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

Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 1205–1210

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Antidepressant properties of the 5-HT4 receptor partial agonist, SL65.0155: Behavioral and neurochemical studies in rats Alessandra Tamburella, Vincenzo Micale, Andrea Navarria, Filippo Drago ⁎ Department of Experimental and Clinical Pharmacology, Faculty of Medicine, University of Catania, Viale A. Doria 6, 95125, Catania, Italy

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Article history: Received 8 February 2009 Received in revised form 2 July 2009 Accepted 2 July 2009 Available online 19 July 2009 Keywords: 5-HT4 receptors Forced swim test Neurotrophic factors SL65.0155

a b s t r a c t 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. © 2009 Elsevier Inc. All rights reserved.

1. 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 Abbreviations: Bcl-2, B-cell lymphoma-2; BDNF, Brain Derived Neurotrophic Factor; CNS, Central Nervous System; FST, Forced Swim Test; HRP, Horseradish Peroxidase; i.p., intraperitoneal; NA, Noradrenaline; p-CREB, phosphorilated cAMP-Response Element Binding Protein; SDS-PAGE, Sodium Dodecil Sulphate-Poliacrilamyde Gel Electrophoresis; 5-HT, Serotonin; SSRI, Selective Serotonin Reuptake Inhibitor; TCA, Tricyclic Antidepressant; TTBS, Tween-Tris-Buffered saline; VEGF, Vascular Endothelium Growth Factor. ⁎ Corresponding author. Tel.: +39 095 7384236; fax: +39 095 7384238. E-mail address: [email protected] (F. Drago). 0278-5846/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2009.07.001

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; Warner-Schmidt and Duman, 2007). The B-cell lymphoma-

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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. 2. Material and methods 2.1. 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. The experiments were performed in a laboratory maintained at a temperature of 22 ± 1 °C between 10.00 and 17.00 according to the behavioral procedures of test used. All the experiments were carried out according to the European Community Council 86/609/EEC and efforts were made to minimize animal suffering and to reduce the number of animal used. The rationale, design and methods of this study have been approved by the Ethical Committee for Animal Research, University of Catania. 2.2. Behavioral tests 2.2.1. Forced swim test (FST) The procedure was based on the behavioral test described by Porsolt et al. (1978). A single experiment consisted of a pre-swim and a test swim. Naive rats were individually placed inside vertical cylinders (height: 40 cm, diameter: 30 cm) containing 25 cm of water at 23–25 °C for 15 min. Following this pre-swim, animals were removed and allowed to dry in a heated enclosure before returning to their home cages. After 24 h, the test swim occurred in which the rats were replaced in the cylinder for 5 min and the total duration of immobility and escape behaviors was measured. Test swims were videotaped and subsequently assessed for the following behaviors: immobility (the animal remains floating passively in the water without struggling and shows the minimal movements necessary to keep its head above water), climbing (very vigorous, active move-

ments with animal's forepaws breaking the water surface usually against the walls of the water container), and swimming (described as active movements more than those necessary to keep the head of the rat above the water, and mainly distinguished as propelling the rat around the cylinder). Total time spent engaged in each activity was recorded and analyzed by two “blind” observers. 2.2.2. Open-field test (OFT) Exploratory activity was evaluated in the open field test (OFT) in order to ensure that the decreased immobility or the increased active behaviors in the FST were not secondary to a non-specific increase in motor activity produced by the treatments (Rex et al., 2004; Cryan et al., 2005). The experiment was performed in a soundproof and moderately illuminated (~50 lx) cubic observation chamber (2× 2 × 2 m) between 10:00 and 17:00 h, using a white wooden open field (100 × 100 cm, walls 40 cm high). At the beginning of the test, animals were placed gently in the centre of the arena and allowed to explore. The exploratory activity in the open field, i.e. the number of squares crossed with all paws (crossing) was counted in a 5 min session, recorded on a tape using video camera (Hitachi Videocam) and then scored by a video tracking software (Ugo Basile, Italy). SL65.0155 (0.1, 0.5 and 1 mg/kg), clomipramine (50 mg/kg), citalopram (15 mg/kg) or vehicle (VHC) were injected intraperitoneally (i.p.) to naïve animals at 24, 5 and 1 h before the test (like in the FST). 2.3. Drugs and experimental design All compounds were administered in a volume of 1 ml/kg body weight. Clomipramine hydrochloride (50 mg/kg) and citalopram hydrobromide (15 mg/kg) (Sigma, USA) were prepared freshly by solution in distilled water. The serotonin 5-HT4 receptor partial agonist, SL65.0155 [5-(8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-3-[1-(2-phenylethyl)-4-piperidinyl]-1,3,4-oxadiazol-2 (3H)-one-monohydrochloride] (Sanofi-Aventis, France) was suspended in 1% Tween 80 (in bi-distilled water) and administered at the doses of 0.1, 0.5 or 1 mg/kg. All rats (n = 70) received i.p. injections of drugs 24, 5 and 1 h prior to the behavioral procedures performed between 10:00 h and 17:00 h. For this experiment, the doses of clomipramine and citalopram, selected as positive controls, have been chosen as they are well-known antidepressant agents active in the despair model of depression (Micale et al., 2008; Kuśmider et al., 2007). Immediately after the FST, animals were killed, the brains removed and whole hippocampus was dissected, frozen on dry ice and stored at − 80 °C until ready for analysis. 2.4. Western blotting analysis of p-CREB, BDNF, Bax, Bcl-2 and VEGF protein expressions To examine hippocampal plasticity following administration of SL65.0155, Western Blot analysis was used to measure p-CREB, BDNF, VEGF, Bax and Bcl-2 proteins levels. Rats hippocampi were dissolved in the lysis buffer containing 40 mM Tris (pH 7.5), 1% Tryton, 0.2% SDS, 0.2% Desoxycholate and 1.6% NaCl. A protease inhibitor cocktail (P8340, Sigma) consisting of 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, bestatin, leupeptin, E-64 and aprotinin was added to prevent protease activity. Tissues were sonicated for around 30 s at medium power in a cold pack and lysates were centrifuged 30 min at 10.000 g. The supernatant was used for SDS-PAGE and the pellet discarded. Protein concentration was determined with the Bradford assay (Bradford, 1976). Equal amounts of hippocampal protein per lane (60 µg) were diluted with SDS sample buffer and loaded onto gels (15%) for SDS-PAGE. Proteins were electroblotted to nitrocellulose membrane (Bio-Rad, Hercules, CA) and the efficiency of transfer was confirmed by staining the membrane with ponceau S red (Sigma). Gel retention was assessed by staining with Coomassie blue (Pierce, Rockford, IL). Nonspecific binding was blocked for 1 h at 4 °C

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with 3% nonfat dry milk in Tween-Tris-buffered saline (TTBS). Membranes were incubated overnight at 4 °C with antibodies to p-CREB (rabbit monoclonal IgG, 1:1000, Santa Cruz Biotechnology), BDNF (rabbit polyclonal IgG, 1: 750, Santa Cruz Biotechnology), VEGF (rabbit polyclonal IgG, 1:250, Calbiochem) Bax (rabbit polyclonal IgG, 1:250, Calbiochem) and Bcl-2 (rabbit polyclonal IgG, 1:500, Santa Cruz Biotechnology). All the antibodies were prepared in 3% nonfat dry milk solution in TTBS. Actin was used to verify equal loading of protein, by using the anti-β-actin antibody (mouse monoclonal IgG, 1:5000, Sigma). Appropriate horseradish (HRP)-conjugated secondary antibodies, which were anti-rabbit (goat IgG; Santa Cruz Biotechnology), or anti-mouse (goat IgG, Sigma), were applied at 1:10.000. Negative controls were performed without primary antibody. Visualization was performed with the ECL kit (Amersham Pharmacia Biotec). Protein

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bands were visualized and quantified digitally with an acquisition and analysis program (Scion Image). 2.5. Statistical analysis Data were analyzed using the one-way ANOVA followed by the posthoc Student–Newman–Keuls for multiple comparisons. A p-value of 0.05 or less was considered as indicative of a significant difference. 3. Results 3.1. 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 b 0.001), climbing time (F6,63 = 40.74; p b 0.001) and swimming time (F6,63 = 19.81; p b 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 b 0.05) and a significant increase in swimming and climbing behavior (p b 0.01; p b 0.001) as compared to vehicle-(VHC) treated group. The positive control SSRI citalopram (15 mg/kg) also induced a significant effect in immobility (p b 0.05) and swimming (p b 0.001) but not in climbing (p N 0.05) as compared to VHC-treated group. However, the TCA clomipramine (50 mg/kg) was the most potent to affect the immobility time (p b 0.01 vs VHC, SL65.0155 1 mg/kg or citalopram 15 mg/kg) and the climbing (p b 0.001 vs VHC, SL65.0155 1 mg/kg or citalopram 15 mg/kg). No difference in the swimming behavior was found between SSRI and TCA drugs, although both induced a stronger effect to increase the swimming time (p b 0.05 vs SL65.0155 1 mg/kg). The locomotor activity paradigm served as an internal control for possible unspecific stimulant effects, which may confound the interpretation of the FST results. Thus, animals treated with SL65.0155 (0.1, 0.5 and 1 mg/kg) clomipramine (50 mg/kg) or citalopram (15 mg/kg) failed to show any change in motor ability assessed in the OFT, as compared to VHC-treated group (F6,63 = 0.61; p N 0.05) (Fig. 2). 3.2. Neurochemical effects of SL65.0155, clomipramine and citalopram The effects of treatment with SL65.0155 (0.1, 0.5 or 1 mg/kg) on hippocampal levels of p-CREB or VEGF protein expression using western blot analyses are shown in Fig. 3. One-way ANOVA revealed a main effect of treatment for p-CREB (F6,63 = 11.88; p b 0.001) or VEGF (F6,63 = 13.53; p b 0.001) protein expression. Post-hoc analysis revealed that already at the lowest dose tested (0.1 mg/kg), SL65.0155 significantly enhanced hippocampal p-CREB and VEGF (p b 0.05 vs VHC) expressions. The higher doses (0.5 or 1 mg/kg) were also able to induce a significant effect on p-CREB and VEGF protein levels (p b 0.05; p b 0.01 vs VHC). However, these effects were apparently not dose dependent since the doses of 0.1 and 0.5 mg/kg or 0.5 and 1 mg/kg

Fig. 1. Effects of SL65.0155, a serotonin 5-HT4 receptor partial agonist on rats in the FST. SL65.0155 (SL; 0.1, 0.5 and 1 mg/kg), clomipramine (CMP; 50 mg/kg), citalopram (CIT; 15 mg/kg) or their vehicles (VHCs) were administered i.p. 24, 5 and 1 h prior to the behavioral testing. In this experiment, two groups of control animals were injected i.p. with SL65.0155 VHC or with clomipramine and citalopram VHC. As similar results were obtained from these two control groups, data were combined. Values are mean ± S.E.M. of time measures expressed in sec. of (n = 10) animals. ⁎p b 0.05, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs VHC-injected controls (Student–Newman–Keuls post-hoc test). #p b 0.05 vs SL 1 (Student–Newman–Keuls post-hoc test). ##p b 0.01, ###p b 0.001 vs SL 1 and CIT (Student–Newman–Keuls post-hoc test).

Fig. 2. Effects of SL65.0155, a serotonin 5-HT4 receptor partial agonist, on the locomotor activity of rats in the OFT. Values are mean ± S.E.M. of (n = 10) animals.

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Fig. 3. Effects of SL65.0155, a 5-HT4 receptor partial agonist, on hippocampal expression of p-CREB (A) or VEGF (B). The graphics of p-CREB and VEGF expression show the mean ± S.E.M. expressed as the percentage of change from (n = 10) animals injected i.p. with vehicles (VHCs), SL65.0155 (SL; 0.1, 0.5 and 1 mg/kg), clomipramine (CMP; 50 mg/kg) and citalopram (CIT; 15 mg/kg). ⁎p b 0.05 and ⁎⁎p b 0.01 vs VHCinjected controls (Student–Newman–Keuls post-hoc test).

induced similar effects on the VEGF and p-CREB expression respectively, with a slight, non-significant prevalence of lover doses. Clomipramine and citalopram, used here as positive control failed to induce any change in the protein expressions, as compared to VHC-injected controls. Fig. 4 shows the effects of treatment on hippocampal BDNF, Bax and Bcl-2 protein expression. Only SL65.0155 (1 mg/kg) was able to increase the hippocampal BDNF protein expression (F6,63 = 3.035; p b 0.05). In Bcl-2 protein expression again ANOVA revealed a main effect of treatment (F6,63 = 38.19; P b 0.0001). In the post-hoc analysis all doses of SL65.0155 enhanced Bcl-2 levels in a not dose dependent manner (p b 0.001 vs VHC). However, SL65.0155 (0.1 or 0.5 mg/kg) induced a stronger effect, as compared to the dose of 1 mg/kg (p b 0.001). No effect was found in the hippocampal Bax expression (F6,63 = 0.9; p N 0.05). 4. 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 many antidepressant-sensitive paradigms) is the fact that short-term antidepressant treatments may reverse the

Fig. 4. Effects of SL65.0155, a 5-HT4 receptor partial agonist, on hippocampal expression of BDNF (A) Bcl-2 (B) or Bax (C). The graphics of BDNF, Bcl-2 and Bax expression show the mean ± S.E.M. expressed as the percentage of change from (n = 10) animals injected i.p. with vehicles (VHCs), SL65.0155 (SL; 0.1, 0.5 and 1 mg/kg), clomipramine (CMP; 50 mg/kg) and citalopram (CIT; 15 mg/kg). ⁎p b 0.05 and ⁎⁎⁎p b 0.001 vs VHC-injected controls (Student–Newman–Keuls post-hoc test). ###p b 0.001 vs SL 1 (Student–Newman–Keuls post-hoc test).

immobility of rats, whereas in patients it can take weeks for the same antidepressants to elevate mood (Cryan et al., 2002). It is noteworthy that using relatively low doses of SL65.0155 (0.5–1 mg/ kg), the effects on immobility and swimming time were similar to that induced by the TCA clomipramine (50 mg/kg) or the SSRI citalopram (15 mg/kg), suggesting that the latter could have properties common to those of classical antidepressants. We should keep in mind that although doses of antidepressants found to be active in the FST are in the range of 20 mg/kg (Jacobson and Cryan, 2007), the dose of clomipramine considered as the minimum effective dose in the same paradigm is rather high (Porsolt et al., 1978). SL65.0155 and clomipramine, but not citalopram, increased climbing behaviors of rats. These effects are in agreement with previous studies showing that SSRIs tend to enhance horizontal swimming movements in the cylinder, whereas antidepressants increasing NA neurotransmission predominantly increase climbing behavior (Cryan et al., 2005). The

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mechanism underlying the effects of SL65.0155 on swimming and climbing behavior is unknown, but it could be due to a facilitator effect on the monoaminergic (5-HT and NA) neurotransmission. More specifically, the acute exposure to swim stress has been found to lead to a decreased 5-HT release in the lateral septum, which innervations derive from the dorsal raphé nucleus (DRN), favoring passive (immobility) over active (swimming and climbing) responses (Kirby et al., 1995). By contrast, activation of serotonin 5-HT4 receptors by partial agonists, such as RS 67333 or prucalopride, elicited behavioral (i.e. decreased immobility time in the FST) and neurochemical (i.e. increased firing activity of DRN-5-HT neurons and hippocampal pCREB expression) antidepressant-like effects (Lucas and Debonnel, 2002; Lucas et al., 2005, 2007). Thus, the swimming-enhancing effect of SL65.0155 in the FST could be linked to an activation of the 5HTergic neurotransmission. Nevertheless, in our study the serotonin 5-HT4 stimulation was also able to augment the climbing behavior in FST, in agreement with Lucas et al. (2007). To date, there is no evidence that SL65.0155 acts on the NAergic system, but it is not to exclude a direct action of the serotonin 5-HT4 receptors on NA release since they are positively coupled to adenylate cyclase via Gs proteins. (Molderings et al., 2006). However, further studies (electrophysiology and microdialysis) are required to confirm these hypotheses. Another point deserving mention is that none of the compounds tested affected the rat motor behavior in the OFT, although all produced a reduction in FST immobility. One of the specific concerns in antidepressant research with the FST is that compounds reducing immobility may produce psychomotor stimulation rather than an antidepressant action per se (Jacobson and Cryan, 2007). As such, compounds that induce locomotor activity may provide a false positive result for an antidepressant-like profile. Thus, our results suggest that the decreased immobility time of treated animals in FST is not due to an effect on locomotor activity. Although the neurobiological basis of depression and the precise mechanism of antidepressant action remain unknown, increasing evidence linking stress, depression and antidepressant action suggests that mood disorders are associated with a fall of the 5-HTergic function and with impairment of cellular mechanisms governing neuronal plasticity (for a review, see Schmidt and Duman, 2007). It is well accepted that antidepressants could exert delayed long-term beneficial effects via neurotrophic and/or anti-apoptotic action on neuronal function, particularly through a stimulation of BDNF expression. As BDNF expression is regulated by CREB, the activity of this factor could thereby underlie some of the effects of antidepressant treatment (Tardito et al., 2006). Antidepressant-induced changes in BDNF expression have been extensively studied, although they are not fully understood. Interestingly, in our study SL65.0155, but not clomipramine or citalopram was able to change p-CREB and BDNF expression after acute administration. More specifically, only the highest dose of the drug was able to affect BDNF expression, while the lower dose already increased the p-CREB expression. However, we did not find any dose-response effect, since the doses of 0.5 and 1 mg/kg induced similar effect in p-CREB expression (with a slight, non-significant higher effect for the 0.5 mg/kg dose). The reason for this discrepancy is unclear, but it could be due to different pathways involved in the activation of these proteins. Alternatively, unmatched doses may explain the different findings. Several factors including detection method, age of animals, class of antidepressants drugs, method of drug delivery, time interval between the last dose and the sacrifice and the length of administration could be involved in the different findings described (Duman and Monteggia, 2006). We are aware that the present results are different from those of previous studies (Nibuya et al., 1996; Thome et al., 2000; Tiraboschi et al., 2004; WarnerSchmidt and Duman, 2007) showing elevated hippocampal p-CREB or BDNF expression following chronic, but not acute antidepressant treatment. The delayed efficacy exerted by classical antidepressants is due to both cellular and molecular adaptations at several levels of brain

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neurons, as well as to the activation of signaling pathways (i.e. activation of protein Kinase A following cAMP elevation) leading to an up-regulation and activation of CREB and to an overexpression of BDNF (Xu et al., 2003; Gass and Riva, 2007). Several data, but not all, confirm the role of BDNF levels in stress-related disorder as depression. In postmortem samples of human brains of depressed patients, BDNF levels have been found to be reduced (Chen et al., 2001). Furthermore, BDNF infusion in the hippocampus exerts antidepressant properties in the FST and learned helplessness paradigms (Shirayama et al., 2002), while mice lacking BDNF fail to have antidepressant responses (Monteggia et al., 2004). In contrast, reduction in BDNF levels or BDNF signaling did not produce depression-like symptoms, as well as injection of BDNF into the dopamine mesolimbic pathway induced depressive-like phenotype (Eisch et al., 2003; Duman and Monteggia, 2006). Taken together these results suggest that although BDNF is implicated in the mechanism of the activity of antidepressant drugs in preclinical models of depression, its involvement appears to depend on several factors as stress procedure applied, antidepressant drug used and brain region involved. However additional studies are required to further support the role of BDNF in stress-related depression disorders. Another point in our results deserving mention is the effects of SL65.0155 on hippocampal Bcl-2, Bax and VEGF expression. Increasing evidence suggests that apoptosis is implicated in the decreased hippocampal volume occurring in depressed patients (Lucassen et al., 2001a,b, 2006). This process is programmed and controlled by the cellular balance between pro- (Bax) and anti- (Bcl-2) apoptotic proteins. Several studies demonstrated that treatment with mood stabilizers (e.g. lithium or valproate), antidepressants (e.g.. venlafaxine, amitriptyline or desipramine) and atypical antipsychotics (e.g. olanzapine or clozapine) increased Bcl-2 levels in the brain (for a review, see Czéh and Lucassen, 2007). These findings link affective disorders and mood regulation to the cellular resilience and the ability to withstand a variety of insults. The effects of antidepressants on neurogenesis have been explained through an action on the survival of newborn cells, increasing hippocampal Bcl-2 expression (Murray and Hutson, 2007). In our study hippocampal Bcl-2 expression was significantly increased following acute treatment with SL65.0155 but not with SSRIs or TCAs, suggesting that the former has cell survival properties already after acute administration. Furthermore, in the present experiments any treatment was not able to influence Bax expression as already found by Murray and Hutson (2007). Unlike p-CREB expression, in Bcl-2 expression the lowest dose induced the stronger change. The reason for this difference is unclear, but it could be due to different pathways involved in the activation of these proteins. However, few studies have focused on apoptotic processes in animal stress models and the relationship between 5-HT4 receptor activation and apoptotic processes remain to be fully elucidated. As recently reviewed by Warner-Schmidt and Duman (2008), VEGF is a potent mitogen and survival factor for endothelial cells and neurons, as well as a modulator of synaptic transmission. It could also be a potential target for the antidepressants treatment. In details, VEGF has neurogenic effects in the hippocampal areas which are associated with antidepressants effects and memory improvement (Warner-Schmidt and Duman, 2007; Segi-Nishida et al., 2008). To further confirm the potential role of hippocampal VEGF in pathophysiology of neuropsychiatric disorders, Blumberg et al. (2008) have suggested that specific variations in human VEGF might contribute to individual differences in hippocampus structure and, potentially, function. In our study, SL65.0155 was able to increase hippocampal VEGF expression already after acute treatment, suggesting that also hippocampal VEGF overexpression could mediate the behavioral response to antidepressant treatment. However, it is noticeable that these neurotrophic factors have been evaluated in rats subjected to FST, thus it is not to exclude that their enhanced expression could provide same resistance to the damaging effects of acute stressor exposure as suggested by Arunrut et al. (2009).

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5. 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 anti-apoptotic factors involved in neuronal function (Warner-Schmidt and Duman, 2007). However, we cannot also exclude an involvement of monoaminergic systems (i.e. 5-HT and NA), since the activation of 5-HT4 receptors increased the firing activity of DRN-5-HT neurons (Cryan et al., 2005). The clinical extension of these findings may be relevant for the pharmacological treatment of depressive disorders since unlike the 5-HT4 full agonists, this compound is devoid of cardiovascular and gastrointestinal effects in line with its antagonistic activity in isolated peripheral tissues (Moser et al., 2002). Further studies are now required to investigate the effects of SL65.0155 in other animal models of depression following long-term treatment (i.e. chronic mild stress), as well as to characterize the drug treatment at the cellular levels, by using other molecular techniques such as in situ hybridization or immunohistochemistry for the proteins examined. 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. References Arunrut T, Alejandre H, Chen M, Cha J, Russo-Neustadt A. Differential behavioral and neurochemical effects of exercise, reboxetine and citalopram with the forced swim test. Life Sci 2009;84:584–9. Blumberg HP, Wang F, Chepenik LG, Kalmar JH, Edmiston E, Duman RS, et al. Influence of vascular endothelial growth factor variation on human hippocampus morphology. Biol Psychiatry 2008;64:901–3. Bockaert J, Claeysen S, Compan V, Dumuis A. 5-HT(4) receptors: history, molecular pharmacology and brain function. Neuropharmacology 2008;55:922–31. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. Chen B, Dowlatshahi D, MacQueen GM, Wang JF, Young LT. Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. Biol Psychiatry 2001;50:260–5. Cryan JF, Markou A, Lucki I. Assessing antidepressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci 2002;23:238–45. Cryan JF, Valentino RJ, Lucki I. Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swim test. Neurosci Biobehav Res 2005;29:547–69. Czéh B, Lucassen PJ. What causes the hippocampal volume decrease in depression? Are neurogenesis, glial changes and apoptosis implicated? Eur Arch Psychiatry Clin Neurosci 2007;257:250–60. Duman RS. A silver bullet for the treatment of depression? Neuron 2007;55:679–81. Duman RS, Monteggia LM. A neurotrophic model for stress-related mood disorders. Biol Psychiatry 2006;59:1116–27. Duman RS, Malberg J, Thome J. Neural plasticity to stress and antidepressant treatment. Biol Psychiatry 1999;46:1181–91. Eisch AJ, Bolaños CA, de Wit J, Simonak RD, Pudiak CM, Barrot M, et al. Brain-derived neurotrophic factor in the ventral midbrain-nucleus accumbens pathway: a role in depression. Biol Psychiatry 2003;54:994-1005. Gass P, Riva MA. CREB, neurogenesis and depression. Bioessays 2007;29:957–61. Heninger GR, Delgrado PR, Charney DS. The revised monoamine theory of depression: a modulatory role for monoamines, based on new findings from monoamine depletion experiments in humans. Pharmacopsychiatry 1996;29:2-11. Jacobson LH, Cryan JF. Feeling strained? Influence of genetic background on depressionrelated behavior in mice: a review. Behav Gen 2007;37:171–213. Kirby LG, Allen AR, Lucki I. Regional differences in the effects of forced swimming on extracellular levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. Brain Res 1995;682:189–96.

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