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Behavioural Brain Research
j ourna l h o mepa ge: www.elsev ier .com/ locate /bbr
esearch report
he anxiolytic buspirone shifts coping strategy in novelnvironmental context of mice with different anxious phenotype
ános Horvátha, Titanilla Szögia, Géza Müllera, Viktor Szegedib,∗
Department of Medical Chemistry, University of Szeged, Szeged, HungaryBiological Research Center, Institute of Biochemistry, Szeged, Temesvári krt. 32, Hungary
i g h l i g h t s
Anxious (AX) and nonanxious (nAX) mice were studied in elevated plus maze for 8 days.Both strains showed decreased open-arm exploration over time.Exposing mice to a novel environmental context changed behavioral profile.AX mice were passive, while nAX mice were active.This behavioral profile was reversed with chronic 2.5 mg/kg buspirone treatment.
a r t i c l e i n f o
rticle history:eceived 18 January 2013eceived in revised form 10 April 2013ccepted 13 April 2013vailable online xxx
eywords:nxietylevated plus mazeontext change
nbred mice
a b s t r a c t
Patients suffering from anxiety disorders show increased fear when encounter a novel environment.Rodents, placed in new environmental context may respond either with increased novelty seeking(active), or enhanced anxiety (passive coping style), which may depend on the trait anxiety of the animal.Here, the connection between the initial level of anxiety and the behavioral responses in a novel environ-ment was investigated. Two inbred mouse strains having either high- or low-anxiety related behavior(AX and nAX) were exposed to elevated plus maze (EPM), a standard test for assessing anxiety level, for8 consecutive days. The initial anxiety level was modulated by chronic treatment with buspirone (bus)treatment, a clinically effective anxiolytic, using 2.5 mg/kg and 5.0 mg/kg doses. Both strains showed agradual decrease of open-arm exploration, which was not prevented by bus treatment. Another cohortof animals was exposed to EPM for 2 days, and then we changed to blue light illumination and used a
XAX
different cleaning substance with citrus odor (context change, CC). It was found that upon CC AX miceexhibited increased, while nAX mice showed decreased anxiety. Bus in 2.5 mg/kg changed the copingstrategy from passive to active exploration after CC in the AX mice; however, the same treatment ren-dered nAX mice passive upon CC. Bus in 5.0 mg/kg failed to alter the overall coping style in the novelenvironment of both strains. These results suggest that these mouse lines use different coping strategyin novel context, which can be changed with bus treatment.
. Introduction
Anxiety disorders, the most common forms of mental illness16], are often accompanied by increased fear in novel contextsnd patients were reported to have difficulties in habituating to
new environment. In preclinical research, elevated plus maze
EPM) is a widely used model to assess anxiety-related behav-or of mice (for review see [8,21]). The time spent in the aversivepen arm is an inverse measure of fear, because anxious animals
∗ Corresponding author at: Biological Research Center – Biochemistry, Temesvárirt. 32, Szeged H-6726, Hungary. Tel.: +36 70 2418260.
prefer the safe closed arm. An intriguing feature of EPM is that miceshow profound behavioral changes when re-exposed to the plus-maze. Typically, undrugged animals exhibit a significant decreaseof open arm exploration upon re-exposure to the EPM, a findingoften referred to as one-trial tolerance (OTT) (e.g. [3,6,18,25]). Thereare conflicting data about the physiological background of OTT: itmay represent an increased anxiety response (phobic-like) to theopen arms during the first trial [3,5], or alternatively, mice couldlose the motivation for exploring the aversive open arms [26]. Insupport of this hypothesis, when an aversive stimulus is introduced
in the closed arm during re-exposure, animals did not show OTT[22]. Although the readout would be the same (decreased timespent in the open-arms), these possibilities are different in termsof anxiety expression and coping style.
Inbred rodent models selected for extremes in anxious pheno-ypes are valuable models of anxiety research. Differences in therait anxiety level may be accompanied by different coping strat-gy or resilience to stress. Indeed, rats bred for extremes in anxietyere shown to use active (less anxious) or passive (more anxious)
oping strategy in the forced swim [14,15] and hole-board test [20].reviously, we have developed two mouse strains having eitherigh- or low-anxiety related behavior (AX and nAX, respectively)s was shown in the EPM, open-field, and light/dark test [28,31].he initial level of anxiety may determine the coping style or theabituation of the animals in new environment, thus those miceere exposed to EPM for eight consecutive days. Furthermore, in
rder to enhance the motivation for re-exploring the open arms, change in the environmental context of the EPM apparatus wasntroduced after 2 days of EPM exposure, and the behavior was fol-owed for 6 days. As the animals have a choice between safe andovel, but aversive compartments, it was expected that mice with
ow trait anxiety would exhibit a preference for novelty, whereasigh trait anxiety subjects would prefer safety. Rather introduc-
ng a negative stimulus, like hot air stream in one closed arm [22],e aimed to enhance the exploratory motivation by changing the
nvironmental context. We asked whether changing some externalues would modify the coping strategy of the animals in the EPM.urthermore, the effect of a clinically effective anxiolytic, buspironebus) was tested in two doses on the behavioral profiles.
. Materials and methods
.1. Animals
Inbred mouse strains having either high- or low anxiety level (AX and nAX)ere bred in our animal facility. Male mice of 2.5–3 month were housed individuallynder a light/dark 12 h cycle (lights on at 08:00) at 24 ± 1 ◦C and given ad libitum foodnd water. Mice were handled for 7 days prior to testing, the procedure described byurst and West [13]. For the experiments, 45th–48th inbred generations were usedf both strains. The study conformed to EU directive 2010/63/EU and was approvedy the regional Station for Animal Health and Food Control under Project LicenseXXI/2012.
.2. Treatment
Mice were injected chronically (daily injection for 4 weeks before and duringhe tests) with buspirone (Sigma–Aldrich, Budapest) at 2.5 mg/kg and 5.0 mg/kgoncentrations. Drug was dissolved in phosphate buffer saline (PBS) pH 7.2 and wasdministered intraperitoneally (i.p.) with 0.1 ml. The control group was treated withaline of the same volume.
.3. Behavioral testing
The EPM was made of stainless steel (painted matt black) consisting of twopposite open arms (35 cm × 7 cm) and two opposite closed arms surrounded by5 cm high walls of the same dimensions. The middle section that allows the animalo transit from arm to arm consisted of a square with dimension of 7 cm × 7 cm. Thepparatus was elevated 50 cm from the floor and the open arms were equipped with.5 cm × 1.5 cm ledges to ensure, that no animals would fall of the maze. The animalouse and the testing location were at the same room. Inside the room the maze waseparated with black curtains. The behavior of the mice was recorded and analyzedith an EthoVision software package (EthoVision XT 8.5 Noldus Technology, Theetherlands). Animals were exposed to EPM for eight consecutive days. Trials werearried out under low white light condition (open arms 45 lux, closed arms 15 lux).ll tests were conducted during the light phase of light/dark period between 13:00nd 17:00 h. The mice were placed individually in the center of the maze facing thepen arm and allowed 5 min to explore the apparatus. The maze was cleaned with0% ethyl alcohol before each trial. The white illumination/ethanol cleaning scenarioas defined as context A. In certain experiments, blue-light illumination combinedith cleaning the apparatus with a substance having light citrus-fragrance was used,
nd this was defined as context B. The strength of illuminance did not differ inontext B (open arms 40 lux, closed arms 14 lux).
.4. Statistical analysis
The data were compared by Repeated Measures of ANOVA (RM ANOVA) withost hoc Bonferroni, unless otherwise stated. Results were considered to be signifi-antly different at a probability level of p < 0.05. Data are presented as means ± SEM.
Research 250 (2013) 32– 38 33
3. Results
The first cohort of mice (AX n = 9, nAX n = 10) was exposedto EPM daily for 8 days. The two strains showed clearly distinctphenotype at the first day (open arms: 120.4 ± 15.1 s for AX vs.193.1 ± 5 s for nAX; p < 0.001; closed arms: 130.2 ± 9.8 s for AX vs.57.2 ± 2.8 s for nAX; p ≤ 0.001, independent samples t-test). Bothstrains showed a continuously decreasing open-arm exploration,however this decline was stronger in the nAX mice (p = 0.020 forAX, and p < 0.001 for nAX; paired samples t-test; Fig. 1). Exemplartracks are shown at Fig. 1 and Supplementary Table 1 contains amore detailed dataset of other behavioral parameters.
Next, using another cohort of mice (AX n = 8, nAX n = 10), theeffect of introducing a change in the surrounding context on thebehavioral profile was investigated. Mice were exposed to the EPMusing normal illumination and ethanol to clear the apparatus beforeeach test. At day 3, the environmental context was changed to blue-light illumination and a citrus-fragrance cleaning substance, whichwe refer to as context B. It was found that the time spent with open-arms exploration increased in context B of the nAX, but significantlydecreased of the AX mice (RM ANOVA, normal condition vs. CC: AXopen arms: F(1,17) = 8.036, p = 0.011; closed arms: F(1,17) = 10.507,p = 0.005, nAX open arms: F(1,18) = 19.947, p < 0.001; closed arms:F(1,18) = 6.974, p = 0.017; Fig. 2). These behavioral patterns suggestthat AX mice were more anxious in the novel environment, andavoided the stressful open-arms. NAX mice, however, acted differ-ently, because the time spent in open-arms did not decrease overtime upon context B introduction in such a rate, as in the normalcondition). It might reflect a proactive behavioral pattern of thenAX mice, e.g. they tried to explore the environment in context B.The actual numbers can be found in Supplementary Table 2.
The same behavioral profile was found in mice which wereexposed first to context B, then to context A (AX n = 7 and nAX n = 6;Fig. 3. and Supplementary Table 3). Using this schema, context A isthe novel environment, where AX mice showed increased anxi-ety (RM ANOVA, open arms: F(1,14) = 1.711, p = 0.212; closed arms:F(1,14) = 7.373, p = 0.017), whereas nAX mice showed increasedexploratory behavior (RM ANOVA, open arms: F(1,14) = 6.674,p = 0.022; closed arms: F(1,14) = 1.712, p = 0.212), suggesting that itwas not the blue-light illumination per se that elicited changes inthe behavioral pattern, but instead the change in the surroundingcontext of the EPM apparatus.
3.1. The effect of buspirone
Buspirone, a clinically effective anxiolytic was injected i.p. intwo different doses for 4 weeks daily before, and during the exper-iments, and the behavior of mice were investigated in the EPM.The anxiolytic effect of bus was evident at the first day, whentreated AX mice (n = 8 for 2.5 mg/kg and n = 14 for 5.0 mg/kg)showed reduced anxiety, and this was dose-dependent (closedarms: 130.3 ± 9.8 s for control AX, 118.4 ± 10.4 s for AX 2.5 mg/kgbus, p = 0.947 vs. control and 90.7 ± 3.9 s for AX 5.0 mg/kg bustreated mice, p = 0.002 vs. control; open arms: 120.4 ± 15.1 s forcontrol AX, 109.5 ± 11.2 s for AX 2.5 mg/kg bus, p = 1.000 vs. control,138.9 ± 9.4 s for AX 5.0 mg/kg bus, p = 0.003 vs. control, one-wayANOVA post hoc Bonferroni). Interestingly, bus increased anxiety-like behavior of nAX mice (n = 10 for 2.5 mg/kg and n = 18 for5.0 mg/kg; closed arms: 57.2 ± 2.8 s for control nAX, 69.3 ± 6.1for nAX 2.5 mg/kg bus, p = 0.170 vs. control, 69.9 ± 2.8 for nAX5.0 mg/kg bus, p = 0.076 vs. control; one-way ANOVA post hoc Bon-ferroni; open arms: 193.2 ± 5.1 s for control nAX mice, 169.9 ± 8.4 s
for nAX 2.5 mg/kg bus, p = 0.705 vs. control, 172.6 ± 4.3 s for nAX5.0 mg/kg bus mice, p < 0.001 vs. control; one-way ANOVA post hocBonferroni Fig. 4. and Supplementary Table 4). Investigating thechange of kinetics of open-arms exploration over the 8-day long
34 J. Horváth et al. / Behavioural Brain Research 250 (2013) 32– 38
Fig. 1. Bar charts showing the total time spent in the open (A) and closed arms (B) of AX and nAX mice during the 8 day long trial. Representative tracks of the days indicatedas red rectangles are shown at panel C and D. The closed arms of the EPM are denoted with green borders. * P ≤ 0.05, RM ANOVA. (For interpretation of the references tocolour please see to the web version of article.)
Fig. 2. Mice responded differently to context change (indicated as a blue background between day 3 and 8 at panel A and B). Representative tracks from day 1 and day 3 (firstday at the novel context), as indicated as a red rectangle, are shown below (C and D). The closed arms of the EPM are denoted with green borders. * P ≤ 0.05, RM ANOVA. (Forinterpretation of the references to colour please see to the web version of article.)
J. Horváth et al. / Behavioural Brain Research 250 (2013) 32– 38 35
F nel B)2 o the w
ttFnnFi8
e
Fa
ig. 3. Bar charts showing the total time spent in the open (Panel A) and closed (Pa. * P ≤ 0.05, RM ANOVA. (For interpretation of the references to colour please see t
est session using RM ANOVA showed no difference between con-rol vs. 2.5 mg/kg bus group neither in AX or nAX mice (open arms:(1,16) = 0.653, p = 0.432 for AX mice and F(1,17) = 0.007, p = 0.937 forAX mice; Fig. 4.). However the time spent in the closed arms for theAX 2.5 mg/kg bus group was significantly different (RM ANOVA:(1,17) = 4.447, p = 0.049 vs. control; Fig. 4.). Similarly, bus treatmentn 5.0 mg/kg altered the time spent in closed-arms throughout the
days of AX mice (RM ANOVA: F(1,21) = 4.511, p = 0.046 vs. control).Mice treated with 2.5 mg/kg bus (AX n = 11, nAX n = 10), how-
ver, showed dramatic change in the novel context. AX mice spent
ig. 4. The effect of chronic buspirone administration (2.5 and 5.0 mg/kg) on the behaviorrms). Significance is indicated in the main text.
arms. Blue backgrounds indicate context B (see main text for details) at day 1 andeb version of article.)
much more time in the open arm compared to control group, indi-cating that low dose of bus changed the coping style of AX mice(RM ANOVA: F(1,20) = 19.170, p < 0.001 vs. control; Fig. 5. and Sup-plementary Table 5.). In contrast, treated nAX mice spent muchless time exploring the open arm than control counterparts, sug-gesting that anxiety has developed in the novel context due to bustreatment (RM ANOVA: F = 5.395, p = 0.032 vs. control).
(1,19)
AX mice treated with the higher dose of bus (n = 9) showed con-tinuously decreasing open-arm exploration upon context change,similarly to control mice (Fig. 5.). Although the difference was
of AX (A for open and B for closed arms) and nAX mice (C for open and D for closed
36 J. Horváth et al. / Behavioural Brain Research 250 (2013) 32– 38
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ig. 5. The effect of chronic administration (2.5 and 5.0 mg/kg) on the behavioral pice upon context change. Blue background indicate novel context. * P ≤ 0.05, RM
rticle.)
ignificant (RM ANOVA: F(1,18) = 4.831, p = 0.042), there was no dif-erence between the time spent in the closed-arms of control vs. bus.0 mg/kg AX mice (RM ANOVA: F(1,18) = 2.428, p = 0.138), suppor-ing the notion that this dose of bus does not modify dramaticallyhe behavioral pattern of AX mice in a novel environment.
Similar results were found for nAX treated with 5.0 mg/kg busn = 9; Fig. 5.). Although there was significant difference in thepen-arms time between 5.0 mg/kg bus vs. control (RM ANOVA:(1,18) = 5.755, p = 0.028) the closed-arm time did not differ (RMNOVA: F(1,18) = 0.871, p = 0.364). This also supports the hypoth-sis, that 5.0 mg/kg bus does not alter profoundly the coping stylef nAX mice upon context change.
. Discussion
The conflicting hypotheses about the reasons of OTT assumehat different coping strategy is being used by the animals. Theres a strong correlation between trait anxiety and passive copingtyle with stressful situations, like a novel environmental context.lso, animal studies have found that inadequate coping strategy istrongly correlated with anxious phenotype. However, trait anxi-ty, by definition, does not change rapidly, thus we hypothesizedhat the reasons behind the decreasing open arm exploration maye different in the two strains. Thus one cohort of mice was exposedo the EPM for 8 consecutive days and an environmental contexthange was also introduced in another cohort of mice.
It was found that besides showing OTT on the second day, AX
ice showed no further decrease of exploratory motivation. In
ontrast, nAX mice exhibited a continuous decrease of open-armxploration, which is, as we suggest, not due to increased anxiety,ut to habituation. The lack of similar habituation in the AX strain
of AX (A for open and B for closed arms) and nAX (C for open and for closed arms)A. (For interpretation of the references to colour please see to the web version of
may be the consequence of elevated anxiety. This is supported byfindings, that in anxious rats, the level of plasma corticosteroidsdoes not habituate between trials [4]. These differences implydifferent coping styles of the two strains, thus we investigatedthe responses upon environmental context change. The observedeffects were not due to the blue illumination per se, because whenthe context was switched from blue light to normal white illumi-nation, the behavioral responses of AX and nAX were the same,suggesting that the change of familiar environmental to a novelone caused these behavioral patterns.
The readout for anxiety in the EPM is based on the conflictbetween the drive to explore (open arm) and the drive to avoid(closed arm), thus highly anxious mice are expected to prefer thesafe, and familiar closed arm in the novel context. Indeed, AXmice responded with a significantly reduced exploratory behav-ior (passive coping style) to the novel context. On the contrary,the response of nAX mice was manifested in increased open armexploration (active coping style). These results suggest that strainshaving different trait anxiety utilize different coping strategy in anexploration/aversive conflict. This proposal is supported by reportsshowing that the highly anxious DBA2 mouse shows decreasedopen arm exploration upon retest in changed environment (mazeorientation or laboratory environment) [24]. The available data isvery limited in the literature about the reactions of non-reactivestrains in a second EPM exposure combined with environmentalchanges. However, we expect that a less anxious strain, such asBALB/c [30] would behave similarly as our nAX mice. The only avail-
able data we are aware of shows that wild mice, who exhibit amarked decreased closed-arm activity compared to standard labo-ratory mice, do not show signs of OTT as is measured by open/closedarm occupancy upon the second EPM exposure [12]. Likewise, nAX
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ice exhibited a reduced OTT at day 2, compared to AX mice. Thendings of Thiel et al. [29] also supportive: they report that ratsith high- or low-rearing behavior in a novel open-field test showifferent behavior upon re-exposure to the test apparatus. High-earing rats (active) respond with more time spent in the center,hile low-rearing rats (passive) show no change in this respect.
.1. The effect of buspirone
Buspirone, a 5-HT1A partial agonist is widely used in the clinics anxiolytic. Similarly to most antidepressants, the onset of actionequires several weeks of medication in human patients. There-ore we have used chronic, 4 weeks of bus treatment in the presentxperiments. It was found that chronic bus treatment in low dose2.5 mg/kg) did not alter the behavioral profiles of AX in the 8-dayPM session, but the larger dose (5.0 mg/kg) induced a graduallyncreasing closed-arm stay. Interestingly, mice treated with theower dose showed increased open-arm activity in the novel con-ext, but here the larger dose did not alter the profile of behavior.n the contrary, bus treatment in any of the doses did not dras-
ically modify open/closed arm profile of nAX in the 8-day EPM.trikingly, low dose bus treated mice exhibited a shift in the copingtrategy in the novel context, namely nAX mice showed a greaterreference for the closed arm, while AX mice exhibited increasedxploratory drive upon context change. The reason behind the dif-erence between the responses for the two applied doses is not easyo explain. First, it is known that activating the 5-HT1A receptor
ay induce anxiogenic and anxiolytic responses in laboratory ani-als. Bouwknecht et al. reported that a 5-HT1A agonist increasedeasures of anxiety in open-field activity test and the light-dark
xploration test of C57BL/6J (B6) mice, a strain regarded to be lessnxious. In contrast, the same treatment had minimal effect onhe highly anxious 129S6/SvEvTac (S6) strain [2]. Second, 5-HT1As localized pre- and postsynaptically, having opposite effects onerotonin neurosignalling [9,23]. The intricate mechanism gover-ing serotonin release is further complicated by the fact, that 5-HTutoreceptors, which provide a negative feedback loop inhibitingerotonin release [10], tend to desensitize, but even at high synap-ic serotonin concentration, they are capable of responding to largeeaps in serotonin level, e.g. at stressful situations [11]. This may beeflected in the dose-dependent effect of 5-HT1A agonist on anxietyeasured in unconditioned anxiety tests: low doses of were anx-
olytic, while high doses were anxiogenic [1,7,19]. Low doses mayesult in different 5-HT release pattern and extracellular level andonsequently different activation of 5-HT receptor subsets whichs manifested in decreased anxiety [17,27].
Based on these results we hypothesize that 5-HT1A stimulationattern plays a role in the development of coping strategy of micelaced in a new environmental context. These findings may haveelevance in treating patients suffering from pathological fears ofovel environments.
cknowledgements
This study was supported by the following grants: OTKA PD3581 from the Hungarian National Scientific Fund and TÁMOP-.2.2.A-11/1/KONV-2012-0052 from the National Developmentgency (NFÜ) grant. The authors wish to thank to Botond Penke
or his support and to Emoke Borbély for her excellent technicalssistance. The authors are also grateful to Kseniya Toropova forruitful discussions.
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.bbr.2013.04.014.
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Research 250 (2013) 32– 38 37
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