The 5-HT2 receptor antagonist, MDL 28,133A, disrupts the serotonergic-dopaminergic interaction mediating the neurochemical effects of 3,4-methylenedioxymethamphetamine.

The selective 5-HT₂ receptor antagonist MDL 28,133A dose dependently blocked the long-term deficits in rat brain 5-HT concentrations produced by the substituted amphetamine analogue 3,4-methylenedioxymethamphctamine (MDMA). This protective effect of MDL 28,133A could be abolished by coadministration of the dopamine precursor, L-dihydroxyphenylalanine (L-DOPA). Electrophysiological experiments demonstrated that the ability of MDL 28,133A to block the MDMA-induced slowing of A⁹ dopaminergic neurons was also sensitive to L-DOPA administration. Both sets of experiments suggest an interaction of MDL 28,133A at the level of dopamine synthesis. Consistent with this explanation, MDL 28,133A antagonized the MDMA-induced stimulation of dopamine synthesis in vivo. MDMA-induced 5-HT release did not reduce the firing rate of dopaminergic neurons as assessed by dopamine depletion following synthesis inhibition with -mcthyl-p-tyrosinc (-MPT). This indicates that the effect of 5-HT₂ receptor antagonists on MDMA-induced dopamine synthesis is not due simply to the removal of an inhibitory serotonergic input followed by an increase in dopamine cell firing and autoreceptor activation. MDL 28,133A was also shown to be without effect on the sensitivity of terminal dopamine autoreceptors. The results are consistent with the hypothesis that 5-HT₂ receptors are permissive for the stimulation of dopamine synthesis necessary to support MDMA-induced transmitter efflux.     

5-HT2 receptors exert a state-dependent regulation of dopaminergic function: studies with MDL 100,907 and the amphetamine analogue, 3,4-methylenedioxymethamphetamine.

The highly selective 5-HT2 receptor antagonist, MDL 100,907, was used to explore the role of serotonin in the stimulation of dopaminergic function produced by the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA). MDL 100,907 blocked MDMA-stimulated dopamine synthesis in vivo without affecting basal synthesis. The long-term deficits in 5-HT concentrations believed to be a consequence of MDMA-induced dopamine release were also blocked by MDL 100,907 over the same dose range. In vivo microdialysis confirmed that 5-HT2 receptor blockade with MDL 100,907 attenuated MDMA-induced increases in extracellular concentrations of striatal dopamine. In contrast to its effect on MDMA-induced synthesis, MDL 100,907 did not alter dopamine synthesis stimulated by haloperidol or reserpine. In vivo dopamine release produced by haloperidol was also unaffected by MDL 100,907. The results suggest a permissive role for 5-HT2 receptors in the activation of the dopamine system which occurs during states of high serotonergic activity or during conditions of elevated dopamine efflux with high D2 receptor occupancy.     

5-HT modulation of auditory and visual sensorimotor gating: II. Effects of the 5-HT2A antagonist MDL 100,907 on disruption of sound and light prepulse inhibition produced by 5-HT agonists in Wistar rats

Increasing evidence suggests an important role of 5-HT, and 5-HT_2A receptors in particular, in the etiology and treatment of schizophrenia. The prepulse inhibition paradigm is used as a model for sensorimotor gating processes that are disrupted in schizophrenia. The present study used the selective serotonin_2A (5-HT_2A) antagonist and putative antipsychotic agent MDL 100,907 to evaluate the contribution of 5-HT_2A receptors to the disruptions of prepulse inhibition produced by several 5-HT agonists. The D₂ antagonist haloperidol was used to evaluate a possible interaction with dopamine neurons. Sound or light prepulses were used to measure the generality of these drug effects on cross-modal prepulse inhibition. In the first study, MDL 100,907 antagonized the disruptions of auditory prepulse inhibition produced by the 5-HT releasing agents fenfluramine and 3,4-methyle-nedioxymethamphetamine (MDMA). These effects on prepulse inhibition were modality-specific in that MDL 100,907 did not reverse the effects of the 5-HT releasers on visual prepulse inhibition. Haloperidol did not alter the disruptive effects of MDMA or fenfluramine on either auditory or visual prepulse inhibition. In the second study, the direct acting 5-HT_2A/2C receptor agonist/hallucinogen (+)1-4-iodo-2,5-dimethoxyphenyl-2-aminopropane (DOI) consistently disrupted auditory prepulse inhibition, and this effect was blocked by MDL 100,907 but not by haloperidol. A dose-response analysis demonstrated that MDL 100,907 potently antagonized DOI disrupted auditory prepulse inhibition, with an ED₅₀ of 0.04 mg/kg, IP. DOI did not consistently disrupt visual prepulse inhibition. In summary, these data indicate that, at least under the conditions of the present studies, the disruptions of auditory prepulse inhibition produced by fenfluramine, MDMA, and DOI result from stimulation of 5-HT_2A receptors. Furthermore, these disruptions do not involve direct or indirect stimulation of D₂ receptors. The identity of the 5-HT receptor(s) underlying the disruptive effects of fenfluramine or MDMA on visual prepulse inhibition has not yet been identified. MDL 100,907 may be generally useful in CNS disorders in which excessive 5-HT_2A receptor tone disrupts sensory gating processes.     

The gastrointestinal prokinetic benzamide derivatives are agonists at the non-classical 5-HT receptor (5-HT4) positively coupled to adenylate cyclase in neurons

Summary We have previously shown that a non-classical 5-hydroxytryptamine (5-HT₄) receptor mediates the stimulation of adenylate cyclase activity in mouse embryo colliculi neurons in primary culture. The pharmacological characteristics of this receptor exclude the possibility that it belongs to the known 5-HT₁, 5-HT₂ or 5-HT₃ receptor types. Here we report that this 5-HT receptor can be stimulated by 4-amino-5-chloro-2-methoxy substituted benzamide derivatives. All these compounds have been reported to be potent stimulants of gastrointestinal motility and some of them are 5-HT₃ receptor antagonists. The rank order of potency of these substituted benzamide derivatives in stimulating cAMP formation was: cisapride > BRL 24924 > 5-HT > zacopride > BRL 20627 > metoclopramide. The non-additivity of benzamide and 5-HT activities suggests that 5-HT and the substituted benzamide derivatives act on the same receptor. Only ICS 205930, a recognized 5-HT₃ receptor antagonist, competitively antagonized the stimulatory effect of cisapride, zacopride and BRL 24924. However, its pK _i (6–6.3) for this new receptor was very different from its pK _i for 5-HT₃ receptors (pK _i = 8 –10). Other selective 5-HT₃ receptor antagonists with an indole group (BRL 43694 and GR 38032F), with a benzoate group (cocaïne, MDL 72222) or with a piperazine group (quipazine) were ineffective in reversing the stimulatory effect of benzamide derivatives. Exposure of neuronal cells to potent agonists at this receptor such as BRL 24924 rapidly reduces its capacity to stimulate cAMP production. For example, a preincubation of 10 min with BRL 24924 (100 mol/l) reduced by 42% the ability of 5-HT to stimulate cAMP production. Cross-desensitization occurs between the effects of 5-HT and benzamides. The unique pharmacology of these nonclassical 5-HT receptors that we propose to call 5-HT₄ is very close and even identical to the pharmacology of the high affinity 5-HT receptors involved in the indirect stimulation of smooth muscle in the guinea pig ileum. These receptors are different from the 5-HT₃ receptors also present in guinea pig ileum.Send offprint requests to A. Dumuis at the above address     

Clozapine blocks dopamine, 5-HT2 and 5-HT3 responses in the medial prefrontal cortex: an in vivo microiontophoretic study

Clozapine is an atypical antipsychotic drug active on both positive and negative symptoms of schizophrenia which has a unique serotonergic and dopaminergic profile. Given the putative role of the medial prefrontal cortex (mPFC) in negative symptoms of schizophrenia, the aim of this study was to assess the effects of clozapine on the dopamine- and serotonin-responsive neurons in that particular brain structure. D₁ and D₂ agonists (SKF 38393 and quinpirole) as well as 5-HT₂ and 5-HT₃ agonists (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, DOI, and phenylbiguanide) were applied by microiontophoresis alone and concurrently with clozapine while recording extracellularly mPFC neurons. Dopamine ejections inhibited firing activity while D₁ and D₂ agonists were ineffective. Clozapine did not change basal firing by itself, but was able to suppress the inhibition produced by dopamine and by the 5-HT₂/5-HT₃ receptor agonists. It is concluded that clozapine at the mPFC level exerts a complex modulatory activity on dopamine receptors, that is directly at the dopaminergic receptors and through 5-HT receptors on the same neurons.     

Role of 5-HT<Subscript>2A</Subscript> and 5-HT<Subscript>2C/B</Subscript> receptors in the acute effects of 3,4-methylenedioxymethamphetamine (MDMA) on striatal single-unit activity and locomotion in freely moving rats

Rationale Like amphetamine, a locomotor-activating dose of 3,4-methylenedioxymethamphetamine (MDMA) predominantly excites striatal single-unit activity in freely moving rats. Although both D₁- and D₂-like dopamine (DA) receptors play important roles in this effect, MDMA, unlike amphetamine, strongly increases both DA and serotonin (5-HT) transmission. Objectives This study was conducted to investigate the 5-HT receptor mechanisms underlying the striatal effects of MDMA. Methods We recorded the activity of >200 single units in the striatum of awake, unrestrained rats in response to acute MDMA administration (5 mg/kg) combined with the selective blockade of either 5-HT_2A or 5-HT_2C/B receptors. Results Prior administration of SR-46349B (a 5-HT_2A antagonist 0.5 mg/kg) blocked nearly all MDMA-induced striatal excitations, which paralleled its significant attenuation of MDMA-induced locomotor activation. Conversely, prior administration of SB-206553 (a 5-HT_2C/B antagonist 2.0 mg/kg) had no effect on the amount of MDMA-induced locomotor activation or the distribution of single-unit responses to MDMA. However, a coefficient-of-variation analysis indicated significantly less variability in the magnitude of both MDMA-induced neuronal excitations and inhibitions in rats that were pretreated with SB-206553 compared to vehicle. Analysis of concurrent single-unit activity and behavior confirmed that MDMA-induced striatal activation was not merely due to behavioral feedback, indicating a primary action of MDMA. Conclusion These results support and extend our previous findings by showing that 5-HT_2A and 5-HT_2C/B receptors differentially regulate the expression of MDMA-induced behavioral and striatal neuronal responses, either directly or through the modulation of DA transmission.     

Modulation of the firing activity of noradrenergic neurones in the rat locus coeruleus by the 5-hydroxtryptamine system

1. The aim of the present study was to investigate the putative modulation of locus coeruleus (LC) noradrenergic (NA) neurones by the 5-hydroxytryptaminergic (5-HT) system by use of in vivo extracellular unitary recordings and microiontophoresis in anaesthetized rats. To this end, the potent and selective 5-HT_1A receptor antagonist WAY 100635 (N-{2-[4(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide trihydroxychloride) was used.
2. In the dorsal hippocampus, both local (by microiontophoresis, 20 nA) and systemic (100 μg kg⁻¹, i.v.) administration of WAY 100635 antagonized the suppressant effect of microiontophorectically-applied 5-HT on the firing activity of CA₃ pyramidal neurones, indicating its antagonistic effect on postsynaptic 5-HT_1A receptors.
3. WAY 100635 and 5-HT failed to modify the spontaneous firing activity of LC NA neurones when applied by microiontophoresis. However, the intravenous injection of WAY 100635 (100 μg kg⁻¹) readily suppressed the spontaneous firing activity of LC NA neurones.
4. The lesion of 5-HT neurones with the neurotoxin 5,7-dihydroxytryptamine increased the spontaneous firing activity of LC NA neurones and abolished the suppressant effect of WAY 100635 on the firing activity of LC NA neurones.
5. In order to determine the nature of the 5-HT receptor subtypes mediating the suppressant effect of WAY 100635 on NA neurone firing activity, several 5-HT receptor antagonists were used. The selective 5-HT₃ receptor antagonist BRL 46470A (10 and 100 μg kg⁻¹, i.v.), the 5-HT_1D receptor antagonist GR 127935 (100 μg kg⁻¹, i.v.) and the 5-HT_1A/1B receptor antagonist (−)-pindolol (15 mg kg⁻¹, i.p.) did not prevent the suppressant effect of WAY 100635 on the firing activity of LC NA neurones. However, the suppressant effect of WAY 100635 was prevented by the non-selective 5-HT receptor antagonists spiperone (1 mg kg⁻¹, i.v.) and metergoline (1 mg kg⁻¹, i.v.), by the 5-HT₂ receptor antagonist ritanserin (500 μg kg⁻¹, i.v.). It was also prevented by the 5-HT_1A receptor/α_1D-adrenoceptor antagonist BMY 7378 (1 mg kg⁻¹, i.v.) and by the α₁-adrenoceptor antagonist prazosin (100 μg kg⁻¹, i.v.).
6. These data support the notion that the 5-HT system tonically modulates NA neurotransmission since the lesion of 5-HT neurones enhanced the LC NA neurones firing activity and the suppressant effect of WAY 100635 on the firing activity of NA neurones was abolished by this lesion. However, the location of the 5-HT_1A receptors involved in this complex circuitry remains to be elucidated. It is concluded that the suppressant effect of WAY 100635 on the firing activity of LC NA neurones is due to an enhancement of the function of 5-HT neurones via a presynaptic 5-HT_1A receptor. In contrast, the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurones appears to be of the 5-HT_2A subtype.

     

Effects of chronic administration of ondansetron (GR38032F), a selective 5-HT3 receptor antagonist,on monoamine metabolism in mesolimbic and nigrostriatal dopaminergic neurons and on striatal D2-receptor binding

The effects of chronic administration of the selective 5-HT₃ receptor antagonist ondansetron (GR38032F) on dopamine (DA) and 5-hydrotryptamine (5-HT) metabolism in the major ascending dopaminergic neurons and on striatal D2-receptor binding characteristics were investigated. The metabolism of 5-HT was also studied in a number of other brain areas. Chronic ondansetron (0.2 mg/kg/day and 1.0 mg/kg/day SC for 16 days) did not change DA or 5-HT metabolism in the nigrostriatal or mesolimbic dopaminergic areas, although the larger dose of ondansetron slightly and statistically significantly reduced basal concentrations of DA and 5-HT in the nucleus caudatus. D2-receptor binding characteristics were not affected in the caudateputamen. Ondansetron did not change 5-HT metabolism in the nucleus raphé dorsalis, amygdala, hippocampus or in habenula. It is concluded that chronic administration of ondansetron does not change DA or 5-HT metabolism in the major ascending dopaminergic neurons. This suggest that unlike chronic D2-receptor blockade, chronic blockade of central 5-HT₃ receptors does not result in a similar reduction in the activity of nigrostriatal and mesolimbic dopaminergic neurons.     

Blockade of 5-HT2 Receptor Selectively Prevents MDMA-Induced Verbal Memory Impairment

3,4-Methylenedioxymethamphetamine (MDMA) or ‘ecstasy'' has been associated with memory deficits during abstinence and intoxication. The human neuropharmacology of MDMA-induced memory impairment is unknown. This study investigated the role of 5-HT_2A and 5-HT_1A receptors in MDMA-induced memory impairment. Ketanserin is a 5-HT_2A receptor blocker and pindolol a 5-HT_1A receptor blocker. It was hypothesized that pretreatment with ketanserin and pindolol would protect against MDMA-induced memory impairment. Subjects (N=17) participated in a double-blind, placebo-controlled, within-subject design involving six experimental conditions consisting of pretreatment (T1) and treatment (T2). T1 preceded T2 by 30 min. T1–T2 combinations were: placebo–placebo, pindolol 20 mg–placebo, ketanserin 50 mg–placebo, placebo–MDMA 75 mg, pindolol 20 mg–MDMA 75 mg, and ketanserin 50 mg–MDMA 75 mg. Memory function was assessed at Tmax of MDMA by means of a word-learning task (WLT), a spatial memory task and a prospective memory task. MDMA significantly impaired performance in all memory tasks. Pretreatment with a 5-HT_2A receptor blocker selectively interacted with subsequent MDMA treatment and prevented MDMA-induced impairment in the WLT, but not in the spatial and prospective memory task. Pretreatment with a 5-HT_1A blocker did not affect MDMA-induced memory impairment in any of the tasks. Together, the results demonstrate that MDMA-induced impairment of verbal memory as measured in the WLT is mediated by 5-HT_2A receptor stimulation.     

Effects of 5-HT3 agonists on reproductive behaviors in rats

Activity at 5-HT₁ and 5-HT₂ receptor sites influences sexual behavior in male and female rats. 5-HT₃ antagonists reportedly have no effect on copulatory activity in rats of either sex although they influence a variety of other behaviors. The effects of 5-HT₃ agonists on sexual behavior are unknown. The following experiments were undertaken to assess the influence of the 5-HT₃ agonists 1-phenylbiguanide (PBG) and 2-methyl-serotonin (2-Me-5-HT) on sexual behavior, when administered intracerebroventricularly. Consistent with earlier reports indicating that 5-HT₁ and 5-HT₂ receptor activity influences reproductive activity in a sex-dependent manner, PBG was found to facilitate male, but not female, rat sexual behavior. 2-Me-5-HT, however, failed to modify either female or male rat sexual activity. Evidence that PBG, but not 2-Me-5-HT, induces carrier-mediated dopamine release suggests that the effect of PBG in male rats is due to dopaminergic mediation. Overall, the present data indicate that 5-HT₃ receptor activation has only slight effects on rat sexual behavior.     

Electrophysiological impact of trazodone on the dopamine and norepinephrine systems in the rat brain

Previous study has documented the long-term effects of the antidepressant trazodone on the serotonin (5-HT) system. The present work examined the impact of sustained trazodone on ventral tegmental area (VTA) dopamine (DA) and locus ceruleus (LC) norepinephrine (NE) neurons firing activity, and characterized its effects at 5-HT_2C, 5-HT_2A receptors and α₁- and α₂-adrenoceptors. Electrophysiological recordings were carried out in anesthetized rats. Subcutaneously implanted minipumps delivered vehicle or trazodone (10 mg/kg/day) for 2 or 14 days. Administration of trazodone for 2 and 14 days did not alter the firing activity of DA neurons. Systemic injection of trazodone, however, reversed the inhibitory effect of the 5-HT_2C receptor agonist Ro 60,0175 on the DA neuronal firing, suggesting an antagonistic action of trazodone at this receptor. Administration of trazodone for 2 days significantly enhanced the NE neurons firing. Despite a return of the NE neurons firing rate to the baseline following 14-day trazodone, the percentage of neurons discharging in burst was increased by this regimen. Administration of trazodone for 14 days enhanced the tonic activation of postsynaptic α₂-adrenoceptors, as indicated by the disinhibitory effect of the α₂-adrenoceptor antagonist idazoxan on hippocampus pyramidal neurons firing. The inhibitory effect of acute trazodone on dorsal raphe (DR) 5-HT neurons firing was shown to be through the 5-HT_1A receptor. Systemic injection of trazodone reversed the inhibitory action of 5-HT_2A agonist DOI on the NE neurons firing rate, indicating its antagonistic action at 5-HT_2A receptors. The enhancement in α₂-adrenergic transmission by trazodone, and its 5-HT_2A and 5-HT_2C receptor antagonism may contribute to its therapeutic action in major depression.     

Electrophysiological Effects of Repeated Administration of Agomelatine on the Dopamine,Norepinephrine, and Serotonin Systems in the Rat Brain

Agomelatine is a melatonergic MT1/MT2 agonist and a serotonin (5-HT) 5-HT_2C antagonist. The effects of 2-day and 14-day administration of agomelatine were investigated on the activity of ventral tegmental area (VTA) dopamine (DA), locus coeruleus (LC) norepinephrine (NE), and dorsal raphe nucleus (DRN) 5-HT neurons using in vivo electrophysiology in rats. The 5-HT_1A transmission was assessed at hippocampus CA3 pyramidal neurons. After a 2-day regimen of agomelatine (40 mg/kg/day, i.p.), an increase in the number of spontaneously active VTA-DA neurons (p<0.001) and in the firing rate of LC-NE neurons (p<0.001) was observed. After 14 days, the administration of agomelatine induced an increase in: (1) the number of spontaneously active DA neurons (p<0.05), (2) the bursting activity of DA neurons (bursts/min, p<0.01 and percentage of spikes occurring in bursts, p<0.05), (3) the firing rate of DRN-5-HT neurons (p<0.05), and (4) the tonic activation of postsynaptic 5-HT_1A receptors located in the hippocampus. The increase in 5-HT firing rate was D2 dependent, as it was antagonized by the D2 receptor antagonist paliperidone. The enhancement of NE firing was restored by the 5-HT_2A receptor antagonist MDL-100,907 after the 14-day regimen. All the effects of agomelatine were antagonized by a single administration of the melatonergic antagonist {"type":"entrez-protein","attrs":{"text":"S22153","term_id":"100005","term_text":"pir||S22153"}}S22153 (except for the increase in the percentage of spikes occurring in burst for DA neurons). The present results suggest that (1) agomelatine exerts direct (2 days) and indirect (14 days) modulations of monoaminergic neuronal activity and (2) the melatonergic agonistic activity of agomelatine contributes to the enhancement of DA and 5-HT neurotransmission.     

Characterization of the contraction to 5-HT in the canine colon longitudinal muscle

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle.
2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of α-adrenoceptors. Tetrodotoxin (0.3 μM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT₄ receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT₁ and 5-HT₂ receptor antagonist methysergide (0.1 μM) depressed the curve to 5-HT, but the selective 5-HT₃ receptor antagonist granisetron (0.3 μM) had no effect.
3. Besides 5-HT, α-methyl-5-HT (α-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC₅₀ values in parentheses): 5-HT (6.9)=α-methyl-5-HT (6.9)>2-Me-5-HT (5.8)=5-MeOT (5.7)=5-CT (5.6), indicative of 5-HT₂ receptor involvement. α-Me-5-HT produced a bell-shaped curve, which was not affected by α-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.
4. The selective 5-HT_2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pK_b values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μM) shifted the curve to 5-MeOT rightward yielding an apparent pA₂ of 7.1. Other antagonists at 5-HT_2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA₂ value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo-irreversible'' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT_2A receptors.
5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT_2A receptors. The presence of inhibitory 5-HT₄ receptors cannot be ruled out.

     

5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor

Background and Purpose

The 5-HT₃ receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT₃ receptor.

Experimental Approach

5-HT₃ receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT₃A receptor and also the mouse native 5-HT₃ receptor that increases neuronal contraction of bladder smooth muscle.

Key Results

Cl-indole (1–100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT₃A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT₃ receptor. Radioligand-binding studies identified that Cl-indole induced a small (∼twofold) increase in the apparent affinity of 5-HT for the h5-HT₃A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT₃ receptors. In contrast to its effect on the 5-HT₃ receptor, Cl-indole did not alter human nicotinic α7 receptor responses.

Conclusions and Implications

The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT₃ receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT₃ receptor and may assist the discovery of novel therapeutic drugs targeting this receptor.

Linked Articles

Recent reviews on allosteric modulation can be found at:Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2. British Journal of Pharmacology 168: 554–575. doi: 10.1111/j.1476-5381.2012.02223.xRoche D, Gil D and Giraldo J (2013). Mechanistic analysis of the function of agonists and allosteric modulators: reconciling two-state and operational models. British Journal of Pharmacology 169: 1189–1202. doi: 10.1111/bph.12231     

Genetic Deletion of Fatty Acid Amide Hydrolase Alters Emotional Behavior and Serotonergic Transmission in the Dorsal Raphe, Prefrontal Cortex, and Hippocampus

Pharmacological blockade of the anandamide-degrading enzyme, fatty acid amide hydrolase (FAAH), produces CB₁ receptor (CB₁R)-mediated analgesic, anxiolytic-like and antidepressant-like effects in murids. Using behavioral and electrophysiological approaches, we have characterized the emotional phenotype and serotonergic (5-HT) activity of mice lacking the FAAH gene in comparison to their wild type counterparts, and their response to a challenge of the CB₁R antagonist, rimonabant. FAAH null-mutant (FAAH^−/−) mice exhibited reduced immobility in the forced swim and tail suspension tests, predictive of antidepressant activity, which was attenuated by rimonabant. FAAH^−/− mice showed an increase in the duration of open arm visits in the elevated plus maze, and a decrease in thigmotaxis and an increase in exploratory rearing displayed in the open field, indicating anxiolytic-like effects that were reversed by rimonabant. Rimonabant also prolonged the initiation of feeding in the novelty-suppressed feeding test. Electrophysiological recordings revealed a marked 34.68% increase in dorsal raphe 5-HT neural firing that was reversed by rimonabant in a subset of neurons exhibiting high firing rates (33.15% mean decrease). The response of the prefrontocortical pyramidal cells to the 5-HT_2A/2C agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ((±)-DOI) revealed desensitized 5-HT_2A/2C receptors, likely linked to the observed anxiolytic-like behaviors. The hippocampal pyramidal response to the 5-HT_1A antagonist, WAY-100635, indicates enhanced tonus on the hippocampal 5-HT_1A heteroreceptors, a hallmark of antidepressant-like action. Together, these results suggest that FAAH genetic deletion enhances anxiolytic-like and antidepressant-like effects, paralleled by altered 5-HT transmission and postsynaptic 5-HT_1A and 5-HT_2A/2C receptor function.     

BIMT 17, a 5-HT1A receptor agonist/5-HT2A receptor antagonist,directly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex

BIMT 17 (1-[2-[4-(3-trifluoromethyl phenyl) piperazin-1-yl] ethyl] benzimidazol- [1H]-2-one), a 5-HT_1A receptor agonist/5-HT_2A receptor antagonist (see Borsini et al., accompanying paper), in a dose range of 1–10 mg/kg i.v., dose-dependently inhibited the electrical activity of rat medial prefronto-cortical neurons, whereas buspirone, in a dose range of 0.1–1000 g/kg, increased it. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and 1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphthyl)] piperazine (S 14671) presented biphasic patterns of response; they increased electrical activity at doses in the range of 0.1–10 g/kg and 0.1–3 g/kg i.v. respectively, and reduced it at higher doses, 30–300 g/kg and 10–30 g/kg i.v., respectively.The inhibitory effect of BIMT 17 on the firing rate of neurons in the frontal cortex was antagonized by the 5-HT_1A antagonists tertatolol and WAY 100135, and was still present after destruction of serotonin (5-HT) containing neuronal endings by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 150 g/rat, given intraventricularly), which reduced the cortical 5-HT content by 85%. This destruction of 5-HT neurons, while suppressing the ability of 8-OH-DPAT to inhibit the firing rate at high doses, did not change the excitatory action of this compound at low doses. The addition of ritanserin, a 5-HT2A receptor antagonist, potentiated both the excitatory and inhibitory effects of 8-OHDPAT on neuronal electrical activity. Direct microiontophoretic application (100 nA/20 s) of 5-HT and BIMT 17, but not that of 8-OH-DPAT, onto medial prefronto-cortical neurons, decreased the firing rate of these neurons.These findings suggest that BIMT 17 directly inhibits the electrical activity of medial prefronto-cortical neurons through its dual mode of receptor interaction.     

Effects of naltrexone on the accumulation of L-3, 4-dihydroxyphenylalanine and 5-hydroxy-L-tryptophan and on the firing rate induced by acute ethanol administration

In order to characterize the effects of naltrexone, a mu-opioid receptor antagonist, on acute ethanol-induced functional modification of dopaminergic neurons in the nigrastriatal and mesolimbic dopamine systems, the accumulation of L-3, 4-dihydroxyphenylalanine (L-DOPA) in the cerebral cortex, dorsal striatum and nucleus accumbens and of 5-hydroxy-L-tryptophan (5-HTP) in the hippocampus was measured in normal rats using the mu-hydroxybenzylhydrazine dihydrochloride (NSD-1015) enzymatic inhibition method. In addition, the firing rates of dopaminergic neurons were recorded in the substantia nigra and ventral tegmental area. Naltrexone resulted in a decrease in the dopaminergic neuronal firing rates activated by ethanol and eventually in a reduction of the dopamine synthesis induced by ethanol in the dorsal striatum and nucleus accumbens, but not in the cerebral cortex. Mesolimbic dopamine neurons were slightly more sensitive to ethanol and naltrexone than were nigrostriatal dopamine neurons. The widespread inhibitory action of naltrexone also decreased the ethanol-induced stimulation of hippocampal serotonin synthesis.     

(+)-WAY 100135, a partial agonist,at native and recombinant 5-HT1B/1D receptors

1. We have studied the effects of the purportedly selective 5-HT_1A receptor antagonist (+)-WAY100135 on electrically stimulated 5-hydroxytryptamine (5-HT) efflux in the ventrolateral geniculate nucleus (vLGN), and its affinity at human 5-HT_1B and 5-HT_1D receptors stably expressed in Chinese hamster ovary (CHO) cells.
2. On short ‘pseudo single pulse'' stimulations (20 pulses at 100 Hz, 190 ms train duration), (+)-WAY100135 (1.0 μM) decreased 5-HT efflux in the vLGN to 68±8% of pre-drug values (P<0.01). This decrease could be blocked by the 5-HT_1D/1B receptor antagonist GR 127935 (50 nM). Conversely, when long stimulations (20 pulses at 20 Hz, 950 ms train) were used, (+)-WAY100135 had no effect on 5-HT efflux (84±8% of pre-drug values) although both methiothepin (200nM) and GR 127935 (50 nM) caused significant increases (to 175±18 and 130±10% of pre-drug values, respectively).
3. Paroxetine (100 nM), the selective 5-HT reuptake inhibitor, increased stimulated 5-HT efflux and re-uptake half-life (to 145±18% and 649±121%, respectively) on pseudo single pulse stimulations. When (+)-WAY 100135 was added in combination with the uptake blocker, the effect of paroxetine on stimulated 5-HT efflux was potentiated to 282±48% (P<0.01) without further effect on the 5-HT re-uptake half-life.
4. The affinity and intrinsic activity of (+)-WAY 100135 were determined at recombinant human 5-HT_1B and 5-HT_1D receptors expressed in CHO cells, by use of radioligand binding and [³⁵S]-GTPγS binding. (+)-WAY 100135 was a partial agonist at human 5-HT_1B and 5-HT_1D receptors with moderately high affinity for 5-HT_1D receptors (pEC₅₀=7.61).
5. In conclusion, (+)-WAY 100135 was found to be not a selective 5-HT_1A autoreceptor antagonist but may act as a partial agonist at the 5-HT_1B/1D receptor, displaying agonist or antagonist properties depending on the stimulation protocol used and the resultant 5-HT ‘tone'' at the receptor.

     

Side effect profile of 5-HT treatments for Parkinson's disease and L-DOPA-induced dyskinesia in rats

BACKGROUND AND PURPOSE

Treatment of Parkinson''s disease (PD) with L-DOPA eventually causes abnormal involuntary movements known as dyskinesias in most patients. Dyskinesia can be reduced using compounds that act as direct or indirect agonists of the 5-HT_1A receptor, but these drugs have been reported to worsen PD features and are known to produce ‘5-HT syndrome’, symptoms of which include tremor, myoclonus, rigidity and hyper-reflexia.

EXPERIMENTAL APPROACH

Sprague-Dawley rats were given unilateral nigrostriatal dopamine lesions with 6-hydroxydopamine. Each of the following three purportedly anti-dyskinetic 5-HT compounds were administered 15 min before L-DOPA: the full 5-HT_1A agonist ±-8-hydroxy-2-dipropylaminotetralin (±8-OH-DPAT), the partial 5-HT_1A agonist buspirone or the 5-HT transporter inhibitor citalopram. After these injections, animals were monitored for dyskinesia, 5-HT syndrome, motor activity and PD akinesia.

KEY RESULTS

Each 5-HT drug dose-dependently reduced dyskinesia by relatively equal amounts (±8-OH-DPAT ≥ citalopram ≥ buspirone), but 5-HT syndrome was higher with ±8-OH-DPAT, lower with buspirone and not present with citalopram. Importantly, with or without L-DOPA, all three compounds provided an additional improvement of PD akinesia. All drugs tempered the locomotor response to L-DOPA suggesting dyskinesia reduction, but vertical rearing was reduced with 5-HT drugs, potentially reflecting features of 5-HT syndrome.

CONCLUSIONS AND IMPLICATIONS

The results suggest that compounds that indirectly facilitate 5-HT_1A receptor activation, such as citalopram, may be more effective therapeutics than direct 5-HT_1A receptor agonists because they exhibit similar anti-dyskinesia efficacy, while possessing a reduced side effect profile.     

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.