In this interesting study the authors explore mRNA regulation of key glycolytic enzymes and proteins involved in glucose homeostasis in the brain of the American bullfrog during 2 and 30 days of cold-induced hibernation. The authors surprisingly found few consistent hibernation-induced changes in the expression of individual genes but report markedly increased correlation between the genes studied with prolonged hibernation. The authors suggest that this correlation may underlie enhanced glycolytic capacity post-hibernation. Overall, the manuscript is very well-written, and the model has significant potential, although the authors somewhat oversell the uniqueness of the metabolic responses in the brain of this organism. I think this study has value, but I am not yet convinced that the increased correlation in the glycolytic genes reported is adaptive per se, or that such changes are even constrained to just the genes related to the glycolytic pathways that were examined in this study. I think that follow-up work to demonstrate that similar changes in gene correlation do NOT occur in other metabolic pathways is required before the authors can adequately support the conclusions that they have drawn regarding glycolysis. I have the following specific comments.

Major Comments

1. A decrease in genes encoding glycolytic enzymes and/or increased correlation between these genes may or may not indicate a change in the proportional use of this pathway during hibernation. Indeed, the evidence for correlated regulation of gene expression related to glycolysis with prolonged hibernation due to cold exposure is interesting but perhaps not unexpected. What is the overall metabolic decrease in this species while hibernating? If overall metabolic demand is greatly reduced, wouldn’t one expect all metabolic pathways to be reduced to some degree relative to the non-hibernating condition? I.e., if metabolism decreases 80% then glycolytic throughput could decrease by 40% and still be “upregulated” relative to baseline metabolic needs (all made up examples of course). The authors may want to consider this possibility in discussing the “unexpected” downregulation or lack of change in glycolytic genes.

2. In the same vein, one might predict that all metabolic pathways may be downregulated during hibernation, and thus one might expect matching and paired correlations between genes in any given metabolic pathway with prolonged hibernation. The use of oxidative phosphorylation may thus still be important here. The animals are hibernating and cold but are not in hypoxia. So, why would glycolysis be expected to be used over oxidative metabolism in this system when O2 is presumably available? The authors do discuss ischemia tolerance post-hibernation but there’s no indication that ischemia tolerance is needed or useful while IN hibernation. The point I am getting at is that there may be increased correlation between all metabolic genes within each individual metabolic pathway with prolonged hibernation, and not just in the glycolytic pathway. Thus, the exact same relationship regarding correlated genes might exist between genes within the oxphos, TCA, b-oxidation, and other pathways.
   These changes, if uniform across other pathways, might also match the paired changes in glucose transporters, etc (which could also fuel oxidative metabolism of glucose). Such changes might also be expected also to match changes/correlations with AMPK, since this mechanism regulates most of these pathways too! Thus, the conclusions that the authors draw regarding putative benefits from increased gene pair correlation, regulation by AMPK, and their potential contribution to enhanced glycolytic efficiency, despite general downregulation or lack of change in individual glycolytic genes, cannot be supported without also analyzing markers from other key metabolic pathways. Specifically, the authors must demonstrate that the same relationship does not emerge broadly across other metabolic pathways before they can claim that this change is specific to glycolysis.

3. Measurements of blood glucose and lactate changes in hibernation would be useful in supporting the conclusions of this paper regarding the need/use of glycolysis over other metabolic pathways during hibernation. Is this data available in the literature?

4. Discussion – conclusions – There is no evidence presented that these animals are hypoxia-tolerant while hibernating. More importantly, the data does not demonstrate a role for the change in glycolytic organization in hypoxia-tolerance per se. These changes may be correlative to emerging tolerance to hypoxia/ischemia but changes to other pathways may also underlie this tolerance. I suggest the authors wrap up their paper with less sweeping conclusions that are better supported by their results and/or which present alternative possibilities with equal weight.

Minor comments:

• Intro line 6 – Oxidative metabolism is only necessary if brain cannot reduce energy expenditure sufficiently to match energy production from glycolysis alone in anoxia/ischemia/hypoxia. There are many anoxia-tolerant species that achieve this degree of metabolic suppression in brain (e.g., freshwater turtles as the authors note in the next paragraph). This sentence should be modified to include this alternative strategy or to point out that this is only the case in hypoxia-intolerant species or those that cannot achieve such deep reductions in brain metabolic demand.

• Intro line 9 – high-altitude flight is not an ‘energetic pathology of the brain’.

• Intro lines 13-15 – Most “intrinsically hypoxia-tolerant” brains also employ metabolic and functional plasticity in adapting to the onset of hypoxia…for example, channel and spike arrest in turtle brain, downregulation of ATPase activity in the brain of many of the organisms listed, changes in mitochondrial efficiency, etc. Thus, this sentence doesn’t really make sense as a way to define two “types” of hypoxia-tolerant organisms or brains.

• Intro line 23-25 - All the animals discussed above this point are examples in which profound metabolic plasticity has been demonstrated at the tissue and organismal level, and notably in brain. This is thus a common strategy in such tolerant species and not particularly unique to the bullfrog. Please revise your intro to more fairly present the bullfrog as an important but not necessarily unique model in these aspects.

• Intro line 22 – most brains have negligible glycogen stores. Please provide evidence/citations that frogs possess high glycogen stores in brain to support this hypothesis.

• Intro – the abstract and intro focus on hypoxia/ischemia but the experimental design and hypothesis are focused on cold-acclimation. The authors might consider more thoroughly introducing the hibernation phenotype in the intro. It is also not clear how hypoxia relates to hibernation as most hibernating animals do not actually experience hypoxia while hibernating. Certainly, both situations may involve hypometabolism and hibernation may confer protection against subsequent ischemia (preconditioning is widely studied in many models) but these ideas could be more clearly and effectively linked.

• Methods – were animals treated (i.e., hibernation induced) during the typical seasonal period of hibernation for this species? If not, do you expect circannual issues with this approach? Is hibernation in this species dependent at all on light cycle? The 12/12 light cycle may also be problematic and would eliminate seasonal photo cues.

• Fig. 4 – I am colour-blind and the red boxes are nearly invisible to me…the authors might consider a different colour pattern or presentation format to highlight this data.

• Discussion line 282 – What evidence is there that tissue pO2 changes with hibernation in this species? HIF can be activated by many processes, including changes in redox homeostasis and the immune system, to name a few. Please provide evidence of hypoxia in frog tissues while hibernating or discuss studies that demonstrate that this occurs in this species and using this hibernation approach. The reference you refer to studied submerged frogs, which presumably have a very different access to O2 than animals hibernating in terrestrial conditions as described in this study.

• Discussion line 326-329 – This statement is not accurate. Most hypoxia-tolerant species (outside of those that experience lifelong hypoxia at altitude) do tolerate transitions in and out of hypoxia. Some are regular (divers, some subterranean species) while others are seasonal (acute changes from anoxia to normoxia in turtles and fish, some hibernating squirrels, etc.) but this is hardly unique to frogs. Please be more careful to avoid presenting the frog model as a unique brain model when there are many other well-studied models of brain hypoxia-tolerance that undergo similar changes. This doesn’t undercut the value of the frog model.

Matthew Pamenter

Thank you for your detailed attention to my comments. I think the manuscript is much improved by your revision and it's fantastic that the new data fit nicely within this very interesting story. I have no further concerns and support publication of your work in PRSB.