Richard Alonso

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 μM) 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.

Interleukin-2 Modulates Evoked Release of [3H]Dopamine in Rat Cultured Mesencephalic Cells

Abstract: Mesencephalic cell cultures were used as a model to investigate the effects of interleukin‐2 (IL‐2) on evoked release of [3H]dopamine ([3H]DA) and γ‐[3H]‐aminobutyric acid ([3H]GABA). At low concentrations (10−13‐10−12M), IL‐2 potentiated [3H]DA release evoked by the excitatory amino acids N‐methyl‐D‐aspartate (NMDA) and kainate, whereas higher IL‐2 concentrations (10−9‐10−8M) had no effect. IL‐2 (10−14‐10−8M) modulated K+‐evoked [3H]DA release in a biphasic manner, with low concentrations (10−12‐10−11M) of IL‐2 potentiating and higher concentrations (10−9‐10−8M) inhibiting K+‐induced [3H]DA release. IL‐2 (10−14‐10−8M) by itself failed to alter spontaneous [3H]DA release. The inhibition by IL‐2 of K+‐evoked [3H]DA release was reversible and not due to neurotoxicity, as preexposure to IL‐2 (10−8M) had no significant effect on the subsequent ability of dopaminergic cells to take up and to release [3H]DA. Under our experimental conditions, IL‐2 (10−8 M) did not alter Ca2+‐independent [3H]GABA release evoked by either K+ or NMDA. The results of this study indicate that IL‐2 is able to potentiate [3H]DA release evoked by a number of different stimuli, including K+ depolarization and activation of both NMDA and non‐NMDA receptor subtypes in mesencephalic cell cultures. IL‐2 is active at very low concentrations, a finding that indicates a potent effect of IL‐2 on dopaminergic neurons and implicates a physiological role for this cytokine in the modulation of DA release.

Characterization of SSR103800, a selective inhibitor of the glycine transporter-1 in models predictive of therapeutic activity in schizophrenia.

On native human, rat and mouse glycine transporter-1(GlyT1), SSR130800 behaves as a selective inhibitor with IC50 values of 1.9, 5.3 and 6.8 nM, respectively. It reversibly blocked glycine uptake in mouse brain cortical homogenates, increased extracellular levels of glycine in the rat prefrontal cortex, and potentiated NMDA-mediated excitatory postsynaptic currents in rat hippocampal slices. SSR103800 (30 mg/kg, p.o.) decreased MK-801- and PCP-induced locomotor hyperactivity in rodents. SSR103800 (1 and 10 mg/kg, p.o.) attenuated social recognition deficit in adult rats induced by neonatal injections of PCP (10 mg/kg, s.c., on post-natal day 7, 9 and 11). SSR103800 (3 mg/kg, p.o.) counteracted the deficit in short-term visual episodic-like memory induced by a low challenge dose of PCP (1 mg/kg, i.p.), in PCP-sensitized rats (10 mg/kg, i.p.). SSR103800 (30 mg/kg, i.p.) increased the prepulse inhibition of the startle reflex in DBA/1J mice. SSR103800 decreased defensive- and despair-related behaviors in the tonic immobility test in gerbils (10 and 30 mg/kg, p.o.) and in the forced-swimming procedure in rats (1 and 3 mg/kg, p.o.), respectively. These findings suggest that SSR103800 may have a therapeutic potential in the management of the core symptoms of schizophrenia and comorbid depression states.

SSR181507, a dopamine D2 receptor antagonist and 5-HT1A receptor agonist. I: Neurochemical and electrophysiological profile

SSR181507 ((3-exo)-8-benzoyl-N-[[(2S)7-chloro-2,3-dihydro-1,4-benzodioxin-1-yl]methyl]-8-azabicyclo[3.2.1]octane-3-methanamine monohydrochloride) is a novel tropanemethanamine benzodioxane derivative that possesses high and selective affinities for D2-like and 5-HT1A receptors (KI=0.8, 0.2, and 0.2 nM for human D2, D3, and 5-HT1A, respectively). In vivo, SSR181507 inhibited [3H]raclopride binding to D2 receptors in the rat (ID50=0.9 and 1 mg/kg, i.p. in limbic system and striatum, respectively). It displayed D2 antagonist and 5-HT1A agonist properties in the same concentration range in vitro (IC50=5.3 nM and EC50=2.3 nM, respectively, in the GTPγS model) and in the same dose range in vivo (ED50=1.6 and 0.7 mg/kg, i.p. on striatal DA and 5-HT synthesis, respectively, and 0.03–0.3 mg/kg, i.v. on dorsal raphe nucleus firing rate). It selectively enhanced Fos immunoreactivity in mesocorticolimbic areas as compared to the striatum. This regional selectivity was confirmed in electrophysiological studies where SSR181507, given acutely (0.1–3 mg/kg, i.p.) or chronically (3 mg/kg, i.p., o.d., 22 days), increased or decreased, respectively, the number of spontaneous active DA cells in the ventral tegmental area, but not in the substantia nigra. Moreover, SSR181507 increased both basal and phasic DA efflux (as assessed by microdialysis and electrochemistry) in the medial prefrontal cortex and nucleus accumbens, but not in the striatum. This study shows that the combination of D2 receptor antagonism and 5-HT1A agonism, in the same dose range, confers on SSR181507 a unique neurochemical and electrophysiological profile and suggests the potential of this compound for the treatment of the main dimensions of schizophrenia.

Blockade by the cannabinoid CB1 receptor antagonist, rimonabant (SR141716), of the potentiation by quinelorane of food-primed reinstatement of food-seeking behavior.

It has been shown previously that the selective cannabinoid CB1 receptor antagonist, rimonabant (SR141716), reduced the intake of palatable food as well as the self-administration of several drugs of abuse, suggesting that endocannabinoid systems play a role in brain reward function. The present study investigated whether a cannabinoid step was involved in food-seeking behavior induced by explicit stimuli, using an operant reinstatement procedure in rats. Experimental sessions consisted of a 15-min food rewarded period, followed by a 45-min extinction period. Rimonabant did not affect the response reinstatement induced by noncontingent delivery of food pellets, but prevented (0.03–0.3 mg/kg) the potentiation by quinelorane, a dopamine D3 receptor-preferring agonist, of food-seeking behavior. A possible link between cannabinoid processes and D3- and/or D2-mediated dopaminergic transmission was further investigated by studying Fos protein expression in cortico-limbic structures in D3 (D3−/−) and D2 (D2−/−) knockout mice. Rimonabant (10 mg/kg) increased Fos immunoreactivity in the prefrontal cortex (pFCortex) and in the shell but not the core of the nucleus accumbens (NAcc). Fos induction by this dose of rimonabant was not seen in mice lacking CB1 receptors, providing clear evidence for the involvement of CB1 receptors. In the NAcc shell, the effect of rimonabant was suppressed in D3−/−, but remained unchanged in D2−/− mice. In contrast, Fos expression by rimonabant in the pFCortex was impervious to D2 or D3 receptor deletion. In conclusion, these data indicate first that rimonabant prevented the enhancement by quinelorane of the appetitive value of food pellets unexpectedly delivered during extinction and second that rimonabant effects might involve D3 receptor-mediated processes. Overall, these results are consistent with the notion that endocannabinoid functions control brain reward processes and in particular the capacity of explicit stimuli to precipitate food-seeking behavior.

SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-Bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide], a Centrally Active Nonpeptide Antagonist of the Tachykinin Neurokinin 1 Receptor: II. Neurochemical and Behavioral Characterization

SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide], a new nonpeptide tachykinin neurokinin 1 (NK1) receptor antagonist, was evaluated against the neurochemical, electrophysiological, and behavioral effects provoked by direct activation of brain tachykinin NK1 receptors or by stress in guinea pigs. SSR240600 (0.1–10 mg/kg i.p. or p.o.) antagonized the excitatory effect of i.c.v. infusion of [Sar9,Met(O2)11]substance P (SP) on the release of acetylcholine in the striatum of anesthetized and awake guinea pigs. This antagonistic action was still observed after repeated administration of SSR240600 (5 days, 10 mg/kg p.o., once a day). SSR240600 (10 mg/kg i.p.) inhibited the phosphorylation of the cAMP response element-binding protein in various brain regions induced by i.c.v. administration of [Sar9,Met(O2)11]SP. In slice preparations, neuronal firing of the locus coeruleus (LC) neurons elicited by the application of [Sar9,Met(O2)11]SP was suppressed by SSR240600 at 100 nM. Norepinephrine release in the prefrontal cortex, elicited either by an intra-LC application of [Sar9,Met(O2)11]SP or by an i.c.v administration of corticotropin-releasing factor, was reduced by SSR240600 (0.3–1 mg/kg and 1–10 mg/kg i.p., respectively). SSR240600 (1–10 mg/kg i.p.) inhibited vocalizations induced in adult guinea pigs by an i.c.v. administration of the NK1 receptor agonist, GR73632 [d-Ala-[l-Pro9,Me-Leu8]substance P(7-11)]. Furthermore, SSR240600 (1–10 mg/kg i.p.) inhibited distress vocalizations produced in guinea pig pups by maternal separation. SSR240600 also reduced maternal separation-induced increase in the number of neurons displaying NK1 receptor internalization in the amygdala. Finally, SSR240600 counteracted the increase in body temperature induced by isolation stress. In conclusion, SSR240600 is able to antagonize various NK1receptor-mediated as well as stress-mediated effects in the guinea pig.

[3H]N‐Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Abstract: Saturation experiments with the muscarinic antagonist [3H]N‐methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax= 1.85 ± 0.01 pmol/mg of protein at 10 days in culture; KD= 0.128 ± 0.01 nM The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki= 273 ± 13 nM), whereas the M2/M3 muscarinic antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H)= 31 ± 5 nM; Ki(L)= 2,620 ± 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N‐methyl‐D‐aspartate (100 μM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.

Effects of L- and N-type Ca2+ channel antagonists on excitatory amino acid-evoked dopamine release.

In the present study we tested the effect of dihydropyridine (DHP) Ca2+ channel antagonists and of omega-conotoxin GVIA on [3H]dopamine (DA) release evoked by the activation of excitatory amino acid (EAA) receptors in cultures of fetal rat ventral mesencephalon, in order to investigate the role of voltage-sensitive L- and N-type Ca2+ channels in these EAA-mediated processes. Micromolar concentrations (10-30 microM) of DHP L-type Ca2+ channel antagonists inhibited [3H]DA release evoked by N-methyl-D-aspartate (NMDA), kainate, quisqualate or veratridine. [3H]DA release evoked by the L-type Ca2+ channel agonist, Bay K 8644, was inhibited by lower concentrations (0.1-1 microM) of the DHP antagonist, nitrendipine, than was the release evoked by EAAs. The DHP antagonist, (+)-PN 200-110, was more potent than (-)-PN 200-110 in inhibiting [3H]DA release evoked by Bay K 8644, but the two stereoisomers were equipotent in inhibiting NMDA-evoked release. These results indicate that activation of L-type Ca2+ channels is able to evoke [3H]DA release. However activation of L-type channels is not involved in EAA-induced [3H]DA release and therefore inhibition of EAA-induced [3H]DA release by micromolar concentrations of DHPs must be mediated by actions other than inhibition of L-type Ca2+ channels. omega-Conotoxin GVIA (3 microM) had no effect on [3H]DA release evoked by Bay K 8644, indicating that the toxin may selectively inhibit N-type channels in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of glucocorticosteroid receptor expression in rat hippocampal cell cultures by nerve growth factor.

Dispersed hippocampal cells cultured in serum-free conditions were used to study the effects of nerve growth factor (NGF) on the expression of type I (mineralocorticosteroid or MR) and type II (glucocorticosteroid or GR) corticosteroid receptors. Cells, plated at a density of 1.2 x 10(6) cells/ml in 60 mm Petri dishes, were mainly identified as neurons (90-95%) and maintained for at least 2 weeks. A 7-day treatment with 10-50 ng NGF/ml induced a concentration-dependent decrease of GR binding (40% decrease) compared to untreated cells. In contrast, MR density was unaffected by a 7-day treatment with 50 ng NGF/ml. Data are discussed as possible direct and/or indirect effects of NGF at the level of both neuronal and glial cells.

Further evidence for the sleep-promoting effects of 5-HT2A receptor antagonists and demonstration of synergistic effects with the hypnotic, zolpidem in rats

5-Hydroxytryptamine (5-HT)2A antagonists are promising therapeutic agents for the treatment of sleep maintenance insomnias, but unlike hypnotics, they have limited effects on sleep initiation. This study evaluated the effects of several 5-HT₂A antagonists (eplivanserin, volinanserin and AVE8488) alone and/or in combination with the short-acting hypnotic, zolpidem, on the rat sleep profile. A repeated-measures design was used in which rats were treated with eplivanserin (3 and 10 mg/kg, i.p. or p.o.), volinanserin (0.3-3 mg/kg, i.p.), AVE8488 (0.1-3 mg/kg, i.p.) and zolpidem (3 and 10 mg/kg, p.o.). In addition, animals received a combination of eplivanserin (3 mg/kg, p.o.) and zolpidem (3 mg/kg, p.o.). Electroencephalogram was analyzed for 6 h after administration. Eplivanserin did not modify wakefulness and non-rapid eye movement sleep (NREMS), while zolpidem (10 mg/kg po) induced a marked increase in NREMS duration. Volinanserin (1 and 3 mg/kg) and AVE8488 (0.3 mg/kg) similarly increased NREMS, while reducing wakefulness. Moreover, the 5-HT₂A antagonists and, to a lesser extent, zolpidem, increased duration of NREMS episodes, while decreasing their frequency. When eplivanserin was co-administered with zolpidem, a synergistic effect was observed as the combination produced an increase in NREMS time and bouts duration. These findings confirm further that 5-HT₂A antagonists promote the maintenance of sleep, and suggest that combining a 5-HT₂A antagonist with a short-acting hypnotic may be a useful strategy for the treatment of insomnia.