tor channel without affecting the current-voltage (IIV) et al. 1991). Both voltage-clamp studies and radioligand­ ratio. However, the homomeric cloned 5-HT3 receptor binding analysis indicate that 5-HT binds to the 5-HT3 expressed in Xenopus oocytes differs from the native receptor in a cooperative manner (Neijt et al. 1988a; Kil­ 5-HT3 receptor (Maricq et al. 1991); in the presence of patrick et al. 1987; Hoyer et al. 1988; Maricq et al. 1991). Ca2+ or Mg2+, the IN relation of the cloned receptor Thus, biochemical, molecular biological (see above), has a region of negative slope-conductance (the current and electrophysiologic evidence (Hoyer and Neijt 1988; increases in amplitude as the cell progressively depolar· McKernan et al. 1990; Maricq et al. 1991) indicates that izes). In this context, the 5-HT3 receptor shares some the 5-HT3 receptor belongs to the family of ligand­ properties with the N-methyl-D-aspartate (NMDA)­ gated ion channel receptors: 1) the 5-HT3 receptor is sensitive , the conductance of which activated by 5-HT even in the absence of is blocked by Mg2+ (Nowak et al. 1984). triphosphate or (GTP) (Yang The Ca2+ permeability and single-channel conduc­ 1990); 2) the response to 5-HT persists even after long tance of the 5-HT3 receptor-linked Na+-K+ channel in periods of internal dialysis with CsF, AIF4-, or GTP in NGI08-15 cells are similar to those of the non-NMDA order to activate G-proteins (Yang 1990); 3) the response type of excitatory amino-acid receptor but lower than to 5-HT remains unaffected by treatment of cells with those of the nicotinic ACh receptor (Yakel et al. 1990). pertussis toxin to prevent G-protein-mediated re­ In neuroblastoma N18 cells, however, the permeabil· sponses (Derkach et al. 1989); and 4) application of 5-HT ity ratio of the 5-HT3 receptor channel for Ca2+ is simi- NEUROPSYCHOPHARMACOLOGY 1993 - VOL. 8, NO. 2 5-HT3 Receptors in Brain 123

larto that of the nicotinic receptor (Adams injection of 6-hydroxydopamine in the substantia nigra et al. 1980; Yang 1990). fails to reduce the number of 5-HT3 recognition sites Two types of desensitization kinetics after 5-HT ap­ in the striatum, suggesting a lack of 5-HT3 binding plication have been observed for the 5-HT3 receptor. sites in the nerve terminals of the nigrostriatal In NIB-115 cells and in oocytes expressing cloned system (Hamon, personal communi­ S-HT3 receptors, desensitization of the receptor is cation). rapid (Neijt et al. 1988a; Maricq et al. 1991) and both Acute in vivo administration of 5-HT3 receptor the onset and recovery from desensitization can be selective antagonists failed to modify dopaminergic identifIed with a single exponential function (Neijt et neuronal activity (Koulu et al. 1989; Imperato and An­ aI.19 88a). In NGI08-15 cells and rat hippocampal neu­ gelucci 1989; Hagan et al. 1990). However, the activa­ rons, a complex biphasic desensitizing response to 5-HT tion of dopaminergic turnover in the mesolimbic is observed (Yakel and Jackson 1988). Classically, the dopaminergic pathway by the administration of mor­ channelsactivated by excitatory have phine (Imperato and Angelucci 1989), nicotine and eth­ been described as voltage independent (Kandel 1985); anol (Carboni et al. 1989), or the neurokinin receptor however, in NGI08-15 cells, but not in NIE-U5 cells, agonist DiMe-C 7 (Hagan et al. 1990), but not that in­ the time course of desensitization after 5-HT applica­ duced by (a DA receptor blocker) (Koulu tion is voltage dependent (Yakel and Jackson 1988). et al. 1989), is blocked by selective 5-HT3 receptor an­ A pharmacologically induced increase in the con­ tagonists, including ICS 205-930, GR38032F, GR65630, centration of cyclic adenosine monophosphate (cAMP) and MOL 72222. in NG108-15 cells or in hippocampal neurons enhances The cellular site at which 5-HT3 antagonists act to the rate of desensitization of the 5-HT3 receptor in­ block the stimulation of DA turnover in vivo is not duced by 5-HT (Yakel and Jackson 1988). Although the known. The increase in DA turnover in the nucleus ac­ mechanism by which 5-HT induces 5-HT3 receptor cumbens induced by either systemic administration of desensitization is unknown, it can be speculated that morphine (Imperato and Angelucci 1989) or by injec­ modulation of 5-HT3 receptor desensitization by cAMP tion of DiMe-C 7 in the ventral tegmental area (VT A) may be mediated by phosphorylation of the receptor (Hagan et al. 1990) can be selectively blocked by sys­ by a cAMP-dependent protein kinase (Sibley and Lef­ temic or intra-VTA injection of various 5-HT3 receptor kowitz 1985). antagonists. However, when ICS 205-930 is injected in the nucleus accumbens, it fails to counteract the increase in DA release induced by morphine administration (Im­ BIOCHEMICAL RESPONSES TO 5-HT 3 perato and Angelucci 1989). In contrast, the increase RECEPTOR ACTIVATION in DA release in the nucleus accumbens induced either by intracerebroventricular administration of 2-methyl- Neurotransmitter release and metabolism in the CNS 5-HT (Jiang et al. 1990) or by direct perfusion of I-phen­ is modified by 5-HT3 receptor-selective drugs (Blan­ ylbiguanide, a purported 5-HT3 receptor agonist, in dina et al. 1988, 1989; Barnes et al. 1989b; Imperato and the nucleus accumbens (Chen et al. 1991), is effectively Angelucci 1989; Hagan et al. 1990). In superfused rat blocked by selective 5-HT3 receptor antagonists in­ striatal slices, Blandina et al. (1988, 1989) demonstrated fused directly into the nucleus accumbens (Chen et al. that 5-HT and the selective 5-HT3 agonist 2-methyl-5- 1991). These results suggest that, according to the mech­ HT stimulated both basal and K + -induced anism of activation of the mesolimbic doparninergic (DA) release. The DA release induced by 5-HT3 recep­ neurons, 5-HT3 receptor antagonists modulate DA re­ tor stimulation was shown tQ be Ca2+-dependent and lease through an action either on the VT A, the area in partialIy sensitive to tetrodotoxin (Blandina et al. 1989). which the cell bodies of the mesolimbic dopaminergic The selective 5-HT3 receptor antagonist ICS 205-930, system are located, or at the level of the nucleus accum­ but not the 5-HT1/5-HT2 receptor antagonists methi­ bens, where the dopaminergic nerve terminals are pres­ othepin and , inhibited the effectof both ent. Whether the action of 5-HT3 receptor antagonists S-HT and 2-methyl-5-HT on DA release; the Ki value within the VTA is exerted on a postsynaptic 5-HT3 was similar to that expected for an effect mediated by receptor located either on the dopaminergic cell S-HT3 receptors (Blandina et al. 1989). perikarya or on intra-VTA interneurons, which in tum, The in vitro experiments performed with super­ regulate the activity of the dopaminergic cells, is not fused striatal slices (Blandina et al. 1989) suggest that clear. In the nucleus accumbens, the 5-HT3 receptors S-HT3 receptor antagonists either block postsynaptic that regulate DA release may be located inpresynaptic receptors located on intrastriatal neurons involved in dopaminergic nerve terminals. the regulation of DA release or block presynaptic 5-HT3 The 5-HT3 receptors also seem to participate in the receptors located on the nigrostriatal dopaminergic ter­ regulation of noradrenaline release from nerve termi­ minals. Recent experiments, however, have shown that nals. In fact, both MOL 72222 and ICS 205-930 selec- 124 J. A. Apud NEUROPSYCHOPHARMACOLOGY 1993- VOL. 8, NO.2

3 tively block the stimulatory effect of 5-HT on [ H]nor­ havioral tests designed to evaluate the effectof CNS­ release. This action of the 5-HT3 receptor active drugs on the performance of rodents and pri­ antagonists is only apparent, however, at concentra­ mates, has allowed an assessment of the potential role tions that are two to three orders of magnitude higher of 5-HT3 receptors in brain function. The 5-HT3 recep­ than those required to block 5-HT3 receptors (Feuer­ tor antagonists are highly effective in various models stein and Hertting 1986), and so the effect may not be of anxiety, psychosis, cognitive impairment, and drug specifIc. dependence and withdrawal and have minimal side 3 The K + -induced increase in [ H]ACh release from effects (Costall et al. 1990). the rat entorhinal cortex can be effectively reduced by A great deal of information on the potential of activation of 5-HT3 receptors with 5-HT or 2-methyl- 5-HT3 receptor antagonists for modulating brain func­ 5-HT (Barnes et al. 1989b). Both GR38032F and zaco­ tion has been obtained from both experimentaland clin­ pride fail to modify basal and evoked ACh release, but ical studies on cytotoxic drug-induced and radiation­ these agents antagonize in a concentration-dependent induced emesis (Cunningham et al. 1987; Leibundgut manner the inhibitory action of 2-methyl-5-HT (Barnes and Lancranjan 1987; Andrews et al. 1988; Dubois et et al. 1989b). Whether this facilitatory action of 5-HT3 al. 1988). Historically, the ability of metoclopramide to receptor antagonists is due to a direct blockade of the inhibit both - and cytotoxic drug-induced presynaptic inhibitory effectof 5-HT 3 receptor agonists emesis was ascribed to the ability of the drug to block on ACh release or to a blockade of 5-HT3 receptors lo­ D2 DA receptors in the trigger zone of the hindbrain. cated on inhibitory afferent fIbersor interneurons im­ More recently, the development of a series of sub­ pinging on cortical cholinergic neurons is not yet clear. stituted benzamides and other drugs with activity on Thus biochemical, pharmacologic, and electrophys­ radiation- and cytotoxic drug-induced emesis, but not iologic evidence indicates that 5-HT3 receptors in ner­ on apomorphine-induced vomiting, led to the sugges­ vous system tissue may participate in the modulation tion that the new compounds were acting through of both neuronal electrical activity and neurotransmit­ mechanisms other than those linked to the D2 recep­ ter release from nerve terminals. Apparently, 5-HT3 tor. In fact, the newly developed drugs with activity receptors are not simply restricted to presynaptic ter­ on emesis proved to be potent antagonists of the 5-HT3 minals (where they modulate transmitter release) but receptor (Fozard and Gittos 1983; Fake et al. 1987; Smith are also distributed more widely over the cell surface et al. 1988). The inability of 5-HT3 receptor antagonists (where they modulate membrane potential). to block apomorphine-induced emesis showed that The modulation of neurotransmitter release by 5-HT these compounds could not be exerting their effects seems to be mediated by a 5-HT3 receptor-gated Na+­ through postsynaptic D2 receptors. Because 5-HT in­ K + channel that is functionally linked to a selective creases the release of DA in various brain areas through Ca2+ channel. Whether the same molecular mecha­ a 5-HT3 receptor-mediated mechanism, 5-HT3 receptor nisms apply to both the stimulation of DA release in antagonists may exert their antiemetic action by block­ the nigrostriatal and mesolimbic dopaminergic systems ing the release of DA induced by radiation or cyto­ and the inhibition of ACh release in cortical tissue by toxic drugs in the trigger zone of the hindbrain. The 5-HT and 5-HT3 receptor agonists is not clear. In fron­ development of 5-HT3 receptor antagonists therefore tocingulate and entorhinal cortices, 5-HT3 receptor offered an alternative to conventional antiemetic ther­ agonists activate PI metabolism (Edwards et al. 1991). apy, especially in view of the fact that these drugs Moreover, in medial prefrontal cortex, the 5-HT3 have signifIcantly fewer side effects than D2 receptor receptor agonist 2-methyl-5-HT selectively depresses, blockers. rather than increases, the fIring rate of both spontane­ It has been postulated that classic DA receptor an­ ously active and glutamate-activated cells (Ashby et al. tagonists exert their antipsychotic action by blocking 1989). If, as suggested by the latter experimental evi­ the hyperactivity of mesolimbic dopaminergic neurons dence, more than one functional mechanism is linked that was presumed, but never demonstrated, to occur to 5-HT3 receptor action, DA and ACh release may be in psychosis. Interestingly, GR38032 and ICS 205-930 differentially regulated by distinct subtypes of 5-HT3 are highly effective in blocking the increase in mesolim­ receptors or by different intracellular mechanisms bic DA turnover induced by administration of DiMe-C linked to Ca2+ function. 7 and morphine, respectively (Imperato and Angelucci 1989; Hagan et al. 1990). Furthermore, behavioral studies have shown that the locomotor hyperactivity induced by selective pharmacologic stimulation of the 5-HT3 RECEPTORS AND CENTRAL mesolimbic system and the rebound hyperactivity that NERVOUS SYSTEM DISORDERS follows chronic administration in rats is selectively reduced by specifIc 5-HT3 receptor antago­ The development of drugs that are selective for the nists (Costall et al. 1987, 1990; Butler et al. 1988; Hagan 5-HT3 receptor, together with the availability of be- et al. 1990). NEUROPSYCHOPHARMACOLOGY 1993 - VOL. 8, NO. 2 5-HT3 Receptors in Brain 125

In normal rats, the failure of a single injection of hyperactivation of the dopaminergic system induced 5·HT3 receptor antagonist to modify either locomotor by drugs of abuse is unknown. activity (Costall et al. 1990) or mesolimbic and nigro­ A common feature of many experimental models striatalDA turnover (Imperato and Angelucci 1989; Ha­ that are responsive to 5-HT3 receptor antagonists is an gan et al. 1990) indicates the lack of a tonic serotonergic increased activity of presynaptic dopaminergic neurons stimulation of 5-HT3 receptors. In agreement with (Costall et al. 1987a, 1987c, 1990; Carboni et al. 1989b; these results, intracerebroventricular administration of Imperato and Angelucci 1989; Hagan et al. 1990). This 5,7·dihydroxytryptamine, a selective neurotoxin for common characteristic suggests that the activation of serotonergic neurons, fails to modify 5-HT3 receptors a serotonergic that impinges on a dopaminer­ in rat brain (Leysen, personal communication). The gic neuron (either at the presynaptic terminal, or on the 5·HT3 antagonist GR38032 is also ineffective at reduc­ cell body or a dendrite) represents a necessary step for ing the increasein dihydroxyphenylacetic acid concen­ the subsequent depolarization of the dopaminergic cell. trations, a reliable index of DA metabolism, in the Thus, 5-HT3 receptor antagonists, by blocking the ac­ mesolimbic and nigrostriatal dopaminergic pathways tion of 5-HT at the 5-HT3 receptor-gated Na+-K+ chan­ induced by a single haloperidol injection (Koulu et al. nel, would reduce Na+ influx through the channel and 1989). prevent changes in the resting membrane potential of Long-term administration of both MDL 73,147EF, the dopaminergic cell. The resultant blockade of Ca2+ a selective 5-HT3 receptor antagonist, and haloperidol channel activation by the 5-HT3 receptor antagonists produces a signifIcantreduction in the number of spon­ would prevent the activation of intracellular mecha­ taneously active dopaminergic neurons in the VTA and nisms that mediate DA release. substantia nigra (Sorensen et al. 1989). These results In most tests that assess anxiolytic activity (with the clearly indicate that 5-HT3 receptor antagonists exert exception of the Vogel test [Costall et al. 1990]), 5-HT3 a neuroleptic-like activity and support the view that receptor blockers have pharmacologic profiles similar 5·HT3 receptors regulate central dopaminergic func­ to those of anxiolytic benzodiazepines, even though the tion (Tricklebank 1989). 5-HT3 receptor antagonists fail to bind to GABAA The pharmacologic profile of 5-HT3 receptor receptor-associated benzodiazepine recognition sites blockers in the mesolimbic system suggests a therapeu­ (Jones et al. 1988). In contrast to benzodiazepines, how­ tic potential of these compounds as antipsychotic ever, 5-HT3 receptor antagonists lack sleep-inducing, agents. Moreover, the ability of these drugs to reduce muscle-relaxant, and anticonvulsant effects.Moreover, the rebound hyperactivation after long-term treatment 5-HT3 receptor blockers fail to induce dependence af­ with classic neuroleptics suggests that 5-HT3 receptor ter long-term treatment (Jones et al. 1988). Interestingly, antagonists ameliorate the undesirable side effectsob­ the 5-HT3 receptor antagonist GR38032F, but not the served after chronic administration of DA receptor anxiolytic nonbenzodiazepine drug , exerts blockers. In this line, although preliminary clinical its anxiolytic effecteven in rodents with an underlying studiesdo not support a role for 5-HT3 antagonists as withdrawal syndrome (Costall et al. 1990). The mecha­ potential antipsychotic drugs, zacopride seemed to be nism by which 5-HT3 receptor antagonists exert their effective in reducing abnormal motor movements in a anxiolytic action in rodents is still unclear. In most tests patient with a moderate degree of tardive dyskinesia that evaluate anxiety-related pharmacologic profiles, (Newcomer et al. 1990). 5-HT3 receptor antagonists show a dose-response re­ The mesolimbic system also seems to be important lation within the range of those observed in other in inthe mechanisms by which substances with abuse vivo studies that evaluate 5-HT3 receptor function potential produce their pharmacologic effects. The in­ (Butler et al. 1988). Moreover, the fact that structurally creasein DA turnover in the nucleus accumbens after different 5-HT3 receptor antagonists are endowed with administration of morphine (Costa et al. 1973; Imper­ anxiolytic activity strongly supports a role for 5-HT3 ato and Angelucci 1989), (Costa and receptors in the mediation of this activity in experimen­ Gropetti 1970; Costa et al. 1972; Costall et al. 1980), or tal animals. In this line, preliminary clinical studies have cocaine(Costa et al. 1973) may contribute to the reward­ shown that zacopride exerts a mild anxiolytic effect ingmechanisms (Wise and Bogharth 1988) and to the without sedation and reduces the distress associated maintenance of psychologic dependency after adminis­ with psychotic symptoms (Newcomer et al. 1990). trationof these drugs of abuse (Costall et al. 1990). In­ The ability of GR38032F to facilitate ACh release in terestingly, 5-HT3 receptor antagonists proved to be cortical tissue (Barnes et al. 1989b) may indicate the bio­ highly effective in reducing the consequences of with­ chemical basis by which 5-HT3 receptor antagonists drawalafter long-term treatment with a number of these improve cognitive performance in behavioral tests that drugs, including , anxiolytics, cocaine, ampheta­ evaluate cognitive function (Costall et al. 1989). Con­ mine, and nicotine (Oakley et al. 1988a, 1988b; Costall sistent with the observation that 5-HT3 receptor antag­ et al. 1990). Whether the effects of 5-HT3 receptor an­ onists reverse -induced cognitive deficits tagonists are mediated through a reduction in the (Barneset al. 1989c) isa preliminary report showing that 126 J.A. Apud NEUROPSYCHOPHARMACOLOGY 1993-VOL . 8, NO.2

zacopride partially reverses the cognitive impairment receptor-gated Na+-K+ channel from rapid desensiti· induced by scopolamine in normal human volunteers. zation follows either monophasic or complex biphasic kinetics depending on the tissue involved (Yakel and Jackson 1988). Moreover, in NG108-15 cells, but not in CONCLUSIONS NlE-115 cells, the time course of desensitization is volt· age dependent (Yakel and Jackson 1988). Progress in the understanding of the functional role of Studies of 5-HT3 receptor function also support 5-HT3 receptors in the brain will evolve from the pi­ the concept of 5-HT3 receptor heterogeneity. Thus, in oneering efforts of different research teams. With the the striatum and the nucleus accumbens, activation of use of various structure-related predictive strategies, 5-HT3 receptors stimulates the release of DA (Blandina researchers have successfully developed potent and et al. 1988, 1989; Imperato and Angelucci 1989), whereas specifIc 5-HT3 receptor agonists and antagonists. in cortical tissue the stimulation of 5-HT3 receptors in­ Moreover, the availability of labeled receptor ligands hibits ACh release (Barnes et al. 1989b). The recent with relatively high specifIc activity has allowed the identifIcationof a second transducing system linked to identifIcation of 5-HT3 binding sites in mammalian 5-HT3 receptor function, namely PI hydrolysis (Ed­ brain. The 5-HT3 recognition site in the brain is simi­ wards et al. 1991), further supports the existence of lar to that in the periphery and is probably located on different 5-HT3 receptor subpopulations in neural neurons. Distribution studies have indicated that the tissues. The possibility, however, that the increase area postrema possesses the highest concentration of of PI metabolism after 5-HT3 receptor stimulation is 5-HT3 binding sites, although high concentrations are due to a still unknown indirect effect cannot be totally also present in cortical and limbic brain st ructures. ruled out. Biochemical, molecular biological, and electrophys­ Biochemical and behavioral studies have provided iologic studies have suggested that the 5-HT3 receptor data on the therapeutic potential of 5-HT3 receptor an­ belongs to the superfamily of ligand-gated ion chan­ tagonists. The high concentration of 5-HT3 binding nels with molecular masses of 250 to 300 kD, which in­ sites in the area postrema, a brain area related to the cludes GABAA, glycine, and neuronal nicotinic ACh control of emesis, suggests a potential role for 5-HT3 receptors. The 5-HT3 receptor thus appears to be un­ receptor antagonists as potent antiemetics during tu­ like other 5-HT receptors in that it does not belong to mor chemotherapy or radiotherapy. On the other hand, the G-protein-linked receptor superfamily but appears the ability of 5-HT3 receptor antagonists to reduce to comprise an ion channel that is permeable to Na + drug-induced hyperactivity of mesolimbic and nigro· and K+. The 5-HT3 receptor has a heterooligomeric striatal dopaminergic neurons implicates these com­ structure composed of four or fIve subunits, which to­ pounds as prospective antipsychotic agents and as gether form a transmembrane ion channel complex that potential alternatives for the treatment of neuroleptic­ is susceptible to transmitter gating. In oocytes express­ induced side effects. The ability of various 5-HT3 ing the cloned 5-HT3 receptor as well as in primary cul­ receptor antagonists to exert anxiolytic effects in ani­ tures and cell lines of neuronal origin, 5-HT3 receptor mal models and to effectivelylimit the severity of symp­ activation results in a fast depolarizing excitatory re­ toms caused by morphine withdrawal in dependent in· sponse that can be selectively blocked by 5-HT3 recep­ dividuals point to the potential use of 5-HT3 receptor tor antagonists. antagonists in a wide variety of psychiatric disorders. A growing body of evidence suggests the presence A signifIcant number of issues still need to be ad­ of distinct 5-HT3 receptor subpopulations in the ner­ dressed to reach a better understanding of the role of vous system. Thus, neurophysiologic studies have the 5-HT3 receptor in mammalian physiology and shown a 35- to 40-fold difference in single-channel con­ pathology. Clinically useful selective agonists with less ductance and at least a twofold difference in ion selec­ polar properties are still required for pharmacologic tivity among clonal cell line and enteric neuron 5-HT3 studies in humans. IdentifIcation of the cellular loca­ receptors (Derkach et al. 1989; Lamberts et al. 1989; tions of 5-HT3 receptors, as well as studies on their Yang 1990). In addition, at odds with the failure of the pharmacologic and biochemical regulation in brain, 5-HT3 receptor antagonist MDL 72222 to counteract the should provide valuable information to permit the tar­ effect of 5-HT in enteric neurons, this compound rever­ geting of specifIc neuronal that contribute to sibly blocks the 5-HT-evoked current in Xenopus 00- the genesis of symptoms of specifIc psychiatric dis­ cytes expressing the cloned 5-HT3 receptor (Maricq et orders. The generation of currents with a single 5-HT3 al. 1991). These data are consistent with the apparent receptor clone demonstrates that a functional single unit absence of 5-HT3 receptor rnRNA in the mouse intes­ can be expressed in Xenopus oocytes. Some of the func­ tine (Maricq et al. 1991). Further evidence for the exis­ tional properties of this receptor, however, seem to tence of multiple 5-HT3 receptors has been provided differfrom the native multimeric receptor; thus, it will by studies showing that the recovery of the 5-HT3 be crucial to determine the additional subunit compo- NEUROPSYCHOPHARMACOLOGY 1993-VOL . 8, NO. 2 5-HT3 Receptors in Brain 127

sition of the native 5-HT3 receptor to understand the mine release in the rat nucleus accumbens. Brain Res molecular mechanisms of receptor regulation. More­ 543:354-357 over, further molecular biological studies should help Conn PI, Sanders-Bush E (1984): Selective 5-HT2 antagonists elucidate whether there is only one 5-HT3 receptor or inhibit serotonin stimulated phosphatidylinositol metab­ olism in cerebral cortex. Neuropharmacology 23:993-996 whether receptor subtypes exist. Conn PI, Sanders-Bush E, HoffmanBI, Hartig PR (1986): A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover. Proc Natl Acad Sci USA REFERENCES 83: 4086-4088 Costa E, Gropetti A (1970): Biosynthesis and storage of AdamsJD, Dwyer TM, Hille B (1980): The permeability of in tissues of rats inj ected with various endplate channels to monovalent and divalent metal cat­ doses of d-amphetamine. In Costa E, Garattini S (eds), ions. J Gen Physiol 75:493-5lO and Related Compounds. New York, Ra­ Andrews PLR, Rapeport WG, Sanger GJ (1988): Neurophar­ ven Press, pp 231-255 macology of emesis induced by anti-cancer therapy. Costa E, Gropetti A, Naimzada MK(1972): Effectsof ampheta­ Trends Pharmacol Sci 9:334-341 mine on the turnover rate of brain catecholamines and Bames JM, Barnes NM, Costall B, Ironside JW, Naylor RJ motor activity. Br J Pharmacol 44:742-751 (1989a) : Identibcation and characterization of 5-hydroxy­ Costa E, Carenz i A, Guidotti A, Revuelta A (1973): Narcotic tryptamine3 recognition sites in human brain tissue. J anal gesics and the regulation of neuronal Neurochem 53:1787-1793 stores. In Us din E, Snyder SH (eds), Frontiers of Cate­ BamesJM, Barnes NM , Costall B, Naylor RJ, Tyers MB (1989b): cholamine Research. London, Pergamon Press, pp 5-HT3 receptors mediate inhibition of acetylcholine re­ 1003-1010 lease in cortical tissue. Nature 338:762-763 Costall B, Fortune DH, Hui SCG, Naylor RJ (1980): Neurolep­ BamesJM, Costall B, Kelly ME, Naylor RI, Onaivi ES, Tom­ tic antagonism of the motor inhibitory eff ects of apomor­ kins DM, Tyers MB (1989c): GR38032 improves perfor­ phine within the nucleus accumbens: drug interaction mance in a mouse habituation test, and inhibits cholin­ at presynaptic receptors? Eur J Pharmacol 63:347-358 ergic- linked defects. Br J Pharmacol 98:693P (abstract) Costall B, Domeney AM , Naylor RI, Tyers MB (1987): Effects Bames JM, Barnes NM, Champaneria S, Costall B, Naylor of the 5-HT3 receptor antagonist, GR38032F, on raised RJ (1990): Characterization and autoradiographic local­ dopaminergic activity in the mesolimbic system of the ization of 5-HT3 receptor recognition sites identifiedwith rat and marmoset brain. Br J Pharmacol 92:881-894 [3 HJ-(S)-zacopride in the forebrain of the rat. Neu­ Costall B, Domeney AM, Kelly ME, Naylor RI, Onaivi ES, ropharmacology 29:1037-1045 Tomkins OM, Tyers MB (1989): Effects of GR38032F on 3 Bames NM, Costall B, Naylor RJ (1988): [ H)Zacopride: performance of common marmosets in an obj ect discrimi­ ligand for the identification of 5-HT3 recognition sites. nation reversal task. Br J Pharmacol 98:637P (abstract) J Pharm PharmacoI 40:548-551 Costall B, Naylor RJ, Tyers MB (1990): The psychopharma­ Blandina P, Goldfarb J, Green JP (1988): Activation of a 5-HT3 cology of 5-HT3 receptors. Pharmacol Ther 47:181-202 receptor releases dopamine from striatal slices. Eur J Phar­ Cunningham D, Pople A, Grazet J-C, Ford HT, Challoner T, macoI 155:349-350 Coombes RC (1987): Prevention of emesis in patients BlandinaP, Goldfarb I, Craddock-Royal B, Green JP (1989): receiving cytotoxic drugs by GR38032F, a selective 5-HT3 Release of endogenous dopamine by stimulation of 5-hy­ receptor antagonist. Lancet 1:1461-1463 droxytryptamine3 receptor in rat striatum. J Pharmacol Derkach V, Suprenant A, North RA (1989): 5-HT3 receptors Exp Ther 251:803-809 are membrane ion channels. Nature 339:706-709 BolanosFJ, Schechter LE, Miquel Me, Emerit MB, Rumigny Dubois A, Fiala N, Boward GA, Bogo V (1988): Prevention JF, Hamon M (1990): Common pharmacological and and treatment of the gastric symptoms of rad iat ion sick­ ph ysico-chemical properties of 5-HT3 binding sites in the ness. Radiat Res 115:595-604 rat cerebral cortex and NG 108-15 cells. Biochem Phar­ macoI 40:1541-1550 Dumuis A, Bouhelal R, SebbenM, Cory R, BockaertJ (1988): A nonclassical 5-hydroxytryptamine receptor positively Bradley PB, Engel G, Feniuk W, Fozard J, Humphrey P, Mid­ coupled with adenylate cyclase in the central nervous sys­ dlemiss DN, Mylecharane E, Richardson B, Saxena P tem. Mol PharmacoI 34:880-887 (1985): Proposals for the classifIcation and nomenclature of functional receptors for 5-hydroxytryptamine. Neu­ Edwards E, Harkins H, Ashby CR, Wang RY (1991): Effect ropharmacology 25:563-576 of 5-hydroxytryptamine3 receptor agonists on phos­ ButlerA, Hill JM, Ireland SJ, Jordan Ce, Tyers MB (1988): phoinositides hydrolysis in the rat fronto-cingulate and Pharmacological properties of GR38032F, a novel an­ entorhinal cortices. J Pharmacol Exp Ther 256:1025-1032 tagonist at 5-HT3 receptors. Br J Pharmacol 94:397-412 Fake CS, King FO, Sanger GJ (1987): BRL 43694: a potent and Carboni E, Acq uas E, Frau R, DiChiara GD (1989): Differen­ novel 5-HT3 antagonist. Br J Pharmacol 91:335P (ab­ tial inhib itory effects of a 5-HT3 antagonist on drug­ stract) induced stimulation of dopamine release. Eur J Pharmacol Fargin A, Raymond JR, Lohse MI, Kobilka BK, Caron MG, 164 : 514-520 Lefkowitz RJ (1988): The genomic clone G-21 which Chen J, van Pragg HM, Gardner EL (1991): Activation of resembles a beta- receptor sequence encodes 5-HT3 receptor by 1-phenylbiguanide increases dopa- a 5-HTla receptor. Nature 335:358-360 128 J.A. Apud NEUROPSYCHOPHARMACOLOGY 1993 - VOL. 8, NO.2

Feuerstein TJ, Hertting G (1986): Serotonin (5-HT) enhances Imperato A, Angelucci L (1989): 5-HT3 receptors control hippocampal noradrenaline release: Evidence for facilita­ dopamine release in the nucleus accumbens of freely tory 5-HT receptors within the CNS. Naunyn Schmiede­ moving rats. Neurosci Lett 101:214-217 bergs Arch Pharmacol 333:191-198 Jiang LH, Ashby CR, Kasser RJ, Wang RY (1990): The effect Fozard JR (1983): Differences between receptors for 5-HT on of intraventricular administration of the 5-HT3 receptor autonomic neurons revealed by nor-( - )-cocaine . J Au­ agonist 2-methylserotonin on the release of dopamine ton Pharmacol 3:21-26 in the nucleus accumbens: An in vivo chronocoulomet· ric study. Brain Res 513:156-160 Fozard JR (1984): Neuronal 5-HT receptors in the periphery. Neuropharmacology 23:1473-1486 Jones BJ, Costall B, Domeney A, Kelly AM, Naylor RJ, Oak· ley NR, Tyers MB (1988): The potential anxiolytic activo Fozard JR (1984a): MDL72222, a potent and highly selective ity of GR38032F, a 5-HT3 receptor antagonist. Br J Phar­ antagonist at neuronal 5-hydroxytryptamine receptors. macoI 93 :985-993 Naunyn Schmiedebergs Arch Pharmacol 326:36-44 Julius D, MacDermott A, Axel R, Jessel TM (1988): Molecular Fozard JR, Gittos MW (1983): Selective blockade of 5-hy­ characterization of a functional cDNA encoding the droxytryptamine neuronal receptors by benzoic acid es­ serotonin1c receptor. Science 241:558-564 ters of tropine . Br J Pharmacol 80:511P (abstract) Kandel ER (1985): Chemically gated ion channels at central Fozard JR, Mobarok Ali ATM (1978): Blockade of neuronal synapses. In Kandel ER, Schwartz JH (eds), Principles tryptamine receptors by metoclopramide. Eur J Phar­ of Neural Science, ed 2. New York, Elsevier, pp 108-119 macoI 49:109-112 Kilpatrick GJ, Jones BJ, Tyers MB (1987): IdentifIcation and Fozard JR, Mobarok Ali AT, Newgrosh G (1979): Blockade distribution of 5-HT3 receptors in rat brain using radio­ of serotonin receptors on autonomic neurons by (-)­ ligand binding. Nature 330:746-748 cocaine and some related compounds. Eur J Pharmacol 59:195-210 Kilpatrick GJ, Jones BJ, Tyers MB (1989): Binding of the ligand, 3 [ H]GR65630, to rat area postrema, vagus nerve and the Gaddum JH, Hameed KA (1954): Drugs which antagonize brains of several species. Eur J Pharmacol 159:157-164 5-hydroxytryptamine. Br J Pharmacol 9:240-248 Kilpatrick GJ, Butler A, Hagan RM, Jones BJ, Tyers MB (1990): Gaddum JH, Piccarelli ZP (1957): Two kinds of tryptamine 3 [ H]GR67330, a very high affinity ligand for 5-HT3 recep­ receptors. Br J Pharmacol Chemother 12:323-328 tors. Naunyn Schmiedebergs Arch PharmacoI 342:22-30 Glaum SR, Anderson E (1988): IdentifIcation of 5-HT3 bind­ Koulu M, Sjoholm B, Lappalainen J, Virtanen R (1989): Effects ing sites in rat spinal cord synaptosomal membranes. Eur of acute GR38032F (odansetron), a 5-HT3 receptor an­ J Pharmacol 156:287-290 tagonist, on dopamine and serotonin metabolism in Glennon RA (1987): Central serotonin receptors as targets for mesolimbic and nigrostriatal dopaminergic neurons. Eur drug research . J Med Chern 30:1-9 J Pharmacol 169:321-324 Glennon RA, Slusher MR, Lyon RA, Titeler M, McKenney Lambert J, Peters JA, Hales TG, Dempster J (1989): The prop­ JD (1986): 5-HT1 and 5-HT2 binding characteristics of erties of 5-HT receptors in clonal cell lines studied by some quipazine analogs. J Med Chern 29:2375-2379 patch-clamp techniques. Br J Pharmacol 97:27-40

Hagan RM, Jones BJ, Jordan CC, Tyers MB (1990): Effect of Leibundgut U, Lancranjan I (1987): First results with ICS 205- 5-HT3 receptor antagonists on responses to selective ac­ 930 (5-HT3 receptor antagonist) in prevention of chemo­ tivation of mesolimbic dopaminergic pathways in the rat. therapy induced emesis. Lancet 1:1198 (letter) Br J Pharmacol 99 :227-232 Leonhardt S, Herrick-Davis K, Titeler M (1989): Detection of Hamblin MW,Metcalf MA (1991): Primary structure and func­ a novel serotonin receptor subtype (5-HT1E) in human tional characterization of a human 5-HTm-type seroto­ brain: interaction with a GTP-binding protein. J Neu­ nin receptor. Mol Pharmacol 40: 143-148 rochem 53:465-471 Tar­ Hamon M, Gallissot MC, Menard F, Gozlan H, Bourgoin S, Lummis SCR, Nielsen M, Kilpatrick GJ, Martin IL (1990): Verge D (1989): 5-HT3 receptor binding sites are on get size of 5-HT3 receptors in N1E-115 neuroblastoma capsaicin-sensitive fIbers in the rat spinal cord. Eur J Phar­ cells and rat brain. Eur J Pharmacol 189:229-232 macol 164:315-322 Mamalaki C, Barnard EA, Stephenson FA (1989): Molecular size of the gamma-aminobutyric acid A receptor puribed Heuring RE, Peroutka SJ (1987): Characterization of a novel 3 from mammalian cerebral cortex_ J Neurochem 52:124- H-5-HT binding subtype in bovine brain membranes. 134 J Neurosci 7:894-903 Maricq AV, Peterson AS, Brake AI, Myers RM, Julius D (1991): Hoyer D, Neijt H (1987): IdentifIcation of 5-HT3 recognition Primary structure and functional expression of the 5-HT3 sites by radioligand binding in NG108-15 neuroblastoma­ receptor, a serotonin-gated ion channel. Science 254: glioma cells. Eur J Pharmacol 143:291-293 432-437.

Hoyer D, Neijt HC (1988): IdentifIcation of serotonin 5-HT3 McKernan RM, Quirk K, Jackson RG, Ian Ragan C (1990a): recognition sites in membranes of N1E-115 neuroblas­ Solubilization of the 5-hydroxytryptarnine 3 receptor from toma cells by radioligand binding. Mol Pharmacol 33: pooled rat cortical and hippocampal membranes. J Neu­ 303-309 rochem 54:924-930

Hoyer D, Gozlan H, Bolanos F, Schechter LE, Hamon M McKernan RM, Gilard NP, Quirk K, Kneen CO, Stevenson (1989): Interaction of psychotropic drugs with central GI, Swain CJ, Ian Ragan C (1990b): PurifIcation of the 5-HT3 recognition sites: Fact or artifact? Eur J Pharmacol 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells. 171:137-139 J Bioi Chern 265:13572-13577 NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO. 2 5-HT3 Receptors in Brain 129

3 Milburn CM, Peroutka SJ (1989): Characterization of [ H]qui­ Peroutka SJ, Lebovitz RM, Snyder SH (1981): Two distinct pazine binding to 5-hydroxytryptamine3 receptors in rat central serotonin receptors with different physiological brain membranes. J Neurochem 52:1787-1792 functions. Science 212:827-829 Miquel MC, Emerit MB, Bolanos FJ, Schechter LE, Gozlan Peters JA, Usherwood PNR (1983): 5-Hydroxytryptamine re­ H, Hamon M (1990): Physicochemical properties of ser­ sponses of murine neuroblastoma cells: ions and puta­ otonin 5-HT3 binding sites solubilized from membranes tive antagonists. Br J Pharmacol 80:523P (abstract) of NG 108-15 neuroblastoma-glioma cells. J Neurochem Peters JA, Lambert JJ (1989): Electrophysiology of 5-HT3 55:1526-1536 receptors in neuronal cell lines. Trends Pharmacol Sci Nakaki T, Roth BL, Chuang D-M, Costa E (1984) : Phasic and 10:172-174 tonic components in 5-HT receptor-mediated rat aorta 2 Peters JA, Hales TG, Lambert JJ (1988): Divalent cations modu­ contraction: participation of Ca2+ channels and phospho­ late 5-HT3 receptor-induced currents in N1E115-neuro­ lipase C. J Pharmacol Exp Ther 243:442-446 blastoma cells. Eur J Pharmacol 151:491-495 Neijt He, Vijverberg HPM, van der Bercken J (1986): The Pfeiffer F, Graham D, Betz H (1982): PurifIcation by affInity dopamine response in mouse neuroblastoma cells is chromatography of the glycine receptor of the rat spinal mediated by serotonin 5-HT3 receptors. Eur J Pharmacal cord. J BioI Chern 257:9389-9393 127:271-274 Pratt GD, Bowery NG (1989): The 5-HT3 receptor ligand Neijt HC, Te Duits II, Vijverberg HPM (1988a): Pharmaco­ 3 [ H]BRL 43694 binds to presynaptic sites in the nucleus logical characterization of serotonin 5-HT3 receptor­ tractus solitarius of the rat. Br J Pharmacol 97:414P (ab­ mediated electrical response in cultured mouse neuro­ stract) blastoma cells. Neuropharmacology 27:301-307 Pratt GD, Bowery NG, Kilpatrick JG, Leslie RA, Barnes NM, Neijt HC, Karpf A, SchoeffterP, Engel G, Hoyer D (1988b): Naylor R, Jones BI, Nelson DR, Palacios JM, Slater P, Reyn­ Characterization of 5-HT3 recognition sites in mem­ olds JM (1990): Consensus meeting agrees distribution branes of NG-108-15 neuroblastoma-glioma cells with of 5-HT3 receptors in mammalian hindbrain. Trends [3H]ICS 205-930. Naunyn Schmiedebergs Arch Phar­ Pharmacol Sci 11:135-137 macoI 337:493-499 Pritchett DB, Bach AWl, Wozny AWl, Taleb 0, Dal Toso R, Newcomer JW, Faustman WO, Roth B, Bierley RA, Moses Shih JC, Seeburg PH (1988): Structure and functionalex­ JA, Csernansky JG (1990): Zacopride in schizophrenia: pression of cloned rat serotonin 5-HT2 receptor. EMBO a single-blind 5-HT3 antagonist trial. Proc Soc Neurosci J 7:4135-4140 16:752 (Abstract) Resier G, Hamprecht B (1989): Serotonin raises the cyclic GMP Nowak L, Bregestovski P, Ascher P, Herbert A, Prochiantz levels in a neuronal cell line via 5-HT3 receptors. Eur J A (1984) : Magnesium gates glutamate activated channels PharmacoI 172:195-198 in mouse central neurons. Nature 307:462-465 Reynolds DJM, Andrews PLR, Leslie RA,Harvey JM, Grasby Oakley NR, Jones BJ, Tyers MB (1988a): Tolerance and with­ PM, Grahame-Smith DC (1989): Bilateral abdominal va­ drawal studies with diazepam and GR38032F in the rat. 3 gotomy abolishes binding of H BRL 43694 in ferret dor­ Br J Pharmacol 95 :764P (abstract) sovagal complex. Br J Pharmacol 98:692P (abstract) Oakley NR, Jones BJ, Tyers MB, Costall B, Domeney AM (1988b): The effectof GR38032F on alcohol consumption Richardson BP, Engel G, Donatsch P, Stadler PA (1985): in the marmoset. Br J Pharmacol 95:870P (abstract) IdentifIcationof serotonin-M rec�ptor subtypes and their specifIc blockade by a new class of drugs. Nature 316: Pazos Hoyer D, Palacios TM (1984): , a selec­ A, 126-131 tive serotonin 2 ligand in the rat cortex, does not label receptors in porcine and human cortex: evidence for spe­ Rizzi JP, Nagel AA, Rosen T, McLean S, Seeger T (1990): An cies differences on brain serotonin 2 receptors. Eur J Phar­ initial three-component pharmacophore for specifIc macol 106:531-538 serotonin-3 receptor ligands. J Med Chern33: 2721-2725

Pedigo NW, Yamamura DL, Nelson J (1981): Discrimination Rocha e Silva M, Valle JR, Piccarelli ZP (1953): A pharmaco­ 3 of multiple H-5-hydroxytryptamine binding sites by the logical analysis of the mode of action of serotonin upon neuroleptic spiperone in rat brain. J Neurochem 36: the guinea pig ileum. Br J Pharmacol 8:378-388 220-226 Schmidt AW, Peroutka SJ (1989): Antidepressant interactions Peroutka SJ (1986): Pharmacological differentiationand char­ with 5-hydroxytryptamine3 receptor binding sites. Eur acterization of 5-HT1B and 5-HTlC binding sites in rat J Pharmacol 163 :397-398 frontal cortex. J Neurochem 47:529-540 Schmidt AW, Siok q, Seeger TF (1988): 5-HT3 binding sites Peroutka SJ (1988): Species variations in 5-HT3 recognition 3 3 identifIed in rat brain using [ H]ICS 205-930. Soc Neu­ sites labeled by H-quipazine in the central nervous sys­ rosci Abstr 14:846 tem. Naunyn Schmiedebergs Arch Pharmacol 338: 472-475 Sibley DR, Lefkowitz RJ (1985): Molecular mechanisms of receptor desensitization using the beta-adrenergic recep­ Peroutka SI, Snyder SH (1979): Multiple serotonin receptors: 3 3 tor-coupled adenylate cyclase system as a model. Nature differential binding of [ H]-5-hydroxytryptamine, [ H]_ 317:124-129 lysergic acid diethylamine and [3H]-spiroperidol. Mol Pharmacol 16:687-699 Sigel E, Stephenson FA, Mamalaki C, Barnard EA (1983): A 3 garnrna-aminobutyric acid/benzodiazepine receptor com­ Peroutka SJ, Hamik A (1988): [ H]Quipazine labels 5-HT3 rec­ plex of bovine cerebral cortex. J BioI Chern258: 6965-6971 ognition sites in rat cortical membranes. Eur J Pharmacol 148:297-299 Smith WL, Sanci1io LF, Owera-Atepo JB, Naylor JR, Lambert 130 J.A. Apud NEUROPSYCHOPHARMACOLOGY 1993 -VOL. 8, NO.2

L (1988): Zacopride: a potent 5-HT3 antagonist. J Ph arm sites in rat brain. EurJ Pharmacol 149:397-398 PharmacoI 40 :301-302 Whiting PI, Lindstrom JM (1986): Puribcation and character· Sorensen SM, Humphreys TM, Palfreyman MG (1989): Effect ization of a nicotinic from chick of acute and chronic MDL 73, 147EF, a 5-HT3 receptor an­ brain. Biochemistry 25 :2082-2093 tagonist, on A9 and A10 dopamine neurons. Eur J Phar­ Wise RA, Bogarth MA (1982): Action of drugs of abuse on macol 163:115-118 brain reward systems: an update with specibc attention Stephenson FA (1988): Understanding the GABAA receptor: to opiates. Pharmacol Biochem Behav 17:239-243 a chemically gated ion channel. Biochem J 249:21-32 Yakel JL, Jackson MB (1988): 5-HT3 receptors mediate rapid Tricklebank MD (1989): Interactions between dopamine and responses in cultured hippocampus and a clonal cell line. 5-HT3 receptors suggest new treatments for psychosis Neuron 1:615-621 and drug addiction. Trends Pharmacol Sci 10: 127-129 Yakel JL, Trussell LO, Jackson MB (1988): Three serotonin Waeber C, Hoyer D, Palacios JM (1989): 5-Hydroxytryptarnine responses in cultured mouse hippocampus and striatal 3 receptors in the human brain: autoradiographic visu­ 3 neurons. J Neurosci 8:1273-1285 alization using [ H]ICS 205-930. Neuroscience 31:393- 400 Yakel JL, Shao XM, Jackson MB (1990): The selectivity of the Watling KJ (1988): Radioligand binding studies identify channel coupled to the 5-HT3 receptor. Brain Res 533: 5-HT3 recognition sites in neuroblastoma cell lines and 46-52 mammalian CNS. Trends Pharmacol Sci 9:227-229 Yang J (1990): Ion permeation through 5-hydroxytryptamine­ 3 Watling KJ, Aspley S, Swain q, Saunders J (1988): [ H]_ gated channels in neuroblastoma N18 cells. J Gen Phys· Quaternised ICS 205-930 labels 5-HT3 receptor binding ioI 96:1177-1198