Allosteric modulation of the effect of escitalopram, paroxetine and fluoxetine: in-vitro and in-vivo studies

Clinical and preclinical studies have shown that the effect of citalopram on serotonin (5-HT) reuptake inhibition and its antidepressant activity resides in the S-enantiomer. In addition, using a variety of in-vivo and in-vitro paradigms, it was shown that R-citalopram counteracts the effect of escitalopram. This effect was suggested to occur via an allosteric modulation at the level of the 5-HT transporter. Using in-vitro binding assays at membranes from COS-1 cells expressing the human 5-HT transporter (hSERT) and in-vivo electrophysiological and microdialysis techniques in rats, the present study was directed at determining whether R-citalopram modifies the action of selective serotonin reuptake inhibitors (SSRIs) known to act on allosteric sites namely escitalopram, and to a lesser extent paroxetine, compared to fluoxetine, which has no affinity for these sites. In-vitro binding studies showed that R-citalopram attenuated the association rates of escitalopram and paroxetine to the 5-HT transporter, but had no effect on the association rates of fluoxetine, venlafaxine or sertraline. In the rat dorsal raphe nucleus, R-citalopram (250 mg/kg i.v.) blocked the suppressant effect on neuronal firing activity of both escitalopram (100 mg/kg i.v.) and paroxetine (500 mg/kg i.v.), but not fluoxetine (10 mg/kg i.v.). Interestingly, administration of R-citalopram (8 mg/kg i.p.) attenuated the increase of extracellular levels of 5-HT ([5-HT] ext) in the ventral hippocampus induced by both escitalopram (0.28 mM) and paroxetine (0.75 mM), but not fluoxetine (10 mM). In conclusion, the present in-vitro and in-vivo studies show that R-citalopram counteracts the activity of escitalopram and paroxetine, but not fluoxetine, by acting at the allosteric binding site of the 5-HT transporter, either located in the dorsal raphe nucleus or post-synaptically in the ventral hippocampus. This conclusion is strengthened by the observation that the inhibitory effect of fluoxetine, which has no stabilizing effect on the radioligand/hSERT complex, was not blocked by co-administration of R-citalopram

Selective serotonin reuptake inhibitors potentiate the rapid antidepressant-like effects of serotonin4 receptor agonists in the rat.

We have recently reported that serotonin(4) (5-HT(4)) receptor agonists have a promising potential as fast-acting antidepressants. Here, we assess the extent to which this property may be optimized by the concomitant use of conventional antidepressants.We found that, in acute conditions, the 5-HT(4) agonist prucalopride was able to counteract the inhibitory effect of the selective serotonin reuptake inhibitors (SSRI) fluvoxamine and citalopram on 5-HT neuron impulse flow, in Dorsal Raphé Nucleus (DRN) cells selected for their high (>1.8 Hz) basal discharge. The co-administration of both prucalopride and RS 67333 with citalopram for 3 days elicited an enhancement of DRN 5-HT neuron average firing rate, very similar to what was observed with either 5-HT(4) agonist alone. At the postsynaptic level, this translated into the manifestation of a tonus on hippocampal postsynaptic 5-HT(1A) receptors, that was two to three times stronger when the 5-HT(4) agonist was combined with citalopram. Similarly, co-administration of citalopram synergistically potentiated the enhancing effect of RS 67333 on CREB protein phosphorylation within the hippocampus. Finally, in the Forced Swimming Test, the combination of RS 67333 with various SSRIs (fluvoxamine, citalopram and fluoxetine) was more effective to reduce time of immobility than the separate administration of each compound.These findings strongly suggest that the adjunction of an SSRI to a 5-HT(4) agonist may help to optimize the fast-acting antidepressant efficacy of the latter

Synergistic antidepressant-like action of gaboxadol and escitalopram

International audienc

Serotonin 4 receptor (5-HT4R) internalization is isoform-specific: Effects of 5-HT and RS67333 on isoforms A and B

Serotonin 4 receptors (5-HT4Rs) are particularly abundant within the limbic system, where they constitute potential targets for the development of novel, rapid acting antidepressants. However, the population of limbic 5-HT4Rs is not homogenous, comprising various isoforms of which 5-HT4(a) and 5-HT4(b) are among the most abundant variants. Sequence divergence at their C-termini is predictive of specificity in isoform signalling and regulation, but the differences, if any, remain ill-defined. The present study compared isoforms 5-HT4(a) and 5-HT4(b) in their ability to undergo endocytic regulation following exposure to 5-HT and to the putatively fast acting antidepressant RS67333. Both ligands differed in their ability to induce internalization of either isoform, 5-HT being more effective than RS67333 in HEK293 cells and in neurons. In contrast, trafficking induced by 5-HT was isoform-specific. In particular, while PKC, GRK2 and βarrestin were necessary for 5-HT4(a)R internalization, sequestration of 5-HT4(b)Rs required PKC but not GRK2 and relied significantly less on βarrestin. After endocytosis, isoform (b) appeared scattered throughout the intracellular compartment and efficiently recycled to the membrane upon agonist removal. Isoform (a) accumulated in the perinuclear compartment and displayed little recycling. Isoform-specific subcellular distribution was present in HEK293 cells and in neurons. In neurons, where internalization by RS67333 was more pronounced than in HEK293 cells, receptors internalized by this ligand followed the same distribution pattern as observed with 5-HT. These results point to isoform-related differences in the way that 5-HTRs respond to different ligands. Such diversity should be taken into account when developing therapeutic agents that target 5-HT4Rs. © 2009 Elsevier Inc. All rights reserved.These studies were supported by research funds from the Canadian Institutes of Health Research to GP and by funds from the Spanish Plan Nacional de l+D+l (SAF2002-03408) to MTV.Peer Reviewe

Axonal Segregation and Role of the Vesicular Glutamate Transporter VGLUT3 in Serotonin Neurons

International audienceA subset of monoamine neurons releases glutamate as a cotransmitter due to presence of the vesicular glutamate transporters VGLUT2 or VGLUT3. In addition to mediating vesicular loading of glutamate, it has been proposed that VGLUT3 enhances serotonin (5-HT) vesicular loading by the vesicular monoamine transporter (VMAT2) in 5-HT neurons. In dopamine (DA) neurons, glutamate appears to be released from specialized subsets of terminals and it may play a developmental role, promoting neuronal growth and survival. The hypothesis of a similar developmental role and axonal localization of glutamate co-release in 5-HT neurons has not been directly examined. Using postnatal mouse raphe neurons in culture, we first observed that in contrast to 5-HT itself, other phenotypic markers of 5-HT axon terminals such as the 5-HT reuptake transporter (SERT) show a more restricted localization in the axonal arborization. Interestingly, only a subset of SERT-and 5-HT-positive axonal varicosities expressed VGLUT3, with SERT and VGLUT3 being mostly segregated. Using VGLUT3 knockout mice, we found that deletion of this transporter leads to reduced survival of 5-HT neurons in vitro and also decreased the density of 5-HT-immunoreactivity in terminals in the dorsal striatum and dorsal part of the hippocampus in the intact brain. Our results demonstrate that raphe 5-HT neurons express SERT and VGLUT3 mainly in segregated axon terminals and that VGLUT3 regulates the vulnerability of these neurons and the neurochemical identity of their axonal domain, offering new perspectives on the functional connectivity of a cell population involved in anxiety disorders and depression

Effect of a 3-day co-treatment with citalopram, and either prucalopride or RS 67333, on the mean firing rate of DRN 5-HT neurons.

<p>In each rat, both citalopram (or its vehicle) and the 5-HT₄ agonist (or its vehicle) were administered via osmotic minipumps inserted subcutaneously, and recordings were performed with the two minipumps still in place. <b>A</b>: Integrated firing rate histogram showing samples of DRN descents in different experimental groups; indicated doses refer to the total daily dosage. <b>B</b>: Summary of the results: bar histograms represent the mean (± S.E.M.) firing activity of 5-HT neurons, calculated on the basis of successive recording tracks performed along the DRN. Values at the bottom of each column indicates the total number of neurons recorded per group (vehicle-vehicle: n = 7 animals, citalopram-RS 67333: n = 5 animals, and n = 4 animals in the other groups). ** p<0.01 and *** p<0.001 vs vehicle/vehicle, Tukey's test.</p

Effect of the combination of citalopram with a 5-HT₄ agonist on the postsynaptic 5-HT_1A neurotransmission.

<p>Cumulative intravenous doses of the selective 5-HT_1A antagonist WAY 100635 were performed in rats continuously treated with <b>A</b>, the citalopram (10 mg/kg/d) + prucalopride (2.5 mg/kg/d) or <b>B</b>, the citalopram + RS 67333 (1.5 mg/kg/d) combination for three days, and their effects on the firing activity of hippocampal pyramidal neurons were recorded in the CA3 sub-field. Single-cell extracellular recordings were performed in chloral-hydrate anaesthetized animals, by using multiple-barrel glass microelectrodes combined with microiontophoretic pumps, and results expressed as the mean (± S.E.M.) percentage elevation of the firing rate with respect to pre-drug values (n = 4 animals in each group). All compounds (or their vehicle) were administered through the use of osmotic minipumps, inserted subcutaneously in the region of the back. Recordings were performed with the minipumps still in place. * p<0.05, ** p<0.01 and *** p<0.001 vs respective vehicle/vehicle values; + p<0.05, ++ p<0.01 and +++ p<0.001 vs respective citalopram/vehicle values; & p<0.05 and && p<0.01 vs respective vehicle/prucalopride values; # p<0.05 vs respective vehicle/RS 67333 values (Tukey's test). The “<i>a</i>” symbol indicates a p<0.05 significant interaction between the citalopram “pre-treatment” and the 5-HT₄ agonist “treatment”, as revealed by the use of a two-way ANOVA.</p

Effect of the continuous co-administration of citalopram (10 mg/kg/d) and RS 67333 (1.5 mg/kg/d) for 3 days on pCREB.

<p>The activation of CREB in hippocampal tissue was assessed by measuring phosphoCREB (pCREB) immunoreactivity. CREB phosphorylation was normalized according to the amount of protein present in each sample by expressing the data as a ratio of pCREB over total CREB immunoreactivity. Results represent mean ± SEM for the number of experiments indicated, for each treatment, by the value at the bottom of the column. Inset shows representative examples of pCREB immunoreactivity for different treatment conditions indicated on the histogram. All compounds were administered through the use of osmotic minipumps, inserted subcutaneously in the region of the back. * p<0,05 vs vehicle/vehicle, + p<0.05 vs citalopram/vehicle and # p<0.05 vs vehicle/RS 67333, Student's <i>t</i>-test.</p