Pharmacology and expression analysis of glycine transporter GlyT1 with [3H]-(N-[3-(4'-fluorophenyl)-3-(4'phenylphenoxy)propyl])sarcosine

In the central nervous system, re-uptake of the neurotransmitter glycine is mediated by two different glycine transporters, GlyT1 and GlyT2. GlyT2 is found in brainstem and spinal cord, whereas GlyT1 is expressed in rat forebrain regions where it is responsible for most glycine transport activity. Initially, GlyT1 and GlyT2 were pharmacologically differentiated by sarcosine, a weak selective inhibitor of GlyT1. The recently described selective and potent GlyT1 antagonist, NFPS/ALX-5407 provided an important additional tool to further characterize GlyT1 pharmacology. In the present study, we have radiolabeled the racemic form of NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (also known as ALX-5407) to investigate its interaction with GlyT1, as well as define GlyT1 expression in the rat central nervous system. Kinetic studies indicated that [3H]NFPS binds rapidly to rat forebrain membranes and dissociates with a t(1/2) of 28 +/- 5 min. [3H]NFPS labeled a saturable population of sites in rat forebrain with a Kd of 7.1+/-1.3 nM and a B(max) of 3.14 +/- 0.26 pmol/mg protein. Bound [3H]NFPS was fully and potently displaced by unlabeled NFPS, whereas glycine and sarcosine were weak, Na+-dependent inhibitors with IC50 of 1,008 and 190 microM, respectively. Additional saturation experiments indicated that glycine and sarcosine were non-competitive antagonists of [3H]NFPS binding. Functional studies revealed that NFPS was a non-competitive inhibitor of [3H]glycine uptake and does not interact with Na+ and Cl- binding sites of GlyT1. Overall, this work shows that [3H]NFPS is a valuable tool in studying GlyT1 expression and pharmacology and that NFPS interacts with GlyT1 at a site different from the transporter translocation and ion binding sites.     

Protection with phencyclidine against inactivation of 5-HT2 receptors by sulfhydryl-modifying reagents

We investigated whether phencyclidine (PCP)-induced head-twitch was antagonized in rats by ritanserin, a selective serotonin2 (5-HT2) receptor antagonist, to confirm the involvement of 5-HT neurons in PCP action and to discover whether PCP could protect the binding sites of [3H]PCP and [3H]ketanserin from the inhibitory effect of protein-modifying reagents which affect sulfhydryl groups. PCP (7.5, 10 and 12.5 mg/kg, i.p.)-induced head-twitch was completely antagonized by ritanserin (1 mg/kg, s.c.). Scatchard plots of specific [3H]PCP and [3H]ketanserin binding showed that sulfhydryl-modifying reagent, N-ethylmaleimide (NEM, 100 microM) caused a significant decrease in Bmax without changing Kd. PCP (10 microM) and ritanserin (1 microM) protected [3H]PCP and [3H]ketanserin binding sites from the decrease in the number induced by NEM (100 microM). 5-HT protected [3H]5-HT binding sites from inactivation by NEM, but PCP and ritanserin did not show any effect. On the basis of the present findings, it is concluded that PCP can interact with 5-HT2 receptors directly or allosterically, and 5-HT2 receptors may locate at PCP binding sites in membranes.     

Inhibitors of GlyT1 affect glycine transport via discrete binding sites

In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Because glycine is a coagonist of the N-methyl-D-aspartate (NMDA) receptor (NMDAR), which has been implicated in schizophrenia, inhibition of GlyT1 is thought to provide an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N-methyl-glycine (sarcosine) derivatives (e.g., (R)-N[3-(4'fluorophenyl)-3-(4'phenyl-phenoxy)propyl]-sarcosine [NFPS], (R)-N[3-phenyl-3-(4'-(4-toluoyl)phenoxy)-propyl]sarcosine [(R)-NPTS], and (R,S)-(+/-)N-methyl-N-[(4-trifluoromethyl)phenoxy]-3-phenyl-propylglycine [Org24589]), and non-sarcosine-containing inhibitors, such as 2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride (SSR504734), have been described. In the present study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus laevis oocytes, and with two binding assays, using [(3)H](R)-NPTS or 2-chloro-N-[(S)-phenyl[(2S)-N-methylpiperidin-2-yl]-methyl]-3-trifluoromethyl benzamide monohydrochloride ([(3)H]N-methyl-SSR504734) as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently irreversible and independent of glycine concentration. The latter indicates a noncompetitive mode of action. In contrast, both SSR504734 and N-methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the binding of the novel radioligand [(3)H]N-methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734 but noncompetitively by sarcosine-based compounds. Inversely, [(3)H](R)-NPTS binding was competitively inhibited by sarcosine-based compounds, whereas glycine, SSR504734, and N-methyl-SSR504734 noncompetitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a noncompetitive or competitive mode of inhibition. The divergent modes of inhibition may significantly affect the efficacy and tolerability of these drugs.     

A novel glycine transporter-1 (GlyT1) inhibitor, ASP2535 (4-[3-isopropyl-5-(6-phenyl-3-pyridyl)-4H-1,2,4-triazol-4-yl]-2,1,3-benzoxadiazole), improves cognition in animal models of cognitive impairment in schizophrenia and Alzheimer's disease

Hypofunction of brain N-methyl-d-aspartate (NMDA) receptors has been implicated in psychiatric disorders such as schizophrenia and Alzheimer's disease. Inhibition of glycine transporter-1 (GlyT1) is expected to increase glycine, a co-agonist of the NMDA receptor and, consequently, to facilitate NMDA receptor function. We have identified ASP2535 (4-[3-isopropyl-5-(6-phenyl-3-pyridyl)-4H-1,2,4-triazol-4-yl]-2,1,3-benzoxadiazole) as a novel GlyT1 inhibitor, and here describe our in vitro and in vivo characterization of this compound. ASP2535 potently inhibited rat GlyT1 (IC(50)=92 nM) with 50-fold selectivity over rat glycine transporter-2 (GlyT2). It showed minimal affinity for many other receptors except for μ-opioid receptors (IC(50)=1.83 μM). Oral administration of ASP2535 dose-dependently inhibited ex vivo [(3)H]-glycine uptake in mouse cortical homogenate, suggesting good brain permeability. This profile was confirmed by pharmacokinetic analysis. We then evaluated the effect of ASP2535 on animal models of cognitive impairment in schizophrenia and Alzheimer's disease. Working memory deficit in MK-801-treated mice and visual learning deficit in neonatally phencyclidine (PCP)-treated mice were both attenuated by ASP2535 (0.3-3mg/kg, p.o. and 0.3-1mg/kg, p.o., respectively). ASP2535 (1-3mg/kg, p.o.) also improved the PCP-induced deficit in prepulse inhibition in rats. Moreover, the working memory deficit in scopolamine-treated mice and the spatial learning deficit in aged rats were both attenuated by ASP2535 (0.1-3mg/kg, p.o. and 0.1mg/kg, p.o., respectively). These studies provide compelling evidence that ASP2535 is a novel and centrally-active GlyT1 inhibitor that can improve cognitive impairment in animal models of schizophrenia and Alzheimer's disease, suggesting that ASP2535 may satisfy currently unmet medical needs for the treatment of these diseases.     

Anxiolytic-like action of MTEP expressed in the conflict drinking Vogel test in rats is serotonin dependent

The purpose of the present study was to investigate whether the anxiolytic-like action of a selective and brain penetrable group I metabotropic glutamate (mGlu5) receptor antagonist 3-[(2-methyl-1,3-tiazol-4-yl)ethynyl]-pyridine (MTEP) is dependent upon the serotonergic system. Experiments were performed on male Wistar rats. The Vogel conflict drinking test was used to detect anxiolytic-like activity. MTEP administered intraperitoneally at doses of 1, 3 and 6 mg/kg induced anxiolytic-like effect. The potential anxiolytic effect of MTEP (1 mg/kg) was inhibited by a nonselective 5-HT receptor antagonist metergoline (2 mg/kg i.p.) and 5-HT2A/2C receptor antagonist ritanserin (0.5 mg/kg i.p.), but not by a 5-HT1A receptor antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl)cyclohexane-carboxamide (WAY 100635) (0.1 mg/kg i.p). The anxiolytic effect of MTEP (6 mg/kg) was attenuated by ritanserin (1 mg/kg i.p.). Moreover, MTEP-induced a dose-dependent release of serotonin in the frontal cortex. The obtained results suggest that the potential anxiolytic effect of the mGlu5 receptor antagonist MTEP is due to the increased serotonin release with subsequent activation of 5-HT2A/2C receptors, most probably located postsynaptically, but not by the 5-HT1A receptors.     

2,5-Dimethoxy-4-iodoamphetamine (DOI) inhibits Delta9-tetrahydrocannabinol-induced catalepsy-like immobilization in mice

The effect of the serotonin 5-HT(2A/2C)-receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on Delta(9)-tetrahydrocannabinol (THC)-induced catalepsy-like immobilization was studied in mice. DOI (0.3 and 1 mg/kg, i.p.) significantly inhibited the catalepsy-like immobilization induced by THC (10 mg/kg, i.p.). In contrast, the selective 5-HT(2C)-receptor agonist 8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one (WAY 161503) had no effect on this catalepsy-like immobilization. Moreover, the 5-HT(2A)-receptor antagonist ketanserin (0.3 mg/kg, i.p.) reversed the inhibition of THC-induced catalepsy-like immobilization caused by DOI (1 mg/kg), whereas the selective 5-HT(2C)-receptor antagonist 6-chloro-2,3-dihydro-5-methyl-N-[6-(2-methyl-3-pyridinyl)oxyl]-3-pyridinyl]-1H-indole-1-carboxyamide (SB 242084) did not affect this inhibitory effect of DOI. On the other hand, ketanserin (0.3 and 1 mg/kg, i.p.) enhanced the catalepsy-like immobilization induced by THC (6 mg/kg, i.p.). Thus, on the basis of these results, it appears that 5-HT(2A)-receptor mechanisms might be responsible for the inhibitory effect of DOI on THC-induced catalepsy-like immobilization.     

Effects of a 5-HT7 receptor antagonist DR4004 on the exploratory behavior in a novel environment and on brain monoamine dynamics in mice

The present study examined whether serotonin (5-hydroxytryptamine; 5-HT)7 receptors play a role in the modulation of emotionality in mice using the selective 5-HT7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydrobenzo (c,d)indol-2-(1H)-one (DR4004). The emotionality of mice was evaluated in terms of exploratory activity in the hole-board test. The mice treated with DR4004 (2.5-10 mg/kg, i.p.) displayed a dose-dependent decrease in locomotor activity by moving less distance in the hole-board, and statistically significant decreases were observed at 5 and 10 mg/kg. On the other hand, DR4004 (10 mg/kg, i.p.) did not affect spontaneous motor activity. In a neurochemical study, decreases in amygdaloid dopamine and 5-HT turnover were observed in mice in which locomotor activity in the hole-board test was attenuated following the administration of DR4004 (10 mg/kg, i.p.). Also, a simple linear regression analysis revealed that locomotor activity on the hole-board was significantly correlated with dopamine and 5-HT turnover in amygdala. Furthermore, co-injection of the selective dopamine reuptake inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR12909; 1.25-5 mg/kg, i.p.) or the selective 5-HT reuptake inhibitor fluvoxamine (20 mg/kg, i.p.) significantly reversed the DR4004 (10 mg/kg, i.p.)-induced decrease in locomotor activity in the hole-board test. These findings constitute the behavioral evidence that 5-HT7 receptors may play a role in the modulation of emotionality. Furthermore, it is also suggested that amygdaloid dopamine and 5-HT neuronal systems may be involved in this modulation.     

Neurochemical, electrophysiological and pharmacological profiles of the selective inhibitor of the glycine transporter-1 SSR504734, a potential new type of antipsychotic.

Noncompetitive N-methyl-D-aspartate (NMDA) blockers induce schizophrenic-like symptoms in humans, presumably by impairing glutamatergic transmission. Therefore, a compound potentiating this neurotransmission, by increasing extracellular levels of glycine (a requisite co-agonist of glutamate), could possess antipsychotic activity. Blocking the glycine transporter-1 (GlyT1) should, by increasing extracellular glycine levels, potentiate glutamatergic neurotransmission. SSR504734, a selective and reversible inhibitor of human, rat, and mouse GlyT1 (IC50=18, 15, and 38 nM, respectively), blocked reversibly the ex vivo uptake of glycine (mouse cortical homogenates: ID50: 5 mg/kg i.p.), rapidly and for a long duration. In vivo, it increased (minimal efficacious dose (MED): 3 mg/kg i.p.) extracellular levels of glycine in the rat prefrontal cortex (PFC). This resulted in an enhanced glutamatergic neurotransmission, as SSR504734 potentiated NMDA-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal slices (minimal efficacious concentration (MEC): 0.5 microM) and intrastriatal glycine-induced rotations in mice (MED: 1 mg/kg i.p.). It normalized activity in rat models of hippocampal and PFC hypofunctioning (through activation of presynaptic CB1 receptors): it reversed the decrease in electrically evoked [3H]acetylcholine release in hippocampal slices (MEC: 10 nM) and the reduction of PFC neurons firing (MED: 0.3 mg/kg i.v.). SSR504734 prevented ketamine-induced metabolic activation in mice limbic areas and reversed MK-801-induced hyperactivity and increase in EEG spectral energy in mice and rats, respectively (MED: 10-30 mg/kg i.p.). In schizophrenia models, it normalized a spontaneous prepulse inhibition deficit in DBA/2 mice (MED: 15 mg/kg i.p.), and reversed hypersensitivity to locomotor effects of d-amphetamine and selective attention deficits (MED: 1-3 mg/kg i.p.) in adult rats treated neonatally with phencyclidine. Finally, it increased extracellular dopamine in rat PFC (MED: 10 mg/kg i.p.). The compound showed additional activity in depression/anxiety models, such as the chronic mild stress in mice (10 mg/kg i.p.), ultrasonic distress calls in rat pups separated from their mother (MED: 1 mg/kg s.c.), and the increased latency of paradoxical sleep in rats (MED: 30 mg/kg i.p.). In conclusion, SSR504734 is a potent and selective GlyT1 inhibitor, exhibiting activity in schizophrenia, anxiety and depression models. By targeting one of the primary causes of schizophrenia (hypoglutamatergy), it is expected to be efficacious not only against positive but also negative symptoms, cognitive deficits, and comorbid depression/anxiety states.     

Comparison of the effects of benzodiazepines and other anticonvulsant drugs on synthesis and utilization of 5-HT in mouse brain

Acute administration of clonazepam (0.5-8.0 mg/kg, i.p.), diazepam (2-32 mg/kg, i.p.), chlordiazepoxide (1-40 mg/kg, i.p.) or diphenylhydantoin (5-320 mg/kg, i.p.), caused a dose-related elevation of the concentrations of, 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and tryptophan in whole mouse brain. Carbamazepine (5-100 mg/kg, i.p.), and phenobarbitone (10-80 mg/kg, i.p.) raised the concentrations of 5-HT and 5-HIAA in the brain, whereas flurazepam (5-80 mg/kg, i.p.) only elevated the level of 5-HIAA. After administration of L[G-3H]tryptophan (25 microCi, s.c.), clonazepam (4 mg/kg), diazepam (32 mg/kg, i.p.), chlordiazepoxide (40 mg/kg) or diphenylhydantoin (40 mg/kg), but not carbamazepine (50 mg/kg), flurazepam (40 mg/kg) or phenobarbitone (80 mg/kg), increased the content of labelled tryptophan in brain. However, administration of drugs did not alter the incorporation of the label into [3H]5-HT, suggesting that the synthesis of 5-HT was unaffected. When incorporation of [3H]tryptophan into [3H]5-HT was complete and the pool of labelled 5-HT was decreasing, clonazepam, diazepam, chlordiazepoxide and diphenylhydantoin elevated the content of [3H]5-HT in brain. Flurazepam, phenobarbitone and carbamazepine were without apparent effect. Calculation of the rate of utilization of 5-HT (Km) showed that all drugs, apart from flurazepam, reduced the utilization of 5-HT. Using the rate of disappearance of 5-HT after inhibition of tryptophan hydroxylase by p-chlorophenylalanine (PCPA), all drugs, except flurazepam, diphenylhydantoin and phenobarbitone, decreased the utilization of 5-HT. The major action of the anticonvulsant drugs on the function of 5-HT in brain appears to be a decrease in the utilization of 5-HT without altering synthesis.     

Effect of acute and chronic administration of buspirone on serotonin and benzodiazepine receptor subtypes in the rat brain: an autoradiographic study.

The affinity of buspirone and its main metabolite 1-(2-pyrimidinyl)piperazine (PmP) for serotonin1 (5-HT1) and benzodiazepine receptors was first evaluated by computerized receptor autoradiography. The results confirmed that buspirone is a selective 5-HT1A ligand, since it inhibited the binding of [3H]5-HT with lower IC50 values (about 100 nM) in regions of the brain of the rat where this receptor subtype is predominant (such as hippocampal areas). Larger IC50 values than 3 microM were found in areas of the brain richer in 5-HT1 receptors, other than the 5-HT1A subtype (e.g. striatum, substantia nigra and the ventricles). The PmP was not selective, inhibiting the binding of [3H]5-HT with similar affinity (about 4-10 microM) in all the regions of the brain examined. Neither buspirone nor PmP, up to 100 microM, were active on benzodiazepine receptors. The autoradiographic technique was therefore used to evaluate the effects of acute (10 mg/kg, p.o., 1 hr before killing) and chronic (10 mg/kg, i.p., twice a day for 21 days, 24 hr washout) treatment with buspirone in male rats. Acute treatment reduced the binding of [3H]5-HT in all the regions of the brain studied, including those with low levels of 5-HT1A receptors, indicating the occupancy of 5-HT1 receptors by either buspirone or its metabolite. The binding of [3H]flunitrazepam was decreased (16%) only in the substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism of fenfluramine-induced hyperthermia in rats by some, but not all, selective inhibitors of 5-hydroxytryptamine uptake

The injection of fenfluramine (7.5 mg kg-1,i.p.) to rats housed at 27-28 degrees C was associated with an elevation of core body temperature which peaked at approximately 1 h post-injection. One h pretreatment with citalopram (20 mg kg-1, i.p.), chlorimipramine (10 mg kg-1, i.p.), femoxetine (10 mg kg-1, i.p.) and fluoxetine (20 mg kg-1, i.p.) resulted in an attenuated response to fenfluramine. In contrast, Org 6582 (20 mg kg-1) and zimelidine (20 mg kg-1) were devoid of an effect on fenfluramine-induced hyperthermia. The response to fenfluramine was was also blocked by i.p. injections of metergoline (0.2 mg kg-1), methysergide (5 mg kg-1) and mianserin (0.5 mg kg-1). Rectal temperature was unaltered by both the 5-hydroxytryptamine (5-HT) uptake inhibitors and the 5-HT receptor antagonists. The IC50 values (nM) for in vitro inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes were for citalopram 2.4, chlorimipramine 8.8, femoxetine 14, fluoxetine 16, Org 6582 75 and zimelidine 250. The injection of all six compounds (20 mg kg-1, i.p.) 1 h before death was associated with an inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes which ranged from 47.2% for chlorimipramine to 83.3% for citalopram. Rat hypothalamic 5-HT levels were decreased by approximately 50% 3 h after the injection of fenfluramine (15 mg kg-1, i.p.). This effect was blocked by a 1 h pretreatment with fluoxetine, Org 6582 and zimelidine (all 20 mg kg-1, i.p.). Ki values for displacement of specifically bound [3H]-5-HT (1 nM) to rat hypothalamic membranes were for metergoline 26 nM, methysergide 1.1 microM, mianserin 3.6 microM, chlorimipramine 9.2 microM and fluoxetine 32.7 microM. Values for citalopram, femoxetine, Org 6582 and zimelidine were in excess of 65.4 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delta(9)-tetrahydrocannabinol prolongs the immobility time in the mouse forced swim test: involvement of cannabinoid CB(1) receptor and serotonergic system

In the present study, we investigated the effect of Delta(9)-tetrahydrocannabinol (THC), the principal psychoactive component of marijuana, on immobility time during the forced swim test. THC (2 and 6 mg/kg, i.p.) significantly prolonged the immobility time. In addition, THC at the same doses did not significantly affect locomotor activity in the open-field test. The selective cannabinoid CB(1) receptor antagonist rimonabant (3 mg/kg, i.p.) significantly reduced the enhancement of immobility by THC (6 mg/kg). Similarly, the selective serotonin (5-HT) reuptake inhibitor (SSRI) citalopram (10 mg/kg, i.p.) and 5-HT(1A/7) receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.3 mg/kg, i.p.) significantly reduced this THC-induced effect. Moreover, the selective 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide dihydrochloride (WAY100635, 1 mg/kg, i.p.) and the postsynaptic 5-HT(1A) receptor antagonist MM-77 (0.1 mg/kg, i.p.) reversed this reduction effect of 8-OH-DPAT (0.3 mg/kg). In contrast, the selective 5-HT(7) receptor antagonist (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulfonyl]phenol hydrochloride (SB269970) had no effect on this reduction effect of 8-OH-DPAT. WAY100635 (1 mg/kg) also reversed the reduction effect of citalopram (10 mg/kg). These findings suggest that the 5-HT(1A) receptors are involved in THC-induced enhancement of immobility.     

Antagonism of the 5-HT1A receptor stimulus in a conditioned taste aversion procedure.

The conditioned taste aversion procedure in mice was used to test for blockade of the drug stimulus of the 5-HT1A receptor agonists (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr (8-OH-DPAT), 1-(4-trifluoromethyl-2-pyridinyl)-4- [4-[2-oxo-1-pyrrolidinyl]butyl]piperazine (E)-2-butenedioate (Org 13011) and the 5-HT reuptake inhibitor fluoxetine. The conditioned taste aversion induced by 8-OH-DPAT (0.22 mg/kg) and Org 13011 (0.5 mg/kg) was readily blocked by the 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY-100635) (0.1 mg/kg). The conditioned taste aversion induced by fluoxetine could not be antagonized by WAY-100635 nor by the 5-HT2 receptor antagonist mianserin. It is concluded that the conditioned taste aversion induced by 8-OH-DPAT or Org 13011 is mediated via 5-HT1A receptors. The results suggest that the conditioned taste aversion induced by fluoxetine is not exclusively mediated by 5-HT1A receptors nor exclusively by 5-HT2 receptors. The results also indicate that the conditioned taste aversion paradigm can be used to test for antagonism of stimulus properties of compounds.     

The role of the 5-HT1D receptor as a presynaptic autoreceptor in the guinea pig

The present study investigated the role of the 5-hydroxytryptamine (5-HT, serotonin)1D receptor as a presynaptic autoreceptor in the guinea pig. In keeping with the literature, the 5-HT1B selective antagonist, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro [furo[2,3-f]indole-3,4'-piperidine]oxalate (SB224289) potentiated [3H]5-HT outflow from pre-labelled slices of guinea pig cerebral cortex confirming its role as a presynaptic autoreceptor in this species. In addition, the 5-HT1D receptor-preferring antagonists, 1-[2-[4-(6-fluoro-1H-indol-3-yl)-3,6-dihydro-2H-pyridin-1-yl]-ethyl]-3-pyridin-4-yl-methyl-tetrahydro-pyrimidin-2-one (LY367642), (R)-1-[2-(4-(6-fluoro-1H-indol-3-yl-)-3,6-dihydro-1(2H)-pyridinyl)ethyl]-3,4-dihydro-1H-2-benzopyran-6-carboxamide (LY456219), (S)-1-[2-(4-(6-fluoro-1H-indol-3-yl-)-3,6-dihydro-1(2H)-pyridinyl)ethyl]-3,4-dihydro-1H-2-benzopyran-6-carboxamide (LY456220) and 1-[2-[4-(4-fluoro-benzoyl)-piperidin-1-yl]-ethyl]-3,3-dimethyl-1,2-dihydro-indol-2-one (LY310762), potentiated [3H]5-HT outflow from this preparation with potencies (EC50 values=31-140 nM) in the same range as their affinities for the guinea pig 5-HT1D receptor (Ki values=100-333 nM). The selective 5-HT1D receptor agonist, R-2-(4-fluoro-phenyl)-2-[1-[3-(5-[1,2,4]triazol-4-yl-1H-indol-3-yl)-propyl]-piperidin-4-ylamino]-ethanol dioxylate (L-772,405), inhibited [3H]5-HT outflow. In microdialysis studies, administration of either SB224289 or LY310762 at 10 mg/kg by the intraperitoneal (i.p.) route, potentiated the increase in extracellular 5-HT concentration produced by a maximally effective dose of the selective serotonin re-uptake inhibitor, fluoxetine (at 20 mg/kg i.p.). In addition, the 5-HT1D receptor-preferring antagonist and 5-HT transporter inhibitor, LY367642 (at 10 mg/kg i.p.), elevated extracellular 5-HT concentrations to a greater extent than a maximally effective dose of fluoxetine. It is concluded that the 5-HT1D receptor, like the 5-HT1B receptor, may be a presynaptic autoreceptor in the guinea pig.     

Phencyclidine-induced head-twitch response in rats treated chronically with methysergide

This study was designed to assess whether phencyclidine (PCP)-induced behaviors in rats were potentiated after two days' withdrawal from chronic methysergide (a 5-HT2 receptor blocker) treatment (10 mg/kg per day i.p. for 12 days), in order to confirm the involvement of 5-hydroxytryptamine (5-HT) neurons in PCP actions. The PCP (10 mg/kg)-induced behaviors (head-twitch, head-weaving, turning and backpedalling) were attenuated by successive pretreatment with PCP (10 mg/kg per day i.p. for 12 days), while PCP- and 5-methoxy-N,N-dimethyltryptamine (2 mg/kg)-induced head-twitch increased significantly after the repeated methysergide treatment was stopped. The development of tolerance to PCP-induced head-twitch was antagonized by pretreatment with methysergide. Furthermore, Scatchard plots of specific [3H]ketanserin binding at the 5-HT2 receptors and [3H]PCP binding at the PCP receptors in the methysergide group revealed significant increases in binding capacity (Bmax) with no change in affinity (Kd). On the contrary, after development of tolerance to PCP, there were significant decreases in Bmax of [3H]ketanserin binding with no change in affinity. PCP can thus displace [3H]ketanserin at the 5-HT2 receptor site, but not [3H]5-HT at the 5-HT1 receptor site. These facts indicate that PCP may produce head-twitch via an agonistic interaction with 5-HT2 receptor sites.     

Modulation of striatal dopamine release by glycine transport inhibitors.

Traditional models of schizophrenia have focused primarily upon dopaminergic (DA) dysregulation. In contrast, more recent models focus on dysfunction of glutamatergic systems, acting particularly through N-methyl-D-aspartate (NMDA) receptors. NMDA receptors in brain are regulated by glycine, acting via a strychnine-insensitive regulatory site, and by glycine (GlyT1) transporters that maintain low glycine levels in the immediate vicinity of the NMDA receptor complex. The present study investigates the role of NMDA receptors in the modulation of striatal dopamine release in vitro, and of glycine transport inhibitors (GTIs) as potential psychotherapeutic agents in schizophrenia. In striatum, NMDA receptors exert dual excitatory/inhibitory effects, with inhibition reflecting activity of local GABAergic feedback regulation. We have previously demonstrated effectiveness of glycine in regulating [3H]DA release both in vivo and in vitro, consistent with its beneficial clinical effects. In the present study, similar effects were observed for the high-affinity GTI (+)N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy-)propyl]sarcosine (NFPS), and for a range of high-affinity GTIs with appropriate rank order of potency. In addition, (+)NFPS significantly stimulated NMDA-induced [3H]GABA release. Effects, of GTIs, were blocked by the glycine-site antagonists L689,560 and HA-966, and the GABA(B) antagonists phaclofen and CGP 52432, confirming the roles of both the NMDA-associated glycine-site and presynaptic GABA(B) receptors in NMDA receptor-mediated regulation of striatal DA release in vitro. Endogenous DA hyperactivity is associated with prominent positive symptoms in schizophrenia. The present results are consistent with recent clinical studies showing significant effectiveness of glycine-site agonists and GTIs in reduction of persistent positive, as well as negative, symptoms in schizophrenia.     

Antiemetic activity of FK1052, a 5-HT3- and 5-HT4-receptor antagonist, in Suncus murinus and ferrets

We investigated the effect of FK1052 [(+)-8,9-dihydro-10-methyl-7-[(5-methyl-1H-imidazol-4-yl)methyl]pyrido[1,2-a]indol-6(7H)-one hydrochloride], a 5-HT3- and 5-HT4-receptor antagonist, on the emesis induced by motion stimuli, copper sulfate, or cisplatin in either Suncus murinus or ferrets and also clarified the role of the 5-HT3 and 5-HT4 receptors in these models. In Suncus murinus, oral administration of FK1052 (100 microg/kg) completely prevented emesis induced by cisplatin (18 mg/kg, i.p.). Intraperitoneal injection of scopolamine (10 mg/kg) and promethazine (32 mg/kg), but not FK1052 (1 mg/kg), significantly reduced the emetic responses by motion stimuli. In ferrets, copper sulfate (40 mg/kg, p.o.)-induced emesis was moderately prevented by FK1052 (3.2 mg/kg), but not by granisetron (3.2 mg/kg). Cisplatin-induced acute (10 mg/kg, i.v.) and delayed (5 mg/kg, i.p.) emesis were significantly reduced by single and multiple intravenous injection of both FK1052 (3.2 mg/kg) and granisetron (3.2 mg/kg), respectively. The present study suggests that FK1052 may be useful against both acute and delayed emesis induced by cancer chemotherapy. Moreover, it is suggested that blockades of 5-HT3 and 5-HT4 receptors are not relevant to the control of motion sickness; and furthermore, it suggested that blocking 5-HT4 receptors in addition to 5-HT3 receptors does not have an additional effect on the control of cisplatin-induced emesis, but that 5-HT4 receptors are at least partly involved in the mechanism of emesis induced by copper sulfate.     

Chronic effects of imipramine and lithium on 5-HT receptor subtypes in rat frontal cortex, hippocampus and choroid plexus: quantitative receptor autoradiographic analysis

The effects of chronic treatment with imipramine or lithium on serotonin (5-HT) receptor subtypes were analyzed in the frontal cortex, hippocampus and choroid plexus of rat brain by quantitative receptor autoradiographic procedures, using radioligands [3H]-5-HT, [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT), [125I]-iodocyanopindolol ([125I]-CYP), [3H]-mesulergine and [125I]-7-amino-8-iodo-ketanserin ([125I]-ketanserin) or [3H]-spiperone. Chronic i.p. administration of imipramine (20 mg/kg/day for 21 days) decreased the densities of 5-HT1, 5-HT1A, 5-HT1C and 5-HT2 sites in the frontal cortex, hippocampus and choroid plexus. Lithium (2 mEq/kg/day for 21 days) also decreased the densities of 5-HT1, 5-HT1C and 5-HT2 sites in the frontal cortex, and the densities of those including 5-HT1A sites in the hippocampus and choroid plexus. Imipramine and lithium very markedly decreased the density of 5-HT1C sites in the choroid plexus. We propose that methods employing quantitative receptor autoradiographic analysis can be used to characterize and understand the local effects of these drugs on 5-HT receptor subtypes.     

Evidence that imipramine activates 5-HT1C receptor function.

The anti-immobility effect of imipramine (15 mg/kg) in the forced swimming test in mice was antagonized by the non-selective 5-hydroxytryptamine (5-HT) antagonist, metitepine (0.5 mg/kg), by the 5-HT1C/5-HT2 antagonist, mesulergine (15 mg/kg), and by the dopamine D2 antagonist, d,l-sulpiride (50 mg/kg). These three antagonists did not alter the behaviour of imipramine-treated mice in an open-field and did not reduce imipramine brain levels. The 5-HT2 antagonist, ritanserin (0.06 mg/kg), the 5-HT1A/5-HTB antagonist, l-propranolol (20 mg/kg), and the 5-HT3 antagonists, endo-2,3-dihydro-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2-oxo-1H- benzimidazole-1-carboxamide hydrochloride (DAU 6215; 0.1 mg/kg) and 1,2,3,9-tetrahydro-9-methyl-3[(2-methyl-1H-imidazol-1-yl)methyl]-4H- carbazol-4-one, HCl.2H2O) (GR 38032F; 0.1 mg/kg), failed to reduce imipramine-induced anti-immobility. Subthreshold doses of 8-hydroxy-2-(di-n-propylamino)tetralin hydrochloride (8-OH-DPAT; 0.5 mg/kg) and imipramine (7.5 mg/kg) did not synergize in reducing immobility. d,l-Sulpiride, but not mesulergine, antagonized the effect of desipramine (15 mg/kg) in the forced swimming test. All compounds were administered i.p. 6 min before imipramine or desipramine, given i.p. 30 min before the testing. Imipramine produced 50% inhibition of [3H]mesulergine binding to 5-HT1C receptors at 10 microM, a concentration below that obtained following i.p. imipramine administration. The results suggest a contribution of 5-HT1C receptors in the mechanism of the imipramine effect in the forced swimming test.     

Regulation of 5-HT2 receptors in rat cortex. Studies with a putative selective agonist and an antagonist

The phenylisopropylamine derivative 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) has been suggested recently as a selective serotonin2 (5-HT2) receptor agonist. Because of the potential importance of such a tool for investigations of 5-HT2 receptor regulation, receptor binding studies were performed in rats after acute and chronic treatment with DOI, the selective 5-HT2 antagonist ketanserin, or vehicle. Single injections of 5 or 10 mg/kg DOI reduced the Bmax of cortical sites labeled with [3H]1-(2,5-dimethoxy-4-bromo-phenyl)-2-aminopropane and [3H]ketanserin (9-32 or 32-46%, respectively). Chronic daily treatment with DOI (3-9 mg/kg) further down-regulated 5-HT2 sites in cortex identified with either [3H]ketanserin (-60%) or with [3H]DOB (-75%), without altering Kd values or affecting 5-HT1 sites. In vitro addition to the [3H]ketanserin or [3H]DOB binding assay of 10 nM to 1 microM DOI resulted in competitive inhibition, suggesting that down-regulation found in vivo was not secondary to residual drug. Chronic treatment with ketanserin (10 mg/kg) also down-regulated both [3H]ketanserin (-38%) and [3H]DOB (-58%) sites in cortex without charges in 5-HT1 sites. In naive cortex, competition experiments revealed a Ki (nM) for ( +/- )-DOI of 1.7 +/- 0.02 at sites labeled by [3H]DOB, and a KH and KL of 4.8 +/- 1.5 and 53 +/- 2 nM at sites labeled by [3H]ketanserin. These data indicate that in chronic treatment, DOI, like ketanserin, is highly selective for 5-HT2 vs 5-HT1 sites at behaviorally useful doses. However, a representative putative 5-HT2 selective agonist and antagonist have similar effects on 5-HT2 receptors labeled by agonist or antagonist radioligands.