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 betaarrestin were necessary for 5-HT4(a)R internalization, sequestration of 5-HT4(b)Rs required PKC but not GRK2 and relied significantly less on betaarrestin. 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.

Effects of bifeprunox and aripiprazole on rat serotonin and dopamine neuronal activity and anxiolytic behaviour.

The atypical antipsychotic bifeprunox is a partial dopamine D(2) and 5-HT(1A) receptor agonist. Using in-vivo electrophysiological and behavioural paradigms in the rat, the effects of bifeprunox and aripiprazole were assessed on ventral tegmental area (VTA) dopamine and dorsal raphe serotonin (5-HT) cell activity and on foot shock-induced ultrasonic vocalisation (USV). In VTA, bifeprunox and aripiprazole decreased (by 20-50%) firing of dopamine neurons. Interestingly, bursting activity was markedly reduced (by 70-100%), bursting being associated with a larger synaptic dopamine release than single spike firing. Both ligands reduced inhibition of firing rate induced by the full dopamine receptor agonist apomorphine, whereas the D(2) receptor antagonist haloperidol prevented these inhibitory effects, confirming partial D(2)-like agonistic properties. On 5-HT neurons, bifeprunox was more potent than aripiprazole to suppress firing activity. The 5-HT(1A) receptor antagonist WAY-100,635 prevented their effects. In the USV test of anxiolytic-like activity, bifeprunox had higher potency than aripiprazole to reduce vocalisations. Both WAY-100,635 and haloperidol reversed the effects of both agonists. The present in-vivo study shows that bifeprunox is a potent partial D(2)-like and 5-HT(1A) receptor agonist reducing preferentially the phasic activity of dopamine neurons. Thus, bifeprunox would be expected to be an effective compound against positive and negative symptoms of schizophrenia.

[Escitalopram: a selective inhibitor and allosteric modulator of the serotonin transporter]. / L'escitalopram: un inhibiteur sélectif et un modulateur allostérique du transporteur de la sérotonine.

Citalopram (Séropram) is an antidepressant of the selective serotonin (5-HT) reuptake inhibitor (SSRI) class, composed of equal amounts of S-enantiomer, escitalopram, and R-enantiomer, R-citalopram. Both clinical and preclinical studies have reported that escitalopram is a potent SSRI that possesses a faster onset of antidepressant activity in comparison with citalopram. Conversely, R-citalopram, although devoid of 5-HT reuptake inhibition property, was reported to counteract the effect of the S-enantiomer in several in vitro and in vivo experiments. For instance, microdialysis studies have shown that escitalopram increased the extracellular 5-HT levels in the frontal cortex and the ventral hippocampus, and this effect was prevented by concomitant injection of R-citalopram. The in vivo relevance of the antagonistic effect of R-citalopram on escitalopram efficacy was confirmed in dorsal raphe nucleus, a brain region known to be a target for SSRIs. In the later region, escitalopram was four times more potent than citalopram in suppressing the firing activity of 5-HT neurons and this effect of escitalopram was significantly prevented by R-citalopram. The antagonizing effect of R-citalopram on escitalopram efficacy was also observed in behavioural tests predictive of anxiolytic or antidepressant properties. In adult rats, R-citalopram reduced the anxiolytic-like effect of escitalopram obtained in the footshock-induced ultrasonic vocalization model, the conditioned fear model or the Vogel conflict and elevated plus maze tests. In validated chronic models with high predictive value for antidepressant activity, when escitalopram was administered for five weeks, either alone or with twice as much R-citalopram, the effect of the treatment regimens on reversal of hedonic deficit was significantly different. Importantly, chronic treatment with escitalopram reversed the decrease in cytogenesis in the rat dentate gyrus, induced by chronic mild stress. However, in naïve rats, while chronic treatment with R-citalopram did not modify the basal proliferation rate in the dentate gyrus, it blocked the increase induced by escitalopram when coadministered. This suggests that neuronal adaptive changes, which are essential for antidepressant response, are rapidly induced by escitalopram but prevented by R-citalopram coadministration. The attenuating effect of R-citalopram was suggested to underlie the delayed recovery of 5-HT neuronal activity following long-term treatment with citalopram versus escitalopram. This is confirmed since a treatment with R-citalopram antagonized the recovery of firing observed in escitalopram-treated rats. The exact mechanism by which R-citalopram exerts its action is not yet fully defined; however, an allosteric interaction between the enantiomers and the 5-HT transporter (SERT) has been proposed. In this context, in vitro studies have revealed the existence of at least two binding sites on SERT: (1) a primary high-affinity binding site or orthosteric site that mediates the inhibition of 5-HT reuptake and (2) an allosteric low-affinity binding site that modulates the binding of ligands at the primary site. In presence of escitalopram alone, both the primary and the allosteric sites are occupied. Thus, escitalopram exerts a stabilizing effect on this association to SERT, resulting in an effective inhibition of 5-HT reuptake activity. On the other hand, in the presence of the two enantiomers, R-citalopram binds to the allosteric site and decreases the escitalopram action on SERT. Such an innovative mechanism of action can constitute a basis for development of new allosteric antidepressants that demonstrate higher efficacy and earlier onset of therapeutic effect.