Behavioural Brain Research 195 (2008) 187191

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Behavioural Brain Research

journa l homepage: www.e lsev ier .co

Research

Acute r tedseroton m

Sara Eug ra, MGeorgina ngua Psychopharm ob Biology of Rep

a r t i c l

Article history:Received 14 FeAccepted 14 FeAvailable online 20 February 2008

Keywords:SerotoninMemoryStressRestraintElevated T-mStriatumRats

ors sut rolelarati

on the type of stressor, their magnitude, and the type of memory. The striatum has been associated with

1. Introdu

Acute avariety ofbehavior anervous sytonin (5-hyphysiologicthem. Expeinvolved in[2] and in fory [57] aaddition, thserotonerg

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non-declarative procedural memory, while the information about stress effects on procedural memoryand their relationwith striatal serotonin is scarce. The objective of this studywas to evaluate the effects ofstress on themodications of the striatal serotonergic system. In Experiment 1, the effects of either 60minof restraint (R) or exposure to the elevated T-maze (ETM) was assessed. Exposure to ETM decreased 5-HTconcentration and to R increased 5-HT activity ([metabolite]/[neurotransmitter]). In Experiment 2, weevaluated the effects of restraint on ETM trained immediately, 24 or 48h after restraint. No effects weredetected in acquisition or escape latencies, while retention latencies were lower in all groups comparedwith the non-restrained group, although signicant effects were detected immediately and 24h afterrestraint. The memory impairment seems to be associated with changes in striatal serotonergic system,given that 5-HT concentration increased, while serotonergic activity decreased. The differences in theactivity of 5-HT detected in each experiment could be explained by the effects of different stressors onthe serotonergic neurons ability to synthesize the neurotransmitter. Thus, we suggest that exposure tostress impairs procedural memory and that striatal serotonin modulates this effect.

2008 Elsevier B.V. All rights reserved.

ction

nd prolonged exposure of experimental animals to astressors is related to signicant changes in animalnd monoamines levels in different zones of the centralstem. According to Jacobs and Azmitia [1] though sero-droxytryptamine, 5-HT) has been involved in severalal and behavioral responses is not fundamental part ofrimental evidence shows that the serotonergic system isthe modulation of emotional and behavioral processesunctions such like sleep [3], eating behavior [4], mem-nd inmental disorders like anxiety anddepression [8]. Inere is substantial evidence supporting the role of centralic system in stress [9,10]., Selye dened stress as the nonspecic response ofany demand and the general adaptation syndrome as

logic development of the response to stressors when

nding author. Tel.: +52 55 5623 1298x136; fax: +52 55 5390 7604.dress: saracruz@servidor.unam.mx (S.E. Cruz-Morales).

their action is prolonged [11]. Stressors are any internal or exter-nal stimuli (physical, psychological) that induce stress or disrupthomeostasis. Physical stressors include disturbances of the inter-nal environment and psychological stressors are stimuli that affectemotion and induce fear, anxiety, or frustration; stressors may bework in combination [12].

The hypothalamicpituitaryadrenal axis (HPA) and thesympatheticadrenal system play an important role in the stressresponse. In response to stress, the corticotropin-releasing factor(CRF) increases resulting in adrenocorticotropin hormone (ACTH)releases by the pituitary, which in turn stimulates glucocorticoidsrelease by the adrenal gland. CRF also functions as a neuromodula-tor within the central nervous system [13]. Neuroendocrine andhormonal responses also include an increase in adrenaline andnoradrenaline release from the adrenal medulla and the sympa-thetic nervous system [14]. In response to stress, there are alsochanges in dopamine and serotonin systems [15,16]. The serotoner-gic systemplays a role in the regulation of the sympathetic nervoussystem and the hypothalamopituitaryadrenal axis and in thesame way glucocorticoids and catecholamines affect central sero-tonergic systems [9,16]. Several cerebral structures, including the

see front matter 2008 Elsevier B.V. All rights reserved.bbr.2008.02.022report

estriction impairs memory in the elevaergic activity in the dorsolateral striatu

enia Cruz-Moralesa,, Norma Laura Garca-SaldvaCastillo-Robertoa, Juana Monroyb, Roberto Dom

acology, FES-Iztacala, UNAM, P.O. Box 314, Tlalnepantla, Mexico, Zip Code 54090, Mexicroduction, FES-Zaragoza, UNAM, P.O. Box 9-020, Mexico, Zip Code 15000, D.F., Mexico

e i n f o

bruary 2008bruary 2008

a b s t r a c t

Serotonin (5-HT) is involved in behaviand depression and plays an importanstress inuence learning as well as decm/locate /bbr

T-maze (ETM) and modies

ara Reyes Gonzalez-Lopeza,ezb

ch as sleep, eating, memory, in mental disorders like anxietyin the modulation of stress. On the other hand, exposure to

ve and non-declarative memory. These effects are dependent

188 S.E. Cruz-Morales et al. / Behavioural Brain Research 195 (2008) 187191

striatum play a role in the stress responses [1719]. Serotonin is amonoamine neurotransmitter widely distributed in the brain, withpathways in different areas [1], including the basal ganglia. Thecorpus striaraphe nuclemedium spstriatum [2

Differenmemory, beThe serotoncognition [5chloroamphserotonin, ithere aremstriatal seroserotonin rthat memo21-day strea maze propus [29]. I(stress) impformation i[31,32]; theblink respolarly, footshfacilitatedpsychologicother subjeonly whenforced swimexposure tosion in coldin two straexposed todiately befowas associa[35].

Thus, thvariables likconditions(declarativeeral reportsresponses.of forced swin the striaseptum andin all ve-bThese resulrotransmissstudy the eswimmingstriatum, halevels whilepocampusexposure tomodify extrpocampus.suggestingserotonergithe hypothrestraint strished in the5-HT activitincreased inonstriatal 5

time. In the nighttime, striatal 5-HT release and themotor activitiesincrease after immobilization stress [37]. In contrast, restraint orrestraint in a cold environment did not modify the concentration

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TM tesprocedterva

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o subsobtaie triarawalidered

Testinr; aftetum receives serotonergic innervations from the dorsalus (DRN) and caudal linear nucleus (CLN) [1] being theiny neuron the major target of serotonergic input in the0,21].t cerebral structures participate in different types ofing the striatum related with procedural memory [22].ergic system participates in learning, memory and,7]. Intrastriatal administration of serotonin [7] or p-etamine (PCA), a drug that increase the release ofnduces amnesia on inhibitory avoidance [23]. Becauseultiple 5-HT receptors [24,25], these results suggest thattonin participates in memory, acting through differenteceptor subtypes [23,26]. Available evidence suggestsry is altered by stress [27,28]. For instance, chronicss by immobilization impaired memory evaluated incedure and reduced 5-HT and 5-HIAA in hippocam-n rats trained in water maze task, the cat-exposedaired spatial memory [30]. Stress effects on memoryn responses conditioned classically has been studiedresults show that the facilitatory effect of stress in eyense is dependent upon the type of stressor [32]. Simi-ock administration before and after passive avoidancelearning, consolidation and evocation [33]. However,al stress (e.g., exposure to an adjacent room in whichcts were exposed to footshock) produced facilitationsubjects were exposed to stress before training, andming onlywhenwas presented before test. In contrast,the combination of restraint and 60min of immer-

-water bath, produced impairment in passive avoidanceins of rats [34]. Similar effects occurred in Wistar ratsthe same stress conditions, exposure to stress imme-re of after training impaired passive avoidance, whichted with an increase in ACTH and corticosterone levels

e effects of stress on memory are inuenced by somee the type of stressor (psychological or physical), stress(time of exposition, acute or chronic), type of memoryor non-declarative memory) and gender [28]. Sev-have documented the striatal involvement in stress

Microdialysis measurements in rats exposed to 30minimming showed elevated extracellular levels of 5-HT

tum, reduced 5-HT levels in the amygdala and lateralno effects in the hippocampus and frontal cortex;

rain regions, forced swimming reduced 5-HIAA [18].ts suggest that forced swimming modulates 5-HT neu-ion in a regionally specic manner [18]. In anotherffects of different stressors were evaluated [19]. Forcedelevated extracellular 5-HT and reduced 5-HIAA in thed not effect on hippocampal 5-HT and reduced 5-HIAAtail pinch increased 5-HT levels in striatum and hip-

and had not effect on 5-HIAA levels; immobilization,a cold environment, and forced motor activity did notacellular 5-HT levels in either the striatum or the hip-These stressors increased plasma corticosterone levels,no relationship between the effects of stressors on thec system and the magnitude of their ability to activatealamicpituitaryadrenal axis [19]. In rats exposed toess 2h daily for 4 consecutive days, 5-HT levels dimin-locus coeruleus, hypothalamus and amygdala, while

y or turnover did not change in the locus coeruleus andthe amygdala [36]. The effects of immobilization stress-HT releasearedifferent in thedaytimeregardingnight-

of 5-HT[38]. Futryptopstriaturole ofconcermemoof stria[7,23,2exposivatedTThe elethe eleety [40prepararms, tavoidaarm, anarm. Thand thtraininond da(R) orneuroclike dr

2. Mate

2.1. Sub

ExpeFES-Iztaand wat08:00h)

2.2. App

Thevated50in an iso(20%), alon the uto avoidavoidancholes to

2.3. Pro

2.3.1. ReThe

stress caimmobilsubjects

2.3.2. EThe

a 24-h in

2.3.2.1.arm, the(BL); twlatenciesavoidancto withdare cons

2.3.2.2.24h lateended.5-HIAA in striatum, accumbens, and prefrontal cortexrmore, in rats immobilized for 2hper dayduring 7days,, 5-HT, and 5-HIAAwere normal in cortex,midbrain and9]. Thus, though there is body of evidence about thetonin in stress and memory, there are inconsistenciesthe effect of different stressors and their inuence onn the other hand, there is evidence of the participationerotonin system on the consolidation of aversive taskence, this study was aimed to evaluate the effect of theto two stressors, restraint and the exposure to the ele-e, on striatal 5-HT systemand its relationwithmemory.d T-maze (ETM) is a behavioral procedure derived fromplus-maze, an extensively used animal model of anxi-ich allows measuring anxiety and memory in the same[41]. The ETM is composed of one closed and two openpen arms are an aversive stimulus that promotes theesponse when the subjects are placed on the enclosedescape response when subjects are placed in the openbjects are trained in inhibitory avoidance and in escapetention is tested 24h later, the responses measured onunconditioned while the responses recorded the sec-conditioned [42]. Importantly, movement restriction

obilization is one of the stressor more used to studyical changes induced by stress and other manipulationsdministration [43].

and methods

tally nave Wistar male rats (250270g) from the animal house ofNAM were distributed in groups of four in acrylic cages with foodlibitum. The colony was in a 12-h light:12-h dark cycle (light on at

s

onsisted of an enclosed arm and two open arms (50 cm10 cm) ele-ove theground; thewall of theclosearmwas40 cmhigh. TheETMwasoomwith low light. After each test themazewas cleanedwith alcoholriments were conducted between 12 and 17h. The procedure is baseditioned behavior showed in the rst day, in which the subjects learnscape from the open arms. During the second day, the conditionedescape responses were evaluated. Acrylic cylindrical restrainers withadjusting to the size of the subjects were used.

tts were handled during 8 days prior the experiments to diminish theor experimental manipulations. After handling period, subjects wereor 60min in the restrainers. Immediately, 24 or 48h after restraint thetrained in the ETM.

ture of the ETM consisted of two sessions, training and a testing with

l.

g. The rst day each subject was placed at the end of the enclosedremaining in this arm was registered and considered the baselineequent avoidance (AV) trials were given (AV1, AV2). The avoidancenedduring therst day are consideredas an indexof acquisition. Afterls, each subject was placed at the end of an open arm, and the timefrom it was considered as escape latency (ES1). The escape latenciesas an index of anxiety.

g. The retention of avoidance (AV3) and escape (ES2) was measuredr 300 s if the subjects did not leave any of the arms, the trial was

S.E. Cruz-Morales et al. / Behavioural Brain Research 195 (2008) 187191 189

2.4. Serotonin measurement

After thebehavioral procedures all subjectsweredecapitated, thebrain removedand kept in liquid nitrogen, samples of dorsal striatumwere taken, following param-eters of the stereotaxic atlas [44]. The amount of 5-HT and 5-hydroxyindoleaceticacid (5-HIAA) was measured following the methodology previously used [4548].In brief, the tissue samples were weighed and homogenized in 300l of 0.1N per-chloric acid, centrifuged at 12,500 rpm for 30min at 4 C the supernatant wasltered and 20l of this extract was injected into the HPLC system for analysis.The HPLC apparatus consisted of an isocratic pump (PerkinElmer Co., Norwalk,CT, USA, model LC-250), a Rheodyne injection valve and a C18 column (Bionalyt-ical Systems, Inc., USA), protected by a pre-column cartridge lter (BionalyticalSystems, Inc.). The concentration of 5-HT and 5-HIAA were quantied electro-chemically, using a BAS LC-4C amperometric detector and an LC-4A glassy carbontransducer cell (Bionalytical Systems, Inc.). The mobile phase consisted of 0.1M cit-rate buffer (pH 3.0), with 100mg of 1-octane-sulfonic acid (Sigma, St. Louis, MO,USA), 20ml of acetonitrile and 15ml of tetrahydrofuran (Omnisolve and Science,Canada). Thephasewaspumpedat aowrate of 1.2ml/min. Results are expressed aspicograms of neurotransmitter per milligram of wt. tissue (pg/mg). Neuronal activ-ity was calculaactivity = [5-H

3. Experim

Almost aaversive situso probablyimportant tcedure. Thedifferent strergic activit

3.1. Procedu

Rats wergroup thatvatedT-mazbehavioral p

3.2. Data an

Indepenof 5-HT, wewere detectThe level of

3.3. Results

In the stration was[F(2,17) =4.2centration.striatum ofnon-restrairestriction

Table 1Concentration (ng/kg) of 5-HT, HIAA and 5-HT turnover in the dorsal striatumof ratsnon-restrained (NR), restrained (R) for 60min or submitted to the elevated T-maze(ETM)

N 5-HT HIAA Turnover

NR 8 1.240.21 0.730.10 0.660.09R 6 1.070.03 0.540.11 0.620.14ETM 6 0.350.09* 0.380.05 1.890.63*

ANOVA F(2,17) =4.27, p0.05 F(2,17) =4.19, p

190 S.E. Cruz-Morales et al. / Behavioural Brain Research 195 (2008) 187191

Fig. 1. Exposufor 60min and(AV). Restraintor 48h after re24h after restrsignicant diff

group (p

S.E. Cruz-Morales et al. / Behavioural Brain Research 195 (2008) 187191 191

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Acute restriction impairs memory in the elevated T-maze (ETM) and modifies serotonergic activity in the dorsolateral striatumIntroductionMaterials and methodsSubjectsApparatusProcedureRestraintETM testTrainingTesting

Serotonin measurement

Experiment 1ProcedureData analysisResults

Experiment 2ProcedureData analysisResults

DiscussionAcknowledgementsReferences