## 2 Results  
### Changes in NMDA Receptors Subunits and NOS Activities Related to FL (Experiment I)  
NMDA receptors and downstream NOS signalling play an important role in activity-dependent modifications in synaptic connectivity and plasticity mechanisms. As a first step, we set out to investigate if changes in these molecular pathways can be detected in the cortex of Wistar rats exposed to FL, and whether these effects are age-dependent. Control experiments for the non-specific effects of the FL apparatus were also carried out, and used as a reference. Figure 1A-C presents a summary histogram of NR1, NR2A, and NR2B response to FL in the frontal cortex (Figure 2 shows representative images of Western blots from these experiments), while Figure 3A-C summarizes the response of downstream NOS isoforms in the R and L parietal cortex separately. Table 1 summarizes the indexes of laterality for particular NOS isoform activities. Quantification of NR1, NR2A, and NR2B expressions in old compared to young rats showed significant age-dependent decreases of NR1 and NR2B (to 89.2% and 95.0%, respectively), while the expression levels of NR2A were age-insensitive. In the same series of experiments, young animals exposed to FL showed a significant decrease in expression of all NR1, NR2A, and NR2B subunits when compared to age-related controls (the drops to 95.7%, to 97.9% and finally to 97.9%). Surprisingly, in old age groups, exposure to FL caused a notable increase in NR1 and NR2B (to 110.2% and 106.1%, respectively) while NR2A expression remained unchanged (Figure 1A-C). Similarly, we analysed the effects of FL on the activity of NOS isoforms, given that NO is one of the key downstream messengers of NMDA receptors. The results are summarized in Figure 3A-C. Measurements with ANOVA with repeated measures, one-way ANOVA, and t-test suggest more pronounced age-related decrease in nNOS activity on the R side (the drop to 71.1%). No age-related changes were observed in eNOS or iNOS activity. Moreover, we did not detect changes in the activity of any of these NOS isoforms related to FL. On the other hand, mild laterality in activity of all NOS isoforms, measured as lateralization index, were found in young adult rats (Table 1). Figure 2: Representative images of Western blots from experiments on young adult and old rats exposed to FL (all data are presented in Figure 1). Representative Western blots showing the expression of NMDA receptor subunits, used for quantification of their changes in the frontal cortex. Anti-NMDA-NR1, anti-NMDA-NR2A and anti-NMDA-NR2B (all fromMerck) were used as primary antibodies. The loading control was incubated with an anti-\(\alpha\)-tubulin antibody (Exbio). FL—forced locomotion, YCTRL—young adult control, YFL—young adult rat exposed to FL, OCTRL—old control, OFL—old rat exposed to FL. Figure 1: Expressions of N-methyl-D-aspartate receptor (NMDA) receptor subunits in the frontal cortex from both hemispheres in young adult and old rats exposed to forced locomotion. Optical density of respective NMDA subunit bands (**A**–**C**) related to that of \(\alpha\)-tubulin. Results are presented as means \(\pm\) SEM. Statistical significance (Student’s \(t\)-test) was calculated with respect to young adult or old controls (* \(p\) < 0.05, *** \(p\) < 0.001). FL—forced locomotion, YCTRL—young adult controls (\(n\) = 8), YFL—young adult rats exposed to FL (\(n\) = 10), OCTRL—old controls (\(n\) = 8), OFL—old rats exposed to FL (\(n\) = 9).