postnatal fluoxetine-evoked anxiety is prevented by...

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Postnatal Fluoxetine-Evoked Anxiety Is Prevented byConcomitant 5-HT2A/C Receptor Blockade andMimicked 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 inrodent 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-HT2Areceptor antagonist, MDL100907, the 5-HT2C receptor antagonist, SB242084, or the 5-HT1A receptor antagonist, WAY-100635, and weretested for behavior in adulthood. The effect of postnatal treatment with the 5-HT2A/C receptor agonist, DOI, on anxiety behavior wasexamined in adulthood.

Results: Postnatal 5-HT2A/C receptor blockade prevented PNFlx-evoked anxiety, attenuated depressive behavior, and normalized specificgene expression changes in the prefrontal cortex. Postnatal, selective 5-HT2A receptor antagonist treatment blocked PNFlx-evokedanxiety 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 prefrontalcortex, were prevented by 5-HT2A/C receptor blockade. Adult anxiety was evoked by either 5-HT2A/C receptor stimulation or 5-HT1A receptorblockade of naive control pups. Our findings implicate serotonin 2 receptors in the development of perturbed emotionality following PNFlxand suggest that an altered balance of signaling through 5-HT1A and 5-HT2A/C receptors in early life influences anxiety behavior.

Key Words: Antidepressant, DOI, 5-HT1A, 5-HT2A, 5-HT2C,ketanserin, Prozac, SSRI, WAY-100635

Serotonin (5-HT) neurotransmission influences the develop-ment of anxiety (1). Perturbations of postnatal 5-HT levels inrodents evoke lasting consequences on emotionality (2). The

selective serotonin reuptake inhibitor (SSRI), fluoxetine, is amongthe first-line therapies for gestational, postpartum, childhood, andadolescent depression, given its favorable side-effect profile (3–6).However, clinical evidence links perinatal SSRI exposure to neonatalserotonin withdrawal symptoms and disrupted neurobehavioraldevelopment (7–9), and childhood and adolescent fluoxetinetreatment is associated with enhanced anxiety, depression, andsuicidal ideation (10,11). Postnatal fluoxetine (PNFlx) treatment inmouse models, considered equivalent to the third trimester ofhuman gestation (12), results in enhanced anxiety and depressivebehavior that persist across the life span (11,13–15). This is para-doxical to the anxiolytic and antidepressant responses observed inhumans (16) and in rodents (17), following fluoxetine administra-tion in adulthood. Perturbations of 5-HT levels evoke differing con-sequences on mood behavior based on the temporal window (11).

The temporal window of postnatal life is critical for defining thetrajectory of emotional development (18–20). It is important tounveil the role of specific 5-HT receptors in mediating the

Authors AS and PC contributed equally to this work.

From the Department of Biological Sciences, Tata Institute of FundamentalResearch, Mumbai, India.

Address correspondence to Vidita A. Vaidya, Ph.D., Tata Institute of Funda-mental Research, Department of Biological Sciences, 1, Homi BhabhaRoad, Colaba, Mumbai 400005, India; E-mail: [email protected].

Received Apr 5, 2013; revised and accepted Nov 1, 2013.

0006-3223/$36.00http://dx.doi.org/10.1016/j.biopsych.2013.11.005

behavioral effects of PNFlx. Serotonergic neurotransmission ismediated via a large family of 14 receptor subtypes that exhibitdistinct developmental expression patterns and functionalresponses (21–23). Genetic perturbations and pharmacologicstudies in rodent models indicate an important role for 5-HT1A,5-HT2A, and 5-HT2C receptors in the development of anxiety(2,24–34). Cortical 5-HT2A receptor knockouts (27) and loss of5-HT2C receptor function (28) exhibit anxiolytic effects. Serotonin1A receptors, besides their postsynaptic expression, also serve aspresynaptic autoreceptors that regulate 5-HT neuron firing andrelease (35,36). Conditional, forebrain-specific 5-HT1A heterorecep-tor, or raphe 5-HT1A autoreceptor, loss of function during postnatallife, as well as postnatal pharmacologic 5-HT1A receptor blockade,evoke enhanced anxiety in adulthood (25,29–33). We hypothe-sized that PNFlx-evoked anxiogenic responses involve a key rolefor 5-HT1A and 5-HT2A/5-HT2C (5-HT2A/C) receptors, strongly impli-cated in the development of anxiety. Using studies in rat models,we show that 5-HT2A/C receptor blockade prevents PNFlx-evokedanxiety and attenuates depressive behavior. Our results indicatethat postnatal 5-HT2A/C receptor stimulation evokes adult anxiety,and the anxiogenic effects of postnatal 5-HT1A receptor antagonisttreatment are attenuated by concomitant 5-HT2A/C receptor block-ade. Our findings implicate 5-HT2A/C receptors in the developmentof perturbed emotionality following PNFlx and suggest that thebalance between 5-HT1A and 5-HT2A/C receptor-driven signalingwithin the postnatal temporal window could play an importantrole in the establishment of baseline anxiety states.

Methods and Materials

AnimalsMale Sprague-Dawley rats bred in the Tata Institute of Funda-

mental Research animal facility and maintained on a 12-hour

BIOL PSYCHIATRY 2014;76:858–868& 2014 Society of Biological Psychiatry

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dx.doi.org/10.1016/j.biopsych.2013.11.005



A. Sarkar et al. BIOL PSYCHIATRY 2014;76:858–868 859

light-dark cycle with ad libitum access to food and water wereused for all experiments. Experimental procedures followed theNational Institute of Health Guide for the care and use of animalsand the Committee for the Purpose of Control and Supervisionof Experimental Animals and were approved by the Tata Instituteof Fundamental Research Animal Ethics Committee (56/1999/CPCSEA).

Drug TreatmentsLitters from primiparous dams were randomly assigned to

control (Ctrl) or test groups, with each treatment group contain-ing pups from two or more litters. Pups were weaned atpostnatal day (P)28, and male pups were group housed intoadulthood. Pups received oral administration via a feedingneedle of fluoxetine (10 mg/kg; Sigma-Aldrich, St. Louis, Missouri)or vehicle (5% sucrose, Sigma-Aldrich) once daily from P2 to P21,within 3 minutes to minimize handling stress. To examine theinfluence of concomitant 5-HT2A/C, 5-HT2A, 5-HT2C, or 5-HT1Areceptor blockade on the behavioral consequences of postnatalfluoxetine, pups received daily pretreatment with the 5-HT2A/Creceptor antagonist, ketanserin tartarate (5 mg/kg, Sigma-Aldrich), the selective 5-HT2A receptor antagonist, MDL100907(1 mg/kg; Toronto Research Chemicals, North York, Ontario,Canada), the selective 5-HT2C receptor antagonist, SB242084 (.5mg/kg, Sigma-Aldrich), the selective 5-HT1A receptor antagonist,WAY-100635 (.5 mg/kg, Sigma-Aldrich), or vehicle (5% sucrosesolution) orally, 30 minutes before vehicle or PNFlx treatmentdaily from P2 to P21. Behavioral testing on the open field test(OFT), elevated plus maze (EPM), and forced swim test (FST) wasperformed in adulthood. Treatment groups were 5-HT2A/C recep-tor blockade experiment: Ctrl, PNFlx, ketanserin (Ket), and PNFlx� Ket (OFT, EPM: n = 6–12 per group; FST: n = 5–11 per group);5-HT2A receptor blockade experiment: Ctrl, PNFlx, MDL100907(MDL), and PNFlx � MDL (OFT: n = 7–13 per group; FST: n = 7–9per group); 5-HT2C receptor blockade experiment: Ctrl, PNFlx,SB242084 (SB), and PNFlx � SB (OFT: n = 6–11 per group; FST: n= 7–11 per group); 5-HT1A receptor blockade experiment: Ctrl,PNFlx, WAY-100635 (WAY), and PNFlx � WAY (OFT: n = 5–7 pergroup). To examine the influence of chronic postnatal treatmentwith the 5-HT2A/C receptor agonist, DOI (2,5-dimethoxy-4-iodo-amphetamine), on anxiety behavior in adulthood, pups receivedDOI (2 mg/kg, Sigma-Aldrich) orally from P2 to P21 (PNDOI) orvehicle (5% sucrose) (Ctrl) (n = 6–8 per group). To assess theinfluence of concomitant 5-HT2A/C receptor blockade on anxietybehavior following postnatal 5-HT1A receptor antagonist treat-ment, pups received vehicle (5% sucrose solution) or ketanserin30 minutes before administration of WAY100635 and the treat-ment groups were Ctrl, WAY, and WAY � Ket (n = 8–9 pergroup). Drug doses were selected based on prior reports(13,25,26,37,38).

Behavioral Tests: Open Field, Elevated Plus Maze, and ForcedSwim Test

Animals were subjected to behavioral tests in adulthood. Openfield test analysis for anxiety behavior (39) was performed for thefollowing experiments: postnatal ketanserin, MDL100907,SB242084, or WAY100635 with PNFlx, postnatal DOI, and post-natal ketanserin with WAY100635 treatment. For the OFT, thearena (100 cm � 100 cm � 70 cm) was placed in a dimlyilluminated room, and behavioral tests were carried out withinthe light phase. Animals were released at one corner of the arenaand exploratory behavior was recorded with an automatedtracking system for 5 minutes. All behavioral tracks were analyzed

using the Ethovision tracking system (Noldus, Wageningen,Netherlands). We assessed total distance covered within thearena, path length, and time spent in the center; number ofvisits to the center; and number of rears in the arena. Elevatedplus maze analysis for anxiety behavior (40) was performed forthe following experiments: PNFlx � Ket and PNDOI. The EPMconsisted of a platform elevated 50 cm from the ground with twoopen and closed arms (45 � 10 cm). Animals were released intothe center of the maze facing the open arm, and behavioralmeasures were assessed for 5 minutes. Analysis was performedfor total distance covered within the maze, path length, and timespent in the open and closed arms and the number of rears in themaze. Forced swim test analysis for depressive behavior (41) wasperformed for the following experiments: postnatal ketanserin,MDL100907, or SB242084 with PNFlx. A modified version of theFST involved habituation on day 1 for 15 minutes to a plasticcylinder (50 cm tall, 21 cm in diameter) filled with water, followedby testing on day 2 for 5 minutes. Video recordings were assessedfor immobility time, latency to immobility, and number ofimmobility events by an investigator blind to the treatmentgroups.

Quantitative Polymerase Chain Reaction AnalysisQuantitative polymerase chain reaction analysis was per-

formed on RNA extracted from prefrontal cortex using the Trizolmethod (Trizol, Sigma-Aldrich) in the following experiment: Ctrl,PNFlx, Ket, PNFlx � Ket (n ¼ 7–12 per group) in adulthood.Complementary DNA synthesis with the High Capacity Comple-mentary DNA Reverse Transcription Kit (Applied Biosystems,Foster City, California) was performed on RNA (2 μg/sample)following RNA quality assessment by analysis of the 260/280optical density ratio using Nanodrop (Eppendorf, Hamburg,Germany). Quantitative polymerase chain reaction was performedwith primers for the genes of interest (Table S1 in Supplement 1).Quantitation was performed using the ΔΔCt method as previ-ously described (42) and data were normalized to hypoxanthine-guanine phosphoribosyltransferase 1 (Hprt1).

Statistical AnalysisExperiments with two groups were analyzed using the unpaired

Student t test and experiments with three or four groups wereanalyzed with one- or two-way analysis of variance (ANOVA),respectively, followed by the Bonferroni post hoc test. Parametricdistribution of data was assessed using the Kolmogorov-Smirnovtest. Significance was determined at p � .05 (Instat and Prism,Graphpad Software Inc., San Diego, California).

Results

Postnatal 5-HT2A/C Receptor Antagonist Treatment Preventsthe Development of PNFlx-Evoked Adult Anxiety

We examined whether concomitant 5-HT2A/C receptor antag-onist treatment altered the development of adult anxiety follow-ing PNFlx on the OFT and EPM (Figure 1A–I). Postnatal fluoxetinetreatment enhanced anxiety behavior on the OFT (Figure 1B–E) asindicated by decreased percent path length, percent time spent,and number of visits to the center of the arena. Postnatalfluoxetine-evoked anxiety on the OFT was completely preventedby concomitant ketanserin treatment (Figure 1B–E). Two-wayANOVA analysis indicated a significant PNFlx � Ket interactionfor percent path length traversed (F1,31 ¼ 6.78, p ¼ .014), percenttime (F1,31 ¼ 5.99, p ¼ .02), and number of visits (F1,31 ¼ 6.34, p ¼

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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) orketanserin (Ket) administration daily from postnatal day (P)2 to P21 and were assessed for anxiety behavior on the open field test (OFT) (P90) and elevatedplus maze (EPM) (P100) and in a separate cohort for depressive behavior on the forced swim test (FST) (P90). Shown are representative tracks on the OFTfrom adult Ctrl, PNFlx, Ket, and PNFlx � Ket animals (B). Postnatal Ket treatment prevented the PNFlx-evoked anxiety observed in the percent path lengthtraversed (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 pathlength 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 schematicrepresentation for the FST (J). PNFlx animals showed a significant increase in percent immobility time (K) on the FST, whereas the Ket group showed asignificant decrease in immobility time (K). Latency to first immobility (L) and number of immobility events (M) were unaltered across groups. Data are themean � 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 ofthe 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 theFST (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).

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



.017) to the center of the OFT arena. Ketanserin treatmentblocked the PNFlx-evoked decline in number of rears in theOFT (Figure S1 in Supplement 1). Postnatal ketanserin treatmentalso prevented the emergence of PNFlx-evoked anxiety on theEPM (Figure 1F). Postnatal fluoxetine animals exhibited a signifi-cant decline in percent path length and percent time in the openarms of the maze, which was normalized in the PNFlx � Ketgroup (Figure 1G–I). We noted a significant two-way ANOVAPNFlx � Ket interaction for percent path length in the open arms(F1,34 ¼ 4.76, p ¼ .036) and a strong trend for a PNFlx � Ketinteraction on percent time in the open arms of the maze (F1,34 ¼3.76, p ¼ .06). Analysis of percent time spent in the outer twothirds of the open arms of the EPM indicated a significant PNFlx� Ket interaction (F1,34 ¼ 4.49, p ¼ .041). Ketanserin treatment didnot alter the number of rears observed in the PNFlx group in theEPM (Figure S1 in Supplement 1). Total distance traversed inthe OFT and EPM was unaltered across groups (Figure S1 inSupplement 1).

We next assessed whether postnatal ketanserin treatmentinfluenced PNFlx-evoked depressive behavior on the FST(Figure 1A,J–M). We did not observe a significant two-way ANOVAPNFlx � Ket interaction for percent immobility time (Figure 1K) onthe FST. We noted significant main effects of PNFlx (F1,27 ¼ 6.28,p ¼ .018) and Ket (F1,27 ¼ 14.86, p ¼ .0006) for percent immobilitytime. The latency to immobility and number of immobility events(Figure 1L,M) were unaltered across groups. These results indicatethat postnatal ketanserin treatment to naive control pups evokesantidepressant-like behavioral responses on percent immobility inthe FST in adulthood.

Postnatal 5-HT2A Receptor Blockade Prevents both PNFlx-Evoked Anxiety and Depressive Behavior, Whereas 5-HT2CReceptor Blockade Prevents PNFlx-Evoked Anxiety but notDepressive Behavior

We next examined the effects of postnatal treatment withselective 5-HT2A or 5-HT2C receptor antagonists on PNFlx-evokedanxiety and depressive behavior in adulthood on the OFT andFST (Figure 2A,F). Postnatal fluoxetine-evoked adult anxiety onthe OFT was prevented by both the 5-HT2A receptor antagonist,MDL100907 (Figure 2B,C), and the 5-HT2C receptor antagonist,SB242084 (Figure 2G,H). Two-way ANOVA analysis indicated asignificant PNFlx � MDL interaction for percent path lengthtraversed (F1,36 ¼ 8.06, p ¼ .007) (Figure 2B), percent time (F1,36¼ 5.52, p ¼ .024) (Figure 2C), and number of visits to the centerof the arena (F1,36 ¼ 5.47, p ¼ .025) (Figure S2 in Supplement 1).Two-way ANOVA analysis indicated a significant PNFlx � SBinteraction for percent path length traversed (F1,33 ¼ 8.09,p ¼ .008) (Figure 2G) and percent time in the center (F1,33 ¼5.10, p ¼ .031) (Figure 2H). Number of visits to the center of thearena was unaltered across groups in the PNFlx � SB experi-ment groups (Figure S2 in Supplement 1). Total distancetraversed in the OFT was unaltered across all groups (FigureS2 in Supplement 1).

Postnatal fluoxetine-evoked depressive behavior on the FSTwas blocked by postnatal 5-HT2A, but not 5-HT2C, receptorblockade (Figure 2D,E,I,J). Two-way ANOVA analysis indicated asignificant PNFlx � MDL interaction for percent immobility time(F1,27 ¼ 5.21, p ¼ .030) (Figure 2D), latency to immobility (F1,27 ¼4.39, p ¼ .045) (Figure 2E), and number of immobility events (F1,27¼ 27.19, p � .0001) (Figure S2 in Supplement 1). Two-way ANOVAanalysis indicated no PNFlx � SB interaction for percent immo-bility time (Figure 2I), latency to immobility (Figure 2J), or numberof immobility events (Figure S2 in Supplement 1).

Postnatal 5-HT2A/C Receptor Stimulation Evokes AnxietyBehavior in Adulthood

Given we observed that postnatal 5-HT2A/C receptor block-ade prevented PNFlx-evoked anxiety, we next addressed theinfluence of postnatal 5-HT2A/C receptor stimulation on adultanxiety. Postnatal treatment with the 5-HT2A/C receptor agonistDOI (PNDOI) increased anxiety on the OFT and EPM inadulthood (Figure 3A–K). Postnatal DOI animals showed asignificant decline in percent path length (Figure 3C), percenttime spent (Figure 3D), and a trend (p ¼ .08) toward a declinein the number of visits (Figure 3E) to the center of the OFTarena. On the EPM, PNDOI animals showed a signifi-cant decrease in percent path length in the open arms(Figure 3H), increased percent path length in the closed arm(Figure 3I), and a trend (p ¼ .07) toward an increase in timespent in the closed arms (Figure 3J), with no change in percenttime in the open arms (Figure 3K). Total distance traversed inthe OFT (Figure 3F) and EPM (Figure S3 in Supplement 1) didnot differ across groups.

Postnatal 5-HT1A Receptor Blockade Does Not Alter PNFlx-Evoked Anxiety but Evokes Anxiety in Control Animals, anEffect Attenuated by Concomitant 5-HT2A/C ReceptorBlockade

We next examined whether concomitant treatment with theselective 5-HT1A receptor antagonist, WAY-100635, influencedPNFlx-mediated anxiety behavior on the OFT (Figure 4A). PostnatalWAY-100635 treatment did not alter the significant PNFlx-evokedanxiety on the OFT (Figure 4B–D). Two-way ANOVA analysisrevealed no significant PNFlx � WAY interaction for percent pathlength traversed (Figure 4C) and percent time spent (Figure 4D) inthe center of the arena. Postnatal WAY-100635 treatment to naiveanimals induced significant anxiety with a decline in the percentpath length (Figure 4C) (main effect of WAY: F1,20 ¼ 4.51, p ¼ .016)and percent time spent (Figure 4D) (main effect of WAY: F1,20 ¼9.90, p ¼ .005) in the center of the arena. Total distance traversedin the entire arena did not differ across groups (Figure 4E).

We then assessed whether the anxiety evoked by postnatalWAY-100635 administration was influenced by concomitant5-HT2A/C receptor antagonist treatment. Postnatal ketanserinadministration partially attenuated the anxiogenic effects ofpostnatal WAY100635 on the OFT in adulthood (Figure 4F–I).One-way ANOVA and post hoc Bonferroni analysis indicated thatwhile the WAY group exhibited a significant decline in percentpath length traversed (Figure 4G), percent time spent (Figure 4H),and number of visits (Figure 4I) to the center of the arena, theWAY � Ket group did not differ from control animals on thesemeasures. Total distance traversed in the arena was unalteredacross groups (Figure 4J).

Postnatal Ketanserin Treatment Blocks SpecificTranscriptional Changes Observed in the Prefrontal CortexFollowing PNFlx Treatment

We examined the influence of PNFlx on 5-HT1A (Htr1a),5-HT2A (Htr2a), and 5-HT2C (Htr2c) receptor expression in theprefrontal cortex, a region strongly implicated in the regulationof anxiety behavior. Quantitative polymerase chain reaction anal-ysis revealed a significant increase in prefrontal 5-HT2A messengerRNA (mRNA) levels in PNFlx animals in adulthood, with no changeobserved in the other 5-HT receptors examined (Figure 5A,B). ThePNFlx-evoked increase in 5-HT2A receptor mRNA was prevented bypostnatal ketanserin treatment. Two-way ANOVA analysis indicateda significant PNFlx � Ket interaction for 5-HT2A receptor expression


Figure 2. Postnatal serotonin 2A receptor blockade prevents both postnatal fluoxetine (PNFlx)-evoked anxiety and depressive behavior, whereasserotonin 2C receptor blockade prevents PNFlx-evoked anxiety but not depressive behavior. Shown is a schematic representation of the treatmentparadigm (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 treatmentprevented 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 pathlength traversed (G) and percent time spent (H) in the center of the OFT arena. Postnatal SB treatment did not alter the PNFlx-evoked depressive behavioron 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 ¼6–11 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 7–11animals/group). (*p � .05 as compared with Ctrl, $p � .05 as compared with PNFlx, &p � .05 as compared with SB, two-way analysis of variance andBonferroni post hoc test).



Figure 3. Chronic postnatal treatment with the serotonin 2A/serotonin 2C agonist DOI evokes anxiogenic responses in adulthood. Shown is a schematicrepresentation 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 tracksof 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 animalsexhibited significant decreases in the percent path length traversed (C) and percent time spent (D) in the center of the arena. Postnatal DOI animalsexhibited 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 unalteredbetween groups (F). Shown are representative tracks of exploratory behavior in the open (oa) and closed (ca) arms of the EPM from adult Ctrl and PNDOIanimals (G). Postnatal DOI treatment evoked anxiety behavior in the EPM test in adulthood with significant decreases in the percent path length traversedin 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 theclosed (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 forpercent 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, andtotal 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 ascompared with Ctrl (Student t test).


(F1,36 ¼ 5.43, p ¼ .026). We next addressed whether the expressionof the immediate early gene, activity-regulated cytoskeleton-asso-ciated protein, Arc, whose levels within the prefrontal cortex arereported to be decreased in animal models of depression (43), wasinfluenced by PNFlx treatment. Postnatal fluoxetine treatmentevoked a significant decline in prefrontal Arc mRNA levels in

adulthood, which was prevented by postnatal ketanserin treat-ment. Two-way ANOVA analysis indicated a significant PNFlx � Ketinteraction for Arc expression in the prefrontal cortex (F1,36 ¼ 7.14,p ¼ .011). These results indicate that postnatal ketanserin treatmentblocks the regulation of specific PNFlx-regulated genes in theprefrontal cortex in adulthood.


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 treatmentparadigm (A). Control (Ctrl) and postnatal fluoxetine-treated (PNFlx) pups received vehicle (Veh) or WAY-100635 (WAY) from postnatal day (P)2 to P21and 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 inducedanxiety 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 totaldistance moved in the OFT was unaltered across groups (E). Shown is a schematic of the treatment paradigm for the combined postnatal 5-HT2A/Cand serotonin 1A receptor antagonist experiment (F). Treatment with the 5-HT2A/C antagonist, ketanserin (Ket) ameliorated the WAY-evokeddecreases 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 wasunaltered 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 Bonferronipost hoc test).


Discussion

The major novel finding of our study indicates that PNFlx-evoked anxiety is prevented by postnatal 5-HT2A/C receptor


blockade and that postnatal 5-HT2A/C receptor stimulation behav-iorally mimics the anxiogenic effects of PNFlx. Our results withselective 5-HT2A and 5-HT2C receptor antagonists indicate that bothPNFlx-evoked adult anxiety and depressive behavior are normalized

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) pupsreceived 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 theprefrontal 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 ascompared 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 receptorblockade prevents the development of anxiety, but not depressivebehavior, in PNFlx animals. Further, we demonstrate that postnatal5-HT1A receptor blockade does not alter the development of anxietyfollowing PNFlx but is sufficient to evoke adult anxiety in naivecontrol animals, an effect diminished by 5-HT2A/C receptor antago-nist co-administration. Our results implicate 5-HT2A/C receptors inmediating the behavioral effects on anxiety and depressive behaviornoted in response to administration of the SSRI fluoxetine in earlylife. These observations provide strong support to the emergingview that a balance between 5-HT1A and 5-HT2A/C receptor-mediated signaling during the critical time window of postnatal lifeis of import in the emergence of anxiety.

Postnatal life represents a critical period wherein serotonergicneurotransmission is hypothesized to regulate the maturation ofemotional neurocircuitry, thus programming lasting effects onemotionality (1,2,44). Genetic knockout experiments with 5-HTtransporter knockout (5-HTT�/�) mice (14,45–47), as well aspharmacologic perturbations with SSRIs in rodents (13,14,48,49),indicate that elevated postnatal 5-HT levels evoke persistentincreases in anxiety and depressive behavior. However, thecontribution of specific 5-HT receptors to the persistent anxio-genic and depressive behaviors programmed by elevated 5-HTlevels during postnatal development is poorly understood. Ourpharmacologic studies provide novel evidence that implicate5-HT2A and 5-HT2C receptors in mediating specific behavioralconsequences of SSRI treatment in postnatal life.

Mouse mutant studies strongly implicate 5-HT1A, 5-HT2A, and5-HT2C receptors in the development of anxiety (2,50–52).Anxiolytic effects have been noted in both 5-HT2A and 5-HT2Creceptor knockout mice (27,28). Conditional reactivation of thecortical 5-HT2A receptor prevented the anxiolytic effects in5-HT2A receptor knockout mice; however, it is unknown whetherthere is a critical temporal window for such a rescue (27). In thisregard, it is of note that our studies with postnatal 5-HT2A/C,

5-HT2A, or 5-HT2C antagonist treatment did not influence anxietybehavior in adulthood in naive control animals but were eachcapable of preventing the development of PNFlx-evoked anxiety.In contrast, PNFlx-mediated depressive behavior on the FST wasprevented by selective 5-HT2A, but not 5-HT2C, receptor blockadein postnatal life. Baseline FST behavior in naive control animalswas not significantly altered following postnatal 5-HT2A or 5-HT2Creceptor blockade; however, we did observe significantantidepressant-like responses in control animals postnatallyadministered the 5-HT2A/C receptor antagonist. These resultssupport the notion that while both 5-HT2A and 5-HT2C receptorscontribute to the emergence of anxiety, 5-HT2A receptors maymediate the emergence of depressive behaviors following earlySSRI exposure. In addition to genetic knockout studies thatimplicate 5-HT2A/C receptors in the regulation of anxiety (2,27–30), recent reports demonstrate that models of psychiatricvulnerability based on early adverse experience, includingmaternal separation (53) and prenatal exposure to influenza(54), exhibit enhanced 5-HT2A/C receptor mediated signaling.Strikingly, chronic postnatal ketanserin treatment overlappingwith the period of maternal separation prevents the develop-ment of anxiety and the dysregulated cortical immediate-earlygene responses in maternally separated animals (26). Thissuggests the possibility that both adverse early experience, aswell as pharmacologic treatments with SSRIs in postnatal life,may impinge on 5-HT2A/C receptor signaling to program adultanxiety.

Further support for a role of 5-HT2A/C receptors in PNFlx-evoked anxiety comes from our results demonstrating thatchronic postnatal treatment with the 5-HT2A/C receptor partialagonist, DOI, also elicits adult anxiety. In contrast to theanxiogenic effects observed with chronic postnatal DOI treat-ment, acute, but not chronic, adult DOI administration is thoughtto evoke anxiolytic effects (55–57). The effects of 5-HT2A/Creceptor stimulation on anxiety behavior during postnatal versus



adult life appear distinct in nature, though the caveat that theduration of treatments varies has to be kept in mind. Adult-onsettreatment with 5-HT2A/C receptor antagonists (58–61) or intra-cerebroventricular administration of 5-HT2A antisense oligonu-cleotides also evokes anxiolytic effects (62). In addition to cortical5-HT2A receptors, 5-HT2A/C receptors within other brain regions,including the hypothalamus, amygdala, and periaqueductal gray,are also implicated in the regulation of anxiety (63–65). Theanxiolytic effects observed with both adult 5-HT2A/C receptoragonists and antagonists may involve specific contributions ofthese receptors in multiple brain circuits that regulate anxiety. Ourresults demonstrate that during the postnatal window, 5-HT2A/Creceptors may play an important role in programming adultanxiety. However, at present, it is difficult to delineate the specificneuronal circuits that underlie the behavioral effects of postnatal5-HT2A/C receptor agonist treatment and contribute to the 5-HT2A/Creceptor antagonist-mediated normalization of PNFlx-evoked anxi-ety and depressive behaviors. The pattern of expression of the5-HT1A, 5-HT2A, and 5-HT2C receptors shows a developmentalregulation with high levels observed in several cortical brainregions during postnatal life (66–72). Cortical excitatory responsesto 5-HT, driven via the 5-HT2A receptor, are noted during postnatallife within layer V output neurons of the prefrontal cortex, whichgradually tip into predominantly inhibitory responses to 5-HT,mediated by the 5-HT1A receptor, as animals mature (70–73). Thissuggests a transient postnatal window wherein elevated levels of5-HT, possibly through cortical 5-HT2A receptor-mediated respon-ses, may exert a strong influence on the development of emotionalneurocircuitry, such as the prefrontal cortex. Enhanced prefrontal5-HT2A receptor expression in PNFlx animals in adulthood, whichwas prevented by postnatal 5-HT2A/C receptor blockade, raises theintriguing possibility of a tipping toward enhanced 5-HT2A receptor-driven responses, as has been noted in other models of psychiatricvulnerability (53,54). Postnatal fluoxetine animals also exhibited asignificant decline in the expression of the activity responsiveimmediate early gene, Arc, in the prefrontal cortex, which wasprevented by concomitant 5-HT2A/C receptor blockade. Previousstudies indicate a decline in prefrontal Arc expression in susceptiblemice following social defeat and in depressed patients (43). Suchan alteration in immediate early gene expression may be indicativeof altered cortical excitability following PNFlx. Future studies arerequired to identify the contribution of specific neuronal circuits tothe behavioral consequences of PNFlx, and to address whetheradult-onset 5-HT2A/C receptor blockade can alter the trajectory ofPNFlx-evoked emotional behaviors.

Genetic perturbations and pharmacologic studies indicate arole for cortical 5-HT1A heteroreceptors and raphe 5-HT1A autor-eceptors in the developmental programming of anxiety anddepressive behaviors (29,30,32). While our results confirmed priorreports (25) that postnatal 5-HT1A receptor antagonist treatmentenhances anxiety in control groups, it did not alter PNFlx-evokedanxiety behavior. Interestingly, the postnatal WAY-100635-evokedanxiety in naive control animals was attenuated by concomitant5-HT2A/C receptor blockade, suggesting an interaction betweenthese 5-HT receptors during postnatal life in the development ofanxiety. Immunohistochemical (74) and electrophysiological stud-ies (75,76) indicate co-localization of 5-HT1A and 5-HT2A receptorsin prefrontal glutamatergic neurons, and prior studies suggest afunctional interaction between these receptors in the regulationof threat perception and prefrontal regulation of amygdalareactivity (77).

Taken together, our results indicate that 5-HT2A and 5-HT2Creceptors contribute to the anxiety and depressive behaviors


evoked by PNFlx and suggest that an optimal balance between5-HT1A and 5-HT2A/C receptor-mediated drive during the postnataltemporal window may be required for the programming ofnormal levels of anxiety. We posit that SSRI treatments withinthis temporal window may disrupt the balance between 5-HT1Aand 5-HT2A/C receptor function and could serve to tip the systemtoward enhanced 5-HT2A/C receptor-mediated drive, thus contri-buting to the establishment of persistent increases in anxietynoted long after the cessation of SSRI treatment. It is tempting tospeculate that multi-action drugs that exhibit combined 5-HT1Aagonist and 5-HT2A/C antagonist properties may serve as putativetherapeutic agents for the treatment of early-onset anxietydisorders (78,79). Our results provide impetus for future studiesto address the key role of 5-HT2A and 5-HT2C receptors and theirdownstream targets in contributing to the behavioral effects ofearly SSRI administration.

This research was supported by a Tata Institute of FundamentalResearch intramural grant (VAV).

The authors report no biomedical financial interests or potentialconflicts of interest.

Supplementary material cited in this article is available online athttp://dx.doi.org/10.1016/j.biopsych.2013.11.005.

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