Genes, Brain and Behavior (2015) 14: 292–300
doi: 10.1111/gbb.12208
Effect of chronic corticosterone application on depression-like behavior in C57BL/6N and C57BL/6J mice M. Sturm, A. Becker, A. Schroeder, A. Bilkei-Gorzo and A. Zimmer∗ Institute of Molecular Psychiatry, University of Bonn, Bonn, Germany *Corresponding author: A. Zimmer, Institute of Molecular Psychiatry, University of Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany. E-mail: [email protected]
Many studies using genetic mouse models are performed with animals on either one of the two closely related genetic backgrounds, C57BL/6J or C57BL/6N. These strains differ only in a few genetic loci, but have some phenotypic differences that also affect behavior. In order to determine the effects of chronic stress hormone exposure, which is relevant for the pathogenesis of psychiatric disorders, we investigated here the behavioral manifestations of long-term increase in corticosterone levels. Thus, male mice from both sub-strains were subcutaneously implanted with corticosterone (20 mg) or placebo pellets that released the hormone for a period of 21 days and resulted in significantly elevated plasma corticosterone levels. Corticosterone significantly increased food intake in B6N, but not in B6J mice. At various time points after pellet implantation, we performed tests relevant to activity and emotional behaviors. B6J mice displayed a generally higher activity in the home cage and the open field. Corticosterone decreased the activity. In B6N mice, corticosterone also decreased sucrose preference, worsened the coat state and increased forced swim immobility, while it had no effect in the B6J strain. Altogether, these results indicate that B6N mice are more sensitive to some of the effects of chronic corticosterone treatment than B6J mice. Keywords: Anhedonia, behavioral changes, C57BL/6 mice, corticosterone, immobility, locomotor activity, major depression, stress, sub-strain, sucrose preference Received 7 October 2014, revised 21 February 2015, accepted for publication 26 February 2015
C57BL/6J (B6J) mice were established at The Jackson Laboratory along with other sub-strains such as C57BL/10 from parental C57BL mice in the 1930s and 1940s. The National Institutes of Health received C57BL/6 mice in 1951 and maintained them, thus resulting in a C57BL/6N (B6N) sub-strain, which was passed on to Charles River in 1974
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and Taconic Farms in 1991. Although the B6J and B6N sub-strains are separated by well over 200 generations, they are genetically very similar (Bothe et al. 2004; Bryant et al. 2008; Mekada et al. 2009; Mulligan et al. 2008). A recent sequence comparison of B6J and B6N from Taconic Farms showed that these sub-strains differ only in 34 single nucleotide polymorphisms, 2 indels in coding regions and 15 structural gene variants. Despite their genetic similarity, both sub-strains differ in a number of phenotypes including behaviors (Bechard et al. 2012; Blum et al. 1982; Bothe et al. 2004; Bryant et al. 2008; Crusio et al. 1991; Hager et al. 2014; Kumar et al. 2013; Matsuo et al. 2010; Mogil & Wilson 1997; Mouri et al. 2012; Mulligan et al. 2008; Ramachandra et al. 2007; Siegmund et al. 2005; Simon et al. 2013; Stiedl et al. 1999), circadian rhythms (Banks et al. 2014), metabolic functions and responses to high-fat diet (Andersson et al. 2010; Heiker et al. 2014; Podrini et al. 2013; Simon et al. 2013; Walker et al. 2014), as well as the physiology and pathology of the cardiovascular (Cardin et al. 2014; Kararigas et al. 2014; Moreth et al. 2014), visual (Mattapallil et al. 2012; Simon et al. 2013) and auditory systems (Kendall & Schacht 2014; Schnabolk et al. 2014). These phenotype differences are important, because many studies use genetic mouse models on these two C57BL/6 genetic backgrounds. Thus, many laboratories have used knockout mice to investigate the cellular and molecular mechanisms contributing to stress responses and affective disorders. As one of the most common and distressing neuropsychiatric disorders, major depression is among the main causes for disability-adjusted life years (WHO 2005). The etiology of depression is clearly multifactorial with contributions of genetic and environmental factors (Kalia 2005; Nestler et al. 2002). Clinical experience and experimental studies have implicated a dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis as one of the major factors in the pathogenesis of depression (Claes 2004; Sullivan et al. 2000; Zunszain et al. 2011). The HPA axis is activated upon stress exposure, which triggers the secretion of the hypothalamic corticotropin releasing hormone (CRH) and the subsequent release of adrenocorticotropin hormone (ACTH) from the pituitary. Adrenocorticotropin hormone in turn stimulates the secretion of cortisol (humans) or corticosterone (mice) from the adrenal cortex. These glucocorticoids activate glucocorticoid and mineralocorticoid receptors in several brain areas, as well as in peripheral tissues, and are responsible for the feedback inhibition of CRH and Arginine vasopressin (AVP) release. Patient suffering from major depression often show increased secretion and reactivity to cortisol, as well as elevated CRH levels (Zunszain et al. 2011). In this study, we were interested to determine if B6N
© 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society
Effect of chronic corticosterone application in C57BL/6N and C57BL/6J mice
and B6J mice respond differently to chronically elevated glucocorticoid levels, which is known to produce many neurobiological and behavioral changes in rodents that parallel those associated with human depression (Sterner & Kalynchuk 2010). We have thus implanted mice from both strains with corticosterone-releasing pellets and investigated behaviors that are relevant to anxiety and depression.
Materials and methods Animals Experiments were carried out on 48 male B6J (n = 24) and B6N (n = 24) mice that were bought at the same time from the same breeder (Charles River Laboratories, Sulzfeld, Germany; B6N strain code 027: B6J stock number: 000064). We used only male animals, because most previous studies comparing B6J and B6N mice were performed with males. All animals were kept under identical housing condition. At the beginning of the experiments, mice were 12 weeks old. All animals were single housed in transparent plastic cages (30 × 12 × 12 cm3 ) under a 12 h/12 h light/dark cycle (lights on at 2100 h) at constant room temperature (22∘ C). Food and water were available ad libitum and animals were allowed to habituate to the novel environment for 14 days after arrival. Behavioral testing was performed between 0900 and 2100 h during the dark cycle. Animals were treated according to the German Animal Protection Law.
Drugs and surgery For corticosterone administration, hormone pellets (Innovative Research of America, Sarisota, FL, USA) were used. Pellets (0.5 × 0.5 cm2 diameter) consisted of corticosterone (20 mg/pellet) in a matrix of cholesterol, lactose, celluloses, phosphates and stearates and had a releasing time of 21 days. Placebo pellets consisted of the same matrix and size. Mice were divided into four groups, each of which containing 12 animals, which were implanted with either corticosterone or placebo pellets (B6JCort, B6JPla, B6NCort and B6NPla). For pellet implantation, mice were briefly anesthetized with isoflurane (0.4 ml/min). The fur was removed above the incision side and a 0.6 cm cut in the skin was performed at the left side of the neck in an area between ear and foreleg. The pellet was placed under the skin and the wound was closed with an absorbable thread (VicrylTM Plus, 3-0; Johnson & Johnson International, New Brunswick, NJ, USA). Subsequently, the mice were intraperitoneally injected with Carprofen (Sigma-Aldrich, Henstedt-Ulzburg, Germany) (4 mg per 1 kg body weight). The surgery required approximately 10 min per animal. Subsequently, animals were returned to their home cages and allowed to recover for 14 days before behavioral tests were performed. Activity in the home cage was recorded during the recovery period between days 6 and 9.
Pre-testing In order to assess the baseline behavior of untreated B6J and B6N mice, sucrose preference test (SPT), forced swim test (FST) and open field test (OFT) were performed with a total of 18 B6J and B6N mice prior to the main experiment under equal experimental conditions (see below). We used independent groups of animals for pre-testing and the main experiment in order to avoid confounding effects of habituation.
in the following order: days 6–9: home cage activity (HCA), day 14: SPT, day 15: OFT, day 16: light–dark test (LDT), day 17: FST, day 21: coat state, decapitation, organ extraction and blood sampling. All behavioral tests were performed between 0900 and 2100 h.
Home cage activity Activity in the home cage of the mice was recorded by means of an infrared sensor system (Mouse-E-Motion; Infra-e-motion, Germany). The sensors were applied on the top of the cages 14 cm above the floor level and the activity of the mice was recorded with a sampling rate of 1 Hz, starting from 2100 h for the next 72 h continuously. The activity recordings were averaged every 4 min and expressed as ‘arbitrary activity index’. Please note that the animals remained in their cages and were not moved to a new one. A habituation to a recording system was therefore not necessary. The activity during the dark phases, the bright phases and the overall activity during 72 h was analyzed and compared between the groups.
Sucrose preference test Sucrose preference test was performed according to Strekalova and Steinbusch (2010) to assess anhedonia in mice. Sucrose preference test was performed in a two-bottle paradigm for 3 h at the beginning of the dark phase, where the mice received two bottles, one filled with 1.5% sucrose solution and one with tap water. After half of the time (90 min), the places of the bottles were changed to avoid side bias. The weight of each bottle was determined before and after the SPT. In order to familiarize the mice to the sucrose taste, they received a 2.5% sucrose solution instead of water for 2.5 h (0900–1130 h) 1 day before the actual test. The SPT was performed before and after the surgery. The sucrose preference was calculated in percent as the amount of consumed sucrose solution divided by the total liquid intake.
Open field test The open field arena (45 × 25 × 22 cm3 ) was illuminated with 5 lx. Each mouse was placed in the right corner of the arena and total distance traveled (cm), visits to the center and rearings were recorded for 5 min to test the reactivity of the animals to novel environment. Between every run, arenas were cleaned carefully with antiseptic solution. The movements were recorded using an automatic infrared movement sensor (ActiMot, TSE-Systems GmbH, Bad Homburg, Germany).
Light–dark test The LDT was performed to assess anxiety as it has a high comorbidity with depression. A dark compartment was inserted into the open field box, which took one third of the area and contained a mouse hole (entrance) in the middle. The light intensity in the bright area was set to 650 lx to represent an aversive environment. The dark compartment was completely shielded from light providing a less threatening environment for the mouse. The mouse was placed into the right corner of the bright area facing the entrance of the dark compartment. The activity of the mouse was recorded for 5 min by the software ACTIMOT v2.0 (TSE-Systems GmbH). The time spent and the distance traveled in the bright and dark areas were taken as parameters for the analysis.
Forced swim test
After pellet implantation, the weight and the food intake of every mouse were measured twice a week over the period of 21 days.
Three plastic cylinders (25 × 19 × 13 cm) were filled with water (23∘ C) up to a height of 12.5 cm and located in a testing room, which was illuminated with 5 lx. Mice were placed in the cylinders and immobility time was measured manually during the test by the experimenter using a stopwatch. Immobility was determined for a period of 4 min, which started after 2 min of habituation. The habituation period was chosen to eliminate the influence of the initial arousal.
Behavioral tests
Coat state
Four days before pellet implantation, sucrose preference of the animals was tested. After pellet implantation, tests were performed
The total score of the coat state resulted from the sum of scores from seven different body parts: head, neck, dorsal and ventral
Body weight and food consumption
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Figure 1: Pre-testing results. (a) There was no significant baseline difference in sucrose consumption between the two sub-strains at baseline. (b) The two sub-strains showed no difference in the immobility time in the forced swim test. (c) Exploratory locomotor activity in open field test was lower in B6N strain compared with B6J animals. Experiments were carried out with 12 animals per group.
coat, tail, forepaws and hindpaws. For each area, a score of 2 was given for well-groomed coat and 0 for an unkempt coat (maximum score 14 = very good condition; minimum score 0 = poor condition).
Blood sampling and adrenal gland weighting After judging the coat state, mice were decapitated, brains were removed and shock-frozen in isopentane/dry ice for further investigation. Adrenal glands were extracted from the remaining mice and stored at −80∘ C until weighing. For detection of corticosterone concentrations, blood sera were isolated. The trunk blood was allowed to clot for 1 h at room temperature. Thereafter, it was kept at 4∘ C for 24 h and centrifuged at 1000 rpm (rounds per min) for 10 min. These sera were analyzed for corticosterone concentrations with commercially available corticosterone HS EIA kit (IDS GmbH, Frankfurt am Main, Germany). For the analysis, all mice were killed between 2200 and 2300 h to minimize the variance due to ultradian variations of endogenous corticosterone levels.
Results Pre-tests Prior to the main behavioral experiments, pre-testing was carried out to evaluate baseline differences in the behavior of the two untreated sub-strains. We found no strain effects in the SPT (t 15 = 1.596, P = 0.13) (Fig. 1a) and FST (t 18 = 0.992, P = 0.33) (Fig. 1b). However, B6J mice showed a significantly higher locomotor activity in the open field than B6N animals (F (1,33) = 30.75, P < 0.001) (Fig. 1c). Before the implantation of corticosterone pellets, mice of both sub-strains consumed more sucrose than water (B6N mice: 1.3 ± 0.4 ml, 0.3 ± 0.1 ml; P < 0.05; B6J mice: 1.4 ± 0.6 ml, 0.2 ± 0.1 ml; P < 0.05).
Blood serum corticosterone Statistical analysis Statistical analysis was carried out using EXCEL 2011 (Microsoft Corp., Redmond, WA, USA), PRISM v.4.0.1 (GraphPad Software, San Diego, CA, USA) and STATISTICA Version 7 (StatSoft Inc., Tulsa, OK, USA) software. For normally distributed data, statistical significance was determined by one- or two-way analysis of variance (ANOVA), as appropriate, followed by post hoc t-tests with Bonferroni correction. In cases where the data were not normally distributed, they were analyzed by Kruskal–Wallis ANOVA followed by Dunn’s post hoc test.
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Serum corticosterone levels of the trunk blood were measured. Two-way ANOVA showed a significant main treatment effect (F (1,24) = 22.24, P < 0.0001), but no strain effect (F (1,24) = 0.75, P = 0.396) and no interaction (F (1,24) = 0.78, P = 0.3863). Post hoc analyses confirmed that serum corticosterone levels of B6NCort mice (P < 0.05) and B6JCort mice (P < 0.005) were significantly higher compared with placebo groups (Fig. 2). Genes, Brain and Behavior (2015) 14: 292–300
Effect of chronic corticosterone application in C57BL/6N and C57BL/6J mice
test. During the dark phase, when mice were active, we found significant strain (F (1,15) = 6.32, P = 0.0238) and treatment (F (1,15) = 4.65, P = 0.0477) effects, but no interaction (F (1,15) = 0.37, P = 0.5527).
Open field test
Figure 2: Serum corticosterone levels after pellet implantation. Corticosterone levels were significantly increased in mice that have been implanted with pellets releasing corticosterone. There was no significant strain difference. The data are represented as mean ± SEM. Experiments were carried out with nine animals per group. **P < 0.001.
Food intake and body weight To investigate the effect of corticosterone on food intake and body weight, we measured the food intake and body weight on days 1, 4, 8 and 11 after the surgery. Repeated measures three-way ANOVA for body weight showed a significant strain × treatment interaction (F (1,55) = 17.37, P = 0.0001), which was also significant over time (strain × treatment × time: F (3,165) = 19.74, P < 0.0001). A subsequent repeated measures two-way ANOVA for both strains showed a significant time × treatment effect in both strains (B6N: F (3,84) = 30.62, P < 0.0001; B6J: F (3,81) = 3.06, P < 0.05), although post hoc testing showed no significant weight differences between the treatment groups for any of the individual measurements in B6J animals (Fig. 3a). Repeated measures three-way ANOVA for food intake also showed a significant strain × treatment interaction (F (1,56) = 36.92, P < 0.0001), which did not change over time (strain × treatment × time: F (2,112) = 1.90, P = 0.154). A subsequent one-way ANOVA showed a significant treatment effect in B6N mice (F (1,28) = 75.85, P < 0.0001), but not in B6J animals (F (1,28) = 0.065, P = 0.80). Thus, corticosterone-treated B6N mice consumed significantly more and gained more weight. In contrast, corticosterone treatment had no effect on food intake in the B6J strain (Fig. 3b).
Home cage activity In order to control for the locomotor activity, being important for interpretation of other behavioral results, the activity of the mice was measured in their familiar environment. For this purpose, an infrared system was attached to the cages and the activity was recorded for 72 h (Fig. 4a,b). We found neither strain (F (1,15) = 1.86, P = 0.1923) nor treatment (F (1,15) = 0.01, P = 0.9244) effects and no interaction (F (1,15) = 0.10, P = 0.7568) during the light phase of the Genes, Brain and Behavior (2015) 14: 292–300
Two-way ANOVA showed significant main effects for strain (F (1,38) =23.95, P < 0.0001) and treatment (F (1,38) = 37.24, P < 0.0001) on locomotor activity, but no interaction (F (1,38) = 0.52, P = 0.46). Bonferroni post hoc testing showed that corticosterone treatment reduced locomotor activity in both sub-strains, when compared with placebo-treated controls (B6JCort: 48.4 ± 16.1 m; B6JPla: 73.2 ± 5.2 m; B6NCort: 39.2 ± 15.2 m; B6NPla: 47.0 ± 26.9 m; P < 0.01; Fig. 5a). Two-way ANOVA of center visits also showed significant main effects for strain (F (1,38) = 10.74, P < 0.01) and treatment (F (1,38) = 14.42, P < 0.001), but no interaction (F (1,38) = 0.54, P = 0.47). The Bonferroni post hoc test showed a significant reduction of the number of center visits within the B6N strain. Corticosterone-treated B6N mice visited the center less frequently than the placebo-treated B6N mice (B6NCort: 5.6 ± 8.6; B6NPla: 17.5 ± 20.0; P < 0.01) (Fig. 5b). The B6J sub-strain showed no significant reduction of center visits (B6JCort: 17.5 ± 9.3; B6JPla: 30.8 ± 12.1; P = 0.1). No significant effects could be detected in the number of rearings in both sub-strains for strain (F (1,38) = 1.79, P = 0.19) treatment (F (1,38) = 0.60, P = 0.44) and interaction (F (1,38) = 0.16, P = 0.69) (data not shown). Two-way ANOVA showed significant effects for treatment (F (1,38) = 7.04, P < 0.01) and strain (F (1,38) = 8.03, P = 0.007) on the ratio of center visits/locomotor activity, but no interaction (F (1,38) = 2.64, P = 0.11). Bonferroni post hoc testing showed a reduced ratio of center visits/locomotor activity only in the corticosterone-treated B6N sub-strain (P = 0.02) (Fig. 5c).
Sucrose preference test Two-way ANOVA showed a significant main effect of strain (F (1,44) = 6.87, P < 0.05), but not treatment (F (1,44) = 2.99, P = 0.09) and no interaction (F (1,44) = 0.90, P = 0.347). However, separate analysis of the data for the two strains showed that B6J mice showed no difference in sucrose preference, when compared with the placebo-implanted B6J group (B6JCort: 78.1 ± 19.2%; B6JPla: 82.0 ± 17.7%; F (1,22) = 1.87, P = 0.1) (Fig. 6a). In contrast, corticosterone-treated B6N mice had a significantly smaller preference to the sucrose solution compared with placebo-treated mice (B6NCort: 60.0 ± 17.7%; B6NPla: 73.6 ± 15.5%; F (1,22) = 8.48, P < 0.001) (Fig. 6b) and showed no significant sucrose preference (one sample t-test: t (11) = 1.958, P = 0.0761).
Light–dark test The LDT was performed to assess anxiety-related behaviors. The distance and the time spent in the bright area were analyzed during 5 min. No significant differences could be detected between the groups (P > 0.05) (data not shown).
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(a) Figure 3: Food intake and body weight gain. All data are represented as mean ± SEM. B6J animals are shown in the left panels, B6N in the right panels. (a) Body weight was measured on days 1, 4, 8 and 11 after pellet implantation. Please note that B6NCort mice had a significantly higher body weight than B6NPla animals on days 8 and 11. (b) The average daily food consumption was calculated for three intervals after the surgery (days 1–4, 5–8 and 9–11). Please note that corticosterone had no effects on food intake in B6J mice, whereas B6NCort consumed significantly more than B6NPla mice. Experiments were carried out with 15 animals per group. ***P < 0.0001; **P < 0.001.
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Figure 4: Home cage activity. Home cage activity was measured during a 72-h period with an infrared system. The activity was separately analyzed during the dark phase (a), during the light phase (b). The data are represented as mean ± SEM (n = 5, BL6N Placebo: n = 4). **P < 0.01.
Forced swim test Two-way ANOVA showed a significant main effect for treatment (F (1,43) = 5.30, P = 0.48) but not for strain (F (1,43) = 0.51, P > 0.05) and no interaction (F (1,43) = 2.89, P = 0.09). Bonferroni post hoc testing showed that the treatment effect was only significant in the B6N strain (B6NCort: 112.1 ± 27.8 seconds; B6NPla: 60.9 ± 43.1 seconds), but not in the B6J strain (B6JCort: 81.2 ± 44.1 seconds; B6JPla: 73.5 ± 55.6 seconds; P < 0.05) (Fig. 7). Thus, corticosterone administration resulted in an increased immobility time in B6N, but not in B6J mice.
Judgment of coat state Compared with placebo, corticosterone treatment yielded a highly significantly reduced outer appearance in the B6N but not in the B6J sub-strain (Kruskal–Wallis ANOVA U = 32.63, P < 0.001). The fur of the corticosterone-treated
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B6N mice became beamless and shaggy (B6NCort: range 6–11 points; B6NPla: range 9–14 points). Within the B6J group, corticosterone treatment did not have a significant effect on the outer appearance of the animals. Placebo- and corticosterone-treated mice did not differ in quality of fur and general body condition (B6JCort: range 11–14 points; B6JPla: range 13–14 points) (Fig. 8).
Adrenal gland weight Additionally, we weighed the adrenal glands to see if they were differently affected by corticosterone application in the sub-strains. Analysis showed significant main genotype (F (1,20) = 16.63, P = 0.00059) and treatment effects (F (1,20) = 139.11, P < 0.0001), but no interaction (F (1,32) = 2.44, P = 0.128). Post hoc analysis showed that the adrenal glands of both B6Cort groups are significantly smaller compared with placebo groups [B6JPla: mean 4.02 mg (range: Genes, Brain and Behavior (2015) 14: 292–300
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Figure 5: Open field test. (a) Locomotor activity in the open field test was assessed 16 days after pellet implantation as the distance traveled. (b) The number of center visits in the open field was assessed as a measure of anxiety-related behavior. (c) Ratio of center visits and distance traveled for further assessment of anxiety-related behavior. Experiments were carried out with 12 animals per group. **P < 0.01; ****P < 0.0001; *P < 0.05 (two-way ANOVA followed by Bonferroni post hoc test).
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Figure 6: Sucrose preference test. Sucrose preference was assessed 4 days before and 14 days after pellet implantation: Corticosterone-implanted B6J mice showed no difference to placebo-implanted mice (a), whereas corticosterone-treated B6N mice showed a significantly decreased sucrose preference (b). Experiments were carried out with 12 animals per group. *P < 0.05
3.2–4.6 mg); B6JCort: mean 2.58 mg (range: 2.0–3.4 mg); B6NPla: mean 3.69 mg (range: 2.3–4.9 mg); B6NCort: mean 1.70 mg (range: 1.1–2.3 mg; P < 0.0001)].
Discussion In this study, we have investigated the manifestation of behavioral changes after chronic corticosterone exposure in Genes, Brain and Behavior (2015) 14: 292–300
B6J and B6N mice. We found that subcutaneous implantation of corticosterone-releasing pellets increased food intake, reduced open field center visits, decreased sucrose preference, increased immobility time in the FST and worsened the coat state in B6N, but not in B6J mice. These findings are consistent with a substantial body of evidence showing that the chronic administration of corticosterone produces robust changes in several emotional and depression-like behaviors (Gourley et al. 2008; Gregus et al. 2005; Johnson et al.
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Figure 7: Forced swim test. The immobility of B6J mice in the forced swim test did not differ between placebo- and corticosterone-implanted animals, whereas the immobility of corticosterone-implanted B6N mice was significantly higher compared with placebo-implanted mice. Experiments were carried out with 12 animals per group. *P < 0.05 (two-way ANOVA followed by Bonferroni post hoc test).
Figure 8: Coat state. B6J mice showed no changes in the coat status at day 21 after corticosterone pellet implantation, whereas the coat condition of corticosterone-implanted B6N mice was rated lower compared with the placebo group. Experiments were carried out with 12 animals per group. ***P < 0.001 (Kruskal–Wallis ANOVA followed by Dunn’s test).
2006; Kalynchuk et al. 2004; Murray et al. 2008; Sterner & Kalynchuk 2010; Stone & Lin 2008). This difference between the two strains was not due to differences in the pharmacokinetics of corticosterone, because plasma levels were similarly elevated in both strains after corticosterone pellet implantation. These findings therefore suggest that B6N mice are more sensitive to the effects of chronic corticosterone than B6J animals.
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B6J and B6N mice showed a number of subtle differences in emotional behaviors under non-stressed conditions (Bryant et al. 2008; Hager et al. 2014; Matsuo et al. 2010; Simon et al. 2013). However, for some behaviors such as open field activity and center time, the differences between the two strains were inconsistent. While Matsuo et al. (2010) described increased activity and center times for B6J mice, Bothe et al. (2004) found no significant difference between the strain. One study even examined the two strains using standardized protocols in different sites and reported opposite findings, depending on where the animals were tested (Simon et al. 2013). The present results support as modestly higher exploratory activity of untreated and placebo-treated B6J mice. Corticosterone reduced the activity of both strains to a similar extend. However, the number of center visits was only decreased in B6N, but not in B6J mice. We did not find a significant strain effect when testing for HCA during the light phase, when the animals were resting. However, during the active dark phase, B6J mice were slightly more active than B6N mice and corticosterone-treated animals were less active than the placebo group. Home cage activity was also investigated by Hager et al. (2014) in a slightly different experimental setting with a DualCage system. They also found an increased locomotor activity of B6J mice during the first part of the dark (active) phase. Thus, B6J mice are somewhat more active than B6N animals. B6N mice treated with corticosterone also showed significant decrease in sucrose preference and immobility in the FST, two behaviors that are often considered to be associated with a depression-like emotional state (Deussing 2006; Russo & Nestler 2013; Yacoubi & Vaugeois, 2007). However, it should be noted that, although the FST has predictive validity for antidepressant drugs, the behavior has not been related to any pathophysiology (Nestler & Hyman 2010; Sterner & Kalynchuk 2010). Untreated or placebo-treated mice showed no strain differences in forced swim immobility. Previously, one study reported no difference between the two strains in this test (Mouri et al. 2012), whereas another study showed a higher immobility in B6N mice (Matsuo et al. 2010). B6N mice also showed changes in the appearance of their coat state after chronic corticosterone application. Their fur became shaggy and dull, which was not the case in the placebo-treated controls nor in B6J animals. A worsening of the coat state due to either metabolic changes or a depression-induced reduction of grooming behavior has been regarded an indicator for a depression-like emotional state (Griebel et al. 2002; Santarelli et al. 2003). Similar changes in the coat state have been observed previously with B6N mice after chronic corticosterone application in drinking water (David et al. 2009). Our results are in agreement with previous demonstrations that depression-like behaviors developed in B6N mice in response to corticosterone treatment or other stress-dependent paradigms resulting in elevated blood corticosterone levels (Stone & Quartermain 1999; Zhao et al. 2008). Strekalova et al. (2004) have shown increased forced swim immobility, reduced exploratory behavior and reduced sucrose preference after exposure of B6N mice to mild chronic stress for 4 weeks. Our results indicate that despite their high genetic similarity, the N and J sub-strains of B6 mice react differently to Genes, Brain and Behavior (2015) 14: 292–300
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chronic corticosterone application. A previous study examining avoidance behavior and risk assessment in these two strains found a high degree of variation among individual B6 animals, although the study reconfirmed a higher degree of fear behavior in B6N compared with B6J mice (Hager et al. 2014). The high degree of individuality and the resulting high variance may necessitate relatively large group sizes in order to detect treatment effects. In this respect, the size of our groups, which ranged from 12 to 15 mice per group and was thus similar to the typical group sizes used by comparable studies (Bryant et al. 2008; Matsuo et al. 2010; Mouri et al. 2012), may have still been too small to detect moderate treatment effects in B6J animals. Our findings have implications on the interpretation of experimental results addressing the function of specific genes in depression using transgenic or knockout animals on these backgrounds. Genetic manipulations that result in an enhanced resistance to depression-like pathologies may be more difficult to ascertain in mice on a B6J background, while the opposite might be true for B6N animals. The genetic background used for the analysis of mutant phenotypes should therefore be carefully selected and reported.
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Acknowledgement This work was supported by a grant from the Deutsche Forschungsgemeinschaft (FOR926, SP6). A.Z. is a member of the Excellence Cluster ‘Immunosensation’.
Genes, Brain and Behavior (2015) 14: 292–300
doi: 10.1111/gbb.12208
Effect of chronic corticosterone application on depression-like behavior in C57BL/6N and C57BL/6J mice M. Sturm, A. Becker, A. Schroeder, A. Bilkei-Gorzo and A. Zimmer∗ Institute of Molecular Psychiatry, University of Bonn, Bonn, Germany *Corresponding author: A. Zimmer, Institute of Molecular Psychiatry, University of Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany. E-mail: [email protected]
Many studies using genetic mouse models are performed with animals on either one of the two closely related genetic backgrounds, C57BL/6J or C57BL/6N. These strains differ only in a few genetic loci, but have some phenotypic differences that also affect behavior. In order to determine the effects of chronic stress hormone exposure, which is relevant for the pathogenesis of psychiatric disorders, we investigated here the behavioral manifestations of long-term increase in corticosterone levels. Thus, male mice from both sub-strains were subcutaneously implanted with corticosterone (20 mg) or placebo pellets that released the hormone for a period of 21 days and resulted in significantly elevated plasma corticosterone levels. Corticosterone significantly increased food intake in B6N, but not in B6J mice. At various time points after pellet implantation, we performed tests relevant to activity and emotional behaviors. B6J mice displayed a generally higher activity in the home cage and the open field. Corticosterone decreased the activity. In B6N mice, corticosterone also decreased sucrose preference, worsened the coat state and increased forced swim immobility, while it had no effect in the B6J strain. Altogether, these results indicate that B6N mice are more sensitive to some of the effects of chronic corticosterone treatment than B6J mice. Keywords: Anhedonia, behavioral changes, C57BL/6 mice, corticosterone, immobility, locomotor activity, major depression, stress, sub-strain, sucrose preference Received 7 October 2014, revised 21 February 2015, accepted for publication 26 February 2015
C57BL/6J (B6J) mice were established at The Jackson Laboratory along with other sub-strains such as C57BL/10 from parental C57BL mice in the 1930s and 1940s. The National Institutes of Health received C57BL/6 mice in 1951 and maintained them, thus resulting in a C57BL/6N (B6N) sub-strain, which was passed on to Charles River in 1974
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and Taconic Farms in 1991. Although the B6J and B6N sub-strains are separated by well over 200 generations, they are genetically very similar (Bothe et al. 2004; Bryant et al. 2008; Mekada et al. 2009; Mulligan et al. 2008). A recent sequence comparison of B6J and B6N from Taconic Farms showed that these sub-strains differ only in 34 single nucleotide polymorphisms, 2 indels in coding regions and 15 structural gene variants. Despite their genetic similarity, both sub-strains differ in a number of phenotypes including behaviors (Bechard et al. 2012; Blum et al. 1982; Bothe et al. 2004; Bryant et al. 2008; Crusio et al. 1991; Hager et al. 2014; Kumar et al. 2013; Matsuo et al. 2010; Mogil & Wilson 1997; Mouri et al. 2012; Mulligan et al. 2008; Ramachandra et al. 2007; Siegmund et al. 2005; Simon et al. 2013; Stiedl et al. 1999), circadian rhythms (Banks et al. 2014), metabolic functions and responses to high-fat diet (Andersson et al. 2010; Heiker et al. 2014; Podrini et al. 2013; Simon et al. 2013; Walker et al. 2014), as well as the physiology and pathology of the cardiovascular (Cardin et al. 2014; Kararigas et al. 2014; Moreth et al. 2014), visual (Mattapallil et al. 2012; Simon et al. 2013) and auditory systems (Kendall & Schacht 2014; Schnabolk et al. 2014). These phenotype differences are important, because many studies use genetic mouse models on these two C57BL/6 genetic backgrounds. Thus, many laboratories have used knockout mice to investigate the cellular and molecular mechanisms contributing to stress responses and affective disorders. As one of the most common and distressing neuropsychiatric disorders, major depression is among the main causes for disability-adjusted life years (WHO 2005). The etiology of depression is clearly multifactorial with contributions of genetic and environmental factors (Kalia 2005; Nestler et al. 2002). Clinical experience and experimental studies have implicated a dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis as one of the major factors in the pathogenesis of depression (Claes 2004; Sullivan et al. 2000; Zunszain et al. 2011). The HPA axis is activated upon stress exposure, which triggers the secretion of the hypothalamic corticotropin releasing hormone (CRH) and the subsequent release of adrenocorticotropin hormone (ACTH) from the pituitary. Adrenocorticotropin hormone in turn stimulates the secretion of cortisol (humans) or corticosterone (mice) from the adrenal cortex. These glucocorticoids activate glucocorticoid and mineralocorticoid receptors in several brain areas, as well as in peripheral tissues, and are responsible for the feedback inhibition of CRH and Arginine vasopressin (AVP) release. Patient suffering from major depression often show increased secretion and reactivity to cortisol, as well as elevated CRH levels (Zunszain et al. 2011). In this study, we were interested to determine if B6N
© 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society
Effect of chronic corticosterone application in C57BL/6N and C57BL/6J mice
and B6J mice respond differently to chronically elevated glucocorticoid levels, which is known to produce many neurobiological and behavioral changes in rodents that parallel those associated with human depression (Sterner & Kalynchuk 2010). We have thus implanted mice from both strains with corticosterone-releasing pellets and investigated behaviors that are relevant to anxiety and depression.
Materials and methods Animals Experiments were carried out on 48 male B6J (n = 24) and B6N (n = 24) mice that were bought at the same time from the same breeder (Charles River Laboratories, Sulzfeld, Germany; B6N strain code 027: B6J stock number: 000064). We used only male animals, because most previous studies comparing B6J and B6N mice were performed with males. All animals were kept under identical housing condition. At the beginning of the experiments, mice were 12 weeks old. All animals were single housed in transparent plastic cages (30 × 12 × 12 cm3 ) under a 12 h/12 h light/dark cycle (lights on at 2100 h) at constant room temperature (22∘ C). Food and water were available ad libitum and animals were allowed to habituate to the novel environment for 14 days after arrival. Behavioral testing was performed between 0900 and 2100 h during the dark cycle. Animals were treated according to the German Animal Protection Law.
Drugs and surgery For corticosterone administration, hormone pellets (Innovative Research of America, Sarisota, FL, USA) were used. Pellets (0.5 × 0.5 cm2 diameter) consisted of corticosterone (20 mg/pellet) in a matrix of cholesterol, lactose, celluloses, phosphates and stearates and had a releasing time of 21 days. Placebo pellets consisted of the same matrix and size. Mice were divided into four groups, each of which containing 12 animals, which were implanted with either corticosterone or placebo pellets (B6JCort, B6JPla, B6NCort and B6NPla). For pellet implantation, mice were briefly anesthetized with isoflurane (0.4 ml/min). The fur was removed above the incision side and a 0.6 cm cut in the skin was performed at the left side of the neck in an area between ear and foreleg. The pellet was placed under the skin and the wound was closed with an absorbable thread (VicrylTM Plus, 3-0; Johnson & Johnson International, New Brunswick, NJ, USA). Subsequently, the mice were intraperitoneally injected with Carprofen (Sigma-Aldrich, Henstedt-Ulzburg, Germany) (4 mg per 1 kg body weight). The surgery required approximately 10 min per animal. Subsequently, animals were returned to their home cages and allowed to recover for 14 days before behavioral tests were performed. Activity in the home cage was recorded during the recovery period between days 6 and 9.
Pre-testing In order to assess the baseline behavior of untreated B6J and B6N mice, sucrose preference test (SPT), forced swim test (FST) and open field test (OFT) were performed with a total of 18 B6J and B6N mice prior to the main experiment under equal experimental conditions (see below). We used independent groups of animals for pre-testing and the main experiment in order to avoid confounding effects of habituation.
in the following order: days 6–9: home cage activity (HCA), day 14: SPT, day 15: OFT, day 16: light–dark test (LDT), day 17: FST, day 21: coat state, decapitation, organ extraction and blood sampling. All behavioral tests were performed between 0900 and 2100 h.
Home cage activity Activity in the home cage of the mice was recorded by means of an infrared sensor system (Mouse-E-Motion; Infra-e-motion, Germany). The sensors were applied on the top of the cages 14 cm above the floor level and the activity of the mice was recorded with a sampling rate of 1 Hz, starting from 2100 h for the next 72 h continuously. The activity recordings were averaged every 4 min and expressed as ‘arbitrary activity index’. Please note that the animals remained in their cages and were not moved to a new one. A habituation to a recording system was therefore not necessary. The activity during the dark phases, the bright phases and the overall activity during 72 h was analyzed and compared between the groups.
Sucrose preference test Sucrose preference test was performed according to Strekalova and Steinbusch (2010) to assess anhedonia in mice. Sucrose preference test was performed in a two-bottle paradigm for 3 h at the beginning of the dark phase, where the mice received two bottles, one filled with 1.5% sucrose solution and one with tap water. After half of the time (90 min), the places of the bottles were changed to avoid side bias. The weight of each bottle was determined before and after the SPT. In order to familiarize the mice to the sucrose taste, they received a 2.5% sucrose solution instead of water for 2.5 h (0900–1130 h) 1 day before the actual test. The SPT was performed before and after the surgery. The sucrose preference was calculated in percent as the amount of consumed sucrose solution divided by the total liquid intake.
Open field test The open field arena (45 × 25 × 22 cm3 ) was illuminated with 5 lx. Each mouse was placed in the right corner of the arena and total distance traveled (cm), visits to the center and rearings were recorded for 5 min to test the reactivity of the animals to novel environment. Between every run, arenas were cleaned carefully with antiseptic solution. The movements were recorded using an automatic infrared movement sensor (ActiMot, TSE-Systems GmbH, Bad Homburg, Germany).
Light–dark test The LDT was performed to assess anxiety as it has a high comorbidity with depression. A dark compartment was inserted into the open field box, which took one third of the area and contained a mouse hole (entrance) in the middle. The light intensity in the bright area was set to 650 lx to represent an aversive environment. The dark compartment was completely shielded from light providing a less threatening environment for the mouse. The mouse was placed into the right corner of the bright area facing the entrance of the dark compartment. The activity of the mouse was recorded for 5 min by the software ACTIMOT v2.0 (TSE-Systems GmbH). The time spent and the distance traveled in the bright and dark areas were taken as parameters for the analysis.
Forced swim test
After pellet implantation, the weight and the food intake of every mouse were measured twice a week over the period of 21 days.
Three plastic cylinders (25 × 19 × 13 cm) were filled with water (23∘ C) up to a height of 12.5 cm and located in a testing room, which was illuminated with 5 lx. Mice were placed in the cylinders and immobility time was measured manually during the test by the experimenter using a stopwatch. Immobility was determined for a period of 4 min, which started after 2 min of habituation. The habituation period was chosen to eliminate the influence of the initial arousal.
Behavioral tests
Coat state
Four days before pellet implantation, sucrose preference of the animals was tested. After pellet implantation, tests were performed
The total score of the coat state resulted from the sum of scores from seven different body parts: head, neck, dorsal and ventral
Body weight and food consumption
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(a)
(b)
(c)
Figure 1: Pre-testing results. (a) There was no significant baseline difference in sucrose consumption between the two sub-strains at baseline. (b) The two sub-strains showed no difference in the immobility time in the forced swim test. (c) Exploratory locomotor activity in open field test was lower in B6N strain compared with B6J animals. Experiments were carried out with 12 animals per group.
coat, tail, forepaws and hindpaws. For each area, a score of 2 was given for well-groomed coat and 0 for an unkempt coat (maximum score 14 = very good condition; minimum score 0 = poor condition).
Blood sampling and adrenal gland weighting After judging the coat state, mice were decapitated, brains were removed and shock-frozen in isopentane/dry ice for further investigation. Adrenal glands were extracted from the remaining mice and stored at −80∘ C until weighing. For detection of corticosterone concentrations, blood sera were isolated. The trunk blood was allowed to clot for 1 h at room temperature. Thereafter, it was kept at 4∘ C for 24 h and centrifuged at 1000 rpm (rounds per min) for 10 min. These sera were analyzed for corticosterone concentrations with commercially available corticosterone HS EIA kit (IDS GmbH, Frankfurt am Main, Germany). For the analysis, all mice were killed between 2200 and 2300 h to minimize the variance due to ultradian variations of endogenous corticosterone levels.
Results Pre-tests Prior to the main behavioral experiments, pre-testing was carried out to evaluate baseline differences in the behavior of the two untreated sub-strains. We found no strain effects in the SPT (t 15 = 1.596, P = 0.13) (Fig. 1a) and FST (t 18 = 0.992, P = 0.33) (Fig. 1b). However, B6J mice showed a significantly higher locomotor activity in the open field than B6N animals (F (1,33) = 30.75, P < 0.001) (Fig. 1c). Before the implantation of corticosterone pellets, mice of both sub-strains consumed more sucrose than water (B6N mice: 1.3 ± 0.4 ml, 0.3 ± 0.1 ml; P < 0.05; B6J mice: 1.4 ± 0.6 ml, 0.2 ± 0.1 ml; P < 0.05).
Blood serum corticosterone Statistical analysis Statistical analysis was carried out using EXCEL 2011 (Microsoft Corp., Redmond, WA, USA), PRISM v.4.0.1 (GraphPad Software, San Diego, CA, USA) and STATISTICA Version 7 (StatSoft Inc., Tulsa, OK, USA) software. For normally distributed data, statistical significance was determined by one- or two-way analysis of variance (ANOVA), as appropriate, followed by post hoc t-tests with Bonferroni correction. In cases where the data were not normally distributed, they were analyzed by Kruskal–Wallis ANOVA followed by Dunn’s post hoc test.
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Serum corticosterone levels of the trunk blood were measured. Two-way ANOVA showed a significant main treatment effect (F (1,24) = 22.24, P < 0.0001), but no strain effect (F (1,24) = 0.75, P = 0.396) and no interaction (F (1,24) = 0.78, P = 0.3863). Post hoc analyses confirmed that serum corticosterone levels of B6NCort mice (P < 0.05) and B6JCort mice (P < 0.005) were significantly higher compared with placebo groups (Fig. 2). Genes, Brain and Behavior (2015) 14: 292–300
Effect of chronic corticosterone application in C57BL/6N and C57BL/6J mice
test. During the dark phase, when mice were active, we found significant strain (F (1,15) = 6.32, P = 0.0238) and treatment (F (1,15) = 4.65, P = 0.0477) effects, but no interaction (F (1,15) = 0.37, P = 0.5527).
Open field test
Figure 2: Serum corticosterone levels after pellet implantation. Corticosterone levels were significantly increased in mice that have been implanted with pellets releasing corticosterone. There was no significant strain difference. The data are represented as mean ± SEM. Experiments were carried out with nine animals per group. **P < 0.001.
Food intake and body weight To investigate the effect of corticosterone on food intake and body weight, we measured the food intake and body weight on days 1, 4, 8 and 11 after the surgery. Repeated measures three-way ANOVA for body weight showed a significant strain × treatment interaction (F (1,55) = 17.37, P = 0.0001), which was also significant over time (strain × treatment × time: F (3,165) = 19.74, P < 0.0001). A subsequent repeated measures two-way ANOVA for both strains showed a significant time × treatment effect in both strains (B6N: F (3,84) = 30.62, P < 0.0001; B6J: F (3,81) = 3.06, P < 0.05), although post hoc testing showed no significant weight differences between the treatment groups for any of the individual measurements in B6J animals (Fig. 3a). Repeated measures three-way ANOVA for food intake also showed a significant strain × treatment interaction (F (1,56) = 36.92, P < 0.0001), which did not change over time (strain × treatment × time: F (2,112) = 1.90, P = 0.154). A subsequent one-way ANOVA showed a significant treatment effect in B6N mice (F (1,28) = 75.85, P < 0.0001), but not in B6J animals (F (1,28) = 0.065, P = 0.80). Thus, corticosterone-treated B6N mice consumed significantly more and gained more weight. In contrast, corticosterone treatment had no effect on food intake in the B6J strain (Fig. 3b).
Home cage activity In order to control for the locomotor activity, being important for interpretation of other behavioral results, the activity of the mice was measured in their familiar environment. For this purpose, an infrared system was attached to the cages and the activity was recorded for 72 h (Fig. 4a,b). We found neither strain (F (1,15) = 1.86, P = 0.1923) nor treatment (F (1,15) = 0.01, P = 0.9244) effects and no interaction (F (1,15) = 0.10, P = 0.7568) during the light phase of the Genes, Brain and Behavior (2015) 14: 292–300
Two-way ANOVA showed significant main effects for strain (F (1,38) =23.95, P < 0.0001) and treatment (F (1,38) = 37.24, P < 0.0001) on locomotor activity, but no interaction (F (1,38) = 0.52, P = 0.46). Bonferroni post hoc testing showed that corticosterone treatment reduced locomotor activity in both sub-strains, when compared with placebo-treated controls (B6JCort: 48.4 ± 16.1 m; B6JPla: 73.2 ± 5.2 m; B6NCort: 39.2 ± 15.2 m; B6NPla: 47.0 ± 26.9 m; P < 0.01; Fig. 5a). Two-way ANOVA of center visits also showed significant main effects for strain (F (1,38) = 10.74, P < 0.01) and treatment (F (1,38) = 14.42, P < 0.001), but no interaction (F (1,38) = 0.54, P = 0.47). The Bonferroni post hoc test showed a significant reduction of the number of center visits within the B6N strain. Corticosterone-treated B6N mice visited the center less frequently than the placebo-treated B6N mice (B6NCort: 5.6 ± 8.6; B6NPla: 17.5 ± 20.0; P < 0.01) (Fig. 5b). The B6J sub-strain showed no significant reduction of center visits (B6JCort: 17.5 ± 9.3; B6JPla: 30.8 ± 12.1; P = 0.1). No significant effects could be detected in the number of rearings in both sub-strains for strain (F (1,38) = 1.79, P = 0.19) treatment (F (1,38) = 0.60, P = 0.44) and interaction (F (1,38) = 0.16, P = 0.69) (data not shown). Two-way ANOVA showed significant effects for treatment (F (1,38) = 7.04, P < 0.01) and strain (F (1,38) = 8.03, P = 0.007) on the ratio of center visits/locomotor activity, but no interaction (F (1,38) = 2.64, P = 0.11). Bonferroni post hoc testing showed a reduced ratio of center visits/locomotor activity only in the corticosterone-treated B6N sub-strain (P = 0.02) (Fig. 5c).
Sucrose preference test Two-way ANOVA showed a significant main effect of strain (F (1,44) = 6.87, P < 0.05), but not treatment (F (1,44) = 2.99, P = 0.09) and no interaction (F (1,44) = 0.90, P = 0.347). However, separate analysis of the data for the two strains showed that B6J mice showed no difference in sucrose preference, when compared with the placebo-implanted B6J group (B6JCort: 78.1 ± 19.2%; B6JPla: 82.0 ± 17.7%; F (1,22) = 1.87, P = 0.1) (Fig. 6a). In contrast, corticosterone-treated B6N mice had a significantly smaller preference to the sucrose solution compared with placebo-treated mice (B6NCort: 60.0 ± 17.7%; B6NPla: 73.6 ± 15.5%; F (1,22) = 8.48, P < 0.001) (Fig. 6b) and showed no significant sucrose preference (one sample t-test: t (11) = 1.958, P = 0.0761).
Light–dark test The LDT was performed to assess anxiety-related behaviors. The distance and the time spent in the bright area were analyzed during 5 min. No significant differences could be detected between the groups (P > 0.05) (data not shown).
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(a) Figure 3: Food intake and body weight gain. All data are represented as mean ± SEM. B6J animals are shown in the left panels, B6N in the right panels. (a) Body weight was measured on days 1, 4, 8 and 11 after pellet implantation. Please note that B6NCort mice had a significantly higher body weight than B6NPla animals on days 8 and 11. (b) The average daily food consumption was calculated for three intervals after the surgery (days 1–4, 5–8 and 9–11). Please note that corticosterone had no effects on food intake in B6J mice, whereas B6NCort consumed significantly more than B6NPla mice. Experiments were carried out with 15 animals per group. ***P < 0.0001; **P < 0.001.
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Figure 4: Home cage activity. Home cage activity was measured during a 72-h period with an infrared system. The activity was separately analyzed during the dark phase (a), during the light phase (b). The data are represented as mean ± SEM (n = 5, BL6N Placebo: n = 4). **P < 0.01.
Forced swim test Two-way ANOVA showed a significant main effect for treatment (F (1,43) = 5.30, P = 0.48) but not for strain (F (1,43) = 0.51, P > 0.05) and no interaction (F (1,43) = 2.89, P = 0.09). Bonferroni post hoc testing showed that the treatment effect was only significant in the B6N strain (B6NCort: 112.1 ± 27.8 seconds; B6NPla: 60.9 ± 43.1 seconds), but not in the B6J strain (B6JCort: 81.2 ± 44.1 seconds; B6JPla: 73.5 ± 55.6 seconds; P < 0.05) (Fig. 7). Thus, corticosterone administration resulted in an increased immobility time in B6N, but not in B6J mice.
Judgment of coat state Compared with placebo, corticosterone treatment yielded a highly significantly reduced outer appearance in the B6N but not in the B6J sub-strain (Kruskal–Wallis ANOVA U = 32.63, P < 0.001). The fur of the corticosterone-treated
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B6N mice became beamless and shaggy (B6NCort: range 6–11 points; B6NPla: range 9–14 points). Within the B6J group, corticosterone treatment did not have a significant effect on the outer appearance of the animals. Placebo- and corticosterone-treated mice did not differ in quality of fur and general body condition (B6JCort: range 11–14 points; B6JPla: range 13–14 points) (Fig. 8).
Adrenal gland weight Additionally, we weighed the adrenal glands to see if they were differently affected by corticosterone application in the sub-strains. Analysis showed significant main genotype (F (1,20) = 16.63, P = 0.00059) and treatment effects (F (1,20) = 139.11, P < 0.0001), but no interaction (F (1,32) = 2.44, P = 0.128). Post hoc analysis showed that the adrenal glands of both B6Cort groups are significantly smaller compared with placebo groups [B6JPla: mean 4.02 mg (range: Genes, Brain and Behavior (2015) 14: 292–300
Effect of chronic corticosterone application in C57BL/6N and C57BL/6J mice
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Figure 5: Open field test. (a) Locomotor activity in the open field test was assessed 16 days after pellet implantation as the distance traveled. (b) The number of center visits in the open field was assessed as a measure of anxiety-related behavior. (c) Ratio of center visits and distance traveled for further assessment of anxiety-related behavior. Experiments were carried out with 12 animals per group. **P < 0.01; ****P < 0.0001; *P < 0.05 (two-way ANOVA followed by Bonferroni post hoc test).
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Figure 6: Sucrose preference test. Sucrose preference was assessed 4 days before and 14 days after pellet implantation: Corticosterone-implanted B6J mice showed no difference to placebo-implanted mice (a), whereas corticosterone-treated B6N mice showed a significantly decreased sucrose preference (b). Experiments were carried out with 12 animals per group. *P < 0.05
3.2–4.6 mg); B6JCort: mean 2.58 mg (range: 2.0–3.4 mg); B6NPla: mean 3.69 mg (range: 2.3–4.9 mg); B6NCort: mean 1.70 mg (range: 1.1–2.3 mg; P < 0.0001)].
Discussion In this study, we have investigated the manifestation of behavioral changes after chronic corticosterone exposure in Genes, Brain and Behavior (2015) 14: 292–300
B6J and B6N mice. We found that subcutaneous implantation of corticosterone-releasing pellets increased food intake, reduced open field center visits, decreased sucrose preference, increased immobility time in the FST and worsened the coat state in B6N, but not in B6J mice. These findings are consistent with a substantial body of evidence showing that the chronic administration of corticosterone produces robust changes in several emotional and depression-like behaviors (Gourley et al. 2008; Gregus et al. 2005; Johnson et al.
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Figure 7: Forced swim test. The immobility of B6J mice in the forced swim test did not differ between placebo- and corticosterone-implanted animals, whereas the immobility of corticosterone-implanted B6N mice was significantly higher compared with placebo-implanted mice. Experiments were carried out with 12 animals per group. *P < 0.05 (two-way ANOVA followed by Bonferroni post hoc test).
Figure 8: Coat state. B6J mice showed no changes in the coat status at day 21 after corticosterone pellet implantation, whereas the coat condition of corticosterone-implanted B6N mice was rated lower compared with the placebo group. Experiments were carried out with 12 animals per group. ***P < 0.001 (Kruskal–Wallis ANOVA followed by Dunn’s test).
2006; Kalynchuk et al. 2004; Murray et al. 2008; Sterner & Kalynchuk 2010; Stone & Lin 2008). This difference between the two strains was not due to differences in the pharmacokinetics of corticosterone, because plasma levels were similarly elevated in both strains after corticosterone pellet implantation. These findings therefore suggest that B6N mice are more sensitive to the effects of chronic corticosterone than B6J animals.
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B6J and B6N mice showed a number of subtle differences in emotional behaviors under non-stressed conditions (Bryant et al. 2008; Hager et al. 2014; Matsuo et al. 2010; Simon et al. 2013). However, for some behaviors such as open field activity and center time, the differences between the two strains were inconsistent. While Matsuo et al. (2010) described increased activity and center times for B6J mice, Bothe et al. (2004) found no significant difference between the strain. One study even examined the two strains using standardized protocols in different sites and reported opposite findings, depending on where the animals were tested (Simon et al. 2013). The present results support as modestly higher exploratory activity of untreated and placebo-treated B6J mice. Corticosterone reduced the activity of both strains to a similar extend. However, the number of center visits was only decreased in B6N, but not in B6J mice. We did not find a significant strain effect when testing for HCA during the light phase, when the animals were resting. However, during the active dark phase, B6J mice were slightly more active than B6N mice and corticosterone-treated animals were less active than the placebo group. Home cage activity was also investigated by Hager et al. (2014) in a slightly different experimental setting with a DualCage system. They also found an increased locomotor activity of B6J mice during the first part of the dark (active) phase. Thus, B6J mice are somewhat more active than B6N animals. B6N mice treated with corticosterone also showed significant decrease in sucrose preference and immobility in the FST, two behaviors that are often considered to be associated with a depression-like emotional state (Deussing 2006; Russo & Nestler 2013; Yacoubi & Vaugeois, 2007). However, it should be noted that, although the FST has predictive validity for antidepressant drugs, the behavior has not been related to any pathophysiology (Nestler & Hyman 2010; Sterner & Kalynchuk 2010). Untreated or placebo-treated mice showed no strain differences in forced swim immobility. Previously, one study reported no difference between the two strains in this test (Mouri et al. 2012), whereas another study showed a higher immobility in B6N mice (Matsuo et al. 2010). B6N mice also showed changes in the appearance of their coat state after chronic corticosterone application. Their fur became shaggy and dull, which was not the case in the placebo-treated controls nor in B6J animals. A worsening of the coat state due to either metabolic changes or a depression-induced reduction of grooming behavior has been regarded an indicator for a depression-like emotional state (Griebel et al. 2002; Santarelli et al. 2003). Similar changes in the coat state have been observed previously with B6N mice after chronic corticosterone application in drinking water (David et al. 2009). Our results are in agreement with previous demonstrations that depression-like behaviors developed in B6N mice in response to corticosterone treatment or other stress-dependent paradigms resulting in elevated blood corticosterone levels (Stone & Quartermain 1999; Zhao et al. 2008). Strekalova et al. (2004) have shown increased forced swim immobility, reduced exploratory behavior and reduced sucrose preference after exposure of B6N mice to mild chronic stress for 4 weeks. Our results indicate that despite their high genetic similarity, the N and J sub-strains of B6 mice react differently to Genes, Brain and Behavior (2015) 14: 292–300
Effect of chronic corticosterone application in C57BL/6N and C57BL/6J mice
chronic corticosterone application. A previous study examining avoidance behavior and risk assessment in these two strains found a high degree of variation among individual B6 animals, although the study reconfirmed a higher degree of fear behavior in B6N compared with B6J mice (Hager et al. 2014). The high degree of individuality and the resulting high variance may necessitate relatively large group sizes in order to detect treatment effects. In this respect, the size of our groups, which ranged from 12 to 15 mice per group and was thus similar to the typical group sizes used by comparable studies (Bryant et al. 2008; Matsuo et al. 2010; Mouri et al. 2012), may have still been too small to detect moderate treatment effects in B6J animals. Our findings have implications on the interpretation of experimental results addressing the function of specific genes in depression using transgenic or knockout animals on these backgrounds. Genetic manipulations that result in an enhanced resistance to depression-like pathologies may be more difficult to ascertain in mice on a B6J background, while the opposite might be true for B6N animals. The genetic background used for the analysis of mutant phenotypes should therefore be carefully selected and reported.
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Acknowledgement This work was supported by a grant from the Deutsche Forschungsgemeinschaft (FOR926, SP6). A.Z. is a member of the Excellence Cluster ‘Immunosensation’.
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