Westrich et al., 2015 (Neuropharmacology) - Involvement of 5-HT7 receptors in vortioxetine's modulation of circadian rhythms and episodic memory in rodents

Involvement of 5-HT7 receptors in vortioxetine's modulation of
circadian rhythms and episodic memory in rodents
Ligia Westrich a, *
, Nasser Haddjeri b, c
, Ouria Dkhissi-Benyahya b, c
, Connie Sanchez a
a
Lundbeck Research USA, 215 College Avenue, Paramus, NJ 07652, USA
b
INSERM U846, Stem Cell and Brain Research Institute, F-69500 Bron, France
c
Universite de Lyon, F-69373 Lyon, France
a r t i c l e i n f o
Article history:
Received 6 June 2014
Received in revised form
14 October 2014
Accepted 18 October 2014
Available online 31 October 2014
Keywords:
Vortioxetine
Biological rhythms
Episodic memory
5-HT7 receptor
Behavior
Suprachiasmatic nucleus
Serotonin
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 and
cognitive 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 of
PER2::LUC expression, circadian behavior, and episodic memory, using tissue explants from genetically
modified 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 with
0.1 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 antagonist
SB269970 (30 mg/kg, s.c.), just prior to activity onset revealed significant delays in wheel-running
behavior. The increase in circadian period length and the phase delay produced by vortioxetine were
abolished in the presence of the 5-HT7 receptor partial agonist AS19. Finally, in the object recognition
test, vortioxetine (10 mg/kg, i.p.) increased the time spent exploring the novel object during the retention
test and this effect was prevented by AS19 (5 mg/kg, i.p.). In conclusion, the present study shows that
vortioxetine, partly via its 5-HT7 receptor antagonism, induced a significant effect on circadian rhythm
and 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 functions
are 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 originate
mainly from the dorsal and median raphe nuclei of the brainstem
and are extensively arborized, thereby innervating most of the
brain. A total of fifteen 5-HT receptor subtypes mediate seroto-
nergic neurotransmission; some of them are located on 5-HT
neurons regulating serotonergic neurotransmission, while others
are involved in controlling other neurotransmitter systems, such as
the glutamatergic, noradrenergic and dopaminergic. The 5-HT7
receptor was first cloned in 1993 and its physiological roles have
mainly been related to regulation of circadian rhythms, sleep and
mood, but also to the regulation of cognitive function (Cowen and
Sherwood, 2013; Hedlund, 2009; Hedlund and Sutcliffe, 2004).
Vortioxetine (Lu AA21004; 1-[2-(2,4-dimethylphenyl-sulfanyl)-
phenyl]-piperazine) is an antidepressant with a multimodal
mechanism of action approved for the treatment of major depres-
sive disorders (Adell, 2010; Alvarez et al., 2012). In in vitro studies in
cell lines expressing cloned human 5-HT receptors and the 5-HT
transporter (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-HT1B
receptor partial agonist (Ki ¼ 33 nM; IA 55%), 5-HT1A receptor
agonist (Ki ¼ 15 nM) and inhibitor of the 5-HT transporter (SERT;
* Corresponding author. Present address: Fairleigh Dickinson University, School
of Pharmacy, 230 Park Avenue, Florham Park, NJ 07932, USA. Tel.: þ1 201 694 5752.
E-mail addresses: westrich@fdu.edu (L. Westrich), nasser.haddjeri@inserm.fr
(N. Haddjeri), ouria.benyahya@inserm.fr (O. Dkhissi-Benyahya), cs@lundbeck.com
(C. Sanchez).
Contents lists available at ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
http://dx.doi.org/10.1016/j.neuropharm.2014.10.015
0028-3908/© 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/).
Neuropharmacology 89 (2015) 382e390

Ki ¼ 1.6 nM) (Bang-Andersen et al., 2011; Westrich et al., 2012). In
preclinical rodent studies, vortioxetine showed the potential to
improve cognitive function (Mork et al., 2013). Furthermore, in two
clinical studies vortioxetine has been shown to be superior to
placebo in pre-defined cognitive outcome measures in patients
with major depressive disorder (Katona et al., 2012; McIntyre et al.,
2013).
We have previously shown that concomitant inhibition of 5-HT
reuptake and 5-HT7 receptors resulted in period lengthening of
PERIOD2::LUCIFERASE (PER2::LUC) bioluminescence in an in vitro
preparation of the suprachiasmatic nucleus (SCN) of the hypo-
thalamus from knock-in mice, and in a phase delay in the circadian
rhythm of freely behaving rats (Westrich et al., 2013). In the present
study, we investigated the effect of vortioxetine on the period
length 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 in
combination with the 5-HT1A receptor agonist flesinoxan, and the
5-HT7 receptor antagonist SB269970. Furthermore, we have pre-
viously shown that vortioxetine improved recognition memory in a
time delay object recognition test in rats (Mork et al., 2013). Here
we assess a putative role of vortioxetine's 5-HT7 receptor antago-
nism with respect to its action on episodic memory using the object
recognition 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 a
12 h:12 h light:dark (12:12 LD) cycle for at least 2 weeks prior to dissection and
tissue collection. Two-month-old SpragueeDawley male rats purchased from
Charles River Laboratories (Wilmington, MA) were housed individually with free
access to running wheels (model 80820, Lafayette Instruments, Lafayette, IA) for the
duration 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 water
available ad libitum. All animal protocols were approved by the Lundbeck Research
USA, Inc. Institutional Animal Care and Use Committee.
The experiments involving the object recognition test were carried out with
male SpragueeDawley OFA rats (Charles River, FRANCE) weighing 250e310 g at the
time of the experiment. The animals were housed four per cage under standard
laboratory conditions; 12 h:12 h light:dark cycle with food and water available ad
libitum. Experiments were performed in compliance with the European Commu-
nities Council Directive 86/609 ECC for the care and use of laboratory animals and
with 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 in
DMSO; AS19 ((2S)-(þ)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin),
a reported 5-HT7 receptor partial agonist (Bonaventure et al., 2007; Roenneberg
et 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 and
sterile 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, drugs
were diluted into the culture media to final concentrations. The concentrations of
the working stock solutions were such that each treatment condition had the same
final 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 and
vehicles were administered subcutaneously (s.c.) in a volume of 2 ml/kg body
weight. All doses used are expressed as mg/kg of the base. Acute dosing occurred at
the 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 constant
darkness immediately after treatment, which coincided with the time of lights-off
(ZT12). This method was employed to minimize light cues and thus allow the
expression of endogenous rhythms. Animals were kept in constant darkness (DD)
for approximately 10e14 days following acute drug administration. Dosing between
ZT11 and ZT12 was chosen to mimic the early morning dose commonly used by
patients 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 from
Sequoia Research Products Lt (Pangbourne, UK) and was dissolved in distilled water.
Drugs and vehicles were administered intraperitoneally (i.p.). All doses used are
expressed 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 were
anesthetized with isoflurane, brains were rapidly removed and 300 mm coronal
sections through the SCN were cut on a vibratome (Campden Instruments, Lafayette,
IN) in chilled Hank's balanced salt solution (Invitrogen, Carlsbad, CA). Sections were
trimmed by hand to include the SCN and a minimum of surrounding tissue and
immediately 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's
medium (SigmaeAldrich, St. Louis, MO) supplemented with 1Â 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 mM beetle luciferin (Promega, Madison, WI, USA)
and were sealed with vacuum grease (Dow, Midland, MI) and a glass coverslip. Two
sections from SCN tissue were collected from a single animal; multiple samples of
the 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 bioluminescence
was collected for 60 s every 10 min with a commercial luminometer (LumiCycle,
Actimetrics, Wilmette, IL) before and after treatment. Bioluminescence analysis was
performed using LumiCycle Analysis software. Data from the dissection day (Day 0)
and from the days with media changes or day 1 of treatment were excluded from the
analysis. All tissues were incubated in the aforementioned media for the first week
to 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 each
week, media changes consistently reset the phase and amplitude of the cultures.
Bioluminescence traces were de-trended by baseline subtraction of a 24 h moving
average and then smoothed with a 2 h running average. De-trended and smoothed
data were analyzed for periodicity with a c2
periodogram (Lumicycle Analysis
software; Actimetrics Inc., Evanston, IL). Chi-square tests for the period of Per2-luc
expression were considered significant at p 0.001. The period of the PER2::LUC
expression rhythms was determined using the sine fit curve-fitting method; mean
values obtained in the control (vehicle) groups ranged from 24.75 ± 0.07 to
24.83 ± 0.07 h (Figs. 1 and 2), in keeping with numerous published reports (Ehlen
et al., 2001; Mullins et al., 1999; Yu and Weaver, 2011). A minimum of 3 days of
bioluminescence data was included in the analysis. Period values were compared
using one-way analysis of variance (ANOVA) followed by a NewmaneKeuls multiple
comparison 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 Activity
Software (AWM V12.0, Lafayette Instruments, Lafayette, IN) via activity wheel
counters (Model 86061) interfaced with computers, prior to and following drug
administration. Total revolutions of the activity wheel were continuously recorded
in 2-min epochs. Collected data were split using AWM software to obtain interval
count data for further analysis by Clocklab software (Actimetrics). Cumulative ac-
tivity was represented in actograms, from which activity onsets and associated
circadian parameters were calculated. Least-squares lines were fitted to the onsets of
activity before and after dosing. The difference between the intersections of the two
lines on the first day after treatment was used to calculate the shift in phase (by
convention a minus (À) symbol indicates a delay and a positive (þ) symbol indicates
an advance). Statistical analyses of the phase changes comprised an ANOVA followed
by 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 Â 15 Â 33 cm). It is based on the natural propensity of rats to explore novelty in
their environment. More specifically, rodents are able to discriminate between a
novel and a previously seen (i.e., familiar) object. The Y-maze was placed in a room
illuminated 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 and walls) and the objects were washed with
ethanol 10% and dried thoroughly after each trial. The objects were different in
shape, color and texture. They were made of glass (white) and metal (gray), around
14 cm high and too heavy to be displaced by rats. Each pair of objects was previously
tested for absence of spontaneous preference for one member of the pair. In each
experimental group, the role (familiar versus novel object) and the relative position
of the two objects were counterbalanced and randomly permuted. Rats were placed
in the experimental room for at least 30 min before testing.
L. Westrich et al. / Neuropharmacology 89 (2015) 382e390 383

The experiment consisted of 4 sessions. On days 1 and 2, rats received two
habituation sessions to the area and test room environment: animals were allowed
to freely explore the apparatus for 6 min per day. For the test session, rats were
submitted 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 objects
and left for 5 min to explore the objects. Any rat not exploring the objects for at least
8 s was excluded from the experiments. Exploration was defined as the animal
having its head within 2 cm of the object while looking at, sniffing, or touching it. In
the second trial (test trial, T2), which lasted 5 min, animals were exposed to an
identical copy of one of the objects previously seen during the first trial and a novel
object. Results were presented as time (sec) spent in active exploration of the
familiar (F) or novel (N) object during T2 ± SEM. Recognition memory was estimated
using a recognition index (RI): [(N À F)/(N þ F)] Â 100. Animals with low level of
object exploration (novel þ familiar 5 s) were excluded from the data analysis. The
statistical analysis was undertaken using an ANOVA followed by NewmaneKeuls
post hoc test; p 0.05 was considered significant. Using a 24 h retention interval, we
assessed the object memory performance of rats after acute administrations of
vehicle, 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. Dosing
occurred 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 tissue
explants from mPER2::LUC mice
Incubation of SCN tissue explants from PER2::LUC mice with
0.1 mM vortioxetine significantly and reproducibly increased the
period length (t) of PER2 bioluminescence (F(4,46) ¼ 7.228,
p 0.0001). Higher or lower vortioxetine concentrations had no
significant effect on the period length (Fig. 1A). Vortioxetine did not
affect the amplitude of PER2 bioluminescence. Combining vorti-
oxetine with the 5-HT1A receptor agonist flesinoxan or the 5-HT7
receptor antagonist SB269970 did not produce further changes
(data not shown). When 0.1 mM vortioxetine was combined with
the partial 5-HT7 receptor agonist AS19 0.01 mM (an active con-
centration that shortens period length, Westrich et al., 2013), the
effect of vortioxetine was abolished (F(3,44) ¼ 10.38, p 0.0001)
and the period length of PER2 bioluminescence was not different
from that of controls (Fig. 2).
3.2. Effects of vortioxetine alone and in combination with AS19 on
circadian 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 freely
behaving rats with access to running wheels. A significant main
effect of treatments was observed following the one-way ANOVA
analysis (F(3,27) ¼ 11.33, p 0.0001). Vortioxetine (10 mg/kg, s.c.)
alone produced a significant phase delay (DF ¼ À0.82 ± 0.07 h,
p 0.001). AS19 (10 mg/kg, s.c.) administered alone at similar time
points as vortioxetine produced no change in the occurence of ac-
tivity onsets (DF ¼ À0.12 ± 0.08 h) compared to vehicle
(DF ¼ À0.10 ± 0.14 h). When vortioxetine was combined with AS19
in paired injections, its effect on the circadian phase was markedly
attenuated (DF ¼ À0.36 ± 0.10 h, p 0.001) compared to vorti-
oxetine alone (Fig. 3).
3.3. Effects of vortioxetine alone and in combination with
flesinoxan 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 corresponds
to the top end of the clinically relevant dose range (defined as
50e90% 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. 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 at
higher 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 mPER2
bioluminescence in the presence and absence of vortioxetine (0.1 mM).
Fig. 2. Combining vortioxetine (0.1 mM) with AS19 (10 nM) abolishes the effect of
vortioxetine on mPER2 bioluminescence (mean ± SEM; *p 0.05 for AS19 versus
control and vortioxetine alone, **p 0.05 for vortioxetine compared to other treatment
groups; NewmaneKeuls post-hoc test; n ¼ 9e12 slices/treatment group).
L. Westrich et al. / Neuropharmacology 89 (2015) 382e390384

has approximately 10 fold lower affinity at the rat 5-HT1A and 5-HT7
receptors compared to human receptors, in cellular in vitro assays
(Bang-Andersen et al., 2011; Westrich et al., 2012). In order to
investigate whether the comparatively higher 5-HT1A receptor ac-
tivity expected in humans at a similar dose leads to any additional
effects on circadian rhythm, the 5-HT1A receptor selective agonist
flesinoxan was co-administered with vortioxetine (Fig. 4). Although
flesinoxan alone did not affect the circadian phase, addition of
flesinoxan (2.5 mg/kg s.c.) to vortioxetine produced a phase delay
that was significantly different from vehicle (DF ¼ À0.85 ± 0.22 h,
p 0.05), but similar in magnitude to that of vortioxetine alone
(DF ¼ À0.74 ± 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 was
significantly different from vehicle (DF ¼ À0.72 ± 0.15 h, p 0.05),
but not from that of vortioxetine alone (DF ¼ À0.79 ± 0.16 h,
F(3,12) ¼ 7.16, p 0.05). SB269970 alone had no effect on the
circadian phase (DF ¼ À0.28 ± 0.04 h, p 0.05 versus vehicle).
3.4. Combined effects of flesinoxan and SB269970 on circadian
rhythm 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 selective
agonist flesinoxan (2.5 mg/kg, s.c.) and the 5-HT7 receptor agonist
SB269970 (30 mg/kg, s.c.). When each compound was administered
alone to rats with access to running wheels at similar time points as
vortioxetine, neither had an effect on the circadian phase (Fig. 5);
however, their combination produced a robust and significant
phase delay (DF ¼ À0.71 ± 0.11 h, F(3,12) ¼ 5.77, p 0.05). The
magnitude of this phase delay was slightly smaller, but similar to
the average phase delay induced by vortioxetine alone in our ex-
periments (DF ¼ À0.78 h).
3.5. Effects of vortioxetine alone and in combination with AS19 on
recognition memory in the object recognition task
In the object recognition task (Fig. 7), one-way ANOVA showed a
significant main effect of treatments (F(5,54) ¼ 6.23, p 0.001).
While control rats spent no more time exploring the novel object
than the familiar one (recognition index or RI ¼ 19.1 ± 4.3%; Fig. 7),
vortioxetine (10 mg/kg, i.p.) increased the recognition index
significantly compared to vehicle controls (RI ¼ 46.7 ± 4.5%;
p 0.001). Although AS19 (5 mg/kg, i.p.) alone increased the
recognition index (RI ¼ 35.5 ± 4.4%; p 0.05), the effect of vorti-
oxetine was no longer present when vortioxetine was combined
with AS19 (RI ¼ 12.4 ± 6.0%; p 0.001 for vortioxetine þ AS19
versus 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 did
not 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 modulation
of 5-HT7 receptors may play a role in its modulatory effects on
circadian rhythms (in tissue culture and behaving rodents) and
memory-improving effects on a time delay-induced deficit of
recognition memory.
Vortioxetine increased the period length (t) of PER2 biolumi-
nescence of SCN tissue explants from PER2::LUC mice and induced
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 following
acute 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. The
shaded 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).

a phase delay (i.e., later timing) in the rat circadian wheel-running
rhythm when administered within 1 h of activity onset (before the
beginning 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. This
result was reproducible in experiments in which both higher and
lower concentrations were used. The fact that vortioxetine acts
through 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 vortioxetine
concentration increases. Vortioxetine's regulation of circadian
rhythms likely involves a complex interplay between 5-HT tone
and inhibition/activation of 5-HT receptor subtypes, i.e., 5-HT1A, 5-
HT1B, 5-HT3, and 5-HT7 receptors (Graff et al., 2007; Lovenberg
et al., 1993; Pickard et al., 1999; Roca et al., 1993). This complex
mechanism of action makes it difficult to assess the importance of
contributions from the individual receptor subtypes. Various
intricate and quite opposite interactions between different 5-HT
receptors may be involved in the observed effects of vortioxetine
on circadian rhythms. Thus, vortioxetine yields different effects at
different concentrations. AS19, described as 5-HT7 receptor partial
agonist with a 77% intrinsic efficacy (Bosker et al., 2009; Vela
Hernandez et al., 2008) shortened the period length as previously
shown (Westrich et al., 2013). When combined with vortioxetine
0.1 mM, AS19 abolishes the period lengthening induced by vorti-
oxetine alone (Fig. 2), suggesting that vortioxetine's effects on PER2
bioluminescence may be mediated through the 5-HT7 receptor at
this 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 circadian
rhythm. However, when AS19 is co-administered with vortioxetine,
the change induced by vortioxetine alone in the circadian phase is
abolished (Fig. 3). Even though AS19 alone does not induce a phase
advance, as hypothesized based on our ex-vivo results (Fig. 2) and
work of other colleagues (Adriani et al., 2012; Sprouse et al., 2004),
the present findings are consistent with the fact that activation of
5-HT7 receptors in the presence of AS19 opposes the phase delay
induced 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 the
highest clinically used dose, 20 mg/day (Areberg et al., 2012;
Stenkrona et al., 2013). Ex vivo binding studies in rats indicate
that an acute vortioxetine dose of 10 mg/kg, s.c. corresponds to
more than 80% occupancy of 5-HT3 and 5-HT1B receptors and SERT
and approximately 35e40% occupancy of 5-HT1A and 5-HT7 re-
ceptors (Leiser et al., 2014). Moreover, since vortioxetine has
approximately 10 fold lower affinity at the rat 5-HT1A and 5-HT7
receptors 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 flesinoxan
and SB269970 alone had no effect on the circadian phase, their
combination produced a robust phase delay (Fig. 5) equivalent in
magnitude (mean D4 ¼ À0.71 h) to that induced by vortioxetine
(mean D4 ¼ À0.78 h) alone. This observation could suggest that a
relatively low level of 5-HT1A and 5-HT7 receptor occupancy is
required for these targets to produce a maximum response on
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: Representative
double-plotted actograms showing a phase delay following acute administration of vortioxetine alone and in combination with flesinoxan at ZT 11. The shaded areas represent daily
wheel-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).

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-plotted
actograms 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 other
treatment 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. The
shaded 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).

circadian regulation. This hypothesis would also be compatible
with the observation that the active flesinoxan dose in the present
study (2.5 mg/kg, s.c.) corresponds to approximately the same level
of 5-HT1A receptor occupancy (30e40%) reported by Leiser et al.
(2014) and probably a low 5-HT7 receptor occupancy with
SB269970 (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 circadian
phase (Fig. 6 e reproduced with permission from Westrich et al.
(2013)).
The fact that escitalopram alone does not affect circadian
rhythm (Westrich et al., 2013), while vortioxetine does, must be
ascribed to its multimodal action involving inhibition of the SERT
and direct effects at 5-HT receptors, in particular 5-HT1A, 5-HT1B
and 5-HT7 receptors, all 3 of which have been implicated in the
control of circadian rhythms (Graff et al., 2007; Lovenberg et al.,
1993; Pickard et al., 1999; Roca et al., 1993). Thus, several lines of
evidence support the hypothesis that 5-HT tone together with
activation/blockade of several 5-HT receptors can modulate circa-
dian rhythms, while blockade of the SERT (i.e., increase in 5-HT
tone) may not be sufficient to cause a measurable effect.
Taken together, our data consistently show that the effect of
vortioxetine 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's
effects on circadian rhythms. Since poor circadian synchrony has
been linked to maladaptive changes in unipolar and bipolar
depression (Wirz-Justice, 2003, 2006), antidepressants that target
key 5-HT receptor subtypes involved in circadian rhythm regula-
tion may have therapeutic utility. The putative impact of this
property of vortioxetine remains to be studied. The melatonin re-
ceptor agonist and 5-HT2C receptor antagonist agomelatine (Tardito
et al., 2012) is thought to exert its antidepressant activity, at least in
part, through regulation of circadian rhythms (Lanfumey et al.,
2013). Whereas vortioxetine likely affects mood and circadian
rhythms via different mechanisms than agomelatine, they may
overlap in their ability to restore the proper phase alignment be-
tween the SCN pacemaker and the lightedark cycle (Dallaspezia
and Benedetti, 2011; Duncan, 1996; Healy and Waterhouse, 1995;
Wirz-Justice, 2003, 2006), which is potentially disrupted during a
depressive episode. But a recent clinical study in which MDD
patients with an inadequate response to SSRI/SNRI monotherapy
were randomized to flexible-dose treatment with vortioxetine
(10e20 mg/day) or agomelatine (25e50 mg/day) showed that
vortioxetine 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 seen with the pro-cognitive agent donepezil, vortioxetine has
been shown to increase the time that rats spend investigating the
novel object in the 24-h object recognition test (Mork et al., 2013).
Using the same retention interval, the present study confirms that
acute 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 vortioxetine
in the object recognition task. Intriguingly, AS19 alone also exerts a
memory enhancing effect in the object recognition task. Since AS19
is a potent (subnanomolar in vitro potency) and selective 5-HT7
receptor agonist (Di Pilato et al., 2014), its effects on memory are
likely mediated by 5-HT7 receptors. This is indeed confirmed by the
fact that the 5-HT7 receptor antagonist SB269970 prevented the
enhancing 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) have
been reported to have memory enhancing or memory impairing
properties 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 receptors
has been proposed for which the acquisition phase requires a
silencing of 5-HT7 receptors while the consolidation phase requires
a high activation of 5-HT7 receptors (Freret et al., 2014). Similar to
the coupling of 5-HT1A and 5-HT7 receptors in the regulation of
circadian rhythms, a study by Eriksson et al. (2012) reported a
complex, brain region dependent crosstalk between 5-HT1A and 5-
HT7 receptors in the regulation of memory function (also see the
review by Meneses, 2014). Thus, while the 5-HT1A and 5-HT7 re-
ceptor agonist 8-OH-DPAT impaired memory function in a passive
avoidance learning test, co-administration of 8-OH-DPAT and the 5-
HT1A receptor antagonist NAD-299 facilitated memory (Eriksson
et 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
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 acute
administration 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 object
compared 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).

receptors, a change in signaling through one them would be likely
to affect the function of the other (Eriksson et al., 2012). As vorti-
oxetine is a 5-HT7 receptor antagonist and a 5-HT1A receptor
agonist (Mork et al., 2012) and vortioxetine at the dose tested oc-
cupies 5-HT1A as well as 5-HT7 receptors, it can be hypothesized
that AS19 interferes negatively with the complex interaction of
vortioxetine at 5-HT1A and 5-HT7 receptors through its interaction
at 5-HT7 receptors.
A limitation of our studies relates to the fact that vortioxetine
acts through multiple interdependent targets, which implies that
determination of the precise function at each of its individual tar-
gets is extremely difficult. Furthermore, target-specific potency
differences between rodents and human offers another layer of
complexity to our studies. As aforementioned, the in vitro affinity of
vortioxetine for rat 5-HT1A and 5-HT7 receptors is approximately
10-fold lower compared to the human receptors. In order to avoid
underestimating the effects of vortioxetine at these receptors, we
co-administered it with the 5-HT1A receptor agonist flesinoxan or
the 5-HT7 receptor antagonist SB269970. However, this approach is
only an approximation, since there are no available methods to
assess vortioxetine's occupancy at the 5-HT1A and 5-HT7 receptors
in the human brain. Taken together, the engagement of multiple
targets and the species differences add to the complexity and
interpretation of our research. To complement our pharmacological
studies, a future direction could be to study the effects of vortiox-
etine in genetically engineered mice with induced mutations, such
as knockout of a functional target, or expression of a human target
(e.g. 5-HT1A receptors). However, the ultimate understanding of
vortioxetine's effects on circadian rhythms and cognitive function
must come from clinical studies in humans.
5. Conclusion
Vortioxetine has the potential to modulate circadian rhythms by
prolonging the period length and producing a phase delay. In line
with published preclinical and clinical data vortioxetine has the
potential to positively affect memory function. Finally, the study
indicates that 5-HT7 receptors may be involved in these significant
effects on circadian rhythm and recognition memory in rodents.
The potential clinical implications of these preclinical findings
remain to be explored.
Acknowledgments
This research was supported by H. Lundbeck A/S and the Takeda
Pharmaceutical Company, Ltd.
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