Acute and Chronic Sleep Deprivation-Related Changes in N-methyl-D-aspartate Receptor—Nitric Oxide Signalling in the Rat Cerebral Cortex with Reference to Aging and Brain Lateralization

Round  1

Reviewer 1 Report

The authors have answered my specific comments #1 and #3 and touched upon my general comment in their answer to comment #3..

As for my comment #2,  the added detail makes the sleep deprivation paradigm more understandable to readers who are not in this specific field.

However, “rest for 4 hours a day (from 8.00 a.m. to 11.00 a.m.)” still does not make sense.

Author Response

1) We performed extensive English revision (see two versions of our manuscript - cleen and corrected with tracked changes.

2) We moderately changed details of chronic sleep deprivation experiment in our manuscript. However, we do not fully understand why it was not clear for your. Animals were exposed to sleep deprivation totally 12 days (20 hours a day, animals relaxed for 4 hours a day). During 20 hours of exposition, slow and interupted rotation was applied.    

Reviewer 2 Report

The authors need to show FULL blots with LOADING controls and demonstrate that the same conditions plotted on graphs are on the same blot and demonstrate the same pattern can be seen by readers of the manuscript. Individual cutouts of blots are not permitted bc they do not allow for direct comparison. 

Author Response

1) Extensive English revision was made (see our two versions of our manuscript - clean version and corrected version with tracked changes).

2) Table 1 was totally changed. Now, blots containig also alpha-tubulin are presented here. We hope that it is OK because we had problems with this table. Co-author Jana Sirova - responsible for Western blotting experiments - is in the maternity leave in the present time.     

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round  1

Reviewer 1 Report

In this manuscript, Kristofikova et al. analyzed the Acute and Chronic Sleep Deprivation-Related changes in N-methyl-D-aspartate receptor-Nitric Oxide Signaling in the Rat Cerebral Cortex with Reference to Aging and Brain Lateralization. Authors described age and sleep deprivation alterations in the prevalence of NMDA receptors and NOS could contribute towards the cognitive decline in elderly as well as in the pathobiology of AD and neurodegenerative process. Although this is an interesting study and still I am having a few suggestions.

I did not see any blots for the work done. 

Immunostaining is essential to support the data.

Many important methodological parts are missing such as Golgi cox, qRT-PCR.

References are not cited properly in the methodology (e.g., Sleep deprivation experiments )

Reviewer 2 Report

Kristofikova et al undertook a very ambitious goal, to study the effects of acute (24 h) and chronic (12 day) sleep deprivation on young adult and aged rats: NMDAR subunit expression in the frontal cortex and nitric oxide synthase activities in the right and left parietal cortex. They touch on many interesting questions, but including so much descriptive data makes it very difficult to reach any conclusions. It may have been more effective to study less experimental conditions but more in depth, for example by including cognitive behavioral tests or experimentally connecting to Alzheimer’s Disease by including Ab.

1) Numbers of animals are only mentioned in the summary table 2. Please clarify n/experimental group either in Materials & Methods if consistent between the experiments or in each figure legend if variable.

2) The description of Sleep Deprivation Experiments is not understandable.

Experiment I, forced locomotion: “test time: 7 m/min for 3 hours, from 8.00 a.m. to 11.00 a.m.”

Experiment II, acute SD: “test time: 4 m/min for 4 sec, rest time: 60 sec, from 8.00 a.m. to 8.00 a.m.”

Experiment III, chronic SD: “test time: 4 m/min for 4 sec, rest time: 60 sec, from 11.00 a.m. through the night to 8.00 a.m.), rest for 4 hours a day (from 8.00 a.m. to 11.00 a.m.)”

3) Discussion line 349; If the aim was to reproduce increased NMDAR expression after 4 h SD or reduced NMDAR after 72 h, one or the other pf those time points should have been chosen. One could expect that if the cited results are true, at some time point between 4 and 72 h, the balance will move from increase to decrease.

Reviewer 3 Report

Overall the manuscript by Kristofikova et al provides some interesting molecular clues as to how risk factors may affect cognitive function over time. Although for the most part the observations reported do not show causational relationships, the observations remain compelling. The manuscript would benefit from some minor revisions

1) Western blots should be shown with all loading controls. This is standard practice for the majority of journals. Quantification without the blots themselves is not sufficient. 

2) The rationale for the FL is not clearly described in the introduction or the results section. Further description is required. 

3) The term "biomarker" in Table 2 is not truly accurate. None of these measurements can currently be assessed in vivo, so they are after the fact markers of function. A different and more descriptive term would be helpful.