In this study we observed that after REMSD the sizes of DR neurons increased in experimental rats as compared to that of the control rats. The effects were specific to REMSD and not non-specific effects because i) the neurons remained unaffected in LPC, where all other conditions remained identical as those of the experimental group; ii) the REMSD induced changes in neuronal size returned to the reference level (the FMC group) after recovery from REMSD (the REC group of rats); and iii) the affected neurons were located within the anatomical region of the brain, the DR in this case, where functional REM-OFF neurons have been reported [28, 29]. This view may be supported by the fact that adjacent neurons in IC, where such REMS-related neurons have not been reported, remained unaffected. In our previous studies  rats were REMS deprived for 4, 6 and 10 days; significant changes were observed in the brain areas including the LC after 6 and 10 days of REMSD, whereas 4 days of REMSD was ineffective. To prevent unnecessary discomfort to the animals and to follow a uniform pattern of deprivation in this study also we used 6 days of REMSD paradigm.
To avoid estimating the same neuron more than once we analyzed every third serial section. Since the sections were 40 μ m thick it is unlikely that a neuronal soma would span three sections. Also, we selected neurons where the nuclear boundaries were clearly visible. The sampling error was minimized by counting a large number of neurons (900 ± 70) at random across each of the anatomical locations from 4 rat brains in each group. The intra-group data were not significantly different and hence the values were pooled for further analysis.
The flowerpot method  is reliable and it has been reported that when continued beyond 24 hrs it deprives animals maximally of REMS whereas non-REMS is least affected ; therefore, by far this is the best available method which has been extensively used globally for experimental REMSD studies. Notwithstanding, like most other experimental procedures this method also has some limitations, which are taken care of by using various control groups. A common criticism of the use of flowerpot method is that the animal may experience stress due to enforced immobilization and social isolation. Our previous studies have shown that stress, if at all, induced by restricted movement or muscular over-activity, by either keeping them in restricted space or making the rats swim for various lengths of time, respectively, were not the causes of flowerpot method induced REMSD-associated increase in Na-K ATPase and chloride-sensitive Mg-ATPase activities [9, 32]. In some studies multiple platforms have been used to avoid possible social isolation , however, in our studies before the experiments, normally the rats were acclimatized for a few days by maintaining them individually in cages; hence, isolation is unlikely to have influenced the results. We have conducted LPC to rule out non-specific effects. In one study large platform rats also showed elevated level of plasma corticosterone . However, in our studies since LPC rats did not show significantly increased Na-K ATPase and chloride- sensitive Mg-ATPase activities [9, 32] nor did they show significant changes in neuronal sizes (neither earlier  nor in this study), it may be safe to say that changes observed by us in the experimental rats are most likely due to REMSD. Notwithstanding, it is also important to keep in mind that whichever method we use, in principle, experimental animals are subjected to a new environment, which by itself, may have some bearing on the results ; designing suitable control experiment is always a challenge to the experimenter. We are also aware of the other limitation of this study e.g. the same neuron could not be estimated before and after the treatment(s), as suffered by most cellulo-behavioral studies; hence, for confirmation one needs to study the same neuron before and after REMSD.
It was observed in this study that following REMSD, the neurons in DR increased in size as compared to controls; whereas neurons in IC were not affected. It has been reported that REMSD alters membrane fluidity , neuronal excitability [4, 22], cytomorphometric changes  and neuronal death . The increased excitability after REMSD is associated with increased Na-K ATPase activity , which among other functions, maintains the transmembrane potential and the osmotic balance within cells to stabilize cell volume [12, 35]. On the other hand, studies have shown correlation between neuronal size and neuronal excitability [35, 36]. Although it might offer some explanation for observing increased neuronal size in the DR after REMSD, it does not explain the possible mechanism of action and why IC neurons were not affected by the REMSD.
There is evidence that suggests the presence of REM-OFF type of cells in the DR nucleus  and those neurons do not cease firing during REMS without atonia . It has been reported that the LC possesses REM-OFF as well as non-REM-OFF neurons and the former do not cease firing but instead continue firing during REMSD . Since DR neurons apparently behave like REM-OFF neurons in relation to REMS, it is expected that those neurons might continue firing during REMSD. Tonic firing of neurons has been proposed to build up a significant metabolic and/or ionic debt . Therefore, it is possible that upon REMSD an increased activity in DR neurons will cause increased Na+ concentration inside the neurons. REMSD-induced increased intracellular positivity, a reflection of depolarization of neurons, supports this view . Increased Na+ concentration and metabolites inside a cell would cause increased water influx into the neurons due to osmosis, thus resulting in swelling and increased cell size [4, 39]. However, recently it has been shown that a small fraction of DR neurons may not behave as REM-OFF type  and the DR contains serotonergic as well as non-serotonergic neurons . Therefore, as a caution, it must be emphasized that all the DR neurons may not behave identically and such cellular and physiological differences cannot be distinguished from the results of this study.
We observed that a REMSD-induced increase in neuronal size was prevented by PRZ, suggesting that NA acting on α1-adrenoceptor was responsible for REMSD-induced increase in DR neuronal size. Although there is no direct evidence to suggest that NA increases DR neuronal size, the following circumstantial evidences support our contention; i) the LC NA-ergic neurons project to DR neurons and regulate their activity ; ii) the activity of DR neurons is turned off by the withdrawal of inputs from the LC during REMS ; iii) cessation of firing of the LC- NA-ergic neurons is a necessity for occurrence and maintenance of REMS ; iv) the NA-ergic neurons in LC cease activity during REMS but are continuously active during REMSD ; v) the NA-synthetic machinery are stimulated after REMSD . Therefore, it is likely that since after REMSD the NA level increases, it in turn modulates the DR neuronal size by acting on α1- adrenoceptors. Our contention may be supported by the fact that REMSD did not affect the IC neurons, which do not possess α1- adrenoceptors . Our earlier findings that REMSD increased Na-K ATPase activity [44, 45] as well as size of LC-NA-ergic neurons  in the rat brain and both the effects were due to increased NA acting on α1- adrenoceptor further support the findings of this study. However, it needs to be investigated if the REMSD induced increased NA would have differential effects on neurons releasing different types of neurotransmitters because after REMSD although the size of the NA-ergic neurons increased, the size of cholinergic neurons showed a decrease .
The soma FF of neurons was calculated as reported earlier; neurons having FF value closer to 1 would be relatively rounder in shape than those closer to zero . It is interesting to note that, in DR, although both the neuronal area and perimeter increased after REMSD, the FF values remained unaltered. This may happen only if the increase in area was proportional to the increase in square of perimeter. Such a condition is most likely to take place in case of smooth and regular-shaped neurons as compared to rough (undulated)-surfaced neurons. This observation is in sharp contrast to the effect of REMSD on LC neurons, where the FF values decreased . It has been reported that morphologically neuronal soma in DR are smooth without any spines on the cell bodies , whereas the perikarya of many LC neurons possess many somatic protrusions, appendages or spines which render irregular surface to those neurons [47, 48]. Hence, it is most likely that after REMSD, the rough or irregular surfaces of the LC neurons stretched or unfolded to become smoother and therefore, the increase in surface area of LC neurons was not proportional to the increase of square of the perimeter, whereas smoother surfaced DR neurons swelled proportionately. Subject to experimental confirmation we propose that DR neurons would be more vulnerable to damage and lysis after REMSD than that of LC neurons.
It has been reported that alterations in the size and shape of cells precede cellular degeneration . It may be argued that neurons upon exposure to conditions detrimental to their survival try to compensate by altering sizes; however, in case of extreme insults, the compensatory mechanism(s) fail and the neurons undergo degeneration. Our finding suggests that neurons in DR increase in size after REMSD, which if not compensated could lead to neurodegeneration and contribute to memory loss, tremors, aggression, depression, psychosis and possibly increase the risk for neurodegenerative conditions, such as Alzheimer's disease. The findings of this study may be relevant to at least Alzheimer's disease because reduced levels of serotonin and imbalance between the serotonergic-cholinergic systems has been reported in patients with Alzheimer's disease  where REMS is also reduced . These findings along with our previous results [7, 12] show significant consequences of REMSD on public health, especially for nightshift workers, viz. nurses, frequent fliers to various time zones, BPO employees, truck drivers and others such as students and elderly people in whom sleep is significantly compromised.