Impact of voluntary exercise and housing conditions on hippocampal glucocorticoid receptor, miR-124 and anxiety
© Pan-Vasquez et al. 2015
Received: 15 May 2015
Accepted: 11 June 2015
Published: 2 July 2015
Lack of physical activity and increased levels of stress contribute to the development of multiple physical and mental disorders. An increasing number of studies relate voluntary exercise with greater resilience to psychological stress, a process that is highly regulated by the hypothalamic-pituitary-adrenal (HPA) axis. However, the molecular mechanisms underlying the beneficial effects of exercise on stress resilience are still poorly understood. Here we have studied the impact of long term exercise and housing conditions on: a) hippocampal expression of glucocorticoid receptor (Nr3c1), b) epigenetic regulation of Nr3c1 (DNA methylation at the Nr3c1-1F promoter and miR-124 expression), c) anxiety (elevated plus maze, EPM), and d) adrenal gland weight and adrenocorticotropic hormone receptor (Mc2r) expression.
Exercise increased Nr3c1 and Nr3c1-1F expression and decreased miR-124 levels in the hippocampus in single-housed mice, suggesting enhanced resilience to stress. The opposite was found for pair-housed animals. Bisulfite sequencing showed virtually no DNA methylation in the Nr3c1-1F promoter region. Single-housing increased the time spent on stretch attend postures. Exercise decreased the time spent at the open arms of the EPM, however, the mobility of the exercise groups was significantly lower. Exercise had opposite effects on the adrenal gland weight of single and pair-housed mice, while it had no effect on adrenal Mc2r expression.
These results suggest that exercise exerts a positive impact on stress resilience in single-housed mice that could be mediated by decreasing miR-124 and increasing Nr3c1 expression in the hippocampus. However, pair-housing reverses these effects possibly due to stress from dominance disputes between pairs.
Lack of physical activity and increased levels of stress strongly contribute to the development of physical and mental disorders . Psychological stress has been associated with multiple medical conditions, from coronary heart disease  to depression . Conversely, exercise has been shown to improve and prevent both physical and mental diseases [4–6] and thus it is increasingly used for the treatment and prevention of mental disorders . There are numerous advantages when using exercise to improve mental health, such as cost-effectiveness, negligible side effects, additional health benefits and sustainability after the end of the treatment.
The mechanisms underlying the mental health benefits of physical exercise are thought to be related, in part, to reduced levels of anxiety and increased stress coping ability . Nonetheless, the molecular processes mediating the beneficial effects of exercise on stress remain unclear. The glucocorticoid receptor (GR) could play a very important role. Both, stress and exercise increase the activation of the hypothalamic-pituitary-adrenal axis (HPA axis), which is regulated by glucocorticoids via a negative feedback loop . However, stress and exercise differ on the timing and strength of HPA activation and therefore on the consequent impact on mental health. While exercise produces an acute increase of circulating glucocorticoids, stress leads to chronic activation of the HPA axis. Indeed the acute elevations in glucocorticoids linked to exercise seem to have a protective effect against some of the adverse consequences of chronic exposure to high glucocorticoid concentrations, such as insulin resistance and development of diabetes .
Glucocorticoids maintain HPA homeostasis after stress via activation of GR in the hippocampus. Increased GR expression at the hippocampus is highly correlated to enhanced resilience to stress [11, 12]. Conversely removal of circulating glucocorticoids increases GR expression in the hippocampus . Thus, we hypothesized that the beneficial effects of exercise on stress resilience might be related to increased expression of hippocampal GR.
While the GR is widely expressed throughout the body, its expression is tightly regulated in different tissues and developmental stages [12, 14]. This regulation can happen at the transcriptional or translational level . There are multiple splice variants of the GR gene (Nr3c1) resulting from alternative first exon usage . The promoters for many of these alternative first exons lie in regions with high CpG content (CpG islands). Therefore their transcription can be modulated via changes in DNA methylation . In the rat, it has been shown that early life events have significant impact on gene expression and DNA methylation of Nr3c1, in particular the hippocampus-specific exon 1.7 (exon 1 F in mouse and human) [18, 19].
A second layer of regulation of Nr3c1 expression is via RNA silencing by microRNAs. The 3′ untranslated region of the Nr3c1 gene contains multiple microRNAs’ seed regions, including miR-124 and miR-18 . While miR-18 is expressed in multiple tissues, miR-124 is particularly enriched in the brain  and has been shown to inhibit Nr3c1 expression in cultured cells and in vivo . Interestingly, miR-124 expression in the hippocampus peaks during the stress hyporesponsive period, when mild stressors do not produce elevation in glucocorticoid levels in neonates .
A third level of regulation of Nr3c1 expression comes from the adrenal gland where glucocorticoids are produced in response to activation of the adrenocorticotropic hormone receptor (ACTH-R, also known as melanocortin 2 receptor, MC2R). Physical exercise acutely activates the adrenal gland resulting in increased release of glucocorticoids. Long-term physical activity produces biochemical changes and hypertrophy in the adrenal gland [23, 24], which is also observed in chronic stress .
It is widely believed that, for young rodents, group-housing is less stressful than single-housing. For example, post-weaning isolation rearing is a preclinical rodent model for schizophrenia . However, the impact of single-housing, particularly in mice, differs when animals are single-housed in adulthood [27, 28]. Additionally, for male mice pair-housing seems to be a more stressful experience than group-housing (3 or more animals per cage), particularly when the mice do not belong to the same litter, since they fight for dominance [29, 30]. For example learning is affected by the acquisition of dominance status in pair-housed male mice . Conversely environmental enrichment, where animals are housed in larger groups with toys and running wheels, has been shown to be beneficial for multiple conditions (e.g. brain injury, ageing, prenatal alcohol exposure) . However it seems that many of the positive effects of environmental enrichment depend on the mice getting free access to running wheels [33, 32].
These observations have led us to hypothesize that long-term exercise could improve resilience to stress through increased hippocampal Nr3c1, which might be mediated via epigenetic modifications. Thus, the aim of this study was to investigate the impact of exercise and housing conditions on: a) Nr3c1 expression in the hippocampus, in particular Nr3c1-1F, b) Nr3c1 epigenetic regulation (miR-124 expression and DNA methylation at the promoter region of Nr3c1-1F), c) anxiety and d) adrenal gland weight and Mc2r expression.
Results & discussion
Using a rodent model of long-term voluntary exercise we studied, at the molecular level, the mechanisms by which exercise improves mental health.
Distance run and impact of housing and exercise on weight gain and food intake
Animals that exercised ate significantly more during the study, without any significant increase in body weight [F (1,25) = 19.896 p < 0.000] (Fig. 1b). Indeed there was a significant correlation between food eaten and distance run [Pearson’s r (17) =0.706, p = 0.002]. Housing had a significant effect on body weight increase during the study (pair-housed mice showed a higher increase in body weight) [F (1,25) = 15.023 p = 0.001] (Fig. 1c).
The effect of exercise on hippocampal Nr3c1 expression differs depending on the housing conditions
Exercise increases Nr3c1-1F expression at the hippocampus without changing DNA methylation at its promoter
The Nr3c1 gene contains multiple alternative first promoters where transcription can start. We focused on the hippocampus-specific promoter 1 F, which is involved in the regulation of Nr3c1 expression by environmental factors [16, 39]. In agreement with our Nr3c1 findings pair-housed animals expressed lower levels of Nr3c1-1F mRNA in their hippocampus [Two-way ANOVA, F (1,25) = 65.95 p < 0.0001] (Fig. 2c). Moreover, a significant housing*exercise interaction was observed [F (1,25) = 6.124 p = 0.020]. Post hoc comparisons indicated that: a) single-housed mice that exercised had significantly higher Nr3c1-1F expression than single-housed sedentary mice [t (14) = 2.577 p = 0.022] (Fig. 2c) and pair-housed mice that exercised [t (11.55) = −7.368 p < 0.0001] (Fig. 2c); while b) single-housed sedentary mice had significantly higher Nr3c1-1F expression than pair-housed sedentary mice [t (13) = −5.325 p < 0.0001] (Fig. 2c). This suggests that the effects of exercise upon Nr3c1 expression are, at least in part, mediated by modulating the expression of the hippocampus-specific Nr3c1-1F splice variant. As for Nr3c1 there was a significant negative correlation between the weight gained and Nr3c1-1F [Pearson’s r (29) = −0.482, p = 0.0081] expression in the hippocampus (Fig. 2d).
The effect of exercise on hippocampal miR-124 levels differs depending on the housing conditions
We may hypothesize that the different effects of exercise on Nr3c1 and miR-124 expression in single versus pair-housed animals could be due to differences in pulsatile/continuous levels of corticosterone and distance run. As mentioned previously, exercise increases glucocorticoids in a pulsatile manner, while stress leads to overall increase of glucocorticoid levels. This could lead to different effects of housing on Nr3c1 and miR-124 as, in the hippocampus, transient release of glucocorticoids promotes neurogenesis, learning and memory [42, 43]. Conversely prolonged secretion of glucocorticoids during chronic stress has the opposite effect [44, 45]. Finally, despite having two wheels per cage, pair-housed animals run approximately half the distance than the single-housed. This difference in distance run could also have affected expression levels. Future studies should investigate the role of circadian glucocorticoid release on exercise-induced changes of Nr3c1 expression.
Impact of exercise and housing on anxiety
Higher hippocampal levels of Nr3c1 are correlated with a better ability to cope with stress, [11, 12]. Thus, we studied the impact of exercise and housing on anxiety using the EPM test. The EPM is widely used to study anxiety when the animals are exposed to unfamiliar, open and elevated locations. However, some of the EPM measures might be influenced by external factors (such as time of testing or prior exposure to behavioural tests). Thus, in order to reduce the chances that our results could be affected by differences in the motility of the animals (due to exercise-induced fatigue), we used ethological measures (stretch attend postures and unprotected head dips) as well as the conventional ratio “time in the open arms vs. all arms”.
Physical exercise led to a significant decrease in the percentage of time spent at the open arms versus all arms [F (1,28) = 9.251 p = 0.005] (Fig. 5e) and the frequency of entrance to the open arms versus all arms [F (1,28) = 6.331 p = 0.017] (Fig. 5f). No housing differences or housing*exercise interactions were found in these tests. These results are in agreement with Fuss et al. [50, 34] and Onksen et al.  who reported a decrease on the time spent in the open arms in animals that exercised. Interestingly the decrease in open arm entry/time by exercise was mechanistically related to hippocampal neurogenesis [50, 34, 51]. The significant decrease in the open/all arms ratio would suggest that exercise increases anxiety. However, based on previously published literature and motility at the EPM (see below), we could argue that performing the EPM during the light phase of the light/dark cycle might not be a good measure of anxiety when mice run 4–10 km/night (as the motility and tendency of the animal to move to open spaces might be affected by its physical activity during the night). Indeed Santos-Soto et al., using a protocol similar to ours, but performing the EPM test during the first hour of the dark phase (active period for the mouse), reported that exercise significantly increased the ratio of time open/all arms . Likewise, similar results were found for the rat when the EPM test was performed during the dark period . It could be argued, as critically reviewed in , that despite its multiple positive outcomes, wheel running has also some potential negative side effects, which might affect the behaviour of the animal at the EPM. For example the tail of the rodent can get bent upwards, particularly when the animals have access to the wheel for extended periods of time (8 weeks or more ). As rodents require their tail for balance, this physical transformation can affect the performance of the rodents on the open arms of the EPM, where good balance is required. However none of our mice developed an “upward bent” tail, probably due to the fact that the surface of our running wheels was made of a continuous plastic sheet and the mice run for less that 5 weeks. Furthermore, our results show that the distance moved [F (1,28) = 5.79 p = 0.023] (Fig. 5g) and speed [F (1,28) = 6.110 p = 0.020] in the EPM was also significantly lower in the exercise group. Indeed, for single-housed mice that exercised, there was a significant negative correlation between the average distance run in the wheels and the speed [Pearson’s r (9) = 0.667, p = 0.049] and distance walked on the EPM [Pearson’s r (9) = 0.676, p = 0 .045] (Fig. 5h). Furthermore, as mentioned before, the percentage of time the mice spent on stretch attend postures, a sign of low stress levels, shows a strong tendency towards higher time stretching in single-housed mice that exercised.
Impact of exercise and housing on adrenal weight and Mc2r expression
In summary, we conclude that exercise in single-housed mice causes an upregulation of Nr3c1 expression in the hippocampus and exerts a positive impact on stress resilience. This increase was correlated with a downregulation of miR-124, a known epigenetic regulator of Nr3c1. Conversely; pair-housing reverses these effects possibly due to increased dominance disputes over access to the wheels. Overall, the results presented here provide new information about the consequences of exercise and housing on HPA axis regulation and their potential impact in stress resilience.
33, C57BL/6 mice (8 weeks old, purchased from Charles River, UK) were randomly distributed into 4 groups: single-housed exercise (n = 9), single-housed sedentary (n = 7), pair-housed exercise (n = 8) and pair-housed sedentary (n = 8). Mice were on a 12 h light-dark cycle with food and water available ad libitum. Mice in the exercise group had free access to a running wheel for the entire duration of the study. Two running wheels were placed in the cages of pair-housed mice that exercised. Mice in the sedentary group were housed with a non-functional wheel to control for environmental enrichment. Distance and time run by each animal were recorded daily. After 4 weeks mice underwent behavioural tests. 5 weeks after the start of the experiment mice were humanely killed by cervical dislocation and their brains and adrenal glands extracted and immediately frozen.
All procedures and animal care were in accordance with the UK Animals (Scientific procedures) Act 1986 and with the ethics committee guidelines of the School of Life Sciences, University of Nottingham.
Elevated plus maze (EPM)
Four weeks after the start of the experiment (i.e. being single/pair | having access, or not to a running wheel) mice were evaluated using the elevated plus-maze (EPM) test. The EPM consisted of two opposing closed arms (surrounded by high walls) and two open arms (36 × 6 cm) that extend from a central platform (6 × 6 cm) elevated 89 cm above the floor. The mice were placed individually on the central platform facing a closed arm and were allowed to freely explore the maze for 5 min. The behaviour of each mouse was monitored using a video camera, and the movements of the mice were automatically registered and analysed with a computerized tracking system (Ethovision 9, Noldus IT, The Netherlands). Entry into an arm was defined as entry of all four paws into the arm. Measurements included: total distance moved, speed, time spent in the open and closed arms, number of times the animal entered each type of arm, latency before entering an open arm and number of defecations. The mice’s behaviour was recorded and later scored with in-house produced behaviour observation software (Clicker v1.13) by an observer blind to the treatment (housing/exercise). Stretch attend posture from the central square into the open arms was defined as postures where both rear legs of the mouse remain in the central square and the forepaws go forward into the open arm. Unprotected head dip was defined as times when the head of the mouse (or nose of the mouse) went below the edge of the platform. Times were automatically transformed to percentage values.
Gene expression profiling by Q-PCR
RNA was isolated with the AllPrep DNA/RNA/Protein mini Kit (Qiagen, Germany) following the manufacturer’s instructions. 500 ng of RNA were reverse transcribed with Superscript III (Life Technologies, UK) and 15-mer random primers (Sigma, UK). Q-PCR reactions were then performed in triplicates with the SensiMix Plus SYBR Green PCR kit (Bioline, UK) and a RotorGene 3000 cycler (Qiagen, Germany). PCR primers for Nr3c1 [NM_008173.3] [forward primer 5′- AGG CCG CTC AGT GTT TTC TA-3′ and reverse primer 5′- TAC AGC TTC CAC ACG TCA GC-3′], Nr3c1-1F [XM_006525663.2] [forward primer 5′- AGC CAG GGA GAA GAG AAA CT -3′ and reverse primer 5′- TAC CAG GGG GAG CTA AGG AT -3′] and Mc2r transcript variants 1–4 [NM_001271716.1, NM_008560.3, NM_001271717.1, NM_001301372.1] [forward primer 5′- TGG CAG TTT TGA AAG CAC AG -3′ and reverse primer 5′- GCA ATG ACA GAC AGG CTG AA -3′] were designed with the Primer3 software (www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). Primer’s efficiency and specificity were verified using melting curve analysis (single melt curve peak). Gene expression was normalized to the geometric average of two control genes (S18 and Hprt1) according to the GeNorm normalization . The relative expression levels of each mRNA were calculated using a modified 2 Delta-Delta-Ct algorithm .
Genomic DNA was isolated with the AllPrep DNA/RNA/Protein mini Kit (Qiagen, Germany) following the manufacturer’s instructions. gDNA from animals from the same group was pooled (adding 200 ng gDNA from each animal) and 500 ng of the pooled gDNA from each group was bisulfited with the EZ DNA methylation kit (ZYMO research, USA), according to the manufacturer’s protocol. Afterwards, the promoter of the gene encoding Nr3c1-1F was amplified (forward primer: 5′- AGTTTTTTTGTTAGAGTGATATATTT -3′ and reverse primer: 5′- ATTTCTTTAATTTCTCTTCTCCCTAACTC -3′), subcloned into the pGEM-T easy cloning vector (Promega, UK) and 10–15 clones per experimental group were sequenced as previously described in . Primers were designed using “MethPrimer” (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi)
cDNA was synthesised from mature miRNAs using the miRCURY LNA™ Universal cDNA synthesis kit (Exiqon A/S, Denmark), which uses a poly-T primer. Samples were run in triplicate using primers for U6 and mmu-miR-124-3p (MIMAT0000134) (Exiqon A/S, Denmark). Q-PCR was undertaken using the ExiLENT SYBR® Green mastermix kit (Exiqon A/S, Denmark), and the Applied Biosystems 7900HT Fast thermocycler was used in standard mode using cycling parameters recommended by Exiqon. These commercial primers, cDNA synthesis and QPCR amplification kits are specifically optimised for miRNAs detection. Data was acquired using Applied Biosystems SDS2.3 programme. ROX™ (Life Technologies, UK) was used as a passive reference for normalising for non-PCR related fluorescence variations, and was incorporated by SDS2.3 to calculate Ct values. The Ct values were analysed using the comparative Ct method, with U6 as the endogenous control and single-housed and sedentary animals as the calibrator. Spike in samples containing UniSp6 were used as inter plate calibrators and variation of readings between replicate plates was <1.5 %.
Data was analysed using two-way ANOVA. For single comparison between groups we used Student’s t-test. Pearson’s correlation was employed to study the relationship between gene expression, running, weight and/or behaviour. The frequency of methylated CpGs was examined using log-linear analysis. SPSS 22.0 (IBM) Statistical package was used for the analysis. Statistics reported in the text and figures represent the mean ± S.E.M. For all tests, null hypotheses were rejected at probability level of p < 0.05.
This work was supported by BBSRC (Research Experience Placement, N.R. BB/J014508/1), the School of Life Sciences (Small Grant Funding) and the University of Nottingham (FRF4294 and start up funds). We would like to thank Dr. MC Pardon for initial advise on the running wheels and the members of Bio Support Unit (University of Nottingham) for their expert and helpful support along the project.
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