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The anxiolytic buspirone shifts coping strategy in novel environmental context of mice with different anxious phenotype
Behavioural Brain Research 250 (2013) 32–38
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The anxiolytic buspirone shifts coping strategy in novel environmental context of mice with different anxious phenotype János Horváth a , Titanilla Szögi a , Géza Müller a , Viktor Szegedi b,∗ a b
Department of Medical Chemistry, University of Szeged, Szeged, Hungary Biological Research Center, Institute of Biochemistry, Szeged, Temesvári krt. 32, Hungary
h 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.
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Article history: Received 18 January 2013 Received in revised form 10 April 2013 Accepted 13 April 2013 Available online 20 April 2013 Keywords: Anxiety Elevated plus maze Context change Inbred mice AX nAX
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 environment 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, for 8 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 a gradual decrease of open-arm exploration, which was not prevented by bus treatment. Another cohort of animals was exposed to EPM for 2 days, and then we changed to blue light illumination and used a different cleaning substance with citrus odor (context change, CC). It was found that upon CC AX mice exhibited increased, while nAX mice showed decreased anxiety. Bus in 2.5 mg/kg changed the coping strategy from passive to active exploration after CC in the AX mice; however, the same treatment rendered nAX mice passive upon CC. Bus in 5.0 mg/kg failed to alter the overall coping style in the novel environment of both strains. These results suggest that these mouse lines use different coping strategy in novel context, which can be changed with bus treatment. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Anxiety disorders, the most common forms of mental illness [16], are often accompanied by increased fear in novel contexts and patients were reported to have difficulties in habituating to a new environment. In preclinical research, elevated plus maze (EPM) is a widely used model to assess anxiety-related behavior of mice (for review see [8,21]). The time spent in the aversive open arm is an inverse measure of fear, because anxious animals
∗ Corresponding author at: Biological Research Center – Biochemistry, Temesvári krt. 32, Szeged H-6726, Hungary. Tel.: +36 70 2418260. E-mail address: [email protected] (V. Szegedi). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.04.014
prefer the safe closed arm. An intriguing feature of EPM is that mice show profound behavioral changes when re-exposed to the plusmaze. Typically, undrugged animals exhibit a significant decrease of open arm exploration upon re-exposure to the EPM, a finding often referred to as one-trial tolerance (OTT) (e.g. [3,6,18,25]). There are conflicting data about the physiological background of OTT: it may represent an increased anxiety response (phobic-like) to the open arms during the first trial [3,5], or alternatively, mice could lose the motivation for exploring the aversive open arms [26]. In support 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 time spent in the open-arms), these possibilities are different in terms of anxiety expression and coping style.
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Inbred rodent models selected for extremes in anxious phenotypes are valuable models of anxiety research. Differences in the trait anxiety level may be accompanied by different coping strategy or resilience to stress. Indeed, rats bred for extremes in anxiety were shown to use active (less anxious) or passive (more anxious) coping strategy in the forced swim [14,15] and hole-board test [20]. Previously, we have developed two mouse strains having either high- or low-anxiety related behavior (AX and nAX, respectively) as was shown in the EPM, open-field, and light/dark test [28,31]. The initial level of anxiety may determine the coping style or the habituation of the animals in new environment, thus those mice were exposed to EPM for eight consecutive days. Furthermore, in order to enhance the motivation for re-exploring the open arms, a change in the environmental context of the EPM apparatus was introduced after 2 days of EPM exposure, and the behavior was followed for 6 days. As the animals have a choice between safe and novel, but aversive compartments, it was expected that mice with low trait anxiety would exhibit a preference for novelty, whereas high trait anxiety subjects would prefer safety. Rather introducing a negative stimulus, like hot air stream in one closed arm [22], we aimed to enhance the exploratory motivation by changing the environmental context. We asked whether changing some external cues would modify the coping strategy of the animals in the EPM. Furthermore, the effect of a clinically effective anxiolytic, buspirone (bus) was tested in two doses on the behavioral profiles. 2. Materials and methods 2.1. Animals Inbred mouse strains having either high- or low anxiety level (AX and nAX) were bred in our animal facility. Male mice of 2.5–3 month were housed individually under a light/dark 12 h cycle (lights on at 08:00) at 24 ± 1 ◦ C and given ad libitum food and water. Mice were handled for 7 days prior to testing, the procedure described by Hurst and West [13]. For the experiments, 45th–48th inbred generations were used of both strains. The study conformed to EU directive 2010/63/EU and was approved by the regional Station for Animal Health and Food Control under Project License XXXI/2012. 2.2. Treatment Mice were injected chronically (daily injection for 4 weeks before and during the tests) with buspirone (Sigma–Aldrich, Budapest) at 2.5 mg/kg and 5.0 mg/kg concentrations. Drug was dissolved in phosphate buffer saline (PBS) pH 7.2 and was administered intraperitoneally (i.p.) with 0.1 ml. The control group was treated with saline of the same volume. 2.3. Behavioral testing The EPM was made of stainless steel (painted matt black) consisting of two opposite open arms (35 cm × 7 cm) and two opposite closed arms surrounded by 15 cm high walls of the same dimensions. The middle section that allows the animal to transit from arm to arm consisted of a square with dimension of 7 cm × 7 cm. The apparatus was elevated 50 cm from the floor and the open arms were equipped with 1.5 cm × 1.5 cm ledges to ensure, that no animals would fall of the maze. The animal house and the testing location were at the same room. Inside the room the maze was separated with black curtains. The behavior of the mice was recorded and analyzed with an EthoVision software package (EthoVision XT 8.5 Noldus Technology, The Netherlands). Animals were exposed to EPM for eight consecutive days. Trials were carried out under low white light condition (open arms 45 lux, closed arms 15 lux). All tests were conducted during the light phase of light/dark period between 13:00 and 17:00 h. The mice were placed individually in the center of the maze facing the open arm and allowed 5 min to explore the apparatus. The maze was cleaned with 20% ethyl alcohol before each trial. The white illumination/ethanol cleaning scenario was defined as context A. In certain experiments, blue-light illumination combined with cleaning the apparatus with a substance having light citrus-fragrance was used, and this was defined as context B. The strength of illuminance did not differ in context B (open arms 40 lux, closed arms 14 lux). 2.4. Statistical analysis The data were compared by Repeated Measures of ANOVA (RM ANOVA) with post hoc Bonferroni, unless otherwise stated. Results were considered to be significantly different at a probability level of p < 0.05. Data are presented as means ± SEM.
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3. Results The first cohort of mice (AX n = 9, nAX n = 10) was exposed to EPM daily for 8 days. The two strains showed clearly distinct phenotype 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). Both strains showed a continuously decreasing open-arm exploration, however this decline was stronger in the nAX mice (p = 0.020 for AX, and p < 0.001 for nAX; paired samples t-test; Fig. 1). Exemplar tracks are shown at Fig. 1 and Supplementary Table 1 contains a more detailed dataset of other behavioral parameters. Next, using another cohort of mice (AX n = 8, nAX n = 10), the effect of introducing a change in the surrounding context on the behavioral profile was investigated. Mice were exposed to the EPM using normal illumination and ethanol to clear the apparatus before each test. At day 3, the environmental context was changed to bluelight illumination and a citrus-fragrance cleaning substance, which we refer to as context B. It was found that the time spent with openarms exploration increased in context B of the nAX, but significantly decreased of the AX mice (RM ANOVA, normal condition vs. CC: AX open 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 suggest that AX mice were more anxious in the novel environment, and avoided the stressful open-arms. NAX mice, however, acted differently, because the time spent in open-arms did not decrease over time upon context B introduction in such a rate, as in the normal condition). It might reflect a proactive behavioral pattern of the nAX 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 were exposed 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 is the novel environment, where AX mice showed increased anxiety (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 increased exploratory 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 it was not the blue-light illumination per se that elicited changes in the behavioral pattern, but instead the change in the surrounding context of the EPM apparatus. 3.1. The effect of buspirone Buspirone, a clinically effective anxiolytic was injected i.p. in two different doses for 4 weeks daily before, and during the experiments, and the behavior of mice were investigated in the EPM. The anxiolytic effect of bus was evident at the first day, when treated 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 (closed arms: 130.3 ± 9.8 s for control AX, 118.4 ± 10.4 s for AX 2.5 mg/kg bus, p = 0.947 vs. control and 90.7 ± 3.9 s for AX 5.0 mg/kg bus treated mice, p = 0.002 vs. control; open arms: 120.4 ± 15.1 s for control 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-way ANOVA post hoc Bonferroni). Interestingly, bus increased anxietylike behavior of nAX mice (n = 10 for 2.5 mg/kg and n = 18 for 5.0 mg/kg; closed arms: 57.2 ± 2.8 s for control nAX, 69.3 ± 6.1 for nAX 2.5 mg/kg bus, p = 0.170 vs. control, 69.9 ± 2.8 for nAX 5.0 mg/kg bus, p = 0.076 vs. control; one-way ANOVA post hoc Bonferroni; 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 nAX 5.0 mg/kg bus mice, p < 0.001 vs. control; one-way ANOVA post hoc Bonferroni Fig. 4. and Supplementary Table 4). Investigating the change of kinetics of open-arms exploration over the 8-day long
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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 indicated as 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 to colour 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 (first day 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. (For interpretation of the references to colour please see to the web version of article.)
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Fig. 3. Bar charts showing the total time spent in the open (Panel A) and closed (Panel B) arms. Blue backgrounds indicate context B (see main text for details) at day 1 and 2. * P ≤ 0.05, RM ANOVA. (For interpretation of the references to colour please see to the web version of article.)
test session using RM ANOVA showed no difference between control vs. 2.5 mg/kg bus group neither in AX or nAX mice (open arms: F(1,16) = 0.653, p = 0.432 for AX mice and F(1,17) = 0.007, p = 0.937 for nAX mice; Fig. 4.). However the time spent in the closed arms for the nAX 2.5 mg/kg bus group was significantly different (RM ANOVA: F(1,17) = 4.447, p = 0.049 vs. control; Fig. 4.). Similarly, bus treatment in 5.0 mg/kg altered the time spent in closed-arms throughout the 8 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), however, showed dramatic change in the novel context. AX mice spent
much more time in the open arm compared to control group, indicating 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 Supplementary Table 5.). In contrast, treated nAX mice spent much less time exploring the open arm than control counterparts, suggesting that anxiety has developed in the novel context due to bus treatment (RM ANOVA: F(1,19) = 5.395, p = 0.032 vs. control). AX mice treated with the higher dose of bus (n = 9) showed continuously decreasing open-arm exploration upon context change, similarly to control mice (Fig. 5.). Although the difference was
Fig. 4. The effect of chronic buspirone administration (2.5 and 5.0 mg/kg) on the behavior of AX (A for open and B for closed arms) and nAX mice (C for open and D for closed arms). Significance is indicated in the main text.
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Fig. 5. The effect of chronic administration (2.5 and 5.0 mg/kg) on the behavioral profile of AX (A for open and B for closed arms) and nAX (C for open and for closed arms) mice upon context change. Blue background indicate novel context. * P ≤ 0.05, RM ANOVA. (For interpretation of the references to colour please see to the web version of article.)
significant (RM ANOVA: F(1,18) = 4.831, p = 0.042), there was no difference between the time spent in the closed-arms of control vs. bus 5.0 mg/kg AX mice (RM ANOVA: F(1,18) = 2.428, p = 0.138), supporting the notion that this dose of bus does not modify dramatically the behavioral pattern of AX mice in a novel environment. Similar results were found for nAX treated with 5.0 mg/kg bus (n = 9; Fig. 5.). Although there was significant difference in the open-arms time between 5.0 mg/kg bus vs. control (RM ANOVA: F(1,18) = 5.755, p = 0.028) the closed-arm time did not differ (RM ANOVA: F(1,18) = 0.871, p = 0.364). This also supports the hypothesis, that 5.0 mg/kg bus does not alter profoundly the coping style of nAX mice upon context change. 4. Discussion The conflicting hypotheses about the reasons of OTT assume that different coping strategy is being used by the animals. There is a strong correlation between trait anxiety and passive coping style with stressful situations, like a novel environmental context. Also, animal studies have found that inadequate coping strategy is strongly correlated with anxious phenotype. However, trait anxiety, by definition, does not change rapidly, thus we hypothesized that the reasons behind the decreasing open arm exploration may be different in the two strains. Thus one cohort of mice was exposed to the EPM for 8 consecutive days and an environmental context change was also introduced in another cohort of mice. It was found that besides showing OTT on the second day, AX mice showed no further decrease of exploratory motivation. In contrast, nAX mice exhibited a continuous decrease of open-arm exploration, which is, as we suggest, not due to increased anxiety, but to habituation. The lack of similar habituation in the AX strain
may be the consequence of elevated anxiety. This is supported by findings, that in anxious rats, the level of plasma corticosteroids does not habituate between trials [4]. These differences imply different coping styles of the two strains, thus we investigated the responses upon environmental context change. The observed effects were not due to the blue illumination per se, because when the context was switched from blue light to normal white illumination, the behavioral responses of AX and nAX were the same, suggesting that the change of familiar environmental to a novel one caused these behavioral patterns. The readout for anxiety in the EPM is based on the conflict between the drive to explore (open arm) and the drive to avoid (closed arm), thus highly anxious mice are expected to prefer the safe, and familiar closed arm in the novel context. Indeed, AX mice responded with a significantly reduced exploratory behavior (passive coping style) to the novel context. On the contrary, the response of nAX mice was manifested in increased open arm exploration (active coping style). These results suggest that strains having different trait anxiety utilize different coping strategy in an exploration/aversive conflict. This proposal is supported by reports showing that the highly anxious DBA2 mouse shows decreased open arm exploration upon retest in changed environment (maze orientation or laboratory environment) [24]. The available data is very limited in the literature about the reactions of non-reactive strains in a second EPM exposure combined with environmental changes. However, we expect that a less anxious strain, such as BALB/c [30] would behave similarly as our nAX mice. The only available data we are aware of shows that wild mice, who exhibit a marked decreased closed-arm activity compared to standard laboratory mice, do not show signs of OTT as is measured by open/closed arm occupancy upon the second EPM exposure [12]. Likewise, nAX
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mice exhibited a reduced OTT at day 2, compared to AX mice. The findings of Thiel et al. [29] also supportive: they report that rats with high- or low-rearing behavior in a novel open-field test show different behavior upon re-exposure to the test apparatus. Highrearing rats (active) respond with more time spent in the center, while low-rearing rats (passive) show no change in this respect. 4.1. The effect of buspirone Buspirone, a 5-HT1A partial agonist is widely used in the clinic as anxiolytic. Similarly to most antidepressants, the onset of action requires several weeks of medication in human patients. Therefore we have used chronic, 4 weeks of bus treatment in the present experiments. It was found that chronic bus treatment in low dose (2.5 mg/kg) did not alter the behavioral profiles of AX in the 8-day EPM session, but the larger dose (5.0 mg/kg) induced a gradually increasing closed-arm stay. Interestingly, mice treated with the lower dose showed increased open-arm activity in the novel context, but here the larger dose did not alter the profile of behavior. On the contrary, bus treatment in any of the doses did not drastically modify open/closed arm profile of nAX in the 8-day EPM. Strikingly, low dose bus treated mice exhibited a shift in the coping strategy in the novel context, namely nAX mice showed a greater preference for the closed arm, while AX mice exhibited increased exploratory drive upon context change. The reason behind the difference between the responses for the two applied doses is not easy to explain. First, it is known that activating the 5-HT1A receptor may induce anxiogenic and anxiolytic responses in laboratory animals. Bouwknecht et al. reported that a 5-HT1A agonist increased measures of anxiety in open-field activity test and the light-dark exploration test of C57BL/6J (B6) mice, a strain regarded to be less anxious. In contrast, the same treatment had minimal effect on the highly anxious 129S6/SvEvTac (S6) strain [2]. Second, 5-HT1A is localized pre- and postsynaptically, having opposite effects on serotonin neurosignalling [9,23]. The intricate mechanism governing serotonin release is further complicated by the fact, that 5-HT autoreceptors, which provide a negative feedback loop inhibiting serotonin release [10], tend to desensitize, but even at high synaptic serotonin concentration, they are capable of responding to large leaps in serotonin level, e.g. at stressful situations [11]. This may be reflected in the dose-dependent effect of 5-HT1A agonist on anxiety measured in unconditioned anxiety tests: low doses of were anxiolytic, while high doses were anxiogenic [1,7,19]. Low doses may result in different 5-HT release pattern and extracellular level and consequently different activation of 5-HT receptor subsets which is manifested in decreased anxiety [17,27]. Based on these results we hypothesize that 5-HT1A stimulation pattern plays a role in the development of coping strategy of mice placed in a new environmental context. These findings may have relevance in treating patients suffering from pathological fears of novel environments. Acknowledgements This study was supported by the following grants: OTKA PD 83581 from the Hungarian National Scientific Fund and TÁMOP4.2.2.A-11/1/KONV-2012-0052 from the National Development Agency (NFÜ) grant. The authors wish to thank to Botond Penke ˝ Borbély for her excellent technical for his support and to Emoke assistance. The authors are also grateful to Kseniya Toropova for fruitful discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2013.04.014.
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Research report
The anxiolytic buspirone shifts coping strategy in novel environmental context of mice with different anxious phenotype János Horváth a , Titanilla Szögi a , Géza Müller a , Viktor Szegedi b,∗ a b
Department of Medical Chemistry, University of Szeged, Szeged, Hungary Biological Research Center, Institute of Biochemistry, Szeged, Temesvári krt. 32, Hungary
h 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
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Article history: Received 18 January 2013 Received in revised form 10 April 2013 Accepted 13 April 2013 Available online 20 April 2013 Keywords: Anxiety Elevated plus maze Context change Inbred mice AX nAX
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 environment 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, for 8 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 a gradual decrease of open-arm exploration, which was not prevented by bus treatment. Another cohort of animals was exposed to EPM for 2 days, and then we changed to blue light illumination and used a different cleaning substance with citrus odor (context change, CC). It was found that upon CC AX mice exhibited increased, while nAX mice showed decreased anxiety. Bus in 2.5 mg/kg changed the coping strategy from passive to active exploration after CC in the AX mice; however, the same treatment rendered nAX mice passive upon CC. Bus in 5.0 mg/kg failed to alter the overall coping style in the novel environment of both strains. These results suggest that these mouse lines use different coping strategy in novel context, which can be changed with bus treatment. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Anxiety disorders, the most common forms of mental illness [16], are often accompanied by increased fear in novel contexts and patients were reported to have difficulties in habituating to a new environment. In preclinical research, elevated plus maze (EPM) is a widely used model to assess anxiety-related behavior of mice (for review see [8,21]). The time spent in the aversive open arm is an inverse measure of fear, because anxious animals
∗ Corresponding author at: Biological Research Center – Biochemistry, Temesvári krt. 32, Szeged H-6726, Hungary. Tel.: +36 70 2418260. E-mail address: [email protected] (V. Szegedi). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.04.014
prefer the safe closed arm. An intriguing feature of EPM is that mice show profound behavioral changes when re-exposed to the plusmaze. Typically, undrugged animals exhibit a significant decrease of open arm exploration upon re-exposure to the EPM, a finding often referred to as one-trial tolerance (OTT) (e.g. [3,6,18,25]). There are conflicting data about the physiological background of OTT: it may represent an increased anxiety response (phobic-like) to the open arms during the first trial [3,5], or alternatively, mice could lose the motivation for exploring the aversive open arms [26]. In support 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 time spent in the open-arms), these possibilities are different in terms of anxiety expression and coping style.
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Inbred rodent models selected for extremes in anxious phenotypes are valuable models of anxiety research. Differences in the trait anxiety level may be accompanied by different coping strategy or resilience to stress. Indeed, rats bred for extremes in anxiety were shown to use active (less anxious) or passive (more anxious) coping strategy in the forced swim [14,15] and hole-board test [20]. Previously, we have developed two mouse strains having either high- or low-anxiety related behavior (AX and nAX, respectively) as was shown in the EPM, open-field, and light/dark test [28,31]. The initial level of anxiety may determine the coping style or the habituation of the animals in new environment, thus those mice were exposed to EPM for eight consecutive days. Furthermore, in order to enhance the motivation for re-exploring the open arms, a change in the environmental context of the EPM apparatus was introduced after 2 days of EPM exposure, and the behavior was followed for 6 days. As the animals have a choice between safe and novel, but aversive compartments, it was expected that mice with low trait anxiety would exhibit a preference for novelty, whereas high trait anxiety subjects would prefer safety. Rather introducing a negative stimulus, like hot air stream in one closed arm [22], we aimed to enhance the exploratory motivation by changing the environmental context. We asked whether changing some external cues would modify the coping strategy of the animals in the EPM. Furthermore, the effect of a clinically effective anxiolytic, buspirone (bus) was tested in two doses on the behavioral profiles. 2. Materials and methods 2.1. Animals Inbred mouse strains having either high- or low anxiety level (AX and nAX) were bred in our animal facility. Male mice of 2.5–3 month were housed individually under a light/dark 12 h cycle (lights on at 08:00) at 24 ± 1 ◦ C and given ad libitum food and water. Mice were handled for 7 days prior to testing, the procedure described by Hurst and West [13]. For the experiments, 45th–48th inbred generations were used of both strains. The study conformed to EU directive 2010/63/EU and was approved by the regional Station for Animal Health and Food Control under Project License XXXI/2012. 2.2. Treatment Mice were injected chronically (daily injection for 4 weeks before and during the tests) with buspirone (Sigma–Aldrich, Budapest) at 2.5 mg/kg and 5.0 mg/kg concentrations. Drug was dissolved in phosphate buffer saline (PBS) pH 7.2 and was administered intraperitoneally (i.p.) with 0.1 ml. The control group was treated with saline of the same volume. 2.3. Behavioral testing The EPM was made of stainless steel (painted matt black) consisting of two opposite open arms (35 cm × 7 cm) and two opposite closed arms surrounded by 15 cm high walls of the same dimensions. The middle section that allows the animal to transit from arm to arm consisted of a square with dimension of 7 cm × 7 cm. The apparatus was elevated 50 cm from the floor and the open arms were equipped with 1.5 cm × 1.5 cm ledges to ensure, that no animals would fall of the maze. The animal house and the testing location were at the same room. Inside the room the maze was separated with black curtains. The behavior of the mice was recorded and analyzed with an EthoVision software package (EthoVision XT 8.5 Noldus Technology, The Netherlands). Animals were exposed to EPM for eight consecutive days. Trials were carried out under low white light condition (open arms 45 lux, closed arms 15 lux). All tests were conducted during the light phase of light/dark period between 13:00 and 17:00 h. The mice were placed individually in the center of the maze facing the open arm and allowed 5 min to explore the apparatus. The maze was cleaned with 20% ethyl alcohol before each trial. The white illumination/ethanol cleaning scenario was defined as context A. In certain experiments, blue-light illumination combined with cleaning the apparatus with a substance having light citrus-fragrance was used, and this was defined as context B. The strength of illuminance did not differ in context B (open arms 40 lux, closed arms 14 lux). 2.4. Statistical analysis The data were compared by Repeated Measures of ANOVA (RM ANOVA) with post hoc Bonferroni, unless otherwise stated. Results were considered to be significantly different at a probability level of p < 0.05. Data are presented as means ± SEM.
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3. Results The first cohort of mice (AX n = 9, nAX n = 10) was exposed to EPM daily for 8 days. The two strains showed clearly distinct phenotype 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). Both strains showed a continuously decreasing open-arm exploration, however this decline was stronger in the nAX mice (p = 0.020 for AX, and p < 0.001 for nAX; paired samples t-test; Fig. 1). Exemplar tracks are shown at Fig. 1 and Supplementary Table 1 contains a more detailed dataset of other behavioral parameters. Next, using another cohort of mice (AX n = 8, nAX n = 10), the effect of introducing a change in the surrounding context on the behavioral profile was investigated. Mice were exposed to the EPM using normal illumination and ethanol to clear the apparatus before each test. At day 3, the environmental context was changed to bluelight illumination and a citrus-fragrance cleaning substance, which we refer to as context B. It was found that the time spent with openarms exploration increased in context B of the nAX, but significantly decreased of the AX mice (RM ANOVA, normal condition vs. CC: AX open 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 suggest that AX mice were more anxious in the novel environment, and avoided the stressful open-arms. NAX mice, however, acted differently, because the time spent in open-arms did not decrease over time upon context B introduction in such a rate, as in the normal condition). It might reflect a proactive behavioral pattern of the nAX 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 were exposed 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 is the novel environment, where AX mice showed increased anxiety (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 increased exploratory 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 it was not the blue-light illumination per se that elicited changes in the behavioral pattern, but instead the change in the surrounding context of the EPM apparatus. 3.1. The effect of buspirone Buspirone, a clinically effective anxiolytic was injected i.p. in two different doses for 4 weeks daily before, and during the experiments, and the behavior of mice were investigated in the EPM. The anxiolytic effect of bus was evident at the first day, when treated 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 (closed arms: 130.3 ± 9.8 s for control AX, 118.4 ± 10.4 s for AX 2.5 mg/kg bus, p = 0.947 vs. control and 90.7 ± 3.9 s for AX 5.0 mg/kg bus treated mice, p = 0.002 vs. control; open arms: 120.4 ± 15.1 s for control 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-way ANOVA post hoc Bonferroni). Interestingly, bus increased anxietylike behavior of nAX mice (n = 10 for 2.5 mg/kg and n = 18 for 5.0 mg/kg; closed arms: 57.2 ± 2.8 s for control nAX, 69.3 ± 6.1 for nAX 2.5 mg/kg bus, p = 0.170 vs. control, 69.9 ± 2.8 for nAX 5.0 mg/kg bus, p = 0.076 vs. control; one-way ANOVA post hoc Bonferroni; 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 nAX 5.0 mg/kg bus mice, p < 0.001 vs. control; one-way ANOVA post hoc Bonferroni Fig. 4. and Supplementary Table 4). Investigating the change of kinetics of open-arms exploration over the 8-day long
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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 indicated as 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 to colour 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 (first day 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. (For interpretation of the references to colour please see to the web version of article.)
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Fig. 3. Bar charts showing the total time spent in the open (Panel A) and closed (Panel B) arms. Blue backgrounds indicate context B (see main text for details) at day 1 and 2. * P ≤ 0.05, RM ANOVA. (For interpretation of the references to colour please see to the web version of article.)
test session using RM ANOVA showed no difference between control vs. 2.5 mg/kg bus group neither in AX or nAX mice (open arms: F(1,16) = 0.653, p = 0.432 for AX mice and F(1,17) = 0.007, p = 0.937 for nAX mice; Fig. 4.). However the time spent in the closed arms for the nAX 2.5 mg/kg bus group was significantly different (RM ANOVA: F(1,17) = 4.447, p = 0.049 vs. control; Fig. 4.). Similarly, bus treatment in 5.0 mg/kg altered the time spent in closed-arms throughout the 8 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), however, showed dramatic change in the novel context. AX mice spent
much more time in the open arm compared to control group, indicating 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 Supplementary Table 5.). In contrast, treated nAX mice spent much less time exploring the open arm than control counterparts, suggesting that anxiety has developed in the novel context due to bus treatment (RM ANOVA: F(1,19) = 5.395, p = 0.032 vs. control). AX mice treated with the higher dose of bus (n = 9) showed continuously decreasing open-arm exploration upon context change, similarly to control mice (Fig. 5.). Although the difference was
Fig. 4. The effect of chronic buspirone administration (2.5 and 5.0 mg/kg) on the behavior of AX (A for open and B for closed arms) and nAX mice (C for open and D for closed arms). Significance is indicated in the main text.
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Fig. 5. The effect of chronic administration (2.5 and 5.0 mg/kg) on the behavioral profile of AX (A for open and B for closed arms) and nAX (C for open and for closed arms) mice upon context change. Blue background indicate novel context. * P ≤ 0.05, RM ANOVA. (For interpretation of the references to colour please see to the web version of article.)
significant (RM ANOVA: F(1,18) = 4.831, p = 0.042), there was no difference between the time spent in the closed-arms of control vs. bus 5.0 mg/kg AX mice (RM ANOVA: F(1,18) = 2.428, p = 0.138), supporting the notion that this dose of bus does not modify dramatically the behavioral pattern of AX mice in a novel environment. Similar results were found for nAX treated with 5.0 mg/kg bus (n = 9; Fig. 5.). Although there was significant difference in the open-arms time between 5.0 mg/kg bus vs. control (RM ANOVA: F(1,18) = 5.755, p = 0.028) the closed-arm time did not differ (RM ANOVA: F(1,18) = 0.871, p = 0.364). This also supports the hypothesis, that 5.0 mg/kg bus does not alter profoundly the coping style of nAX mice upon context change. 4. Discussion The conflicting hypotheses about the reasons of OTT assume that different coping strategy is being used by the animals. There is a strong correlation between trait anxiety and passive coping style with stressful situations, like a novel environmental context. Also, animal studies have found that inadequate coping strategy is strongly correlated with anxious phenotype. However, trait anxiety, by definition, does not change rapidly, thus we hypothesized that the reasons behind the decreasing open arm exploration may be different in the two strains. Thus one cohort of mice was exposed to the EPM for 8 consecutive days and an environmental context change was also introduced in another cohort of mice. It was found that besides showing OTT on the second day, AX mice showed no further decrease of exploratory motivation. In contrast, nAX mice exhibited a continuous decrease of open-arm exploration, which is, as we suggest, not due to increased anxiety, but to habituation. The lack of similar habituation in the AX strain
may be the consequence of elevated anxiety. This is supported by findings, that in anxious rats, the level of plasma corticosteroids does not habituate between trials [4]. These differences imply different coping styles of the two strains, thus we investigated the responses upon environmental context change. The observed effects were not due to the blue illumination per se, because when the context was switched from blue light to normal white illumination, the behavioral responses of AX and nAX were the same, suggesting that the change of familiar environmental to a novel one caused these behavioral patterns. The readout for anxiety in the EPM is based on the conflict between the drive to explore (open arm) and the drive to avoid (closed arm), thus highly anxious mice are expected to prefer the safe, and familiar closed arm in the novel context. Indeed, AX mice responded with a significantly reduced exploratory behavior (passive coping style) to the novel context. On the contrary, the response of nAX mice was manifested in increased open arm exploration (active coping style). These results suggest that strains having different trait anxiety utilize different coping strategy in an exploration/aversive conflict. This proposal is supported by reports showing that the highly anxious DBA2 mouse shows decreased open arm exploration upon retest in changed environment (maze orientation or laboratory environment) [24]. The available data is very limited in the literature about the reactions of non-reactive strains in a second EPM exposure combined with environmental changes. However, we expect that a less anxious strain, such as BALB/c [30] would behave similarly as our nAX mice. The only available data we are aware of shows that wild mice, who exhibit a marked decreased closed-arm activity compared to standard laboratory mice, do not show signs of OTT as is measured by open/closed arm occupancy upon the second EPM exposure [12]. Likewise, nAX
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mice exhibited a reduced OTT at day 2, compared to AX mice. The findings of Thiel et al. [29] also supportive: they report that rats with high- or low-rearing behavior in a novel open-field test show different behavior upon re-exposure to the test apparatus. Highrearing rats (active) respond with more time spent in the center, while low-rearing rats (passive) show no change in this respect. 4.1. The effect of buspirone Buspirone, a 5-HT1A partial agonist is widely used in the clinic as anxiolytic. Similarly to most antidepressants, the onset of action requires several weeks of medication in human patients. Therefore we have used chronic, 4 weeks of bus treatment in the present experiments. It was found that chronic bus treatment in low dose (2.5 mg/kg) did not alter the behavioral profiles of AX in the 8-day EPM session, but the larger dose (5.0 mg/kg) induced a gradually increasing closed-arm stay. Interestingly, mice treated with the lower dose showed increased open-arm activity in the novel context, but here the larger dose did not alter the profile of behavior. On the contrary, bus treatment in any of the doses did not drastically modify open/closed arm profile of nAX in the 8-day EPM. Strikingly, low dose bus treated mice exhibited a shift in the coping strategy in the novel context, namely nAX mice showed a greater preference for the closed arm, while AX mice exhibited increased exploratory drive upon context change. The reason behind the difference between the responses for the two applied doses is not easy to explain. First, it is known that activating the 5-HT1A receptor may induce anxiogenic and anxiolytic responses in laboratory animals. Bouwknecht et al. reported that a 5-HT1A agonist increased measures of anxiety in open-field activity test and the light-dark exploration test of C57BL/6J (B6) mice, a strain regarded to be less anxious. In contrast, the same treatment had minimal effect on the highly anxious 129S6/SvEvTac (S6) strain [2]. Second, 5-HT1A is localized pre- and postsynaptically, having opposite effects on serotonin neurosignalling [9,23]. The intricate mechanism governing serotonin release is further complicated by the fact, that 5-HT autoreceptors, which provide a negative feedback loop inhibiting serotonin release [10], tend to desensitize, but even at high synaptic serotonin concentration, they are capable of responding to large leaps in serotonin level, e.g. at stressful situations [11]. This may be reflected in the dose-dependent effect of 5-HT1A agonist on anxiety measured in unconditioned anxiety tests: low doses of were anxiolytic, while high doses were anxiogenic [1,7,19]. Low doses may result in different 5-HT release pattern and extracellular level and consequently different activation of 5-HT receptor subsets which is manifested in decreased anxiety [17,27]. Based on these results we hypothesize that 5-HT1A stimulation pattern plays a role in the development of coping strategy of mice placed in a new environmental context. These findings may have relevance in treating patients suffering from pathological fears of novel environments. Acknowledgements This study was supported by the following grants: OTKA PD 83581 from the Hungarian National Scientific Fund and TÁMOP4.2.2.A-11/1/KONV-2012-0052 from the National Development Agency (NFÜ) grant. The authors wish to thank to Botond Penke ˝ Borbély for her excellent technical for his support and to Emoke assistance. The authors are also grateful to Kseniya Toropova for fruitful discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2013.04.014.
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