Young-Adult Male Rats' Vulnerability to Chronic Mild Stress Is Reflected by Anxious-Like instead of

13 Pages·2016·1.5 MB·English

Hindawi Publishing Corporation
Neuroscience Journal
Volume 2016, Article ID 5317242, 12 pages
http://dx.doi.org/10.1155/2016/5317242
Research Article
Young-Adult Male Rats’ Vulnerability to Chronic Mild Stress Is
Reflected by Anxious-Like instead of Depressive-Like Behaviors
Herrera-PérezJoséJaime,1Benítez-CoronelVenus,1Jiménez-RubioGraciela,1
Hernández-HernándezOliviaTania,2andMartínez-MotaLucía1
1LaboratoriodeFarmacolog´ıaConductual,Direccio´ndeInvestigacionesenNeurociencias,
InstitutoNacionaldePsiquiatr´ıaRamo´ndelaFuenteMun˜iz,CalzadaMe´xico-Xochimilco101,ColoniaSanLorenzoHuipulco,
Delegacio´nTlalpan,14370CiudaddeMe´xico,DF,Mexico
2ConsejoNacionaldeCienciayTecnolog´ıa,InstitutoNacionaldePsiquiatr´ıaRamo´ndelaFuenteMun˜iz,
CalzadaMe´xico-Xochimilco101,ColoniaSanLorenzoHuipulco,Delegacio´nTlalpan,14370CiudaddeMe´xico,DF,Mexico
CorrespondenceshouldbeaddressedtoMart´ınez-MotaLuc´ıa;[email protected]
Received11April2016;Accepted1June2016
AcademicEditor:NashatAbumaria
Copyright©2016Herrera-Pe´rezJose´Jaimeetal. This is an open access article distributed under the Creative Commons
AttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkis
properlycited.
Inapreviousstudy,wefoundthatchronicmildstress(CMS)paradigmdidnotinduceanhedoniainyoung-adultmaleratsbutit
reducedtheirbodyweightgain.Thesecontrastingresultsencouragedustoexploreotherindicatorsofanimal’svulnerabilitytostress
suchasanxious-likebehaviors,sincestressisanetiologicfactoralsoforanxiety.Thus,inthisstudy,weevaluatedthevulnerability
oftheseanimalstoCMSusingbehavioraltestsofdepressionoranxietyandmeasuringserumcorticosterone.MaleWistarrats
wereexposedtofourweeksofCMS;theanimals’bodyweightandsucrosepreference(indicatorofanhedonia)wereassessedafter
threeweeks,and,afterthefourthweek,someanimalswereevaluatedinabehavioralbattery(elevatedplusmaze,defensiveburying
behavior,andforcedswimmingtests);meanwhile,otherswereusedtomeasureserumcorticosterone.WefoundthatCMS(1)
didnotaffectsucrosepreference,immobilitybehaviorintheforcedswimmingtest,orserumcorticosterone;(2)decreasedbody
weightgain;and(3)increasedtherat’sentriesintoclosedarmsoftheplusmazeandthecumulativeburyingbehavior.Thesedata
indicatethatyoungmalerats’vulnerabilitytoCMSisreflectedaspoorbodyweightgainandanxious-likeinsteadofdepressive-like
behaviors.
1.Introduction response, which in turn would cause depressive or anxious
events[3,4].
AccordingtoSeyle[1,2],stressisdefinedasthenonspecific Depression is a mood disorder characterized by anhe-
responseofthebodytoanydemand.Thisresponseisinduced donia (incapacity to experience pleasure), feelings of sad-
by any new situation (chemical, physical, environmental, ness and guilt, depressed mood, hopelessness, and suicidal
emotional, and psychosocial stressor) which disturbs the thoughts[5];meanwhile,anxietyisdistinguishedbyexcessive
homeostasisandinducesageneraladaptiveresponseaimed andinappropriateworrying(i.e.,persistentandnotrestricted
at restoring the initial level of stability (adaptation). All the to particular circumstances), restlessness, difficulty concen-
manifestationsoftheadaptiveresponsearebeneficialtothe trating, irritability, muscle tension, and disturbed sleep [5].
organismwhenlimitedintime,butwhenthedurationofthe To study these pathologies, researchers have used animal
stressisexcessiveitcontributestothedevelopmentofpatho- models to simulate a symptom of the human disease [6];
logicalconditions[1,2].Thus,thediathesis-stresshypothesis most of these models are based on rodent exposure to a
ofaffectivedisordersstatesthatchronicstresscouldinduce stressor.Chronicmildstress(CMS)isananimalmodelthat
physiologicalalterationsthatpromoteanexaggeratedstress simulates anhedonia; in this paradigm, constant exposure
2 NeuroscienceJournal
of the animal to several stressors of moderate intensity 2.2.2. Baseline Sucrose Consumption. The baseline sucrose
duringaperiodofseveralweeksleadstoareductionofthe consumption was determined two days after the training
consumption of a palatable sucrose solution, indicating an periodatthebeginningofthedarkphase(at10:00a.m.).For
anhedonic state [7]. It has been established that this model thispurpose,theratswerewaterandfooddeprivedfor20h
hashighvalidity(face,predictive,andconstructvalidity)that andthereafterpresentedwithtwobottlesduringaperiodof
makesitausefultooltostudytheneurobiologyofdepression onehour:onecontainingsucrosesolution(1%)andtheother
[8]. In agreement with the diathesis-stress hypothesis of tap water. The objective of the food and water deprivation
affective disorders, several studies have found that rodent’s periodistostimulatetheliquidconsumptionintheanimals
exposure to CMS can induce several behavioral alterations inordertohaveagooddynamicrangetoevaluatedifferences
(differentfromanhedonia)thatmimichumansymptomsof in fluid intake [16]. Fluid (sucrose solution or tap water)
depression [8–10] and anxiety [11, 12]. Recently, using this consumption was calculated by weighing the bottles before
andafterexposuretotheanimals.Forbaselinebodyweight
model,wefoundthatahighproportionofyoung-adultmale
measurements, animals were weighed immediately before
rats did not develop anhedonia when exposed to a CMS
waterandfooddeprivation.
paradigm,suggestingthattheseanimalsareresilienttothis
kind of stress [13]. Contrary to this, we also found that the
CMS reduced the rats’ body weight gain as compared to 2.2.3.ExperimentalDesignforBehavioralStudy. Afterdeter-
unstressedrats[13].Thesedataindicatethatthechronicstress minationofbaselinesucroseconsumption,young-adultmale
scheduleaffectsthephysiologyoftheanimal,butthisisnot ratswererandomlyassignedtoacontrolorstressgroup.The
enoughtoinduceananhedonicstate.Theseresultsencourage malesinthecontrolgroup(𝑛 = 13)weremaintainedduring
finding other indicators to evaluate the vulnerability of a period of four weeks without stress, but under similar
young-adult rats to CMS; this could be done by scanning handling and storage conditions to the stressed animals.
a broader spectrum of behaviors or phenotypes potentially The stress group (𝑛 = 13) was exposed, during a period
impaired or induced by this kind of stress and by mea- of four weeks, to several stressors: white noise (∼90dB),
suringtheactivityofhypothalamic-pituitary-adrenal(HPA) overcrowding (2-3 animals per cage), continuous lighting,
axis. soiledcage(250mLwaterspilledintobedding),stroboscopic
∘
Onthesebases,wehypothesizethattheyoungratsthat light (300flashes/min), 45 cage tilt along the vertical axis,
did not develop anhedonia when exposed to CMS present andwaterdeprivation.Thestressorschedulefollowedinthis
other behavioral alterations that suggest depression and/or studywaspreviouslyused[13,17,18]anditisshowninTable1.
anxiety.Thus,theobjectiveofthisstudywastoevaluatethe To determine anhedonia development, we used a within-
vulnerability of young-adult rats to CMS using behavioral subject design since the effect of stress on hedonic state is
tests that detect depressive- or anxious-like behaviors and progressive and the kinetics of the changes in fluid intake
measuringcorticosteroneserumlevels. are importantto determine theestablishment ofanhedonia
in the animals. Thus, during the first three weeks of CMS,
sucroseandwaterintakewasdeterminedweekly,asindicated
2.MaterialsandMethods
below. At the fourth week (day 27), the 20h of water and
food deprivation indicated in Table1 was omitted and at
2.1. Animals. Young (3-month-old) male Wistar rats were
this time the CMS was finished. Afterwards, a behavioral
obtained from the vivarium of the Instituto Nacional de
tests battery was performed to evaluate stress vulnerability
Psiquiatr´ıaRamo´ndelaFuenteMun˜iz (INPRFM).Animals
wereindividuallyhousedincagesmeasuring27×16×23cm beyond anhedonia; we used a between-subject design in
order to avoid ceiling effects or reduction of exploration
andmaintainedonaninverted12hdark-lightcyclecondition
originatedbyrepeatedanimals’exposuretothetests.Then,
(lights off at 10:00a.m.), under controlled temperature and
onday28,thecontrolandstressedgroupswereexposedto
humidity. The animals had free access to water and food,
the elevated plus maze test and, immediately after, to the
except for the periods required by the CMS procedure.
defensiveburyingbehaviortest.Ondays29and30,animals
Animal management was done according to the general
were evaluated in the forced swimming test (pretest and
principles of laboratory animal care [14]. All experimental
test,resp.,at2:00p.m.).Severalstudiesindicatethatanimal’s
procedureswereperformedinaccordancewiththeMexican
performanceinthesebehavioraltestsissensibletoprevious
officialnormforanimalcareandhandling[15]andapproved
stressexposure,includingCMS[19–22];thus,theycouldbe
by the Ethical Committee of the INPRFM. All efforts were
good indicators of stress vulnerability in the nonanhedonic
made to minimize the number of animals used and their
rats. The tests are described below and the time flow of
suffering.
experimentalmanipulationsisindicatedinFigure1.
2.2.ChronicMildStressModel
2.3. Sucrose Preference Test and Body Weight Measurement.
2.2.1. Sucrose Consumption Training. Animals were allowed Incontrolandstressedanimals,thesucrosesolutionandtap
to adapt to the taste of a palatable sucrose solution (1%) waterconsumptionweredeterminedweeklyasindicatedin
for two weeks. During this period, a bottle containing the baseline sucrose consumption section. These measure-
sucrose solution was presented to the rats daily for one ments (for the control and stressed animals) were done at
hour. 10:00a.m.,aftera20hperiodofwaterandfooddeprivation.
NeuroscienceJournal 3
Table1:Chronicmildstressschedule.
Time(hours) FirstWed. Thr. Fri. Sat. Sun. Mon. Tue. EverysubsequentWed.
7:00-8:00 SC/CL WD O/CL FD/WD
8:00-9:00 SC/CL WD CT/CL FD/WD
9:00-10:00 CL WD CT/CL FD/WD
10:00-11:00 Baseline SL WN CT/CL SPT
11:00-12:00 O SL WN CT/CL
12:00-13:00 O SL WN SL CT
13:00-14:00 WN O SL WN SL CT WN
14:00-15:00 WN O SL SL FD/WD WN
15:00-16:00 WN O/CL SL FD/WD WN
16:00-17:00 SC/CL WD SL FD/WD
17:00–7:00 SC/CL WD O/CL FD/WD
WN:whitenoise(∼90dB);O:overcrowding(2-3ratspercage);CL:continuouslighting;SC:soiledcage(250mLwaterspilledintobedding);SL:stroboscopic
∘
light(300flashes/min);WD:waterdeprivation;CT:cagetilt(45);FD:fooddeprivation;SPT:sucrosepreferencetest.
Elevated plus maze
and defensive burying
Sucrose consumption
Control or CMS
training
Time −14 0 21 28 29 30
(days)
Baseline Determination Pretest Test
sucrose of anhedonia
Forced swimming
intake
test
Figure1:Timelineforexperimentalmanipulations.
Rat’sbodyweightwasmeasuredweekly,immediatelybefore periodof10minforposterioranalysis.Themazewascleaned
the 20h of water and food deprivation. To avoid changes aftereachtesting.Thenumberofentriesandthetimespent
determined by baseline differences, individual sucrose and intheclosedoropenarmsweremeasured.Anincrementof
water consumption were expressed as relative sucrose or rat’s anxiety-like state induced by CMS would be indicated
water intake; this parameter was calculated by dividing the byincreasingthetimespentorthenumberofentriesinthe
sucroseorwaterconsumptionatanytimebytherespective closedarms[24].
baselineingestion;thisparameterhasstatisticalsensibilityto
reductionsinsucrosesolutionintake[18].Sucrosepreference
2.5. Defensive Burying Behavior Test. The burying behavior
was calculated weekly by dividing the sucrose intake by
test was used to assess experimental anxiety and it is based
total fluid (sucrose plus water) consumption and the result
ontherat’sinnatebehaviortoburyaversivestimuli[25].The
was expressed as percentage. Finally, rat’s body weight was
expressedasapercentagerelativetobaselinebodyweightin test was performed in a cage measuring 27 × 16 × 23cm
ordertodiscardeffectsofbaselinedifferences. withanelectrifiedprobe(7cmlong,2cmabovethebedding
The anhedonic state generated by the exposure to CMS material) placed in one of the cage walls. When the rat
schedule would be indicated by a reduction in the weekly touched the electrified probe with its snout or forepaws, it
sucroseconsumptionorpreferenceafterthreeweeksofstress received a 0.3mA shock. The experiments were performed
[7,13,18]. under dim red light. The rats were exposed to this cage
during a period of 10min, and the test was videotaped for
2.4. Elevated Plus Maze Test. The elevated plus maze is an posterioranalysis.Thenumberofprodshocks,theburying
animal model of anxiety based on rodents’ aversion for behaviorlatency(timebetweenthefirstshockandthedisplay
openspaces[23].Thetestsettingconsistedofaplus-shaped of burying behavior), and the cumulative burying behavior
apparatus(armssize:50×10cm)placed50cmabovethefloor (total amount of time spent spraying the bedding material
withtwoopenandtwoclosedarms(enclosedby40cmhigh towardsandontopoftheshockprobe)weremeasured.An
walls).Theexperimentswereperformedunderdimredlight. anxiogenic effect of CMS would be indicated by a decrease
Theratswereplacedattheintersectionofthefourarmsofthe of the burying behavior latency and/or an increase of the
mazefacinganopenarm.Thetestwasvideotapedduringa cumulativeburyingbehavior.
4 NeuroscienceJournal
2.6.ForcedSwimmingTest. Theforcedswimmingtest(FST) test; and (e) corticosterone serum levels. Statistical analysis
is a commonly used animal model of depression that was carried out using the Sigma Plot software, version 11.
evaluates behavioral despair. In this study, we used the A value of 𝑝 < 0.05 was considered statistically signifi-
modified FST [26]. Swimming sessions were conducted by cant.
placing the rat in a glass cylinder (46cm tall × 20cm in
∘
diameter) containing water (30cm depth) at 23–25 C. The
3.Results
FST consisted of two swimming sessions 24h apart. In the
first session (pretest), rats were allowed to swim for 15min. 3.1.AnhedoniaDevelopmentintheCMS. Figure2showsthe
Twenty-four hours later, animals were subjected to a 5min sucrose intake (a), the tap water consumption (b), and the
swimmingsession(test).Attheendofeachsession,ratswere sucrosepreference(c)ofratsexposedtoCMS(𝑛=13,black
removed from the jar, dried with paper towel, and placed circles)ormaintainedwithoutstress(𝑛 = 13,whitecircles).
in a cage for 15min before returning them to the home The two-way RM ANOVA indicated that sucrose solution
cages.Thetestwasvideotapedandatime-samplingtechnique intakewasnotaffectedbystress(𝐹1,24 = 2.537,𝑝 = 0.124),
was used to score, every 5s, the presence of immobility timeofexposure(𝐹3,72 =2.175,𝑝=0.098),ortheinteraction
behavior(floatingwithoutstrugglingandmakingonlythose ofbothfactors(𝐹3,72 =0.975,𝑝=0.409).Inasimilarway,tap
movements necessary to keep the head above the water). waterconsumptiondidnotshowsignificantdifferences(RM
Behavioral despair would be indicated by high levels of ANOVA,stress:𝐹1,24 = 0.172,𝑝 = 0.682;timeofexposure:
immobility. 𝐹3,72 = 2.151,𝑝 = 0.101;andinteraction:𝐹3,72 = 1.248,𝑝 =
0.299).Regardingsucrosepreference,thestatisticsindicated
2.7. Corticosterone Serum Measurements. To evaluate the differences determined by time (𝐹3,72 = 3.011, 𝑝 = 0.036)
effectofCMSonHPAactivity,wemeasuredcorticosterone butnotbystress(𝐹1,24 =1.385,𝑝=0.251)ortheinteraction
serumlevels.Ithasbeendemonstratedthatrats’exposureto of both factors (𝐹3,72 = 0.584, 𝑝 = 0.627); the main effect
oftimeisduetoincreaseofpreferencealongtheexperiment
behavioral tests such as the FST could increase their HPA
(Figure2(c)).
axis activity [27]; this effect could veil the impact of the
CMS protocol on the function of this axis. Thus, in the
current study, the corticosterone levels were measured in 3.2. Relative Body Weight. Relative body weight of rats
two independent groups of rats that were experimentally exposedtoCMS(𝑛=13,blackcircles)ormaintainedwithout
manipulated as indicated above (see Figure1), except that stress (𝑛 = 13, white circles) is shown in Figure3. Data
theywerenotexposedtotheelevatedplusmaze,thedefensive analysisindicatesasignificanteffectofstress(𝐹1,24 =37.919,
behavior, or the forced swimming tests. During a period 𝑝 < 0.001),timeofexposure(𝐹3,72 =6.762,𝑝 < 0.001),and
of four weeks, one of these groups was exposed to CMS theinteractionofbothfactors(𝐹3,72=10.213,𝑝<0.001).Post
(𝑛 = 7);meanwhile,theotherwaskeptoncontrolcondition hocanalysisindicatedthatthebodyweightofcontrolratsat
(𝑛 = 6); according to the sucrose preference, after three week 1, 2, or 3 is higher than their basal value (𝑝 < 0.001
weeksofCMS,noneofthestressedratsdevelopedanhedonia inallcases);meanwhile,inthestressedgroup,onlythebody
(data not shown). On day 28, the animals of both groups weightatweek1waslowerthantheirbasallevel(𝑝=0.041).
were sacrificed by decapitation and their trunk blood was Tukeytestalsoevidencedthenotionthatbodyweightofthe
collectedincoldtubes.Thebloodwascentrifuged(4000rpm stressedgroupwaslowerthantheweightinthecontrolgroup
for25minat4∘C)toobtainserumsamplesthatwerestored atweeks1,2,and3(𝑝<0.001inallcases).
at −4∘C until analysis. Corticosterone concentrations were
measured by radioimmunoassay using a commercial kit 3.3. Anxiety-Like Behavior in the Elevated Plus Maze. The
(Siemens Rat Corticosterone Coat-A-Count Kit, TKRC1); performance of stressed (𝑛 = 13, gray bars) or unstressed
125
this procedure used antibody-coated tubes in which I- (𝑛 = 13,blackbars)maleratsontheelevatedplusmazetest
labelledratcorticosteronecompetedwiththefreehormone is shown in Figure4. The time spent in the open or closed
in the sample for antibody sites. Corticosterone (ng/mL) arm (Figure4(a)) did not present significant differences
totalquantitywasdeterminedusingacalibrationcurve.The determinedbyCMS(𝑝 = 0.606,𝑝 = 0.445,resp.).Similarly,
interassay and intra-assay variabilities were 4.8 and 4.0%, thenumberofentriesintotheopenarm(Figure4(b))didnot
respectively. showsuchdifference(𝑝 = 0.667);incontrast,thestatistical
analysisofthenumberofentriesintotheclosedarmindicated
that the stressed group showed more entrances than the
2.8.Statistics. Tapwaterorsucrosesolutionintake,sucrose
unstressedgroup(𝑝=0.010).
preference, and relativebody weight were analyzed by two-
way repeated measures analysis of variance (RM ANOVA)
including the factors stress exposure and time, followed 3.4.DefensiveBuryingBehavior. Animalsthatdidnotreceive
by Tukey as the post hoc test. Student’s 𝑡-test was used to a shock (2 stressed and 4 control rats) throughout the test
analyze (a) the time spent and the number of entrances were omitted from the analysis; there was no difference
in the open or closed arm of the elevated plus maze; (b) betweentheproportionsofstressedandcontrolanimalsthat
the number of shocks, the burying behavior latency, or did not receive the shock (𝑝 = 0.645, Fisher exact test).
the cumulative burying behavior in the defensive burying Figure5 shows the number of shocks (a), burying behavior
behavior test; (c) the immobility in the forced swimming latency (b), and cumulative burying behavior (c) of rats
NeuroscienceJournal 5
2.0 2.0
e
k
a
nt ke
olution i 1.5 ater inta 1.5
ose s 1.0 ap w 1.0
ucr ve t
e s 0.5 ati 0.5
ativ Rel
Rel 0.0 0.0
Baseline 1 2 3 Baseline 1 2 3
Time (weeks) Time (weeks)
Control Control
Stressed Stressed
(a) (b)
100
%)
e (
c
n
e
er 80
ef
pr
n
o
uti 60
ol
e s
os
cr
Su 40
Baseline 1 2 3
Time (weeks)
Control
Stressed
(c)
Figure2:CMSfailedtoinduceanhedoniaintheanimals.Thesucrosesolution(a)andtapwaterintake(b)aswellasthesucrosesolution
preference(c)ofratexposedtoCMS(blackcircles)ormaintainedwithoutstress(whitecircles)didnotchangethroughouttheexperiment.
Fluidintakeofsucrosesolutionandtapwaterwasexpressedasrelativetobasalconsumptioninordertoavoidbaselinedifferences.Dataare
expressedasmean±SEM.
previouslyexposed(𝑛 = 11,blackbars)ornonexposed(𝑛 = 4.Discussion
9,graybars)toCMSandevaluatedinthedefensiveburying
behavior test. Data analysis did not indicate differences 4.1. CMS as a Paradigm of Depressive-Like State. CMS
determined by stress in the shock number or the burying paradigm is a highly validated animal model that uses
latency (𝑝 = 0.419 and 𝑝 = 0.818, resp.); in contrast, this stressors of moderate intensity (to give a realistic analogue
analysis showed that the cumulative burying behavior was ofthestressorfoundineverydaylife)togenerateanhedonia
higherinthestressedgroupcomparedtotheunstressedone inrodents[7,8];thisstateisindicatedbyareductionofthe
(𝑝=0.035). intake of a sucrose solution, which is found to be palatable
for most rats. The sensitivity of this parameter to detect
anhedoniaincreaseswhentheanimalsaredeprivedoffood
3.5. Behavioral Despair in the FST. Figure6 illustrates the
and water for several hours (14 to 20) before the sucrose
immobilitybehaviorintheFSTofratspreviouslyexposedto
solution intake test; this deprivation reduces the within-
4weeksofCMS(𝑛 = 13,blackbar)orthosewhowerekept
group variability, resulting in a wider difference between
unstressed(𝑛=13,graybar).Statisticalanalysisofthesedata
stressed and control animals with respect to nondeprived
indicatednosignificantdifferencesbetweenthecontroland
subjects [16]. In our laboratory, the mild stressors used in
stressedgroup(𝑝=0.971).
the CMS schedule have been effective to induce anhedonia
in male rats, showing age differences, insomuch that high
3.6.CorticosteroneSerumLevels. Corticosteroneserumlev- andsmallpercentagesofmiddle-agedandyoungratsexpress
els were 242.21 ±45.56ng/mL (𝑛 = 6) for control rats and thiscoresignofdepression,respectively[13].Inthepresent
197.35 ± 17.14ng/mL (𝑛 = 7) for the animals exposed to study, we found that young-adult male rats did not reduce
CMS;statisticalanalysisdidnotshowsignificantdifferences the sucrose consumption or sucrose preference when they
(𝑝=0.349,𝑡-test). were exposed to the CMS paradigm during a period of
6 NeuroscienceJournal
110 stressschedules,developedanhedonia[7,29,30].However,
therearealsodescriptionsthatsomeanimalsareresistantto
105 ∗∗∗ ∗∗∗ ∗∗∗ aparticularCMSschedule[31,32];althoughthisproportion
%) isrelativelylower,itsuggeststhatintrastraindifferencesare
ht ( important regarding stress resilience. According to these
eig 100 ### authors, the difference in vulnerability to CMS could be
y w ### determinedbyvariationsinfactorssuchashippocampalneu-
d ∗
o rogenesisorcellularplasticitypathways[31,32].Alternatively,
b 95 ###
ve it could be argued that 3 weeks of CMS is not adequate
elati or enough to induce anhedonia in the animals; however, a
R
90 similarstressprotocolwasshowntobeeffectivetoinducethis
stateinyoung-adultSpragueDawleyrats[33];furthermore,
in our laboratory, this scheme of stressors has effectively
85
Baseline 1 2 3 inducedanhedoniainmiddle-aged[13,18]andyoung-adult
Time (weeks) [17] Wistar male rats. Additionally, in our experience, the
exposureofnonanhedonicyoung-adultmaleratstoalonger
Control
periodofCMS(7weeks)didnotchangethethresholdofrats
Stressed
toexhibitahedonicstate(unpublisheddata).
Figure 3: The rats’ growing rate is reduced by CMS exposure. Incontrastwithresultsofanhedonia,herewefoundthat
Compared to unstressed rats (white circles), the relative body the body weight gain of the rats was reduced by the CMS
weights of rats exposed to CMS (black circles) are lower at the paradigm.Thesedataareinlinewithapreviousstudymade
threeweeksofstressexposure.Valuesareexpressedaspercentageof
on Sprague Dawley and Long Evans rats where CMS did
baselinetoavoideffectsdeterminedbybaselinedifferences.Dataare
expressedasmean±SEM.Tukeytest:∗𝑝 < 0.05and∗∗∗𝑝 < 0.001 notinduceanhedoniabutreducedrats’bodyweight[34].It
versusbaseline;###𝑝<0.001versuscontrolgroup. has been proposed that body weight changes indicate male
rats’ vulnerability to the stress since they reflect the overall
impactofachronicstressfulsituation[35,36].Thissignmay
beinterpretedasamanifestationofadepressive-likestatein
threeweeks,whichagreeswithourpreviousstudy[13].The ratssincereductionofbodyweightisconsideredasymptom
lack of difference in the anhedonic state between stressed of the human disorder, mainly in comorbidity with anxiety
and nonstressed rats cannot be explained by differences disorders[5].
in conditions of housing or handling, since they were the Restriction of body weight in rats exposed to chronic
same for both groups along the experiment (except for the stressors is a physiologic correlate evoked by changes in a
stressors), including the periods of deprivation of food and central level. Stress response is mediated by a main secre-
waterbeforethesucroseconsumptiontest. tagogue, the 41-aminoacid peptide corticotrophin releasing
Inthesameline,presentresultscouldsuggestvariations factor (CRF), which is synthesized in neurons of hypotha-
in the spectrum of physiological or behavioral responses lamic or extrahypothalamic nuclei (i.e., the paraventricular
in rodents after application of chronic stressors. Besides nucleusandcentralamygdala,resp.).CFRinteractswithtwo
anhedonia,CMSparadigmgeneratesotheroutcomesrelated main receptors (CRF1 and CRF2), which are differentially
todepression(increasedimmobilityintheFST,potentiation expressedindistinctbrainstructuresandperipheraltissues
of learned helplessness, decreased male sexual behavior, [37]. A huge number of studies demonstrate that central
and decreased REM sleep latency) and other stress-related administration of CRF replicates signs of depression and
disorders such as anxiety (for review, see [10]). In the same anxiety in rodents, such as reduced exploration, increased
way,severalreportsindicatethatestablishmentofhighlevels despair, decreased appetite, disrupted sleep, and reduced
of anhedonia by means of a CMS schedule could show gainofbodyweight[38–42].Furthermore,experimentswith
variationsbyfactorsas(1)intrastrainvariation;(2)epigenetic drugsthatselectivelyantagonizetheCRF1receptorssuggest
or genetic aspects; (3) the laboratory environment; and (4) that these play an important role in the initiation of events
adverseorstimulatingearlylifeevents[28].Forthesimilitude that lead to reduction of body weight in rodents without
with the multifactorial etiology of human depression, these stress exposure (i.e., poor food intake) [43] as well as in
observationsfurthersupportthehypotheticalvalidityofthe downregulationofbodyweightofanimalsunderCMS[44].
CMSmodel. ResultsshownheresuggestthatthevulnerabilitytoCMSof
someyoung-adult malesseems to be expressed by different
4.2.Depressive-LikeBehaviorsInducedbyCMS. Inthisstudy, signs associated with the depressive spectrum and is not
we found that young-adult male rats did not reduce the limitedtoanhedonia.
sucrose consumption or sucrose preference when exposed Given the contrasting results described above and con-
to the CMS paradigm during a period of three weeks and sidering that CMS is able to generate behavioral despair
couldsuggestthatyoungmaleratsareresilienttothiskindof in the FST (for review, see [10]), another animal model of
stress.TheresilienceofyounganimalstotheCMSparadigm depression, we decided to evaluate whether this behavior
foundinthecurrentstudycontrastswiththereportsshowing would better indicate the vulnerability of young rats to the
that rats from the same age and strain, exposed to similar CMSparadigm.ResultsfromtheFSTindicatedthatchronic
NeuroscienceJournal 7
500 12 ∗
10
econds) 340000 of entries 68
me (s 200 mber 4
Ti u
100 N 2
0 0
Open arm Closed arm Open arm Closed arm
Control Control
Stressed Stressed
(a) (b)
Figure4:EffectofCMSonmalerat’sperformanceintheelevatedplusmaze.TheCMSparadigmdidnotaffectthetimespentintheopenor
closedarms(a),butitincreasedthenumberofentriesintotheclosedarms(b),suggestingananxiogeniceffectofCMS.Dataareexpressed
asmean±SEM.Student’s𝑡-test:∗𝑝<0.05versuscontrolgroup.
6 300
5 250
mber 4 onds) 200
ock nu 3 me (sec 150
Sh 2 Ti 100
1 50
0 0
Control Stressed Control Stressed
(a) (b)
120
100
∗
ds) 80
n
o
ec 60
e (s
m
Ti 40
20
0
Control Stressed
(c)
Figure5:EffectofCMSontheperformanceofmaleratsinthedefensiveburyingbehaviortest.CMSdidnotaffectthenumberofshocks(a)
orburyingbehaviorlatency(b);however,itincreasedthecumulativeburyingbehavior(c),suggestingananxiogeniceffectofCMS.Dataare
expressedasmean±SEM.Student’s𝑡-test:∗𝑝<0.05versuscontrolgroup.
stress did not increase the behavioral despair, suggesting currentstudyisinagreementwithotherreportswhereCMS
again that the young-male rats are resilient to CMS. This didnotincreasebehavioraldespairofyoung-adultratsinthe
result contrasts with studies that describe an increase of FST [45–47]. A putative explanation for the inconsistency
immobility behavior of young animals in the FST after of result among the effect of CMS on FST could be related
previousexposuretoaCMSschedule[19,20];however,the to variations in the intensity, unpredictability, or duration
8 NeuroscienceJournal
60 test, these animals presented higher cumulative burying
behaviorwithrespecttothenonstressedanimals;thisresult
50 indicates that CMS induces an increased aversion for the
noxiousstimulusinthemalerats.
n
mi These data together indicate that CMS induced an
40
5unts/ aannxhieedtyo-nliiake(satsateinindiycoautendg-baydutlhteainrimsuaclsrothseatpdridefneroetndceev;elsoepe
o 30
of c Figure2(c)).Theseresultsareinlinewithpreviousreports[11,
er 12]whereCMSincreasedanxious-likebehaviorsinrodents
mb 20 evaluated in several tests. The anxiogenic effect of CMS on
u
N young male rats indicates that they are not resilient to this
10 kindofstressor,aspreviouslyreported[13];instead,itappears
that young animals’ vulnerability to CMS is not reflected
as the core symptoms of depression. The anxiogenic-like
0
Control Stressed effects of CMS found in the current study contrast with
the study of Kompagne et al. [19] who found ambiguous
Figure6:EffectofCMSonthebehavioraldespairintheFST.There
were no significant differences between the control and stressed effectsofthisparadigmonanxious-likebehaviorsinWistar
group in the immobility behavior. Data are expressed as mean ± rats(CMSinducedsocialavoidanceandincreasedgrooming
SEM. but acted as if it was anxiolytic in the elevated plus maze).
Interestingly, in the study referred to, a clear effect of CMS
ondepressive-likebehaviorswasobserved.Inline,Karsonet
al.[49]foundthatWistaryoung-adultmaleratsexposedto
of stressors used in the stress regime; also, the rat strain
theCMSparadigmexhibiteddepressive-likebehaviorwhen
used could be a factor of variability. In the current study,
evaluatedinthesucrosepreferencetestandtheFST;however,
immobilitylevelspresentedbythemaleratscouldcontribute
theanimalsdidnotdevelopbehavioralalterationsrelatedto
to the lack of effect of CMS on FST; as shown in Figure6,
anxiety in the elevated plus maze test. Compared with the
control male rats presented very high levels of immobility
currentCMSschedule,Karson’sprotocolincludedperiodsof
(∼50countsfromatotalof60);thischaracteristicimportantly
restraint (4h), level shaking (10min), and nip tail (10min);
reducesthedynamicrangetoobserveputativestress-related
meanwhile,itdidnotincludestroboscopiclight,whitenoise,
magnification of the depressive-like behavior (an increase
ordeprivationoffoodorwater.Thus,allthesedatasuggest
of immobility levels) in the animals; thus, it is difficult to
thatCMSinduceseitheranxietyordepressive-likebehaviors
detecttheimpactofCMSonbehavioraldespair.Thiseffect
inyoungmalerats;thisdifferentialeffectcouldbedetermined
could be related to the large period (around 2 months) of
bytheintensityordurationofthestressorsemployedinthe
animal isolation required by the CMS schedule, since this
CMS schedule. This idea is supported by a clinical report
condition alters the animals’ performance in the FST [48];
suggestingthatthenatureofthestressorisimportantinthe
thishypothesisremainstobeproved.
developmentofdepressionoranxiety[50].
Italsomightbethoughtthattheanimalsrequiredmore Itisknownthatthestressorandtheabilitytoeffectively
than three weeks of CMS exposure to develop high levels deal with it contribute to the development of anxious and
of behavioral despair in the FST; however, the exposure of depressivesymptoms[3,4].Thisrelationshipconducestothe
3 to 4 weeks of CMS has been shown to be enough to coexistenceofanxietyanddepressionsymptomatologyinthe
effectivelyincreaseimmobilityintheFST[19,20].Thus,the same patients; this fact has led authors to propose a con-
discrepancies in the expression of depressive-like behaviors tinuum model, from anxiety syndromes to mild, moderate,
maybedeterminedbydifferencesinstressorsintensityand and severe depression. However, an alternative hypothesis
animals’abilitytocopewiththem. that needs to be explored is whether the comorbidity of
Since anxiety is another psychiatric disease where the anxiety and depression symptomatology is due to linked
stressfuleventsplayanimportantetiologicrole,wedecidedto neurobiological abnormalities that are nevertheless distinct
evaluatetheimpactofthiskindofstressonrodent’sanxious- intermsofwhethertheymediatetheanxietyorthedepressive
likebehaviors. symptoms[51].Inthisrespect,thecurrentstudysuggeststhat
CMS paradigm could be a useful tool to selectively study
4.3. Anxious-Like Behaviors Induced by CMS. In this study, neurobiologicalbasisofanxietyanddepression.Thus,itwill
weevaluatedanxious-likebehaviorsusingtwotestssensitive be interesting to study different CMS schedules varied in
to previous stress exposure: elevated plus maze [21] and duration,intensity,ornumberofstressorsperdayinorderto
defensive burying behavior test [22]. In the elevated plus differentially generate anxious- or depressive-like behaviors
mazetest,wefoundthatCMSonlyincreasedthenumberof inyoungmalerats.
entries to the closed arms, suggesting that stressed animals
have an increased preference for the closed spaces which 4.4.CorticosteroneSerumLevels. Inthecurrentstudy,wedid
is considered an anxious-like behavior [23, 24]. In line, notfinddifferencesbetweencorticosteronelevelsofstressed
althoughthestressedanimaldidnotavoidtheelectrode(no andcontrolrats,suggestinghabituationoftheanimalstothe
differences in the shock number) in the defensive burying stressors employed in our CMS schedule. This result is in
NeuroscienceJournal 9
agreementwithpreviousdatafoundinourlaboratorywhere tochronicstress[43].Interestingly,someoftheseeffectsare
seven weeks of CMS induced a reduction of corticosterone independentofadrenals,sincecorticosteronelevelswerenot
serumlevels(345.98±47.73ng/mL,unstressedgroup,versus directly corelated to increases or decreases in CRF [42, 43,
185.41 ± 56.85ng/mL, stressed group, 𝑛 = 8, 𝑝 < 0.01, 68].Accordingly,someauthorsproposethatthisdissociation
unpublished data); however, the failure of CMS to increase isduetotheactionsofcorticosteroneondifferentneuronal
corticosterone levels in young adults contrasts with reports populations: on PVN neurons for blunting the HPA axis
indicating that chronic stress induces elevated levels of activity(viarestrictionofCRFproduction)andonneurons
glucocorticoids[52,53].Thisdiscrepancymaybeexplained ofamygdalaforstimulatingCRFexpressionandinductionof
byvarianceinintensityofstressorssinceinthosereportsthe anxiety-likebehaviors.Resultspresentedhereseemtobein
stressorsweremoresevere(e.g.,restraint)thantheonesused linewiththesecondoption;however,furtherexperimentsare
inthecurrentstudy;insupportofthisidea,oneoftherules neededtotestthishypothesis.
ofhabituationthatappeartogovernadaptationtoachronic Present data together indicate that the relationship
stressorstatesthat“theprogressivereductionoftheresponses between stress and affective disorders is not as simple as
to a repeated stimulus is negatively related to its intensity” it appears; thus, to elucidate the impairments induced by
[54]. chronicstressoncentralnervoussystemthatcouldgenerate
Contrastingwiththecurrentresults,therearealsoseveral thesedisorders,anevaluationofthedifferentcomponentsof
reportsinwhichyoung-adultratsexposed toseveralweeks HPAaxisandtheireffectonlimbicstructuresisneeded.
ofCMSshowedelevatedcorticosteronelevelscomparedwith
theunstressedanimals[34,55–57];however,itisimportant 5.Conclusions
to highlight the notion that in these studies there was a
significantproportionofanhedonicrats(∼70%accordingto The CMS schedule used in this study induced behavioral
[58]),asindicatedbythereductioninsucroseintake,which alterationsthatsuggestanxietybutnotthecoresymptomof
is different from the one found in the current study, where depression, anhedonia, in young-adult males. These results
no animal developed anhedonia. Thus, such dissimilarities indicatethatnonanhedonicyoungmaleratsarenotresilient
maybe,atleastinpart,explainedbydifferencesinbehavioral to this kind of stressor, but rather the alterations are not
phenotypesinceinthecurrentstudytheanimalsexpressed expressedinthesphereofmotivationalbehaviors.Thisstudy
anxious-likeinsteadofdepressive-likebehaviors. highlights the need to use several physiologic measures
In the current study, the animals exposed to CMS (suchasbodyweight)andbehavioraltests,whenevaluating
displayed an anxious-like phenotype which was not related animal’svulnerabilitytostress.Thisstudyalsosuggeststhat
to hyperactivity of HPA axis; this result contradicts the CMScouldbeananimalmodeltostudytheneurobiologyof
diathesis-stresshypothesisforanxietyanddepression;how- depressionandanxiety.
ever, in some cases, especially after chronic stress, low
corticosteronelevelsarethefeatureofHPAaxisdysfunction CompetingInterests
[54,59]thatcouldberelatedtoaffectivedisorders,asinthe
The authors declare that there are no competing interests
caseofatypicaldepressioninhumans[59,60].Furthermore,
regardingthepublicationofthispaper.
thecorticosteroneprofilefoundinourstressedanimalsisin
agreementwitharecentstudy[61]thatdescribesareduction
incorticosteronelevelsofyoungratsafterchronicstress(21 Acknowledgments
days of social isolation); despite this, the animals displayed
anxious-anddepressive-likephenotypesintheelevatedplus TheauthorswishtothankbachelorBeatrizCruz-Lo´pezfor
mazeandtheFST,respectively;thesebehavioralresultswere technicalassistanceinexperiments.Thisworkwassupported
relatedtoalterationsinbrainplasticityinthestressedanimals by Instituto Nacional de Psiquiatr´ıa Ramo´n de la Fuente
(asmeasuredbydeficiencyinsomecelladhesionmolecules), Mun˜iz(NC093370.2).
which are characteristic of depressive/anxious states [62].
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