Serotonin 2C receptor agonists and the behavioural satiety sequence in mice

The studies reported here examined the role of the 5-hydroxytryptamine (5-HT)(2C) receptor subtype in the control of ingestive behaviour in mice. Behavioural satiety sequence (BSS) and food intake measurements were taken, comparing the selective 5-HT(2C) receptor agonist (S)-2-(6-chloro-5-fluoro-indol-l-yl)-l-methylethylamine hydrochloride (Ro 60-0175; 1.0, 3.0 and 10.0 mg/kg) and D-fenfluramine (3.0 mg/kg). Ro 60-0175 produced a dose-dependent decrease in food intake. The effects of Ro 60-0175 (3.0 mg/kg) on the BSS were similar to the hypophagic effects of D-fenfluramine (3.0 mg/kg). In a second experiment, the specific effects on feeding produced by Ro 60-0175 (5.6 mg/kg) were attenuated by pretreatment with the selective 5-HT(2C) receptor antagonist 6-chloro-5-methyl-1-[2(2-methylpyridyl-3-oxy)-pyrid-5-yl carbamoyl] indoline (SB 242084; 0.5 mg/kg). The 5-HT(1B/2C) receptor agonist 1-(m-chlorophenyl)piperazine (mCPP; 3 mg/kg) also produced a substantial decrease in food intake, which was attenuated by SB 242084 (0.5 mg/kg). A dose of the selective 5-HT(1B/1D) antagonist 2'-methyl-4'(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-(5-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]amide (GR 127935; 3.0 mg/kg) that successfully attenuated the action of the 5-HT(1B) agonist 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969; 5.0 mg/kg) failed to attenuate mCPP-induced hypophagia. These data suggest that Ro 60-0175- and mCPP-induced hypophagia in mice are mediated via activation of 5-HT(2C) receptors and that stimulation of 5-HT(1B) receptors plays only a minor role in mCPP-induced hypophagia.     

5-HT 2A receptor activation of the external urethral sphincter and 5-HT 2C receptor inhibition of micturition: a study based on pharmacokinetics in the anaesthetized female rat

Central and peripheral 5-hydroxytryptamine (5-HT) receptors play a critical role in the regulation of micturition. Bolus doses of 5-HT(2A/2C) receptor agonists have been shown to activate the external urethral sphincter (EUS) and to inhibit micturition. This study was designed to determine the contribution of these two 5-HT receptor subtypes to activation of the EUS and inhibition of micturition utilising pharmacokinetic knowledge to better control drug exposure. Recordings of urethral and bladder pressure, EUS-Electromyogram (EMG), the micturition reflex induced by bladder filling, blood pressure and heart rate were made in anaesthetized female rats. The effects of intravenous (i.v.) infusions of the 5-HT(2) receptor agonist (2S)-1-(6-chloro-5-fluoroindol-1-yl)propan-2-amine fumarate (Ro 60-0175) in the absence or presence of the selective 5-HT(2C) receptor antagonist 6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yl)oxypyridin-3-yl]-2,3-dihydroindole-1-carboxamide dihydrochloride (SB 242084) or 5-HT(2A) receptor antagonist (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (MDL-100,907) were studied on these variables. Continuous infusion of increasing concentrations of Ro 60-0175 only evoked EUS-EMG activity at the highest concentration, which was blocked by co-infusion of MDL-100,907 but not SB 242084. Urethral pressure was unaffected by any drug infusion. Ro 60-0175 at the lowest concentration inhibited the micturition reflex but as the concentration increased this was reversed to facilitation. SB 242084 blocked the inhibition while MDL-100,907 blocked the excitation. Activation of 5-HT(2A) not 5-HT(2C) receptors evoked EUS-EMG activity. In conclusion, 5-HT(2A) receptor activation facilitated the micturition reflex and evoked EUS-EMG while 5-HT(2C) receptor activation only inhibited the micturition reflex.     

Effects of the serotonin 5-HT2A and 5-HT2C receptor ligands on the discriminative stimulus effects of nicotine in rats

The present study tested the hypothesis that serotonergic (5-HT) 5-HT2A or 5-HT2C receptors or their pharmacological stimulation modulated the discriminative stimulus effects of nicotine in male Wistar rats. To this end the selective 5-HT2A receptor antagonist R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol (M100,907; 0.5-1 mg/kg, i.p.), the functional 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI; 0.1-1 mg/kg, s.c.), the selective 5-HT2C receptor antagonist 6-chloro-5-methyl-1-{[2-(2-methylpyrid-3-yloxy)pyrid-5-yl]carbamoyl}indoline (SB 242,084; 0.25-1 mg/kg, i.p.) and the 5-HT2C receptor agonists (S)-2-chloro-5-fluoro-indol-1-yl)-1-methylethylamine fumarate (Ro 60-0175; 0.3-1 mg/kg, s.c.) and (7bR, 10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole (WAY 163,909; 0.75-1.5 mg/kg, i.p.) were used. Additionally, the effects of the selective alpha4beta2 nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA; 0.01 mg/kg, s.c.) were investigated. In rats trained to discriminate (-)-nicotine (0.4 mg/kg, s.c.) from saline in a two-lever, water-reinforced fixed ratio 10 task, substitutions were not observed with 5-HT2 receptor ligands (<32% nicotine-lever responding), conversely 5-IA induced a full substitution (100% nicotine-lever responding). In combination studies, fixed doses of M100,907 (0.5-1 mg/kg) or SB 242,084 (0.25-1 mg/kg) did not alter the dose-response curve of nicotine, while DOI (0.3 mg/kg), Ro 60-0175 (1 mg/kg) and WAY 163,909 (1 and 1.5 mg/kg) attenuated the discriminative stimulus effects of nicotine. The decrease in the expression of the discriminative stimulus effects of nicotine produced by DOI was blocked by M100,907 (1 mg/kg), but not by SB 242,084 (1 mg/kg), while that evoked by Ro 60-0175 or WAY 163,909 was blocked by SB 242,084 (1 mg/kg), but not by M100,907 (1 mg/kg). Further studies showed that DOI (0.3 mg/kg) and Ro 60-0175 (1 mg/kg), but not WAY 163,909 (1.5 mg/kg) blocked full substitution of 5-IA (0.01 mg/kg) for nicotine. Our pharmacological analyses indicate that tonic activation of 5-HT2A or 5-HT2C receptors is not required for subjective effects of nicotine, however these receptors appear to have inhibitory influence on nicotine cue, since pharmacological stimulation of either receptor attenuates the discriminative stimulus effects of nicotine.     

Thermogenic effect of YM348, a novel 5-HT2C-receptor agonist, in rats

We have investigated the effect of S-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine (YM348), a novel 5-HT(2C)-receptor agonist, on body temperature and energy expenditure in Wistar rats. m-Chlorophenylpiperazine (mCPP) and S-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (RO 60-0175) were used as reference 5-HT(2C)-receptor agonists. Administration of YM348, mCPP and RO 60-0175 dose-dependently and significantly increased body temperature in rats. YM348- or RO 60-0175-induced hyperthermia was significantly attenuated by the non-selective 5-HT(2)-receptor antagonist methysergide and the selective 5-HT(2C)-receptor antagonist SB242084, but not by the selective 5-HT(2A)-receptor antagonist MDL100907. mCPP-induced hyperthermia was significantly attenuated by methysergide, SB242084 and MDL100907. In addition to the increase in body temperature, YM348, mCPP and RO 60-0175 produced dose-related and significant increases in energy expenditure. YM348-, mCPP- and RO 60-0175-induced increases in energy expenditure were significantly attenuated by methysergide and SB242084 but not by MDL100907. These results suggested that 5-HT(2C)-receptor stimulation increased body temperature and energy expenditure and that the 5-HT(2C)-receptor was the target receptor in the thermogenic effect of YM348 in Wistar rats.     

Differential effects of 5-HT2C receptor ligands on place conditioning and locomotor activity in rats

Effects of the 5-hydroxytryptamine (5-HT)(1A/1B/2C) receptor agonist N-[3-(trifluoromethyl)phenyl] piperazine (TFMPP, 0-3.0 mg/kg s.c.) and the 5-HT2C receptor agonist 8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one (WAY 161503, 0-3.0 mg/kg s.c.) in place conditioning were measured in male Sprague-Dawley rats. Effects of TFMPP, alone and with the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N-2-pyridinyl-cyclohexanecarboxamine (WAY 100635), the 5-HT(1B) receptor antagonist N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-1,1'-biphenyl-4-carboxamide (GR 127935) or the 5-HT2C receptor antagonist 6-chloro-5-methyl-1-[[2-(2-methylpyrid-3-yloxy)pyrid-5-yl]carbamoyl]indoline (SB 242084) and of WAY 161503 alone and with SB 242084 on locomotor activity were also assessed. Neither TFMPP nor WAY 161503 induced place conditioning. WAY 161503 (1.0 and 3.0 mg/kg s.c.) decreased locomotor activity; SB 242084 (1.0 mg/kg i.p.) blocked this effect. Reduced locomotor activity following TFMPP was blocked by SB 242084 but not WAY 100635 (0.1 mg/kg s.c.) or GR 127935 (3.0 mg/kg s.c.). Behaviourally relevant levels of 5-HT2C receptor stimulation may not exert reinforcing effects, although other studies indicate that such manipulations alter reinforcing effects of drugs of abuse.     

Pharmacological profile of YM348, a novel, potent and orally active 5-HT2C receptor agonist

YM348, (S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine, showed a high affinity for cloned human 5-HT(2C) receptors (K(i): 0.89 nM). The functional selectivity for 5-HT(2C) receptors in the 5-HT(2) receptor family was the highest among 5-HT(2C) receptor agonists, including m-chlorophenylpiperazine (mCPP) and Ro60-0175 ((S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine). Oral administration of YM348 induced penile erections and hypolocomotion in rats, being completely inhibited by a selective 5-HT(2C) receptor antagonist, SB242084 (6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy) pyridin-3-yl carbamoyl] indoline). The dose-response curve for penile erections, unlike that for hypolocomotion, was an inverted U-shape in the dose range of 0.0677-2.03 mg/kg. A selective 5-HT(2A) receptor antagonist, MDL100907 (R(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol), and a selective 5-HT(2B) receptor antagonist, RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine), had no effect on the decline in penile erection frequency at 2.03 mg/kg of YM348. YM348 did not affect blood pressure at 2.03 mg/kg. In conclusion, YM348 is a novel, potent and orally active 5-HT(2C) receptor agonist, and neither the activation of 5-HT(2A) or 5-HT(2B) receptors nor a cardiovascular effect is likely to contribute to the inverted U-shape dose-response curve for penile erections.     

Effects of RO 60 0175, a 5-HT(2C) receptor agonist, in three animal models of anxiety

There is some controversy as to whether 5-HT(2C) receptor agonists are anxiogenic or anxiolytic. The effects of the novel 5-HT(2C) receptor agonist, (S)-2-chloro-5-fluoro-indol-1-yl)-1-methyl ethylamine fumarate (RO 60 0175), in three models of anxiety were therefore tested. RO 60 0175 was found to induce hypolocomotion in rats at doses greater than 0.5 mg/kg s.c., an effect reversed by the selective 5-HT(2C) receptor antagonist, SB-242084. RO 60 0175 did not elicit anxiolytic-like responses in the social interaction test under high light unfamiliar conditions, but suppressed both time spent in social interaction and locomotion at doses of 1 and 3 mg/kg s.c., suggesting a sedative response. In the Vogel conflict test, RO 60 0175 had no significant action on the number of shocks taken. In the Geller-Seifter test, RO 60 0175 (0.3 and 1 mg/kg s.c.) simultaneously reduced both unpunished and punished lever pressing, a profile consistent with sedation. Finally, RO 60 0175 was tested in a rat social interaction test under low light familiar conditions optimal for the detection of anxiogenic-like responses. At 1 and 3 mg/kg s.c., RO 60 0175 reduced both time spent in social interaction and concurrent locomotion, a profile more consistent with sedation than anxiogenesis. In conclusion, RO 60 0175 induced sedative-like responses via 5-HT(2C) receptor activation, but was neither anxiolytic, nor clearly anxiogenic at the doses tested.     

Influence of the 5-HT2C receptor antagonist, SB-242084, in tests of anxiety

The 5-HT2C antagonist SB-242084 was examined in various anxiety tests at doses based on reversal of mCPP-induced hypoactivity (0.1--3 mg/kg ip). In the elevated plus-maze task, SB-242084 exhibited signs of anxiolysis (time spent, distance travelled, and entries into open arms), but this was potentially confounded by its general increase of locomotion; alprazolam selectively affected open-arm parameters. In a Geller--Seifter conflict test, SB-242084 produced a modest, nonsignificant increase in punished responding compared to the significant effect produced by diazepam. None of the treatments significantly affected unpunished responding. In the conditioned emotional response (CER) test, SB-242084 produced an increase in the suppression ratio (SR, smaller than diazepam). Since this 5-HT2C antagonist also increased lever pressing, an additional test was conducted with amphetamine that stimulated lever pressing but, nonetheless, failed to produce any change in SR. In the fear-potentiated startle task, SB-242084 was inactive in comparison to a significant effect of diazepam. The previously described reduction of schedule-induced polydipsia by fluoxetine and 5-HT2C receptor agonist Ro60-0175 was attenuated by SB-242084 pretreatment, however, the latter compound exhibited a potent increase in polydipsia when given alone. The present results demonstrate an anxiolytic potential of SB-242084, as well as an intrinsic response-enhancing property, however, both of these effects are task dependent.     

mCPP-induced hyperactivity in 5-HT2C receptor mutant mice is mediated by activation of multiple 5-HT receptor subtypes

The serotonin receptor agonist mCPP induces hyperlocomotion in 5-HT2C receptor knockout (KO) mice or in the presence of a 5-HT2C receptor antagonist. In the present group of experiments, we evaluate the role of 5-HT1A, 5-HT1B and 5-HT2A receptors in mCPP-induced hyperactivity in 5-HT2C KO mice. We also assess the ability of agonists at these receptors to induce hyperactivity in wildtype (WT) mice pre-treated with a selective 5-HT2C receptor antagonist. As previously reported, mCPP (3 mg/kg) induced hyperactivity in 5-HT2C KO mice. A combination of the 5-HT1B receptor agonist CP-94,253 (20 mg/kg) and the 5-HT1A receptor agonist 8-OH-DPAT (0.5 mg/kg) induced marked hyperactivity in WT but not in 5-HT2C KO mice, nor in mice treated with the selective 5-HT2C receptor antagonist, SB 242084 (1.5 mg/kg). Neither CP-94,253 nor 8-OH-DPAT had any intrinsic effect on locomotion in WTs. mCPP-induced hyperactivity was attenuated in 5-HT2C KO mice by the 5-HT1B receptor antagonist SB 224289 (2.5 mg/kg), and the 5-HT2A receptor antagonists ketanserin (0.3 mg/kg) and M100907 (0.01 mg/kg) but not by the 5-HT1A receptor antagonist WAY 100635 (1 mg/kg). The 5-HT(2A/2B/2C) receptor agonist, Ro 60-0175 (3 mg/kg), induced a modest increase in locomotor activity in WT mice pre-treated with SB 242084. However, the combination of Ro 60-0175 with CP-94,253 induced a substantial increase in activity in 5-HT2C KO mice, an effect comparable to mCPP-induced hyperactivity. Thus, joint activation of 5-HT1A and 5-HT1B receptors stimulates locomotion in WT mice but this response is dependent on a functional 5-HT2C receptor population and hence is absent in 5-HT2C KO mice. By contrast, mCPP-induced hyperactivity depends on the inactivation of a separate 5-HT2C receptor population and is mediated by 5-HT2A and 5-HT1B receptor activation.     

Role of 5-HT(2) receptors in the tryptamine-induced 5-HT syndrome in rats

We distinguished the functions of the different 5-hydroxytryptamine-2 (5-HT(2)) receptor (5-HT(2)R) subtypes in the tryptamine-induced 5-HT syndrome in rats using (1) the 5-HT(2A)R antagonist R93274 (N-[(3-p-fluorophenyl-1-propyl)-4-methyl-4-piperidinyl]-4-amino-5-iodo-2-methoxybenzamide), the 5-HT(2A/C)R antagonist R99647 (2-(dimethylaminomethyl)2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine), the 5-HT(2B/C)R antagonist SB-242084 (6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline), and several 5-HT(2)R antagonists (ketanserin, risperidone, pipamperone and mianserin); and (2) chronic 5-HT(2)R activation by 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast to SB-242084, the selective 5-HT(2A)R antagonist R93274 as well as the non-selective 5-HT(2A)R antagonists (R99647, ketanserin, risperidone, pipamperone and mianserin) significantly inhibited tryptamine-induced forepaw treading and tremors, and reversed peripherally mediated cyanosis into hyperaemia; only the 5-HT(2A/C)R antagonists R99647 and mianserin inhibited the tryptamine-induced hunched back. Intermittent DOM administration (intravenously every 48 h for 12 days) did not change the centrally mediated tryptamine-induced forepaw treading, tremors and hunched back at 1, 4 or 7 days after the last DOM pretreatment. The DOM-induced head twitch response, measured immediately after every DOM injection, was not affected. In contrast, peripherally mediated cyanosis was reversed into hyperaemia in 75, 11 and 20% of all pretreated rats at 1, 4 and 7 days, respectively, after the last DOM administration. Taken together, these finding suggest that central 5-HT(2A)Rs mediate tryptamine-induced forepaw treading and tremors, that peripheral 5-HT Rs mediate tryptamine-induced cyanosis, and that 5-HT(2A)Rs mediate tryptamine-induced hunched back. Peripheral 5-HT(2C)Rs are more sensitive to desensitization after intermittent treatment with an agonist than central 5-HT(2A)Rs.     

Similarities in the action of Ro 60-0175, a 5-HT2C receptor agonist, and d-fenfluramine on feeding patterns in the rat

RATIONALE: Activation of 5-HT2C receptors is thought to enhance satiety and to mediate the action of the prototypical anorectic drug d-fenfluramine. OBJECTIVE: Four experiments investigated the role of the 5-HT2C receptor in the modulation of feeding by comparison of the effects of the putative selective 5-HT2C receptor agonist Ro 60-0175 and d-fenfluramine on feeding behaviour. METHODS: Microstructural analyses of meal patterning and drinking of a palatable solution were made over a range of drug doses administered to male Lister hooded rats. RESULTS: Ro 60-0175 increased the latency to the first meal (3 mg/kg) and reduced meal size (1 mg/kg). d-Fenfluramine (1 mg/kg) produced a similar behavioural pattern, but 3 mg/kg produced a more profound hypophagia that persisted for 10-12 h. Ro 60-0175 (1, 3 mg/kg) and d-fenfluramine (1.5 mg/kg) reduced ingestion of a palatable glucose/saccharin solution, by a reduction in the number of bouts of licking, with little effect on the size of individual bouts. d-Fenfluramine-induced hypophagia (2.1 mg/kg) was challenged by the administration of the selective 5-HT2C receptor antagonist SB 242084 (1, 3 mg/kg) in the meal patterning paradigm. SB 242084 significantly attenuated the decrease in feeding rate and increase in latency to feed produced by d-fenfluramine, but had no effect on the fenfluramine-induced reduction in meal size. A similar pattern of results was obtained when Ro 60-0175-induced hypophagia (3 mg/kg) was challenged by SB 242084 (1, 3 mg/kg). CONCLUSIONS: These results demonstrate that Ro 60-0175 is a useful probe of the importance of 5-HT2C activation in the control of food intake and support the hypothesis that activation of 5-HT2C receptors is a critical aspect of the hypophagic action of d-fenfluramine. The 5-HT2C receptor may prove to be a useful target in the development of clinically effective drugs for the treatment of obesity.     

Opposing effects of 5-HT2A and 5-HT2C receptor antagonists in the rat and mouse on premature responding in the five-choice serial reaction time test

Rationale Serotonin (5-HT) has been linked to impulsivity with recent data suggesting that different receptor sub-types exert opposing influences on this behaviour. Objectives This work characterised the effects of 5-HT_2A (ketanserin, (±)2,3-dimethoxyphenyl-1-[2–4-(piperidine)-methanol] [M100907]), 5-HT_2B (6-chloro-5-methyl-1-(5-quinolylcarbamoyl) indoline [SB215505]) and 5-HT_2C (6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl carbomyl] indoline [SB242084]) receptor antagonists on impulsive behaviour, measured in the five-choice serial reaction time test (5CSRTT), in rats and mice. The effects of (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) and (S)-2-(chloro-5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C₄H₄O₄ (Ro60-0175), two compounds that have been used extensively as agonists for the 5-HT_2A and 5-HT_2C receptor, were also measured. Materials and methods Rats and mice were trained on the 5CSRTT in which reinforcement is earned for detecting and correctly responding to brief presentations of a stimulus light. Impulsivity in this task is measured as premature responding, before stimulus presentation. Several variants of the task were used in which the inter-trial interval (ITI) length was manipulated to alter basal levels of premature responding. Results In the rat, ketanserin and M100907 reduced and SB242084 enhanced premature responding. SB215505 had no effect. DOI generally disrupted responding, while Ro60-0175 reduced premature responding when a long ITI was used. In mice, M100907 reduced and SB242084 increased premature responding when the ITI was lengthened. The effects of these drugs on other aspects of performance were less robust. M100907 and ketanserin did not affect response accuracy but tended to slow speed of responding; SB242084 occasionally increased speed of responding and slightly reduced accuracy. Conclusions Serotonin exerts both excitatory and inhibitory influences on motor impulsivity via 5-HT_2A and 5-HT_2C receptors in both rats and mice.     

Efficacy of selective 5-HT6 receptor ligands determined by monitoring 5-HT6 receptor-mediated cAMP signaling pathways

1. Two novel selective 5-HT6 receptor ligands E-6801 (6-chloro-N-(3-(2-(dimethylamino)ethyl)-1H-indol-5-yl)imidazo[2,1-b]thiazole-5-sulfonamide) and E-6837 (5-chloro-N-(3-(2-(dimethylamino)ethyl)-1H-indol-5-yl)naphthalene-2-sulfonamide) were investigated and compared to the putative 5-HT6 receptor antagonists SB-271046 (5-chloro-N-(4-methoxy-3-(piperazin-1-yl)phenyl)-3-methylbenzo[b]thiophene-2-sulfonamide) and Ro 04-06790 (N-(2,6-bis(methylamino)pyrimidin-4-yl)-4-aminobenzenesulfonamide) using a cAMP-mediated pathway. 2. Forskolin stimulation, to increase the magnitude of agonist cAMP responses, and site-directed mutagenesis of the 5-HT6 receptor, in order to yield constitutively active receptor, were applied. 3. 5-HT (E(max), % over basal: 200), E-6801 (120) and E-6837 (23) induced cAMP formation at the rat 5-HT6 receptor. In the copresence of forskolin, cAMP responses were more potent and enhanced to 294 (5-HT, % over forskolin), 250 (E-6801) and 207 (E-6837), respectively. 5-HT-mediated cAMP formation was dose-dependently blocked by SB-271046 (pA(2): 8.76+/-0.22) and Ro 04-6790 (pA(2): 7.89+/-0.10) and not affected by the copresence of forskolin. Both E-6801 and E-6837 yielded partial antagonism of the 5-HT response in the absence of forskolin, whereas antagonism was either completely absent (E-6801) or attenuated (E-6837) in the copresence of forskolin. Intrinsic activity of these 5-HT6 receptor ligands at a constitutively active human S267K 5-HT6 receptor in Cos-7 cells indicated similar efficacy (E(max), % over basal) for 5-HT (97), E-6801 (91) and E-6837 (100), while Ro 04-6790 (-33) and SB-271046 (-39) were equi-efficacious inverse agonists. 4. The use of either forskolin or a constitutively active S267K 5-HT6 receptor enhances the resolution for monitoring the efficacy of 5-HT6 receptor ligands. E-6801 and E-6837 are potent partial agonists at the 5-HT6 receptor. Ro 04-6790 and SB-271046 appear to act as inverse agonists/antagonists.     

Control of hippocampal theta rhythm by serotonin: role of 5-HT2c receptors

The hippocampus plays an important role in learning and memory and has been implicated in a number of diseases, including epilepsy, anxiety and schizophrenia. A prominent feature of the hippocampal network is the capability to generate rhythmic oscillations. Serotonergic modulation is known to play an important role in the regulation of theta rhythm. 5-HT2c receptors represent a specific target of psychopharmacology and, in particular, the behavioral effects of the 5-HT2c receptor agonist mCPP have been thoroughly tested. The present study used this compound and the selective 5-HT2c receptor antagonist SB-242084 to elucidate the role of 5-HT2c receptors in the generation of hippocampal oscillations. Hippocampal EEG was recorded and the power in the theta frequency range was monitored in different behaviors in freely-moving rats and after brainstem stimulation in anesthetized animals. We found that in freely-moving rats, mCPP suppressed hippocampal theta rhythm and the effect was stronger during REM sleep than during waking theta states. Under urethane anesthesia, mCPP decreased the power for both spontaneous and elicited theta rhythm in a dose-dependent manner and the 5-HT2c antagonist reversed this effect. The results of this study demonstrate that 5-HT2c receptors are important element of the serotonergic modulation of hippocampal theta oscillations and thus pharmacological interactions with these receptors can modulate physiological and pathological processes associated with limbic theta activity.

SB-242084 = 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl carbamoyl]indoline mCPP = 1-(3-chlorophenyl)piperazine dihydrochloride     

Further pharmacological characterization of 5-HT2C receptor agonist-induced inhibition of 5-HT neuronal activity in the dorsal raphe nucleus in vivo

Background and purpose:

Recent experiments using non-selective 5-hydroxytryptamine (5-HT)_2C receptor agonists including WAY 161503 suggested that midbrain 5-HT neurones are under the inhibitory control of 5-HT_2C receptors, acting via neighbouring gamma-aminobutyric acid (GABA) neurones. The present study extended this pharmacological characterization by comparing the actions of WAY 161503 with the 5-HT_2C receptor agonists, Ro 60-0275 and 1-(3-chlorophenyl) piperazine (mCPP), as well as the non-selective 5-HT agonist lysergic acid diethylamide (LSD) and the 5-HT releasing agent 3,4-methylenedioxymethamphetamine (MDMA).

Experimental approach:

5-HT neuronal activity was measured in the dorsal raphe nucleus (DRN) using extracellular recordings in anaesthetized rats. The activity of DRN GABA neurones was assessed using double-label immunohistochemical measurements of Fos and glutamate decarboxylase (GAD).

Key results:

Ro 60-0175, like WAY 161503, inhibited 5-HT neurone firing, and the 5-HT_2C antagonist SB 242084 reversed this effect. mCPP also inhibited 5-HT neurone firing (∼60% neurones) in a SB 242084-reversible manner. LSD inhibited 5-HT neurone firing; however, this effect was not altered by either SB 242084 or the 5-HT_2A/C receptor antagonist ritanserin but was reversed by the 5-HT_1A receptor antagonist WAY 100635. Similarly, MDMA inhibited 5-HT neurone firing in a manner reversible by WAY 100635, but not SB 242084 or ritanserin. Finally, both Ro 60-0275 and mCPP, like WAY 161503, increased Fos expression in GAD-positive DRN neurones.

Conclusions and implications:

These data strengthen the hypothesis that midbrain 5-HT neurones are under the inhibitory control of 5-HT_2C receptors, and suggest that the 5-HT_2C agonists Ro 60-0175, mCPP and WAY 161503, but not LSD or MDMA, are useful probes of the mechanism(s) involved.     

A comparison of the behavioural effects of 5-HT2A and 5-HT2C receptor agonists in the pigeon

Activity at the 5-HT2A receptor versus that of the 5-HT2C receptor was studied in three behavioural paradigms. In pigeons trained to discriminate 0.32 mg/kg of 1-(2,5-diemethoxy-4-iodophenyl)-2-aminopropane (DOI) (a mixed 5-HT2A/C receptor agonist) from vehicle, quipazine (0.1-1 mg/kg) and m-chlorophenylpiperazine (mCPP) (1-3 mg/kg) substituted for DOI in a dose-related manner, and this generalization was blocked by MDL100907 (0.0001-0.01 mg/kg), a selective 5-HT2A receptor antagonist. RO60-0175 (a relatively selective 5-HT2C agonist) induced partial substitution at 3 mg/kg that was antagonized by both MDL100907 and by 3 mg/kg of SB242084, a relatively selective 5-HT2C antagonist. MK212 (a mixed 5-HT2C/A agonist) induced partial substitution that was antagonized by SB242084, but not by MDL100907. On a progressive ratio 5 operant schedule (PR5) for food reinforcement, DOI, quipazine, mCPP, MK212 and R060-0175 decreased the break point; mCPP, DOI, MK212 and quipazine also induced vomiting. Although MDL100907 antagonized both the reductions of break point and vomiting, SB242084 only partially attenuated the decrease in break point observed with MK212 and DOI, and was unable to eliminate vomiting. Thus pharmacological activity at the 5-HT2A receptor can be behaviourally distinguished from pharmacological activity at the 5-HT2C receptor in the pigeon. Furthermore, the decrease in the break point of a PR5 schedule induced by 5-HT2C receptor agonists may be related to decreased appetite, whereas that induced by 5-HT2A receptor agonists may be due to unrelated factors, such as emesis.     

Effects of the 5-HT2C receptor agonist Ro60-0175 and the 5-HT2A receptor antagonist M100907 on nicotine self-administration and reinstatement

The reinforcing effects of nicotine are mediated in part by brain dopamine systems. Serotonin, acting via 5-HT(2A) and 5-HT(2C) receptors, modulates dopamine function. In these experiments we examined the effects of the 5-HT(2C) receptor agonist Ro60-0175 and the 5-HT(2A) receptor antagonist (M100907, volinanserin) on nicotine self-administration and reinstatement of nicotine-seeking. Male Long-Evans rats self-administered nicotine (0.03 mg/kg/infusion, IV) on either a FR5 or a progressive ratio schedule of reinforcement. Ro60-0175 reduced responding for nicotine on both schedules. While Ro60-0175 also reduced responding for food reinforcement, response rates under drug treatment were several-fold higher than in animals responding for nicotine. M100907 did not alter responding for nicotine, or food, on either schedule. In tests of reinstatement of nicotine-seeking, rats were first trained to lever press for IV infusions of nicotine; each infusion was also accompanied by a compound cue consisting of a light and tone. This response was then extinguished over multiple sessions. Injecting rats with a nicotine prime (0.15 mg/kg) reinstated responding; reinstatement was also observed when responses were accompanied by the nicotine associated cue. Ro60-0175 attenuated reinstatement of responding induced by nicotine and by the cue. The effects of Ro60-0175 on both forms of reinstatement were blocked by the 5-HT(2C) receptor antagonist SB242084. M100907 also reduced reinstatement induced by either the nicotine prime or by the nicotine associated cue. The results indicate that 5-HT(2C) and 5-HT(2A) receptors may be potential targets for therapies to treat some aspects of nicotine dependence.     

Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells.

1. The goal of this study was to characterize the agonist pharmacology of human 5-HT2A, 5-HT2B and 5-HT2C (VSV) receptors expressed in CHO-K1 (Chinese hamster ovary) cells. 2. We used a fluorometric imaging plate reader (FLIPR) which allows rapid detection of rises in intracellular calcium levels upon the addition of agonists. 3. Stimulation of all three receptors by 5-HT caused a robust concentration dependent increase in intracellular calcium levels. No such effect was observed from non-transfected control CHO-K1 cells. 4. The rank order of potency of agonists at the different receptor subtypes varied. Tryptamines, BW-723C86, d-norfenfluramine, Ro 60-0175 and LSD exhibited the following rank order of potency; 5-HT2B>5-HT2C>5-HT2A. Piperazines such as m-Chlorophenylpiperazine (mCPP), ORG-12962, MK-212 and also ORG-37684 exhibited a rank order of potency of 5-HT2C>5-HT2B>5-HT2A. The phenylisopropylamines DOI and DOB had a rank order of 5-HT2A>5-HT2B>5-HT2C. 5. Many agonists tested had partial agonist actions when compared to 5-HT, and a wide range of relative efficacies were exhibited, which was cell line dependent. For example, mCPP had a relative efficacy of 65% at 5-HT2C receptors but <25% at either 5-HT2A or 5-HT2B receptors. 6. Interpretation of literature values of functional assays using different cell lines, different receptor expression levels and different receptor isoforms, is complex. Species differences and the previous use of antagonist radioligands to characterize agonist potency in binding assays emphasizes the importance of studying agonists in the same experiment using the same assay conditions and parental cell lines.     

Serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in human neocortex slices: involvement of 5-HT(2C) and 5-HT(1A) receptors

The NMDA receptor/nitric oxide (NO)/cyclic GMP pathway and its modulation by 5-hydroxytryptamine (5-HT) was studied in slices of neocortical samples obtained from patients undergoing neurosurgery. The cyclic GMP elevation produced by 100 microM NMDA was blocked by 100 microM of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) or by 10 microM of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha] quinoxaline-1-one (ODQ). The NMDA effect was prevented by 5-HT or by the 5-HT(2) agonist (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane ((+/-)-DOI; EC(50)=22 nM). The (+/-)-DOI inhibition was insensitive to the 5-HT(2A) receptor antagonist MDL 100907 or the 5-HT(2B) antagonist rauwolscine; it was largely prevented by 1 microM of the non-selective 5-HT(2C) antagonists mesulergine (5-HT(2A,B,C)), ketanserin (5-HT(2A,C)) or SB 200646A (5-HT(2B,C)); it was completely abolished by 0.1 microM of the selective 5-HT(2C) receptor antagonist SB 242084. The NMDA-induced cyclic GMP elevation also was potently inhibited by the selective 5-HT(2C) agonist RO 60-0175 and by the antidepressant trazodone, both added at 1 microM, in an SB 242084-sensitive manner. Finally, the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 1 microM) inhibited the NMDA-evoked cyclic GMP response, an effect blocked by the selective 5-HT(1A) receptor antagonist WAY 100635. In conclusion, the NMDA receptor/NO/cyclic GMP pathway in human neocortex slices can be potently inhibited by activation of 5-HT(2C) or 5-HT(1A) receptors.     

5-HT7, but not 5-HT2B, receptors mediate hypotension in vagosympathectomized rats

This study evaluated the possible involvement of 5-HT(2B) receptors in long-lasting hypotension to 5-hydroxytryptamine (5-HT), which is predominantly mediated by 5-HT7 receptors, in anaesthetised vagosympathectomized rats. Intravenous injections of 5-HT and 5-carboxamidotryptamine (5-CT) elicited a dose-dependent hypotension that was dose-dependently antagonised by (R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl) ethyl] pyrrolidine (SB-269970; a selective 5-HT7 receptor antagonist), but not by saline. Interestingly, alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine (BW723C86; a 5-HT(2B) receptor agonist) produced vasopressor responses without affecting hypotension to 5-HT. These results suggest that hypotension to 5-HT and 5-CT is mainly mediated by 5-HT7 receptors, whilst the role of 5-HT(2B) receptors seems unlikely.