INCREASED GENE EXPRESSION OF SELECTED VESICULAR AND GLIAL GLUTAMATE TRANSPORTERS IN THE FRONTAL CORTEX IN RATS
EXPOSED TO VOLUNTARY WHEEL RUNNING

There is no doubt that physical exercise is a part of a healthy life style being beneficial for both metabolic and cardiovascular functions as well as emotional well being. The mechanisms of positive somatic effects of exercise are under intensive investigation and several new regulatory pathways have been revealed (1-3). In contrast, though positive effects of exercise on mood are well recognised (4), the central regulatory mechanisms are still not fully understood. The issue is further complicated by the fact that extreme physical demands on an individual may result in neurohormonal changes involved in the stress response (5, 6).

There are several animal models of forced physical activity and one animal model of voluntary exercise based on the access to a running wheel (7). With respect to the central regulatory mechanisms, the majority of findings have been obtained in studies employing the model of voluntary wheel running. There are observations suggesting the involvement of glutamate, serotonin, norepinephrine and dopamine. The changes in glutamatergic system may be suggested based on the findings of increased gene expression of glutamate receptor GluR1 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptors in the ventral tegmental area as well as the prefrontal cortex following voluntary running of 3 weeks (8, 9). Prolonged voluntary wheel running for 6 – 10 weeks resulted in a decrease (10) or increase (11) of hippocampal glutamate in mice. However, increased concentrations of glutamate in the extracellular fluid were observed during static muscle contraction (12) and increased brain glutamate signals were observed following endurance exercise in rats (13). We have recently demonstrated increased excitability and firing activity of serotonin but not noradrenaline and dopamine neurons voluntary wheel running rats (14).

Next to modeling prolonged exercise, the voluntary wheel running may be considered a model of mild stress (8). It is known that under stress conditions glutamate release in the brain is increased. The stress-induced rise in glutamate in the hippocampus seems to be dependent upon corticosterone (15). Furthermore, stress stimuli were found to influence also brain glutamate transporters (16, 17).

The regulation of glutamate is mediated primarily by re-uptake and packaging of glutamate into vesicles for release. There are two types of glutamate transporters. The homeostasis of glutamatergic system is maintained by vesicular glutamate transporters (VGLUTs) present in the membrane of synaptic vesicles. The concentration of glutamate in the synaptic cleft is determined by its clearance through high affinity sodium dependent glutamate transporters. Postsynaptic neuronal excitatory amino acid carrier 1 (EAAC1) (18) as well as glial glutamate transporters (glial glutamate transporter GLT-1; glutamate aspartate transporter GLAST) rapidly terminate the action of glutamate and maintain its extracellular concentrations bellow cytotoxic levels (19).

The effects of physical exercise on glutamate transporter expression were investigated mainly in the context of Parkinson's disease and aging in relevant brain regions such as the striatum and the substantia nigra (20, 21). Less information is available on potential changes in glutamate transporters in brain structures related to stress and mood disorders, such as the brain cortex. The present study was aimed to testing the hypothesis that voluntary wheel running activates the gene expression of glutamate transporters in the brain cortex of rats. Because of the known interrelationships among glutamate neurotransmission, brain plasticity and stress (22), mRNA levels coding for brain derived neurotrophic factor (BDNF) and plasma corticosterone were also measured.

MATERIALS AND METHODS

Animals

Twenty male Sprague-Dawley rats (Velaz, Praha, Czech Republic), weighing 250 g (aged 8 weeks) at the beginning of the experiments were used. The rats were allowed to habituate to the housing facility for 7 days. The animals were housed under standard laboratory conditions with free access to food and water. A constant light-dark cycle was maintained with light on at 06.00 h and off at 18.00 h. Temperature was maintained at 22 ± 2 °C and humidity at 55 ± 10%. All experimental procedures were approved by the Animal Health and Animal Welfare Division of the State Veterinary and Food Administration of the Slovak Republic (permission No Ro 331/16-221) and conformed to the Directive 2010/63/EU.

Voluntary wheel running

The animals were randomly assigned to the control (n = 10) and voluntary wheel running (n = 10) groups. Voluntary wheel running rats were housed individually in plastic cages (35 × 20 × 15 cm) with the free access to a stainless steel activity wheel (diameter = 35 cm; Tecniplast Gazzada, Buguggiate, Italy) as described previously (8, 9). The animals were exposed to running for 3 weeks. The running distance and speed of running were monitored daily. Control sedentary rats were housed two per cage in standard laboratory cages (35 × 20 × 15 cm).

Blood and organ collection

Following 3 weeks of running, the animals were quickly decapitated with a guillotine between 08.00 and 10.30 h in the morning. Trunk blood was collected into cooled polyethylene tubes containing EDTA as anticoagulant and centrifuged immediately at 4°C to separate plasma, which was then stored at –20°C until analyzed. The right frontal cortex was quickly removed, frozen in liquid nitrogen and stored at –80°C until analyzed.

Plasma corticosterone measurements

Plasma corticosterone concentrations were analyzed by a radioimmunoassay after dichloromethane extraction of the steroid from 10 µl aliquots of plasma as described previously (23). The assay had a sensitivity of 0.1 µg/100 ml. The intra- and inter-assay coefficients of variances were 6 and 8%, respectively.

Reverse transcription and quantitative real time-polymerase chain reaction (RT-PCR)

In the samples of right frontal cortex, the total mRNA was isolated by TRIzol® Reagent (Life technologies, California, USA) according to manufacturer protocol. Concentration and purity of mRNA preparations was measured by absorption spectroscopy (Nanodrop 2000). The isolated mRNA (1 µg) was reverse-transcribed to cDNA using oligo (dT) nucleotides by M-MuLV reverse transcription system. Primer BLAST NCBI (24) software was used to design primers specific for studied genes (Table 1). Real time qPCR was used for evaluation of mRNA concentrations. Analysis was performed in a reaction volume of 20 µl by GoTaq qPCR Master Mix (Promega, USA) as described previously (25, 26). Primers (Table 1) were used at a concentrations of 0.25 pmol/µl. Reaction mix consisted of 10 µl GoTaq qPCR Master Mix, 0.2 µl ROX (Promega, USA), 1 µl of forward and reverse primer (5 µM stock solution), 5 – 50 ng cDNA and water was added to the final reaction volume of 20 µl. qPCR was performed by Fast Real-Time PCR System 7900 HT (Applied Biosystems, USA). Initial denaturation at 95°C for 10 min was followed by 40 cycles at 95°C for 15 s, 60°C (VGLUT1-3, GLAST, GLT-1, EAAC1 primers) or 66°C (BDNF, Tubb and PPIA primers) for 90 s of hybridization/extension. Melting curve analysis for 20 min was performed and did not show any unspecific products of PCR. All data obtained by qPCR analysis were evaluated as ng of mRNA (cDNA) according to a standard curve and was normalized to gene expression of peptidyl prolyl isomerase A (PPIA) and beta tubulin gene (Tubb) as reference genes. Gene expressions were evaluated by ΔΔCt calculation and normalized to PPIA and Tubb housekeeping genes as arbitrary units.

Table 1. List of gene specific primers for qPCR.

Statistical analysis

The values were checked for the normality of distribution using the Shapiro-Wilks test. Data not normally distributed were subjected to natural logarithm transformations to normalize the distributions before analyses. One-way ANOVA for factor time followed by Bonferoni post hoc test, was used to compare the daily running distance and speed. The statistical analysis of data on gene expression was performed by Student´s t-test for independent groups. The Pearson correlation was computed to determine relationships among the running distance and other parameters measured. Values are expressed as means ± S.E.M.

RESULTS

The daily running distance was 2.06 ± 0.28 km on the day one and this gradually increased to 6.21 ± 1.05 km by day 21. One-way ANOVA for repeated measures demonstrated a significant effect of time (F(19,171) = 8.27; P < 0.001) and the Bonferoni posthoc test revealed significant (P < 0.001) differences between running distances on day 1 and days 5 – 21 (Fig. 1). Average speed was 2.86 ± 0.03 km/h, and this did not change over the time course (data not shown).

Fig. 1. Daily running distance (km) presented in average values per day ± S.E.M. (n = 10 rats/group). Statistical significance as revealed by one-way ANOVA for factor time. *P < 0.05 in comparison with day 1 (Bonferroni post-hoc test).

The expression of VGLUT1 (Fig. 2A) and VGLUT2 (Fig. 2B) in the frontal cortex did not differ between running and control animals. VGLUT3 mRNA levels (Fig. 2C) were significantly elevated in the group of running animals compared to the values in sedentary controls (P < 0.05). The concentrations of mRNA coding for GLT-1 were significantly increased in the running group (Fig. 2D). GLAST, EAAC1 and GluR1 mRNA levels were not affected by running (data not shown). Rats exposed to voluntary running exhibited significantly increased gene expression of BDNF in the frontal cortex (Fig. 2E) compared to that in controls (P < 0.05).

Fig. 2. The effect of voluntary wheel running on gene expression of vesicular transporters VGLUT1, VGLUT2, VGLUT3, glial transporter GLT-1 and BDNF in the frontal cortex as well as on corticosterone concentrations in plasma. Each value represents mean ± S.E.M. (n = 10 rats/group). Statistical significance as revealed by Student´s t-test: *P < 0.05.

Voluntary running resulted in a rise in plasma corticosterone concentrations (Fig. 2F). Corticosterone concentrations were significantly elevated in voluntary running rats compared to those in control animals (P < 0.05).

Pearson correlation analysis revealed a significant positive correlation between BDNF and GLT-1 mRNA concentrations in running rats (r = 0.72, P < 0.05; Fig. 3). In addition, GLT-1 mRNA concentrations positively correlated with VGLUT1 (r = 0.93, P < 0.05) and GLAST (r = 0.81, P < 0.05) mRNA concentrations. A positive correlation was found also between VGLUT1 and GLAST mRNA levels (r = 0.88, P < 0.05). None of the parameters measured correlated significantly with the running activity.

Fig. 3. The positive correlation between BDNF and GLT-1 mRNA concentration in the frontal cortex of voluntary wheel running rats as calculated by Pearson correlation analysis.

DISCUSSION

The results of the present study demonstrate that voluntary wheel running in rats results in the activation of one vesicular and one glial glutamate transporter gene expression in the brain cortex. The working hypothesis was only partially confirmed. In addition, a rise in BDNF expression in the frontal cortex and increased release of corticosterone induced by voluntary running were observed.

In contrast to our expectation, the gene expression of VGLUT1 failed to be modified by voluntary running. This glutamate transporter has been found to mediate glutamate release under stress conditions and has been implicated in the effects of some antidepressant drugs (17, 27). As voluntary wheel running in rats is a mild stress situation and exercise in general is inducing positive effects on the mood (28), the present observation is somewhat surprising. However, the present study was performed in male animals and Brancato et al. (29) have recently demonstrated alterations in VGLUT1 containing synapses that may contribute to stress susceptibility in female mice. The present findings are consistent with the results of Caffino and colleagues (30) showing no change in VGLUT1 mRNA levels in the prefrontal cortex of male adolescent rats to a swim stress test, unless the rats were pretreated with cocaine.

Similarly, gene expression of glutamate transporter VGLUT2 was not affected by three week voluntary wheel running. The information on the expression of this subtype of glutamate transporter in response to environmental stimuli is scarce. Swedish authors have recently described down-regulated gene expression of VGLUT2 in the cortex of adult rats exposed to early life stress (31) using a different experimental design.

Voluntary wheel running resulted in a significant increase in the gene expression of VGLUT3 in the frontal cortex. Again, very little information is available on the physiological role of this unique glutamate transporter. Interestingly, increased anxiety behaviour was observed in mice lacking VGLUT3 (32). The observation of increased expression of VGLUT3 in response to voluntary running in the present study is thus consistent with positive effects of exercise on anxiety (33).

Present experiments also revealed increased expression of glial glutamate transporter GLT-1 but not GLAST and EAAC1. These results are consistant with increased GLT-1 levels in the striatum due to treadmill exercise in an animal model of Parkinson's disease (21). In another study treadmill exercise resulted in increased EAAC1 expression in the substantia nigra but not in the striatum of aged rats (20). Acute but not long term voluntary wheel running led to increased EAAC1 mRNA levels in the hippocampus, while the gene expression of GLT-1 and GLAST was unchanged (34). Six week prior voluntary wheel running, which improved consequences of peripheral nerve injury, was able to prevent the decrease in GLT-1 protein expression in the spinal cord (35). The results of studies on GLT-1 expression under stress conditions are inconsistent. Some authors reported an increase in GLT-1 in the hippocampus (16, 17). More recent studies demonstrated a reduction of GLT-1 in the hippocampus and other parts of the brain in models of chronic stress and depression (36, 37).

Interestingly, the gene expression of both vesicular transporter VGLUT3 and glial transporter GLT-1 was simultaneously increased by voluntary running. As brain glutamate may increase in response to exercise (13), the upregulation of VGLUT1 and GLT-1 (EAAT2) expression may occur as a compensatory response to exercise- or stress-induced elevations in glutamate (17).

Three-week voluntary running led to increased gene expression of cortical BDNF. It is well consistent with the general knowledge that exercise can induce various growth factors with neuroprotective potential including the BDNF (38). Increased BDNF is likely to contribute to neuroprotective effects of voluntary physical activity (7, 39, 40). The results of the present correlation analysis showing positive correlation between BDNF and GLT-1 mRNA concentrations as well as between several glutamate transporter mRNA levels are in favour of the involvement of glutamate neurotransmission in effects of voluntary exercise.

The finding of increased plasma corticosterone concentrations in voluntary running rats is in agreement with our previous results demonstrating an activation of the hypothalamic-pituitary-adrenocortical axis in response to chronic wheel running (8, 41). In contrast, Uysal and colleagues (7) reported lower serum corticosterone concentrations in voluntary running rats compared to those in sedentary controls. However, because the methodology of corticosterone measurement is not described and the values of serum corticosterone are unrealistically low, a verification of this finding is needed. A limitation of the present study is the fact that sedentary control animals were housed 2 per cage, while the wheel was available for rats caged individually. We cannot exclude some influence of isolation housing of running rats, though the isolation must not necessarily activate the hypothalamic-pituitary-adrenocortical axis (42). The same model was used also in our previous (8, 9) as well as current studies (14).

In conclusion, chronic voluntary wheel running associated with increased brain BDNF expression and elevated plasma corticosterone, results in increased gene expression of VGLUT3 and GLT-1 in the brain cortex without changes in other glutamate transporter subtypes. To our knowledge, this is the first report of increased gene expression of selected vesicular and glial glutamate transporters induced by physical exercise.

Acknowledgements: The authors thank Ludmila Zilava for excellent laboratory assistance.

This work was supported by the grant of Slovak Research and Development Agency (APVV-15-0388) and by bilateral project between Slovak and Hungarian Academy of Sciences.

Conflict of interests: None declared.

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