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Bupropion induces social anxiety in adolescent mice: Influence of housing conditions
Pharmacological Reports 69 (2017) 806–812
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Original article
Bupropion induces social anxiety in adolescent mice: Influence of housing conditions Carmen Gómez, Rosa Redolat, Carmen Carrasco* Departamento de Psicobiología, Facultad de Psicología, Universitat de València, Valencia, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 December 2016 Accepted 15 March 2017 Available online 22 March 2017
Background: The antidepressant bupropion has received increasing attention as a pharmacological tool to treat addiction although little is known about its effects on social behaviour in adolescents. The present study aimed to evaluate if environmental housing conditions influence bupropion’s actions on social behaviour of adolescent mice. Methods: Mice were either group- or individually housed for 2-weeks and then randomly divided into 2 cohorts: half of the mice remained in the initial housing condition and the other half were changed to isolated conditions for further 2-weeks. The following groups were compared: isolated/isolated (ISO/ ISO), isolated/group-housed (ISO/GR), group-housed/isolated (GR/ISO), and group-housed/group-housed (GR/GR). The effects of bupropion (40, 20, 10 mg/kg) or saline on social interaction were assessed for each housing condition. Social encounters were evaluated using ethological analysis. Results: Data showed significant effects of bupropion on grooming and digging. This drug diminished time mice allocated to these behavioural categories in all housing conditions. In ISO/GR and GR/ISO conditions, bupropion increased environmental exploration (non-social exploration and exploration from a distance), reduced social investigation and increased avoidance/flee and defence/submission behaviours. An augment of avoidance/flee during social interactions was observed in bupropion-treated mice in GR/GR housing condition. Conclusion: These results suggest that this drug exhibits anxiogenic-like properties in social encounters between adolescent mice, especially when a transition in housing conditions has been experienced during this period. Changes in housing conditions may be a useful model for evaluating the effects of bupropion on social behaviour and the role of environmental housing conditions. © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Keywords: Adolescents Bupropion Housing Mice Social behaviour
Introduction It is known that a lack of appropriate stimulation during development can have important consequences on neural maturation. Increasing experimental evidence suggests that environmental conditions have a clear influence on emotionality [1]. Adolescence is a time of brain maturation during which there are critical neurobiological changes in the regulation of social behaviour [2]. This period is sensitive to affective-related disorders
Abbreviations: BUP, Bupropion; BUP-10, 10 mg/kg of bupropion; BUP-20, 20 mg/ kg of bupropion; BUP-40, 40 mg/kg of bupropion; GR/GR, group-housed/grouphoused; GR/ISO, group-housed/isolated-housed; ip, : intraperitoneal injection; ISO/ ISO, isolated-housed/isolated-housed; ISO/GR, isolated-housed/group-housed; PND, post-natal day; SAL, physiological saline. * Corresponding author. E-mail addresses: [email protected] (C. Gómez), [email protected] (R. Redolat), [email protected] (C. Carrasco).
and the risk of problems of maladaptation augments, including alterations in social behaviour, stress control and drug addiction [3,4]. Isolation rearing is a paradigm commonly applied for studying the influence of environmental conditions on social behaviour in rodents [5]. Deprivation of social contacts in mice induces changes in neuroplasticity although its effects depend on the timing of housing conditions [6]. The age at which the social isolation is initiated seems to be a critical factor and early isolation elicits neurobiological and behavioural changes that continue into adulthood [7]. Social isolation during adolescence has been proposed as an animal model for identifying vulnerability to anxiety disorders [8] and for researching developmental psychopathology [9]. One pharmacological treatment that has received increasing attention in the area of addiction is the antidepressant bupropion, a monoamine (dopamine and norepinephrine) reuptake inhibitor transporter [10,11] and non-competitive antagonist of nicotinic acetylcholine receptors [12]. Bupropion is used for the
http://dx.doi.org/10.1016/j.pharep.2017.03.010 1734-1140/© 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
C. Gómez et al. / Pharmacological Reports 69 (2017) 806–812
treatment of dependence on tobacco and of different drugs of abuse [13–15]. Earlier evidence indicates that acute administration of bupropion antagonizes muricidal behaviour and augments latency to mouse-killing in rats [16]. In mice, chronic treatment with this drug did not to induce effects in the clonidine-induced aggression model [17]. Bupropion increases attack during social interactions in mice with lower aggression levels [18] although it didn’t have significant influence on aggression in group-housed adults [19] or on the time devoted to social investigation [18], but following a 2weeks of social isolation adolescents were more sensitive to bupropion than individually-housed adults [20]. Previous findings support investigating bupropion’s effects on social behaviour and the modulatory influence of housing conditions in adolescent rodents since no prior study has evaluated the effects of this drug when housing conditions change. Indeed, little it is known concerning the consequences of early exposure to bupropion on social behaviour and no studies have evaluated the influence of the re-socialization of adolescents on the effects of bupropion. The main aim in this work was to evaluate if environmental housing conditions influence bupropion’s effects on social behaviour of adolescent mice. We also intend to investigate the impact of transition between different housing conditions since this factor may be relevant for future investigations including the isolation of experimental animals. The evaluation of this animal model during adolescence may be particularly useful since it is a developmental period characterized by vulnerability to psychiatric disorders and drug addiction and, consequently, of the initiation to pharmacological treatments. Materials and methods
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isolated-housed/isolated-housed (ISO/ISO): mice were individually-housed for 4-weeks (PND 24-52); isolated-housed/grouphoused (ISO/GR): mice individually-housed for 2-weeks (PND 24-37) and then group-housed for 2-weeks (PND 38-52). At the end of the housing periods, mice received acute treatment with BUP40, BUP20, BUP10 and SAL, and the 16 groups were evaluated in the social interaction test (Fig. 1). Social interaction test Social encounters took place in a neutral cage (60 33 30 cm) in which mice were confronted during 10-min with a “standard opponent” after 1-min of adaptation to the apparatus. Responses were video-recorded during the social interaction test: grooming, digging, non-social exploration, exploration from a distance, social investigation, threat, attack, avoidance/flee, defence/submission and immobility (more detailed explanation of categories in [23,24]). After each encounter, the apparatus was cleaned (ethanol and water) and the sawdust bedding was changed. The responses during social encounters were examined by a trained observer blind to the treatment using a custom-developed “Raton-time” software [25,26], a program that allows ethological analysis of behavioural categories [18,20,23]. Statistical analyses The time allocated to each behavioural category is indicated as a median with ranges. Due to great variability in the expression of each behavioural category, data were not normally distributed and only drug effects were analyzed and housing conditions were not compared directly. Data were analyzed using Kruskal-Wallis test and two-tailed Mann-Whitney U-test at a significance of p < 0.05.
Subjects Results OF1 male mice (Charles-River, Spain) aged 24 days (post-natal day, PND-24) on arrival at the laboratory were used. Animals were randomly assigned to isolated or group-housed conditions: i.e. 72 subjects were individually-housed in cages (24 13.5 13 cm) and 78 group-housed in 5 per cage (25 25 15 cm). After 2-weeks, half the subjects were maintained in the same housing condition (isolated-housed vs. group-housed) and the remainder were challenged by the other housing condition (group-housed vs. isolated-housed), in both cases for a period of 2-weeks before test. 200 grouped mice were rendered anosmic (intranasal lavage with 4% zinc-sulphate solution) 1-day before test and used as “standard opponents”.
Analysis revealed a main effect of Treatment in the following categories: grooming [x2 = 65.674 df = 15 p < 0.001], digging [x2 = 60.902 df = 15 p < 0.001], non-social exploration [x2 = 62.471 df = 15 p < 0.001], exploration from a distance [x2 = 35.425 df = 15 p < 0.002], social investigation [x2 = 38.057 df = 15 p < 0.001], threat [x2 = 67.320 df = 15 p < 0.001], attack [x2 = 53.886 df = 15 p < 0.001], avoidance/flee [x2 = 45.630 df = 15 p < 0.001], defence/submission [x2 = 45.564 df = 15 p < 0.001].
Drugs Bupropion hydrochloride (Sigma–Aldrich, Spain) was dissolved in physiological saline (SAL). Mice received 30-min before testing 40, 20, 10 mg/kg (BUP-40, BUP-20, BUP-10, respectively) or SAL by intraperitoneal injection (ip) in a volume of 10 ml/kg. Doses employed are habitually used to assess the effects of burpopion in rodents [18,21,22]. Procedure Mice (PND-24) were distributed into two groups on arrival at the laboratory: half the mice were housed during 2-weeks in groups of five and the rest were housed individually. In a second phase initiated on PND-38, subjects were allocated to different groups. Group-housed/group-housed (GR/GR): mice remained group-housed for 4-weeks (PND 24-52); group-housed/isolatedhoused (GR/ISO): mice remained group-housed for 2-weeks (PND 24-37) and then individually-housed for 2-weeks (PND 38-52);
Fig. 1. Experimental design of the experimental procedure. At the end of the housing periods (PND 52) mice received acute treatment with BUP-40, BUP-20, BUP-10 and SAL and were assessed in a social interaction test.
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Consequently, two separate Kruskal–Wallis analyses were performed to evaluate the main effects of housing conditions and of pharmacological treatment. In order to present results more clearly, we will describe the main effects observed in each of the groups maintained in each of the housing conditions provided and the main effects of drug. Analysis of housing conditions 1) ISO/ISO housing (n = 39, 11-9 mice/group). The main factor Treatment had significant effects on grooming [x2 = 15.454 df = 3 p < 0.001]. U-tests confirmed that animals receiving BUP-40 and BUP-20 devoted less time to this category (p < 0.01 and p < 0.001 respectively) (see Fig. 2). Bupropion influenced time allocated to digging [x2 = 19.695 df = 3 p < 0.001]: BUP-40 [Mdn = 3.4 (0.3–5.8)] and BUP-20 [Mdn = 6 (1.6–12.9)] groups spent less time engaged in this behaviour than SAL groups [Mdn = 22.2 (9.3–36.8) p < 0.01]. 2) ISO/GR housing (n = 33, 8–9 mice/group). There were significant differences in time allocated to grooming [x2 = 11.146 df = 3 p < 0.011] and digging [x2 = 10.729 df = 3 p < 0.013]: BUP-40 and BUP-20 diminished grooming (p < 0.003 and p < 0.03 respectively) (see Fig. 2) and digging [BUP-40 Mdn = 3.55 (1.3–7.3) and BUP-20 Mdn = 3.9 (0.2–9.3) p < 0.01 vs. SAL Mdn = 19.9 (2.7–115.1), p < 0.007 and p < 0.01 respectively]. A main effect of Treatment on non-social exploration [x2 = 17.837 df = 3 p < 0.001] and exploration from a distance [x2 = 8.576 df = 3 p < 0.035] was also found. Mice treated with BUP-40 spent more time engaged in non-social exploration [BUP-40 Mdn = 559.8 (517.5–571.2) vs. SAL Mdn = 428.7 (317.1–544.1) p < 0.001] and exploration from a distance [BUP-40
Mdn = 14.75 (12.3–24) vs. SAL Mdn = 9.3 (4.3–25.1) p < 0.02]. Bupropion reduced time allocated to social investigation [x2 = 12.755 df = 3 p < 0.005], reaching statistical significance at the dose BUP-40 (p < 0.02) (see Fig. 3). There were also differences in avoidance/flee [x2 = 11.380 df = 3 p < 0.01] and defence/submission categories [x2 = 15.885 df = 3 p < 0.001] (see Table 1). Bupropiontreatment increased the amount of time allocated to avoidance/flee [BUP-40 Mdn = 0.8 (0–4.6) vs. SAL Mdn = 0 (0–0) p < 0.002] and defence/submission [BUP-40 Mdn = 2.6 (0–6.8) and BUP-20 Mdn = 1.75 (0–100.6) vs. SAL Mdn = 0 (0–0) p < 0.002]. 3) GR/GR housing (n = 39, 11-9 mice/group). The factor Treatment influenced time allocated to grooming [x2 = 20.907 df = 3 p < 0.001]. Post-hoc tests confirmed that bupropion reduced time allocated to this behaviour (BUP-40 p < 0.001; BUP-20 p < 0.003; BUP-10 p < 0.01) (see Fig. 2). Analysis detected a significant effect on time mice spent digging [x2 = 16.100 df = 3 p < 0.001] showing that bupropion-treated mice [BUP-40 Mdn = 3 (1–9) and BUP-20 Mdn = 5.2 (1.1–24.5)] devoted less time to this category than SAL [Mdn = 16.1 (2–120.8) p < 0.005]. Bupropion’s effects on avoidance/flee were significant [x2 = 7.795 df = 3 p < 0.05]: BUP-40 group spent more time engaged in avoidance/ flee behaviours than SAL [BUP-40 Mdn = 0.0 (0–18) vs. SAL Mdn = 0.0 (0–0) p < 0.04]. No effects of bupropion on any other category were detected. 4) GR/ISO housing (n = 39, 10-9 mice/group). Bupropion influenced grooming [x2 = 15.580 df = 3 p < 0.001] and digging [x2 = 11.172 df = 3 p < 0.011]. BUP-40 and BUP-20 reduced time spent grooming (p < 0.001 and p < 0.005 respectively) (see Fig. 2) and digging [BUP-40 Mdn = 4.1 (0.4–12.6) and BUP-20 Mdn = 3.1
Fig. 2. Mean time (SEM) allocated by groups to the grooming in housing conditions: isolated/isolated (ISO/ISO), isolated/group-housed (ISO/GR), group-housed/isolated (GR/ISO), and group-housed/group-housed (GR/GR). Mice were injected ip with SAL or BUP-40, BUP-20 and BUP-10. Thirty minutes later mice performed social encounters for 10-min. U-tests +p < 0.005 vs. SAL; ***p < 0.001 vs. SAL; **p < 0.01 vs. SAL; *p < 0.05 vs. SAL.
C. Gómez et al. / Pharmacological Reports 69 (2017) 806–812
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Fig. 3. Mean time ( SEM) allocated by groups to the social investigation in housing conditions: isolated/isolated (ISO/ISO), isolated/group-housed (ISO/GR), group-housed/ isolated (GR/ISO), and group-housed/group-housed (GR/GR). Mice were injected ip with SAL or BUP-40, BUP-20 and BUP-10. Thirty minutes later mice performed social encounters for 10-min. U-tests * p < 0.05 vs. SAL. Table 1 Effects of acute treatment with bupropion 10 mg/kg (BUP-10), 20 mg/kg (BUP-20), 40 mg/kg (BUP-40) or saline (SAL) on median times (with ranges) with seconds allocated to broad behavioural categories by adolescent mice in ISO/GR housing condition.
a
Grooming Diggingb
Non social explorationc Explore from a distanced Exploración sociale Threat Attack Avoidance/fleef Defense/submissiong Immobility
SAL
BUP-10
BUP-20
BUP-40
14.4 (0.7–43.2) 19.9 (2.7–115.1) 428.7 (317.1–544.1) 9.3 (4.3–25.1) 84.9 (0–194) 0 (0–36.9) 0 (0–27.6) 0 (0–0) 0 (0–0) 0 (0–30.4)
7.4 (0.4–32.3) 14.5 (0.5–36.5) 434.2 (238.4–518.9) 7.85 (4.7–28.8) 117 (55.4–140.2) 0 (0–0) 0 (0–1.6) 0 (0–0.4) 0 (0–1.4) 0 (0–228.7)
1.95* (0 34.9) 3.9** (0.2–9.3) 479.5 (399–537.1) 5.5 (3.1–22.8) 68.3 (2.9–122.4) 0 (0–12.9) 0 (0–14) 0 (0–69.2) 1.75** (0–100.6) 0 (0–20.1)
1.2+ (0.3–2.06) 3.5** (1.3–7.3) 559.8*** (517.5–571.2) 14.75* (12.3–24) 15.2* (5.7–54.8) 0 (0–0.4) 0 (0–0.3) 0.8+ (0–4.6) 2.6+ (0–6.8) 0 (0–2.8)
Kruskal-Wallis test and Mann Whitney U-test showed: a Significant variance p < 0.01: +vs. SAL p < 0.005, *vs. SAL p < 0.05. bSignificant variance p < 0.01: ** vs. SAL p < 0.01. c Significant variance p < 0.001: *** vs. SAL p < 0.001. dSignificant variance p < 0.05: * vs. SAL p < 0.05. eSignificant variance p < 0.005: * vs. SAL p < 0.05. fSignificant variance p < 0.01:+ vs. SAL p < 0.005. gSignificant variance p < 0.001:+ vs. SAL p < 0.005
(0–13.2) p < 0.05; SAL Mdn = 16.15 (0–120) p < 0.003 and p < 0.05 respectively]. An effect of Treatment on non-social exploration [x2 = 15.381 df = 3 p < 0.002] and exploration from a distance [x2 = 8.447, df = 3 p < 0.038] was detected. Post-hoc tests indicated
that more time was spent exploring the environment by bupropion-treated mice. BUP-40 increased non-social exploration [BUP-40 Mdn = 476.4 (231.4–555) vs. SAL Mdn = 386 (239.6–500) p < 0.001] and exploration from a distance [BUP-40 Mdn = 20.8
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(8.1–42.6) vs. SAL Mdn = 16.1 (2.6–37.6) p < 0.014] (see Table 2). In contrast, bupropion reduced time allocated to social investigation [x2 = 9.460 df = 3 p < 0.024] reaching statistical significance at the dose of BUP-40 (p < 0.019) (see Fig. 3).
(p < 0.01), and increasing threat and attack in GR/GR vs. ISO/GR (p < 0.05) and in GR/ISO vs. GR/GR (p < 0.01).
Analysis of pharmacological treatment
This study aimed to assess if housing conditions modify the effects of bupropion on social behaviour in adolescent male mice. Results suggest that this drug induces behavioural changes modulated by social housing conditions. In ISO/GR and GR/ISO, bupropion increased environmental exploration, avoidance/flee and defence/submission behaviours whereas it reduced social investigation. In GR/GR, an augment of avoidance/flee during social interactions was observed in bupropion-treated mice. In all housing conditions, bupropion decreased time allocated to grooming and digging. One interesting finding is that bupropion disrupted social responses displayed by mice when social housing conditions were changed, affecting defensive and social behaviours. In mice reared in the ISO/GR, BUP-40 and BUP-20 elicited defensive behaviours such as avoidance/flee and defence/submission towards the opponent. The highest dose increased time devoted to avoidance/flee by animals kept in the GR/GR condition without increasing defence/submission. This effect could be associated to its anxiogenic actions. An increase of the defensive response during social encounters has been interpreted as a coping strategy that reflects an anxiogenic response [25] and as a consistent predictor of the anxiogenic effects of drugs [18,26,27]. Our study suggests that bupropion elicits an emotional coping strategy in mice maintained in group-housed conditions. Preclinical research shows that aggressive and non-aggressive responses may be related to diverse behavioural and physiological processes of stress adaptation [28], whereas sociability during social encounters is considered a sign of diminished anxiety [29]. In our study, bupropion reduced social exploration in ISO/GR and GR/ISO, as said groups displayed a low level of pro-social behaviour. The highest dose clearly affected social investigation augmenting the time spent exploring the environment. The less time allocated to social exploration exhibited by bupropiontreated mice could reflect anxiogenic actions. This increased
There were differences in BUP-40 innon-social exploration [x2 = 16.967 df = 3 p < 0.001], threat [x2 = 16.114 df = 3 p < 0.001], attack [x2 = 14.363 df = 3 p < 0.002] and avoidance/flee [x2 = 9.379 df = 3 p < 0.025]. We observed more time devoted to these behaviours in ISO/ISO vs. ISO/GR and GR/ISO (p < 0.005 and p < 0.05 respectively). This dose augmented non-social exploration in GR/GR vs. ISO/GR and GR/ISO (p < 0.001 and p < 0.05 respectively), threat in GR/GR vs. ISO/GR (p < 0.05) and avoidance/flee in GR/GR vs. GR/ISO (p < 0.05), whereas it reduced threat in GR/GR vs. GR/ISO (p < 0.05). Analyses of BUP-20 revealed differences in nonsocial exploration [x2 = 12.335 df = 3 p < 0.006], exploration from a distance [x2 = 8.576 df = 3 p < 0.036], threat [x2 = 17.312 df = 3 p < 0.001], attack [x2 = 12.590 df = 3 p < 0.006] and defence/ submission [x2 = 14.214 df = 3 p < 0.003]. This dose increased exploration from a distance in ISO/ISO vs. ISO/GR (p < 0.01) and threat in ISO/ISO vs. GR/ISO (p < 0.01), reducing non-social exploration in ISO/ISO vs. GR/GR (p < 0.01) and attack in ISO/ISO vs. GR/ISO (p < 0.05). An augment of non-social exploration and a decrease of defence/submission in GR/GR vs. GR/ISO were observed (p < 0.005 and p < 0.05 respectively). Mice increased threat and attack in GR/GR vs. ISO/GR (p < 0.01). In GR/ISO, mice were more aggressive than GR/GR (p < 0.01 and p < 0.05 respectively). Analyses of BUP-10 showed differences in threat [x2 = 18.190 df = 3 p < 0.001] and attack [x2 = 14.023 df = 3 p < 0.003]. Time allocated to these behaviours was increased in ISO/ISO vs. ISO/GR and GR/ISO (p < 0.005), and GR/GR vs. ISO/GR (p < 0.05). Differences in attack were also identified in GR/GR vs. ISO/GR (p < 0.05) and in threat between GR/ISO vs. ISO/GR (p < 0.05). Differences in SAL were also detected in threat [x2 = 10.759 df = 3 p < 0.01] and attack [x2 = 10.777 df = 3 p < 0.01], showing mice more threat in ISO/ISO vs. GR/ISO (p < 0.01) and attack in GR/ISO vs. ISO/ISO
Discussion
Table 2 Effects of acute treatment with bupropion 10 mg/kg (BUP-10), 20 mg/kg (BUP-20), 40 mg/kg (BUP-40) or saline (SAL) on median times (with ranges) with seconds allocated to broad behavioural categories by adolescent mice in GR/ISO housing condition.
a
Grooming Diggingb
Non social explorationc Explore from a distanced Social investigatione Threat Attack Avoidance/flee Defense/submission Immobility
SAL
BUP-10
BUP-20
BUP-40
12.6 (5.2–21) 16.1 (0–120) 386 (239.6–500) 16.1 (2.6–37.6) 101.9 (47.5–171.1) 18.1 (0–71.3) 18.3 (0–202.4) 0 (0–0) 0 (0–0) 0 (0–0.5)
7.4 (3.6–9.5) 11.9 (3.2–81.1) 422.5 (358.7–490.6) 10.6 (3.2–79.9) 69.6 (29.7–180.3) 24.6 (0–38.2) 3.9 (0–36.9) 0 (0–1.9) 0 (0–1.4) 0 (0–395,8)
1.6+ (0.3–12.5) 3.1* (0–13.2) 402.2 (183–470.7) 12 (1.2–24.1) 94.7 (24–273.3) 20.4 (0.2–79.1) 33.6 (0–187.9) 0 (0–0) 0 (0–1.5) 0 (0–5.8)
2.4*** (0.3–9.5) 4.1+ (0.4–12.6) 476.4*** (231.4–555) 20.8* (8.1–42.6) 49.5* (2.4–336.7) 3.5 (0–55.3) 0 (0–54.9) 0 (0–18) 0 (0–69.4) 0 (0–25.2)
Kruskal-Wallis test and Mann Whitney U-test showed: a Significant variance p < 0.001: + vs. SAL, p < 0.005, ***vs. SAL p < 0.001. bSignificant variance p < 0.01:+ vs. SAL p < 0.005, *vs. SAL p < 0.05. cSignificant variance p < 0.005: *** vs. SAL p < 0.001. dSignificant variance p < 0.05: * vs. SAL p < 0.05. eSignificant variance p < 0.05: * vs. SAL p < 0.05.
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anxiety was observed especially when the housing conditions of adolescent mice were changed. In the same social interaction paradigm, it has been reported that BUP-40 reduced time spent in social investigation and increased non-social exploration in adolescent (PND 36-37) and adult OF1 mice (PND 65-66) after a 2-weeks isolation [20]. However, bupropion does not alter social investigation in adolescent and adult mice after a 30-day of isolation housing [18,19]. Nevertheless, these results are in line with the present findings in that they suggest that the period of isolation and the transition between different housing conditions are relevant variables in the evaluation of social investigation parameter. In the present research analyses were performed for each housing condition separately and only drug effects were taken into account. Bupropion-treated mice spent less time engaged in grooming and digging behaviours in all housing conditions. BUP40 and BUP-20 disrupted grooming in all groups and BUP-10 also reduced this response in mice reared in GR/GR. The drug also decreased digging in all housing conditions. A decrease in time spent digging and grooming has been regarded as a reliable indicator of the anxiogenic properties of drugs [26]. In fact, an increase of digging and reactivity to unknown environments is observed with anxiolytic drugs [30]. Grooming is a response sensitive to both endogenous and exogenous variables and may be a relevant measure for the evaluation of reactivity to novelty and stress [31]. Grooming can be modulated by anxiety levels although motor aspects of this behaviour should also be considered [32]. A decrease in grooming and digging induced by high doses of bupropion has also been related to the stimulant motor effects of the drug [18]. In our analysis, we took into account the influence of bupropion on time devoted to behavioural categories with motor components such as exploration of the environment (non-social exploration) and towards the opponent mouse (exploration from a distance and social investigation). We found an increase in time devoted to environmental exploration and exploring the opponent from a distance, a behaviour that anticipates social contact and that can precede aggressive behaviour [33]. Although bupropion induces locomotor stimulation in a dose-dependent manner [18,21] it is important to note that non-social exploration was accompanied by a marked decrease in social investigation, which is considered a parameter with significant motor components. This observation would indicate that motor changes are not the only explanation for behaviours displayed by mice during social encounters, suggesting a possible synergy between the anxiogenic and locomotor actions of bupropion. According to the ethopharmacological profile, bupropion exerts anxiogenic-like properties on social encounters, especially when housing conditions change. Findings are in line with reports in adolescent mice (PND 36-37) isolated-housed for 2-weeks [20]. In adults, contrary results have been obtained in isolated and grouphoused, without significant changes in the time devoted to social investigation in bupropion-treated mice [18,19]. Studies using other paradigms of anxiety that assess an innate anxiety response to a novel context offer conflicting results [18,21,34]. Factors such as age, experimental manipulations or housing conditions could underlie these discrepancies. Differences reported in our study between saline-treated groups could suggest that when housing conditions change during adolescence, mice could be more sensitive to this transition. Analysis for each bupropion dose also confirms greater sensitivity to bupropion in those mice housed in changing conditions (ISO/GR and GR/ISO). These observations could be related to different factors since alterations in anxiety-like behaviour, basal levels of corticosterone or social recognition memory have been reported after isolation-housing [35,36].
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The mechanisms involved in the effects of bupropion on social behaviour are not well understood. Early social deprivation induces neuroendocrine and neurobiological effects, which may affect the social behaviour displayed when acute bupropion treatment is administered during adolescence. Socially deprived mice exhibit changes in immune-endocrine responses displaying a hyper-reactivity to novel situations [37] or stressful stimuli [38]. Individual housing increases arousal and vocalizations in social situations [39]. Social stress reduces mesocortical dopamine levels [40] and causes alterations in the central nervous system during adolescence [41]. Several neurotransmitter systems, including monoaminergic systems, are involved in the anxiogenic actions of drugs [42]. Differences in mesolimbic dopaminergic activity also influence the relationship between environmental reactivity and response to drugs [43]. We found that higher doses of bupropion provoke social anxiety, inducing deleterious emotional effects in adolescents. In this sense, it has been observed that bupropion can reverse the effect caused by nicotine and D-amphetamine and abolish their anxiolytic-like action [44]. Present study adds information regarding our understanding about the bupropion actions on social behaviour in adolescents. The transition between housing conditions influences some responses and could have even greater impact than housing conditions themselves. The mechanisms which underlie the influence of differential housing conditions on the responses to bupropion are not well understood. Changes in monoaminergic activity in response to environmental conditions can influence responses to drugs [45]. For example, SSRIs antidepressants induce different effects on aggression depending of the age of the animals as well as the housing conditions [46]. Our findings indicate that not only the dose used but also the housing conditions experienced during adolescence modulate the influence of bupropion on the social behaviour displayed by mice and confirm that effects on social deprivation in mice depends on the timing [6]. The absence of a direct measure of locomotor activity could be a limitation for an adequate interpretation of our findings. Moreover, we have no data of the changes in behaviour when transition between housing conditions takes place and it will of interest to add more experimental groups in order to asses them in this period. Future studies should also include more bupropion doses and evaluate effects of chronic treatments. In conclusion, social behaviour displayed by adolescents is sensitive to bupropion and its ethopharmacological profile suggests an anxiogenic-like effect in adolescents, though its actions depend strongly on housing conditions. Results support the importance of the transition between different housing conditions and have implications for social isolation investigation. Changes in housing conditions may be a useful model for evaluating the bupropion effects on social behaviour. Funding This research was supported by the University of Valencia. The sponsor had no role in the study design; in the collection analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. References [1] Andersen SL. Exposure to early adversity: points of cross-species translation that can lead to improved understanding of depression. Dev Psychopathol 2015;27:477–91, doi:http://dx.doi.org/10.1017/S0954579415000103. [2] Kilford EJ, Garrett E, Blakemore SJ. The development of social cognition in adolescence: an integrated perspective. Neurosci Biobehav Rev 2016;70:106– 20, doi:http://dx.doi.org/10.1016/j.neubiorev.2016.08.016. [3] Holliday E, Gould TJ. Nicotine, adolescence, and stress: a review of how stress can modulate the negative consequences of adolescent nicotine abuse.
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Original article
Bupropion induces social anxiety in adolescent mice: Influence of housing conditions Carmen Gómez, Rosa Redolat, Carmen Carrasco* Departamento de Psicobiología, Facultad de Psicología, Universitat de València, Valencia, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 December 2016 Accepted 15 March 2017 Available online 22 March 2017
Background: The antidepressant bupropion has received increasing attention as a pharmacological tool to treat addiction although little is known about its effects on social behaviour in adolescents. The present study aimed to evaluate if environmental housing conditions influence bupropion’s actions on social behaviour of adolescent mice. Methods: Mice were either group- or individually housed for 2-weeks and then randomly divided into 2 cohorts: half of the mice remained in the initial housing condition and the other half were changed to isolated conditions for further 2-weeks. The following groups were compared: isolated/isolated (ISO/ ISO), isolated/group-housed (ISO/GR), group-housed/isolated (GR/ISO), and group-housed/group-housed (GR/GR). The effects of bupropion (40, 20, 10 mg/kg) or saline on social interaction were assessed for each housing condition. Social encounters were evaluated using ethological analysis. Results: Data showed significant effects of bupropion on grooming and digging. This drug diminished time mice allocated to these behavioural categories in all housing conditions. In ISO/GR and GR/ISO conditions, bupropion increased environmental exploration (non-social exploration and exploration from a distance), reduced social investigation and increased avoidance/flee and defence/submission behaviours. An augment of avoidance/flee during social interactions was observed in bupropion-treated mice in GR/GR housing condition. Conclusion: These results suggest that this drug exhibits anxiogenic-like properties in social encounters between adolescent mice, especially when a transition in housing conditions has been experienced during this period. Changes in housing conditions may be a useful model for evaluating the effects of bupropion on social behaviour and the role of environmental housing conditions. © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Keywords: Adolescents Bupropion Housing Mice Social behaviour
Introduction It is known that a lack of appropriate stimulation during development can have important consequences on neural maturation. Increasing experimental evidence suggests that environmental conditions have a clear influence on emotionality [1]. Adolescence is a time of brain maturation during which there are critical neurobiological changes in the regulation of social behaviour [2]. This period is sensitive to affective-related disorders
Abbreviations: BUP, Bupropion; BUP-10, 10 mg/kg of bupropion; BUP-20, 20 mg/ kg of bupropion; BUP-40, 40 mg/kg of bupropion; GR/GR, group-housed/grouphoused; GR/ISO, group-housed/isolated-housed; ip, : intraperitoneal injection; ISO/ ISO, isolated-housed/isolated-housed; ISO/GR, isolated-housed/group-housed; PND, post-natal day; SAL, physiological saline. * Corresponding author. E-mail addresses: [email protected] (C. Gómez), [email protected] (R. Redolat), [email protected] (C. Carrasco).
and the risk of problems of maladaptation augments, including alterations in social behaviour, stress control and drug addiction [3,4]. Isolation rearing is a paradigm commonly applied for studying the influence of environmental conditions on social behaviour in rodents [5]. Deprivation of social contacts in mice induces changes in neuroplasticity although its effects depend on the timing of housing conditions [6]. The age at which the social isolation is initiated seems to be a critical factor and early isolation elicits neurobiological and behavioural changes that continue into adulthood [7]. Social isolation during adolescence has been proposed as an animal model for identifying vulnerability to anxiety disorders [8] and for researching developmental psychopathology [9]. One pharmacological treatment that has received increasing attention in the area of addiction is the antidepressant bupropion, a monoamine (dopamine and norepinephrine) reuptake inhibitor transporter [10,11] and non-competitive antagonist of nicotinic acetylcholine receptors [12]. Bupropion is used for the
http://dx.doi.org/10.1016/j.pharep.2017.03.010 1734-1140/© 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
C. Gómez et al. / Pharmacological Reports 69 (2017) 806–812
treatment of dependence on tobacco and of different drugs of abuse [13–15]. Earlier evidence indicates that acute administration of bupropion antagonizes muricidal behaviour and augments latency to mouse-killing in rats [16]. In mice, chronic treatment with this drug did not to induce effects in the clonidine-induced aggression model [17]. Bupropion increases attack during social interactions in mice with lower aggression levels [18] although it didn’t have significant influence on aggression in group-housed adults [19] or on the time devoted to social investigation [18], but following a 2weeks of social isolation adolescents were more sensitive to bupropion than individually-housed adults [20]. Previous findings support investigating bupropion’s effects on social behaviour and the modulatory influence of housing conditions in adolescent rodents since no prior study has evaluated the effects of this drug when housing conditions change. Indeed, little it is known concerning the consequences of early exposure to bupropion on social behaviour and no studies have evaluated the influence of the re-socialization of adolescents on the effects of bupropion. The main aim in this work was to evaluate if environmental housing conditions influence bupropion’s effects on social behaviour of adolescent mice. We also intend to investigate the impact of transition between different housing conditions since this factor may be relevant for future investigations including the isolation of experimental animals. The evaluation of this animal model during adolescence may be particularly useful since it is a developmental period characterized by vulnerability to psychiatric disorders and drug addiction and, consequently, of the initiation to pharmacological treatments. Materials and methods
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isolated-housed/isolated-housed (ISO/ISO): mice were individually-housed for 4-weeks (PND 24-52); isolated-housed/grouphoused (ISO/GR): mice individually-housed for 2-weeks (PND 24-37) and then group-housed for 2-weeks (PND 38-52). At the end of the housing periods, mice received acute treatment with BUP40, BUP20, BUP10 and SAL, and the 16 groups were evaluated in the social interaction test (Fig. 1). Social interaction test Social encounters took place in a neutral cage (60 33 30 cm) in which mice were confronted during 10-min with a “standard opponent” after 1-min of adaptation to the apparatus. Responses were video-recorded during the social interaction test: grooming, digging, non-social exploration, exploration from a distance, social investigation, threat, attack, avoidance/flee, defence/submission and immobility (more detailed explanation of categories in [23,24]). After each encounter, the apparatus was cleaned (ethanol and water) and the sawdust bedding was changed. The responses during social encounters were examined by a trained observer blind to the treatment using a custom-developed “Raton-time” software [25,26], a program that allows ethological analysis of behavioural categories [18,20,23]. Statistical analyses The time allocated to each behavioural category is indicated as a median with ranges. Due to great variability in the expression of each behavioural category, data were not normally distributed and only drug effects were analyzed and housing conditions were not compared directly. Data were analyzed using Kruskal-Wallis test and two-tailed Mann-Whitney U-test at a significance of p < 0.05.
Subjects Results OF1 male mice (Charles-River, Spain) aged 24 days (post-natal day, PND-24) on arrival at the laboratory were used. Animals were randomly assigned to isolated or group-housed conditions: i.e. 72 subjects were individually-housed in cages (24 13.5 13 cm) and 78 group-housed in 5 per cage (25 25 15 cm). After 2-weeks, half the subjects were maintained in the same housing condition (isolated-housed vs. group-housed) and the remainder were challenged by the other housing condition (group-housed vs. isolated-housed), in both cases for a period of 2-weeks before test. 200 grouped mice were rendered anosmic (intranasal lavage with 4% zinc-sulphate solution) 1-day before test and used as “standard opponents”.
Analysis revealed a main effect of Treatment in the following categories: grooming [x2 = 65.674 df = 15 p < 0.001], digging [x2 = 60.902 df = 15 p < 0.001], non-social exploration [x2 = 62.471 df = 15 p < 0.001], exploration from a distance [x2 = 35.425 df = 15 p < 0.002], social investigation [x2 = 38.057 df = 15 p < 0.001], threat [x2 = 67.320 df = 15 p < 0.001], attack [x2 = 53.886 df = 15 p < 0.001], avoidance/flee [x2 = 45.630 df = 15 p < 0.001], defence/submission [x2 = 45.564 df = 15 p < 0.001].
Drugs Bupropion hydrochloride (Sigma–Aldrich, Spain) was dissolved in physiological saline (SAL). Mice received 30-min before testing 40, 20, 10 mg/kg (BUP-40, BUP-20, BUP-10, respectively) or SAL by intraperitoneal injection (ip) in a volume of 10 ml/kg. Doses employed are habitually used to assess the effects of burpopion in rodents [18,21,22]. Procedure Mice (PND-24) were distributed into two groups on arrival at the laboratory: half the mice were housed during 2-weeks in groups of five and the rest were housed individually. In a second phase initiated on PND-38, subjects were allocated to different groups. Group-housed/group-housed (GR/GR): mice remained group-housed for 4-weeks (PND 24-52); group-housed/isolatedhoused (GR/ISO): mice remained group-housed for 2-weeks (PND 24-37) and then individually-housed for 2-weeks (PND 38-52);
Fig. 1. Experimental design of the experimental procedure. At the end of the housing periods (PND 52) mice received acute treatment with BUP-40, BUP-20, BUP-10 and SAL and were assessed in a social interaction test.
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Consequently, two separate Kruskal–Wallis analyses were performed to evaluate the main effects of housing conditions and of pharmacological treatment. In order to present results more clearly, we will describe the main effects observed in each of the groups maintained in each of the housing conditions provided and the main effects of drug. Analysis of housing conditions 1) ISO/ISO housing (n = 39, 11-9 mice/group). The main factor Treatment had significant effects on grooming [x2 = 15.454 df = 3 p < 0.001]. U-tests confirmed that animals receiving BUP-40 and BUP-20 devoted less time to this category (p < 0.01 and p < 0.001 respectively) (see Fig. 2). Bupropion influenced time allocated to digging [x2 = 19.695 df = 3 p < 0.001]: BUP-40 [Mdn = 3.4 (0.3–5.8)] and BUP-20 [Mdn = 6 (1.6–12.9)] groups spent less time engaged in this behaviour than SAL groups [Mdn = 22.2 (9.3–36.8) p < 0.01]. 2) ISO/GR housing (n = 33, 8–9 mice/group). There were significant differences in time allocated to grooming [x2 = 11.146 df = 3 p < 0.011] and digging [x2 = 10.729 df = 3 p < 0.013]: BUP-40 and BUP-20 diminished grooming (p < 0.003 and p < 0.03 respectively) (see Fig. 2) and digging [BUP-40 Mdn = 3.55 (1.3–7.3) and BUP-20 Mdn = 3.9 (0.2–9.3) p < 0.01 vs. SAL Mdn = 19.9 (2.7–115.1), p < 0.007 and p < 0.01 respectively]. A main effect of Treatment on non-social exploration [x2 = 17.837 df = 3 p < 0.001] and exploration from a distance [x2 = 8.576 df = 3 p < 0.035] was also found. Mice treated with BUP-40 spent more time engaged in non-social exploration [BUP-40 Mdn = 559.8 (517.5–571.2) vs. SAL Mdn = 428.7 (317.1–544.1) p < 0.001] and exploration from a distance [BUP-40
Mdn = 14.75 (12.3–24) vs. SAL Mdn = 9.3 (4.3–25.1) p < 0.02]. Bupropion reduced time allocated to social investigation [x2 = 12.755 df = 3 p < 0.005], reaching statistical significance at the dose BUP-40 (p < 0.02) (see Fig. 3). There were also differences in avoidance/flee [x2 = 11.380 df = 3 p < 0.01] and defence/submission categories [x2 = 15.885 df = 3 p < 0.001] (see Table 1). Bupropiontreatment increased the amount of time allocated to avoidance/flee [BUP-40 Mdn = 0.8 (0–4.6) vs. SAL Mdn = 0 (0–0) p < 0.002] and defence/submission [BUP-40 Mdn = 2.6 (0–6.8) and BUP-20 Mdn = 1.75 (0–100.6) vs. SAL Mdn = 0 (0–0) p < 0.002]. 3) GR/GR housing (n = 39, 11-9 mice/group). The factor Treatment influenced time allocated to grooming [x2 = 20.907 df = 3 p < 0.001]. Post-hoc tests confirmed that bupropion reduced time allocated to this behaviour (BUP-40 p < 0.001; BUP-20 p < 0.003; BUP-10 p < 0.01) (see Fig. 2). Analysis detected a significant effect on time mice spent digging [x2 = 16.100 df = 3 p < 0.001] showing that bupropion-treated mice [BUP-40 Mdn = 3 (1–9) and BUP-20 Mdn = 5.2 (1.1–24.5)] devoted less time to this category than SAL [Mdn = 16.1 (2–120.8) p < 0.005]. Bupropion’s effects on avoidance/flee were significant [x2 = 7.795 df = 3 p < 0.05]: BUP-40 group spent more time engaged in avoidance/ flee behaviours than SAL [BUP-40 Mdn = 0.0 (0–18) vs. SAL Mdn = 0.0 (0–0) p < 0.04]. No effects of bupropion on any other category were detected. 4) GR/ISO housing (n = 39, 10-9 mice/group). Bupropion influenced grooming [x2 = 15.580 df = 3 p < 0.001] and digging [x2 = 11.172 df = 3 p < 0.011]. BUP-40 and BUP-20 reduced time spent grooming (p < 0.001 and p < 0.005 respectively) (see Fig. 2) and digging [BUP-40 Mdn = 4.1 (0.4–12.6) and BUP-20 Mdn = 3.1
Fig. 2. Mean time (SEM) allocated by groups to the grooming in housing conditions: isolated/isolated (ISO/ISO), isolated/group-housed (ISO/GR), group-housed/isolated (GR/ISO), and group-housed/group-housed (GR/GR). Mice were injected ip with SAL or BUP-40, BUP-20 and BUP-10. Thirty minutes later mice performed social encounters for 10-min. U-tests +p < 0.005 vs. SAL; ***p < 0.001 vs. SAL; **p < 0.01 vs. SAL; *p < 0.05 vs. SAL.
C. Gómez et al. / Pharmacological Reports 69 (2017) 806–812
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Fig. 3. Mean time ( SEM) allocated by groups to the social investigation in housing conditions: isolated/isolated (ISO/ISO), isolated/group-housed (ISO/GR), group-housed/ isolated (GR/ISO), and group-housed/group-housed (GR/GR). Mice were injected ip with SAL or BUP-40, BUP-20 and BUP-10. Thirty minutes later mice performed social encounters for 10-min. U-tests * p < 0.05 vs. SAL. Table 1 Effects of acute treatment with bupropion 10 mg/kg (BUP-10), 20 mg/kg (BUP-20), 40 mg/kg (BUP-40) or saline (SAL) on median times (with ranges) with seconds allocated to broad behavioural categories by adolescent mice in ISO/GR housing condition.
a
Grooming Diggingb
Non social explorationc Explore from a distanced Exploración sociale Threat Attack Avoidance/fleef Defense/submissiong Immobility
SAL
BUP-10
BUP-20
BUP-40
14.4 (0.7–43.2) 19.9 (2.7–115.1) 428.7 (317.1–544.1) 9.3 (4.3–25.1) 84.9 (0–194) 0 (0–36.9) 0 (0–27.6) 0 (0–0) 0 (0–0) 0 (0–30.4)
7.4 (0.4–32.3) 14.5 (0.5–36.5) 434.2 (238.4–518.9) 7.85 (4.7–28.8) 117 (55.4–140.2) 0 (0–0) 0 (0–1.6) 0 (0–0.4) 0 (0–1.4) 0 (0–228.7)
1.95* (0 34.9) 3.9** (0.2–9.3) 479.5 (399–537.1) 5.5 (3.1–22.8) 68.3 (2.9–122.4) 0 (0–12.9) 0 (0–14) 0 (0–69.2) 1.75** (0–100.6) 0 (0–20.1)
1.2+ (0.3–2.06) 3.5** (1.3–7.3) 559.8*** (517.5–571.2) 14.75* (12.3–24) 15.2* (5.7–54.8) 0 (0–0.4) 0 (0–0.3) 0.8+ (0–4.6) 2.6+ (0–6.8) 0 (0–2.8)
Kruskal-Wallis test and Mann Whitney U-test showed: a Significant variance p < 0.01: +vs. SAL p < 0.005, *vs. SAL p < 0.05. bSignificant variance p < 0.01: ** vs. SAL p < 0.01. c Significant variance p < 0.001: *** vs. SAL p < 0.001. dSignificant variance p < 0.05: * vs. SAL p < 0.05. eSignificant variance p < 0.005: * vs. SAL p < 0.05. fSignificant variance p < 0.01:+ vs. SAL p < 0.005. gSignificant variance p < 0.001:+ vs. SAL p < 0.005
(0–13.2) p < 0.05; SAL Mdn = 16.15 (0–120) p < 0.003 and p < 0.05 respectively]. An effect of Treatment on non-social exploration [x2 = 15.381 df = 3 p < 0.002] and exploration from a distance [x2 = 8.447, df = 3 p < 0.038] was detected. Post-hoc tests indicated
that more time was spent exploring the environment by bupropion-treated mice. BUP-40 increased non-social exploration [BUP-40 Mdn = 476.4 (231.4–555) vs. SAL Mdn = 386 (239.6–500) p < 0.001] and exploration from a distance [BUP-40 Mdn = 20.8
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(8.1–42.6) vs. SAL Mdn = 16.1 (2.6–37.6) p < 0.014] (see Table 2). In contrast, bupropion reduced time allocated to social investigation [x2 = 9.460 df = 3 p < 0.024] reaching statistical significance at the dose of BUP-40 (p < 0.019) (see Fig. 3).
(p < 0.01), and increasing threat and attack in GR/GR vs. ISO/GR (p < 0.05) and in GR/ISO vs. GR/GR (p < 0.01).
Analysis of pharmacological treatment
This study aimed to assess if housing conditions modify the effects of bupropion on social behaviour in adolescent male mice. Results suggest that this drug induces behavioural changes modulated by social housing conditions. In ISO/GR and GR/ISO, bupropion increased environmental exploration, avoidance/flee and defence/submission behaviours whereas it reduced social investigation. In GR/GR, an augment of avoidance/flee during social interactions was observed in bupropion-treated mice. In all housing conditions, bupropion decreased time allocated to grooming and digging. One interesting finding is that bupropion disrupted social responses displayed by mice when social housing conditions were changed, affecting defensive and social behaviours. In mice reared in the ISO/GR, BUP-40 and BUP-20 elicited defensive behaviours such as avoidance/flee and defence/submission towards the opponent. The highest dose increased time devoted to avoidance/flee by animals kept in the GR/GR condition without increasing defence/submission. This effect could be associated to its anxiogenic actions. An increase of the defensive response during social encounters has been interpreted as a coping strategy that reflects an anxiogenic response [25] and as a consistent predictor of the anxiogenic effects of drugs [18,26,27]. Our study suggests that bupropion elicits an emotional coping strategy in mice maintained in group-housed conditions. Preclinical research shows that aggressive and non-aggressive responses may be related to diverse behavioural and physiological processes of stress adaptation [28], whereas sociability during social encounters is considered a sign of diminished anxiety [29]. In our study, bupropion reduced social exploration in ISO/GR and GR/ISO, as said groups displayed a low level of pro-social behaviour. The highest dose clearly affected social investigation augmenting the time spent exploring the environment. The less time allocated to social exploration exhibited by bupropiontreated mice could reflect anxiogenic actions. This increased
There were differences in BUP-40 innon-social exploration [x2 = 16.967 df = 3 p < 0.001], threat [x2 = 16.114 df = 3 p < 0.001], attack [x2 = 14.363 df = 3 p < 0.002] and avoidance/flee [x2 = 9.379 df = 3 p < 0.025]. We observed more time devoted to these behaviours in ISO/ISO vs. ISO/GR and GR/ISO (p < 0.005 and p < 0.05 respectively). This dose augmented non-social exploration in GR/GR vs. ISO/GR and GR/ISO (p < 0.001 and p < 0.05 respectively), threat in GR/GR vs. ISO/GR (p < 0.05) and avoidance/flee in GR/GR vs. GR/ISO (p < 0.05), whereas it reduced threat in GR/GR vs. GR/ISO (p < 0.05). Analyses of BUP-20 revealed differences in nonsocial exploration [x2 = 12.335 df = 3 p < 0.006], exploration from a distance [x2 = 8.576 df = 3 p < 0.036], threat [x2 = 17.312 df = 3 p < 0.001], attack [x2 = 12.590 df = 3 p < 0.006] and defence/ submission [x2 = 14.214 df = 3 p < 0.003]. This dose increased exploration from a distance in ISO/ISO vs. ISO/GR (p < 0.01) and threat in ISO/ISO vs. GR/ISO (p < 0.01), reducing non-social exploration in ISO/ISO vs. GR/GR (p < 0.01) and attack in ISO/ISO vs. GR/ISO (p < 0.05). An augment of non-social exploration and a decrease of defence/submission in GR/GR vs. GR/ISO were observed (p < 0.005 and p < 0.05 respectively). Mice increased threat and attack in GR/GR vs. ISO/GR (p < 0.01). In GR/ISO, mice were more aggressive than GR/GR (p < 0.01 and p < 0.05 respectively). Analyses of BUP-10 showed differences in threat [x2 = 18.190 df = 3 p < 0.001] and attack [x2 = 14.023 df = 3 p < 0.003]. Time allocated to these behaviours was increased in ISO/ISO vs. ISO/GR and GR/ISO (p < 0.005), and GR/GR vs. ISO/GR (p < 0.05). Differences in attack were also identified in GR/GR vs. ISO/GR (p < 0.05) and in threat between GR/ISO vs. ISO/GR (p < 0.05). Differences in SAL were also detected in threat [x2 = 10.759 df = 3 p < 0.01] and attack [x2 = 10.777 df = 3 p < 0.01], showing mice more threat in ISO/ISO vs. GR/ISO (p < 0.01) and attack in GR/ISO vs. ISO/ISO
Discussion
Table 2 Effects of acute treatment with bupropion 10 mg/kg (BUP-10), 20 mg/kg (BUP-20), 40 mg/kg (BUP-40) or saline (SAL) on median times (with ranges) with seconds allocated to broad behavioural categories by adolescent mice in GR/ISO housing condition.
a
Grooming Diggingb
Non social explorationc Explore from a distanced Social investigatione Threat Attack Avoidance/flee Defense/submission Immobility
SAL
BUP-10
BUP-20
BUP-40
12.6 (5.2–21) 16.1 (0–120) 386 (239.6–500) 16.1 (2.6–37.6) 101.9 (47.5–171.1) 18.1 (0–71.3) 18.3 (0–202.4) 0 (0–0) 0 (0–0) 0 (0–0.5)
7.4 (3.6–9.5) 11.9 (3.2–81.1) 422.5 (358.7–490.6) 10.6 (3.2–79.9) 69.6 (29.7–180.3) 24.6 (0–38.2) 3.9 (0–36.9) 0 (0–1.9) 0 (0–1.4) 0 (0–395,8)
1.6+ (0.3–12.5) 3.1* (0–13.2) 402.2 (183–470.7) 12 (1.2–24.1) 94.7 (24–273.3) 20.4 (0.2–79.1) 33.6 (0–187.9) 0 (0–0) 0 (0–1.5) 0 (0–5.8)
2.4*** (0.3–9.5) 4.1+ (0.4–12.6) 476.4*** (231.4–555) 20.8* (8.1–42.6) 49.5* (2.4–336.7) 3.5 (0–55.3) 0 (0–54.9) 0 (0–18) 0 (0–69.4) 0 (0–25.2)
Kruskal-Wallis test and Mann Whitney U-test showed: a Significant variance p < 0.001: + vs. SAL, p < 0.005, ***vs. SAL p < 0.001. bSignificant variance p < 0.01:+ vs. SAL p < 0.005, *vs. SAL p < 0.05. cSignificant variance p < 0.005: *** vs. SAL p < 0.001. dSignificant variance p < 0.05: * vs. SAL p < 0.05. eSignificant variance p < 0.05: * vs. SAL p < 0.05.
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anxiety was observed especially when the housing conditions of adolescent mice were changed. In the same social interaction paradigm, it has been reported that BUP-40 reduced time spent in social investigation and increased non-social exploration in adolescent (PND 36-37) and adult OF1 mice (PND 65-66) after a 2-weeks isolation [20]. However, bupropion does not alter social investigation in adolescent and adult mice after a 30-day of isolation housing [18,19]. Nevertheless, these results are in line with the present findings in that they suggest that the period of isolation and the transition between different housing conditions are relevant variables in the evaluation of social investigation parameter. In the present research analyses were performed for each housing condition separately and only drug effects were taken into account. Bupropion-treated mice spent less time engaged in grooming and digging behaviours in all housing conditions. BUP40 and BUP-20 disrupted grooming in all groups and BUP-10 also reduced this response in mice reared in GR/GR. The drug also decreased digging in all housing conditions. A decrease in time spent digging and grooming has been regarded as a reliable indicator of the anxiogenic properties of drugs [26]. In fact, an increase of digging and reactivity to unknown environments is observed with anxiolytic drugs [30]. Grooming is a response sensitive to both endogenous and exogenous variables and may be a relevant measure for the evaluation of reactivity to novelty and stress [31]. Grooming can be modulated by anxiety levels although motor aspects of this behaviour should also be considered [32]. A decrease in grooming and digging induced by high doses of bupropion has also been related to the stimulant motor effects of the drug [18]. In our analysis, we took into account the influence of bupropion on time devoted to behavioural categories with motor components such as exploration of the environment (non-social exploration) and towards the opponent mouse (exploration from a distance and social investigation). We found an increase in time devoted to environmental exploration and exploring the opponent from a distance, a behaviour that anticipates social contact and that can precede aggressive behaviour [33]. Although bupropion induces locomotor stimulation in a dose-dependent manner [18,21] it is important to note that non-social exploration was accompanied by a marked decrease in social investigation, which is considered a parameter with significant motor components. This observation would indicate that motor changes are not the only explanation for behaviours displayed by mice during social encounters, suggesting a possible synergy between the anxiogenic and locomotor actions of bupropion. According to the ethopharmacological profile, bupropion exerts anxiogenic-like properties on social encounters, especially when housing conditions change. Findings are in line with reports in adolescent mice (PND 36-37) isolated-housed for 2-weeks [20]. In adults, contrary results have been obtained in isolated and grouphoused, without significant changes in the time devoted to social investigation in bupropion-treated mice [18,19]. Studies using other paradigms of anxiety that assess an innate anxiety response to a novel context offer conflicting results [18,21,34]. Factors such as age, experimental manipulations or housing conditions could underlie these discrepancies. Differences reported in our study between saline-treated groups could suggest that when housing conditions change during adolescence, mice could be more sensitive to this transition. Analysis for each bupropion dose also confirms greater sensitivity to bupropion in those mice housed in changing conditions (ISO/GR and GR/ISO). These observations could be related to different factors since alterations in anxiety-like behaviour, basal levels of corticosterone or social recognition memory have been reported after isolation-housing [35,36].
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The mechanisms involved in the effects of bupropion on social behaviour are not well understood. Early social deprivation induces neuroendocrine and neurobiological effects, which may affect the social behaviour displayed when acute bupropion treatment is administered during adolescence. Socially deprived mice exhibit changes in immune-endocrine responses displaying a hyper-reactivity to novel situations [37] or stressful stimuli [38]. Individual housing increases arousal and vocalizations in social situations [39]. Social stress reduces mesocortical dopamine levels [40] and causes alterations in the central nervous system during adolescence [41]. Several neurotransmitter systems, including monoaminergic systems, are involved in the anxiogenic actions of drugs [42]. Differences in mesolimbic dopaminergic activity also influence the relationship between environmental reactivity and response to drugs [43]. We found that higher doses of bupropion provoke social anxiety, inducing deleterious emotional effects in adolescents. In this sense, it has been observed that bupropion can reverse the effect caused by nicotine and D-amphetamine and abolish their anxiolytic-like action [44]. Present study adds information regarding our understanding about the bupropion actions on social behaviour in adolescents. The transition between housing conditions influences some responses and could have even greater impact than housing conditions themselves. The mechanisms which underlie the influence of differential housing conditions on the responses to bupropion are not well understood. Changes in monoaminergic activity in response to environmental conditions can influence responses to drugs [45]. For example, SSRIs antidepressants induce different effects on aggression depending of the age of the animals as well as the housing conditions [46]. Our findings indicate that not only the dose used but also the housing conditions experienced during adolescence modulate the influence of bupropion on the social behaviour displayed by mice and confirm that effects on social deprivation in mice depends on the timing [6]. The absence of a direct measure of locomotor activity could be a limitation for an adequate interpretation of our findings. Moreover, we have no data of the changes in behaviour when transition between housing conditions takes place and it will of interest to add more experimental groups in order to asses them in this period. Future studies should also include more bupropion doses and evaluate effects of chronic treatments. In conclusion, social behaviour displayed by adolescents is sensitive to bupropion and its ethopharmacological profile suggests an anxiogenic-like effect in adolescents, though its actions depend strongly on housing conditions. Results support the importance of the transition between different housing conditions and have implications for social isolation investigation. Changes in housing conditions may be a useful model for evaluating the bupropion effects on social behaviour. Funding This research was supported by the University of Valencia. The sponsor had no role in the study design; in the collection analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. References [1] Andersen SL. Exposure to early adversity: points of cross-species translation that can lead to improved understanding of depression. Dev Psychopathol 2015;27:477–91, doi:http://dx.doi.org/10.1017/S0954579415000103. [2] Kilford EJ, Garrett E, Blakemore SJ. The development of social cognition in adolescence: an integrated perspective. Neurosci Biobehav Rev 2016;70:106– 20, doi:http://dx.doi.org/10.1016/j.neubiorev.2016.08.016. [3] Holliday E, Gould TJ. Nicotine, adolescence, and stress: a review of how stress can modulate the negative consequences of adolescent nicotine abuse.
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