1. The effects of combining serotonin reuptake inhibition and 5-HT7 receptor blockade
on circadian rhythm regulation in rodents
Ligia Westrich a,
⁎, Jeffrey Sprouse b
, Connie Sánchez a
a
Lundbeck Research USA, 215 College Avenue, Paramus, NJ 07652, USA
b
Sprouse Consulting, LLC, 513 East 82nd Street, New York, NY 10028, USA
H I G H L I G H T S
► Inhibition of serotonin transporter and 5-HT7 receptor lengthens circadian period.
► Serotonin transporter and 5-HT7 receptor blockade delay phase of circadian activity.
► 5-HT7 receptor activation modulates circadian period length.
a b s t r a c ta r t i c l e i n f o
Article history:
Received 18 May 2012
Received in revised form 1 August 2012
Accepted 20 December 2012
Available online 28 December 2012
Keywords:
Serotonin receptor
Serotonin transporter
Circadian rhythms
Suprachiasmatic nucleus
Period
PER2::LUCIFERASE
Disruption of circadian rhythms may lead to mood disorders. The present study investigated the potential ther-
apeutic utility of combining a 5-HT7 antagonist with a selective serotonin (5-HT) reuptake inhibitor (SSRI), the
standard of care in depression, on circadian rhythm regulation. In tissue explants of the suprachiasmatic nucleus
(SCN) from PER2::LUC mice genetically modified to report changes in the expression of a key clock protein, the
period length of PER2 bioluminescence was shortened in the presence of AS19, a 5-HT7 partial agonist. This
reduction was blocked by SB269970, a selective 5-HT7 antagonist. The SSRI, escitalopram, had no effect alone
on period length, but a combination with SB269970, yielded significant increases. Dosed in vivo, escitalopram
had little impact on the occurrence of activity onsets in rats given access to running wheels, whether the drug
was given acutely or sub-chronically. However, preceding the escitalopram treatment with a single acute dose
of SB269970 produced robust phase delays, in keeping with the in vitro explant data. Taken together, these find-
ings suggest that the combination of an SSRI and a 5-HT7 receptor antagonist has a greater impact on circadian
rhythms than that observed with either agent alone, and that such a multimodal approach may be of therapeutic
value in treating patients with poor clock function.
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
Virtually all organisms exhibit circadian rhythms. Highly conserved
processes are in place to ensure that the timing of daily internal rhythms
matches those of the solar day with the primary clock mechanism in
mammals originating in the suprachiasmatic nucleus (SCN). So-called
retinorecipient neurons receive direct retinal input and dictate the
timing of light to an adjacent population of pacemaker cells [1,2].
Other oscillators located within the brain, as well as those distributed
widely in the periphery, follow this lead in a highly orchestrated manner
[3,4]. The capacity to oscillate is the result of a sequence of transcription-
al/translational feedback loops within the oscillator cells with key pro-
teins (PER2, among others) cycling with a period length of 24 h [5–7].
Non-photic input can also serve as zeitgebers or “time-givers” to oscilla-
tor function and the SCN also receives input from a number of midbrain
and forebrain projections [8,9]. Of these, the serotonergic (5-HT) system
is of particular interest in view of its dense innervation of the SCN [10].
While a number of 5-HT receptors have been implicated in SCN func-
tion, the 5 HT7 subtype has received a great deal of attention. Evidence
for the presence of 5-HT7 receptors in the SCN is clear from anatomical
and functional studies [11–16], but the precise nature of their actions
is not fully understood [17]. Most experimental work suggests that
within the SCN 5-HT7 receptors have a primary role of limiting light-
induced changes in the phase of rest:activity rhythms [15,18]. Their
function in terms of daily adaptation to the prevailing light:dark cycle
is more obscure, although 5-HT7 receptor activation is known to lead
to advances in phase [16,19]. What is perhaps clearer is the potential
value of the 5-HT7 receptor as a target for novel therapeutic approaches
to mental disorders [20]. The concept of 5-HT7 receptor antagonists as
antidepressants, antipsychotics, sleep aids and cognitive enhancers has
received substantial experimental support [21–24] and, in fact, a 5-
HT7 antagonist/SSRI combination has been shown to enhance the in-
crease in cognitive ability observed with the antagonist alone [25]. To
the extent that 5-HT7 receptor-mediated modulation of circadian
Physiology & Behavior 110–111 (2013) 42–50
⁎ Corresponding author at: Fairleigh Dickinson University, School of Pharmacy, 230
Park Avenue, Florham Park, NJ 09732, USA. Tel.: +1 201 694 5752.
E-mail address: westrich@fdu.edu (L. Westrich).
0031-9384/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.physbeh.2012.12.009
Contents lists available at SciVerse ScienceDirect
Physiology & Behavior
journal homepage: www.elsevier.com/locate/phb

2. rhythm function underlies such clinical utility, a better understanding of
its action within the SCN might benefit the field.
Disruptions in circadian rhythm regulation are thought to lead to
increased risk of cardiovascular disease, cancer, diabetes and obesity,
among other health-related consequences [26–28], and there is a grow-
ing link to mood disorders [29]. Perception of mood follows a circadian
pattern in healthy individuals and loss of synchrony may lead to mal-
adaptive changes [30]. Unipolar and bipolar depression have been
most commonly linked to the circadian clock and a new generation of
antidepressants appears poised to take advantage of this connection
[31–33]. Perhaps more speculatively, the question arises as to the role
of traditional pharmacotherapies — the selective serotonin reuptake in-
hibitors (SSRIs) and the serotonin norepinephrine reuptake inhibitors
(SNRIs) — in modulating circadian rhythm function. Given their current
widespread use, there is a growing need to add to the handful of studies
that have examined these agents alone or in combination with selective
modulators that might fine-tune changes in net 5-HT neurotransmis-
sion [34–38].
5-HT7 receptors represent an interesting target in such multimodal
combinations. Selective antagonists of this receptor subtype appear to
be robustly active in behavioral measures of antidepressant-like re-
sponses when administered with an SSRI [39–41]. A combined 5-HT7
receptor antagonist/SSRI effect on cortical 5-HT release may underlie
these observations, although the nature of this interaction in other
brain regions is unknown. A goal of the present investigation was to
gain a better understanding of the respective roles of 5-HT7 receptors
and SSRIs in SCN function. To this end, tissue explants from mice genet-
ically modified to provide a bioluminescent measure of mPER2 cycling
were used as a measure of oscillator function, as determining the pat-
tern of clock gene expression has proven to be a useful means to mon-
itor changes in circadian period length and amplitude [9,42]. Wheel
running activity was used as a means to assess circadian behavior at
the whole animal level.
2. Materials and methods
2.1. Animals
Three to six-month-old male homozygous mPER2::LUCIFERASE
(PER2::LUC) knock-in mice [43] 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 Sprague–Dawley rats purchased from Charles River
Laboratories (Wilmington, MA) were housed individually with free ac-
cess to running wheels (model 80820, Lafayette Instruments, Lafayette,
IA) for the duration of the experiment. All animals were maintained
under controlled environmental 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. Institu-
tional Animal Care and Use Committee.
2.2. Drug treatments
For the in vitro studies, AS19, a reported 5-HT7 receptor partial ago-
nist [7,39], and SB269970, a 5-HT7 receptor antagonist [44] (Tocris Bio-
science, Ellisville, MO) were dissolved in DMSO and sterile water,
respectively; the SSRI escitalopram (H. Lundbeck A/S, DK) was dissolved
in sterile water. At the time of tissue treatment, drugs were diluted into
the culture media to final concentrations. For the acute and subchronic
in vivo treatments, drugs were dissolved as follows: AS19 in 20%
β-cyclodextrin (Sigma-Aldrich, St. Louis, MO), and SB267790 and
escitalopram in saline or 5% β-cyclodextrin. Prior to administration, pH
was adjusted toward pH 5–6 with 0.1 N NaOH; more physiological pH
values caused compounds to precipitate.
Drugs and vehicles were administered subcutaneously in a volume
of 2 ml/kg body weight to investigate the acute effects of serotonergic
modulation, or via osmotic minipumps (ALZET Osmotic Pumps, Cuper-
tino, CA) to investigate phasic effects. Once daily dosing was timed to
occur at either CT6 (Circadian Time 6) or ZT11 (Zeitgeber Time 11).
Prior to the dose, animals were switched from the prevailing 12:12 LD
cycle to a period of constant darkness beginning at either the time of
lights-on (ZT0, or 6 h before the “subjective” midday treatment at
CT6) or switched at the time of lights-off (ZT12 or shortly after the
ZT11 treatment during LD). This method was employed to minimize
light cues and thus allow the expression of endogenous rhythms. Ani-
mals were kept in constant darkness for approximately 10 days follow-
ing acute drug administration; 14 days of constant darkness were
allowed after implantation of osmotic minipumps.
Dosing of escitalopram at CT6 was chosen as earlier studies had
shown this time to be sensitive to serotonergic modulation [14,45]; dos-
ing at ZT11 was chosen to mimic the early morning dose commonly
used by patients and to be less disruptive to the normal sleep–wake
cycle of the rodents.
2.3. Tissue preparation
Tissue explants containing the SCN from PER2::LUC knock-in mice
were prepared according to published methods [43]. Briefly, animals
were anesthetized with isoflurane, brains were rapidly removed and
300 μm coronal sections through the SCN were cut on a vibratome
(Campden Instruments, Lafayette, IN) in chilled Hank's balanced salt so-
lution (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 (Sigma-Aldrich, 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 units/ml penicillin-G sodium (Invitrogen), 34 μM streptomycin sul-
fate (Invitrogen), and 100 μM beetle luciferin (Promega, Madison, WI,
USA) and were sealed with vacuum grease (Dow, Midland, MI) and
glass coverslip. Two SCN tissue sections were collected from a single an-
imal; 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 bio-
luminescence was collected for 60 s every 10 min with a commercial
luminometer (LumiCycle, Actimetrics, Wilmette, IL) before and after
treatments. 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. Bioluminescence traces were de-trended by baseline
subtraction of a 24-hour moving average and then smoothed with a
2-hour running average. De-trended and smoothed data were analyzed
for periodicity with a χ2 periodogram (Lumicycle Analysis software;
Actimetrics Inc., Evanston, IL). Chi-square tests for the period of Per2-
luc expression were considered significant at pb0.001. The period of
the PER2::LUC expression rhythms was determined with a sine-fit
curve [30]; mean values obtained in the control (vehicle) groups ranged
from 24.94±0.10 to 25.15±0.14 (Figs. 1 and 2), in keeping with nu-
merous published reports (e.g. [19,21,28]). A minimum of 3 days of bio-
luminescent data was included in the analysis. Period values were
compared with one-way analysis of variance (ANOVA) followed by
Newman–Keuls multiple comparison test. All comparisons were con-
sidered significant at pb0.05.
2.5. Locomotor activity rhythm monitoring
Daily wheel-running activity of male Sprague–Dawley rats was mon-
itored with Running Wheel Activity Software (AWM V12.0, Lafayette
43L. Westrich et al. / Physiology & Behavior 110–111 (2013) 42–50

3. BA
0 0.01 0.1 1 10
23
24
25
26
* **
AS19 (μM)
Periodlength(h)
23
24
25
26
*
Periodlength(h)
0 10 30 100 300
23
24
25
26
SB269970 (μM)
Periodlength(h)
AS19 (0.01 μM); τ=24.50 h
AS19 (0.01 μM) + SB269970 (100 μM); τ=25.44 h
Bioluminescence
(detrended,cps)
Time (days)
DC
Fig. 1. 5-HT7 receptor-mediated shortening of the period length of PER2 bioluminescence in tissue explants of suprachiasmatic nucleus (SCN) from mPER2::LUC transgenic male mice. (A) Con-
centration–response curve for AS19, a 5-HT7 receptor partial agonist on period length (τ) showing a significant shortening at the two lowest concentrations tested (mean±SEM; ANOVA, [F(4,
50)=5.448, p=0.0010], **pb0.001; *pb0.05 vs. control); (B) concentration–response for SB269970, a 5-HT7 receptor antagonist, revealing no significant changes in τ (mean±SEM; ANOVA,
[F(4, 34)=1.278, p=0.2979]); (C) period shortening by AS19 (0.01 μM) and its blockade by SB269970 (100 μM) (mean±SEM; ANOVA, [F(3, 28)=4.324, p=0.0126], *pb0.01 vs. control,
b0.05 vs. SB269970 alone); (D) representative records of the daily cycling of mPER2 bioluminescence in the presence of AS19 (0.01 μM; τ=24.50 h; black line) and the lengthening of τ in
the presence of the combination of AS19 (0.01 μM) and SB269970 (100 μM; τ=25.44 h; gray line).
23
24
25
26
27
*
Periodlength(hours)
Escitalopram (60 μM); τ=24.00 h
Escitalopram (60 μM) + SB269970 (100 μM); τ=25.50 h
Bioluminescence
(detrended,cps)
Time (days)
BA
Fig. 2. Combined dosing of a selective serotonin reuptake inhibitor (SSRI) and a 5-HT7 receptor antagonist on the period length of mPER2 bioluminescence in tissue explants of suprachiasmatic
nucleus (SCN) from mPER2::LUC. (A) Effect of escitalopram (60 μM) or SB269970 (100 μM) alone, revealing no significant change in τ but a significant lengthening following dosing with the
combination (mean±SEM; ANOVA, [F(3, 31)=4.978, p=0.0062], *pb0.05 vs. control and escitalopram alone). (B) Daily cycling of PER2 bioluminescence in the presence of escitalopram
(60 μM; τ=24.00 h; black line) and the lengthening of τ in the presence of the combination of escitalopram (60 μM) and SB269970 (100 μM; τ=25.50 h; gray line).
44 L. Westrich et al. / Physiology & Behavior 110–111 (2013) 42–50

4. Instruments, Lafayette, IN) via activity wheel counters (Model 86061)
interfaced with computers. 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 only for further analysis
by Clocklab software (Actimetrics). Cumulative activity was represented
in actograms from which activity onsets and associated circadian param-
eters were calculated. Least-squares lines were fitted to the onsets of
activity before and after dosing. The difference between the intersec-
tions of the two lines on the first day after treatment was used to calcu-
late the shift in phase (by convention a minus symbol (−) was used to
indicate delays and a positive symbol (+) to indicate advances). Statis-
tical analyses of the phase changes comprised an ANOVA followed by a
Newman–Keuls multiple comparison test. pb0.05 values were consid-
ered significant.
2.6. Functional assay for the rat 5HT7 receptor
The functional potency of SB269970 at the rat 5-HT7 receptor was de-
termined using a whole cell cAMP HiRange HTRF assay (Cisbio Bioassays,
Bedford, MA, two step protocol, 62AM6PEB). Briefly, polyclonal HEK-293
cell lines expressing the rat 5-HT7 receptor were re-suspended in stimu-
lation buffer (1× Hank's balanced salt solution with CaCl2 and MgCl2,
5 mM HEPES pH 7.4, 0.075% BSA) and counted on a Guava EasyCyte
(Millipore, Billerica, MA). In each well of a 384-well Optiplate, the fol-
lowing were added: 5 μl of cells (30,000 cells), 2.5 μl drug, 2.5 μl 5-HT,
5 μl of cAMP-D2 conjugate and 5 μl cryptate conjugate as recommended
by the kit manufacturer. Reconstituted anti-cAMP-cryptate and cAMP-
D2 lyophilized reagents were used at 1/5 dilutions using the lysis buffer
provided in the kit. Plates were incubated with SB269970 (8× stock) for
30 min (final concentration of 1 μM). The plates were then incubated for
30 min with 5-HT (at EC80 corresponding to 60 nM final) at 37 °C, and
an additional 10 min at room temperature. Following addition of the
conjugates, the plates were incubated for 1 h at room temperature be-
fore the cell cAMP content was measured using EnVision 2104 Multilabel
Reader (Perkin Elmer, Waltham, MA). Results were calculated from the
ratio of absorbance at 665 nm/620 nm and expressed in Delta F. Calcu-
lated Delta F {[(standard or sample ratio−ratio of negative control)/
ratio of negative control]×100} was used by interpolation from the stan-
dard curve to calculate cAMP concentrations. All data points were nor-
malized and analyzed by sigmoidal dose response curve-fitting using
Graph Pad Prism 4 to determine IC50. The Cheng–Prusoff equation was
used to calculate Kb: Kb=IC50/[1+(A/EC50)], where A=concentration
of reference agonist that is being inhibited and EC50=relative EC50 of the
reference agonist.
2.7. Bioanalysis
One hour following acute dosing of escitalopram and SB269970
brains were rapidly removed and frozen, while trunk blood was collect-
ed in EDTA-containing tubes and spun down to obtain plasma. Frozen
brains were weighed and homogenized in 3× (weight/volume) of ho-
mogenization buffer consisting of 50% water, 30% 2-propanol and 20%
DMSO. A 150 μl internal standard solution was added to 50 μl homoge-
nized brain/plasma sample. The samples were mixed and centrifuged.
The supernatant was injected directly into the ThermoFinnigam Quan-
tum Ultra LC/MS/MS (liquid chromatography–tandem mass spectrom-
etry) system for analysis.
3. Results
3.1. Combined effects of 5-HT7 receptor blockade and serotonin reuptake
inhibition on circadian rhythm regulation in SCN tissue explants from
mPER2::LUC mice
Incubation of SCN tissue explants with AS19, a selective 5-HT7 recep-
tor partial agonist, produced a shortening of the period (τ) of mPER2
bioluminescence by approximately 1 h at the two lower concentrations
tested (0.01 and 0.1 μM; pb0.05). In contrast, the two higher concentra-
tions (1 and 10 μM) yielded no significant change in τ (Fig. 1A). In a sep-
arate series of experiments, SB269970, a selective 5-HT7 receptor
antagonist, had no effect on τ within a 30-fold range of concentrations
(10–300 μM) (Fig. 1B). When dosed in combination with an active con-
centration of AS19 (0.01 μM), SB269970 (100 μM) reduced the effects
of the agonist on period shortening (Fig. 1C and D), since the value of
τ with the combination was not significantly different from control ex-
plants. For these experiments a high concentration of SB269970 was se-
lected to ensure complete blockade of 5-HT7 receptors in the presence of
AS19, a compound with a much higher affinity for this 5-HT receptor
subtype (Ki ~30 nM vs. 0.6–4.6 nM; [44,46,47]). Dampening of the bio-
luminescence amplitude over time following AS19 or SB269970 was not
apparent from visual examination of the records.
Incubation of SCN explants with escitalopram, a highly selective
serotonin reuptake inhibitor, yielded no change in PER2 expression
when dosed at a high concentration (60 μM) relative to its potency in
inhibiting 5-HT transport in vitro (IC50 =2.1 nM; [7]) (Fig. 2). When
escitalopram (60 μM) was co-incubated with SB269970 (100 μM),
however, there was a lengthening of τ by approximately 1 h, a value sig-
nificantly greater in magnitude than with either agent alone (Fig. 2A and
B; pb0.05).
3.2. Combined effects of 5-HT7 receptor blockade and serotonin reuptake
inhibition on circadian rhythm regulation in freely behaving rats with
access to running wheels
Plotting of wheel running behavior into daily actograms revealed
evidence of entrainment of the normal rest:activity rhythms to the pre-
vailing light:dark cycle (12:12 LD) within 2 weeks of exposure to the
wheels. Shortly thereafter, a single dose of escitalopram (2.5 mg/kg
s.c.) was given at ZT11 and the animals were released into constant
darkness (DD) at the time of lights-off on the day of dosing (Fig. 3A).
The switch to DD, in this instance, was incorporated into the paradigm
so as to limit the masking effects of light that normally limit locomotor
behavior. Extrapolation of the activity onsets during DD back to the time
of escitalopram dosing yielded no change in phase (ΔΦ=−0.59±0.12
h; p>0.05). Similarly timed dosing with SB269970 (10 mg/kg s.c.)
alone also produced no change in the occurrence of activity onsets
(Fig. 3C).
When the two agents were combined in paired injections at ZT11, a
significant phase delay emerged, in keeping with the period lengthening
observed with the combination in vitro (Fig. 3B and C). The magnitude
of this delay with the dosed combination was significantly different
from that observed with vehicle control or either of the active agents
alone (pb0.05). Dosing timed to occur at CT6 (or circadian time 6, dur-
ing DD, but 6 h after lights-on would have normally occurred) yielded
very similar results as the ZT11 dosing (Fig. 3D).
Subchronic dosing with escitalopram in DD (10 mg/kg/day s.c. via
osmotic minipumps for 14 days) yielded no significant change in
wheel running onsets (Fig. 4C). A single dose of SB269970 (30 mg/kg
s.c.), timed at ZT11 at the beginning of subchronic escitalopram dosing,
produced a significant delay in activity onset (pb0.05; Fig. 4B and C). No
changes in the occurrence of phase were observed with the SB269970
dosing that preceded subchronic dosing of vehicle in DD (Fig. 4A and
C). Although free-running periods were not examined in all animals
prior to minipump implantation, in those experiments for which re-
cords are available (e.g., Fig. 4A and B), there appeared to be a lengthen-
ing of period due to the escitalopram/SB269970 combination.
3.3. Exposures of escitalopram and SB269970 achieved with in vivo dosing
to rats
A satellite group of rats dosed with escitalopram (2.5 mg/kg s.c.) to
mimic the acute experiments on rest:activity rhythms achieved high
45L. Westrich et al. / Physiology & Behavior 110–111 (2013) 42–50

5. brain levels of drug 1 h post dose (Fig. 5A). Transporter occupancies at
this exposure were also high, exceeding 90% based on displacement of
radiolabeled compound, [3
H]DASB (Alan Pehrson, personal communi-
cation); occupancies following sub-chronic dosing of escitalopram via
minipumps (10 mg/kg/day s.c.) were equally robust (data not shown).
Brain exposures 1 h following acute dosing with SB269970 (1–
30 mg/kg s.c.) were low relative to that observed in plasma (Fig. 5B).
Such levels may be sufficient to achieve significant 5-HT7 receptor occu-
pancy, however, based on the potency of SB269970 in reducing agonist-
elicited increases in cAMP production in HEK293 cells (IC50=2.7 nM or
0.95 ng/ml; Ki =0.3 nM or 0.11 ng/ml; Fig. 5C).
4. Discussion
Serotonergic modulation of circadian rhythms has been extensively
studied in a number of experimental paradigms. In vitro approaches
have ranged from monitoring the electrical activity of cultured SCN
slices to bioluminescence measures in PER::LUC explanted tissues (as
in the present work); in vivo approaches have included work in rats,
mice and hamsters, measuring both the effects on period length and
on light-induced shifts in behavioral onsets. Taken as a whole, the re-
sults have been mixed with some research groups providing ample ev-
idence of rhythm regulation (e.g. [35,37]) and others, surprisingly little
(e.g. [48,49]). Studies that have focused more closely on the effects of
antidepressants (citalopram, paroxetine, venlafaxine, fluvoxamine, flu-
oxetine, sertraline) [35,37,38,50] constitute only a small subset of the
total. A persistent question that underlies much of this work can be
simply stated: Why would the SCN, which receives dense serotonergic
innervation involving a host of 5-HT receptor subtypes, be so resistant
to the changes in tone afforded by the selective reuptake inhibitors?
As the results of the present study suggest, the answer may lie in the
mix of receptors activated indirectly by reuptake blockade.
Monitoring the oscillation in clock gene expression, as enabled by
the SCN explants from the mPER2::LUC mice, is a simple means to
detect changes in various circadian parameters [9,42]. As shown in
Fig. 1, the effect of the 5-HT7 receptor partial agonist AS19 on the period
length of mPER2 oscillation was concentration-dependent at lower
doses (b0.1 μM), yielding a shortening of period, with higher doses
(>0.1 μM) having little apparent effect. A shortening was expected —
earlier work monitoring the effect of 8-OH-DPAT, a mixed 5-HT1A/7 re-
ceptor agonist, on the firing rate of cells within cultured SCN slices
showed phase advances, an effect sensitive to 5-HT7 receptor antago-
nists [51]. The reasons for the loss of a period-shortening effect when
AS19 was given at higher concentrations are not known, but may be re-
lated to its level of intrinsic efficacy at the 5-HT7 receptor or its selectiv-
ity relative to other 5-HT receptor subtypes. As to the former, AS19 is
reported to be a 5-HT7 receptor partial agonist (77% intrinsic efficacy;
[46,47]) and such a profile might add to the existing degree of 5-HT
tone at low concentrations of the partial agonist, but detract from it as
exposure rises and its low intrinsic activity prevails. Alternatively, the
reported affinity of AS19 for 5-HT1A receptors (Ki ~75 nM vs. 4.6 nM;
[39]) might mitigate its action at 5-HT7 receptors, as the two subtypes
have opposing effects on intracellular signaling cascades [16,52]. Re-
gardless of the mechanisms that shape the concentration–response
A
ΔΔ Φ = -1.50h
Escitalopram
and SB267790
B
Δ Φ = -0.25h
Escitalopram
Constant darkness
-2.0
-1.5
-1.0
-0.5
0.0
0.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
*
Phasechange(hours)
C
*Phasechange(hours)
D summarized data at CT6summarized data at ZT11
Fig. 3. Combined dosing of a selective serotonin reuptake inhibitor (SSRI) and a 5-HT7 receptor antagonist on daily running wheel behavior as revealed in double-plotted actograms. (A) Rep-
resentative actogram showing the effect of a single acute injection of escitalopram (2.5 mg/kg s.c.) at ZT11 (zeitgeber time 11 or 11 h after lights-on or 1 h before lights-off in a 12:12 light:dark
(LD) cycle) following entrainment to the LD cycle as calculated by extrapolation of activity onsets in constant darkness (DD) back to the time of dosing (change in phase (ΔΦ)=−0.25 h); (B)
effect of combined doses of escitalopram (2.5 mg/kg s.c.) and SB269970 (10 mg/kg s.c.) at ZT11, yielding a more substantial phase change in activity onsets (ΔΦ=−1.50 h); (C) summarized
data with all injections occurring at ZT11 and revealing a significantly greater phase delay following combined dosing with escitalopram and SB269970 (mean±SEM; ANOVA, [F(3, 21)=7.797,
p=0.0011]; *pb0.001 vs. vehicle; b0.05 vs. all other treatment groups); (D) summarized data with all injections occurring at CT6 (circadian time 6, during DD but 6 h after lights-on would have
normally occurred) (mean±SEM; ANOVA, F(3, 12)=3.950, p=0.0358; *pb0.05 vs. vehicle and SB269970 alone).
46 L. Westrich et al. / Physiology & Behavior 110–111 (2013) 42–50

6. curve, the effect of AS19 at the lower concentrations (0.01 μM) is likely
mediated by 5-HT7 receptors as the decrease in period length was re-
duced by SB269970, a 5-HT7 receptor antagonist. Confirmation of this
effect at a lower concentration of SB269970 would strengthen this argu-
ment as would blockade by a second, structurally unrelated, 5-HT7
antagonist.
Having thus defined a 5-HT7 receptor-active dose of SB269970 in the
in vitro explant paradigm, it is interesting to note that blockade of this
receptor subtype does not, by itself, elicit a change in period length, find-
ings similar to those reported previously [53]. One possible explanation
for this lack of an effect again relates to 5-HT tone in the explant culture,
presumably too low for an antagonist to yield a sufficient change in neu-
rotransmission. When tone is increased by the presence of an SSRI, the
combination leads to a significant increase in period length, the opposite
of that observed with AS19. In this sense, the present findings are con-
sistent with reports of 5-HT7-receptor-mediated phase advances [16]
and suggest that an antagonist may have effects as well if tone is suffi-
ciently high. Conversely, the lack of an effect by an antagonist might
be a reasonable sign that serotonin tone is endogenously (or exogenous-
ly) low. It is surprising that escitalopram when given alone was ineffec-
tive in modulating period length as previous studies had shown a
period-shortening effect for SSRIs in tissue culture [37]. The differences
reported here might suggest a distinguishing property of escitalopram
as the earlier work had chosen to focus primarily on sertraline. Alterna-
tively, the differences in experimental protocols (primarily rat-1 fibro-
blasts as in the case of [25] vs. SCN explants in the present study;
mPER1 expression in [37] vs. mPER2 in the present study) might have
had their impact.
Such observations in the in vitro explant system also appear to hold
when roughly equivalent studies are conducted in vivo. Acute doses of
SB269970 (10 mg/kg s.c.) given at ZT11 yielded no significant change
in activity onsets as the animals entered 10 days of constant darkness
(Fig. 3A). Escitalopram also produced no discernable change in the
phase of locomotor rhythms at the dose tested (2.5 mg/kg s.c.). Dosing
at ZT11 was chosen to mimic the morning dose commonly used by
patients and to be less disruptive to the normal sleep–wake cycle of noc-
turnal animals. That a phase change could have been observed experi-
mentally is confirmed by earlier work by our group employing 8-OH-
DPAT [54]. In contrast to single dosing, the net effect of combining
escitalopram and SB269970 amounted to a phase delay larger in magni-
tude than any other treatment group, essentially recapitulating the in
vitro explant results and generalizing such results across (mouse and
rat) species. Dosing the combination at CT6, a time noted by others to
produce a robust effect on phase with 5-HT agonists [45,55] produced
nearly identical results (Fig. 3D) as did sub-chronic inhibition of the
5-HT transporter combined with acute blockade of the 5-HT7 receptor
(Fig. 4). Based on these observations, the effect of phasic and tonic sero-
tonergic modulation appeared to be much the same. The modest effect
of chronic SSRI dosing on period length as reported by others [14] was
not observed, possibly due to differences in exposure. Decreases in
5-HT7 receptor sensitivity that might occur early on as a result of contin-
uous transporter inhibition [56] did not appear to be sufficient to alter
BA
-1.5
-1.0
-0.5
0.0
0.5
*
Phasechange(hours)
Saline
SB269970
Saline Pump
x 14 days
Constant darkness
ΔΔ Φ = -0.45h
Saline
SB267790
Escitalopram
Pump x 14 days
Δ Φ = -0.98h
C
Fig. 4. Subchronic dosing of an SSRI combined with a single acute dose of a 5-HT7 receptor antagonist on daily running wheel behavior as revealed in double-plotted actograms. (A) Represen-
tative actogram showing the effect of a single acute dose of SB269970 at ZT11 followed by subchronic vehicle dosing via minipump during constant darkness (DD) and revealing no apparent
change in the phase of activity onsets (ΔΦ=−0.45 h); (B) representative actogram showing the effect of a single acute dose of SB269970 at ZT11 followed by subchronic escitalopram dosing
via minipump (10 mg/kg/day s.c.) during DD and revealing a more substantial phase delay (ΔΦ=−0.98 h); (C) summarized data showing a significantly greater phase delay following com-
bination dosing (mean±SEM; ANOVA [F(3, 11)=5.438, p=0.0154]; *pb0.05 vs. vehicle and all other treatment groups). Note that in the two actograms shown (A & B) an earlier period of
constant darkness (DD) enabled an assessment of free-running period length both prior to and after drug treatment (although this was undertaken in too few animals for statistical analyses). In
each case, the period of DD was preceded by a dose of saline at CT6 as part of pilot work for later studies. The summary data (C) are derived only from the periods of active drug treatments.
47L. Westrich et al. / Physiology & Behavior 110–111 (2013) 42–50

7. activity onsets. In other words, the decrement in 5-HT7 receptor-specific
neurotransmission that might follow desensitization of this subtype was
modest compared to that achieved with pharmacologic antagonism.
Given the high exposures achieved and the evidence of near com-
plete transporter/receptor occupancy, it is tempting to infer synergy
(as opposed to additivity) in interpreting our results. The precise mech-
anism that would account for synergy between 5-HT transporter inhibi-
tion and 5-HT7 receptor blockade is not known. At best, one can note that
the net effect of an increase in 5-HT tone afforded by an SSRI alone is
modest in terms of circadian rhythm regulation and only becomes im-
portant when the 5-HT7 receptor subtype is no longer functional, that
is, when tone is increased on all of the 5-HT receptor systems with the
exception of 5-HT7. Enhanced increases in net 5-HT release have been
reported for citalopram when it is combined with SB269970 [39], but
the exact nature of this interaction is not known, nor is its potential im-
portance within the SCN. In fact, previous attempts to impact the sup-
pression of light-induced phase advances by citalopram failed to find a
role for SB269970 or 5-HT7 antagonism [36].
The range of 5-HT receptor subtypes that might be activated in the
peri-SCN by an SSRI is impressive — 5-HT1A, 5-HT1B, 5-HT2C, 5-HT3,
5-HT5A, 5-HT7 — and this list essentially expands to include all of the
subtypes when inputs to and outputs from the SCN are factored in
[14,17,51,55,57,58]. There is also the possibility that the site of action
may be some distance from the SCN. For example, the 5-HT-containing
neurons of the raphe nuclei provide substantial input to the SCN and ac-
tivation/blockade of 5-HT7 cell body autoreceptors can impact non-
photic phase-resetting responses [13,59,60]. In either case, the 5-HT7 re-
ceptor would appear to oppose the actions of the other 5-HT receptor
subtypes, such that limiting its function with an antagonist would disin-
hibit some or all of the remaining subtypes. Situated postsynaptically on
cell bodies, the 5-HT7 receptor subtype is unique in being positively
coupled to cAMP production and to that extent may oppose, for exam-
ple, 5-HT1A receptors in terms of intracellular second messenger cas-
cades as noted earlier [16]. An interesting follow-up study might be to
incorporate a 5-HT1A receptor antagonist in combination with AS19 to
determine whether the resulting loss of 5-HT1A receptor function adds
to (or synergizes with) the effect size observed with the 5-HT7 receptor
agonist alone.
The potential clinical advantages of a combined SSRI plus 5-HT7
receptor antagonist are interesting to consider. Circadian rhythm distur-
bances in patients that lead to poor synchrony and an altered mood
state would naturally represent a primary target. The prevailing theory
purports that an abnormal phase relationship between the circadian
system and sleep could be involved in the pathogenesis and in the main-
tenance of depression such that a delay in circadian rhythms (or an ad-
vance in the sleep–wake cycle) might effectively treat mood symptoms
[33,61]. In fact, sleep phase advancement has been shown to alleviate
depressive mood in the majority of patients after 2–3 weeks of therapy
[62]. Theoretically, a delay in the circadian phase to achieve proper
alignment (that did not appreciably alter sleep onset) would also
improve patient symptoms. An important caveat with respect to the
present findings is the artificial nature of the LD cycle employed: where-
as the effects on phase were examined under the constant darkness in
the animal studies, patients obviously experience normal LD cycles.
The actions of SSRIs in this subset of patients would likely be minimal
in light of the present studies but might rise in importance in combination
with a 5-HT7 receptor antagonist. In terms of reported clinical findings,
the atypical antipsychotics risperidone and olanzapine have been
shown to improve the antidepressant potential of SSRIs in unipolar and
bipolar depressed patients [63–67] with possible mechanisms relating
to their actions at a variety of receptors, most often the 5-HT2A receptor
subtype. 5-HT7 receptor binding is also typical of this class of agents
[23,68] and efficacy related to this aspect of their overall pharmacology
might also reveal important synergies in patients. On this last note, it is in-
teresting that olanzapine addition to SSRI therapy in treatment-resistant
patients has been shown to improve sleep continuity along with depres-
sive symptoms [69], an action that might be closely linked to SCN function
given its role in sleep regulation [26,31]. Certainly, many receptor
A
0
100
200
300
400
500
0
1000
2000
3000
4000
5000
1 10 30
SB269970 (mg/kg)
BrainExposure(ng/g)
PlasmaConcnetration(ng/ml)
Brain
Plasma
B
2.5 2.5
Escitalopram(mg/kg)
0
1000
2000
3000
4000
5000
BrainExposure(ng/g)
0
250
500
750
1000
PlasmaExposure(ng/ml)
Brain Plasma
-12 -11 -10 -9 -8 -7 -6 -5 -4
0
25
50
75
100
Log[SB269970], M
%cAMPresponsein
5-HT7R-HEK293cells
C
Fig. 5. Brain and plasma exposures of drugs following in vivo dosing to satellite groups of rats. (A) Levels of escitalopram in brain and plasma 1 h following a single acute dose (2.5 mg/kg s.c.;
mean±SEM, N=8); (B) levels of SB269970 in brain and plasma 1 h following single acute doses (1–30 mg/kg s.c.; mean±SEM, N=4–8); (C) potency of SB269970 in blocking
agonist-stimulated increases in cAMP production in HEK293 cells (N=3).
48 L. Westrich et al. / Physiology & Behavior 110–111 (2013) 42–50

8. subtypes may account for the effects observed with compounds such as
olanzapine that involve multiple and complex pharmacologies, but the
present findings suggest a new and potentially important role for 5-HT7
receptors in therapies designed to improve mood.
In conclusion, the combination of an SSRI and a 5-HT7 receptor
antagonist has the potential to produce an effect on circadian rhythm
regulation greater in magnitude than that observed with either agent
alone. Such a multimodal approach, by targeting many but not all 5-
HT receptor subtypes for an improvement in neurotransmission, may
yield changes in clock function that serve to restore proper rhythms in
those patients suffering from poor synchrony.
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