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Neuropharmacology 65 (2013) 213e222

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Neuropharmacology

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

Adolescent male rats are less sensitive than adults to the anxiogenic andserotonin-releasing effects of fenfluramine

Andrew E. Arrant, Hikma Jemal, Cynthia M. Kuhn*

Department of Pharmacology & Cancer Biology, Duke University, Room 100B Research Park Building 2, Box 3813, Duke University Medical Center, Durham, NC 27710, USA

a r t i c l e i n f o

Article history:Received 28 August 2012Received in revised form14 October 2012Accepted 18 October 2012

Keywords:AdolescenceSerotoninAnxietyMicrodialysis

* Corresponding author. Tel.: þ1 919 684 8828; faxE-mail address: ckuhn@duke.edu (C.M. Kuhn).

0028-3908/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2012.10.010

a b s t r a c t

Risk taking behavior increases during adolescence, which is also a critical period for the onset of drugabuse. The central serotonergic system matures during the adolescent period, and its immaturity duringearly adolescence may contribute to adolescent risk taking, as deficits in central serotonergic functionhave been associated with impulsivity, aggression, and risk taking. We investigated serotonergicmodulation of behavior and presynaptic serotonergic function in adult (67e74 days old) and adolescent(28e34 days old) male rats. Fenfluramine (2 mg/kg, i.p.) produced greater anxiogenic effects in adult ratsin both the light/dark and elevated plus maze tests for anxiety-like behavior, and stimulated greaterincreases in extracellular serotonin in the adult medial prefrontal cortex (mPFC) (1, 2.5, and 10 mg/kg,i.p.). Local infusion of 100 mM potassium chloride into the mPFC also stimulated greater serotonin effluxin adult rats. Adult rats had higher tissue serotonin content than adolescents in the prefrontal cortex,amygdala, and hippocampus, but the rate of serotonin synthesis was similar between age groups.Serotonin transporter (SERT) immunoreactivity and SERT radioligand binding were comparable betweenage groups in all three brain regions. These data suggest that lower tissue serotonin stores in adolescentslimit fenfluramine-stimulated serotonin release and so contribute to the lesser anxiogenic effects offenfluramine.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Adolescence is the period of transition from childhood toadulthood (Spear, 2000). This transitional period includes thetime from around 12 to 18 years of age in humans (reviewed inSpear, 2000). In rodents, adolescence encompasses postnatal days28e42 (PN28e42), though adulthood is not considered to beginuntil around PN60 (reviewed in McCutcheon and Marinelli, 2009;Spear, 2000). Behavior changes during adolescence, with risktaking, novelty seeking, and social behavior expressed at higherlevels than in childhood or adulthood (Stansfield and Kirstein,2006; Steinberg et al., 2008, 2009; reviewed in Spear, 2000).Impulsive, risk taking behavior is part of normal development, butalso contributes to major causes of adolescent injury and mortalitysuch as reckless driving, suicide, unsafe sexual behavior, andexperimentation with drugs (Chen and Kandel, 1995; Eaton et al.,2010; SAMHSA, 2011; Steinberg, 2008; reviewed in Spear, 2000).

Immature function of the neural circuits that mediate goaldirected behavior contributes to adolescent risk taking. The balance

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between systems mediating approach to rewarding stimuli andavoidance of aversive stimuli may be biased toward approachduring adolescence (reviewed in Ernst and Fudge, 2009; Ernst et al.,2006). Prefrontal cortical regulation of limbic brain regions isimmature, limiting the regulation of these approach and avoidancedrives (reviewed in Casey et al., 2011; Ernst and Fudge, 2009; Ernstet al., 2006; Steinberg, 2010; Sturman and Moghaddam, 2011).Immaturity of dopaminergic and serotonergic function in theforebrain may also contribute to this approach/avoidance imbal-ance in adolescents (reviewed in Chambers et al., 2003; Crews et al.,2007; Ernst et al., 2006). Serotonin is an important mediator ofbehavioral inhibition in response to aversive situations, and lowcentral serotonergic function has been associated with risk taking,impulsivity, and aggression (Brown et al., 1979; Crockett et al.,2009; Higley and Linnoila, 1997; Higley et al., 1996; Mehlmanet al., 1994; Soubrie, 1986; Virkkunen et al., 1995). Serotonin alsocontributes to the aversive effects of some drugs of abuse, andadolescents are less sensitive to aversive effects of drugs in animalmodels (Ettenberg and Bernardi, 2006, 2007; Ettenberg et al., 2011;Infurna and Spear, 1979; Jones et al., 2009, 2010; Rocha et al., 2002;Schramm-Sapyta et al., 2006; Serafine and Riley, 2010). Lowerserotonergic function in adolescents could therefore contribute toincreased risk taking behavior and reduce the aversive effects of

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drugs of abuse. These effects could factor into the increasedexperimentation with drugs seen during adolescence (Chen andKandel, 1995; SAMHSA, 2011).

Animal studies suggest that forebrain serotonergic functionduring early adolescence may be lower than in adults, especially inthe cortex. While serotonin receptor expression, dorsal raphe firingrates, and the anatomic pattern of serotonergic innervation appearto be mature by adolescence, neurochemical markers of presyn-aptic serotonergic function increase between adolescence andadulthood (Beique et al., 2004; Daval et al., 1987; Garcia-Alcoceret al., 2006; Lanfumey and Jacobs, 1982; Lidov and Molliver, 1982;Miquel et al., 1994; Pranzatelli and Galvan, 1994; Vizuete et al.,1997; Waeber et al., 1996, 1994). Serotonin transporter (SERT)binding is lower in the cortex of early adolescent rats (PN28e35),and some studies show lower SERT binding in subcortical regionssuch as the amygdala and striatum (Dao et al., 2011; Galineau et al.,2004; Moll et al., 2000; Tarazi et al., 1998). Serotonin tissue contentand synaptosomal uptake are also lower in the cortex and striatumof early adolescent rats compared to adults (Kirksey and Slotkin,1979; Loizou, 1972; Loizou and Salt, 1970; Mercugliano et al.,1996). Studies during later adolescence have reported moreadult-like serotonin function. Older adolescent rats (PN45e50) andadults have similar baseline and methamphetamine-stimulatedextracellular serotonin in medial prefrontal cortex (mPFC) (Staitiet al., 2011). SERT binding and serotonin uptake are also matureby this age (Kirksey and Slotkin, 1979; Tarazi et al., 1998). Thesestudies show that neurochemical markers of presynaptic seroto-nergic function reach adult levels during the adolescent period, andthat these markers are lower than in adults during early adoles-cence from PN28 to PN35.

While the ontogeny of forebrain serotonergic innervation hasbeen described in young adolescents, serotonin release in responseto pharmacologic or behavioral stimuli has not been evaluated overthis critical developmental window. Additionally, the ability ofserotonergic drugs to influence some behaviors has been studied inadolescents of several species, but a direct comparison of seroto-nergic regulation of behavioral inhibition in adolescents and adultshas not been reported (Higley et al., 1996; LeMarquand et al., 1998;Mehlman et al., 1994; Zepf et al., 2008). The purpose of this study isto address this gap in our understanding of how serotonin regulatesbehavior during adolescence by assessing behavioral and neuro-chemical responses to pharmacologic challenge of the serotonergicsystem in adults (PN67e74) and early adolescents (PN28e34). Weused the serotonin-releasing drug fenfluramine to compare theability to mobilize serotonin stores in adults and early adolescents.Fenfluramine mobilizes serotonin stores by a similar mechanism asdrugs of abuse such as methamphetamine and methylenediox-ymethamphetamine (MDMA), but is more selective for the sero-tonergic system than these drugs (Rothman et al., 2001; reviewedin Sulzer et al., 2005). The behavioral effects of fenfluraminetreatment were evaluated in the light/dark (LD) and the elevated

Fig. 1. A description of the timing from arrival in our animals facility to A) behavior testing inAdolescent rats arrived in our facilities as juveniles at PN21, while adult rats arrived as youmicrodialysis testing. All experiments were timed to occur during the first week of adolesc

plus maze (EPM) tests for anxiety-like behavior. Unconditionedtests for anxiety-like behavior such as the LD and EPM are thoughtbe an ethologically relevant model of risk taking behavior becausethey measure inhibition of species-typical behaviors in novel,aversive, and potentially risky environments (Harro, 2002; Macriet al., 2002; Olausson et al., 1999). We then used microdialysis toassess the effect of fenfluramine and potassium on extracellularserotonin in the mPFC, and further investigated presynaptic sero-tonin function by measuring serotonin content, synthesis, inner-vation density, and SERT levels in the prefrontal cortex, amygdala,and hippocampus.

2. Materials and methods

2.1. Animals

Young adult (PN60e63) and juvenile (PN21) male SpragueeDawley (CD) ratswere purchased from Charles River Laboratories (Raleigh, NC). The rats were housedin ventilated plastic cages (Techniplast USA, Exton, PA) or standard rat cages(Allentown Caging, Allentown, NJ) with corn cob bedding on a 12:12 h light/darkcycle with lights on at 06:00 and lights off at 18:00. All rats were allowed to accli-mate to our AALAC accredited facility for at least 7 days before behavior testing orcollection of tissue or microdialysis samples. The acclimation period did not exceed13 days to ensure that adolescent rats were tested during the first week of adoles-cence (PN28e34). The timeline from arrival in our animal facility to behavior ormicrodialysis testing is depicted in Fig. 1. Separate cohorts of animals were used forall behavioral and microdialysis experiments to avoid potential confounding effectsof repeated testing upon behavior or extracellular serotonin levels. All experimentswere approved by the Duke University Institutional Animal Care and UseCommittee.

2.2. Drugs

Fenfluramine hydrochloride, 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015), and potassium chloride were purchased from SigmaeAldrich (St. Louis, MO).Fenfluramine and NSD-1015 were dissolved in saline the morning of testing andinjected i.p. at a volume of 1 mL/kg and 2 mL/kg, respectively. Ketamine (Ketaset)was purchased from Fort Dodge Animal Health (Fort Dodge, IA) and xylazine(Anased) was purchased from Lloyd Inc. (Shenandoah, IA). Ketamine and xylazinewere administered i.m. at a volume of 1 mL/kg.

2.3. Light/dark test

Rats were tested in Kinder locomotor boxes (Kinder Scientific, Inc. Poway, CA)(40 cm � 40 cm � 40 cm) with black plastic inserts (20 cm � 40 cm � 40 cm) thatoccupied half of the locomotor box. Each insert had a sliding door that restrictedaccess to one part of the box. The roomwas lit by two incandescent lamps so that thelighting in the light side of each box was 65 lux on average. Rats were placed in thedark half of the box and testing began as the door to the light side of the box wasraised. Time and distance traveled in each compartment were measured for 15 minwith infrared photobeams and software from the manufacturer. The latency toemerge into the light compartment was determined by dividing the session into 5 sbins and determining the bin of first light entry. Adult and adolescent rats wereinjected with either saline or fenfluramine (2 mg/kg) 30 min prior to testing (n ¼ 12per age, per treatment). This dose of fenfluramine was expected to be non-maximalin terms of increasing extracellular serotonin in the brain (Gundlah et al., 1997;Rocher and Gardier, 2001; Tao et al., 2002). For each age group, saline-treatedanimals were used as a control group to determine baseline behavior. Behavior offenfluramine-treated animals was compared to age-matched saline controls for

the LD or EPM tests, B) microdialysis testing. Animal ages are shown in postnatal days.ng adults aged PN60e63. All animals waited a minimum of 7 days before behavior orence (PN28e34).

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determination of drug effects. A total of 48 rats were used for this experiment (12per experimental group, adult and adolescent rats treated with saline orfenfluramine).

2.4. Elevated plus maze

The elevated plus maze was made of black painted wood and consisted of twoopen arms and two closed arms arranged in a “plus” shape elevated 60 cm from thefloor. The arms were 60 cm � 9 cm. The closed arms were enclosed by a 50 cmwalland the open arms had a 1 cm ledge. Rats were placed in an open field for 5 minimmediately prior to testing to enhance the ability to measure anxiogenic drugeffects (Pellow and File, 1986). Rats were placed in the center square (9 � 9 cm)facing an open arm and allowed to explore the maze for 5 min. White sheets werehung around the maze to create a neutral visual field. Average lighting was 8 lux foropen arms and 2 lux for closed arms. All sessions were recorded with a video cameraand scored for time in each area of the maze, entries into arms, and the latency tofirst open arm entry. Adult and adolescent rats were injected with either saline orfenfluramine (2 mg/kg) 30 min prior to placement in the open field. Behavior offenfluramine-treated animals was compared to age-matched saline controls fordetermination of drug effects. A total of 60 rats were used for this experiment (15 perexperimental group, adult and adolescent rats treated with saline or fenfluramine).

2.5. Stereotaxic surgery

Animalswere anesthetizedwith ketamine (100mg/kg i.m.) and xylazine (20mg/kg i.m.) and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA). Bodytemperature was maintained with a Deltaphase isothermal heating pad (BraintreeScientific, Braintree, MA). Guide cannulae (BAS, inc. West Lafayette, IN) wereimplanted 2 mm over the medial prefrontal cortex, and placed at þ3.2 mm anteriorand þ0.8 mm lateral from bregma and �3.0 mm from the dura based on a rat brainatlas (Paxinos and Watson, 1986). Coordinates for adolescent rats were adjustedtoþ2.8 mm anterior and þ0.8 mm lateral from bregma and�2.5 mm from the dura.Adolescent coordinates were empirically determined in preliminary dye injectionand microdialysis experiments. The cannula was secured using three anchor screwsand dental cement. Animals were allowed to recover for a minimum of 48 h beforeinsertion of the microdialysis probe. Ibuprofen (20 mg/kg/day) was administeredthrough the drinking water from 3 days prior to surgery until the night beforemicrodialysis experiments in accordance with Duke University IACUC standards.

2.6. Microdialysis set up and collection of baseline samples

The evening prior to all experiments, animals were briefly anesthetized withisoflurane and a 2 mm microdialysis probe (BAS, Inc. West Lafayette, IN) wasinserted into the cannula. Animals were placed in a clear plastic microdialysischamber (BAS, Inc. West Lafayette, IN) and the probe was perfused overnight withartificial cerebrospinal fluid (aCSF, 147 mMNaCl, 2.7 mMKCl, 1.2 mM CaCl2, 0.85 mMMgCl2, CMA Microdialysis, Solna, Sweden) at 0.5 mL/min using a syringe pump(Harvard Apparatus, Holliston, MA). The microdialysis cages were bowl-shaped andhad a diameter of approximately 36 cm at the bottom of the cage, and a depth ofapproximately 30 cm with bedding. The same size cages were used for each agegroup. The inlet and outlet lines for the microdialysis probe were run througha liquid swivel (BAS, Inc,West Lafayette, IN), and animals were tethered to the swivelby plastic collars. Food and water were freely available throughout the overnightequilibration and subsequent experiments. The followingmorning the flow ratewasincreased to 1 mL/min for 1 h before collection of baseline samples. Baseline sampleswere then collected for 2 h prior to experimental manipulations. Samples werecollected on ice, then immediately frozen on dry ice for later analysis by HPLC withelectrochemical detection. One adult and one adolescent rat were run simulta-neously each day.

2.7. Fenfluramine dose response

Animals were sequentially injected with 1, 2.5, and 10 mg/kg fenfluramine i.p.with 3 h between each dose. Samples were collected at 30 min intervals. All threedoses were given to the same animal on the same day. Collection of baseline samplesbegan at 7 AM and the experiments concluded around 6 PM to ensure that allsamples were collected during the light phase. The baseline samples from eachanimal were used as an internal control for comparison with fenfluramine-stimulated extracellular serotonin levels. A total of 17 animals were used for thisexperiment (9 adults and 8 adolescents).

2.8. Potassium depolarization

After collection of baseline samples, the aCSF in the syringes was replaced withaCSF containing 100 mM potassium chloride. The high potassium aCSF was perfusedfor 20 min before the syringes were switched back to normal aCSF. Samples werecollected for another 4 h with normal aCSF. All samples were collected in 20 minintervals. A total of 22 animals were used for this experiment (12 adults and 10adolescents).

2.9. Verification of probe placement

After conclusion of microdialysis experiments, animals were anesthetized withisoflurane and decapitated. Brains were postfixed in 10% formalin, cut into 30 mmsections, and stained with cresyl violet to confirm probe placement (SupplementaryFig. 1). Animals with probes placed greater than �0.5 mm anterior or posterior fromthe target region of 3.2 mm anterior from bregma were excluded from furtheranalysis (1 adult and 3 adolescents). These animals are not included in the samplesizes given for the fenfluramine or potassium microdialysis experiments.

2.10. HPLC detection for microdialysis

Dialysates were directly injected onto a 2.1 � 100 mm reversed phase C18column (Phenomenex, Torrance, CA). Themobile phase consisted of 150mM sodiumphosphate, 4.8 mM citric acid, 3 mM sodium dodecyl sulfate, 50 mM EDTA, with 11%methanol and 17% acetonitrile, pH ¼ 5.6 and was run at a flow rate of 0.2 mL/min.Serotonin was measured using an electrochemical detector set to 0.55 V (BAS, Inc.West Lafayette, IN). The sensitivity of the assay was 1 fmol of serotonin in a 15 mLinjection. Samples were quantitated relative to an external standard curve run eachday.

2.11. Serotonin synthesis and tissue serotonin content

Adult and adolescent rats (n ¼ 8 per age, per treatment) were injected withsaline or 100mg/kg NSD-1015 to inhibit l-amino acid decarboxylase and decapitated30 min later. Brains were collected at ages PN28e29 for adolescent rats and PN67e71 for adult rats. Levels of 5-hydroxytryptophan (5-HTP) after treatment with NSD-1015 were used to compare the rate of serotonin synthesis (Carlsson et al., 1972).Serotonin and 5-HIAA were also compared in saline-treated adults and adolescents.Prefrontal cortex, amygdala, and hippocampus were dissected using a brain block(Heffner et al., 1980). The samples were weighed, frozen on dry ice, and stored at�80 �C until HPLC analysis. Tissue samples were homogenized by sonication in 0.2 Nperchloric acid and injected onto a 4.6 � 100 mm reversed phase C18 column(Phenomenex, Torrance, CA). Serotonin, 5-HIAA, dopamine, DOPAC, HVA, and 5-HTPweremeasured using electrochemical detection at 0.70 V (BAS,West Lafayette, IN) ata flow rate of 0.7 mL/min. The mobile phase consisted of 0.1 M sodium phosphate,0.8 mM octanesulfonic acid, 0.1 mM EDTA, and 18% methanol adjusted to pH 3.10(Miguez et al., 1995). Samples were quantitated relative to an external standardcurve run each day. Protein content per tissue wet weight is similar in the forebrainof early adolescent (PN30) and adult rats, so data from each sample were correctedfor tissue weight (Porcher and Heller, 1972).

2.12. Serotonin transporter immunostaining

Adult (PN67e70) and adolescent (PN28) rats were anesthetized and trans-cardially perfused with PBS followed by 10% formalin. Brains were postfixed in 10%formalin overnight at 4 �C, then cryoprotected in 30% sucrose until sectioning. Thebrains were cut into 30 mm sections and mounted onto slides prior to immuno-staining. After heat mediated antigen retrieval in citric acid buffer (10mM citrate, pH6.0), the sections were stained with an anti-SERT primary antibody (Immunostar,Hudson, WI) followed by an anti-rabbit secondary antibody conjugated to AlexFluor594 (Invitrogen, Carlsbad, CA) and counterstained with DAPI (Invitrogen). Thesections were imaged on a widefield fluorescent microscope with a motorized stage(DeltaVision Elite, Applied Precision, Issaquah, WA). Panels of images were taken ofthe prefrontal cortex, amygdala, and dorsal hippocampus using a 20� objective,with 7 z-stacks per panel. The images were deconvoluted, projections were made ofthe z-stacks, and the panels were stitched together to create a single high-resolutionimage. For analysis, the amygdala was subdivided into central and basolateralamygdala, and the hippocampus was subdivided into CA1, CA3, and dentate gyrus.ImageJ software was used for analysis. The images were thresholded to excludebackground, and the percent of the total image area occupied by thresholdedstructures was used to quantitate the density of SERT-immunoreactive innervation.A total of 24 rats were used for this experiment (12 per age group).

2.13. Serotonin transporter radioligand binding

Samples of prefrontal cortex, amygdala, and hippocampus were collected fromadult (PN67e70) and adolescent (PN28) rats using the same procedure as for HPLCanalysis. The samples were immediately frozen on dry ice and stored at�80 �C untilanalysis. Samples were thawed and homogenized by sonication in 20 volumes ofphosphate buffer (8 mM sodium phosphate dibasic, 2 mM sodium phosphatemonobasic, 118 mMNaCl, 5 mM KCl, pH 7.4). The samples (10 mg of protein per tube)were incubatedwith 3H-paroxetine (Perkin Elmer,Waltham,MA) for 90min at roomtemperature. Fluoxetine (10 mM) was used for determination of nonspecific binding.The reactions were terminated by the addition of 3 mL of ice cold buffer, and thenfiltered onto glass fiber filters (Cambridge Technology, Watertown, MA) presoakedin 0.05% polyethylenimine to reduce nonspecific binding.

A single point binding analysis was performed for each sample and saturationbinding analysis was run on pooled adult and adolescent samples from each brain

A.E. Arrant et al. / Neuropharmacology 65 (2013) 213e222216

region. For single point analysis, samples were incubated with 50 pM 3H-paroxetineso that age differences in either the affinity or total number of binding sites could bedetected. The samples were then pooled for saturation analysis, with one pool perage group for each region. Pooled samples were run with a range of 3H-paroxetineconcentrations from 10 pM to 2000 pM. Nonlinear fits of saturation binding curveswere performed with GraphPad Prism 5.0 (Graphpad, La Jolla, CA). Sixteen rats wereused for this experiment (8 per age group).

2.14. Data analysis and statistics

The effects of age and fenfluramine treatment on anxiety-like behavior in the LDand EPM tests were analyzed by two-way ANOVAwith Age and Treatment as factorsusing NCSS 2004 software (NCSS, Kayesville, UT). Data from microdialysis experi-ments were analyzed by repeated measures ANOVA with Age as a between factorand Time and Dose (fenfluramine only) as within factors. Repeated measuresANOVAwith Age as a between factor and Brain Region as awithin factor was used toinvestigate the effect of age on tissue serotonin and 5-HIAA content, SERT immu-nostaining, and 3H-paroxetine binding across brain regions. The effects of age andNSD-1015 treatment on serotonin synthesis in multiple brain regions were analyzedby repeatedmeasures ANOVAwith Age and Treatment as between factors, and BrainRegion as a within factor. For all analyses, significant three-way interactions werefurther investigated by lower-order two-way ANOVA and significant two-wayinteractions were followed by NewmaneKeuls post-hoc testing with significanceset at p < 0.05. All data are shown as mean � SEM.

3. Results

3.1. Light/dark test

The time spent in the light compartment and the latency toemerge into the light were used as measures of anxiety-likebehavior, and the total distance traveled was used to assess loco-motion (Morley et al., 2005; Schramm-Sapyta et al., 2007). Fen-fluramine increased anxiety-like behavior in both ages (n ¼ 12 perexperimental group) as shown by a reduction in time spent inthe light compartment (Fig. 2A) [main effect of Treatment,F(1,39) ¼ 77.92, p < 0.001]. This effect was significantly greater inadult rats, as indicated by a significant Age � Treatment interaction[F(1,39) ¼ 7.75, p < 0.01]. Post-hoc testing showed that fenflur-amine reduced the time spent in the light compartment more inadults than in adolescents. Fenfluramine produced similar age-dependent anxiogenic effects on latency to emerge (Fig. 2B)

Fig. 2. Summary of behavior in the LD test. A) Time spent in the light compartment, B)compartment, D) total distance traveled. * ¼ Significantly different from age-matched conadolescents, p < 0.05 by NewmaneKeuls post-hoc testing.

[Age � Treatment interaction F(1,40) ¼ 5.91, p < 0.05]. Post-hoctesting confirmed that fenfluramine increased latency to emergein adult, but not adolescent, rats relative to controls. Fenfluraminereduced the number of entries into the light compartment (Fig. 2C)[main effect of Treatment F(1,39) ¼ 28.83, p < 0.001] and totaldistance (Fig. 2D) [main effect of Treatment F(1,39) ¼ 50.30,p < 0.001] similarly in both ages.

3.2. Elevated plus maze

Fenfluramine was also more anxiogenic in adult rats thanadolescents in the EPM (n ¼ 15 per experimental group). Thepercent time spent in the open and closed arms of the maze, thenumber of entries into the open arms, and the latency to emergeinto an open arm were used as measures of anxiety-like behavior,while the number of entries into closed arms was used as an indexof locomotion (Cruz et al., 1994; File et al., 2004, 1993; Rogerio andTakahashi, 1992). Fenfluramine produced significantly greateranxiogenic effects in adult rats than adolescents on percent timespent in the closed arms (Fig. 3D) [Age � Treatment interaction[F(1,55) ¼ 4.41, p < 0.05], and the latency to enter an open arm(Fig. 3C) [Age � Treatment interaction F(1,55) ¼ 4.40, p < 0.05].Post-hoc testing showed that fenfluramine-treated adults spentmore time in the closed arms and took longer to enter an open armthan adult controls, while adolescents were unaffected. A maineffect of Age [F(1,55) ¼ 9.89, p < 0.01] was found for the number ofentries into the open arms (Fig. 3B), which was driven by a decreasein open entries in fenfluramine-treated adults. No main effects orinteractions were identified by an ANOVA of percent time spent inthe open arms (Fig. 3A). Several of the adult saline-treated rats hadlow time in open arms and open entries, which could have causeda floor effect for seeing significant treatment effects in thesemeasures. Rat behavior in the EPM is variable and is affected bymany environmental factors (File et al., 2004). Adolescents are lesssensitive than adults to environmental effects on EPM behavior,which may have contributed to the lower variability in saline-treated adolescents (Doremus et al., 2004). There were no signifi-cant effects of fenfluramine on the number of closed entries

latency to emerge into the light compartment, C) number of entries into the lighttrols, ** ¼ significantly different from age-matched controls and fenfluramine-treated

Fig. 3. Summary of behavior in the EPM. A) Percentage of total time spent in the open arms, B) number of open arm entries, C) latency to enter an open arm, D) percentage of totaltime spent in the closed arms, E) number of closed arm entries. * ¼ Significantly different from age-matched controls, p < 0.05 by NewmaneKeuls post-hoc testing.

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(Fig. 3E), but ANOVA revealed a main effect of Age [F(1,55) ¼ 18.81,p < 0.001].

3.3. Fenfluramine microdialysis

Fenfluramine-stimulated serotonin levels were greater in adultrats (n ¼ 9) than adolescents (n ¼ 8). The baseline serotonin levelswere used as a within-animal control for comparison withfenfluramine-stimulated extracellular serotonin. Adolescent ratshad higher baseline extracellular serotonin than adults at the 1 h

Fig. 4. Microdialysis results frommedial prefrontal cortex. Fenfluramine-stimulated serotoniefflux (C) and percent increase from baseline (D). * ¼ Significant age difference at that tim

time point as shown by ANOVA and post-hoc testing [Age � Timeinteraction serotonin F(3,45) ¼ 3.38, p < 0.05, percent increaseF(3,45) ¼ 3.49, p < 0.05]. Fenfluramine injection producedincreases in extracellular serotonin that peaked 1 h after injection.Serotonin levels did not completely return to baseline during the3 h after each dose, resulting in a cumulative increase in serotoninacross time [main effect of Time F(3,45) ¼ 93.76, p < 0.001]. GlobalANOVA indicated differential effects of the fenfluramine doseresponse curve in each age group with an Age � Time � Doseinteraction for serotonin levels in each sample (Fig. 4A)

n levels (A) and percent increase from baseline (B) and potassium-stimulated serotonine point, p < 0.05 by NewmaneKeuls post-hoc testing.

A.E. Arrant et al. / Neuropharmacology 65 (2013) 213e222218

[F(9,135) ¼ 2.64, p < 0.01] and the percent increase from baseline[Fig. 4B, F(9,135) ¼ 2.14, p < 0.05]. This interaction was furtherinvestigated by two factor (Age, Time) repeated measures ANOVAand post-hoc testing for each dose of fenfluramine. SignificantAge � Time interactions were observed at each dose for serotoninlevels [1 mg/kg F(5,74) ¼ 2.73, p < 0.05, 2.5 mg/kg F(5,75) ¼ 4.42,p< 0.01,10mg/kg F(5,75)¼ 3.79, p< 0.01] and the percent increasefrom baseline [1 mg/kg F(5,74) ¼ 2.98, p < 0.05, 2.5 mg/kgF(5,75) ¼ 3.96, p < 0.01, 10 mg/kg F(5,75) ¼ 3.19, p < 0.05], indi-cating differential effects of each dose between age groups. Post-hoc testing showed that adolescents had lower fenfluramine-stimulated serotonin release than adults during the two peaksamples following each dose.

3.4. Potassium evoked serotonin release

A 20 min infusion of 100 mM KCl into the mPFC increasedserotonin levels above baseline for 40 min and produced a greaterincrease in extracellular serotonin in adult rats (n ¼ 12) comparedto adolescents (n ¼ 10). ANOVA revealed an Age � Time interac-tion for serotonin levels (Fig. 4C) [F(15,285) ¼ 2.37, p < 0.01] andpercent increase from baseline (Fig. 4D) [F(15,285) ¼ 3.64,p < 0.001], indicating age differences in the response to potassiuminfusion. Post-hoc testing showed that adolescent extracellularserotonin levels were significantly lower than those of adults atboth time points of elevated serotonin following potassiuminfusion.

3.5. Tissue serotonin content and synthesis

ANOVA for all tissue measures revealed a main effect of Treat-ment with NSD-1015 for 5-HIAA [F(1,28) ¼ 38.99, p < 0.001], soonly saline-treated animals (n ¼ 8 per age group) were used to

Fig. 5. (AeC) Baseline tissue serotonin content, 5-HIAA levels, and 5-HIAA/5-HT for prefrp < 0.05. (DeF) Accumulation of 5-HTP after treatment with NSD-1015 (100 mg/kg) in pre

investigate age differences in serotonin, 5-HIAA, 5-HIAA/5-HT.Adolescent rats had lower serotonin content (Fig. 5AeC) thanadults across the prefrontal cortex, amygdala, and hippocampus[main effect of Age F(1,14) ¼ 49.57, p < 0.001]. ANOVA alsoproduced an Age � Region interaction for serotonin content[F(2,28) ¼ 7.45, p < 0.01], and post-hoc testing confirmed thatadolescents had lower serotonin content than adults in all threebrain regions. No age differences were detected in 5-HIAA levels.ANOVA confirmed brain regional differences in both serotonin[main effect of Region F(2,28) ¼ 210.69, p < 0.001] and 5-HIAA[main effect of Region F(2,28) ¼ 258.98, p < 0.001], with levels ofeach being highest in the amygdala. Adolescent rats had higherserotonin turnover (5-HIAA/5-HT) than adults [main effect of AgeF(1,14) ¼ 52.82, p < 0.001], which reflected the lower serotoninlevels and comparable 5HIAA. Treatment with the decarboxylaseinhibitor NSD-1015 increased 5-HTP levels similarly in each agegroup (Fig. 5DeF, n ¼ 8 per age, per treatment) [main effect ofTreatment F(1,28)¼ 265.54, p< 0.001], indicating comparable ratesof serotonin synthesis.

3.6. Serotonin transporter immunostaining

The density of SERT-immunoreactive innervation was quanti-tated in prefrontal cortex, amygdala, and hippocampus to investi-gate possible age differences (n¼ 12 per age group) (SupplementaryFig. 2). There were significant regional differences in the densityof SERT-immunoreactive axons (Fig. 6A) [main effect RegionF(5, 104) ¼ 192.4, p < 0.001]. SERT immunoreactivity was highestin the basolateral amygdala, as has been seen in previous immu-nostaining and autoradiography studies (Hrdina et al., 1990; Suret al., 1996). No age difference in SERT immunoreactivity wasdetected in these brain regions and there was no interaction ofRegion and Age.

ontal cortex (A), amygdala (B), and hippocampus (C). * ¼ Significant age difference,frontal cortex (D), amygdala (E), and hippocampus (F).

Fig. 6. Density of SERT-immunoreactive projections (A) and single point binding datafor 50 pM 3H-paroxetine (B). A) mPFC ¼ medial prefrontal cortex, BLA ¼ basolateralamygdala, CeA ¼ central amygdala, DG ¼ dentate gyrus, B) PFC ¼ prefrontal cortex,AMG ¼ amygdala, HPC ¼ hippocampus.

A.E. Arrant et al. / Neuropharmacology 65 (2013) 213e222 219

3.7. Serotonin transporter radioligand binding

We performed 3H-paroxetine binding in homogenates ofprefrontal cortex, amygdala, and hippocampus to compare totalSERT levels between age groups (n ¼ 8 per age group). Single pointbinding with 50 pM 3H-paroxetine (Fig. 6B) revealed no effect ofAge on ligand binding, though there were regional differences inbinding [main effect of Region F(2,28) ¼ 84.37, p < 0.001]. Theamygdala had higher binding levels than cortex or hippocampus,which is consistent with the higher serotonin content and SERTimmunoreactivity found in the brain region. Saturation binding ofpooled homogenates revealed no age differences in Bmax or Kd(Table 1).

4. Discussion

This study shows that adolescent male rats are less sensitivethan adult males to the anxiogenic effects of the serotonin-releasing drug fenfluramine, and produces neurochemical datawhich suggest this behavioral effect may result from lowerfenfluramine-stimulated serotonin release in adolescents. Fenflur-amine did not induce anxiety-like behavior as effectively inadolescent rats as in adults in either the LD test or EPM. The anx-iogenic effects of fenfluramine in adult rats are consistent withprevious studies showing anxiogenic effects produced by acutetreatment with indirect serotonergic agonists and may representa form of behavioral inhibition relevant to risk taking (Drapier et al.,2007; Gomes et al., 2009; Graeff et al., 1996; Griebel et al., 1994;Morley et al., 2005; reviewed in Griebel, 1995). Microdialysis inmPFC revealed lower fenfluramine-stimulated increases in extra-cellular serotonin levels in adolescents than in adults, suggestingthat lower serotonin release in adolescents could be a mechanismunderlying the insensitivity to fenfluramine’s behavioral effects.

The lower fenfluramine- and potassium-stimulated serotoninrelease in adolescent mPFC may reflect functional immaturity ofthe serotonergic system. Lower serotonin content in forebrainregions has been observed in several studies of adolescent rats, andmay explain the smaller increase in extracellular serotoninobserved after both fenfluramine and potassium (Loizou, 1972;Loizou and Salt, 1970; Mercugliano et al., 1996). Fenfluramine

Table 1Results of nonlinear fits for saturation binding in adult and adolescent tissuehomogenates. Bmax is shown in fmol/mg of protein and Kd is shown in pM � thestandard error of the mean calculated by the nonlinear fit.

Prefrontal cortex Amygdala Hippocampus

Adolescents Bmax ¼ 289 � 21 Bmax ¼ 592 � 51 Bmax ¼ 231 � 19Kd ¼ 22.4 � 8.4 Kd ¼ 38.2 � 14.5 Kd ¼ 23.5 � 8.6

Adults Bmax ¼ 305 � 32 Bmax ¼ 526 � 57 Bmax ¼ 279 � 28Kd ¼ 26.9 � 13.9 Kd ¼ 35.4 � 16.6 Kd ¼ 32.2 � 12.7

stimulates non-exocytotic serotonin release that is independentof serotonergic neuron firing, and may be limited by serotonincontent (Carboni and Di Chiara, 1989; reviewed in Sulzer et al.,2005). The lower tissue serotonin content seen in adolescentssuggests that stimuli such as fenfluramine and potassium thatstrongly recruit tissue serotonin stores should stimulate greaterserotonin release in adults than adolescents. While lower tissueserotonin content in adolescents may result in lower fenfluramine-stimulated serotonin release, it does not create a deficit in baselineextracellular serotonin. This is consistent with reports that deficitsin tissue serotonin content of at least 60% are needed to see changesin basal extracellular serotonin, as adolescent rats had only 29%lower serotonin content in prefrontal cortex (Hall et al., 1999).Lower tissue serotonin stores are not due to lower synthesis inadolescents, as shown by similar 5-HTP increases in each age groupafter decarboxylase inhibition. This result is consistent with priorstudies showing that tryptophan hydroxylase activity maturesaround PN30 in rats (Deguchi and Barchas, 1972; Park et al., 1986;Schmidt and Sanders-Bush, 1971). The immature serotonin tissuecontent and lower serotonin release in adolescents may reflectlower vesicular serotonin stores in the terminals of serotonergicneurons.

Comparable SERT-immunoreactive innervation density and 3H-paroxetine binding between age groups rule out serotonergicinnervation density as a contributing factor to the lower responseto fenfluramine in adolescents. Mature density of serotonergicinnervation was found by PN21 in previous studies of serotonin-immunoreactive neurons, although some studies have foundlower SERT binding in the adolescent cortex by autoradiography(Dao et al., 2011; Lidov and Molliver, 1982; Loizou, 1972; Moll et al.,2000). The discrepancy between SERT binding in cortical homog-enates in the present study and cortical autoradiography data fromother studies may be explained by differences in the radioligandused or the greater anatomic resolution of autoradiography versushomogenate binding. Studies on SERT binding in subcorticalstructures in adolescence have also produced mixed results, soadolescent deficits in SERTexpression may be relatively subtle (Daoet al., 2011; Galineau et al., 2004; Moll et al., 2000; Tarazi et al.,1998). Data on adolescent tissue serotonin content, innervationdensity, and SERT binding collectively indicate that early adolescentrats have a similar density of forebrain serotonergic innervation asadults, but that these terminals have lower serotonin stores.

The anxiogenic effects of fenfluramine are thought to be causedby increases in extracellular serotonin (Graeff et al., 1996). Seroto-nergic mediation of fenfluramine’s anxiogenic effects is consistentwith observations that acute treatment with indirect serotoninagonists generally produces anxiogenic effects in unconditionedtests for anxiety-like behavior (Drapier et al., 2007; Gomes et al.,2009; Graeff et al., 1996; Griebel et al., 1994; Morley et al., 2005;reviewed in Griebel, 1995). Fenfluramine also releases dopamineand norepinephrine, though at the dose used for behavior testingthere is very little stimulated dopamine release and release ofnorepinephrine is much lower than serotonin (Balcioglu andWurtman, 1998; Rothman et al., 2003). However, some contribu-tion of norepinephrine to age differences in fenfluramine’s anxio-genic effect is possible, as the noradrenergic system is immaturethrough mid-adolescence in rats (reviewed in Bylund and Reed,2007).

There may also be a postsynaptic component to the lesseranxiogenic effects of fenfluramine in adolescents. Most evidenceindicates that serotonin receptor expression is mature duringadolescence, and that adolescents and adults exhibit similarsensitivity to serotonin syndrome induced by serotonin receptoragonists (Beique et al., 2004; Darmani and Ahmad, 1999; Davalet al., 1987; Garcia-Alcocer et al., 2006; Li et al., 2004; Miquel

A.E. Arrant et al. / Neuropharmacology 65 (2013) 213e222220

et al., 1994; Pranzatelli and Galvan, 1994; Vizuete et al., 1997;Waeber et al., 1996, 1994). However, connections between brainregions that mediate anxiety-like behavior such as prefrontalcortex and amygdala continue to mature during adolescence inboth rodents and humans (reviewed in Casey et al., 2008; Cressmanet al., 2010; Cunningham et al., 2002; Ernst et al., 2006). Adolescentbehavioral insensitivity to fenfluramine could be caused byimmaturity of the neural circuits modulated by serotonin, as well aslower fenfluramine-stimulated extracellular serotonin levels.

These findings have implications for neurochemical andbehavioral responses to drugs of abuse during adolescence,particularly psychostimulants. Adolescent rats are less sensitivethan adults to the aversive effects of psychostimulants, and mayfind them more rewarding based on conditioned place preferencestudies (Infurna and Spear, 1979; Schramm-Sapyta et al., 2006;reviewed in Schramm-Sapyta et al., 2009). Psychostimulantsincrease extracellular levels of dopamine, serotonin, and norepi-nephrine in the brain, with the relative increase in each neuro-transmitter differing between stimulants. Dopamine is primarilyresponsible for the reinforcing effects of psychostimulants, whilenorepinephrine and serotonin mostly mediate the aversive effectsof these drugs (Chen et al., 2006; Jones et al., 2009, 2010; Lynesset al., 1979; Roberts et al., 1977; Thomsen et al., 2009). Serotonincontributes to the aversive effects of psychostimulants in animalmodels such as conditioned taste aversion, conditioned placeaversion, and the runway model of cocaine self-administration(Ettenberg and Bernardi, 2006, 2007; Ettenberg et al., 2011; Joneset al., 2009, 2010; Rocha et al., 2002; Serafine and Riley, 2010).Serotonin depletion studies show that serotonin inhibits psychos-timulant self-administration and locomotion in rodents (Hollisteret al., 1976; Loh and Roberts, 1990; Lyness et al., 1980; Mabry andCampbell, 1973). The lower fenfluramine and potassium-stimulated serotonin release observed in adolescents suggest thatadolescents would also have a lower serotonergic response topsychostimulants such as amphetamine, methamphetamine, andMDMA that release serotonin by the same mechanism as fenflur-amine (reviewed in Sulzer et al., 2005). Methamphetamineproduces similar increases in extracellular serotonin in prefrontalcortex of PN45e50 rats and adults, but this response may beimmature in younger adolescent rats around PN28 (Staiti et al.,2011). This potentially lower serotonin response in adolescentscould contribute to the reduced aversive effects of serotonin-releasing psychostimulants observed in this age group (Infurnaand Spear, 1979). Less serotonergically-mediated aversive psy-chostimulant effects in adolescents could facilitate drug abuse byshifting the balance of rewarding and aversive effects towardrewarding effects in adolescents. This could be especially prob-lematic given evidence that adolescents are more sensitive to therewarding effects of psychostimulants in conditioned place pref-erence models (reviewed in Schramm-Sapyta et al., 2009).

The mechanism by which psychostimulants increase extracel-lular monoamines could be an important factor in the balance ofrewarding and aversive effects in adolescents. Psychostimulantsinclude drugs such as cocaine that increase extracellular mono-amines by inhibiting uptake via transporters, as well as drugs suchas amphetamine, methamphetamine, and MDMA that inhibituptake and release monoamines by a similar mechanism as fen-fluramine (reviewed in McMillen, 1983; Sulzer et al., 2005). Moststudies with cocaine, an uptake inhibitor, show that adolescents aremore sensitive to its conditioned rewarding effects (Aberg et al.,2007; Badanich et al., 2006; Balda et al., 2006; Brenhouse andAndersen, 2008; Brenhouse et al., 2008; Campbell et al., 2000;Schramm-Sapyta et al., 2004; Zakharova et al., 2009a,b). However,data from releasing drugs such as amphetamine or methamphet-amine are more equivocal. Studies with these drugs have found

greater rewarding effects in either age group or similar effectsbetween groups, though adolescents self-administer moreamphetamine than adults while acquiring self-administration(Adriani and Laviola, 2003; Mathews and McCormick, 2007;Shahbazi et al., 2008; Torres et al., 2008; Zakharova et al., 2009a).Even if there are no age differences in rewarding effects of releasingdrugs, the reduced aversive effects of these drugs in adolescentscould result in a greater rewarding to aversive effect ratio than inadults (Infurna and Spear, 1979). Comparison of the effects ofuptake inhibitors versus releasers on extracellular dopamine hasrevealed that adolescents aremore sensitive to the effects of uptakeinhibitors such as cocaine upon extracellular dopamine, but not toreleasing drugs such as amphetamine (Walker and Kuhn, 2008;Walker et al., 2010). This could be due to similar mechanisms aswith serotonin and fenfluramine, as early adolescents have lowertissue dopamine content than adults (Porcher and Heller, 1972).Given that the rewarding/aversive effects of uptake-inhibitingpsychostimulants are even more skewed toward rewarding thanreleasing drugs, it will be important for future studies to assess theeffects of uptake inhibition on extracellular serotonin in adult andadolescent rats.

Acknowledgment

This work was supported by National Institute on Drug Abusegrants DA019114 and 1F31DA032532. The authors wish to thankSam Johnson and the Duke Light Microscopy Core Facility fortechnical help with imaging and Jacob Jacobsen for technical helpwith microdialysis.

Appendix A. Supplementary material

Supplementary material related to this article can be found athttp://dx.doi.org/10.1016/j.neuropharm.2012.10.010.

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