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European Neuropsychopharmacology (2010) xx, xxx–xxx

ARTICLE IN PRESSNEUPSY-10272; No of Pages 10

The β3 adrenoceptor agonist, amibegron (SR58611A)counteracts stress-induced behavioral andneurochemical changesAlessandra Tamburella, Vincenzo Micale, Gian Marco Leggio, Filippo Drago⁎

Department of Experimental and Clinical Pharmacology, University of Catania Medical School, Viale A. Doria 6, 95125,Catania, Italy

Received 10 November 2009; received in revised form 13 April 2010; accepted 15 April 2010

⁎ Corresponding author. Tel.: +39 09E-mail address: fdrago@tin.it (F. D

0924-977X/$ - see front matter © 201doi:10.1016/j.euroneuro.2010.04.006

Please cite this article as: Tamburbehavioral and neurochemical chang

KEYWORDSβ3 adrenoceptors;Restraint stress;Neurotrophic factors;Forced swim test;Amibegron

Abstract

These experiments were made to study the mechanisms underlying the antidepressant-likeeffects of the β3 adrenoceptor agonist amibegron (SR58611A). To this purpose, the expressionlevels of the hippocampal cyclic adenosine monophosphate (cAMP)-response element bindingprotein (CREB), brain-derived neurotrophic factor (BDNF), B-cell lymphoma-2 (Bcl-2) and Baxproteins were assessed, by using western blot analysis, in rats tested in the forced swim test

(FST). Under basal conditions (no previous exposure to stressors), different groups of male Wistarrats received acutely or repeatedly (once/day for 7 days) intraperitoneal (i.p.) injections ofamibegron (1, 5 and 10 mg/kg), the tricyclic antidepressant (TCA) clomipramine (50 mg/kg), theselective serotonin reuptake inhibitor (SSRI) citalopram (15 mg/kg) or their vehicles. Theinfluence of stress-related conditions was studied in rats subjected to acute (4 h) or repeated(4 h/day for 7 days) restraint stress, applied prior to the FST procedure. Compared to the controlgroups, both stressor procedures increased the immobility time in the FST and reducedhippocampal BDNF and Bcl-2/Bax ratio proteins expression, which were counteracted byamibegron (5 and 10 mg/kg) treatment. Opposite effects were found in the CREB expression,since it was lower after acute and higher after repeated stress procedure, respectively. Again,these effects were reversed by amibegron treatment. Different results were obtained in animalstreated with clomipramine or citalopram. Hence, it is likely that the observed behavioral effectsof amibegron could be due, at least in part, to its action on hippocampal expression ofneurotrophic and/or anti-apoptotic factors, supporting the hypothesis that β3 adrenoceptorsmay be a therapeutic target for the treatment of stress-related disorders.© 2010 Elsevier B.V. All rights reserved.

5 7384236; fax: +39 095 73842rago).

0 Elsevier B.V. All rights reserv

ella, A., et al., The β3 adrenes, Eur. Neuropsychopharmaco

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oceptor agonist, amibegron (SR58611A) counteracts stress-inducedl. (2010), doi:10.1016/j.euroneuro.2010.04.006

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1. Introduction

The selective β3 adrenoceptor (β3-AR) agonist amibegron(SR58611A), has been demonstrated to exhibit a profile ofantidepressant/anxiolytic activity in rodents, which is quitesimilar to that of classical β1/β2 receptor agonists. However,unlike other adrenergic agonists, it is devoid of side effectssuch as sedation, myorelaxation, sleep/wakefulness alter-ation or memory impairment (Simiand et al., 1992; Consoliet al., 2007; Overstreet et al., 2008; Stemmelin et al., 2008,2010). The mechanisms underlying the amibegron antide-pressant/anxiolytic activity remain still unclear, althoughthe involvement of serotoninergic (5-HTergic) and norad-renergic (NAergic) neurotransmitter systems, whose dysre-gulations are fully involved in the physiopathology of stress-related disorders as well as in the application of severalacute stressors, has been suggested (Lenard et al., 2003;Claustre et al., 2008).

Although the biological bases of stress-related disorderslike depression and the precise mechanism of the antide-pressant action remain partly unknown, increasing evidencesuggests that depression is associated with structuralimpairments as well as disruption in cellular mechanismsgoverning neuronal plasticity (Schmidt and Duman, 2007).Stressful life events are major predisposing risk factors fordepression (McEwen, 2005) and preclinical studies havehighlighted both the effects of pre-training stressors (such asdrug injection procedure, inescapable foot-shocks, re-straint, forced swim) on the behavioral response of animalsand the influence of these stressful stimuli on the action ofanxiolytic and/or antidepressant drugs (Drago et al., 2001;Briones-Aranda et al., 2002; Teixeira and De Lima, 2003;Chaki et al., 2004; Consoli et al., 2005; Micale et al., 2008b).

Several studies with few exceptions have shown thatchronic antidepressant treatment counteracts hippocampalstress-induced atrophy and exerts neurotrophic activityincreasing the expression of factors as the transcriptionfactor cyclic adenosine monophosphate (cAMP)-responseelement binding protein (CREB), the brain-derived neuro-trophic factor (BDNF) and the anti-apoptotic protein B-celllymphoma-2 (Bcl-2) (Nibuya et al., 1996; Duman et al., 1999;Malberg et al., 2000; Xu et al., 2003; Luo et al., 2004; Murrayand Hutson, 2007). Thus, long-term beneficial effects ofthese drugs in the brain could be mediated via neurotrophicand/or anti-apoptotic action on neuronal function.

Based on the above premises, this study was undertakento assess the possible neurobiological mechanisms underlyingthe antidepressant-like effect of the β3-AR agonist amibe-gron in the forced swim test (FST), an experimental modelwidely used for preclinical studies on novel antidepressantdrugs (Porsolt et al., 1978). For this purpose, we haveevaluated, by western blot analysis, the changes in CREB,BDNF, Bcl-2 and Bax proteins levels in the hippocampus, abrain region involved in the expression of emotionalresponses. Since the mechanism of action and the effectsof this compound following earlier exposure to stress are stillunclear (Stemmelin et al., 2008), the influence of stress-related behavior was also assessed by applying acute (4 h) orrepeated (4 h/day for 7 days) restraint stress, as described byLuo et al. (2005), prior to the FST. We also investigatedwhether the effects of acute or repeated restraint stressmight be reversed by the acute or repeated treatment,

Please cite this article as: Tamburella, A., et al., The β3 adrenocebehavioral and neurochemical changes, Eur. Neuropsychopharmacol.

respectively, followed by the study of changes in theexpression of the above described factors. Comparativedata for the tricyclic antidepressant (TCA) clomipramine andthe serotonin selective uptake inhibitor (SSRI) citalopram,obtained under the same experimental conditions, were alsoprovided.

2. Experimental procedures

2.1. Animals

Wistar male rats (aged 8–10 weeks) weighing 220–240 g (obtainedfrom Charles River, Italy) were used throughout all experiments. Forat least 1 week prior to the experiments, the animals were housedfour 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 tapwater available ad libitum.

Randomly assigned to any treatment group, animals were usedonly once in the behavioral experiments and were then sacrificed atthe end of behavioral procedures. The experiments were performedbetween 10.00 and 17.00 in an environment maintained at atemperature of 22±1 °C according to the behavioral procedures oftest used. All the experiments were carried out according to theEuropean Community Council 86/609/EEC and efforts were made tominimize animal suffering and to reduce the number of animal used.The rationale, design and methods of this study have been approvedby the Ethical Committee for Animal Research, University ofCatania.

2.2. Behavioral test

2.2.1. Forced swim test (FST) procedure for ratsFor the FST procedure, each rat was forced to swim inside a

vertical Plexiglas cylinder containing 25 cm of water maintained at25 °C (Porsolt et al., 1978). After 15 min in the water it was removedand allowed to dry for 15 min in a heated container before beingreturned to its home cage. It was then replaced in the cylinders 24 hlater and the total duration of immobility was measured during a 5-min test. The rat was judged to be immobile whenever it remainedpassively floating in the water in a slightly hunched but uprightposition, its head just above the surface.

2.3. Drugs and experimental design

All compounds were administered in a volume of 1 ml/kg bodyweight. Control animals received the vehicles intraperitoneally (i.p.). Clomipramine hydrochloride (50 mg/kg) and citalopram hydro-bromide (15 mg/kg) were prepared freshly daily by solution indistilled water. Amibegron (SR58611A) (N [(2 S)-7-carbethoxy-methoxy-1,2,3,4-tetrahydronaphth-2-yl]-(2R)-2-hydroxy-2-(3-chlor-ophenyl) ethanamine hydrochloride) was diluted in saline containing10% dimethyl sulfoxide (DMSO) and injected i.p. to different groupsof animals at the doses of 1, 5 or 10 mg/kg. In this experiment, twogroups of control animals were injected i.p. with amibegron VHC orwith clomipramine and citalopram VHC. As similar results wereobtained from these two control groups, data were combined. Allchemical substances were purchased from Sigma (USA), except foramibegron that was donated by Sanofi-Aventis, Italy.

Two different experiments were programmed and carried out(Table 1). In the first experiment, different groups of rats (n=10)received i.p. injections of drugs 24, 5 and 1 h prior to the FSTprocedure performed at 15:00 h without stress application (A) orafter a stress-related condition given by an acute restraint stressprocedure as described by Luo et al. (2005) (B). Here, “no stressed”groups are indicated as NST and “stressed” groups are indicated asSTR. Briefly the animals were placed into individual plastic rodent

ptor agonist, amibegron (SR58611A) counteracts stress-induced(2010), doi:10.1016/j.euroneuro.2010.04.006

Table 1 Representation of experimental design.

D: Day; FST: forced swim test; Inj: injection; NST: non-stressed group; RS: restraint stress; STR: stressed group.

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restrainers for 4 h, 30 min prior the pre-test of the FST procedure. Asdescribed previously (Porsolt et al., 1978), the treatment startedright after the pre-test. Immobility time in sec was scored as thebehavioral index. For this experiment, clomipramine and citalopramwere selected as active controls as they are well-known antidepres-sant agents active in the FST, as well as the doses of amibegron wereselected based on results of previous experiments (Micale et al.,2006, 2008a; Consoli et al., 2007; Tamburella et al., 2009).

In the second experiment, different groups of NST rats (n=10)received an i.p. injection of compounds daily for 7 days at the samedoses described above (C). For the measurement of immobility timein the FST after repeated stress procedure, other groups of STR ratswere subjected to restraint stress as described above for 4 h/dayduring 7 consecutive days. Animals received one injection i.p.30 min after the stress procedure. On day seven the last injectionwas made 1 h prior the pre-test of the FST procedure (D).Immediately after the 5 min test, all animals were sacrificed bydecapitation, the brains removed and the whole hippocampus wasdissected, frozen on dry ice and stored at −80 °C until ready for

Please cite this article as: Tamburella, A., et al., The β3 adrenocebehavioral and neurochemical changes, Eur. Neuropsychopharmacol. (

analysis. The influence of acute or repeated restraint stressparadigm on corticosterone levels has been reported by Rademacheret al. (2008), showing an increase of plasma corticosterone levels inboth conditions.

2.4. Biochemical method

2.4.1. Western blotting analysis of CREB, BDNF Bcl-2and Baxproteins expression

To examine stress-related alteration of hippocampal plasticitywestern blot analysis was used to measure CREB, BDNF, Bcl-2 andBax proteins levels. Rats hippocampi were made soluble in a lysisbuffer containing 40 mM Tris (pH 7.5), 1% Triton, 0.2% SDS, 0.2%desoxycholate and 1.6% NaCl. Protease inhibitor cocktail (P8340,Sigma) consisting of 4-(2-aminoethyl) benzenesulfonyl fluoride,pepstatin A, bestatin, leupeptin, E-64, and aprotinin was added toprevent protease activity. Tissues were sonicated for around 30 s atmedium power in a cold pack and lysates were centrifuged 30 min at

ptor agonist, amibegron (SR58611A) counteracts stress-induced2010), doi:10.1016/j.euroneuro.2010.04.006

Figure. 1 Effects of amibegron, a β3 adrenoceptor agonist, onimmobility time of rats in the forced swim test (FST) under basalcondition (NST) (A) or after the application of acute restraintstress (STR) (B). Amibegron (SR; 1, 5 and 10 mg/kg), clomipra-mine (CMP; 50 mg/kg), citalopram (CIT; 15 mg/kg) or theirvehicles (VHC) were administered i.p. 24, 5 and 1 h prior to thebehavioral testing. Values are mean±S.E.M. of time measuresexpressed in sec. of (n=10) animals. *Pb0.05 and **Pb0.01 vsvehicle-injected non-stressed (VHC+NST) controls (Student–Newman–Keuls post-hoc test). #Pb0.05 and ##Pb0.01 vsvehicle-injected stressed (VHC+STR) controls (Student–New-man–Keuls post-hoc test).

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10.000 g. The supernatant was used for SDS-PAGE and the pelletdiscarded. Protein concentration was determined with the Bradfordassay (Bradford, 1976). Equal amounts of hippocampal protein perlane (60 µg) were diluted with SDS sample buffer and loaded ontogels (15%) for SDS-PAGE. Proteins were electroblotted to nitrocel-lulose membrane (Bio-Rad, Hercules, CA) and the efficiency oftransfer was confirmed by staining the membrane with ponceau Sred (Sigma). Gel retention was assessed by staining with Coomassieblue (Pierce, Rockford, IL). Nonspecific binding was blocked for 1 hat 4 °C with 3% nonfat dry milk in Tween-Tris-buffered saline (TTBS).Membranes were incubated overnight at 4 °C with antibodies toCREB (rabbit monoclonal IgG, 1:1000, Sigma), BDNF (rabbitpolyclonal IgG, 1:750, Santa Cruz Biotechnology) Bcl-2 (rabbitpolyclonal IgG, 1:500, Santa Cruz Biotechnology) and Bax (rabbitpolyclonal IgG, 1:250, Calbiochem). All the antibodies wereprepared in 3% nonfat dry milk solution in TTBS. Actin was used toverify 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 performedwithout primary antibody. Visualization was performed with the ECLkit (Amersham Pharmacia Biotec). Protein bands were visualized andquantified digitally with an acquisition and analysis program (ScionImage).

Please cite this article as: Tamburella, A., et al., The β3 adrenocebehavioral and neurochemical changes, Eur. Neuropsychopharmacol.

2.5. Statistical analysis

Data were analyzed using two-way analysis of variance (ANOVA). Thepost-hoc Student–Newman–Keuls test was used for multiplecomparisons. Differences were considered significant at Pb0.05.

3. Results

As depicted in Fig. 1, two-way ANOVA (factor 1: stress, factor 2:treatment) revealed a main effect of stress (F1,126=79.223;Pb0.001), treatment (F6,126=23.575; Pb0.001), and a stress-treatment interaction (F6,126=3.882; Pb0.05). The systemicadministration of amibegron produced a significant dose-depen-dent decrease of immobility time in the FST paradigm in the NSTrats (Fig. 1A). This effectwas statistically significant at the dose of5 (Pb0.05) or 10 mg/kg (Pb0.01), but not at the dose of 1 mg/kg(PN0.05). However, the effect of the highest dose of amibegron toreduce the immobility time was similar to that of bothclomipramine (50 mg/kg) and citalopram (15 mg/kg) (Pb0.01).Application of acute restraint stress, performed theday before the5 min test, modulated the behavioral performance, as describedby the significant increase of immobility time of vehicle-injectedstressed (VHC+STR) rats as compared to VHC injected non-stressed (VHC+NST) group in FST (Pb0.05). In contrast, amibegron(5 or 10 mg/kg), clomipramine (50 mg/kg) and citalopram (15 mg/kg) were able to counteract the behavioral effect of stressprocedure, inducing a decrease of immobility time, as comparedto VHC+STR group (Pb0.05, Pb0.01) (Fig. 1B).

The influence of treatment alone or combined to acuterestraint stress on the hippocampal BDNF, CREB, Bax and Bcl-2protein expression using western blot analyses is described inFig. 2. Two-way ANOVA (factor 1: stress, factor 2: treatment)revealed a main effect of stress (F1,126=36.646; Pb0.001,F1,126=31.191; Pb0.001), treatment (F6,126=15.755; Pb0.001,F6,126=53.888; Pb0.001) and a stress-treatment interaction(F6,126=7.332; Pb0.001, F6,126=27.267 Pb0.001) for BDNF andCREB expression, respectively. For Bcl-2/Bax ratio, a maineffect of stress (F1,126=12.212; Pb0.01), but neither a maineffect of treatment (F6,126=3.321; n.s.) nor a stress-treatmentinteraction (F6,126=2.542; n.s.) was found. Post-hoc analysisrevealed that in NST group, amibegron (10 mg/kg) increasedthe hippocampal BDNF and CREB expression as compared toVHC-treated animals (Pb0.05) (Fig. 2A). No difference wasfound in the Bcl-2/Bax ratio among the groups. Application ofacute restraint stress induced in VHC+STR rats lower hippo-campal expression of BDNF and CREB, as well as a decrease ofBcl-2/Bax ratio as compared to NST group (Pb0.05, Pb0.01)(Fig. 2B). Again, amibegron (10 mg/kg), clomipramine (50 mg/kg) and citalopram (15 mg/kg), increasing the hippocampalBDNF and CREB expression but not the Bcl-2/Bax ratio, reversedthe effects of acute restrain stress procedure (Pb0.01).However, lower doses of amibegron (1 and 5 mg/kg) alsocounteracted the effects of stress increasing both CREBexpression and Bcl-2/Bax ratio (Pb0.05, Pb0.01).

The effects of repeated (7 days) administration of amibegronon the behavioral performance of rats in the FST under basalconditions or after application of repeated restraint stressprocedure are depicted in Fig. 3. Two-way ANOVA (factor 1:stress, factor 2: drug treatment) revealed a main effect ofstress (F1,126 =7.192; Pb0.05), treatment (F6,126 = 64,066;Pb0.001) and a stress-treatment interaction (F6,126=4.527;

ptor agonist, amibegron (SR58611A) counteracts stress-induced(2010), doi:10.1016/j.euroneuro.2010.04.006

Figure. 2 Effects of amibegron, a β3 adrenoceptor agonist, on hippocampal expression of BDNF, CREB and Bcl-2/Bax ratio in ratsunder basal conditions (A) and after the application of acute restraint stress (B). Photographs show representative western blots ofBDNF (upper panel), CREB (middle panel), Bcl-2 and Bax (calculated as the ratio of Bcl-2 and Bax densitometry; lower panel). Thegraphics of BDNF and CREB expression show the mean values±S.E.M. expressed as the percentage of change from the non-stressed(NST) control animals (n=10) injected i.p. with vehicles or stressed (STR) animals (n=10) injected i.p. with vehicles (VHC), amibegron(SR; 1, 5 and 10 mg/kg), clomipramine (CMP; 50 mg/kg) and citalopram (CIT; 15 mg/kg). *Pb0.05, **Pb0.01, vs vehicle-injected non-stressed (VHC+NST) controls (Student–Newman–Keuls post-hoc test). #Pb0.05, ##Pb0.01, vs vehicle-injected stressed (VHC+STR)controls (Student–Newman–Keuls post-hoc test).

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Pb0.01). The application of repeated restraint stress procedureincreased the immobility time in VHC+STR rats as compared toVHC+NST (Pb0.05) (Fig. 3B). Repeated treatment with amibe-gron (5 and 10 mg/kg/day), clomipramine (50 mg/kg/day) orcitalopram (15 mg/kg/day) reduced the immobility time in NSTand STR groups (Pb0.01) (Fig. 3A–B). In Fig. 4 are shown theeffects of repeated treatment alone or combined to theapplication of repeated stress procedure on the hippocampalBDNF, CREB, Bcl-2/Bax ratio protein expression. Two-wayANOVA (factor 1: stress, factor 2: treatment) revealed a main

Please cite this article as: Tamburella, A., et al., The β3 adrenocebehavioral and neurochemical changes, Eur. Neuropsychopharmacol. (

effect of stress (F1,126=42.941, Pb0.001; F1,126=6.876, Pb0.05;F1,126=654.494, Pb0.001), treatment (F6,126=41.548, Pb0.001;F6,126=2.923, Pb0.05; F6,126=9.305, Pb0.001) and a stress-treatment interaction (F6,126=10.166, Pb0.001; F6,126=21.616,Pb0.001; F6,126=10.213, Pb0.001) for BDNF, CREB and the ratioBcl-2/Bax expressions, respectively. In NST rats, post-hocanalysis revealed that the highest dose of amibegron (10 mg/kg/day) was able to increase BDNF protein expression and Bcl-2/Bax ratio (Pb0.05, Pb0.01). No difference among all treatedgroup was found in the hippocampal CREB expression (Fig. 4A).

ptor agonist, amibegron (SR58611A) counteracts stress-induced2010), doi:10.1016/j.euroneuro.2010.04.006

Figure. 3 Effects of amibegron, a β3 adrenoceptor agonist, onimmobility time of rats in the forced swim test (FST) under basalcondition (A) or after the application of repeated (7 days)restraint stress (B). Amibegron (SR; 1, 5 and 10 mg/kg/day),clomipramine (CMP; 50 mg/kg/day), citalopram (CIT; 15 mg/kg/day) or their vehicles (VHC) were administered i.p. once a dayfor 7 days. The repeated restraint stress procedure consisted inplacing the animals into individual plastic rodent restrainers(4 h/day) for 7 days. Values are mean±S.E.M. of time measuresexpressed in sec. of (n=10) animals. *Pb0.05 and **Pb0.01, vsvehicle-injected non-stressed (VHC+NST) controls (Student–Newman–Keuls post-hoc test). #Pb0.05 and ##Pb0.01, vsvehicle-injected stressed (VHC+STR) controls (Student–New-man–Keuls post-hoc test).

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As described in Fig. 4B, repeated restraint stress-induced lowerhippocampal BDNF expression and Bcl-2/Bax ratio, as well ashigher CREB levels as compared to VHC+NST group (Pb0.01),which were reversed by amibegron (1, 5 or 10 mg/kg/day),clomipramine (50 mg/kg/day) and citalopram (15 mg/kg/day)treatment (Pb0.05; Pb0.01).

The acute and repeated treatment as well as the differentstress paradigms did not induce any change in body weight ofanimals (data not shown).

4. Discussion

The first finding of present study confirms that the β3-ARagonist amibegron improves, under basal condition, thebehavioral performance of rats in the FST paradigm.Furthermore, for the first time, our results demonstratethat changes in the hippocampal neurotrophic (i.e. CREBand BDNF) and/or anti-apoptotic (Bcl-2/Bax ratio) proteinsexpression could underlie its antidepressant-like effects.Amibegron is a β3-AR agonist exhibiting a profile of

Please cite this article as: Tamburella, A., et al., The β3 adrenocebehavioral and neurochemical changes, Eur. Neuropsychopharmacol.

antidepressant/anxiolytic-like activity in a wide range ofexperimental animal models, maybe through an involve-ment of the 5-HTergic and NAergic systems (Simiand et al.,1992; Lenard et al., 2003; Consoli et al., 2007; Claustreet al., 2008; Overstreet et al., 2008; Stemmelin et al., 2008,2010).

It is well accepted that the antidepressants could mediatetheir delayed long-term beneficial effects via neurotrophicand/or anti-apoptotic action on neuronal function. Interest-ingly, in our study only amibegron (10 mg/kg), but neitherclomipramine nor citalopram used here as positive controls,changed hippocampal BDNF and CREB expressions even afteracute administration, meaning that the behavioral effects ofa lower dose of amibegron could be independent of changesin the expression of factors cited above. These findings are ofprimary importance, since it is well known that chronic, butnot acute administration of antidepressants is required toupregulate the neurotrophic factors, considered to be keymarkers of antidepressant action (Schmidt and Duman,2007). It is commonly believed that the FST procedureshows specific sensitivity to the acute antidepressanttreatment; in our experiments it was also sensitive to arepeated treatment, according to previous studies (Rénéricet al., 2002; Dulawa et al., 2004; Cryan et al., 2005; Micaleet al., 2008a). However, also after repeated treatment inNST group only the behavioral effect of amibegron (10 mg/kg) was paralleled by changes in the CREB expression and inthe Bcl-2/Bax ratio.

Since the real role of β3-AR in stress-related conditions, aswell as the neurochemical mechanisms underlying amibe-gron activity is still unclear, this raises some questions onboth the effects of this drug after stressor application andthe mechanisms involved. The interactions existing betweenstress and the behavioral and molecular changes found indepressive-like disorders, have become increasingly clear inpreclinical and clinical studies, although details remain to beelucidated. As human stressful life events are associatedwith an increased risk of depression, behavioral models ofdepression utilize acute or repeated stress paradigms toinduce depressive-like symptoms in laboratory animals(McEwen, 2005; Schmidt and Duman, 2007). The exposureto acute or chronic stressors affects the expression ofhippocampal neurotrophic/growth factors, which is counter-acted by the antidepressants, supporting the neurotrophichypothesis of depression and antidepressant action (Warner-Schmidt and Duman, 2006). Thus, in line with previousstudies showing that pre-training stressors could affect thebehavioral response of animals and induce neuronal plastic-ity changes (Drago et al., 2001; Briones-Aranda et al., 2002;Teixeira and De Lima, 2003, Chaki et al., 2004; Consoli et al.,2005; Micale et al., 2008b; Pittenger and Duman, 2008), inthis experiment rats exposed to acute or repeated restraintstress exhibited an increase of immobility time in FST, anindex of impaired behavioral performance. Acute or repeat-ed treatment with amibegron elicited a dose-dependentantidepressant-like activity in the FST, reversing the effectof stress paradigm on immobility time. Interestingly, theeffect of highest dose of amibegron in the FST wascomparable in terms of magnitude to that observed withthe reference antidepressants clomipramine (50 mg/kg) orcitalopram (15 mg/kg). Acute or repeated restraint stressprocedure induced changes in CREB and BDNF expressions,

ptor agonist, amibegron (SR58611A) counteracts stress-induced(2010), doi:10.1016/j.euroneuro.2010.04.006

Figure. 4 Effects of amibegron, a β3 adrenoceptor agonist on hippocampal expression of BDNF, CREB and Bcl-2/Bax ratio in ratsunder basal condition (A) or after the application of repeated (7 days) restraint stress (B). Photographs show representative westernblots of BDNF (upper panel), CREB (middle panel), Bcl-2 and Bax (calculated as the ratio of Bcl-2 and Bax densitometry; lower panel).The graphics of BDNF and CREB expression show the mean values±S.E.M. expressed as the percentage of change from the non-stressed(NST) control animals (n=10) injected i.p. with vehicles (VHC) or stressed (STR) animals (n=10) injected i.p. with vehicles (VHC),amibegron (SR; 1, 5 and 10 mg/kg), clomipramine (CMP; 50 mg/kg) and citalopram (CIT; 15 mg/kg). *Pb0.05 and **Pb0.01, vs vehicle-injected non-stressed (VHC+NST) controls (Student–Newman–Keuls post-hoc test). #Pb0.05 and ##Pb0.01, vs vehicle-injectedstressed (VHC+STR) controls (Student–Newman–Keuls post-hoc test).

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which are commonly considered an index of neurogenesis.The same procedure led to a change in Bcl-2/Bax ratio asindex of anti-apoptotic processes too, suggesting an impair-ment of hippocampal plasticity mechanisms (Duman et al.,1997; Czéh and Lucassen, 2007). However, in this study wedid not evaluate the expression of neurotrophic or anti-apoptotic factors in a sham group (non exposed to FST), sinceunpublished data from our lab partially confirmed by Arunrutet al. (2009), suggest that the FST procedure per se does not

Please cite this article as: Tamburella, A., et al., The β3 adrenocebehavioral and neurochemical changes, Eur. Neuropsychopharmacol. (

affect their expression. Thus, a stronger stressor procedurecould be necessary to affect it.

Interestingly, we found that changes of hippocampalCREB protein expression induced by acute or repeatedrestraint stress (i.e. decrease or increase, respectively)were reversed by acute or chronic treatment (i.e. increase ordecrease, respectively), suggesting that stress-inducedchanges in CREB levels are generally reversed by antide-pressants. More specifically, it was surprising that acute

ptor agonist, amibegron (SR58611A) counteracts stress-induced2010), doi:10.1016/j.euroneuro.2010.04.006

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treatment with amibegron (1, 5 or 10 mg/kg), citalopram(15 mg/kg) or clomipramine (50 mg/kg) increased hippo-campal CREB expression; whereas after repeated treatmentonly the highest dose of amibegron decreased hippocampalCREB expression as well as citalopram or clomipramine.Since a biphasic effect has been proposed, up-regulation inthe initial phase of the treatment followed by down-regulation after chronic treatment, it could explain in partour results (Manier et al., 2002). However, additionalmechanisms as well as several pathways may contribute tothe effects of stress procedure on CREB expression, thusfurther studies could be necessary to identify them.

The effects of antidepressant treatment on BDNF expres-sion have been extensively studied, although they are notfully understood. According to some of previous data, wefound that both stress procedures used in our experimentslowered the hippocampal BDNF protein expression. Thiseffect, which was reversed by the different compounds,supports the hypothesis according to which increased BDNFsignaling is required for the normal behavioral response toantidepressants (Nibuya et al., 1995; Smith et al., 1995;Manji and Chen, 2002; Manji et al., 2003; Luo et al., 2004;Warner-Schmidt and Duman, 2006). Furthermore, we did notfind any difference between the TCA clomipramine and theSSRI citalopram in inducing changes in the BDNF expression,suggesting that they could be a common target of antide-pressant treatment. However, the lack of effects ofclomipramine and citalopram treatment in the BDNFexpression of NST rats suggests that the exposure to a stressparadigm could be a key element in the activation of thisprotein. Several data, but not all confirm the role of BDNFlevels in stress-related disorders like depression. In post-mortem samples of human brains of depressed patients,BDNF levels have been found to be reduced (Chen et al.,2001). Furthermore, whilst BDNF infusion in the hippocam-pus exerts antidepressant properties in the FST and learnedhelplessness paradigms (Shirayama et al., 2002), micelacking BDNF fail to have antidepressant responses (Mon-teggia et al., 2004). In contrast, reduction in BDNF levels orBDNF signaling did not produce depression-like symptoms, aswell as injection of BDNF into mesolimbic dopamine pathwayinduced depressive-like phenotype (Eisch et al., 2003;Duman and Monteggia, 2006). Taken together, these resultssuggest that although BDNF seems to be implicated in thetreatment of stress by antidepressants, its involvement maydepend on several factors as stress procedure applied,antidepressant drug used and brain region involved. Addi-tional studies, however, are required to further support therole of BDNF in stress-related disorders.

Apoptosis also has been demonstrated to play a role in thedecreased hippocampal volume occurring in depressedpatients (for review see Czéh and Lucassen, 2007). Thisprocess is programmed and controlled by the cellularbalance between pro- (Bax) and anti-(Bcl-2) apoptoticproteins. Thus, an increased ratio of pro- versus anti-apoptotic proteins is associated to apoptotic activation andcell death (Cory and Adams, 2002; Lindsten et al., 2005).Stress-related apoptotic phenomena (i.e. reduced Bcl-2levels) in cortex and hippocampus are counteracted byantidepressant treatment and hippocampal apoptosis withincreased Bax protein levels has been found in postmortemstudies of depressed patients (Lucassen et al., 2001, 2004;

Please cite this article as: Tamburella, A., et al., The β3 adrenocebehavioral and neurochemical changes, Eur. Neuropsychopharmacol.

Murray and Hutson, 2007). In our study, acute or repeatedrestraint stress induced a reduction of hippocampal Bcl-2/Bax ratio which was reversed by the different compounds,suggesting that antidepressants have cell survival propertiesin agreement to some of previous data (Xu et al., 2003; Luoet al., 2004; Murray and Hutson, 2007; Zhao et al., 2007;Kosten et al., 2008). It was surprising that lower doses ofamibegron (1 and 5 mg/kg) induced stronger effects toreverse the Bcl-2/Bax ratio in stressed animals and that thisparameter was not correlated to the behavioral performanceof these treated groups in the FST. Thus, further studiescould be necessary to elucidate the signal transductionpathways of apoptotic markers following amibegron admin-istration. However, the relationship between β3-ARs activa-tion and apoptotic processes remain to be fully elucidatedand it has been focused only recently. Lirussi et al. (2008)found that the activation of β3-ARs prevented the lipopoly-saccharide-induced apoptosis and cytokine production invitro model of chorioamnionitis. Interestingly, it has beenseen that SR58611A improved cutaneous wound healingprocess in animal models of type II diabetes, increasing therelease of vascular endothelial growth factor (VEGF) (Tonelloet al., 1999; Schaeffer et al., 2006). Since VEGF should be anessential mediator of the neurotrophic and behavioralactions of antidepressants (Warner-Schmidt and Duman,2007, 2008; Tamburella et al., 2009), our results could bealso due to an increased release of VEGF induced byamibegron.

In conclusion, here we confirm the antidepressant-likeeffects of amibegron, suggesting for the first time that themodulation of the expression of different hippocampalproteins (i.e. BDNF, CREB, Bax and Bcl-2) could underlie itseffect. According to a current view, these results furthersupport the neurotrophic hypothesis of depression and thetherapeutic effects of antidepressants could be hence linkedto changes in the hippocampal expression of neurotrophicand/or anti-apoptotic factors involved in neuronal function(Warner-Schmidt and Duman, 2006). However, other brainareas might be involved since it has been seen that β3-ARsare also localized in cerebral cortex, hypothalamus andamygdala of both rodents and humans, although in smallquantities (Rodriguez et al., 1995; Summers et al., 1995;Strosberg, 1997; Claustre et al., 2008). We are aware thatfurther studies are needed to assess stress-induced changesof β3-ARs at the cellular level by using other moleculartechniques (such as in situ hybridization or immunohisto-chemistry for the proteins examined) and neuroendocrineresponses (such as plasma corticosterone levels) afteramibegron treatment. However, it is important to underliethat the main scope of the current study was to appraise ifthe impaired hippocampal neuroplasticity induced by re-straint stress procedure could be reversed by amibegrontreatment and how these changes could be related to itsbehavioral effects.

Role of the funding source

The authors declare that funding source of the present research is ofthe public type. All financial support to this research came from theDepartment of Experimental and Clinical Pharmacology, Universityof Catania, Italy as part of the institutional research activity.

ptor agonist, amibegron (SR58611A) counteracts stress-induced(2010), doi:10.1016/j.euroneuro.2010.04.006

9β3 adrenoceptor agonist and antidepressant-like effect

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Contributors

The authors declare that contributors of the present research areonly those who appear as authors of the paper. For financialcontributors see declaration concerning role of the funding source.

Conflict of interest

The authors declare that no conflict of interest should be disclosedconcerning the present research by any of the authors.

Acknowledgements

We are grateful to Sanofi-Aventis (Italy) for the generous gift ofamibegron (SR58611A). These experiments were supported by theInternational PhD Program in Neuropharmacology, University ofCatania, Medical School, Catania.

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