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Behavioural Brain Research 270 (2014) 248–255

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

orced swim and chronic variable stress reduced hippocampal cellurvival in OVX female rats

elly M. Vega-Riveraa, Alonso Fernández-Guastib, Gerardo Ramírez-Rodríguezc,rika Estrada-Camarenaa,∗

Laboratorio de Neuropsicofarmacología, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, México, MéxicoDepartamento de Farmacobiología, CINVESTAV-IPN, sede Sur, México, MéxicoLaboratorio de Neurogénesis, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, México, México

i g h l i g h t s

Forced swim test reduce cell survival but not cell proliferation in female rats.CVS reduced both cell proliferation and survival in female rats.The effects of FST on cell survival are analogous to those induced by CVS.

r t i c l e i n f o

rticle history:eceived 13 April 2014eceived in revised form 13 May 2014ccepted 16 May 2014vailable online 24 May 2014

eywords:hronic variable stressorced swimming testell proliferation

a b s t r a c t

Stress and glucocorticoids induce effects on neuronal and behavioral function. These effects may dependon the study design and importantly on the nature and duration of the stressor. We have previouslyobserved that a single exposure to the forced swim test (FST) caused long-lasting effects on the HPA axisresponse and hippocampal cell survival.

In despite that the FST and the chronic variable stress (CVS) paradigm are not strictly comparable;the aim of this study was to compare their effects on the respective depressive-like behavior, the serumcorticosterone levels and cell proliferation and survival in ovariectomized female rats. Cell proliferationwas determined by Ki67-labeling, while cell survival was analyzed with BrdU, a thymidine analog. Theresults showed that FST increased immobility and corticosterone levels at the same time that it decreased

ell survivalorticosteronevariectomy

cell survival without modifying cell proliferation. In contrast, after 5 weeks of CVS there was a sharpreduction in sucrose intake, cell proliferation and survival, but a lack of effect on corticosterone levels.The FST produced a reduction on newborn cell survival analogous to that exerted by CVS. These datasuggest that the FST could be considered as an attractive model to study some kind of stress-relateddisorders.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The forced swim test (FST) and chronic variable stress (CVS) are

ithin the most widely used paradigms to assess antidepressant-

ike effects and to induce depressive-like behaviors [1–3]. In theST after two sessions the animal shows a depressive-like behavior

∗ Corresponding author at: Laboratorio de Neuropsicofarmacología, Dirección deeurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, Calz México-ochimilco 101, San Lorenzo Huipulco, Delegación Tlalpan, México, DF, Mexico.el.: +55 41605053.

E-mail addresses: estrada@imp.edu.mx, monesca71@gmail.comE. Estrada-Camarena).

ttp://dx.doi.org/10.1016/j.bbr.2014.05.033166-4328/© 2014 Elsevier B.V. All rights reserved.

known as immobility [4], while the CVS requires several weeks ofstress exposition to induce a depressive-like behavior called anhe-donia, usually reflected as a decrease in sucrose intake [4]. Bothanimal models are sensitive to antidepressants’ treatments [4–6],and the FST is also sensitive to non-pharmacological manipulations[5,7].

Adult neurogenesis is a commonly used readout of neuro-plasticity. The dentate gyrus (DG) of the hippocampus is oneof the brain areas where new neurons are generated in adult-hood [8,9]. The function of adult hippocampal neurogenesis is

not yet understood; it is proposed to offer enlarged plasticityfor shaping the existing neuronal circuitry in response to newexperiences, and thus contributes to adaptation to environmentalchanges [10–13]. Reduction in neurogenesis has been implicated in

N.M. Vega-Rivera et al. / Behavioural Brain Research 270 (2014) 248–255 249

Table 1Mild stressors exposure schedule used in the CVS.

Time (h) Thursday Friday Saturday Sunday Monday Tuesday Wednesday

7:00–8:00 FD; WD SC WD GH8:00–9:00 FD; WD SC WD GH9:00–10:00 FD; WD EWB CT

10:00–11:00 FD; WD SL WN CT11:00–12:00 TEST SL WN SL CT12:00–13:00 SL WN SL CT WN13:00–14:00 GH SL WN SL CT WN14:00–15:00 GH SL CT WN15:00–16:00 GH SL FD; WD16:00–17:00 GH WD GH FD; WD17:00–18:00 SC WD GH FD; WD18:00–19:00 SC; CL WD GH; CL FD; WD19:00–20:00 SC; CL WD GH; CL FD; WD20:00–21:00 SC; CL WD GH; CL FD; WD21:00–7:00 SC; CL WD GH; CL FD; WD

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H, grouped housing (2–3 rats per cage); SC, soiled cage; CL, continuous light; SL,

T, cage tilt (45◦); FD, food deprivation.

ognitive and mood disorders [13–15]. However, the effects oftress on neurogenesis are not consistent and may depend on studyesign and importantly on the nature and duration of the stressor16,17].

Many studies report that stress, especially when experiencedhronically (in the form of CVS), suppresses one or more phasesf the adult hippocampal neurogenesis process [16,18–22]. It haseen reported that CVS induces signs of depression such as anhe-onia and corticosterone hypersecretion [23–25] that underlie theorphological changes in the hippocampus [26], as well as the

eduction in cell proliferation and survival [27–29]. It is worth men-ioning that most of these data have been obtained in males withemales receiving little attention. Recently, we showed that forcedwim also induces long-lasting effects on the HPA axis responsend some aspects of neurogenesis [30]. For example, a single 15 minorced swim experience decreases hippocampal cell survival par-lleled with an increase in corticosterone levels in ovariectomizedemale rats [30]. This finding is in agreement with the effectseported by other authors using different models of acute stressnd mostly males as experimental subjects [31–35].

Notwithstanding the FST and the CVS paradigm are not strictlyomparable, mainly because of their duration and type of stressors,e aimed to evaluate whether the deleterious effect on cell prolifer-

tion and survival of acute stress induced by the FST is similar to thataused by CVS in young adult ovariectomized rats. One of the mainrotein markers for cell proliferation is Ki67, which labels cells dur-

ng the phases M, G1, G2 and S of the cell cycle [36,37]. Cell survivalas inferred by analyzing the BrdU-positive cells [14,38]. Ovariec-

omized animals were selected because recently we demonstratedhat the lack of ovarian hormones increases the animal’s suscep-ibility to the harmful effects of stress on new hippocampal cellroliferation and survival [30].

. Materials and methods

.1. Animals

Ovariectomized Wistar rats (250–300 g, provided by theational Institute of Psychiatry ‘Ramón de la Fuente’ vivarium) weresed in all experiments. Animals were housed under standard 12-h

ight/dark conditions (12-h light cycle starting at 22:00 h), con-tant temperature (23 ± 1 ◦C) and with access to food and water

d libitum. To do the ovariectomy animals were anesthetized withribromoethanol (200 mg/kg) and a single midline incision in theentral area was made to expose and remove the ovaries. Animalsere allowed to recover for three weeks and thereafter randomly

scopic light; WD, water deprivation; EWB, empty water bottle; WN, white noise;

assigned to an experimental group [39–41]. Behavioral studieswere carried out in accordance with the National Institute of HealthGuide for the Care and Use of Laboratory Animals, the Mexican Offi-cial Norm for animal care and handling (NOM-062-ZOO-1999) andapproved by the local Institutional Ethics Committee. All effortswere made to minimize animal suffering and reduce the numberof subjects.

2.2. Forced swim test (FST)

The FST was conducted by introducing rats in individual Plex-iglass cylinders (height: 46 cm and diameter: 20 cm) filled with30 cm of water at 23 ± 2 ◦C [7,42,43]. Two swim sessions were con-ducted as follows: an initial 15-min pretest followed 24 h later bya 5-min test, which was videotaped for later scoring. After eachsession, rats were towel dried, placed in heated cages for 30 minand returned to their home cages.

The number of immobilities, as a sign of depressive-like behav-ior, was scored during the test session and was defined as theminimal movements to keep the snout above the water in a periodof 5 s during the 5-min test session [7,41,43]. Animals were sacri-ficed 30 min after the last stress session.

2.3. Chronic variable stress (CVS)

Rats were individually housed and randomly exposed duringfive consecutive weeks to the different stressors: white noise,grouped housing (2–3 rats per cage), continuous light, soiled cage,stroboscopic light, water deprivation, cage tilt (45◦) and water andfood deprivation according to the protocol used by Herrera-Pérezet al. [44] and Récamier-Carballo and Fernández-Guasti [4], fordetails see Table 1. Sucrose solution (1%) and tap water consump-tion were weekly determined after a 20 h period of water and fooddeprivation; the procedure consisted of 1-h exposure to two bot-tles: one with sucrose solution and the other one with tap water.The anhedonia was reflected as a reduction in sucrose consump-tion of at least 2 g [4,44]. Animals were sacrificed after the last stresssession. Only those animals that reached the criterion of anhedoniawere included in the data analyses.

2.4. Ki67 and BrdU immunohistochemistry

Rats were anesthetized with ketamine/xilacine (i.p.) andintracardially perfused with 0.9% saline solution, followed by 4%p-formaldehyde (PFA) in 0.1 M sodium phosphate buffer (pH 7.4).Subsequently, brains were removed and post-fixed in PFA for

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4 h. Then, brains were kept in 30% sucrose in phosphate bufferntil sectioned. Brains were cut into 40 �m coronal sections on aibratome (Microm International, Walldorf, Germany) and storedt 4 ◦C in a cryoprotective solution until required [30].

Cell proliferation was determined by rabbit polyclonal anti-Ki67ntibody added to the brain slices in blocking solution (1:100 + 5%D + 0.25% Triton-X; Abcam, San Francisco, CA, USA) and cell sur-ival with mouse monoclonal anti-BrdU also added in blockingolution (2:500 + 5% SD + 0.25% Triton-X100). Specific secondaryiotinylated antibodies were from Abcam (San Francisco, CA, USA)nd Vector laboratories (Burlingame, CA, USA). Sections were clar-fied and mounted in Neomount medium (Merck, Whitehousetation, NJ, USA). Immunoreactive cells were counted along sub-ranular zone (SGZ) in the hippocampus using a 40× objective on

light microscope, as previously described [30,45]. To determineell survival BrdU (75 mg/kg, i.p.) was administered 12 h and 30 minrevious to the first FST session [30] and 12 h/12 h during three daysefore the beginning of the CVS protocol.

.5. Determination of corticosterone levels

Corticosterone (CORT) levels were determined from plasmaamples obtained from the different groups using the cortico-terone immunoassay kit (Assay Designs, Ann Harbor, Ma, USA)ccording to the manufacturer’s instructions. Microplate was readt 405 nm in an ELISA reader (Bio-Tek, Winooski, VT, USA). Inter-nd intra-assay variabilities were 7.8 and 6.6%, respectively.

.6. Statistical analysis

Results are presented as means ± standard error of the meanSEM). Comparisons of Ki67- and BrdU-labeled cells, immobilitynd corticosterone levels were done by a t test using the SigmaS-at 3.1 software (Systat Software Inc, USA). The sucrose preferenceata evaluated in the CVS were analyzed by means of a Repeatedeasures Two-way ANOVA test followed by Holm–Sidak’s test

onsidering as factor A: time and factor B: stress. A Two-wayNOVA test followed by Student–Newman–Keuls was performed

o compare the type of stress effect’s on the number of Ki67- andrdU-labeled cells considering as factors: presence of stress (fac-or A) and type of stressor (factor B). Differences were consideredtatistically significant at p < 0.05.

.7. Experimental design

Ovariectomized (OVX) rats were divided in four groups (twoontrol and two stressed): the control rats were maintained with-ut stress during 2 days for the FST or 5 weeks for the CVS protocol,ut under similar handling and housing conditions than the exper-

mental groups. The stressed groups were exposed to FST (15 minnd 24 h later a 5-min test session) or during 5 weeks for the CVSchedule shown in Table 1 [44]. The FST requires that the rats areubjected to swim to induce the immobility behavior. In view ofhat an additional independent group of OVX rats, which was notsed for Ki-67 and BrdU determinations, was forced to swim for

min considering that this brief session is not enough to induce aepressive-like behavior in female rats [46]. This group served asontrol for that receiving two swim sessions.

We compared the number of Ki67- and BrdU-labeled cells

etween control and stressed OVX rats in the FST and CVSFigs. 1 and 2). In addition we calculated the percentage of reductionf labeled cells from their respective controls in both paradigmsTable 2).

in Research 270 (2014) 248–255

3. Results

Fig. 1 shows representative hippocampal photomicrographs ofKi67 and BrdU positive cells (panel A), immobility behavior (panelB), quantification of BrdU- and Ki67-labeled cells (panel C) and cor-ticosterone levels (panel D) in OVX rats subjected or not to theFST. Animals exposed to two FST sessions showed more immobil-ity behavior than those exposed to a single swim session (controlgroup; p < 0.001, panel B). FST significantly decreased the numberof surviving newborn cells (t = 5.47, df = 6, p = 0.002) but did notaffect cell proliferation (t = 1.28, df = 7, ns) in the dentate gyrus whencompared to the unstressed control group (Fig. 1, panel C). The cor-ticosterone levels of the animals forced to swim were higher thanthose of the unstressed control group (t = −4.74, df = 6, p = 0.003;Fig. 1, panel D).

Fig. 2 shows representative hippocampal photomicrographs ofKi67 and BrdU positive cells (panel A), sucrose intake (panel B),quantification of BrdU- and Ki67-labeled cells (panel C) and cortico-sterone levels (panel D) in OVX rats subjected to CVS protocol. Onlythose animals that reached the anhedonia criterion were includedin the statistical analysis (75%). The analysis of sucrose intakerevealed that after 6 weeks of CVS (F3,21 = 3.35, p = 0.03) the sucroseintake was significantly reduced in the stressed group comparedto the unstressed control group (F1,21 = 47.34, p < 0.01). Likewise,the analysis of the total number of BrdU- and Ki67 labeled cellsin the dentate gyrus of animals exposed to CVS revealed a signif-icant reduction in cell survival and proliferation in comparison tothe unstressed control group (t = −7.54, df = 5; p < 0.001 for BrdU;and t = 3.91, df = 7, p = 0.006 for Ki67; respectively. Fig. 2, panel C).In addition, corticosterone levels did not differ between the ani-mals exposed to the CVS and the unstressed group (t = −0.092 withdf = 8; ns; Fig. 2, panel D).

The number of both the Ki67- and BrdU-labeled cellsin unstressed control group of FST (Ki67 = 1611 ± 95 andBrdU = 781 ± 79) and CVS (Ki67 = 1695 ± 329 and BrdU = 889 ± 95)were similar (Table 2) and considered as the 100% reference tocompare with the stressed groups. Remarkably, both behavioralprotocols similarly reduced the number of BrdU-labeled cells (58%,for FST and in 69%, for CVS, see Table 2). In contrast, the FST pro-duced a marginal reduction of around 17% in Ki67 labeled cells,while the CVS provoked a drastic decrease close to 70%. Two-wayANOVA test yielding the following values: presence of stress (fac-tor A) F1,14 = 17.45, p < 0.001; type of stressor (factor B) F1,14 = 4.48,p = 0.05; and the interaction AXB F1,14 = 6.76, p = 0.02.

4. Discussion

Acute stress provoked by forced swim and chronic variablestress similarly reduced hippocampal cell survival, however, cellproliferation was only diminished in animals exposed to CVS.

Most of the effects of stress on neurogenesis are explainedthrough the very well established inhibitory effect of corticoste-roids on hippocampal cell proliferation and survival [16,20,47].However, the role of glucocorticoids in the regulation of neuroge-nesis is not simple and direct. An increase in corticosteroid levelsseems to be necessary for the effects of stress on neurogenesis,however, it is not sufficient and does not strictly correlate with theeffect on newly generated cells. Importantly, after certain stressors,the raised glucocorticoid levels are not associated with a suppres-sion of hippocampal cell proliferation (vide infra) or may even beaccompanied by an increase in the number of new cells [48]. In

the present study we found that in the CVS model the reduction insucrose intake was accompanied by a decrease in cell proliferationand survival, but not by an increase in corticosterone levels. Thislast finding is in agreement with previous results reporting that

N.M. Vega-Rivera et al. / Behavioural Brain Research 270 (2014) 248–255 251

Fig. 1. Panel A. Representative photomicrographs of the effect of forced swimming on proliferating (panel A, upper part) and surviving cells (panel A, lower part) in the dentategyrus (DG) of the hippocampus. Scale bar = 100 �m, scale bar insert = 40 �m. Images show the hilus, subgranular zone (SGZ), granular cell layer (GCL) and the molecular layer(ML). Panel B. Effect of forced swimming on immobility behavior of animals forced to swim once (control, n = 8) and forced to swim twice (FST, n = 9). Panels C and D. Effectof forced swimming on cell proliferation (Panel C left, n = 4) and survival (Panel C right, n = 4) and corticosterone levels (panel D, n = 4). Data represent the mean ± standarderror of mean (SEM). Student’s t test, *p < 0.01; **p < 0.001; ***p = 0.0001; versus control group.

252 N.M. Vega-Rivera et al. / Behavioural Brain Research 270 (2014) 248–255

Fig. 2. Panel A. Representative photomicrographs of the effect of chronic variable stress on proliferating (panel A, upper part) and surviving cells (panel A, lower part) inthe dentate gyrus (DG) of the hippocampus. Scale bar = 100 �m, scale bar insert = 40 �m. Images show the hilus, subgranular zone (SGZ), granular cell layer (GCL) and themolecular layer (ML). Panel B. Sucrose intake of unstressed (control, white bars, n = 7) and stressed (CVS, dashed bars, n = 7) under basal conditions (before stress, left) and 5weeks after stress (right). Panels C and D. Effect of chronic variable stress on cell proliferation (Panel C left, n = 4) and survival (Panel C right, n = 4) and corticosterone levels(panel D, n = 4). Data represent the mean ± standard error of mean (SEM). RM two-way ANOVA test followed by Holm–Sidak’s test for sucrose intake, *p < 0.01; Student’s ttest for other groups comparisons, **p < 0.001; ***p = 0.0001; versus control group.

N.M. Vega-Rivera et al. / Behavioural Brain Research 270 (2014) 248–255 253

Table 2Number (and percentage) of Ki67- and BrdU-labeled cells in OVX control unstressed females or in rats exposed to acute stress, FST or to chronic variable stress. Date areexpressed as means ± SEM. Student–Newman–Keuls test, *p ≤ 0.05; **p ≤ 0.005 vs control, &&p ≤ 0.005 vs FST.

Acute stress (FST) Chronic stress (CVS)

Without stress With stress Without stress With stress

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Ki67-labeled cells 1611 ± 95 (100%) 1337 ± 1BrdU-labeled cells 781 ± 79 (100%) 330 ± 2

fter relatively long periods of chronic stress there is no changen the serum ACTH or corticosterone levels [49–51]. As previouslyuggested [49,52], the lack of increase in serum corticosterone lev-ls may be based on the desensitization of the HPA axis. In support,honically stressed animals showed lower corticosterone levelshan their acutely stressed counterpart [17,50]. Thus, even if we didot find an increase in corticosterone levels after five weeks of CVS,thers have reported that after two to three weeks of similar proto-ols of CVS there is a strong increase in corticosterone serum levels53,54], particularly in females [55]. These high levels of corticoste-one may have impaired the proliferation and survival of newbornells in the hippocampus [24,25,56], although other factors pos-ibly contribute to the inhibition of these processes (vide infra).resent data showing that CVS importantly inhibited cell prolifera-ion is in line with several previous data showing that proliferation

ost likely decreases as the result of continuous stress exposition16,18–22,57]. However, such result apparently disagrees withhat reported by Lagunas et al. [58] who showed that this paradigmid not further reduce cell proliferation in aged female mice. It isorth noting that in both studies OVX animals were used, thusiscarding that ovarian hormones may mediate the divergence.owever a possible explanation may rely on the subjects’ ageecause seven months old mice were used in that study and itas been shown that in 6–8 months mice there is a constitutive

ow number of BrdU-labeled cells compared with young animals59,60], that could mask the effects of CVS on cell proliferation.

In contrast with CVS, two expositions to FST induced a markedncrease in serum corticosterone levels, a finding in line with thedea that this animal model is a potent acute stressor [30,61]. Suchncrease was associated with a sharp decrease in hippocampal cellurvival, but was unaccompanied by an effect on cell proliferation.nterestingly, the FST-induced decreased on early newborn cell sur-ival confirms previous results [30]. Moreover, the lack of action ofST on cell proliferation is also in agreement with earlier reportshowing that several acute stressors such as tail- or foot-shocks,esident-intruders and restrain did not reduce cell proliferationn the dentate gyrus, albeit if associated with high corticosteroneevels [17,62–66]. The fact that glucocorticoid receptors are barelyxpressed on hippocampal precursor cells [67,68] suggest that cor-icosteroids act via other pathways, possibly through glutamate and-methyl-d-aspartate (NMDA) receptor dependent mechanisms

69]. The lack of effect of FST on cell proliferation contrasts withhe results reported by Heine et al. [70] who showed a reductionn this parameter after acute stress. The possible factors explaininghis discrepancy include the type of stressors: cold restriction andorced swim and the sacrifice time: 24 h later versus 30 min after,mphasizing that the time of euthanasia is also a crucial factor thatay veil the actions of stress on neurogenesis.In recent years, a line of research has focused on the impor-

ance of neurotrophins, which are involved in cellular plasticity,s another important target for stress on the impaired hippocam-al neurogenesis. One of the most important neurotrophin is therain-derived neurotrophic factor (BDNF), which is a target of

rime importance in the neurobiology and treatment of depres-ion [71–73]. In fact, it has been reported that the levels of thiseurotrophin are decreased in serum and in postmortem brainamples from depressed patients [74–76]. Reduced BDNF levels, as

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2.97%) 1695 ± 329 (100%) 516 ± 74 (30.44)**,&&

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well as attenuated neurogenesis, have been found in the brains ofanimals exposed to stress protocols, while antidepressants increasethe expression of BDNF [77,78]. Interestingly, acute stress increasesBDNF protein and mRNA as well as its TrK-B receptor from 15to 60 min whereas chronic stress decreases BDNF protein andincreases TrK-B [79]. If BDNF is working like a buffer to regulate thestress impact [80], it is possible that the increase of BDNF observedafter acute stress exposition [79] contributes to the lack of effectof FST on cell proliferation [81], situation that did not occur [82]after CVS. However, specific experiments focused to explore thishypothesis are warranted.

5. Conclusion

In closing the present series of results show that exposition tostandard forced swim test produces a drastic effect on hippocampalcell survival analogous to that exerted by several weeks of chronicvariable stress, strengthening the idea that the FST is an intenseacute stressor.

Conflict of interest

The authors declare no potential conflict of interests.

Acknowledgments

Authors express special thanks to QFB. Leonardo Ortíz-López forhis technical assistance in corticosterone determinations. V-R NMreceived fellowship from CONACyT (216658) and E-C E received agrant from CONACyT (CB104659).

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