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Neuropharmacology 89 (2015) 382e390

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Involvement of 5-HT7 receptors in vortioxetine's modulation ofcircadian rhythms and episodic memory in rodents

Ligia Westrich a, *, Nasser Haddjeri b, c, Ouria Dkhissi-Benyahya b, c, Connie S�anchez a

a Lundbeck Research USA, 215 College Avenue, Paramus, NJ 07652, USAb INSERM U846, Stem Cell and Brain Research Institute, F-69500 Bron, Francec Universit�e de Lyon, F-69373 Lyon, France

a r t i c l e i n f o

Article history:Received 6 June 2014Received in revised form14 October 2014Accepted 18 October 2014Available online 31 October 2014

Keywords:VortioxetineBiological rhythmsEpisodic memory5-HT7 receptorBehaviorSuprachiasmatic nucleusSerotonin

* Corresponding author. Present address: Fairleighof Pharmacy, 230 Park Avenue, Florham Park, NJ 0793

E-mail addresses: westrich@fdu.edu (L. Westrich(N. Haddjeri), ouria.benyahya@inserm.fr (O. Dkhissi-(C. S�anchez).

http://dx.doi.org/10.1016/j.neuropharm.2014.10.0150028-3908/© 2014 The Authors. Published by Elsevier

a b s t r a c t

Since poor circadian synchrony and cognitive dysfunction have been linked to affective disorders, an-tidepressants that target key 5-HT (serotonin) receptor subtypes involved in circadian rhythm andcognitive regulation may have therapeutic utility. Vortioxetine is a multimodal antidepressant that in-hibits 5-HT1D, 5-HT3, 5-HT7 receptor activity, 5-HT reuptake, and enhances the activity of 5-HT1A and 5-HT1B receptors. In this study, we investigated the effects of vortioxetine on the period length ofPER2::LUC expression, circadian behavior, and episodic memory, using tissue explants from geneticallymodified PER2::LUC mice, locomotor activity rhythm monitoring, and the object recognition test,respectively. Incubation of tissue explants from the suprachiasmatic nucleus of PER2::LUC mice with0.1 mM vortioxetine increased the period length of PER2 bioluminescence. Monitoring of daily wheel-running activity of SpragueeDawley rats treated with vortioxetine (10 mg/kg, s.c.), alone or in combi-nation with the 5-HT1A receptor agonist flesinoxan (2.5 mg/kg, s.c.) or the 5-HT7 receptor antagonistSB269970 (30 mg/kg, s.c.), just prior to activity onset revealed significant delays in wheel-runningbehavior. The increase in circadian period length and the phase delay produced by vortioxetine wereabolished in the presence of the 5-HT7 receptor partial agonist AS19. Finally, in the object recognitiontest, vortioxetine (10 mg/kg, i.p.) increased the time spent exploring the novel object during the retentiontest and this effect was prevented by AS19 (5 mg/kg, i.p.). In conclusion, the present study shows thatvortioxetine, partly via its 5-HT7 receptor antagonism, induced a significant effect on circadian rhythmand presented promnesic properties in rodents.© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

The serotonergic neurotransmitter system comprises an exten-sive and complex network within the central nervous system(reviewed by (Artigas, 2013)). Numerous physiological functionsare influenced by serotonin (5-HT), e.g., aggression/impulse con-trol, anxiety/affect, appetitive/satiety, circadian rhythms/sleep,cognitive function, endocrine regulation, gastrointestinal functions,motor activity, pain, reproduction functions, sensory functions(Murphy et al., 1998). The brain's serotonergic neurons originatemainly from the dorsal and median raphe nuclei of the brainstemand are extensively arborized, thereby innervating most of the

Dickinson University, School2, USA. Tel.: þ1 201 694 5752.), nasser.haddjeri@inserm.frBenyahya), cs@lundbeck.com

Ltd. This is an open access article u

brain. A total of fifteen 5-HT receptor subtypes mediate seroto-nergic neurotransmission; some of them are located on 5-HTneurons regulating serotonergic neurotransmission, while othersare involved in controlling other neurotransmitter systems, such asthe glutamatergic, noradrenergic and dopaminergic. The 5-HT7receptor was first cloned in 1993 and its physiological roles havemainly been related to regulation of circadian rhythms, sleep andmood, but also to the regulation of cognitive function (Cowen andSherwood, 2013; Hedlund, 2009; Hedlund and Sutcliffe, 2004).

Vortioxetine (Lu AA21004; 1-[2-(2,4-dimethylphenyl-sulfanyl)-phenyl]-piperazine) is an antidepressant with a multimodalmechanism of action approved for the treatment of major depres-sive disorders (Adell, 2010; Alvarez et al., 2012). In in vitro studies incell lines expressing cloned human 5-HT receptors and the 5-HTtransporter (SERT), vortioxetine is a 5-HT3A (Ki ¼ 3.7 nM), 5-HT7(Ki ¼ 19 nM) and 5-HT1D (Ki ¼ 54 nM) receptor antagonist, 5-HT1Breceptor partial agonist (Ki ¼ 33 nM; IA 55%), 5-HT1A receptoragonist (Ki ¼ 15 nM) and inhibitor of the 5-HT transporter (SERT;

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

L. Westrich et al. / Neuropharmacology 89 (2015) 382e390 383

Ki ¼ 1.6 nM) (Bang-Andersen et al., 2011; Westrich et al., 2012). Inpreclinical rodent studies, vortioxetine showed the potential toimprove cognitive function (Mork et al., 2013). Furthermore, in twoclinical studies vortioxetine has been shown to be superior toplacebo in pre-defined cognitive outcome measures in patientswith major depressive disorder (Katona et al., 2012; McIntyre et al.,2013).

We have previously shown that concomitant inhibition of 5-HTreuptake and 5-HT7 receptors resulted in period lengthening ofPERIOD2::LUCIFERASE (PER2::LUC) bioluminescence in an in vitropreparation of the suprachiasmatic nucleus (SCN) of the hypo-thalamus from knock-in mice, and in a phase delay in the circadianrhythm of freely behaving rats (Westrich et al., 2013). In the presentstudy, we investigated the effect of vortioxetine on the periodlength of PER2::LUC expression and circadian behavior. Since vor-tioxetine has markedly lower affinity for 5-HT1A and 5-HT7 re-ceptors in rodents than in humans (Bang-Andersen et al., 2011;Mork et al., 2012) we also explored the effects of vortioxetine incombination with the 5-HT1A receptor agonist flesinoxan, and the5-HT7 receptor antagonist SB269970. Furthermore, we have pre-viously shown that vortioxetine improved recognition memory in atime delay object recognition test in rats (Mork et al., 2013). Herewe assess a putative role of vortioxetine's 5-HT7 receptor antago-nismwith respect to its action on episodic memory using the objectrecognition test.

2. Methods

2.1. Animals

Three-to six-month-old male homozygous PER2::LUC knock-in mice (Yoo et al.,2004) purchased from Jackson Laboratory (Bar Harbor, ME) were housed in a12 h:12 h light:dark (12:12 LD) cycle for at least 2 weeks prior to dissection andtissue collection. Two-month-old SpragueeDawley male rats purchased fromCharles River Laboratories (Wilmington, MA) were housed individually with freeaccess to running wheels (model 80820, Lafayette Instruments, Lafayette, IA) for theduration of the experiment. All animals were maintained under controlled envi-ronmental conditions (22 ± 2 �C; lights on at 06:00 to 18:00 h) with food and wateravailable ad libitum. All animal protocols were approved by the Lundbeck ResearchUSA, Inc. Institutional Animal Care and Use Committee.

The experiments involving the object recognition test were carried out withmale SpragueeDawley OFA rats (Charles River, FRANCE) weighing 250e310 g at thetime of the experiment. The animals were housed four per cage under standardlaboratory conditions; 12 h:12 h light:dark cycle with food and water available adlibitum. Experiments were performed in compliance with the European Commu-nities Council Directive 86/609 ECC for the care and use of laboratory animals andwith the approval of the Regional Animal Care Committees (University Lyon 1).

2.2. Drug treatments

For the in vitro studies, vortioxetine (H. Lundbeck A/S, DK) was dissolved inDMSO; AS19 ((2S)-(þ)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin),a reported 5-HT7 receptor partial agonist (Bonaventure et al., 2007; Roenneberget al., 2008), and SB269970 ((2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride), a potent 5-HT7 receptor antagonist(Hagan et al., 2000) (Tocris Bioscience, Ellisville, MO) were dissolved in DMSO andsterile water, respectively; flesinoxan, a 5-HT1A receptor agonist (synthesized by H.Lundbeck A/S, DK) was dissolved in saline. At the time of tissue treatment, drugswere diluted into the culture media to final concentrations. The concentrations ofthe working stock solutions were such that each treatment condition had the samefinal concentration of DMSO, which never exceeded 0.1% (v/v).

For the wheel-running experiments, AS19 was dissolved in 20% 2-hydroxypropyl-b-cyclodextrin (SigmaeAldrich, St. Louis, MO), while flesinoxan,SB267790 and vortioxetine were dissolved in 10% 2-hydroxypropyl-b-cyclodextrin.Prior to administration, the pH was adjusted to pH 5e6 with 0.1 N NaOH. Drugs andvehicles were administered subcutaneously (s.c.) in a volume of 2 ml/kg bodyweight. All doses used are expressed as mg/kg of the base. Acute dosing occurred atthe end of the light period, between Zeitgeber Time 11 and 12 (ZT11 and ZT12).Animals were switched from the prevailing 12:12 LD cycle to a period of constantdarkness immediately after treatment, which coincided with the time of lights-off(ZT12). This method was employed to minimize light cues and thus allow theexpression of endogenous rhythms. Animals were kept in constant darkness (DD)for approximately 10e14 days following acute drug administration. Dosing betweenZT11 and ZT12 was chosen to mimic the early morning dose commonly used bypatients and to be less disruptive to the normal sleepewake cycle of the rodents.

For the object recognition experiments, vortioxetine was dissolved in 10% 2-hydroxypropyl-b-cyclodextrin. AS19 was purchased from Tocris Bioscience (Ill-kirch, France) and dissolved in 0.5% methylcellulose. SB269970 was purchased fromSequoia Research Products Lt (Pangbourne, UK) and was dissolved in distilled water.Drugs and vehicles were administered intraperitoneally (i.p.). All doses used areexpressed as mg/kg of the salt.

2.3. Tissue preparation

Tissue explants containing the SCN from PER2::LUC knock-in mice were pre-pared according to published methods (Yoo et al., 2004). Briefly, animals wereanesthetized with isoflurane, brains were rapidly removed and 300 mm coronalsections through the SCNwere cut on a vibratome (Campden Instruments, Lafayette,IN) in chilled Hank's balanced salt solution (Invitrogen, Carlsbad, CA). Sections weretrimmed by hand to include the SCN and a minimum of surrounding tissue andimmediately transferred to culture inserts (Millipore, Billerica, MA) in 35 mm dishes(BD Falcon, Franklin Lakes, NJ). Dishes contained 1.2 ml Dulbecco's modified Eagle'smedium (SigmaeAldrich, St. Louis, MO) supplemented with 1� B27 (Invitrogen),4 mM L glutamine (Invitrogen), 25 mM glucose (Sigma), 4.2 mM NaHCO3 (Sigma),10 mM HEPES (Sigma), 25 U/ml penicillin-G sodium (Invitrogen), 34 mM strepto-mycin sulfate (Invitrogen), and 100 mMbeetle luciferin (Promega, Madison, WI, USA)and were sealed with vacuum grease (Dow, Midland, MI) and a glass coverslip. Twosections from SCN tissue were collected from a single animal; multiple samples ofthe same tissue from a single animal were placed in different treatment groups.

2.4. Bioluminescence monitoring

SCN tissue cultures were maintained at 35.8 �C, and integrated bioluminescencewas collected for 60 s every 10 min with a commercial luminometer (LumiCycle,Actimetrics, Wilmette, IL) before and after treatment. Bioluminescence analysis wasperformed using LumiCycle Analysis software. Data from the dissection day (Day 0)and from the days withmedia changes or day 1 of treatment were excluded from theanalysis. All tissues were incubated in the aforementioned media for the first weekto obtain a baseline, followed by control media or drug treatment once a week.Although the amplitude of the rhythms slightly damped towards the end of eachweek, media changes consistently reset the phase and amplitude of the cultures.Bioluminescence traces were de-trended by baseline subtraction of a 24 h movingaverage and then smoothed with a 2 h running average. De-trended and smootheddata were analyzed for periodicity with a c2 periodogram (Lumicycle Analysissoftware; Actimetrics Inc., Evanston, IL). Chi-square tests for the period of Per2-lucexpression were considered significant at p < 0.001. The period of the PER2::LUCexpression rhythms was determined using the sine fit curve-fitting method; meanvalues obtained in the control (vehicle) groups ranged from 24.75 ± 0.07 to24.83 ± 0.07 h (Figs. 1 and 2), in keeping with numerous published reports (Ehlenet al., 2001; Mullins et al., 1999; Yu and Weaver, 2011). A minimum of 3 days ofbioluminescence data was included in the analysis. Period values were comparedusing one-way analysis of variance (ANOVA) followed by a NewmaneKeuls multiplecomparison test. All comparisons were considered significant at p < 0.05.

2.5. Locomotor activity rhythm monitoring

Daily wheel-running activity was monitored with Running Wheel ActivitySoftware (AWM V12.0, Lafayette Instruments, Lafayette, IN) via activity wheelcounters (Model 86061) interfaced with computers, prior to and following drugadministration. Total revolutions of the activity wheel were continuously recordedin 2-min epochs. Collected data were split using AWM software to obtain intervalcount data for further analysis by Clocklab software (Actimetrics). Cumulative ac-tivity was represented in actograms, from which activity onsets and associatedcircadian parameters were calculated. Least-squares lines were fitted to the onsets ofactivity before and after dosing. The difference between the intersections of the twolines on the first day after treatment was used to calculate the shift in phase (byconvention a minus (�) symbol indicates a delay and a positive (þ) symbol indicatesan advance). Statistical analyses of the phase changes comprised an ANOVA followedby a NewmaneKeuls multiple comparison test. p < 0.05 was considered significant.

2.6. Object recognition test

The object recognition task was performed in a Plexiglas Y-maze apparatus(45 � 15 � 33 cm). It is based on the natural propensity of rats to explore novelty intheir environment. More specifically, rodents are able to discriminate between anovel and a previously seen (i.e., familiar) object. The Y-maze was placed in a roomilluminated only by a halogen lamp oriented towards the ceiling and giving a uni-form dim light in the apparatus (intensity of 60 lux) (Bartolini et al., 1996; Bertaina-Anglade et al., 2011). The Y-maze (floor andwalls) and the objects werewashed withethanol 10% and dried thoroughly after each trial. The objects were different inshape, color and texture. They were made of glass (white) and metal (gray), around14 cm high and too heavy to be displaced by rats. Each pair of objects was previouslytested for absence of spontaneous preference for one member of the pair. In eachexperimental group, the role (familiar versus novel object) and the relative positionof the two objects were counterbalanced and randomly permuted. Rats were placedin the experimental room for at least 30 min before testing.

Fig. 1. Vortioxetine increases the period length of PER2 bioluminescence. (A) Concentration-response curve showing a significant increase in the period length at 0.1 mM, but not athigher or lower concentrations (mean ± SEM; p < 0.05; NewmaneKeuls post-hoc test; n ¼ 5e11 slices/treatment group); (B) Representative records of the daily cycling of mPER2bioluminescence in the presence and absence of vortioxetine (0.1 mM).

Fig. 2. Combining vortioxetine (0.1 mM) with AS19 (10 nM) abolishes the effect ofvortioxetine on mPER2 bioluminescence (mean ± SEM; *p < 0.05 for AS19 versuscontrol and vortioxetine alone, **p < 0.05 for vortioxetine compared to other treatmentgroups; NewmaneKeuls post-hoc test; n ¼ 9e12 slices/treatment group).

L. Westrich et al. / Neuropharmacology 89 (2015) 382e390384

The experiment consisted of 4 sessions. On days 1 and 2, rats received twohabituation sessions to the area and test room environment: animals were allowedto freely explore the apparatus for 6 min per day. For the test session, rats weresubmitted to two trials with a 24 h inter-trial interval. During the first trial (day 3,learning trial, T1), animals were placed in the Y-maze containing 2 identical objectsand left for 5 min to explore the objects. Any rat not exploring the objects for at least8 s was excluded from the experiments. Exploration was defined as the animalhaving its head within 2 cm of the object while looking at, sniffing, or touching it. Inthe second trial (test trial, T2), which lasted 5 min, animals were exposed to anidentical copy of one of the objects previously seen during the first trial and a novelobject. Results were presented as time (sec) spent in active exploration of thefamiliar (F) or novel (N) object during T2 ± SEM. Recognitionmemory was estimatedusing a recognition index (RI): [(N � F)/(N þ F)] � 100. Animals with low level ofobject exploration (novelþ familiar < 5 s) were excluded from the data analysis. Thestatistical analysis was undertaken using an ANOVA followed by NewmaneKeulspost hoc test; p < 0.05 was considered significant. Using a 24 h retention interval, weassessed the object memory performance of rats after acute administrations ofvehicle, vortioxetine (10 mg/kg, i.p.), AS19 (5 mg/kg, i.p.) or SB269970 (4 mg/kg, i.p.)alone and combinations of vortioxetine and AS19 or SB269960 and AS19. Dosingoccurred just after T1 and 1 h before T2.

3. Results

3.1. Effects of vortioxetine alone and in combination with AS19, a 5-HT7 receptor ligand, on circadian rhythm regulation in SCN tissueexplants from mPER2::LUC mice

Incubation of SCN tissue explants from PER2::LUC mice with0.1 mM vortioxetine significantly and reproducibly increased theperiod length (t) of PER2 bioluminescence (F(4,46) ¼ 7.228,p < 0.0001). Higher or lower vortioxetine concentrations had nosignificant effect on the period length (Fig. 1A). Vortioxetine did notaffect the amplitude of PER2 bioluminescence. Combining vorti-oxetine with the 5-HT1A receptor agonist flesinoxan or the 5-HT7receptor antagonist SB269970 did not produce further changes(data not shown). When 0.1 mM vortioxetine was combined withthe partial 5-HT7 receptor agonist AS19 0.01 mM (an active con-centration that shortens period length, Westrich et al., 2013), theeffect of vortioxetine was abolished (F(3,44) ¼ 10.38, p < 0.0001)and the period length of PER2 bioluminescence was not differentfrom that of controls (Fig. 2).

3.2. Effects of vortioxetine alone and in combination with AS19 oncircadian rhythm regulation in wheel-running behavior

The acute effects of vortioxetine and AS19 (alone and in com-bination) on circadian rhythm regulation were studied in freelybehaving rats with access to running wheels. A significant maineffect of treatments was observed following the one-way ANOVAanalysis (F(3,27) ¼ 11.33, p < 0.0001). Vortioxetine (10 mg/kg, s.c.)

alone produced a significant phase delay (DF ¼ �0.82 ± 0.07 h,p < 0.001). AS19 (10 mg/kg, s.c.) administered alone at similar timepoints as vortioxetine produced no change in the occurence of ac-tivity onsets (DF ¼ �0.12 ± 0.08 h) compared to vehicle(DF ¼�0.10 ± 0.14 h). When vortioxetine was combined with AS19in paired injections, its effect on the circadian phase was markedlyattenuated (DF ¼ �0.36 ± 0.10 h, p < 0.001) compared to vorti-oxetine alone (Fig. 3).

3.3. Effects of vortioxetine alone and in combination withflesinoxan or SB269970 on circadian rhythm regulation in wheel-running behavior

Receptor occupancy studies in rodents suggest that the 10 mg/kg vortioxetine dose used in this study corresponds to approxi-mately 90% SERT occupancy (Leiser et al., 2014), which correspondsto the top end of the clinically relevant dose range (defined as50e90% SERT occupancy; (Areberg et al., 2012; Stenkrona et al.,2013)). However, direct translation of vortioxetine's pharmacolog-ical effects may be difficult from the perspective that vortioxetine

Fig. 3. Vortioxetine (10 mg/kg, s.c.) produces significant changes in daily wheel-running behavior. Top: Representative double-plotted actograms showing a phase delay followingacute administration of vortioxetine at ZT 11, and the lack of an effect on circadian phase when vortioxetine is co-administered with AS19 (10 mg/kg, s.c.) in paired injections. Theshaded areas represent daily wheel-running activity under constant darkness conditions. Bottom: Vortioxetine produces a significant phase delay, which is absent when vorti-oxetine is combined with AS19 (mean ± SEM; *p < 0.0001 versus vehicle and AS19, <0.001 versus vortioxetine þ AS19; NewmaneKeuls post-hoc test; n ¼ 7e8 animals/group).

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has approximately 10 fold lower affinity at the rat 5-HT1A and 5-HT7receptors compared to human receptors, in cellular in vitro assays(Bang-Andersen et al., 2011; Westrich et al., 2012). In order toinvestigate whether the comparatively higher 5-HT1A receptor ac-tivity expected in humans at a similar dose leads to any additionaleffects on circadian rhythm, the 5-HT1A receptor selective agonistflesinoxanwas co-administeredwith vortioxetine (Fig. 4). Althoughflesinoxan alone did not affect the circadian phase, addition offlesinoxan (2.5 mg/kg s.c.) to vortioxetine produced a phase delaythat was significantly different from vehicle (DF ¼ �0.85 ± 0.22 h,p < 0.05), but similar in magnitude to that of vortioxetine alone(DF ¼ �0.74 ± 0.13 h, F(3,12) ¼ 6.55, p < 0.05). Likewise, co-administration of vortioxetine (10 mg/kg, s.c.) and SB269970(30 mg/kg, s.c.) produced a phase delay (data not shown) that wassignificantly different from vehicle (DF ¼ �0.72 ± 0.15 h, p < 0.05),but not from that of vortioxetine alone (DF ¼ �0.79 ± 0.16 h,F(3,12) ¼ 7.16, p < 0.05). SB269970 alone had no effect on thecircadian phase (DF ¼ �0.28 ± 0.04 h, p > 0.05 versus vehicle).

3.4. Combined effects of flesinoxan and SB269970 on circadianrhythm regulation in wheel-running behavior

Based on the mechanisms of action that vortioxetine possesses,we also tested the effect of combining the 5-HT1A receptor selectiveagonist flesinoxan (2.5 mg/kg, s.c.) and the 5-HT7 receptor agonistSB269970 (30mg/kg, s.c.). When each compoundwas administeredalone to rats with access to runningwheels at similar time points asvortioxetine, neither had an effect on the circadian phase (Fig. 5);however, their combination produced a robust and significantphase delay (DF ¼ �0.71 ± 0.11 h, F(3,12) ¼ 5.77, p < 0.05). Themagnitude of this phase delay was slightly smaller, but similar to

the average phase delay induced by vortioxetine alone in our ex-periments (DF ¼ �0.78 h).

3.5. Effects of vortioxetine alone and in combination with AS19 onrecognition memory in the object recognition task

In the object recognition task (Fig. 7), one-way ANOVA showed asignificant main effect of treatments (F(5,54) ¼ 6.23, p < 0.001).While control rats spent no more time exploring the novel objectthan the familiar one (recognition index or RI ¼ 19.1 ± 4.3%; Fig. 7),vortioxetine (10 mg/kg, i.p.) increased the recognition indexsignificantly compared to vehicle controls (RI ¼ 46.7 ± 4.5%;p < 0.001). Although AS19 (5 mg/kg, i.p.) alone increased therecognition index (RI ¼ 35.5 ± 4.4%; p < 0.05), the effect of vorti-oxetine was no longer present when vortioxetine was combinedwith AS19 (RI ¼ 12.4 ± 6.0%; p < 0.001 for vortioxetine þ AS19versus vortioxetine alone). Similarly, the effect of AS19 was signif-icantly prevented when combined with the selective 5-HT7 re-ceptor antagonist SB269970 (4 mg/kg, i.p.) (RI ¼ 14.1 ± 4.6%;p < 0.01 for SB269970 þ AS19 versus AS19 alone), which alone didnot alter the recognition index compared to vehicle controls(RI ¼ 20.1 ± 6.3%; p > 0.05).

4. Discussion

In the present study, we showed that vortioxetine's modulationof 5-HT7 receptors may play a role in its modulatory effects oncircadian rhythms (in tissue culture and behaving rodents) andmemory-improving effects on a time delay-induced deficit ofrecognition memory.

Vortioxetine increased the period length (t) of PER2 biolumi-nescence of SCN tissue explants from PER2::LUC mice and induced

Fig. 4. Vortioxetine alone (10 mg/kg, s.c.) and in combination with flesinoxan (2.5 mg/kg, s.c.) produces significant changes in daily wheel-running behavior. Top: Representativedouble-plotted actograms showing a phase delay following acute administration of vortioxetine alone and in combination with flesinoxan at ZT 11. The shaded areas represent dailywheel-running activity under constant darkness conditions. Bottom: Vortioxetine alone and in combination with flesinoxan produces a significant phase delay (mean ± SEM;*p < 0.05 versus vehicle and all other treatment groups; NewmaneKeuls post-hoc test; n ¼ 4 animals/group).

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a phase delay (i.e., later timing) in the rat circadian wheel-runningrhythmwhen administered within 1 h of activity onset (before thebeginning of the subjective night). The modulation of PER2 biolu-minescence by vortioxetine was concentration-dependent (Fig. 1)with an increase in period length observed only at 0.1 mM. Thisresult was reproducible in experiments in which both higher andlower concentrations were used. The fact that vortioxetine actsthrough multiple 5-HT receptors, in addition to inhibiting the SERT(Mork et al., 2012; Westrich et al., 2012), supports our results,which reflect a gradual engagement of receptors as vortioxetineconcentration increases. Vortioxetine's regulation of circadianrhythms likely involves a complex interplay between 5-HT toneand inhibition/activation of 5-HT receptor subtypes, i.e., 5-HT1A, 5-HT1B, 5-HT3, and 5-HT7 receptors (Graff et al., 2007; Lovenberget al., 1993; Pickard et al., 1999; Roca et al., 1993). This complexmechanism of action makes it difficult to assess the importance ofcontributions from the individual receptor subtypes. Variousintricate and quite opposite interactions between different 5-HTreceptors may be involved in the observed effects of vortioxetineon circadian rhythms. Thus, vortioxetine yields different effects atdifferent concentrations. AS19, described as 5-HT7 receptor partialagonist with a 77% intrinsic efficacy (Bosker et al., 2009; VelaHernandez et al., 2008) shortened the period length as previouslyshown (Westrich et al., 2013). When combined with vortioxetine0.1 mM, AS19 abolishes the period lengthening induced by vorti-oxetine alone (Fig. 2), suggesting that vortioxetine's effects on PER2bioluminescence may be mediated through the 5-HT7 receptor atthis particular ex vivo concentration.

Rats exposed to an acute dose of vortioxetine (10 mg/kg, s.c.)display a shift (phase delay, i.e., delayed timing) in their circadianrhythm. However, when AS19 is co-administeredwith vortioxetine,the change induced by vortioxetine alone in the circadian phase isabolished (Fig. 3). Even though AS19 alone does not induce a phaseadvance, as hypothesized based on our ex-vivo results (Fig. 2) andwork of other colleagues (Adriani et al., 2012; Sprouse et al., 2004),the present findings are consistent with the fact that activation of5-HT7 receptors in the presence of AS19 opposes the phase delayinduced by vortioxetine alone.

The vortioxetine dose of 10 mg/kg was chosen because it pro-duces a similar level of SERT occupancy (~90%) in rodents as thehighest clinically used dose, 20 mg/day (Areberg et al., 2012;Stenkrona et al., 2013). Ex vivo binding studies in rats indicatethat an acute vortioxetine dose of 10 mg/kg, s.c. corresponds tomore than 80% occupancy of 5-HT3 and 5-HT1B receptors and SERTand approximately 35e40% occupancy of 5-HT1A and 5-HT7 re-ceptors (Leiser et al., 2014). Moreover, since vortioxetine hasapproximately 10 fold lower affinity at the rat 5-HT1A and 5-HT7receptors compared to humans (Bang-Andersen et al., 2011;Westrich et al., 2012), we co-administered vortioxetine with flesi-noxan (Fig. 4) or SB269970 (data not shown). Although flesinoxanand SB269970 alone had no effect on the circadian phase, theircombination produced a robust phase delay (Fig. 5) equivalent inmagnitude (mean D4 ¼ �0.71 h) to that induced by vortioxetine(mean D4 ¼ �0.78 h) alone. This observation could suggest that arelatively low level of 5-HT1A and 5-HT7 receptor occupancy isrequired for these targets to produce a maximum response on

Fig. 5. Combining flesinoxan (2.5 mg/kg, s.c.) with SB269970 (30 mg/kg, s.c.) results in significant changes in daily wheel-running behavior. Top: Representative double-plottedactograms showing flesinoxan alone and in combination with SB269970 administered in paired injections at ZT 11. The shaded areas represent daily wheel-running activity un-der constant darkness conditions. Bottom: Combination of flesinoxan with SB269970 produces a significant phase delay (mean ± SEM; *p < 0.05 versus vehicle and all othertreatment groups; NewmaneKeuls post-hoc test; n ¼ 4 animals/group).

Fig. 6. Adapted with permission from Westrich et al., 2013. Combining escitalopram (10 mg/kg, s.c.) with SB269970 (10 mg/kg, s.c.) results in significant changes in daily wheel-running behavior. Top: Representative double-plotted actograms showing escitalopram alone and in combination with SB269970 administered in paired injections at ZT 11. Theshaded areas represent daily wheel-running activity under constant darkness conditions. Bottom: Combination of escitalopram with SB269970 produces a significant phase delay(mean ± SEM; *p < 0.001 versus vehicle, <0.05 versus all other treatment groups; NewmaneKeuls post-hoc test; n ¼ 4e7 animals/group).

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Fig. 7. Vortioxetine reverses deficits in time-delayed object recognition task. (A) This schematic represents the object recognition test as described in Methods. (B) The acuteadministration of vortioxetine (10 mg/kg, i.p.) significantly increased the time exploring the novel object compared to the familiar one (mean ± SEM; **p < 0.01). Interestingly, the 5-HT7 receptor partial agonist AS19 (5 mg/kg, i.p.) alone enhanced the recognition index, but significantly prevented the increase in the recognition index induced by vortioxetine(mean ± SEM; ###p < 0.001). The acute administration of selective 5-HT7 receptor antagonist SB269970 (4 mg/kg, i.p.) did not affect the time spent exploring the novel objectcompared to the familiar one (p > 0.05), but significantly prevented the increase in the recognition index induced by AS19 (mean ± SEM; ##p < 0.01; NewmaneKeuls post-hoc test;n ¼ 8e12 animals/group).

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circadian regulation. This hypothesis would also be compatiblewith the observation that the active flesinoxan dose in the presentstudy (2.5 mg/kg, s.c.) corresponds to approximately the same levelof 5-HT1A receptor occupancy (30e40%) reported by Leiser et al.(2014) and probably a low 5-HT7 receptor occupancy withSB269970 (Westrich et al., 2013) due to its poor bloodebrain bar-rier penetration. However, SB269970 administered with an inef-fective dose of escitalopram, did produce a shift in the circadianphase (Fig. 6 e reproduced with permission from Westrich et al.(2013)).

The fact that escitalopram alone does not affect circadianrhythm (Westrich et al., 2013), while vortioxetine does, must beascribed to its multimodal action involving inhibition of the SERTand direct effects at 5-HT receptors, in particular 5-HT1A, 5-HT1Band 5-HT7 receptors, all 3 of which have been implicated in thecontrol of circadian rhythms (Graff et al., 2007; Lovenberg et al.,1993; Pickard et al., 1999; Roca et al., 1993). Thus, several lines ofevidence support the hypothesis that 5-HT tone together withactivation/blockade of several 5-HT receptors can modulate circa-dian rhythms, while blockade of the SERT (i.e., increase in 5-HTtone) may not be sufficient to cause a measurable effect.

Taken together, our data consistently show that the effect ofvortioxetine in modulating PER2 bioluminescence and wheel-running behavior is abolished in the presence of AS19. This in-dicates a strong involvement of 5-HT7 receptors in vortioxetine'seffects on circadian rhythms. Since poor circadian synchrony hasbeen linked to maladaptive changes in unipolar and bipolardepression (Wirz-Justice, 2003, 2006), antidepressants that targetkey 5-HT receptor subtypes involved in circadian rhythm regula-tion may have therapeutic utility. The putative impact of thisproperty of vortioxetine remains to be studied. The melatonin re-ceptor agonist and 5-HT2C receptor antagonist agomelatine (Tarditoet al., 2012) is thought to exert its antidepressant activity, at least inpart, through regulation of circadian rhythms (Lanfumey et al.,2013). Whereas vortioxetine likely affects mood and circadianrhythms via different mechanisms than agomelatine, they mayoverlap in their ability to restore the proper phase alignment be-tween the SCN pacemaker and the lightedark cycle (Dallaspeziaand Benedetti, 2011; Duncan, 1996; Healy and Waterhouse, 1995;Wirz-Justice, 2003, 2006), which is potentially disrupted during adepressive episode. But a recent clinical study in which MDD

patients with an inadequate response to SSRI/SNRI monotherapywere randomized to flexible-dose treatment with vortioxetine(10e20 mg/day) or agomelatine (25e50 mg/day) showed thatvortioxetine was statistically superior to agomelatine on the pre-defined primary outcome using the Montgomery-Åsberg Depres-sion Rating Scale (MADRS) (H€aggstr€om et al., 2013).

As seenwith the pro-cognitive agent donepezil, vortioxetine hasbeen shown to increase the time that rats spend investigating thenovel object in the 24-h object recognition test (Mork et al., 2013).Using the same retention interval, the present study confirms thatacute administration of vortioxetine (10 mg/kg, i.p.) improved ob-ject memory performance of naïve rats. Our data indicate that 5-HT7 receptor modulation may be involved in vortioxetine's cogni-tive enhancing properties, since the partial 5-HT7 receptor agonist,AS19, attenuated the memory enhancing properties of vortioxetinein the object recognition task. Intriguingly, AS19 alone also exerts amemory enhancing effect in the object recognition task. Since AS19is a potent (subnanomolar in vitro potency) and selective 5-HT7receptor agonist (Di Pilato et al., 2014), its effects on memory arelikely mediated by 5-HT7 receptors. This is indeed confirmed by thefact that the 5-HT7 receptor antagonist SB269970 prevented theenhancing effect of AS19 in the object recognition task. However,the literature is still confusing since both 5-HT7 receptor agonists(e.g., AS19) and 5-HT7 receptor antagonists (e.g., SB269970) havebeen reported to have memory enhancing or memory impairingproperties depending on the animal model and test conditions(such as the 5-HT tone) (Gasbarri and Pompili, 2014; Meneses,2014). Also, a phase-dependent involvement of 5-HT7 receptorshas been proposed for which the acquisition phase requires asilencing of 5-HT7 receptors while the consolidation phase requiresa high activation of 5-HT7 receptors (Freret et al., 2014). Similar tothe coupling of 5-HT1A and 5-HT7 receptors in the regulation ofcircadian rhythms, a study by Eriksson et al. (2012) reported acomplex, brain region dependent crosstalk between 5-HT1A and 5-HT7 receptors in the regulation of memory function (also see thereview by Meneses, 2014). Thus, while the 5-HT1A and 5-HT7 re-ceptor agonist 8-OH-DPAT impaired memory function in a passiveavoidance learning test, co-administration of 8-OH-DPATand the 5-HT1A receptor antagonist NAD-299 facilitated memory (Erikssonet al., 2012). The authors emphasized that since 5-HT has high af-finity for both 5-HT1A and 5-HT7 receptors compared to other 5-HT

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receptors, a change in signaling through one them would be likelyto affect the function of the other (Eriksson et al., 2012). As vorti-oxetine is a 5-HT7 receptor antagonist and a 5-HT1A receptoragonist (Mork et al., 2012) and vortioxetine at the dose tested oc-cupies 5-HT1A as well as 5-HT7 receptors, it can be hypothesizedthat AS19 interferes negatively with the complex interaction ofvortioxetine at 5-HT1A and 5-HT7 receptors through its interactionat 5-HT7 receptors.

A limitation of our studies relates to the fact that vortioxetineacts through multiple interdependent targets, which implies thatdetermination of the precise function at each of its individual tar-gets is extremely difficult. Furthermore, target-specific potencydifferences between rodents and human offers another layer ofcomplexity to our studies. As aforementioned, the in vitro affinity ofvortioxetine for rat 5-HT1A and 5-HT7 receptors is approximately10-fold lower compared to the human receptors. In order to avoidunderestimating the effects of vortioxetine at these receptors, weco-administered it with the 5-HT1A receptor agonist flesinoxan orthe 5-HT7 receptor antagonist SB269970. However, this approach isonly an approximation, since there are no available methods toassess vortioxetine's occupancy at the 5-HT1A and 5-HT7 receptorsin the human brain. Taken together, the engagement of multipletargets and the species differences add to the complexity andinterpretation of our research. To complement our pharmacologicalstudies, a future direction could be to study the effects of vortiox-etine in genetically engineered mice with induced mutations, suchas knockout of a functional target, or expression of a human target(e.g. 5-HT1A receptors). However, the ultimate understanding ofvortioxetine's effects on circadian rhythms and cognitive functionmust come from clinical studies in humans.

5. Conclusion

Vortioxetine has the potential to modulate circadian rhythms byprolonging the period length and producing a phase delay. In linewith published preclinical and clinical data vortioxetine has thepotential to positively affect memory function. Finally, the studyindicates that 5-HT7 receptors may be involved in these significanteffects on circadian rhythm and recognition memory in rodents.The potential clinical implications of these preclinical findingsremain to be explored.

Acknowledgments

This research was supported by H. Lundbeck A/S and the TakedaPharmaceutical Company, Ltd.

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