agomelatine (s20098) modulates the expression of cytoskeletal microtubular proteins, synaptic...

ORIGINAL INVESTIGATION

Agomelatine (S20098) modulates the expression of cytoskeletalmicrotubular proteins, synaptic markers and BDNF in the rathippocampus, amygdala and PFC

Nataly Ladurelle & Cecilia Gabriel & Adela Viggiano &

Elisabeth Mocaër & Etienne E. Baulieu &

Massimiliano Bianchi

Received: 17 June 2011 /Accepted: 18 November 2011 /Published online: 8 December 2011# Springer-Verlag 2011

AbstractRationale Agomelatine is described as a novel and clinicaleffective antidepressant drug with melatonergic (MT1/MT2)agonist and 5-HT2C receptor antagonist properties. Previousstudies suggest that modulation of neuronal plasticity andmicrotubule dynamics may be involved in the treatment ofdepression.Objective The present study investigated the effects of ago-melatine on microtubular, synaptic and brain-derived neuro-trophic factor (BDNF) proteins in selected rat brain regions.Methods Adult male rats received agomelatine (40 mg/kg i.p.)once a day for 22 days. The pro-cognitive effect of agomelatinewas tested in the novel object recognition task and antidepres-sant activity in the forced swimming test. Microtubule dynam-ics markers, microtubule-associated protein type 2 (MAP-2),phosphorylated MAP-2, synaptic markers [synaptophysin,postsynaptic density-95 (PSD-95) and spinophilin] and BDNFwere measured by Western blot in the hippocampus, amygdalaand prefrontal cortex (PFC).Results Agomelatine exerted pro-cognitive and antidepres-sant activity and induced molecular changes in the brain areasexamined. Agomelatine enhanced microtubule dynamics in

the hippocampus and to a higher magnitude in the amygdala.By contrast, in the PFC, a decrease in microtubule dynamicswas observed. Spinophilin (dendritic spines marker) was de-creased, and BDNF increased in the hippocampus. Synapto-physin (presynaptic) and spinophilin were increased in thePFC and amygdala, while PSD-95 (postsynaptic marker)was increased in the amygdala, consistent with the phenom-ena of synaptic remodelling.Conclusions Agomelatine modulates cytoskeletal microtu-bule dynamics and synaptic markers. This may play a role inits pharmacological behavioural effects and may result fromthe melatonergic agonist and 5-HT2C antagonist propertiesof the compound.

Keywords Agomelatine . Depression . Neuronal plasticity .

Cytoskeleton .Microtubules . Synaptic markers . BDNF.

Hippocampus . Amygdala . PFC

Introduction

Growing evidence suggests that the clinical efficacy of antide-pressant drugs may include long-term phenomena of structuralneuronal plasticity including neurogenesis and remodelling ofaxons and dendrites in specific brain areas (Warner-Schmidtand Duman 2006; Bessa et al. 2009). However, such effectshave been mainly observed with conventional antidepres-sant drugs having as main mechanism of action the inhibi-tion of monoamine transporters, and data on novelantidepressant molecules are currently lacking. Agomelatine(S20098) is described as a novel antidepressant (de Bodinatet al. 2010) with a unique profile of MT1/MT2 melatonergicagonist (Audinot et al. 2003) and 5-HT2C receptor antago-nist properties (Millan et al. 2003). Agomelatine displaysantidepressant- and anxiolytic-like activities in several

N. Ladurelle : E. E. BaulieuInstitut National de la Santé et de laRecherche Médicale-UMR788,Le Kremlin-Bicêtre, France

C. Gabriel : E. MocaërInstitut de Recherches International Servier,Courbevoie, France

A. Viggiano : E. E. Baulieu :M. Bianchi (*)Psychopharmacology Team, MAPREG,Bldg. Gregory PINCUS 80,rue du General Leclerc,94276 Le Kremlin-Bicetre Cedex, Francee-mail: [email protected]

Psychopharmacology (2012) 221:493–509DOI 10.1007/s00213-011-2597-5

preclinical animal paradigms (Bertaina-Anglade et al. 2006;Papp et al. 2003; Bourin et al. 2004; Barden et al. 2005;Millan et al. 2005; Tuma et al. 2005; Papp et al. 2006).Clinically, agomelatine has demonstrated efficacy in majordepressive disorders in several trials (Lôo et al. 2002,Goodwin et al. 2009; Olié and Kasper 2007; Kennedy andRizvi 2010).

Synaptic remodelling is correlated with the composition,function and dynamics of the cytoskeletal neuronal microtu-bules which are fundamental for the formation and mainte-nance of axons, dendrites and dendritic spines (for extensivereviews, see Conde and Cáceres 2009; Hoogenraad andBradke 2009). Microtubules are formed by the polymeriza-tion of α/β-tubulin heterodimers and display a dynamicbehaviour by alternating periods of slow assembly and fastdisassembly at the plus end of the polymer (Mitchison andKirschner 1984). Alpha-tubulin undergoes posttranslationalmodifications (i.e. α-tubulin isoforms) including the stableacetylated α-tubulin (Acet-Tub) and the products of the C-terminus enzymatic cycle of detyrosination/tyrosination (foran extensive review, see Janke and Kneussel 2010), specif-ically the dynamic tyrosinated α-tubulin (Tyr-Tub), the sta-ble detyrosinated α-tubulin (Glu-Tub) and the neuron-specific Δ2-tubulin (Δ2-Tub) which is produced by remov-al of the last glutamate residue irrespective of microtubulestabilization (Paturle-Lafanechere et al. 1994). Themicrotubule-associated protein type 2 (MAP-2) is a familyof heat-stable proteins particularly abundant in the mamma-lian brain, consisting of multiple isoforms generated byalternative splicing of a single gene (Kalcheva et al. 1995).The MAP-2 family is divided into two main groups: highmolecular weight MAP-2a and MAP-2b (270–280 kDa;located in neuronal cell bodies, dendrites and dendriticspines) and the low molecular MAP-2c and MAP-2d (70–75 kDa; located throughout the neuron) which are onlyexpressed during brain development but not in the adult-hood (for an extensive review, see Sánchez et al. 2000). Thehigh molecular weight MAP-2 proteins regulate microtu-bule assembly, stability and dynamics, the spacing betweenmicrotubules as well as dendritic and dendritic spinesremodelling (Conde and Cáceres 2009). These functionsare regulated by phosphorylation of MAP-2 at multiple siteswhich are substrates for several families of protein kinases(Sánchez et al. 2000). Emerging preclinical data indicatethat antidepressant drugs affect the composition and func-tion of cytoskeletal neuronal microtubules. We have firstreported that chronic administration of the selective seroto-nin (5-HT) reuptake inhibitor (SSRI) fluoxetine modulatesα-tubulin isoforms in rat hippocampus consistent with thephenomena of neuronal remodelling (Bianchi et al. 2009a,b). The results were later confirmed by other authors eitherex vivo (Yang et al. 2009) or in primary hippocampalneuronal cell cultures (Chen et al. 2010). Additionally,

different classes of antidepressant drugs have been shownto induce MAP-2 phosphorylation in rat brain (Perez et al.1989, 1991; Miyamoto et al. 1997; Iwata et al. 2006; Yanget al. 2009). Moreover, exposure to fluoxetine or desipra-mine decreased microtubule assembly and enhanced thepool of available tubulin dimers in neuroblastoma cells(Donati and Rasenick 2005). The possible relation betweenthe antidepressant-induced changes in microtubules andtheir therapeutical efficacy may be supported by resultsshowing that different models of stress and depressionchange the ratio of hippocampal α-tubulin isoforms (Bian-chi et al. 2003, 2006, 2009b; Yang et al. 2009), alter theexpression of MAP-2 (Bianchi et al. 2006; Iwata et al. 2006;Yang et al. 2009) and that of other microtubular proteins(Piubelli et al. 2011a, b, c). Taken together, these findingsare supporting our initial hypothesis that microtubule dy-namics play a key role in the pathogenesis and treatment ofdepression (Bianchi et al. 2005).

The main objective of the present study was to investigatethe effects of agomelatine on α-tubulin isoforms, MAP-2 andphosphorylated MAP-2 in the hippocampus, amygdala andprefrontal cortex (PFC), brain areas of key clinical importancein depressive disorders. The analysis of the microtubular pro-teins was coupled with that of synaptic markers such as syn-aptophysin, postsynaptic density-95 (PSD-95) and spinophilin.Synaptophysin is themajor integral protein of synaptic vesicles,and its reduction has been associated with a decrease in synap-tic density (Eastwood and Harrison 2001). PSD-95 is a post-synaptic scaffolding protein having immunoreactivity alonghippocampal dendrites in rats and is therefore used as amarker of dendrite branching and remodelling (Hunt et al.1996; Grillo et al. 2005). Spinophilin is highly enriched indendritic spines (Allen et al. 1997), and its expression hasbeen correlated with the density of dendritic spines becauseboth decrease in response to estrogen inhibition (Kretz et al.2004) and 5-HT depletion (Alves et al. 2002). Brain-derivedneurotrophic factor (BDNF) is a neurotrophin modulatingneuronal plasticity which has been frequently associatedwith antidepressant treatment (see Tardito et al. 2006). Ad-ditionally, such molecular analyses were preceded by behav-ioural assays to investigate activities of agomelatine on theforced swimming test (Porsolt et al. 1977), a test used toassay antidepressant drugs, and in the novel object recogni-tion (NOR) task (Ennaceur and Delacour 1988) used toevaluate recognition memory.

Materials and methods

Animal maintenance

Adult male (250–300 g) Sprague–Dawley rats (n028) wereobtained from Janvier (Le Genest St. Isle, France). Animals

494 Psychopharmacology (2012) 221:493–509

were group-housed (n04 per cage) and maintained undercontrolled conditions (21±1°C, 12/12-h light/dark cycleslights on at 7 a.m.) with food and water available ad libitum.The experiments started after 1 week of animal acclimatisa-tion to the facilities environment. The experimental proce-dures were in accordance with the European CommunitiesCouncil Directive (86/609/EEC) and approved by the inter-nal scientific committee of MAPREG and INSERMUMR788.

Experimental design

The experimental design is depicted in Fig. 1. Rats wererandomly allocated to either (n014 for each group): (1)vehicle [hydroxyethylcellulose 1%, 400 μl/rat intraperito-neally (i.p.)] or (2) agomelatine (40 mg/kg i.p.) administra-tion. On day 20 of treatment, locomotor activity (LMA) andrecognition memory (NOR task) were tested in an open-field arena. Antidepressant efficacy was assessed ondays 21–22 of treatment using the forced swimming test(FST). All behavioural tests were performed around 18 hafter last drug administration during morning to early after-noon (11 a.m.–2 p.m.). Due to technical reasons linked tothe video tracking system, LMAwas recorded in six animalsper group. Two animals in the vehicle-treated and threeanimals in the agomelatine-treated groups were excludedfrom the NOR task and FST data analysis for defaults inthe video tracking system recording and concerns abouttreatment allocation. Thus, the NOR task and FST datainclude n012 rats in the vehicle-treated and n011 in theagomelatine-treated groups. All rats were sacrificed 18 hafter the last injection and the hippocampi, amygdala andPFC dissected for molecular analyses.

Drug preparation and administration

Agomelatine (S20098; Servier, France) was suspended inhydroxyethylcellulose 1% (vehicle). The agomelatine sus-pension was prepared freshly each day of treatment and keptin suspension during the period of administration. Agome-latine (40 mg/kg) was injected i.p. once a day in a fixvolume of 400 μl/rat. Rats were treated between 5 and6 p.m. (1–2 h before the dark phase). The dose of agome-latine was chosen on the basis of its property to increase cellsurvival and neurogenesis in the rat dentate gyrus (Banasr et

al. 2006; Soumier et al. 2009). The same dose also showedactivity in different rat models of depression and anxiety(Millan et al. 2005; Morley-Fletcher et al. 2011).

Locomotor activity and novel object recognition task

The NOR task is a test of recognition memory based on thespontaneous preference of rats for novelty. The appliedprocedure was based on that of Ennaceur and Delacour(1988) as previously described (Bianchi et al. 2006) withmodifications. Briefly, on day 20 of treatment, the animalswere individually placed in the open-field arena (70×100×70 cm) for a 3-min period of habituation and then returnedto their “home” cage for 1 min. LMA was detected duringthe 3 min of habituation period using a computerized system(VideoTrack V2.5; ViewPoint, France) and data expressedas distance (centimeters) travelled in the open field.

Rats were returned to the open-field arena for two con-secutive 5-min trials, separated by a specific inter-trial in-terval (ITI). In the first (familiarisation) trial, all rats(vehicle-treated, n012 and agomelatine-treated, n011) wereexposed to two identical objects consisting of brown glassbottles, 21 cm high ×10 cm in diameter filled with equalvolumes of water preventing animals from displacing themduring exploration. The objects were placed at a distance of34 cm between them, at 23 cm from the side wall and 30 cmfrom the rear one. In the second (choice) trial, the animalswere exposed to a familiar object (one of the two identicalbottles explored in the familiarisation trial chosen at ran-dom) and one novel object (modified bottle: brown glassbottle, 21 cm high×10 cm in diameter covered with whitemasking tape with two additional horizontal black 1.5-cm-wide strips of insulating tape), which was placed in eitherposition at random. Two different ITIs were used betweenthe familiarisation and choice trial, and the animals weresplit into two groups: (1) ITI, 1 h (vehicle-treated, n06 andagomelatine-treated, n05) or (2) ITI, 4 h (vehicle-treated,n06 and agomelatine-treated, n06).

The arena and the objects were rinsed with 20% v/vethanol between familiarisation and choice trial to removeany olfactory cue. Exploration of the objects, defined assniffing, touching and having moving vibrissae whilstdirecting the nose towards the object at a distance of≤1 cm, was recorded manually by an experimenter. Theexperimenter was blind to the treatment group. Sitting on,

Fig. 1 Experimental design; see main text for details. LMA locomotor activity, NOR novel object recognition task, FST forced swimming test

Psychopharmacology (2012) 221:493–509 495

leaning against or chewing the objects was not considered asexploration. The basic behavioural measurement was thetime (seconds) spent exploring each of the objects in bothfamiliarisation and choice trial. Additionally, the followingmeasures were calculated according to the method of Ennaceurand Delacour (1988): (1) E1 index, which is the total explo-ration time of the familiarisation trial: object 1 (seconds)+object 2 (seconds); (2) E2 index, which is the total explora-tion time of the choice trial: familiar object (seconds)+novelobject (seconds) and (3) D2 index, which represents theability to discriminate the novel from familiar object: [novelobject (seconds)−familiar object (seconds)]/[novel object(seconds)+familiar object (seconds)].

Forced swimming test

On treatment days 21 (pretest session) and 22 (test session),the animals (vehicle-treated, n012 and agomelatine-treated,n011) were submitted to a modified FST as previouslydescribed (Bianchi et al. 2002) with slight modifications.Briefly, rats were individually placed into a plexiglass cyl-inder (40 cm height, 20 cm diameter) containing 25 cm ofwater at 25±1°C. After a pretest session of 15 min, the ratwas removed, dried using a towel and returned to its "home"cage. Twenty-four hours after the pretest session, the rat wasplaced again in the swim cylinder (under identical condi-tions) for the 5-min test session. The test session wasrecorded using a video camera placed above the cylinderfor subsequent behavioural analysis. A time sampling (5-speriod) method was used as described previously (Detke etal. 1995) in order to score several behaviours during a singleviewing. The following behaviours were measured (Cryanet al. 2005): (1) immobility: measured when no additionalactivity is observed other than that required to keep the rat’shead above the water; (2) swimming: defined as horizontalmovements throughout the cylinder including crossingacross quadrants of the cylinder; and (3) climbing: definedas upward directed movements of the forepaws along theside of the cylinder. Previous studies showed that all refer-ence antidepressants reduced immobility in the modifiedFST, while swimming and climbing were selectively in-creased by antidepressant drugs acting on the serotonergicor noradrenergic transmission, respectively (for an extensivereview, see Cryan et al. 2005).

Western blot detection

Eighteen hours following the last injection, the animals weresacrificed by decapitation and the brain removed. The hip-pocampi and amygdalae were dissected from the wholebrain, while PFC (considered as Cg1, Cg3 and IL subre-gions) was dissected from 2-mm-thick slices according toplates six to ten of the atlas Paxinos and Watson (1996).

The dissection was performed on an ice-chilled plate, thesamples immediately frozen in dry ice and kept at −80°Cuntil analyses. Only samples from animals included in thebehavioural data analysis have been processed for Westernblot analysis. Western blot analyses of α-tubulin isoformswas performed as previously reported (Bianchi et al. 2006)with slight modifications, whereas new protocols were ap-plied for the detection of the other proteins. Brain sampleswere homogenized using a tip sonicator in either 500 (hip-pocampus), 300 (amygdala) or 200 μl (PFC) of lysis buffer[5 mM Tris–HCl containing 2% (v/v) of protease inhibitorcocktail (Sigma)]. Only samples from animals included inthe behavioural data analyses were used in the Western blot(vehicle-treated, n012 and agomelatine-treated, n011). Thesamples of the dissected regions (hippocampi, amygdalaeand PFCs) obtained from each animal were not pooledtogether, but rather run as individual homogenates. Sincegels may hold a maximum of 15 samples, samples fromeach brain area were split into two gels each having n06(vehicle-treated group) and n05–6 (agomelatine-treatedgroup) to ensure direct comparison and accurate quantifica-tion. This loading pattern was repeated two to four times foreach sample set. The densitometric values of each proteinanalysed were expressed as percentages of control (vehicle-treated) values, and data of each replica averaged for statis-tical analysis.

Western blot detection: α-tubulin isoforms, synapticmarkers and BDNF

Protein concentrations in separated aliquots of all homoge-nates were determined using the Bradford colorimetric assay(Sigma) to ensure equal loading onto the gels. Samples werethen diluted 1:2 in Laemmli buffer (Laemmli 1970) andheated for 3 min at 99°C. Proteins were separated on 10%bisacrylamide/trisacrylamide gel electrophoresis (NuPAGE,Invitrogen). After electrophoresis, proteins were transferredonto nitrocellulose membranes (Invitrogen) using a dryblotting system (iBlot, Invitrogen). A pre-stained molecularsize marker (260–3.5 kDa range, Invitrogen) was used tolocate proteins on the basis of their position on the gel.Membranes were blocked for 1 h with 5% skimmed milkpowder in Tris-buffered saline (TBS; 0.05 M Tris, 0.150 MNaCl) and then incubated with primary antibodies diluted in0.1% skimmed milk powder in TBS. Details on primaryantibodies and on their usage in the applied protocols arereported in Table 1. Following incubation with the primaryantibodies against α-tubulin isoforms, synaptic markers andβ-actin, the membranes were incubated with secondary IgGanti-mouse or anti-rabbit antibodies conjugated with alka-line phosphatase (Chemicon), and the reaction was devel-oped using a stabilized substrate for alkaline phosphatase(Western Blue, Promega). The BDNF precursor pro-BDNF


and its mature form (mature BDNF) analyses were doneusing the ECL method with a secondary IgG anti-rabbitantibody conjugated with horseradish peroxidase (JacksonImmunoResearch) and the signals revealed using the ECLWestern Blotting kit (Pierce). The intensity of the proteinbands was quantified by densitometric analyses using theNIH image software. The α-tubulin isoforms data werenormalized to total α-tubulin (TOT-Tub) expression to en-sure detection of changes in posttranslational modificationsbut not to possible alterations of α-tubulin synthesis per se.Tyr-Tub and Glu-Tub data were expressed as Tyr/Glu-Tubratio representing a relative index of microtubule dynamics(Bianchi et al. 2006). Beta-actin expression was used tonormalize the synaptic markers and BDNF results.

Infrared Western blot detection: MAP-2 and phosphorylatedMAP-2

The monoclonal anti-MAP-2 antibody (clone HM-2) waspurchased from Sigma, and it reacts with all MAP-2 iso-forms, namely MAP-2a, MAP-2b and MAP-2c, and allowsselective labelling of dendrites throughout the brain (Huberand Matus 1984). Polyclonal antibody against phosphory-lated MAP-2 (#4541) was purchased from Cell Signaling,and it reacts with all isoforms of MAP-2 only when these arephosphorylated at Ser136 (Cell Signaling, product informa-tion page, http://www.cellsignal.com). Details on the prima-ry antibodies are given in Table 1. Proteins were separatedon 4–20% Tris–Glycine gel electrophoresis (NuPAGE, Invi-trogen). Pre-stained molecular size markers (260–3.5 kDarange, Invitrogen) and a bovine brain MAPs fraction whereMAP-2 constitutes 70% of total protein (Cytoskeleton Inc.,Denver, USA) were used to locate MAP-2 on the basis of

their position on the gel. Following blotting, the membraneswere blocked for 1 h with Odyssey blocking buffer (Li-Cor,Lincoln, USA) to prevent unspecific binding of the primaryantibodies. Based on pilot set-up studies, the following co-immunodetections were done on the same membrane:MAP-2, phosphorylated MAP-2, TOT-Tub and β-actin. Pri-mary antibodies diluted in Odyssey blocking buffer plus0.2% (v/v) Tween 20 were added to the membranes andincubated O/N at 4°C. Following incubation, membraneswere washed (4×15 min) with PBST [PBS plus 0.1% (v/v)Tween 20 (Sigma)] to remove unbound antibodies. Mem-branes were then incubated for 1 h with secondary anti-bodies coupled with IRDyes detectable by the infraredsystem, i.e. goat anti-mouse IRDye 680 and goat anti-rabbit IRDye 800, diluted at 1:10,000 in Odyssey blockingbuffer plus 0.2% (v/v) Tween 20, plus 0.1% SDS. Mem-branes were washed 4×15 min with PBST and scannedusing the Odyssey infrared imaging system (Li-Cor). Intensi-ties of the protein bands were quantified using the OdysseyV3.0 software. The densitometric values were normalized toβ-actin expression.

Statistical analysis

Appropriate multifactorial analysis of variance (ANOVA) wasused to detect statistical significance of dependent variablesfor the results obtained in the NOR task. The Fisher leastsignificant difference (LSD) test was employed for post hocanalyses. The Student’s t test was employed to analyse thebody weight, LMA, FST and Western blot data. Probabilityvalues of P<0.05 were considered as statistically signifi-cant. Statistical analyses were performed using InVivoStatV1.2 (Clark et al. 2011).

Table 1 Details on primaryantibodies and on their usage inthe applied Western blotprotocols

HC hippocampus, AMY amyg-dala, PFC prefrontal cortex, Tyr-Tub tyrosinated α-tubulin, Glu-Tub detyrosinated α-tubulin,Δ2-Tub Δ2 α-tubulin, Acet-Tubacetylated α-tubulin, MAP-2microtubule-associated proteintype 2, P-MAP-2 phosporylatedMAP-2, SYN synaptophysin,PSD-95 postsynaptic density-95,SPIN spinophilin, BDNF brain-derived neurotrophic factor, RTroom temperature, O/NovernightaWhen co-incubated with MAP-2and phosphorylated MAP-2

Antibody Clone Supplier Total proteins loaded (μg) Dilution Incubation

HC AMY PFC

Tyr-Tub 6-11B-1 Sigma 1 0.6 0.5 1:1,000 1 h–RT

Glu-Tub Polyclonal Chemicon 1 0.6 0.5 1:1,000 1 h–RT

Δ2-Tub Polyclonal Chemicon 1 0.6 0.5 1:1,000 1 h–RT

Acet-Tub TUB-1A2 Sigma 1 0.6 0.5 1:1,000 1 h–RT

TOT-Tub DM-1A Sigma 1 0.6 0.5 1:500 O/N–4°C

1:250,000a

SYN 4E-206 Abcam 1 1 1 1:3,000 2 h–RT

PSD-95 7E3-IB8 Sigma 9 6 1 1:10,000 O/N–4°C

SPIN Polyclonal Chemicon 9 6 1 1:5,000 O/N–4°C

BDNF Polyclonal Santa Cruz 25 25 25 1:200 O/N–4°C

β-actin AC-15 Sigma 0.8 0.5 0.6 1:20,000 O/N–4°C

1:1,500,000a

MAP-2 HM2 Sigma 10 14 16 1:2,000 O/N–4°C

P-MAP-2 Polyclonal Cell Signalling 10 14 16 1:2,000 O/N–4°C


http://www.cellsignal.com

Results

Effects of agomelatine on locomotor activity

LMA was not different between vehicle-treated (4,877±320 cm; n06) and agomelatine-treated rats (4,153±326 cm; n06).

Effects of agomelatine on the novel object recognition task

Familiarisation trial

NOR data on object exploration during the familiarisationtrial were analysed by two-way ANOVA with treatments(vehicle or agomelatine) and objects (object 1 or object 2)as dependent variables, while the E1 index was analysedusing the Student’s t test.

The two-way ANOVA did not show significanteffects of the treatments [F(1, 42)03.81, P00.06], the objects[F(1, 42)00.10, P00.75] and the treatments×objects interac-tion [F(1, 42)00.03, P00.87]. Despite the tendency towards asignificant effect of the treatment variable, the clear lack ofsignificance of the treatments×objects interaction showedthat both vehicle- and agomelatine-treated animals had nopreferential exploration of either identical object (object 1and object 2), irrespective of their location, during thefamiliarisation trial (Table 2). Furthermore, agomelatine ad-ministration did not affect the E1 index (object 1+object 2;

P00.16) showing that the total time of exploration of thetwo objects was not influenced by the treatment (Table 2).

Choice trial

The data gathered on object exploration during the choicetrial were analysed by a three-way ANOVAwith treatments(vehicle or agomelatine), ITI group (ITI 1 h or ITI 4 h) andobjects (familiar object and novel object) as dependentvariables. The E2 and D2 indices were analysed using atwo-way ANOVA with treatments (vehicle or agomelatine)and ITI group (ITI 1 h or ITI 4 h) as dependent variables.

The three-way ANOVA performed on the exploration timeof familiar and novel objects yielded a significant effect of theobjects [F(1, 38)044.91, P<0.001], and a tendency towards asignificant effect was detected for the ITI group×objectinteraction [F(1, 38)03.35, P00.07]. In contrast, the ANOVAdid not show main effects of the treatments [F(1, 38)00.05,P00.82], the ITI group×treatments interaction [F(1, 38)00.43, P00.51], the objects×treatments interaction[F(1, 38)01.97, P00.17] and the ITI group×objects×treat-ments interaction [F(1, 38)00.01, P00.91]. The post hocanalysis (Fisher LSD test) revealed that following an ITIof 1 h, both vehicle- and agomelatine-treated rats spentsignificantly (P<0.001) more time exploring the novel thanthe familiar object (Table 2). Following an ITI of 4 h, thetime spent by the vehicle-treated animals in exploring thenovel object did not reach significance (P00.07). In con-trast, agomelatine-treated rats spent significantly (P<0.01)more time in exploring the novel than the familiar object(Table 2). The effects of agomelatine in the NOR task wereconfirmed by expressing the data as a D2 index (to assessthe ability to discriminate between familiar and novel objectas a proportion of total objects exploration). Thus, the two-way ANOVA of the D2 index showed the main effects ofthe treatments [F(1,19)06.84, P<0.05], but not of the ITIgroup [F(1,19)02.36, P00.14] and of the ITI group×treat-ments interaction [F(1,19)00.85, P00.36]. Accordingly, thepost hoc analyses (Fisher LSD test) revealed significantlyhigher (P<0.05) D2 index in agomelatine-treated rats (0.50±0.08) compared to vehicle treatment (0.25±0.03) follow-ing 4 h of ITI (Fig. 2a). Importantly, the two-way ANOVAof the E2 index (familiar+novel object) yielded no signifi-cant main effects of the treatments [F(1,19)00.03, P00.86],the ITI group [F(1,19)01.31, P00.27] and of the ITI group×treatments interaction [F(1,19)00.29, P00.60]. Thus, agome-latine treatment did not affect the total exploration time ofthe objects (Table 2).

Effects of agomelatine on the forced swimming test

Statistical differences between the vehicle- and theagomelatine-treated rats (n012 and n011, respectively)

Table 2 Exploratory activity (seconds) in the NOR task followingchronic (once a day for 20 days) agomelatine (40 mg/kg i.p.)administration

Vehicle Agomelatine

Familiarisation trial

Object 1 13.1±1.8 10.3±0.6

Object 2 12.8±2.0 9.5±1.2

E1 index (object 1+object 2) 25.3±3.6 19.8±1.8

Number of animals 12 11

Choice trial; ITI: 1 h

Familiar 6.6 ±1.3 5.7±1.5

Novel 16.3±1.8** 19.6±3.5**

E2 index (familiar+novel object) 22.9±2.1 23.4±4.3


Choice trial; ITI: 4 h

Familiar 7.9±1.1 5.5±1.5

Novel 12.9±1.8 14.1±2.1*

E2 index (familiar+novel object) 20.8±2.9 19.7±3.6


Data are expressed as mean±SEM

*P<0.01; **P<0.001 (Fisher LSD test) novel vs. familiar object


were assessed for each of the FST behaviours using theStudent’s t test. Agomelatine significantly decreased(P<0.01) the immobility counts (vehicle, 40.3±2.0 vs. ago-melatine, 32.5±1.6; Fig. 2b). Additionally, the swimmingcounts showed a tendency (P00.051) to be increased byagomelatine (21.6±1.5) compared to vehicle-treated ani-mals (15.9±1.9; Fig. 2b). Climbing counts were unchangedby agomelatine administration (Fig. 2b).

Effects of agomelatine on the expression of cytoskeletalmicrotubule dynamics markers, synaptic markersand BDNF in the hippocampus, amygdala and PFC

Hippocampus

The expression of TOT-Tub and β-actin was not changed byagomelatine in the hippocampus (Table 3). Chronic agome-latine administration affected the expression of hippocampalmicrotubule dynamics markers by significantly increasing

(P<0.05) Tyr/Glu-Tub ratio (1.36±0.08) and the neuron-specific Δ2-Tub (1.19±0.03), indicating increased neuronalmicrotubule dynamics. The stable form Acet-Tub was un-changed (Fig. 3a). The expression of MAP-2 and phosphor-ylated MAP-2 proteins was also significantly increased(P<0.001) by agomelatine to 1.98±0.05 and 2.11±0.05,respectively (Fig. 3b). The changes in microtubular proteinswere accompanied by a significant decrease of the postsyn-aptic marker spinophilin to 0.74±0.07, suggesting phenom-ena of dendritic spines remodelling (Fig. 3c). Theexpression of the presynaptic marker synaptophysin wasalso decreased (0.81±0.03), but this failed to reach statisti-cal significance (Fig. 3c). PSD-95 (postsynaptic marker)expression was not affected (Fig. 3c). Finally, agomelatinesignificantly increased (P<0.05) hippocampal matureBDNF to 1.24±0.05, but not pro-BDNF (Fig. 3d).

Amygdala

TOT-Tub expression was significantly decreased (P<0.05)by agomelatine showing an effect on α-tubulin synthesis inthe amygdala, while β-actin expression was not altered(Table 3). Furthermore, agomelatine significantly increased(P<0.001) Tyr/Glu-Tub ratio (1.29±0.02) and showed atendency to increase Δ2-Tub to 1.16±0.06 (Fig. 4a), butthis lacks statistical significance. Acet-Tub was significantlydecreased (P<0.05) by agomelatine to 0.79±0.03 (Fig. 4a).These changes are clearly suggesting increased microtubulardynamics in the amygdala. MAP-2 (3.88±0.49) and phos-phorylated MAP-2 (4.88±0.59) were significantly increased(P<0.01) by agomelatine in the amygdala to a much higherrate compared to the hippocampus (Fig. 4b). Moreover,agomelatine significantly increased (P<0.05) the expressionof synaptophysin (1.52±0.16), PSD-95 (3.94±0.20) andspinophilin (1.69±0.20; Fig. 4c). In contrast, agomelatineadministration induced a decrease in pro-BDNF (0.85±0.04) and mature BDNF (0.79±0.13), but both did not reachstatistical significance due to the high individual variabilitydetected between the samples.

PFC

Agomelatine administration did not change the expressionof TOT-Tub and β-actin in the PFC (Table 3). In contrast tothe hippocampus and amygdala, the changes induced byagomelatine in the PFC were consistent with decreasedneuronal microtubule dynamics. Particularly, agomelatinesignificantly decreased Tyr/Glu-Tub ratio (0.77±0.09) andΔ2-Tub (0.76±0.03), without affecting Acet-Tub (Fig. 5a).The expression of MAP-2 and phosphorylated MAP-2 wasalso not changed by agomelatine in the PFC (Fig. 5b).Additionally, agomelatine significantly increased both syn-aptophysin (1.32±0.09, P<0.05) and spinophilin (1.70±

Vehicle Agomelatine

Fig. 2 Effects of chronic (once a day) agomelatine (40 mg/kg i.p.) onthe D2 index (ability to discriminate between familiar and novelobject) in the choice trial of the novel object recognition task atday 20 of treatment (a) and on immobility, swimming and climbingcounts in the forced swimming test at day 22 of treatment (b). Data areexpressed as mean±SEM. *P<0.05; **P<0.01 (Fisher LSD test) vs.vehicle-treated rats


0.16, P<0.01) in the PFC, but did not changePSD-95 (Fig. 5c). Moreover, the expression of pro-BDNFand mature BDNF was not changed by agomelatine(Fig. 5d).

Discussion

The main findings of the present study are summarized inTable 4: (1) chronic administration of agomelatine (40 mg/

Table 3 Effects of chronic (once a day for 22 days) agomelatine (40 mg/kg i.p.) administration on TOT-Tub and β-actin expression in thehippocampus, amygdala and PFC

TOT-Tub β-Actin

Hippocampus Amygdala PFC Hippocampus Amygdala PFC

Vehicle 100.0±4.7 100.0±5.1 100.0±3.9 101.6±4.0 100.0±5.9 100.0±3.9

Agomelatine 98.5±2.7 81.2±3.7* 98.2±4.7 91.6±4.6 104.0±5.7 91.6±3.2

Data are expressed as percentage of control; mean±SEM

*P<0.05 (t test) vs. vehicle-treated rats

HIPPOCAMPUS

Vehicle Agomelatine

Fig. 3 Western blot analyses of hippocampus homogenates followingchronic (once a day for 22 days) agomelatine (40 mg/kg i.p.) administra-tion. Representative Western blot bands (top inset) and densitometricanalyses of the bands (bottom) of α-tubulin isoforms (a), MAP-2 and

phosphorylated MAP-2 (b), synaptic markers (c), and BDNF (d). Dataare expressed as mean±SEM. *P<0.05; ***P<0.001 (t test) vs.vehicle-treated rats


kg i.p.) exerted pro-cognitive and antidepressant effects,confirming previous results; (2) the behavioural effects wereaccompanied by changes of α-tubulin isoforms, MAP-2,synaptic markers and BDNF in the hippocampus, amygdalaand PFC showing modulation of neuronal plasticity; and (3)the pattern of changes was region-specific.

Effects of agomelatine in the FST and NOR task

Chronic agomelatine administration did not significantly affectLMA in the open field, confirming a previous mouse study(Rainer et al. 2011) and excluding an important confoundingfactor for both the NOR task and the FST. Agomelatineshowed antidepressant efficacy in a number of clinical trials(Lôo et al. 2002; Olié and Kasper 2007; Goodwin et al.2009; Kennedy 2009; Kennedy and Rizvi 2010). The pres-ent study tested antidepressant activity of chronic agomela-tine administration in rats using a well-established modifiedversion of the FST (Cryan et al. 2005). Agomelatine-treated

rats showed significantly decreased immobility counts com-pared to vehicle-treated rats. These findings are in agree-ment with a previous study showing decreased immobilityin the classical version of the FST (Porsolt et al. 1977)following either a single or repeated (once a day for 13 days)administration of agomelatine (2, 10, 50 mg/kg orally (p.o.))in rats (Bourin et al. 2004). Previous studies testing theantidepressant efficacy of either melatonin or 5-HT2C antag-onists showed some inconsistencies. Single administrationof melatonin (0.5 and 1 mg/kg i.p.) decreased immobility inthe FST (Micale et al. 2006) as well as single and a 5-day(once a day) administration of the novel melatonin agonistNEU-P11 (25–100 mg/kg i.p.) (Tian et al. 2010), but onestudy showed no effects of chronic (14 days in drinkingwater) administration of melatonin (4 μg/ml) (Brotto et al.2000). Similarly, results obtained with a single administra-tion of the 5-HT2C antagonist molecules SB206533 (Cryanand Lucki 2000) or SB242084 (Calcagno et al. 2009)showed no effects in the rat FST, while the highly selective

AMYGDALA

Vehicle Agomelatine

Fig. 4 Western blot analyses of amygdala homogenates followingchronic (once a day for 22 days) agomelatine (40 mg/kg i.p.) admin-istration. Representative Western blot bands (top inset) and densito-metric analyses of the bands (bottom) of α-tubulin isoforms (a), MAP-2

and phosphorylated MAP-2 (b), synaptic markers (b), and BDNF (d).Data are expressed as mean±SEM. *P<0.05; **P<0.01; ***P<0.001(t test) vs. vehicle-treated rats


Table 4 Effects of chronic agomelatine (40 mg/kg i.p.): overview summary of the main findings of the study

Behavioural effects

LMA (distance; cm)

Day 20 of treatment

NOR (D2-index)

Day 20 of treatment

FST (counts)

Day 22 of treatment

ITI: 1h ITI: 4h Immobility Swimming Climbing

Molecular effects (18 h after last injection at day 22 of treatment)

Microtubular proteins Synaptic markers BDNF

Tyr/Glu-Tub 2-Tub Acet-Tub MAP-2 P-MAP-2 SYN PSD-95 SPIN Pro Mature

Hippocampus

Amygdala

PFC

↑ significant increase, ↓ significant decrease, ↔ no changes, : tendency to increase, : tendency to decrease, LMA locomotor activity, NOR novelobject recognition task, FST forced swimming test. Tyr/Glu-Tub tyrosinated α-tubulin/detyrosinated α-tubulin ratio, Δ2-Tub Δ2 α-tubulin; Acet-Tub acetylated α-tubulin, MAP-2 microtubule-associated protein type 2, P-MAP-2 phosporylated MAP-2, SYN synaptophysin, PSD-95 postsyn-aptic density-95, SPIN spinophilin, BDNF brain-derived neurotrophic factor, PFC prefrontal cortex

PREFRONTAL CORTEX

Vehicle Agomelatine

Fig. 5 Western blot analyses ofPFC homogenates followingchronic (once a day for 22 days)agomelatine (40 mg/kg i.p.)administration. RepresentativeWestern blot bands (top inset)and densitometric analyses ofthe bands (bottom) of α-tubulinisoforms (a), MAP-2 and phos-phorylated MAP-2 (b), synapticmarkers (c), and BDNF (d). Dataare expressed as mean±SEM.*P<0.05; **P<0.01;***P<0.001 (t test) vs.vehicle-treated rats


S32006 (2.5–40.0 mg/kg, i.p. and p.o.) was effective indecreasing immobility following either single or subchronic(24 h, 17 h and 30 min before the test) administration(Dekeyne et al. 2008). The present study also investigatedswimming and climbing behaviours in the FST which havebeen shown to be selectively increased by serotonergic andnoradrenergic antidepressants, respectively (Cryan et al.2005). Chronic agomelatine showed a clear tendency(P00.051) to increase swimming behaviour, while climbingwas unchanged. These data are in agreement with a recentFST study performed in mice showing that chronic (once aday for 28 days) agomelatine (40 mg/kg i.p.) decreasedimmobility by increasing swimming behaviour (Rainer etal. 2011). Since agomelatine consistently showed no effectsin increasing extracellular 5-HT levels in the dorsal hippo-campus and frontal cortex (Millan et al. 2005), the neuro-chemical meaning of our data remains unclear. However, itis possible to speculate that the observed effect of agomela-tine on immobility and swimming behaviours in the FST islikely to result from the synergy of its MT1 and MT2 agonistand 5-HT2C properties as already demonstrated in a chronicmild stress model of depression (Papp et al. 2003).

Chronic agomelatine administration did not affect explo-ration time of the two identical objects in the familiarisationtrial of the NOR task, showing no adverse effect of agome-latine on exploratory and learning behaviour. Resultsobtained in the choice trial of the NOR task demonstratedthat agomelatine-treated rats discriminate between the noveland the familiar object following 1 h of ITI to a similarextent as vehicle-treated animals, excluding any pro-amnesic effects of the drug in this task. Additionally, ago-melatine increased recognition memory when a 4-h ITI wasapplied since agomelatine-treated animals showed a signif-icantly higher D2 index compared to vehicle. Our dataobtained following chronic administration are in line witha previous study demonstrating that a single injection ofagomelatine (2.5, 10, 40 mg/kg i.p.) significantly enhancedrecognition memory following a 24-h ITI in the rat(Bertaina-Anglade et al. 2011). The pro-cognitive effectsof agomelatine are supported by previous animal studiesshowing that a single administration of agomelatine (1 and10 mg/kg i.p.) improved discrimination performance ofmice in the T-maze (Jaffard et al. 1993), while chronicadministration (once a day for 22 days; 10 mg/kg i.p.)reversed the spatial memory impairments induced by pred-ator exposure in the radial arm water maze in rats (Conboyet al. 2009). Therefore, these findings suggest that agome-latine has a rapid and sustained enhancing cognitive activitywhich may act as an additional contributor to its antidepres-sant effect which is reached following repeated treatment.Such enhancing cognitive activity of agomelatine seemsmore robust compared to traditional antidepressant drugs.Indeed, the relationship between antidepressant activity and

hippocampus-dependent learning task is still a matter ofdebate since contrasting results have been reported follow-ing both single and repeated administrations. Fluoxetine(SSRI; 5 mg/kg i.p.) or imipramine (TCA; 10 mg/kg i.p.)was shown to impair recognition memory when adminis-tered chronically in rats (Naudon et al. 2007; Valluzzi andChan 2007). Chronic administration of paroxetine (SSRI;10 mg/kg i.p.) improved memory performance in a protocolsimilar to the NOR task (Naudon et al. 2007) and partiallyreversed the NOR task deficits induced by chronic mildstress in the mice (Elizalde et al. 2008). Furthermore, asingle injection of fluoxetine (15 mg/kg s.c.) improvedmemory consolidation and retrieval in mice (Flood andCherkin 1987), while chronic fluoxetine (1 mg/kg i.p.)showed no effects on spatial learning in the Morris watermaze (Stewart and Reid 2000). It should be noted that theeffects of agomelatine on the FST and the NOR taskreported in the present study appear to be less potent com-pared to previous reports (Bourin et al. 2004; Bertaina-Anglade et al. 2011; Rainer et al. 2011). However, onlyone dose of agomelatine was employed here, and this mayexplain the differences.

Effects of agomelatine on the expression of cytoskeletalmicrotubule dynamics markers, synaptic markersand BDNF in the hippocampus, amygdala and PFC

The present study reports for the first time the differentialeffects of chronic agomelatine on markers of cytoskeletalmicrotubule dynamics and MAP-2 in the hippocampus,amygdala and PFC. These effects were accompanied byregion-specific changes in synaptic markers and BDNF,indicating possible phenomena of neuronal remodelling.

Chronic agomelatine increased both Tyr/Glu-Tub ratioand the neuron-specific Δ2-Tub in the hippocampus, sug-gesting increased neuronal microtubule dynamics. This is inline with previous studies showing that chronic administra-tion (once a day for 21 or 28 days) of fluoxetine (10 mg/kgi.p.) induced similar changes (Bianchi et al. 2009a, b). Inparticular, in those studies, chronic fluoxetine did actuallyincrease Δ2-Tub, but no apparent changes in Tyr/Glu-Tubratio were detected; a decrease in Acet-Tub was also con-sistently observed. However, the fluoxetine-inducedchanges in Δ2-Tub are very likely to result from disturban-ces at the cycle of detyrosination/tyrosination, as indicatedby the increase in Glu-Tub observed following a singlefluoxetine injection (Bianchi et al. 2009a). This view issupported by another study confirming decreased hippo-campal Acet-Tub following chronic (21 days) fluoxetine(10 mg/kg i.p.) treatment but also paralleled by increasedTyr-Tub (Yang et al. 2009). Sprague–Dawley rats wereemployed in that latter study as well as in the present, incontrast to Lister hooded rats which were used in our


previous works (Bianchi et al. 2009a, b). Thus, strain differ-ences in the time-frame modulation induced by antidepres-sant drugs in the α-tubulin cycle of detyrosination/tyrosination cannot be excluded. Furthermore, agomelatineappears to exert a specific effect on the hippocampal C-terminal cycle of detyrosination/tyrosination since the N-terminal Acet-Tub is not influenced. Our data raise the novelidea that the hippocampal α-tubulin cycle of detyrosination/tyrosination may be a common target for antidepressantaction because both the novel agomelatine and the traditionalSSRI fluoxetine increase hippocampal Δ2-Tub followingchronic administration.

Similarly to what was observed in the hippocampus,chronic agomelatine increased Tyr/Glu-Tub ratio and Δ2-Tub in the amygdala, but this was now accompanied bydecreased Acet-Tub, clearly suggesting increased microtu-bule dynamics. The possibility of a major activity of chronicagomelatine on the microtubular system in the amygdala issupported by the significant decrease of TOT-Tub expres-sion per se. This decrease may be indicative of unknownmechanisms of negative feedback aimed to reduce the avail-able free tubulin dimers and in turn decrease microtubuledynamics.

The expression of the dendrite-specific MAP-2 and itsphosphorylation at Ser136 were both increased by chronicagomelatine in the hippocampus and at much higher level inthe amygdala, consistent with a greater magnitude of effectsof chronic agomelatine on this specific brain region. Theincreased expression of MAP-2 was associated with in-creased Tyr/Glu-Tub and Δ2-Tub in both the hippocampusand amygdala suggesting a possible correlation betweenthese C-terminal α-tubulin modifications and increasedMAP-2 expression.

In contrast to what was observed in the hippocampus andamygdala, Tyr/Glu-Tub ratio and Δ2-Tub were decreased inthe PFC thus suggesting decreased microtubule dynamics.The expression of Acet-Tub, MAP-2 and phosphorylatedMAP-2 was unchanged.

Taken all together, the present data on microtubular pro-teins are therefore showing that chronic agomelatine in-duced three different patterns of changes in microtubuledynamics markers and MAP-2. Such cytoskeletal modifica-tions may reflect phenomena of neuronal remodelling. Thus,α-tubulin detyrosination/tyrosination and acetylation are notonly markers of microtubule dynamics (Khawaja et al.1988; Palazzo et al. 2003) but also have major importancein modulating neuronal development, neuronal organizationand axonal and dendritic trafficking (for an extensive re-view, see Janke and Kneussel 2010). Additionally, MAP-2has fundamental importance in dendritic elongation (for areview, see Sánchez et al. 2000) as well as in the formationand maintenance of dendritic spines (Hu et al. 2008; Jaworskiet al. 2009). Phosphorylation of MAP-2 at Ser136, which is

located at the N-terminus close to the protein kinase A (PKA)RII-binding domain, has been shown to play a major role inthe development and maintenance of dendrite architecture(Riederer et al. 1995; Philpot et al. 1997; Khuchua et al.2003). The agomelatine effects on MAP-2 in the hippocam-pus and amygdala may therefore be related to regulation ofmicrotubule dynamics and/or dendritic remodelling. Thefinding that phosphorylation at Ser136 is kept proportionalto the total expression of MAP-2 indicates that agomelatinedoes not negatively influence MAP-2 phosphorylation andactivity on dendrite remodelling. Our results are in agree-ment with previous studies showing that different classes ofantidepressant drugs can affect MAP-2 expression and/orphosphorylation in rat brain regions. MAP-2 expression wasincreased by subchronic treatment (once a day for 7 days)with the TCA imipramine (20 mg/kg i.p.) in rat hippocam-pus, but not by the SSRI fluvoxamine (20 mg/kgi.p.) (Iwata et al. 2006). Furthermore, increased phosphory-lation of MAP-2 at the serine residues was observed follow-ing 5 or 14 days administration (once a day) of fluvoxamineor desipramine, respectively, in rat cerebral cortex withoutchanges in total MAP-2 expression (Perez et al. 1995;Miyamoto et al. 1997). Interestingly, the increased hippo-campal MAP-2 expression may be due to the melatonergicactivity of agomelatine since melatonin (15 μg/ml) supple-mented in drinking water for 6 or 12months increasedMAP-2expression in the rat hippocampus (Prieto-Gómez et al. 2008).

Our results support the view that modifications in micro-tubule dynamics can be associated with possible phenomenaof neuronal remodelling since parallel changes in the ex-pression of synaptic markers were observed. Nevertheless, aclear correlation between the two phenomena is difficult toreconcile with the current data on microtubular proteinssince their agomelatine-induced increase was associatedwith either increased (amygdala) or decreased (hippocam-pus) synaptic markers. Moreover, in the PFC, agomelatineinduced a decrease of microtubular proteins associated withincreased synaptic markers.

However, as previously underlined, three different pat-terns of changes in terms of modified proteins and magni-tude of effects have been induced by chronic agomelatine onthe microtubular system in the three brain areas examined(see Table 4). This may reflect very different functionaloutputs which can explain the difficulty in correlating thechanges in microtubular proteins with those of the synapticmarkers. The analysis of homogenates using Western blot isa limitation to extrapolate more functional details on theobserved changes, and ex vivo and in vitro immunohisto-chemistry and imaging studies would answer such importantquestions.

Thus, the synaptic markers’ results showed that chronicagomelatine decreased spinophilin in the hippocampuswithout significantly affecting the other synaptic markers


despite a slight tendency to increase PSD-95 was observed.The decrease in spinophilin is in accordance with a previousstudy showing decreased rat hippocampal expression of thepolysialylated neural cell adhesion molecule (PSA-NCAM),a neurite and spine outgrowth-related protein (Dityatev et al.2004), by chronic administration of agomelatine at the samedose, route and sacrifice time point of the present study(Banasr et al. 2006). The decreased spinophilin expressionmight be indicative of a specific transient decrease in den-dritic spine density, which is likely to result from remodel-ling of hippocampal dendritic spines density. Thus, previousstudies using chronic fluoxetine showed remodelling ofdendritic spines towards a mushroom type rather thanchanging dendritic spines density per se (Bessa et al. 2009;Ampuero et al. 2010). In the amygdala, synaptophysin,PSD-95 and spinophilin were all increased by agomelatinetreatment suggesting neuronal remodelling and changes insynaptic connections. In line with the effects of agomelatine inthe amygdala observed here, a recent study in rats showed thatchronic agomelatine reduces long-term memory but not acqui-sition or short-term expression of fear memory (Diaz-Mataix etal. 2010). It should be noted though that chronic stress inanimal studies increased dendritic arborisation and synapto-genesis in the amygdala (Fuchs et al. 2006). Future studiesshould address the effects of agomelatine on amygdalaneuronal plasticity following chronic stress. Finally, chronicagomelatine increased synaptophysin and spinophilin in thePFC, but not PSD-95. These indicate remodelling of synap-tic connections with a possible specific effect on dendriticspines. Similar data showing increased synaptophysin aswell as PSA-NCAM expression (Varea et al. 2009) andremodelling of dendritic spines (Bessa et al. 2009) in ratPFC have been obtained following chronic administration ofthe SSRI fluoxetine indicating these phenomena as a com-mon action of antidepressant drugs.

Concerning BDNF expression, chronic agomelatine in-creased mature BDNF in the hippocampus and had a ten-dency to decrease both pro- and mature BDNF in theamygdala, while the PFC was not affected. The increasedhippocampal expression of mature BDNF is consistent withrecent investigations (Soumier et al. 2009; Calabrese et al.2011) and supports the hypothesis of hippocampal BDNFexpression as a possible common correlate of antidepressantresponse in the hippocampus (Tardito et al. 2006). Thetendency of chronic agomelatine to decrease BDNF expres-sion in the amygdala is here reported for the first time, and itmight relate to a negative feedback mechanism in responseto the high magnitude of neuronal remodelling or as adistinct and yet unknown neurochemical event.

On the other hand, the lack of effects of agomelatine onBDNF expression in the PFC is in contrast with a recentstudy showing that chronic (once a day for 21 days) ago-melatine (40 mg/kg i.p.) increased pro-BDNF expression in

such brain area (Calabrese et al. 2011). The reasons for suchdiscrepancy remain unclear, but inconsistencies on BDNFexpression emerge in the literature following administrationof antidepressant drugs (Tardito et al. 2006).

The BDNF data obtained here are not surprising since theexisting literature supports a dual region-specific role ofBDNF as neuronal plasticity modulator in the hippocampus,amygdala and nucleus accumbens–ventral tegmental areapathway (Castrén et al. 2007; Groves 2007; Castrén andRantamäki 2010). Thus, BDNF exerts different or evenopposite effects on depressive-like behaviours dependingon the involved neural circuit (Groves 2007; Ma et al.2011; Zoladz et al. 2011). Consistently, growing evidencesuggests that antidepressant drugs differently affect BDNFin a region-specific fashion (Tardito et al. 2006; Groves2007; Schulte-Herbrüggen et al. 2009).

Our findings confirm other investigations where the ef-fect of agomelatine in modulating neuronal plasticity phe-nomena was demonstrated. Thus, chronic agomelatineshowed neurogenic effects in the hippocampus in rats(Banasr et al. 2006; Soumier et al. 2009) and reverseddecreased neurogenesis in different models of depressionsuch as prenatally stressed rats (Morley-Fletcher et al.2011), glucocorticoid receptor-impaired mice (Païzanis etal. 2010) and the corticosterone-treated mice (Rainer et al.2011). Agomelatine exposure increased neurite outgrowthof granule cells in hippocampal primary cell culture andaccelerates maturation of newly formed granule cells in rats(Soumier et al. 2009).

Conclusions

The present study shows that agomelatine is a potent mod-ulator of cytoskeletal microtubule dynamics and synapticmarkers in the rat hippocampus, amygdala and prefrontalcortex. Noteworthy, effects of agomelatine on the amygdalahave here been described for the first time. The observedmolecular changes may play a role in the antidepressant andpro-cognitive effect of agomelatine and may result fromsynergy between the 5-HT2C antagonist and melatonergicagonist properties of the drug. According to its 5-HT2C

antagonist properties, agomelatine increased the release ofnoradrenaline and dopamine (Millan et al. 2003). These mayin turn associate to the functional output of β-adrenergic, D1

and D2 receptors and activation or inhibition of the cAMP–PKA pathway which has consistently been shown to mod-ulate microtubule dynamics (Perez et al. 1989; Miyamoto etal. 1997; Bianchi et al. 2005) and synaptic plasticity (Tarditoet al. 2006; Warner-Schmidt and Duman 2006). Additional-ly, the MT1 and MT2 agonistic properties of agomelatinemay also play a role since both receptors modulate severalsignalling pathways [e.g. PKA, protein kinase C (PKC),


ERKs, CREB and JNK] implicated in microtubule and syn-aptic plasticity regulation (D’Sa and Duman 2002; Donatiand Rasenick 2005; Bianchi et al. 2005). Noteworthy, in-creased microtubule dynamics has been shown to preventthe loss of high potency states of the human MT2 receptorand to increase melatonin-induced PKC activity in vitro,while the efficacy of melatonin to phase shift the circadianactivity rhythms in rats is enhanced by microtubule-depolymerizing agents (Jarzynka et al. 2009).

The beneficial effects of chronic agomelatine on recognitionmemory in the NOR taskmay be put in relation to the increasedhippocampal Tyr/Glu-Tub ratio and MAP-2 levels. Thus, iso-lated rats show impaired recognition memory in the NOR taskparalleled by decreased Tyr/Glu-Tub ratio and MAP-2 expres-sion (Bianchi et al. 2006). Moreover, pharmacological dis-ruption of hippocampal microtubules using nontoxic dosesof colchicine induced memory deficits in a number ofhippocampal-dependent tasks (Bensimon and Chermat1991; Nakayama and Sawada 2002; Lee and Solivan2010). Increased Tyr/Glu-Tub ratio in the hippocampusmight also be involved in the effects of agomelatine in theFST. Thus, decreased levels of Tyr-Tub were previouslyreported in the hippocampus of rats submitted to the FSTcompared to control ones (Bianchi et al. 2005). The actionof chronic agomelatine on synaptic markers in the threebrain regions examined may also contribute to the antide-pressant effect exerted by the molecule in the rat FST.Accordingly, the recent work of Sousa and colleaguesshowed that synaptic remodelling in the hippocampus andPFC is associated with the effectiveness of antidepressantdrugs in the FST (Bessa et al. 2009). In conclusion, our datasupport the hypothesis that changes in synaptic connectionsbetween brain areas forming the emotional circuit (i.e. hip-pocampus, amygdala and PFC) may be a common action inresponse to antidepressants (Bessa et al. 2009).

Acknowledgements This study was carried out at MAPREG andwas sponsored by Servier, Paris, France. The authors CG and EM areemployees of Servier and were involved in designing of the study andapproval of the report. The authors received income from their primaryemployer, and no other financial support or compensation has beenreceived from any individual or corporate entity over the past 3 yearsfor research or professional service, and there are no personal financialholdings that could be perceived as constituting a potential conflict ofinterest. The authors are grateful to C. Potard, C. Perier and L. Paresysfor technical assistance. The authors thank Dr. R. Baeurle for reviewingthe very first draft of the manuscript.

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