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The effect of the selective serotonin reuptake inhibitor fluoxetine was examined on the 5-HT4 receptor-mediated relaxation in the rat isolated ileum. Fluoxetine unsurmountably antagonized the relaxation to exogenous 5-HT with abolition of the response at 10 microM. Fluoxetine (10 microM) also caused a gradual loss of the resting tension. These effects of fluoxetine were prevented by a prior addition of the 5-HT4 receptor selective antagonist GR113808 (100 nM), which itself caused a contraction of the tissues when administered alone. Fluoxetine (10 microM) also failed to prevent the relaxation due to exogenous 5-HT and the 5-HT4 receptor agonist 5-methoxytryptamine in tissues taken from the rats treated with para-chlorophenylalanine (300 mg kg-1) for 3 and 6 days, which reduced the 5-HT level in the mucosa by 88 and 97.5% respectively. The contraction of the tissues with GR113808 indicates the presence of an endogenous 5-HT tone at the 5-HT4 receptor in the rat ileum. It is hypothesized that in the presence of fluoxetine, the concentration of endogenous 5-HT at the receptor was increased sufficiently to reduce or abolish the relaxation to 5-HT added exogenously. The inability of fluoxetine to prevent the relaxation to 5-HT in the presence of GR113808 or after the p-CPA treatment supports this hypothesis.

The pharmacological characterization of a 5-HT receptor-mediated contractile response in the mouse isolated ileum is described. In the presence of methysergide (1 microM), 5-hydroxytryptamine (5-HT, 0.3 - 100 microM) produced phasic concentration-dependent contractions of segments of the mouse isolated ileum with a pEC(50) value of 5.47+/-0.09. The 5-HT(3) receptor selective agonists m-chlorophenylbiguanide (0.3 - 100 microM, pEC(50) 5.81+/-0.04), 1-phenylbiguanide (3 - 100 microM, pEC(50) 5.05+/-0.06) and 2-methyl-5-HT (3 - 100 microM, pEC(50) 5.00+/-0.07) acted as full agonists to induce contractile responses. 5-methoxytryptamine (0.1 - 100 microM), RS 67506 (0.1 - 100 microM) and alpha-methyl-5-HT (0.1 - 100 microM) failed to mimic the 5-HT responses. The contractile response to 5-HT was not antagonized by either 5-HT(2) receptor antagonists ritanserin (0.1 microM) or ketanserin (1 microM) nor the 5-HT(4) receptor antagonist SB 204070 (0.1 microM). The 5-HT(3) receptor selective antagonists granisetron (0.3 - 1 nM), tropisetron (1 - 10 nM), ondansetron (10 nM - 1 microM) and MDL 72222 (10 nM - 1 microM) caused rightward displacement of the concentration-response curves to 5-HT. The lower concentrations of the antagonists caused approximate parallel rightward shifts of the concentration-response curves to 5-HT with apparent pK(B) values for granisetron (9.70+/-0. 39), tropisetron (9.18+/-0.20), ondansetron (8.84+/-0.24) and MDL 72222 (8.65+/-0.35). But higher concentrations of antagonists resulted in a progressive reduction in the maximum responses. The contractile response to 5-HT was abolished by tetrodotoxin (0.3 microM); atropine (0.1 and 1 microM) decreased the maximum response of the 5-HT concentration-response curve by approximately 65%. It is concluded that a neuronally located 5-HT(3) receptor mediates a contractile response to 5-HT in the mouse ileum. The 5-HT(3) receptor in the mouse ileum has a different pharmacological profile to that reported for the guinea-pig ileum.

The involvement of 5-HT2 receptor subtypes in mediating a contraction response in the isolated intestine of Suncus murinus was investigated using DOI ((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane, a 5-HT2 receptor agonist) which produced a bell-shaped concentration response curve that was significantly (p < 0.05) reduced by methysergide (a 5-HT1/2 receptor antagonist, 1 microM) but not ketanserin (a 5-HT2A receptor antagonist, 1 microM), yohimbine (a 5-HT2B receptor antagonist, 1 microM) or a combination of ondansetron (a 5-HT3 receptor antagonist, 1 microM) plus SB204070 (8-amino-7-chloro(N-butyl-4-piperidyl) methylbenzo-1,4-dioxan-5-carboxylate hydrochloride, a 5-HT4 receptor antagonist, 1 nM). The contraction response to the lower concentrations of DOI (10 nM-0.3 microM) was reduced in the presence of SB206553 (5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2 ,3-f]indole, a 5-HT2B/2C receptor antagonist, 1 microM), whilst conversely, the reducing response to the higher concentrations of DOI (1-30 microM) was prevented. A repeated challenge with 3 microM DOI produced a smaller response (desensitisation) and also reduced the response to 5-HT (5-hydroxytryptamine, 0.3 microM) that was inhibited by SB206553 (1 microM). Data indicate that 5-HT2C receptors are likely candidates to mediate the contractile response to DOI and demonstrate desensitisation to repeated challenges.

1. The effects of 5-HT and 5-HT agonists to induce contraction and the 5-HT receptors mediating these effects were investigated in the proximal, central and terminal intestinal segments of Suncus murinus. 2. The contraction curves to 5-HT (3 nM - 30 microM) were shifted to the right by methysergide (1 microM) and ritanserin (0.1 microM), without affecting the maximum response. 3. In the central and terminal segments (but not the proximal segments) ondansetron (1 microM) and atropine (1 microM) significantly attenuated the contractions to higher concentrations of 5-HT. The selective 5-HT4 receptor antagonist SB204070 (1 nM), failed to modify 5-HT induced contractions in any segment examined. 4. 5-carboxamidotryptamine, alpha-methyl-5-HT and 5-methoxytryptamine (0.003 - 3.0 microM) induced contractions but unlike 5-HT, higher concentrations of these three agents failed to increase the response or were associated with a decrease in response. 2-methyl-5-HT (0.03 - 1.0 microM) was ten times less potent than 5-HT to induce contraction but achieved the same maximum response. 5. The contractions induced by the lower concentrations of 2-methyl-5-HT (0.03 - 1.0 microM) in all segments were markedly reduced or abolished by methysergide (1.0 microM); the response to the higher concentrations of 2-methyl-5-HT (3 - 30.0 microM) were markedly reduced by atropine (1.0 microM) and ondansetron (1.0 microM). 6. In all segments examined, tetrodotoxin (1 microM) significantly reduced the 5-HT-induced contraction. 7. It is concluded that the 5-HT-induced contraction was mediated via 5-HT2 (ritanserin sensitive) receptors in all regions of the intestine, with 5-HT3 (ondansetron sensitive) receptors mediating an additional major component in the central and terminal regions.

Recent great advances in the neuropharmacology of the emetic pathways have led to better therapy and improved insight into pathophysiological processes in patients undergoing chemo- and radiotherapy. This article gives an overview of the area, outlines current controversies and makes recommendations for future clinical studies.

1. The ability of 5-HT2 and 5-HT4 receptor antagonists to modify the disinhibitory profile of diazepam and other agents was investigated in male BKW mice in the light/dark test box. 2. The 5-HT2A/2B/2C receptor antagonists ritanserin, MDL11939 and RP62203 and also methysergide, which failed to modify mouse behaviour when administered alone, caused dose-related enhancements (4 to 8 fold) in the potency of diazepam to disinhibit behavioural responding to the aversive situation of the test box. 3. Ritanserin was shown to enhance the disinhibitory potency of other benzodiazepines, chlordiazepoxide (4 fold), temazepam (10 fold) and lorazepam (10 fold), the 5-HT1A receptor ligands, 8-OH-DPAT (25 fold), buspirone (100 fold) and lesopitron (500 fold), the 5-HT3 receptor antagonists, ondansetron (100 fold) R(+)-zacopride (100 fold) and S(-)-zacopride (greater than a 1000 fold), the substituted benzamides, sulpiride (10 fold) and tiapride (5 to 10 fold) and the cholecystokinin (CCK)A receptor antagonist, devazepide (100 fold). It also reduced the onset of action of disinhibition following treatment with the 5-HT synthesis inhibitor parachlorophenylalanine. Ritanserin failed to enhance the disinhibitory effects of the CCKB receptor antagonist CI-988, the angiotensin AT1 receptor antagonist losarten or the angiotensin converting enzyme inhibitor ceranapril. 4. The 5-HT4 receptor antagonists SDZ205-557, GR113808 and SB204070 caused dose-related reductions in the disinhibitory effect of diazepam, returning values to those shown in vehicle treated controls. The antagonists failed to modify mouse behaviour when administered alone. 5. GR113808 was also shown to cause a dose-related antagonism of the disinhibitory effects of chlordiazepoxide, lorazepam, 8-OH-DPAT, buspirone, lesopitron, ondansetron, R(+)-zacopride, sulpiride, tiapride, devazepide, CI-988, losarten, ceranapril and parachlorophenylalanine. 6. It was concluded that in BKW mice (a) the failure of 5-HT2 and 5-HT4 receptor antagonists when administered alone to modify behaviour in the light/dark test indicates an absence of an endogenous 5-HT tone at the 5-HT2 and 5-HT4 receptors and (b) the enhancement by the 5-HT2 receptor antagonists and attenuation by the 5-HT4 receptor antagonists of drug-induced disinhibition indicates a plurality of 5-HT receptor involvement in the mediation of drug-induced disinhibitory profiles in the mouse.

1. The 5-HT receptor involved in the effect of mucosal application of 5-HT to facilitate peristalsis was investigated in the isolated guinea pig ileum. 2. An application of 5-HT (3-100 microM) to the mucosal surface (by inclusion of 5-HT in the Krebs-Henseleit solution passing through the lumen of the ileum) caused a concentration related facilitation of peristalsis characterized by a reduction in the peristaltic threshold. 3. Peristalsis was not modified by methiothepine (0.1 microM), ritanserin (0.1 microM), ondansetron (5 microM), granisetron (1 microM) or SB 204070 (0.1 microM) administered alone to the mucosal surface. 4. The concentration-response curve to mucosally applied 5-HT was not altered by the mucosally applied 5-HT1/2 receptor antagonist methiothepine (0.1 microM), the 5-HT2 receptor antagonist ritanserin (0.1 microM) or the 5-HT4 receptor antagonist SB 204070 (0.1 microM). However, the mucosally applied 5-HT3 receptor antagonists ondansetron (5 microM) and granisetron (1 microM) shifted the response curves to mucosally applied 5-HT to the right in a parallel and surmountable manner. The pD2 values in the absence and presence of ondansetron were 5.42 +/- 0.07 and 4.12 +/- 0.10, respectively, (n = 6) and that of granisetron were 5.45 +/- 0.12 and 4.50 +/- 0.10 respectively, (n = 5). 5. Serosally applied ondansetron (5 microM) or granisetron (1 microM) had no effect on the concentration-response curve to mucosally applied 5-HT. However, the serosally applied ondansetron and granisetron antagonised the facilitatory effect of serosally applied 5-HT (10 microM) when administered in the presence of serosally applied SB 204070 (0.1 microM). 6. It is concluded that the facilitatory effect of mucosally applied 5-HT to reduce the peristaltic threshold in the guinea pig ileum is mediated via a 5-HT3 receptor located on the mucosal and not the serosal side of the ileum.

1 The aim of the present study was to investigate a 5-HT4 receptor involvement in the mediation of a 5-HT-induced relaxation response in the rat isolated ileum in vitro. 2 Ileal segments were taken at regular intervals from the ileo-caecal junction to duodenum. 5-HT (1 microM) induced a relaxation or contraction response in segments taken from the terminal ileum: the relaxation decreased and finally disappeared as contractions dominated in the proximal tissues. The 5-HT-induced relaxations were enhanced in the terminal segments and the contractions attenuated in both terminal and proximal segments, in the presence of methysergide (1 microM) and atropine (0.1 microM). 3 In the presence of methysergide (1 microM) and atropine (0.1 microM), a cumulative addition of 5-HT (0.01-1 microM) induced a concentration-dependent relaxation in the terminal (1-20 cm from the ileo-ceacal junction) ileal segments which at higher concentrations of 5-HT (3-30 microM) reverted to contraction. 4 The rank order of potency of indole agonists in inducing a concentration-related relaxation response in tissues of the terminal ileum (pretreated with pargyline (100 microM) and in the presence of methysergide (1 or 100 microM) and atropine (0.1 microM) was 5-hydroxytryptamine (6.97 +/- 0.06), 5-methoxytryptamine (6.50 +/- 0.07), alpha-methyl-5-hydroxytryptamine (5.53 +/- 0.17), 5-carboxamidotryptamine (5.51 +/- 0.12) and 2-methyl-5-hydroxytryptamine (< 5), the pEC50 values (mean +/- s.e.mean) being shown in parentheses. 5 Pretreatment of tissues with pargyline (100 microM) selectively enhanced the potency of 5-methoxytryptamine by a factor of 19 but failed to modify the potency of the other indole agonists. 6 The 5-HT4 receptor antagonists, tropisetron, SDZ 205-557 and GR 113808 antagonized the relaxation response to 5-HT (in the presence of methysergide (1 or 10 microM) and atropine (0.1 microM)) with pKB values (95% CL) of 6.09 (5.94-6.24), 7.0 (6.9-7.09) and 8.95 (8.81-9.1) respectively. Apparent pKB values estimations for tropisetron (1 microM) and GR 113808 (10 nM) using the agonists 5-methoxytryptamine and 5-carboxamidotryptamine were 6.37 +/- 0.31, 5.91 +/- 0.38 and 8.83 +/- 0.11, 8.82 +/- 0.22 respectively. 7 Tropisetron (10 microM), SDZ 205-557 (3 microM) and GR 113808 (10-100 nM) caused an increase in basal tone of the rat terminal ileum when administered in the presence of methysergide and atropine. 8 The relaxation response to 5-HT in the rat terminal ileum was not antagonized by ritanserin (1 microM), ondansetron (1 microM) or N omega-nitro-L-arginine methyl ester (100 microM) and with only a twofold dextral shift of the concentration-response curve by tetrodotoxin (1 microM). 9 It is concluded that the relaxant response to 5-HT in the terminal region of the ileum is mediated directly at the smooth muscle; a ranked indole agonist potency and selective antagonism by 5-HT4 receptor antagonists tropisetron, SDZ 205-557 and GR 113808 indicate a 5-HT4 receptor involvement in the relaxation response.

1. The patterns of intestinal motility and the effect of an increase in intraluminal pressure were studied in vitro on segments obtained from the marmoset small intestine. 2. Segments obtained from the distal half of the marmoset small intestine exhibited segmentation, consisting of narrow annular contractions (lasting for 2-3 s) interposed between the relaxed segments of varying length. The subsequent contractions occurred slightly distal to the previous contraction, with ring-like contractions appearing to move in the aboral direction. Such segmentation was infrequent or absent in the segments obtained from the proximal half of the small intestine. An increase in intraluminal pressure inhibited segmentation and finally produced peristalsis in most of the tissues. 3. The influence of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on the threshold of the peristaltic reflex was investigated in the segments obtained from the distal half of the intestine after segmentation had subsided. The effect of drug application to the serosal surface was measured as a change in threshold pressure required to trigger the peristaltic reflex. A facilitation was defined in two ways (a) as a reduction in the threshold pressure required to trigger the reflex and (b) in those tissues that failed to respond with peristalsis on raising intraluminal pressure to the maximum attainable (1 kPa), as a reduction in threshold pressure compared to this value. 4. 5-HT (7.85 +/- 0.19), 5-methoxytryptamine (7.79 +/- 0.24), 5-carboxamidotryptamine (6.66 +/- 0.13) and 2-methyl-5-HT (6.24 +/- 0.16) caused a concentration related facilitation of the peristaltic reflex, the pD2 values (mean +/- s.e.mean) being shown in parentheses. 5. The concentration-response curves to both 5-HT and 5-methoxytryptamine were dextrally shifted in a surmountable manner in the presence of GR 113808 (30 nM). pD2 values for 5-HT and 5-methoxytryptamine were significantly decreased to 6.98 +/- 0.24 and 6.83 +/- 0.36 respectively in the presence of GR 113808 (30 nM). 6. In the presence of a high concentration of (10 microM) 5-methoxytryptamine the subsequent addition of 2-methyl-5-HT (3-10 microM) but not 5-methoxytryptamine (10 microM) facilitated peristalsis; the effect of 3 microM 2-methyl-5-HT was significantly decreased by 2 microM ondansetron. 7. It is concluded that the facilitation of the peristaltic reflex in the marmoset intestine induced by 5-HT at submicromolar concentrations involves a 5-HT4 receptor stimulation with an additional 5-HT3 receptor activation at higher concentrations.

The main function of emesis is to remove toxins from the body. The emetic response will also be triggered by cancer chemotherapy and radiotherapy or surgery under general anaesthesia. The mechanism of activation of the vomiting system is dependent on stimulation of gastrointestinal (mechanoreceptors and chemoreceptors) and/or central pathways which activate the chemoreceptor trigger zone in the area postrema. Postoperative emesis is activated by a range of factors before, during and after anaesthesia. The precise mechanism of action of any one of the influencing factors can only be speculated as there has been very little basic research into this area, due largely to the lack of an appropriate model for postoperative nausea and vomiting. The range of agents used in the prevention and treatment of emesis are effective to varying degrees, but some are associated with poor side effect profiles making them particularly unsuitable for prophylactic use. Newer antiemetics, which selectively antagonise 5-HT3 receptors, have proved effective and well tolerated in the treatment of chemotherapy-induced emesis and postoperative nausea and vomiting.

1. The influence of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on the emptying phase (circular muscle contraction) of the peristaltic reflex was investigated in the guinea-pig isolated ileum. 2. The effect of drug application to the serosal surface was measured as the changes in threshold pressure required to trigger the peristaltic reflex and the interval between the peristaltic strokes. A facilitation or inhibition of peristalsis was defined as a reduction or increase in threshold pressure respectively. 3. Peristalsis was not modified by the inclusion of methysergide (1 microM) and/or ondansetron (2 microM) in the bathing medium. 5-HT (0.1-1.0 microM) caused a facilitation of the peristaltic reflex; the response curve to 5-HT was not altered by the presence of methysergide (1 microM) and ondansetron (2 microM). 4. In the presence of methysergide (1 microM) plus ondansetron (2 microM), 5-HT (7.36 +/- 0.06), 5-methoxytryptamine (7.01 +/- 0.17), 5-carboxamidotryptamine (5.43 +/- 0.06), renzapride (6.09 +/- 0.17), (S)-zacopride (5.99 +/- 0.11), (R)-zacopride (5.61 +/- 0.13) and metoclopramide (4.8 +/- 0.65) caused a concentration-related facilitation of the peristaltic reflex, the pEC50 values (mean +/- s.e.mean) being shown in parentheses. 2-Methyl-5-HT was ineffective up to 10 microM. 5. The administration of SDZ 205-557 (1 microM) alone failed to modify the peristaltic reflex, but caused a parallel dextral shift in the concentration-effect curve to 5-HT (apparent pKB 7.38 +/- 0.30). It failed to modify the effect of acetylcholine to enhance the peristaltic reflex. 6. It is concluded that the rank order of potency of the 5-HT agonists from the indole and substituted benzamide series to facilitate the emptying phase of the peristaltic reflex in the guinea-pig ileum closely correlates with their published actions as 5-HT4 agonists in other systems. An agonist action on the 5-HT4 receptor is also supported by the potency of the 5-HT3/5-HT4 antagonist SDZ 205-557 (but not the 5-HT3 antagonist ondansetron) to inhibit the effects of 5-HT.

5-HT3 receptors have an exclusive neuronal location and evidence is presented of their involvement in behaviour. 5-HT3 receptor antagonists such as ondansetron, tropisetron and zacopride have provided the critical pharmacological tools to reveal a potent and efficacious ability to regulate disturbed behaviour. Thus the 5-HT3 receptor antagonists will restore to normal rodent and primate behaviour disturbed by increasing limbic dopamine function, aversive situations, cognitive impairments and drug abuse. The remarkable feature of their action is a failure to modify normal behaviour. This unique pharmacological signature has ensured a wide interest in the potential role of the 5-HT3 receptor antagonists in the treatment of schizophrenia, anxiety, age related memory impairment and the problems of withdrawal from drugs of abuse. The preclinical data and preliminary clinical observations are presented.

1. Tritiated derivatives of the potent and selective 5-HT3 receptor antagonists GR65630 and LY278584 were used to identify 5-HT3 recognition sites in the rat gastrointestinal tract. 2. Binding studies were carried out in homogenates of the rat oesophagus, the cardia, fundus, body and antrum of the stomach, regions of the small intestine, caecum and large intestine. The specific binding of a single concentration of GR65630 (0.5 nM) defined by granisetron (10 microM) in these areas indicated that the density of 5-HT3 recognition sites varied from 2.4 +/- 1.0 to 10.1 +/- 1.0 fmol mg-1 protein. 3. Saturable binding of [3H]-GR65630 could only be demonstrated in the terminal regions of the small intestine (Bmax in the range of 13.83 +/- 4.54-21.19 +/- 0.89 fmol mg-1 protein; mean +/- s.e. mean) and of high affinity (Kd in the range of 0.42 +/- 0.18-0.79 +/- 0.24 nM). Use of [3H]-LY278584 revealed a similar binding density (Bmax 19.54 +/- 0.26 fmol mg-1 protein) and affinity (Kd 1.04 +/- 0.07 nM) in the terminal small intestine. 4. Binding of [3H]-GR65630 and [3H]-LY278584 to the terminal region of the small intestine was inhibited by 5-HT3 receptor ligands ondansetron and S-zacopride (and 5-hydroxytryptamine), but not by 5-HT1, 5-HT2, catecholamine, gamma-aminobutyric acid and opioid receptor ligands. 5. These data demonstrate that there are regional variations in the density of 5-HT3 recognition sites within the rat gastrointestinal tract. Such data are relevant to the potential use of 5-HT3 receptor ligands to modify secretory and contraction responses in the gastrointestinal system.

E-4424 (2-(4-[4-(4-chloro-1-pyrazolyl)butyl]-1-piperazinyl)pyrimidine) was shown to be a 5-hydroxytryptamine1A receptor ligand in radioligand binding assays and in an in vitro guinea pig ileum preparation had both 5-hydroxytryptamine1A antagonist and agonist effects. The antagonist/agonist ratio of E-4424 was greater than in the case of buspirone and ipsapirone. E-4424 was compared to diazepam, buspirone and ipsapirone to inhibit the behavioral response to an aversive situation in the mouse black and white test box, the rat social interaction test and a marmoset human threat test. The acute administration of E-4424 (0.0001-0.5 mg/kg, i.p.) to the mouse decreased aversion to the white area of the test box and was as effective as diazepam (0.125-1.0 mg/kg, i.p.) and much more potent than buspirone (0.25-1.0 mg/kg, i.p.) or ipsapirone (0.5-5.0 mg/kg, i.p.). E-4424 was also effective in enhancing rat social interaction and reducing anxiety-related behaviors in the marmoset and was again more potent than diazepam, buspirone or ipsapirone. Withdrawal from a 14-day administration of diazepam, cocaine, nicotine or alcohol exacerbated the response to the aversive situation in the mouse test. This was not observed after withdrawal from a chronic treatment with E-4424, buspirone or ipsapirone. However, E-4424 administered during drug withdrawal prevented the response caused by withdrawal from cocaine, alcohol, nicotine and diazepam: buspirone was ineffective and ipsapirone only attenuated that syndrome after alcohol withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

The anxiolytic-like potential of anpirtoline was assessed in a mouse light/dark aversion test. Anpirtoline (1.0 ng kg(-1)-1.0 micrograms kg-1 i.p.) reduced the aversive responding of mice. This was detected as an increase in the latency to locate the non-aversive compartment and by decreases in the percentage of the time spent in the dark compartment, and the numbers of rears and line crossings in the dark compartment. In radioligand binding studies anpirtoline displayed submicromolar affinity for 5-HT1A, 5-HT1B and 5-HT3 receptor recognition sites (Ki = 151, 28 and 30 nM, respectively) and more modest affinity for 5-HT2 receptor recognition sites (Ki = 1.48 microM). It is concluded that anpirtoline has a unique spectrum of affinity for 5-HT receptor subtypes, its interaction with which may account for its anxiolytic-like activity.

The distribution of the binding of [3H]GR65630 (0.2 nM) to putative 5-HT3 recognition sites in the brain of the common marmoset was assessed using autoradiography. Specific binding was heterogeneously distributed with the highest densities found in discrete nuclei of the lower medulla (nucleus tractus solitarii, dorsal motor nucleus of the vagus nerve, nucleus of spinal trigeminal nerve tract and the area postrema). In forebrain areas, relatively high binding densities were located in the medial habenula nucleus and the hippocampus (CA3, CA4 and fascia dentata). Low levels of specific binding in the rest of the forebrain hindered quantification.

The 5-HT3 receptor antagonists such as ondansetron have been shown to have anxiolytic-like activity in a wide range of preclinical tests: in the mouse black: white test, the rat social interaction test, the rat elevated X-maze and the marmoset human threat test. Ondansetron, like other 5-HT3 receptor antagonists appears to have important advantages over existing anxiolytic therapies. Thus, the 5-HT3 receptor antagonists are not sedative, they do not have addictive liabilities, there are no problems of withdrawing from chronic treatment and they can be used following benzodiazepine withdrawal. Such compounds even inhibit the behavioural syndrome associated with withdrawal from drugs of abuse, nicotine, alcohol, cocaine, opiates. The preclinical data, therefore, indicates the 5-HT3 receptor antagonists as novel anxiolytics of the future, and there is new clinical work in support of this suggestion.

Evidence is reviewed that 5-HT (5-hydroxytryptamine, serotonin) acting through the 5-HT3 receptor subtype can influence behaviour relevant to anxiety, schizophrenia and cognitive disorders, and that 5-HT3 receptor antagonists such as ondansetron (CAS 116002-70-1) can correct behavioural disturbance in the absence of effect on normal behaviour. The 5-HT3 receptor antagonists exert a breadth of action over a wide dose range in rodent and primate models to inhibit aversive behaviour in animal models of anxiety and certain symptoms of withdrawal from drugs of abuse, alcohol, nicotine, diazepam and cocaine, to antagonise increased locomotor activity caused by mesolimbic dopamine excess, and facilitate performance in cognitive tests. The studies reveal an important role for 5-HT3 receptors in the regulation of limbic-cortical functioning, and a critical role for 5-HT3 receptor antagonists to establish the role of 5-HT3 receptors in schizophrenia, anxiety, drug withdrawal phenomena and cognitive disturbance. Preliminary clinical trials indicate a positive effect of ondansetron in anxiety, schizophrenia, alcohol withdrawal and age associated memory impairment.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.5. The nature of the interaction of competing compounds (quipazine, granisetron, PBG, 5-HT, 2-methyl-5-HT) for the [3H]-(S)-zacopride binding site in the rat entorhinal cortex preparation was not altered by the removal of the Krebs ions or the addition of the monoamine oxidase inhibitor, pargyline, to the HEPES/Krebs buffer.6. In conclusion, the present studies provide further evidence towards the presence of cooperativity within the 5-HT3 receptor macromolecule and indicate that either [3H]-(S)-zacopride labels a different site on the receptor complex from [3H]-LY278,584, [3H]-granisetron or [3H]-GR67330, or it binds in such a manner as to prevent the conformatory change in the receptor protein responsible for the cooperative binding of agonists (and quipazine).

5-HT3 receptors or recognition sites have been located in the brain of many animal species and man, and are considered to mediate an increase in cation conductance. The functional relevance of the central 5-HT3 receptors is being established using selective 5-HT3 receptor antagonist ligands, which have been shown to modify behaviour in animal tests that is predictive of an action to reduce anxiety, schizophrenia and cognitive disorders. A remarkable feature of the effect of 5-HT3 receptor antagonists is their ability and potency to control a disturbed behaviour in the absence of effect on normal behaviour. This reveals an important role for 5-HT in different behaviours: indeed, the breadth of action of the 5-HT3 receptor antagonists may be indicative of the critical importance of 5-HT in the control of catecholaminergic, cholinergic and peptidergic systems throughout the forebrain. Precisely how such effects are achieved remains a significant investigative challenge, with the potential for novel therapeutic developments being a major goal.

The pharmacological characterisation and topographical distribution of [3H]-(S)-zacopride recognition sites in the forebrain of the rat was studied using homogenate and autoradiographic radioligand binding techniques. [3H]-(S)-Zacopride labelled a single, saturable, specific binding site (defined by 10.0 microM granisetron) in homogenates prepared from the entorhinal cortex of the rat (pKD = 9.51 +/- 0.08; Bmax = 104 +/- 7 fmol mg-1 protein; mean +/- SEM, n = 8). Pharmacological characterisation of the recognition site, within the entorhinal cortex, suggested that [3H]-(S)-zacopride selectively labelled the recognition site of the 5-HT3 receptor. Specific binding of [3H]-(S)-zacopride (defined by 1.0 microM granisetron) was differentially distributed throughout the forebrain of the rat; highest densities were located within sub-nuclei of the amygdala (cortical amygdaloid nucleus, amygdalohippocampal area, posterior medial cortical amygdaloid nucleus, posterior lateral amygdaloid nucleus), cortical areas (primary olfactory cortex, entorhinal cortex) and hippocampus. Non-specific binding was distributed homogeneously, although lower in myelinated structures. It is concluded that [3H]-(S)-zacopride selectively labels 5-HT3 receptor recognition sites within the forebrain of the rat; the topographical distribution of these sites, within the limbic nuclei, is consistent with the behavioural actions in animal models of the selective 5-HT3 receptor antagonists.

The present studies investigate the possible difference in 5-HT3 receptor recognition site density in amygdaloid and hippocampal homogenates from patients with Alzheimer's disease compared with age-matched controls. The 5-HT3 receptor recognition site radioligand [3H]-(S)-zacopride identified a comparable density of specific sites (defined by granisetron 10.0 microM) in tissues obtained from patients with Alzheimer's disease as compared to age-matched controls (amygdala, Bmax 41 +/- 4 and 43 +/- 9; hippocampus, Bmax 59 +/- 9 and 51 +/- 9 fmol mg-3 protein; tissue from Alzheimer's disease patients and age-matched controls, respectively, mean +/- s.e.m., n = 7-8). These results suggest that the density of 5-HT3 receptor recognition sites labelled by [3H]-(S)-zacopride are not altered in the amygdala and hippocampus of patients with Alzheimer's disease.

The distribution of [3H]zacopride (1.0 nM) to putative 5-HT3 receptor recognition sites in the ferret hindbrain was assessed using autoradiography. Specific binding (defined by the inclusion of granisetron, 1.0 microM) was heterogeneously distributed with highest density within the dorsal vagal complex (area postrema, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve). Lower densities were detected in the spinal trigeminal nerve complex whilst no other significant specific binding was detected ventral to the dorsal vagal complex. The location of 5-HT3 receptor recognition sites within the dorsal vagal complex may provide sites of action for zacopride and other 5-HT3 receptor antagonists to inhibit the emesis induced by cancer chemotherapeutic agents and x-radiation.

The present studies demonstrate the presence of specific [3H]GR65630 binding sites within the human brainstem using the techniques of in vitro receptor autoradiography and ligand binding to homogenates. Autoradiography revealed the greatest accumulation of specific binding in the area postrema and subpostrema (AP/ASP). A lower level of specific binding was identified in the nucleus tractus solitarius (excluding area subpostrema). No specific binding was evident in the remainder of the hindbrain at this level. Discrete dissection followed by ligand binding to homogenates revealed that the specific binding of [3H]GR65630 (defined by the presence of 30 microM metoclopramide) was differentially distributed with highest levels in the AP/ASP (112.1 fmol/mg protein) and lower levels in the dorsal vagal complex (nucleus tractus solitarius--excluding the area subpostrema--dorsal motor nucleus of the vagus and hypoglossal nucleus) (DVC) and olivary nucleus (ON) (22.9 and 3.9 fmol/mg, respectively). No specific binding was detectable in the reticular formation (RF) located ventral to the dorsal vagal complex. The specific [3H]GR65630 binding site was pharmacologically similar to the 5-HT3 receptor since the potent and selective 5-HT3 receptor antagonists ICS 205-930 and zacopride (100 nM) and the agonist 5-HT (10 microM) inhibited binding to the same extent as metoclopramide in each of the individual areas (90, 60 and 20% in the AP/ASP, DVC and ON, respectively). The 5-HT1-like and 5-HT2 receptor antagonist methysergide (10 microM) failed to compete for the binding site. 5-HT3 receptor recognition sites within the AP/ASP and the DVC may be functionally involved in the ability of 5-HT3 receptor antagonists to control emesis.

The review presents evidence that 5-HT3 receptors within the brain may contribute to the control of behavior. 5-HT3 receptor antagonists GR38032F, zacopride, ICS 205-930 and other agents are very potent in reducing mesolimbic dopamine hyperactivity caused by the injection of amphetamine or infusion of dopamine into the rat nucleus accumbens and amygdala, and the ventral striatum of the marmoset. Such actions are distinguished from those of neuroleptic agents by a failure to reduce normal levels of activity or to induce a rebound hyperactivity after discontinuation of treatment. Indeed, the 5-HT3 receptor antagonists can prevent the neuroleptic-induced rebound hyperactivity. Further evidence that 5-HT3 receptors moderate limbic dopamine function is shown by their ability to reduce both the behavioral hyperactivity and changes in limbic dopamine metabolism caused by DiMe-C7 injection into the ventral tegmental area. The 5-HT3 receptor antagonists also have an anxiolytic profile in the social interaction test in the rat, the light/dark exploration test in the mouse, the marmoset human threat test and behavioral observations in the cynomolgus monkey. They differ from the benzodiazepines by an absence of effect in the rat water lick conflict test and a withdrawal syndrome. Importantly, the 5-HT3 receptor antagonists are highly effective to prevent the behavioral syndrome following withdrawal from treatment with diazepam, nicotine, cocaine and alcohol. Intracerebral injection techniques in the mouse indicate that the dorsal raphe nucleus and amygdala may be important sites of 5-HT3 receptor antagonist action to inhibit aversive behavior. Studies with GR38032F indicate an additional effect in reducing alcohol consumption in the marmoset. The identification and distribution of 5-HT3 receptors in the brain using a number of 5-HT3 receptor ligands, [3H]65630, [3H]zacopride and [3H]ICS 205-930 correlates between studies, and the 5-HT3 recognition sites in cortical, limbic and other areas meet the criteria for 5-HT3 receptors to mediate the above behavioral effects. Thus the use of 5-HT3 receptor antagonists reveals an important role for 5-hydroxytryptamine in the control of disturbed behavior in the absence of effect on normal behavior. The profile of action of the 5-HT3 receptor antagonists has generated a major clinical interest in their potential use for schizophrenia, anxiety and in the control of drug abuse.

The 5-HT3 receptor antagonist [3H]zacopride was used to identify 5-HT3 recognition sites in the ferret area postrema. Specific binding was determined by the inclusion of the 5-HT3 receptor antagonist BRL 43694 in the incubation media, and was shown to be much higher in the area postrema than in other brain regions. The increased binding in the area postrema may reflect either a greater number of binding sites, a higher affinity for such sites, or both. The results indicate that 5-HT3 recognition sites are present within the area postrema and may afford an antiemetic site of action for zacopride and other 5-HT3 receptor antagonists.

[3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methylindazole-3- carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD = 2.64 +/- 0.75 and 2.93 +/- 0.41 nmol/L and Bmax = 55 +/- 7 and 44 +/- 9 fmol/mg of protein in the amygdala and hippocampus, respectively). 5-Hydroxytryptamine 3 (5-HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1 = 6.61 X 10(5) and 7.65 X 10(5)/mol/L/s and k-1 = 3.68 X 10(-3) and 3.45 X 10(-3)/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5-HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5-HT3 receptors, may provide the opportunity for 5-HT3 receptor antagonists to modify 5-HT function in the human brain.

The injection of 5-hydroxytryptamine (5-HT) and 2-methyl-5-HT into the perifornical area of the hypothalamus of the guinea-pig reduced the emptying of barium sulphate spheroids from the stomach. In contrast, the injection of the 5-HT M-receptor antagonist ICS 205-930 into the same area of the hypothalamus enhanced gastric emptying and attenuated the inhibitory effect of 5-HT. Similar injections of methysergide and ritanserin were without effect. It is concluded that an endogenous hypothalamic 5-HT system may inhibit gastric emptying through a 5-HT M-receptor mechanism which may afford a site of action for 5-HT agonist and antagonist drugs to modify gastric emptying.

The administration to the ferret of cisplatin, 10mg/kg (i.v.), caused an intense emetic response that was prevented by ICS 205-930 (0.1 and 1.0 mg/kg i.v.) and metoclopramide (4.0 mg/kg i.v.). Smaller doses of ICS 205-930 (0.01 mg/kg i.v.) and metoclopramide (2.0 mg/kg i.v.) attenuated the emetic response to cisplatin. It is concluded that the potent action of ICS 205-930 against cisplatin-induced emesis is the consequence of a 5-hydroxytryptamine M-receptor antagonism which may also contribute to the antiemetic action of metoclopramide.

The release of cerebral acetylcholine from terminals in the cerebral cortex has been shown to be regulated by 5-hydroxytryptamine (5-HT) but it is not known which subtype of the 5-HT receptor is involved. 5-HT receptor agonists increase acetylcholine levels in vivo, indicating a reduced turnover, and reduce release of acetylcholine from striatal slices in vitro. Depleting 5-HT by inhibiting synthesis or by destroying the neurons containing 5-HT potentiates acetylcholine release, and increases acetylcholine turnover in the cerebral cortex and hippocampus. Selective antagonists for the 5-HT3 receptor subtypes which seem to have effects on mood and activity may exert their effect through the regulation of acetylcholine release in the cortex and limbic system. Radioligand binding studies show a high density of 5-HT3 receptors in the cholinergic-rich entorhinal cortex and we provide evidence that a reduction in cortical cholinergic function can be effected in vitro by 5-HT3 receptors.

1. The highly selective 5-HT3-receptor antagonist, GR38032F, has been tested in five animal models predictive for anxiolytic activity. 2. In the social interaction test in the rat and in a light/dark exploration test in the mouse, GR38032F dose-dependently released suppressed behaviour without modifying locomotor activity. 3. In the cynomolgus monkey and the marmoset, GR38032F reduced anxiety-related symptoms without causing sedation. In the marmoset, the effects were clearly dose-related. 4. GR38032F did not have any detectable activity in the water-lick conflict test in the rat. 5. We conclude that GR38032F is potentially a very potent anxiolytic agent without sedative, anticonvulsant or hypnotic activity.

1. The cerebral topography of the action of diazepam and the action of the 5-hydroxytryptamine 5-HT3 receptor antagonists GR38032F and ICS 205-930 in attenuating an aversive response was studied in the mouse. 2. Mice which had been cannulated to allow drug injection into the dorsal and median raphe nuclei, the amygdala, nucleus accumbens or caudate-putamen were placed in a two compartment black (dimly illuminated) and white (brightly illuminated) test box. Measurements were made of the time spent, rearing and line crossings in the two sections and the latency of initial movement from the white to the black area. 3. The injection of diazepam (0.1-10 ng), GR38032F (0.01-1.0 ng) and ICS 205-930 (1.0-10 ng) into the dorsal raphe nucleus and amygdala, and the injection of diazepam (0.1-10 ng) into the median raphe nucleus, reduced an aversive response to the brightly illuminated white area, delaying the initial movement into the black section and increasing the time spent, rearings and line crossings in the white area. Concomitantly such activities were decreased in the black section. 4. The injection of the 5-HT3 agonist 2-methyl-5-hydroxytryptamine (0.1-10 ng) into the dorsal raphe nucleus and amygdala caused the opposite response, decreasing the time taken to move into the black section and increasing the time spent, rearings and line crossings in the black section, decreasing such activities in the white area. 5. The 5-HT3 agonist and antagonists showed little or no effect following injection into the median raphe nucleus and there were no changes in exploratory behaviour following their injection, or injection of diazepam, into the nucleus accumbens or caudate-putamen. 6. It is concluded that in the mouse the cerebral topography of action of GR38032F and ICS 205-930 in attenuating an aversive response follows that of diazepam in the dorsal raphe nucleus and amygdala but that diazepam may have additional effects mediated via the median raphe nucleus.

[3H]Zacopride displayed saturable binding to homogenates of the rat entorhinal cortex as measured by the inclusion of the 5-HT3 receptor antagonist BRL43694 in the incubation media. Scatchard analysis indicated a single high affinity binding site (KD 0.76 +/- 0.08 nM, Bmax 77.5 +/- 6.5 fmol (mg protein)-1) with a Hill slope close to unity. Other 5-HT3 receptor antagonists (zacopride, ICS 205-930, GR38032F, GR65630, metoclopramide and cocaine) also competed for the binding site displacing 60% of the total [3H]zacopride binding. 5-HT and 2-methyl-5-HT also were competitive antagonists for [3H]zacopride binding whereas 5-HT1/5-HT2 agonists and antagonists, and agents acting on other neurotransmitter receptors had Ki values greater than 10(-5) M. It is concluded that [3H]zacopride may prove a useful ligand for the study of 5-HT3 recognition sites.