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If you have any questions, please do not hesitate to contact us. This manuscript from the Lim laboratory follows upon their recent work showing that serine modulates sleep in Drosophila. The focus here is on threonine, which appears to be sleep-promoting, likely through an effect on GABA signaling.
The idea that metabolic factors are important regulators of sleep is gaining recognition, and this study is an interesting addition in that regard. As written though, this big picture view of the work does not come across until the end of the Discussion; laying it out earlier in the manuscript e. Abstract and Introduction would significantly boost the appeal of the work. From a scientific perspective, it would be good to have the mechanism nailed. This will require attention to the major points in each of the reviews below.
Attending to these will likely involve experiments that will take about months in addition to the time needed for re-writing parts of the manuscript. These concerns derive from our consultation on the two reviews. They highlight specific points and need to be seen along with the two reviews.
As pointed out in the summary above, the big overview needs to be linked to the core of every section of the Results. Ideally, they should show how GABA signaling is affected, but at the very least address the points made by the reviewers or satisfactorily exclude clock cells. Yet, the results suggest that an explanation could be at the level of GABA synthesis presynaptically Figure 4—figure supplement 6. This need to be better discussed. Although this may be much more difficult to show. Maybe it should be repeated?
Please particularly note the points about rutabaga made by reviewer 1. Both reviewers feel that this is an important experimental point to address. To deal with potential developmental roles, it will be good to see at least one adult- specific manipulation and phenotype of GABA manipulation. The authors favor the idea that threonine acts through GABA signaling, pointing out that threonine and GABA and their metabolic derivatives are competitive substrates?
For what-for the GABA receptor? However, how does this explain interactions of threonine with presynaptic GABA-CaLexa signals are increased in GAD neurons by threonine and blocking GAD neurons with shibire is stated to attenuate effects of threonine? The manuscript shows specificity for threonine by using another amino acid as control arginine , but what about specificity for GABAergic cells? The blocking effect of GAD-GAL4 driven shibire on threonine action is questionable Figure 4 as the threonine effect in controls in this experiment is even smaller than normal, and even normally it is not great.
Is this really relevant even if significant? The comparisons in Figure 4B are hard to follow. If the point is that threonine has no effects when GABA B -R1 is knocked down, then it should be shown that threonine increases sleep in the GAL4 alone control, but not in the knockdown, relative to the same genotypes not treated with threonine from the data, it looks like threonine does increase sleep, probably significantly, in the knockout.
Instead, control and knockdown are compared with each other, both in the presence of threonine.
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If the learning assay with rutabaga is to show that increased sleep with threonine rescues learning in rutabaga, then the authors should show that blocking the sleep increase with deprivation abrogates rescue. Otherwise, the direct effects of threonine on learning cannot be excluded. Does threonine or the threonine-increasing mutant display any kind of metabolic phenotype, as does the GABAT mutant Maguire et al.
Given that the underlying mechanisms are the same or overlapping, it is worth at least discussing this point. The authors use Student's t-test a lot, which is not ideal or stringent enough for most measurements. These are potentially interesting findings, but additional data are required to strengthen some of the claims.
However, they do not show that these neurons have any role in sleep. Nor do they show any increase in CaLexA signal in known sleep centers in response to Thr. However, it is unclear whether the effect is sufficient to contribute significantly to SPET. To show that a gene is involved in a process, it is important to provide converging evidence from multiple sources: e. Adult specific knockdown or developmental rescue could alleviate the concern. If the reduction is not statistically significant, it may be due to the unusually large error bar in one of the conditions.
Additional data may provide a clearer picture. These unexpected features of the data raise concerns about the validity of the rest of the data. However, it would strengthen the conclusion if additional memory mutants were tested. Please see reviewer 1 's points on this,. It would be informative to show these data in a format where sleep amount per 30min or 1h is plotted as a function of time of day.
Although pharmacological data somewhat alleviate this concern, a genetic demonstration using hypomorphs? Where are the data for light-independence of SPET? In these situations, standard ANOVAs that assume equal variance across conditions are not appropriate. Some of the N's for behavioral experiments are as low as 8. This seems too low.
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As noted in the minor point 7, some of the statistics do not seem appropriate. We modified our title, Abstract, and Introduction to better state our view regarding the metabolic control of sleep behaviors in the revised manuscript. In our revised manuscript, we provided new pieces of evidence that exclude the possible requirement of clock-dependent control of sleep in SPET. Second, a genetic loss of per or Clk shortened or lengthened sleep latency, respectively, in LD cycles Liu et al. Please see our point-by-point responses to additional points made by each reviewer below.
While this could be a compensatory decrease, additional lines of our evidence indicated that suppression of the metabotropic GABA transmission indeed induced SPET-like sleep phenotypes in control-fed flies and displayed non-additive effects with SPET in threonine-fed flies please see our responses to the reviewer comment and the reviewer 2, major point 2 below. We thus reasoned that dietary threonine decreased GABA levels likely as a metabolic consequence important for SPET and better discussed it in our revised manuscript.
Please see our specific responses to the reviewer comments and as well as the reviewer 2, major points 1 and 2 below. Please see our response to the reviewer comment below. In our revised manuscript, we provided new data that dietary threonine rescued memory deficits in another memory mutants Figure 6B. Using caffeine-induced sleep suppression, we further showed that this memory rescue required threonine-induced sleep Figure 6B and C.
Please see our responses to the reviewer comment and the reviewer 2, major point 6. In our revised manuscript, we provided converging evidence from multiple genetic and transgenic sources e. While our pharmacological manipulations of GABA transmission independently confirmed the genetic evidence, oral administration of GABA-relevant inhibitors and agonists to adult flies also excluded the possible developmental effects of GABA manipulations.
Please see our specific responses to reviewer 2, major points 3 and 4 below. The structural homology among threonine, GABA, and their derivatives led us to the hypothesis that these relevant chemicals may act as competitive substrates in enzymatic reactions for their overlapping metabolism. As the reviewer suggested above, they may similarly act as competitive ligands for GABA receptors i.
In our revised manuscript, we further found that threonine-fed flies displayed low levels of GABA and glutamate compared to those in control-fed flies Figure 4A. This model is consistent with our observations that genetic suppression of GABA transmission by the conditional blockade of synaptic transmission in GAD1-expressing neurons or by the transgenic depletion of metabotropic GABA receptors in R2 EB neurons drives sleep in control-fed flies i.
By contrast, high levels of extracellular GABA e. Given this revised model, we reason that some group of GAD1 neurons i. Accordingly, we revised our Results and Discussion to clarify this model and better interpret our results. As the reviewer suggested, we tested additional Gal4 drivers expressed in other sleep-regulatory loci i. As the editor suggested above, we repeated the shibire experiment in a different incubator with better control of the internal temperature and provided more convincing data in our revised manuscript Figure 4B.
We confirmed that overexpression of the temperature-sensitive shibire in GAD1-expressing neurons induced sleep in control-fed condition and masked SPET only at restrictive temperature.
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We also noted that low i. Given the comments from both reviewers please also see the reviewer 2, minor comment 9 , we realized that the different format was rather confusing. Accordingly, we presented all our behavioral data in the same format in the revised manuscript. In our revised manuscript, we showed that dietary threonine also rescued memory deficits in dumb mutants Figure 6B. Co-administration of caffeine with threonine substantially blocked sleep induction as well as memory rescue in dumb mutants Figure 6B and C. By contrast, caffeine alone did not significantly affect short-term memory in control and dumb mutants under our experimental condition.
These data thus support that dietary threonine rescues memory mutants in a sleep-dependent manner. To examine any metabolic phenotypes induced by dietary threonine, we compared the relative levels of free amino acids and energy metabolites between control- and threonine-fed flies. Accordingly, we compared and discussed these metabolic phenotypes in GABA-T mutants and threonine-fed flies in our revised manuscript.
We also clarified our statistical analyses in the text, figures, and figure legends of our revised manuscript.
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As mentioned in our response to the reviewer comment above, we did not see any detectable increase in the CaLexA signals from sleep-regulatory mushroom body or dopaminergic neurons upon threonine diet Figure 4—figure supplement 4D and E. Lack of a specific Gal4 driver that targets these LN only, however, did not allow us to validate that these effects were necessary for SPET.
Nonetheless, it led us to find that a conditional blockade of synaptic transmission in GAD1-expressing neurons, including LN, actually induced sleep in control-fed flies, and masked SPET in threonine-fed flies. We revised our manuscript accordingly. As the reviewer suggested, we provided converging evidence from multiple sources in the revised manuscript. We have been testing adult-specific knockdown or developmental rescue using tubGal80ts.
Unfortunately, heterozygous controls of the key UAS transgenes e. Although it was likely due to their leaky expression, this limited our experimental conditions to genetically validate the developmental defects. Regarding CG, we could not fully exclude the possibility of metabolic compensation or developmental effects using transgenic reagents available to us.
So, we toned down our conclusion and stated these possibilities in our revised manuscript. In our original manuscript, we examined sleep behaviors in male trans-heterozygous mutants of Rdl and could not observe their short sleep latency compared to heterozygous controls in control-fed condition. Since the original paper has shown the latency phenotype in female Rdl mutants Agosto et al. Regarding the transgenic manipulations of circadian pacemaker neurons, we tested male transgenic flies in our original manuscript and could not detect any phenotype in their sleep latency.
As the reviewer mentioned above, these data were not consistent with previous observations in female flies that have suggested the wake-promoting role of PDF-expressing neurons Parisky et al. Accordingly, we examined SPET in female transgenic flies that have been validated for clock-dependent control of sleep latency in PDF neurons. Consistent with the published result Liu et al. These new pieces of our genetic evidence more convincingly demonstrate that SPET requires neither Rdl - nor PDF clock-dependent control of sleep drive.
In our revised manuscript, we further showed that dietary threonine rescued memory deficits in dumb mutants likely in a manner dependent on threonine-induced sleep Figure 6B. Please see our response to the reviewer comment above. We included the sleep profiles of threonine-fed flies as well as their sleep latency at different time-points of the day in the revised manuscript Figure 2C and D, Figure 2—figure supplement 2.
In our revised manuscript, we tested several mutants trans-heterozygous for GABA-T alleles Figure 3—figure supplement 1A and found that those harboring weaker allelic combinations did not display a floor effect on sleep latency, but their sleep behaviors were actually resistant to SPET Figure 3A.
To clarify this, we included this statement in Materials and methods Statistics section of the revised manuscript. To clarify this, we modified our text, figures, and figure legends in the revised manuscript to better describe our statistical comparisons among different genotypes and conditions.
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In our original manuscript, we generally measured the latency to sleep onset right after lights-off in LD cycles ZT12 but we also observed SPET on the sleep latency after mechanical arousal at ZT16 i. So, we modified our text in the revised manuscript.
Error bars in our original violin plots indicate SD although large variations in some genotypes e. We quantified the CaLexA signals from each hemisphere since we often observed asymmetric ones in the whole-brain images. The CaLexA reporter involves a transcriptional amplification step. However, the reviewer 1 also mentioned that the original Figure 4B was rather hard to follow please see the reviewer comment The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.