Antidepressant properties of the 5-HT4 receptor partial agonist, SL65.0155: Behavioral and neurochemical studies in rats

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Abstract

This study was undertaken to investigate the potential antidepressant-like properties of SL65.0155, a serotonin 5-HT4 receptor partial agonist, in male rats of the Wistar strain tested in the forced swim test (FST), an experimental model widely used to assess antidepressant-like activity. The expression of hippocampal neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), the phosphorilated cAMP response element-binding protein (p-CREB), the B cell lymphoma-2 (Bcl-2), the Bax and the vascular endothelium growth factor (VEGF) were also evaluated by Western Blot analysis. Different groups of rats received intraperitoneally (i.p.) injections of SL65.0155 (0.1, 0.5 and 1 mg/kg), clomipramine (50 mg/kg), citalopram (15 mg/kg) or vehicle, respectively, 24, 5 and 1 h prior to the FST. Compared to the control group, SL65.0155 (0.5 and 1 mg/kg), clomipramine or citalopram injected animals showed an increased swimming and climbing behavior and reduced immobility time in the FST. Interestingly, this effect was not due to changes in the locomotor activity since all treated groups failed to show any change in motor ability as assessed in the open field test. Western blot analysis of hippocampal homogenates showed an enhancement of p-CREB, BDNF Bcl-2 and VEGF protein levels in SL65.0155 treated groups, but not in citalopram or clomipramine treated groups, used here as positive control. No change was found in Bax expression in any treated group. These findings give further support to the hypothesis that the stimulation of serotonin 5-HT4 receptors may be a therapeutic target for depression.

Introduction

It is well accepted that a complex dysregulation of the catecholaminergic system, particularly a fall of the serotonergic (5-HTergic) function plays a crucial role in the depressive disorders (Schloss and Williams, 1998). Thus, some of the most widely prescribed antidepressant drugs include the tricyclic antidepressants (TCAs) and the selective serotonin reuptake inhibitors (SSRIs) which affect intrasynaptic concentrations of 5-HT and noradrenaline (NA) (Heninger et al., 1996). However, the role of different serotonin 5-HT receptor subtypes in the pathophysiology and treatment of these disturbances has still to be fully clarified, even though the 5-HT1A/2A, 5-HT1B/1D, 5-HT2C and 5-HT3 receptors could be most involved (Cryan et al., 2005). Actually, neurochemical and behavioral studies support a major role for the serotonin 5-HT4 receptor subtype in depressive disorders (Duman, 2007). The serotonin 5-HT4 receptor is a G-protein-coupled, 7-transmembrane domain protein, positively linked to the activation of adenylate cyclase, located within the central nervous system (CNS) in regions related to emotional processes, such as olfactory tubercules, hippocampus, frontal cortex and amygdala (for a review, see Bockaert et al., 2008). Recently, Lucas et al. (2007) showed that serotonin 5-HT4 receptor partial agonists, such as prucalopride and RS 67333 induce behavioral and neurochemical antidepressant-like effects with a rapid onset of action.

Although synaptic levels of neurotransmitters like 5-HT and NA are increased immediately by antidepressant treatment, there is typically a six-week to eight-week delay before therapeutic efficacy can be found, suggesting that a cascade of events including neuronal adaptations to these treatments is responsible for the relief of depressive symptoms. One of the signaling pathways regulated by chronic antidepressant treatment is the cyclic adenosine monophosphate (cAMP) cascade. This second messenger pathway, leading to an up-regulation of phopshorilated (cAMP)-response element binding protein (p-CREB), may activate downstream targets such as brain-derived neurotrophic factor (BDNF) and vascular endothelium growth factor (VEGF) (Nibuya et al., 1996, Duman et al., 1999, Malberg et al., 2000, Perera et al., 2008). Cerebral infusion of BDNF and VEGF elicited antidepressant-like effects in different animal models, and this finding further supports the role of these factors in the therapeutic action of antidepressants (Shirayama et al., 2002, Schmidt and Duman, 2007). The B-cell lymphoma-2 (Bcl-2) is a membrane-associated protein with both anti-apoptotic and neurotrophic properties, under the transcriptional control of p-CREB (Wilson et al., 1996). Recently, increased hippocampal expression of this protein has been found following chronic treatment with SSRIs or TCAs (Xu et al., 2003, Luo et al., 2004, Murray and Hutson, 2007). Thus, the delayed long-term beneficial effects of antidepressants may be due to their neurotrophic and/or anti-apoptotic action on neurons, although the neural mechanisms following the chronic treatment with these drugs are not fully understood.

SL65.0155 is a benzodioxanoxadiazalone compound acting as a partial agonist with high affinity and good selectivity for human serotonin 5-HT4 receptors (Ki of 0.6 nM). Acting at central serotonin 5-HT4 receptors as an agonist, it showed cognition-enhancing properties in several experimental models of amnesia. Unlike other 5-HT4 agonists, this compound lacks cardiovascular and gastrointestinal effects, in line with its antagonistic activity in isolated peripheral tissues (pKb of 8.81 in rat esophagus) (Moser et al., 2002, Micale et al., 2006). Based on the above premises, the present study was performed to assess the antidepressant profile of SL65.0155 in the FST, an experimental model widely used to assess antidepressant-like activity (Porsolt et al., 1978). The open field test (OFT) was performed in order to make sure that decreased immobility or increased active behaviors in the FST were not secondary to non-specific effects on locomotor activity due to the treatment (Cryan et al., 2005). Given the neurotrophic hypothesis of depression, we also evaluated changes in VEGF, BDNF, p-CREB, Bcl-2 and Bax protein levels in hippocampus, a brain region primarily involved in the pathophysiology of depression and in the antidepressant response. Comparative data for the TCA clomipramine and for the SSRI citalopram, under the same experimental conditions, were also obtained.

Section snippets

Animals

Wistar male rats weighing 220–240 g (obtained from Charles River, Italy) were used throughout all experiments. For at least 1 week prior to the experiments, the animals were housed four to a cage at a temperature of 22 ± 1 °C and under a 12-h light/dark cycle (lights on between 8.00 and 20.00), with food and tap water available ad libitum. Randomly assigned to any treatment group, animals were used only once in the behavioral experiments and then sacrificed at the end of behavioral procedures.

Behavioral effects of SL65.0155, clomipramine and citalopram on FST and OFT

As shown in Fig. 1, ANOVA revealed a significant treatment effect for immobility time (F6,63 = 12.81; p < 0.001), climbing time (F6,63 = 40.74; p < 0.001) and swimming time (F6,63 = 19.81; p < 0.001) of rats tested in the FST. Post-hoc analysis revealed that SL65.0155 (0.5 or 1 mg/kg) induced both a significant reduction of the immobility time (p < 0.05) and a significant increase in swimming and climbing behavior (p < 0.01; p < 0.001) as compared to vehicle-(VHC) treated group. The positive control SSRI

Discussion

The present results clearly show for the first time that the serotonin 5-HT4 receptor partial agonist, SL65.0155 given systemically to rodents is effective in improving the behavioral performance in the FST, an experimental model widely used to assess antidepressant-like activity (Porsolt et al., 1978). A wide variety of antidepressants and compounds with potential antidepressant activity reduce the duration of immobility in the FST procedure. However, one major disadvantage of the FST (as for

Conclusions

For the first time we have described here the antidepressant-like properties of SL65.0155, a 5-HT4 partial agonist in FST, suggesting that changes in the expression of different hippocampal protein (i.e. BDNF, CREB, and Bcl-2) could be involved in this effect. According to a current view, these results further support both the neurotrophic hypothesis of depression and that the therapeutic effects of antidepressants could be due to changes in the hippocampal expression of neurotrophic and/or

Acknowledgements

We are grateful to Sanofi-Aventis (Italy) for the generous gift of SL65.0155. These experiments were supported by the PhD International School Program in Neuropharmacology, University of Catania Medical School.

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