Postnatal Fluoxetine-Evoked Anxiety Is Prevented by Concomitant 5-HT2A/C Receptor Blockade and Mimicked by Postnatal 5-HT2A/C Receptor Stimulation

 
ARCHIVAL REPORT
Postnatal Fluoxetine-Evoked Anxiety Is Prevented by
Concomitant 5-HT2A/C Receptor Blockade and
Mimicked by Postnatal 5-HT2A/C Receptor Stimulation
Ambalika Sarkar, Parul Chachra, and Vidita A. Vaidya
Background: Postnatal treatment with the selective serotonin reuptake inhibitor fluoxetine, evokes anxiety and depressive behavior in
rodent models in adulthood. We examined the role of serotonin 2A (5-HT2A), serotonin 2C (5-HT2C) and serotonin 1A (5-HT1A) receptors,
implicated in the development of anxiety, in the behavioral consequences of postnatal fluoxetine (PNFlx).

Methods: Control and PNFlx rat pups received concomitant treatment with the 5-HT2A/C receptor antagonist, ketanserin, the 5-HT2A
receptor antagonist, MDL100907, the 5-HT2C receptor antagonist, SB242084, or the 5-HT1A receptor antagonist, WAY-100635, and were
tested for behavior in adulthood. The effect of postnatal treatment with the 5-HT2A/C receptor agonist, DOI, on anxiety behavior was
examined in adulthood.
Results: Postnatal 5-HT2A/C receptor blockade prevented PNFlx-evoked anxiety, attenuated depressive behavior, and normalized specific
gene expression changes in the prefrontal cortex. Postnatal, selective 5-HT2A receptor antagonist treatment blocked PNFlx-evoked
anxiety and depressive behavior, whereas 5-HT2C receptor antagonist treatment prevented anxiety but not depressive behavior.
Postnatal 5-HT2A/C receptor stimulation was sufficient to evoke anxiety in adulthood. Serotonin 1A receptor blockade did not alter PNFlx-
evoked anxiety but resulted in anxiety in control animals, an effect attenuated by concomitant 5-HT2A/C receptor blockade.

Conclusions: Postnatal fluoxetine-evoked anxiety and depressive behavior, as well as specific gene expression changes in the prefrontal
cortex, were prevented by 5-HT2A/C receptor blockade. Adult anxiety was evoked by either 5-HT2A/C receptor stimulation or 5-HT1A receptor
blockade of naive control pups. Our findings implicate serotonin 2 receptors in the development of perturbed emotionality following PNFlx
and suggest that an altered balance of signaling through 5-HT1A and 5-HT2A/C receptors in early life influences anxiety behavior.

                                                                              behavioral effects of PNFlx. Serotonergic neurotransmission is
Key Words: Antidepressant, DOI, 5-HT1A, 5-HT2A, 5-HT2C,                       mediated via a large family of 14 receptor subtypes that exhibit
ketanserin, Prozac, SSRI, WAY-100635                                          distinct developmental expression patterns and functional
                                                                              responses (21–23). Genetic perturbations and pharmacologic
                                                                              studies in rodent models indicate an important role for 5-HT1A,

S
      erotonin (5-HT) neurotransmission influences the develop-
      ment of anxiety (1). Perturbations of postnatal 5-HT levels in          5-HT2A, and 5-HT2C receptors in the development of anxiety
      rodents evoke lasting consequences on emotionality (2). The             (2,24–34). Cortical 5-HT2A receptor knockouts (27) and loss of
selective serotonin reuptake inhibitor (SSRI), fluoxetine, is among            5-HT2C receptor function (28) exhibit anxiolytic effects. Serotonin
the first-line therapies for gestational, postpartum, childhood, and           1A receptors, besides their postsynaptic expression, also serve as
adolescent depression, given its favorable side-effect profile (3–6).          presynaptic autoreceptors that regulate 5-HT neuron firing and
However, clinical evidence links perinatal SSRI exposure to neonatal          release (35,36). Conditional, forebrain-specific 5-HT1A heterorecep-
serotonin withdrawal symptoms and disrupted neurobehavioral                   tor, or raphe 5-HT1A autoreceptor, loss of function during postnatal
development (7–9), and childhood and adolescent fluoxetine                     life, as well as postnatal pharmacologic 5-HT1A receptor blockade,
treatment is associated with enhanced anxiety, depression, and                evoke enhanced anxiety in adulthood (25,29–33). We hypothe-
suicidal ideation (10,11). Postnatal fluoxetine (PNFlx) treatment in           sized that PNFlx-evoked anxiogenic responses involve a key role
mouse models, considered equivalent to the third trimester of                 for 5-HT1A and 5-HT2A/5-HT2C (5-HT2A/C) receptors, strongly impli-
human gestation (12), results in enhanced anxiety and depressive              cated in the development of anxiety. Using studies in rat models,
behavior that persist across the life span (11,13–15). This is para-          we show that 5-HT2A/C receptor blockade prevents PNFlx-evoked
doxical to the anxiolytic and antidepressant responses observed in            anxiety and attenuates depressive behavior. Our results indicate
humans (16) and in rodents (17), following fluoxetine administra-              that postnatal 5-HT2A/C receptor stimulation evokes adult anxiety,
tion in adulthood. Perturbations of 5-HT levels evoke differing con-          and the anxiogenic effects of postnatal 5-HT1A receptor antagonist
sequences on mood behavior based on the temporal window (11).                 treatment are attenuated by concomitant 5-HT2A/C receptor block-
                                                                              ade. Our findings implicate 5-HT2A/C receptors in the development
    The temporal window of postnatal life is critical for defining the
                                                                              of perturbed emotionality following PNFlx and suggest that the
trajectory of emotional development (18–20). It is important to
                                                                              balance between 5-HT1A and 5-HT2A/C receptor-driven signaling
unveil the role of specific 5-HT receptors in mediating the
                                                                              within the postnatal temporal window could play an important
                                                                              role in the establishment of baseline anxiety states.
From the Department of Biological Sciences, Tata Institute of Fundamental
   Research, Mumbai, India.
Authors AS and PC contributed equally to this work.                           Methods and Materials
Address correspondence to Vidita A. Vaidya, Ph.D., Tata Institute of Funda-
   mental Research, Department of Biological Sciences, 1, Homi Bhabha         Animals
   Road, Colaba, Mumbai 400005, India; E-mail: vvaidya@tifr.res.in.             Male Sprague-Dawley rats bred in the Tata Institute of Funda-
Received Apr 5, 2013; revised and accepted Nov 1, 2013.                       mental Research animal facility and maintained on a 12-hour

0006-3223/$36.00                                                                                          BIOL PSYCHIATRY 2014;76:858–868
http://dx.doi.org/10.1016/j.biopsych.2013.11.005                                                        & 2014 Society of Biological Psychiatry
A. Sarkar et al.                                                                       BIOL PSYCHIATRY 2014;76:858–868 859

light-dark cycle with ad libitum access to food and water were       using the Ethovision tracking system (Noldus, Wageningen,
used for all experiments. Experimental procedures followed the       Netherlands). We assessed total distance covered within the
National Institute of Health Guide for the care and use of animals   arena, path length, and time spent in the center; number of
and the Committee for the Purpose of Control and Supervision         visits to the center; and number of rears in the arena. Elevated
of Experimental Animals and were approved by the Tata Institute      plus maze analysis for anxiety behavior (40) was performed for
of Fundamental Research Animal Ethics Committee (56/1999/            the following experiments: PNFlx ⫹ Ket and PNDOI. The EPM
CPCSEA).                                                             consisted of a platform elevated 50 cm from the ground with two
                                                                     open and closed arms (45  10 cm). Animals were released into
Drug Treatments                                                      the center of the maze facing the open arm, and behavioral
   Litters from primiparous dams were randomly assigned to           measures were assessed for 5 minutes. Analysis was performed
control (Ctrl) or test groups, with each treatment group contain-    for total distance covered within the maze, path length, and time
ing pups from two or more litters. Pups were weaned at               spent in the open and closed arms and the number of rears in the
postnatal day (P)28, and male pups were group housed into            maze. Forced swim test analysis for depressive behavior (41) was
adulthood. Pups received oral administration via a feeding           performed for the following experiments: postnatal ketanserin,
needle of fluoxetine (10 mg/kg; Sigma-Aldrich, St. Louis, Missouri)   MDL100907, or SB242084 with PNFlx. A modified version of the
or vehicle (5% sucrose, Sigma-Aldrich) once daily from P2 to P21,    FST involved habituation on day 1 for 15 minutes to a plastic
within 3 minutes to minimize handling stress. To examine the         cylinder (50 cm tall, 21 cm in diameter) filled with water, followed
influence of concomitant 5-HT2A/C, 5-HT2A, 5-HT2C, or 5-HT1A          by testing on day 2 for 5 minutes. Video recordings were assessed
receptor blockade on the behavioral consequences of postnatal        for immobility time, latency to immobility, and number of
fluoxetine, pups received daily pretreatment with the 5-HT2A/C        immobility events by an investigator blind to the treatment
receptor antagonist, ketanserin tartarate (5 mg/kg, Sigma-           groups.
Aldrich), the selective 5-HT2A receptor antagonist, MDL100907
(1 mg/kg; Toronto Research Chemicals, North York, Ontario,           Quantitative Polymerase Chain Reaction Analysis
Canada), the selective 5-HT2C receptor antagonist, SB242084 (.5          Quantitative polymerase chain reaction analysis was per-
mg/kg, Sigma-Aldrich), the selective 5-HT1A receptor antagonist,     formed on RNA extracted from prefrontal cortex using the Trizol
WAY-100635 (.5 mg/kg, Sigma-Aldrich), or vehicle (5% sucrose         method (Trizol, Sigma-Aldrich) in the following experiment: Ctrl,
solution) orally, 30 minutes before vehicle or PNFlx treatment       PNFlx, Ket, PNFlx ⫹ Ket (n ¼ 7–12 per group) in adulthood.
daily from P2 to P21. Behavioral testing on the open field test       Complementary DNA synthesis with the High Capacity Comple-
(OFT), elevated plus maze (EPM), and forced swim test (FST) was      mentary DNA Reverse Transcription Kit (Applied Biosystems,
performed in adulthood. Treatment groups were 5-HT2A/C recep-        Foster City, California) was performed on RNA (2 μg/sample)
tor blockade experiment: Ctrl, PNFlx, ketanserin (Ket), and PNFlx    following RNA quality assessment by analysis of the 260/280
⫹ Ket (OFT, EPM: n = 6–12 per group; FST: n = 5–11 per group);       optical density ratio using Nanodrop (Eppendorf, Hamburg,
5-HT2A receptor blockade experiment: Ctrl, PNFlx, MDL100907          Germany). Quantitative polymerase chain reaction was performed
(MDL), and PNFlx ⫹ MDL (OFT: n = 7–13 per group; FST: n = 7–9        with primers for the genes of interest (Table S1 in Supplement 1).
per group); 5-HT2C receptor blockade experiment: Ctrl, PNFlx,        Quantitation was performed using the ΔΔCt method as previ-
SB242084 (SB), and PNFlx ⫹ SB (OFT: n = 6–11 per group; FST: n       ously described (42) and data were normalized to hypoxanthine-
= 7–11 per group); 5-HT1A receptor blockade experiment: Ctrl,        guanine phosphoribosyltransferase 1 (Hprt1).
PNFlx, WAY-100635 (WAY), and PNFlx ⫹ WAY (OFT: n = 5–7 per
group). To examine the influence of chronic postnatal treatment       Statistical Analysis
with the 5-HT2A/C receptor agonist, DOI (2,5-dimethoxy-4-iodo-           Experiments with two groups were analyzed using the unpaired
amphetamine), on anxiety behavior in adulthood, pups received        Student t test and experiments with three or four groups were
DOI (2 mg/kg, Sigma-Aldrich) orally from P2 to P21 (PNDOI) or        analyzed with one- or two-way analysis of variance (ANOVA),
vehicle (5% sucrose) (Ctrl) (n = 6–8 per group). To assess the       respectively, followed by the Bonferroni post hoc test. Parametric
influence of concomitant 5-HT2A/C receptor blockade on anxiety        distribution of data was assessed using the Kolmogorov-Smirnov
behavior following postnatal 5-HT1A receptor antagonist treat-       test. Significance was determined at p ⬍ .05 (Instat and Prism,
ment, pups received vehicle (5% sucrose solution) or ketanserin      Graphpad Software Inc., San Diego, California).
30 minutes before administration of WAY100635 and the treat-
ment groups were Ctrl, WAY, and WAY ⫹ Ket (n = 8–9 per
group). Drug doses were selected based on prior reports              Results
(13,25,26,37,38).
                                                                     Postnatal 5-HT2A/C Receptor Antagonist Treatment Prevents
Behavioral Tests: Open Field, Elevated Plus Maze, and Forced         the Development of PNFlx-Evoked Adult Anxiety
Swim Test                                                                We examined whether concomitant 5-HT2A/C receptor antag-
    Animals were subjected to behavioral tests in adulthood. Open    onist treatment altered the development of adult anxiety follow-
field test analysis for anxiety behavior (39) was performed for the   ing PNFlx on the OFT and EPM (Figure 1A–I). Postnatal fluoxetine
following experiments: postnatal ketanserin, MDL100907,              treatment enhanced anxiety behavior on the OFT (Figure 1B–E) as
SB242084, or WAY100635 with PNFlx, postnatal DOI, and post-          indicated by decreased percent path length, percent time spent,
natal ketanserin with WAY100635 treatment. For the OFT, the          and number of visits to the center of the arena. Postnatal
arena (100 cm  100 cm  70 cm) was placed in a dimly                fluoxetine-evoked anxiety on the OFT was completely prevented
illuminated room, and behavioral tests were carried out within       by concomitant ketanserin treatment (Figure 1B–E). Two-way
the light phase. Animals were released at one corner of the arena    ANOVA analysis indicated a significant PNFlx  Ket interaction
and exploratory behavior was recorded with an automated              for percent path length traversed (F1,31 ¼ 6.78, p ¼ .014), percent
tracking system for 5 minutes. All behavioral tracks were analyzed   time (F1,31 ¼ 5.99, p ¼ .02), and number of visits (F1,31 ¼ 6.34, p ¼

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860 BIOL PSYCHIATRY 2014;76:858–868                                                                                                       A. Sarkar et al.

Figure 1. Postnatal serotonin 2A/serotonin 2C receptor antagonist treatment prevents the development of postnatal fluoxetine-evoked adult anxiety.
Shown is a schematic representation of the treatment paradigm (A). Control (Ctrl) and postnatal fluoxetine-treated (PNFlx) pups received vehicle (Veh) or
ketanserin (Ket) administration daily from postnatal day (P)2 to P21 and were assessed for anxiety behavior on the open field test (OFT) (P90) and elevated
plus maze (EPM) (P100) and in a separate cohort for depressive behavior on the forced swim test (FST) (P90). Shown are representative tracks on the OFT
from adult Ctrl, PNFlx, Ket, and PNFlx ⫹ Ket animals (B). Postnatal Ket treatment prevented the PNFlx-evoked anxiety observed in the percent path length
traversed (C), percent time spent (D), and number of visits to the center (E) of the arena. Shown are representative tracks in the open (oa) and closed arms
(ca) of the EPM from Ctrl, PNFlx, Ket, and PNFlx ⫹ Ket animals (F). Postnatal Ket treatment prevented the PNFlx-evoked anxiety observed in the percent path
length traversed (G) and percent time spent in the open arms (H) and the outer two thirds of the open arms of the maze (I). Shown is a schematic
representation for the FST (J). PNFlx animals showed a significant increase in percent immobility time (K) on the FST, whereas the Ket group showed a
significant decrease in immobility time (K). Latency to first immobility (L) and number of immobility events (M) were unaltered across groups. Data are the
mean ⫾ SEM for percent path length and percent time in the OFT (n ¼ 6–12 animals/group) and percent path length and percent time in the open arms of
the EPM (n ¼ 7–12 animals/group), number of visits in the OFT, percent immobility time, latency to first immobility, and number of immobility events in the
FST (n ¼ 5–11 animals/group). (*p ⬍ .05 as compared with Ctrl, $p ⬍ .05 as compared with PNFlx, two-way analysis of variance and Bonferroni post hoc test).
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A. Sarkar et al.                                                                          BIOL PSYCHIATRY 2014;76:858–868 861

.017) to the center of the OFT arena. Ketanserin treatment              Postnatal 5-HT2A/C Receptor Stimulation Evokes Anxiety
blocked the PNFlx-evoked decline in number of rears in the              Behavior in Adulthood
OFT (Figure S1 in Supplement 1). Postnatal ketanserin treatment             Given we observed that postnatal 5-HT2A/C receptor block-
also prevented the emergence of PNFlx-evoked anxiety on the             ade prevented PNFlx-evoked anxiety, we next addressed the
EPM (Figure 1F). Postnatal fluoxetine animals exhibited a signifi-        influence of postnatal 5-HT2A/C receptor stimulation on adult
cant decline in percent path length and percent time in the open        anxiety. Postnatal treatment with the 5-HT2A/C receptor agonist
arms of the maze, which was normalized in the PNFlx ⫹ Ket               DOI (PNDOI) increased anxiety on the OFT and EPM in
group (Figure 1G–I). We noted a significant two-way ANOVA                adulthood (Figure 3A–K). Postnatal DOI animals showed a
PNFlx  Ket interaction for percent path length in the open arms        significant decline in percent path length (Figure 3C), percent
(F1,34 ¼ 4.76, p ¼ .036) and a strong trend for a PNFlx  Ket           time spent (Figure 3D), and a trend (p ¼ .08) toward a decline
interaction on percent time in the open arms of the maze (F1,34 ¼       in the number of visits (Figure 3E) to the center of the OFT
3.76, p ¼ .06). Analysis of percent time spent in the outer two         arena. On the EPM, PNDOI animals showed a signifi-
thirds of the open arms of the EPM indicated a significant PNFlx         cant decrease in percent path length in the open arms
 Ket interaction (F1,34 ¼ 4.49, p ¼ .041). Ketanserin treatment did    (Figure 3H), increased percent path length in the closed arm
not alter the number of rears observed in the PNFlx group in the        (Figure 3I), and a trend (p ¼ .07) toward an increase in time
EPM (Figure S1 in Supplement 1). Total distance traversed in            spent in the closed arms (Figure 3J), with no change in percent
the OFT and EPM was unaltered across groups (Figure S1 in               time in the open arms (Figure 3K). Total distance traversed in
Supplement 1).                                                          the OFT (Figure 3F) and EPM (Figure S3 in Supplement 1) did
    We next assessed whether postnatal ketanserin treatment             not differ across groups.
influenced PNFlx-evoked depressive behavior on the FST
(Figure 1A,J–M). We did not observe a significant two-way ANOVA          Postnatal 5-HT1A Receptor Blockade Does Not Alter PNFlx-
PNFlx  Ket interaction for percent immobility time (Figure 1K) on      Evoked Anxiety but Evokes Anxiety in Control Animals, an
the FST. We noted significant main effects of PNFlx (F1,27 ¼ 6.28,       Effect Attenuated by Concomitant 5-HT2A/C Receptor
p ¼ .018) and Ket (F1,27 ¼ 14.86, p ¼ .0006) for percent immobility     Blockade
time. The latency to immobility and number of immobility events             We next examined whether concomitant treatment with the
(Figure 1L,M) were unaltered across groups. These results indicate      selective 5-HT1A receptor antagonist, WAY-100635, influenced
that postnatal ketanserin treatment to naive control pups evokes        PNFlx-mediated anxiety behavior on the OFT (Figure 4A). Postnatal
antidepressant-like behavioral responses on percent immobility in       WAY-100635 treatment did not alter the significant PNFlx-evoked
the FST in adulthood.                                                   anxiety on the OFT (Figure 4B–D). Two-way ANOVA analysis
                                                                        revealed no significant PNFlx  WAY interaction for percent path
Postnatal 5-HT2A Receptor Blockade Prevents both PNFlx-                 length traversed (Figure 4C) and percent time spent (Figure 4D) in
Evoked Anxiety and Depressive Behavior, Whereas 5-HT2C                  the center of the arena. Postnatal WAY-100635 treatment to naive
Receptor Blockade Prevents PNFlx-Evoked Anxiety but not                 animals induced significant anxiety with a decline in the percent
Depressive Behavior                                                     path length (Figure 4C) (main effect of WAY: F1,20 ¼ 4.51, p ¼ .016)
    We next examined the effects of postnatal treatment with            and percent time spent (Figure 4D) (main effect of WAY: F1,20 ¼
selective 5-HT2A or 5-HT2C receptor antagonists on PNFlx-evoked         9.90, p ¼ .005) in the center of the arena. Total distance traversed
anxiety and depressive behavior in adulthood on the OFT and             in the entire arena did not differ across groups (Figure 4E).
FST (Figure 2A,F). Postnatal fluoxetine-evoked adult anxiety on              We then assessed whether the anxiety evoked by postnatal
the OFT was prevented by both the 5-HT2A receptor antagonist,           WAY-100635 administration was influenced by concomitant
MDL100907 (Figure 2B,C), and the 5-HT2C receptor antagonist,            5-HT2A/C receptor antagonist treatment. Postnatal ketanserin
SB242084 (Figure 2G,H). Two-way ANOVA analysis indicated a              administration partially attenuated the anxiogenic effects of
significant PNFlx  MDL interaction for percent path length              postnatal WAY100635 on the OFT in adulthood (Figure 4F–I).
traversed (F1,36 ¼ 8.06, p ¼ .007) (Figure 2B), percent time (F1,36     One-way ANOVA and post hoc Bonferroni analysis indicated that
¼ 5.52, p ¼ .024) (Figure 2C), and number of visits to the center       while the WAY group exhibited a significant decline in percent
of the arena (F1,36 ¼ 5.47, p ¼ .025) (Figure S2 in Supplement 1).      path length traversed (Figure 4G), percent time spent (Figure 4H),
Two-way ANOVA analysis indicated a significant PNFlx  SB                and number of visits (Figure 4I) to the center of the arena, the
interaction for percent path length traversed (F1,33 ¼ 8.09,            WAY ⫹ Ket group did not differ from control animals on these
p ¼ .008) (Figure 2G) and percent time in the center (F1,33 ¼           measures. Total distance traversed in the arena was unaltered
5.10, p ¼ .031) (Figure 2H). Number of visits to the center of the      across groups (Figure 4J).
arena was unaltered across groups in the PNFlx ⫹ SB experi-
ment groups (Figure S2 in Supplement 1). Total distance                 Postnatal Ketanserin Treatment Blocks Specific
traversed in the OFT was unaltered across all groups (Figure            Transcriptional Changes Observed in the Prefrontal Cortex
S2 in Supplement 1).                                                    Following PNFlx Treatment
    Postnatal fluoxetine-evoked depressive behavior on the FST               We examined the influence of PNFlx on 5-HT1A (Htr1a),
was blocked by postnatal 5-HT2A, but not 5-HT2C, receptor               5-HT2A (Htr2a), and 5-HT2C (Htr2c) receptor expression in the
blockade (Figure 2D,E,I,J). Two-way ANOVA analysis indicated a          prefrontal cortex, a region strongly implicated in the regulation
significant PNFlx  MDL interaction for percent immobility time          of anxiety behavior. Quantitative polymerase chain reaction anal-
(F1,27 ¼ 5.21, p ¼ .030) (Figure 2D), latency to immobility (F1,27 ¼    ysis revealed a significant increase in prefrontal 5-HT2A messenger
4.39, p ¼ .045) (Figure 2E), and number of immobility events (F1,27     RNA (mRNA) levels in PNFlx animals in adulthood, with no change
¼ 27.19, p ⬍ .0001) (Figure S2 in Supplement 1). Two-way ANOVA          observed in the other 5-HT receptors examined (Figure 5A,B). The
analysis indicated no PNFlx  SB interaction for percent immo-          PNFlx-evoked increase in 5-HT2A receptor mRNA was prevented by
bility time (Figure 2I), latency to immobility (Figure 2J), or number   postnatal ketanserin treatment. Two-way ANOVA analysis indicated
of immobility events (Figure S2 in Supplement 1).                       a significant PNFlx  Ket interaction for 5-HT2A receptor expression

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862 BIOL PSYCHIATRY 2014;76:858–868                                                                                                    A. Sarkar et al.

Figure 2. Postnatal serotonin 2A receptor blockade prevents both postnatal fluoxetine (PNFlx)-evoked anxiety and depressive behavior, whereas
serotonin 2C receptor blockade prevents PNFlx-evoked anxiety but not depressive behavior. Shown is a schematic representation of the treatment
paradigm (A). Control (Ctrl) and postnatal fluoxetine treated (PNFlx) pups received vehicle (Veh) or serotonin 2A receptor antagonist MDL100907 (MDL)
from postnatal day (P)2 to P21 and were assessed on the open field test (OFT) (P90) and the forced swim test (FST) (P100). Postnatal MDL treatment
prevented the PNFlx-evoked anxiety observed in the percent path length traversed (B) and percent time spent (C) in the center of the OFT arena.
Postnatal MDL treatment prevented the PNFlx-evoked depressive behavior on the FST observed in percent immobility time (D) and latency to immobility
(E). Shown is a schematic representation of the treatment paradigm (F). Ctrl and PNFlx pups received Veh or serotonin 2C receptor antagonist SB242084
(SB) from P2 to P21 and were assessed on the OFT and FST. Postnatal SB treatment prevented the PNFlx-evoked anxiety observed in the percent path
length traversed (G) and percent time spent (H) in the center of the OFT arena. Postnatal SB treatment did not alter the PNFlx-evoked depressive behavior
on the FST (I, J). Data are the mean ⫾ SEM for percent path length and percent time in the OFT (PNFlx ⫹ MDL: n ¼ 7–13 animals/group, PNFlx ⫹ SB: n ¼
6–11 animals/group), percent immobility time and latency to first immobility in the FST (PNFlx ⫹ MDL: n ¼ 7–9 animals/group, PNFlx ⫹ SB: n ¼ 7–11
animals/group). (*p ⬍ .05 as compared with Ctrl, $p ⬍ .05 as compared with PNFlx, &p ⬍ .05 as compared with SB, two-way analysis of variance and
Bonferroni post hoc test).

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A. Sarkar et al.                                                                                   BIOL PSYCHIATRY 2014;76:858–868 863

Figure 3. Chronic postnatal treatment with the serotonin 2A/serotonin 2C agonist DOI evokes anxiogenic responses in adulthood. Shown is a schematic
representation of the treatment paradigms (A). Pups received treatment with either vehicle (Ctrl) or DOI (postnatal DOI [PNDOI]) daily from postnatal day
(P)2 to P21 and were assessed for anxiety behavior in the open field test (OFT) (P90) and elevated plus maze (EPM) (P100). Shown are representative tracks
of exploratory behavior in the open field arena from adult Ctrl and PNDOI animals with the center ‘c’ indicated by the gray zone (B). Postnatal DOI animals
exhibited significant decreases in the percent path length traversed (C) and percent time spent (D) in the center of the arena. Postnatal DOI animals
exhibited a trend toward a decline in the number of visits to the center of the OFT arena (E). Total distance moved in the OFT arena was unaltered
between groups (F). Shown are representative tracks of exploratory behavior in the open (oa) and closed (ca) arms of the EPM from adult Ctrl and PNDOI
animals (G). Postnatal DOI treatment evoked anxiety behavior in the EPM test in adulthood with significant decreases in the percent path length traversed
in the open arms (H) and a significant increase in the percent path length traversed in the closed arms (I) of the maze. While percent time spent in the
closed (J) and open arms (K) of the maze did not exhibit significant differences across the groups, we noted a trend (p ¼ .07) toward an increase for
percent time spent in the closed (J) arms of the maze. Data are the mean ⫾ SEM percent path length, percent time and number of visits to the center, and
total distance moved in the OFT and percent path length and percent time in the open and closed arms of the EPM (n ¼ 6–8 animals/group). *p ⬍ .05 as
compared with Ctrl (Student t test).

(F1,36 ¼ 5.43, p ¼ .026). We next addressed whether the expression             adulthood, which was prevented by postnatal ketanserin treat-
of the immediate early gene, activity-regulated cytoskeleton-asso-             ment. Two-way ANOVA analysis indicated a significant PNFlx  Ket
ciated protein, Arc, whose levels within the prefrontal cortex are             interaction for Arc expression in the prefrontal cortex (F1,36 ¼ 7.14,
reported to be decreased in animal models of depression (43), was              p ¼ .011). These results indicate that postnatal ketanserin treatment
influenced by PNFlx treatment. Postnatal fluoxetine treatment                    blocks the regulation of specific PNFlx-regulated genes in the
evoked a significant decline in prefrontal Arc mRNA levels in                   prefrontal cortex in adulthood.

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864 BIOL PSYCHIATRY 2014;76:858–868                                                                                                    A. Sarkar et al.

Figure 4. Postnatal serotonin 1A receptor blockade does not alter postnatal fluoxetine (PNFlx)-evoked anxiety but evokes anxiety in control animals,
an effect attenuated by concomitant serotonin 2A/serotonin 2C (5-HT2A/C) receptor blockade. Shown is a schematic representation of the treatment
paradigm (A). Control (Ctrl) and postnatal fluoxetine-treated (PNFlx) pups received vehicle (Veh) or WAY-100635 (WAY) from postnatal day (P)2 to P21
and were assessed for anxiety behavior in the open field test (OFT) (P90). Shown are representative tracks on the OFT from Ctrl, PNFlx, WAY, and PNFlx
⫹ WAY animals (B). WAY treatment did not alter PNFlx-evoked anxiety behavior on the OFT. Postnatal WAY treatment of naive control pups induced
anxiety behavior in adulthood with a decline in percent path length traversed (C) and percent time spent (D) in the center ‘c’ of the arena, while total
distance moved in the OFT was unaltered across groups (E). Shown is a schematic of the treatment paradigm for the combined postnatal 5-HT2A/C
and serotonin 1A receptor antagonist experiment (F). Treatment with the 5-HT2A/C antagonist, ketanserin (Ket) ameliorated the WAY-evoked
decreases in the percent path length traversed (G), percent time spent (H), and number of visits (I) to the center of the arena, while total distance was
unaltered across groups (J). Data are the mean ⫾ SEM percent path length, percent time, and the total distance covered in the OFT arena. (PNFlx ⫹
WAY: n ¼ 5–7 animals/group; WAY ⫹ Ket: n ¼ 8–9 animals/group). *p ⬍ .05 as compared with Ctrl, one- or two-way analysis of variance Bonferroni
post hoc test).

Discussion                                                                     blockade and that postnatal 5-HT2A/C receptor stimulation behav-
                                                                               iorally mimics the anxiogenic effects of PNFlx. Our results with
   The major novel finding of our study indicates that PNFlx-                   selective 5-HT2A and 5-HT2C receptor antagonists indicate that both
evoked anxiety is prevented by postnatal 5-HT2A/C receptor                     PNFlx-evoked adult anxiety and depressive behavior are normalized

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A. Sarkar et al.                                                                                   BIOL PSYCHIATRY 2014;76:858–868 865

Figure 5. Postnatal ketanserin (Ket) treatment blocks specific transcriptional changes observed in the prefrontal cortex following postnatal fluoxetine
(PNFlx) treatment. Shown is a schematic representation of the treatment paradigm (A). Control (Ctrl) and postnatal fluoxetine-treated (PNFlx) pups
received vehicle or Ket administration from postnatal day (P)2 to P21 and were subjected to gene expression analysis in the prefrontal cortex in adulthood
(P110). Postnatal Ket treatment prevented the PNFlx-evoked increase in Htr2a expression (B) and the PNFlx-evoked decline in Arc expression in the
prefrontal cortex (C). Data are represented as the fold change of control animals and are the mean ⫾ SEM (n ¼ 8–12 animals/group). (*p ⬍ .05 as
compared with Ctrl, $p ⬍ .05 as compared with PNFlx, two-way analysis of variance and Bonferroni post hoc test). 5HT, serotonin; mRNA, messenger RNA.

by concomitant 5-HT2A receptor blockade, whereas 5-HT2C receptor               5-HT2A, or 5-HT2C antagonist treatment did not influence anxiety
blockade prevents the development of anxiety, but not depressive               behavior in adulthood in naive control animals but were each
behavior, in PNFlx animals. Further, we demonstrate that postnatal             capable of preventing the development of PNFlx-evoked anxiety.
5-HT1A receptor blockade does not alter the development of anxiety             In contrast, PNFlx-mediated depressive behavior on the FST was
following PNFlx but is sufficient to evoke adult anxiety in naive               prevented by selective 5-HT2A, but not 5-HT2C, receptor blockade
control animals, an effect diminished by 5-HT2A/C receptor antago-             in postnatal life. Baseline FST behavior in naive control animals
nist co-administration. Our results implicate 5-HT2A/C receptors in            was not significantly altered following postnatal 5-HT2A or 5-HT2C
mediating the behavioral effects on anxiety and depressive behavior            receptor blockade; however, we did observe significant
noted in response to administration of the SSRI fluoxetine in early             antidepressant-like responses in control animals postnatally
life. These observations provide strong support to the emerging                administered the 5-HT2A/C receptor antagonist. These results
view that a balance between 5-HT1A and 5-HT2A/C receptor-                      support the notion that while both 5-HT2A and 5-HT2C receptors
mediated signaling during the critical time window of postnatal life           contribute to the emergence of anxiety, 5-HT2A receptors may
is of import in the emergence of anxiety.                                      mediate the emergence of depressive behaviors following early
     Postnatal life represents a critical period wherein serotonergic          SSRI exposure. In addition to genetic knockout studies that
neurotransmission is hypothesized to regulate the maturation of                implicate 5-HT2A/C receptors in the regulation of anxiety (2,27–
emotional neurocircuitry, thus programming lasting effects on                  30), recent reports demonstrate that models of psychiatric
emotionality (1,2,44). Genetic knockout experiments with 5-HT                  vulnerability based on early adverse experience, including
transporter knockout (5-HTT/) mice (14,45–47), as well as                    maternal separation (53) and prenatal exposure to influenza
pharmacologic perturbations with SSRIs in rodents (13,14,48,49),               (54), exhibit enhanced 5-HT2A/C receptor mediated signaling.
indicate that elevated postnatal 5-HT levels evoke persistent                  Strikingly, chronic postnatal ketanserin treatment overlapping
increases in anxiety and depressive behavior. However, the                     with the period of maternal separation prevents the develop-
contribution of specific 5-HT receptors to the persistent anxio-                ment of anxiety and the dysregulated cortical immediate-early
genic and depressive behaviors programmed by elevated 5-HT                     gene responses in maternally separated animals (26). This
levels during postnatal development is poorly understood. Our                  suggests the possibility that both adverse early experience, as
pharmacologic studies provide novel evidence that implicate                    well as pharmacologic treatments with SSRIs in postnatal life,
5-HT2A and 5-HT2C receptors in mediating specific behavioral                    may impinge on 5-HT2A/C receptor signaling to program adult
consequences of SSRI treatment in postnatal life.                              anxiety.
     Mouse mutant studies strongly implicate 5-HT1A, 5-HT2A, and                   Further support for a role of 5-HT2A/C receptors in PNFlx-
5-HT2C receptors in the development of anxiety (2,50–52).                      evoked anxiety comes from our results demonstrating that
Anxiolytic effects have been noted in both 5-HT2A and 5-HT2C                   chronic postnatal treatment with the 5-HT2A/C receptor partial
receptor knockout mice (27,28). Conditional reactivation of the                agonist, DOI, also elicits adult anxiety. In contrast to the
cortical 5-HT2A receptor prevented the anxiolytic effects in                   anxiogenic effects observed with chronic postnatal DOI treat-
5-HT2A receptor knockout mice; however, it is unknown whether                  ment, acute, but not chronic, adult DOI administration is thought
there is a critical temporal window for such a rescue (27). In this            to evoke anxiolytic effects (55–57). The effects of 5-HT2A/C
regard, it is of note that our studies with postnatal 5-HT2A/C,                receptor stimulation on anxiety behavior during postnatal versus

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866 BIOL PSYCHIATRY 2014;76:858–868                                                                                               A. Sarkar et al.

adult life appear distinct in nature, though the caveat that the         evoked by PNFlx and suggest that an optimal balance between
duration of treatments varies has to be kept in mind. Adult-onset        5-HT1A and 5-HT2A/C receptor-mediated drive during the postnatal
treatment with 5-HT2A/C receptor antagonists (58–61) or intra-           temporal window may be required for the programming of
cerebroventricular administration of 5-HT2A antisense oligonu-           normal levels of anxiety. We posit that SSRI treatments within
cleotides also evokes anxiolytic effects (62). In addition to cortical   this temporal window may disrupt the balance between 5-HT1A
5-HT2A receptors, 5-HT2A/C receptors within other brain regions,         and 5-HT2A/C receptor function and could serve to tip the system
including the hypothalamus, amygdala, and periaqueductal gray,           toward enhanced 5-HT2A/C receptor-mediated drive, thus contri-
are also implicated in the regulation of anxiety (63–65). The            buting to the establishment of persistent increases in anxiety
anxiolytic effects observed with both adult 5-HT2A/C receptor            noted long after the cessation of SSRI treatment. It is tempting to
agonists and antagonists may involve specific contributions of            speculate that multi-action drugs that exhibit combined 5-HT1A
these receptors in multiple brain circuits that regulate anxiety. Our    agonist and 5-HT2A/C antagonist properties may serve as putative
results demonstrate that during the postnatal window, 5-HT2A/C           therapeutic agents for the treatment of early-onset anxiety
receptors may play an important role in programming adult                disorders (78,79). Our results provide impetus for future studies
anxiety. However, at present, it is difficult to delineate the specific    to address the key role of 5-HT2A and 5-HT2C receptors and their
neuronal circuits that underlie the behavioral effects of postnatal      downstream targets in contributing to the behavioral effects of
5-HT2A/C receptor agonist treatment and contribute to the 5-HT2A/C       early SSRI administration.
receptor antagonist-mediated normalization of PNFlx-evoked anxi-
ety and depressive behaviors. The pattern of expression of the
5-HT1A, 5-HT2A, and 5-HT2C receptors shows a developmental                  This research was supported by a Tata Institute of Fundamental
regulation with high levels observed in several cortical brain           Research intramural grant (VAV).
regions during postnatal life (66–72). Cortical excitatory responses        The authors report no biomedical financial interests or potential
to 5-HT, driven via the 5-HT2A receptor, are noted during postnatal      conflicts of interest.
life within layer V output neurons of the prefrontal cortex, which
gradually tip into predominantly inhibitory responses to 5-HT,              Supplementary material cited in this article is available online at
mediated by the 5-HT1A receptor, as animals mature (70–73). This         http://dx.doi.org/10.1016/j.biopsych.2013.11.005.
suggests a transient postnatal window wherein elevated levels of
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