## 3 Discussion  
In this study, we present several sets of experimental results demonstrating age- and SD-related changes in NMDA receptors subunits and downstream NO signalling in the cortex of male Wistar rats. While general alterations concerning these key molecular players are bilateral, some subtle molecular asymmetry was also evident. At this stage, the functional and homeostatic impact of described findings remains unknown. In light of the general impact of NMDA receptors and NOS enzymes on neuronal functions and synaptic plasticity mechanisms, it is, however, clear that described changes could contribute towards age-dependent deficit in synaptic plasticity and cognitive decline, with implications for pathobiology of AD and neurodegenerative process. Prior to discussing aging or SD-related alterations, it is worth noting two interesting traits observed in young adult rats with reference to NMDA receptors and NOS activities: (i) moderate lateralization of nNOS and eNOS activity in the parietal cortex of young adult controls (Table 1) and (ii) mild seasonal variations in the expression levels and activity of NOS. Mild asymmetry of nNOS and eNOS isoforms agrees with our previous study in rats and humans [38, 45]. Interestingly, the molecular asymmetry seems to be sensitive to seasonal changes, which typically is associated with changes in sex hormone levels [38, 46]. Incidentally, functional asymmetry of nNOS and eNOS isoforms corresponds with published data (e.g., testosterone and other androgens are able to activate nNOS [47] and eNOS activity [48]). Seasonal fluctuations could possibly also contribute to the variability of some controls in our study, given that our experiments were performed in different time of the year on different animal cohorts. Our results also show an interesting association between changes in various NMDA subunits (NR1/NR2A, NR1/NR2B, and NR2A/NR2B) with some functional implications, in accordance with the literature [29]. A stronger coupling of NR1/NR2B (but not NR2A) and iNOS activity in the parietal cortex, for example, suggests that NR1/NR2B heteromers could be more tightly bound with iNOS at the postsynaptic side of glutamatergic synapses [49]. Analysis of NR1, NR2A, and NR2B subunits show a considerable difference in their sensitivity to age. NR1 decrease was evident in all three sets of experiments and is in agreement with previously published studies [27; 28; 29]. The age-dependent decrease was also found in NR2A or NR2B, although this effect was less pronounced. Assessment of the activity of NOS isoforms at matching time points revealed less convincing data for age-dependent changes. Thus, only in two of three experiments, alterations in nNOS or eNOS levels have been detected, while the level of iNOS remained stable. Analysis of data from all control rats revealed a decrease in nNOS and in eNOS activity (Table 2), which correlated with age-dependent modifications in NR1 and NR2B subunits. Taken as a whole, while there are consistent age-related changes in expression of NMDA receptor subunits and NOS signalling throughout the rat brain, our data do not fully support the HAROLD model, reporting higher sensitivity of the L hemisphere to processes of normal aging [38; 46]. Similar to aging, NMDA receptor level and NOS signalling showed sensitivity to acute and chronic SD. In young adult rats exposed to acute 24 h SD, we could not reproduce increases in NMDA receptor expression observed by others, after 4 h SD, or reductions observed after 72 h SD [23; 24; 25]. In the older animal group, however, we have found a significant increase in iNOS activity in rats exposed to acute SD when compared to age-related controls, known to be induced by prolonged wakefulness. Data in the literature suggest that acute SD-evoked increase in NO via iNOS is an important homeostatic factor [39; 40].