Scheduled feeding improves behavioral outcomes and reduces inflammation in a mouse model of Fragile X syndrome

Reviewer #1 (Public review):

The authors conducted a comprehensive investigation into sleep and circadian rhythm disturbances in Fmr1 knockout (KO) mice, a model for Fragile X Syndrome (FXS). They began by monitoring daily home cage behaviors to identify disruptions in sleep and circadian patterns, then assessed the mice's adaptability to altered light conditions through photic suppression and skeleton photoperiod experiments. To uncover potential mechanisms, they examined the connectivity between the retina and the suprachiasmatic nucleus. The study also included an analysis of social behavior deficits in the mutant mice and tested whether scheduled feeding could alleviate these issues. Notably, scheduled feeding not only improved sleep, circadian, and social behaviors but also normalized plasma cytokine levels. The manuscript is strengthened by its focus on a significant and underexplored area-sleep deficits in an FXS model-and by its robust experimental design, which integrates a variety of methodological approaches to provide a thorough understanding of the observed phenomena and potential therapeutic avenues.

Reviewer #2 (Public review):

Summary:

In the present study, the authors, using a mouse model of Fragile X syndrome, explore the intriguing hypothesis that restricting food access over the daily schedule will improve sleep patterns and subsequently enhanced behavioral capacities. By restricting food access from 12h to 6h over the nocturnal period (the active period for mice), they show, in these KO mice, an improvement in the sleep pattern accompanied by reduced systemic levels of inflammatory markers and improved behavior. These data, using a classical mouse model of neurodevelopmental disorder (NDD), suggest that modifying eating patterns might improve sleep quality, leading to reduced inflammation and enhanced cognitive/behavioral capacities in children with NDD.

Overall, the paper is well-written and easy to follow. The rationale of the study is generally well introduced. Data are globally sound. The interpretation is overall supported by the provided data.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

The authors investigated sleep and circadian rhythm disturbances in Fmr1 KO mice. Initially, they monitored daily home cage behaviors to assess sleep and circadian disruptions. Next, they examined the adaptability of circadian rhythms in response to photic suppression and skeleton photic periods. To explore the underlying mechanisms, they traced retino-suprachiasmatic connectivity. The authors further analyzed the social behaviors of Fmr1 KO mice and tested whether a scheduled feeding strategy could mitigate sleep, circadian, and social behavior deficits. Finally, they demonstrated that scheduled feeding corrected cytokine levels in the plasma of mutant mice.

Strengths:

(1) The manuscript addresses an important topic-investigating sleep deficits in an FXS mouse model and proposing a potential therapeutic strategy.

(2) The study includes a comprehensive experimental design with multiple methodologies, which adds depth to the investigation.

We thank the reviewer for the positive comments.

Weaknesses:

(1) The first serious issue in the manuscript is the lack of a clear description of how they performed the experiments and the missing definitions of various parameters in the results.

We thank the reviewer for pointing out lapses in the editing of the manuscript. We were trying to keep the descriptions of previously published methods brief but must have gone too far, the manuscript has been carefully checked for grammar and readability. Description of the experimental design has been refined and a graphical presentation has been added as Suppl Fig 3. The sleep and circadian parameters have been thoroughly explained in the methods and briefly in the figure legnds.

(2) Although the manuscript has a relatively long Methods section, some essential information is missing. For instance, the definition of sleep bout, as described above, is unclear. Additional missing information includes

Figure 2: "Rhythmic strength (%)" and "Cycle-to-cycle variability (min)."

Figure 3: "Activity suppression."

Figure 4: "Rhythmic power (V%)" (is this different from rhythmic strength (%)?) and "Subjective day activity (%)."

We have provided definitions for the general audience of the terms used in the field of circadian rhythms, such as sleep bout, rhythm power, cycle-to-cycle, masking, and % of activity during the day in the methods and Fig legends. Most of the techniques used in this study, for example, the behavioral measurement of sleep or locomotor activity, are well established and have been used in multiple published works, including our own. We have made sure to include citations for interested readers.

Figure 5: Clear labeling of the SCN's anatomical features and an explanation for quantifying only the ventral part instead of the entire SCN.

We have added more landmarks (position of the third ventricle and optic chiasm) to Fig 5, and have outlined the shell and core of the SCN in two additional images of the ventral hypothalamus in Suppl fig 4.

We had actually quantified the fluorescence in the whole SCN as well as in the ventral part.This was/is described in the methods as well as reported in the results section and Table 4 “Likewise, a subtle decrease in the intensity of the labelled fibers was found in the whole SCN (Table 4) of the Fmr1 KO mice as compared to WT.“

Methods: ” Two methods of analyses were carried out on the images of 5 consecutive sections per animal containing the middle SCN. First, the relative intensity of the Cholera Toxin fluorescent processes was quantified in the whole SCN, both left and right separately, by scanning densitometry using the Fiji image processing package of the NIH ImageJ software (https://imagej.net). A single ROI of fixed size (575.99 μm x 399.9 μm, width x height) was used to measure the relative integrated density (mean gray values x area of the ROI) in all the images. The values from the left and right SCN were averaged per section and 5 sections per animal were averaged to obtain one value per animal………..”

Since the retinal innervation of the SCN is strongest in the ventral aspect, where the retino-hypothalamic fibers reach the SCN and our goal was to identify differences in the input to the SCN, e.g. defects in the retino-SCN connectivity as suggested by some deficits in circadian behaviour; we also looked at intensity of Cholera Toxin in the fibers arriving to the ventral SCN from the retina.

We have added a sentence in the methods about the rationale for measuring the intensity of the cholera toxin labelled fiber in the whole SCN and also just in the ventral part: “Second, the retinal innervation of the SCN is strongest in the ventral aspect, where the retino-hypothalamic fibers reach the SCN, hence, the distribution….”

Figure 6: Inconsistencies in terms like "Sleep frag. (bout #)" and "Sleep bouts (#)." Consistent terminology throughout the manuscript is essential.

We have now clearly explained that sleep bouts are a measure of sleep fragmentation throughout the manuscript and in the fig legends; in addition, we have corrected the figures, reconciled the terminology, which is now consistent throughout the results and methods.

Methods: “Sleep fragmentation was determined by the number of sleep bouts, which were operationally defined as episodes of continuous immobility with a sleep count greater than 3 per minute, persisting for at least 60 secs.”

(3) Figure 1A shows higher mouse activity during ZT13-16. It is unclear why the authors scheduled feeding during ZT15- 21, as this seems to disturb the rhythm. Consistent with this, the body weights of WT and Fmr1 KO mice decreased after scheduled feeding. The authors should explain the rationale for this design clearly.

We have added to the rationale for the feeding schedule. This protocol was initially used by the Panda group to counter metabolic dysfunction (Hatori et al., 2012). We have used it for many years now (see citations below) in various mouse models presenting with circadian disruption to reset the clock and improve sleep. This study represents our first application/intervention in a mouse model of a neurodevelopmental disease.

Hatori M, Vollmers C, Zarrinpar A, DiTacchio L, Bushong EA, Gill S, Leblanc M, Chaix A, Joens M, Fitzpatrick JA, Ellisman MH, Panda S. Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet. Cell Metab. 2012 Jun 6;15(6):848-60. doi: 10.1016/j.cmet.2012.04.019. Epub 2012 May 17. PMID: 22608008; PMCID: PMC3491655.

Chiem E, Zhao K, Dell'Angelica D, Ghiani CA, Paul KN, Colwell CS. Scheduled feeding improves sleep in a mouse model of Huntington's disease. Front Neurosci. 2024 18:1427125. doi: 10.3389/fnins.2024.1427125. PMID: 39161652.

Whittaker DS, Akhmetova L, Carlin D, Romero H, Welsh DK, Colwell CS, Desplats P. Circadian modulation by time-restricted feeding rescues brain pathology and improves memory in mouse models of Alzheimer's disease. Cell Metab. 2023 35(10):1704- 1721.e6. doi: 10.1016/j.cmet.2023.07.014. PMID: 37607543

Brown MR, Sen SK, Mazzone A, Her TK, Xiong Y, Lee JH, Javeed N, Colwell CS, Rakshit K, LeBrasseur NK, Gaspar-Maia A, Ordog T, Matveyenko AV. Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of β cell function. Sci Adv. 2021 7(51):eabg6856. doi: 10.1126/sciadv.abg6856. PMID: 34910509

Whittaker DS, Loh DH, Wang HB, Tahara Y, Kuljis D, Cutler T, Ghiani CA, Shibata S, Block GD, Colwell CS. Circadian-based Treatment Strategy Effective in the BACHD Mouse Model of Huntington's Disease. J Biol Rhythms. 2018 33(5):535-554. doi: 10.1177/0748730418790401. PMID: 30084274.

Wang HB, Loh DH, Whittaker DS, Cutler T, Howland D, Colwell CS. Time-Restricted Feeding Improves Circadian Dysfunction as well as Motor Symptoms in the Q175 Mouse Model of Huntington's Disease. eNeuro. 2018 Jan 3;5(1):ENEURO.0431-17.2017. doi: 10.1523/ENEURO.0431-17.2017.

Loh DH, Jami SA, Flores RE, Truong D, Ghiani CA, O'Dell TJ, Colwell CS. Misaligned feeding impairs memories. Elife. 2015 4:e09460. doi: 10.7554/eLife.09460.

(4) The interpretation of social behavior results in Figure 6 is questionable. The authors claim that Fmr1 KO mice cannot remember the first stranger in a three-chamber test, writing, "The reduced time in exploring and staying in the novelmouse chamber suggested that the Fmr1 KO mutants were not able to distinguish the second novel mouse from the first now-familiar mouse." However, an alternative explanation is that Fmr1 KO mice do remember the first stranger but prefer to interact with it due to autistic-like tendencies. Data in Table 5 show that Fmr1 KO mice spent more time interacting with the first stranger in the 3-chamber social recognition test, which support this possibility. Similarly, in the five-trial social test, Fmr1 KO mice's preference for familiar mice might explain the reduced interaction with the second stranger.

Thank you for this interesting interpretation of the social behavior experiments. We used the common interpretations for both the three-chamber test and the 5-trial social interaction test, but have now modified the text leaving space for alternative interpretations, have soften the language, and mentioned decreased sociability in the Fmr1 KO mice. “The reduced time spent exploring the novel-mouse chamber suggest that the mutants were, perhaps, unable to distinguish the second novel mouse from the first, now familiar, mouse, along with decreased sociability.”

In Figure 6C (five-trial social test results), only the fifth trial results are shown. Data for trials 1-4 should be provided and compared with the fifth trial. The behavioral features of mice in the 5-trial test can then be shown completely. In addition, the total interaction times for trials 1-4 (154 {plus minus} 15.3 for WT and 150 {plus minus} 20.9 for Fmr1 KO) suggest normal sociability in Fmr1 KO mice (it is different from the results of 3-chamber). Thus, individual data for trials 1-4 are required to draw reliable conclusions.

We have added a suppl figure showing the individual trial results for both WT and Fmr1 KO mice as requested (Suppl. Fig. 2).

In Table 6 and Figure 6G-6J, the authors claim that "Sleep duration (Figures 6G, H) and fragmentation (Figures 6I, J) exhibited a moderate-strong correlation with both social recognition and grooming." However, Figure 6I shows a p-value of 0.077, which is not significant. Moreover, Table 6 shows no significant correlation between SNPI of the three-chamber social test and any sleep parameters. These data do not support the authors' conclusions.

Thanks for pointing out the error with statement about Fig. 6I.

“…. Sleep duration (Fig. 6G, H; Table 6) exhibited a moderate to strong correlation with both social recognition and grooming time, while sleep fragmentation (measured by sleep bouts number) only correlated with the latter (Fig. 6J); the length of sleep bouts (Table 6) showed moderate correlation with both social recognition and repetitive behavior. In addition, a moderate correlation was seen between grooming time and the circadian parameters, rhythmic power and activity onset variability (Table 6). In short, our work suggests that even when tested during their circadian active phase, the Fmr1 KO mice exhibit robust repetitive and social behavioral deficits. Moreover, the shorter and more fragmented the daytime sleep, the more severe the behavioral impairment in the mutants.”

(5) Figure 7 demonstrates the effect of scheduled feeding on circadian activity and sleep behaviors, representing another critical set of results in the manuscript. Notably, the WT+ALF and Fmr1 KO+ALF groups in Figure 7 underwent the same handling as the WT and Fmr1 KO groups in Figures 1 and 2, as no special treatments were applied to these mice. However, the daily patterns observed in Figures 7A, 7B, 7F, and 7G differ substantially from those shown in Figures 2B and 1A, respectively. Additionally, it is unclear why the WT+ALF and Fmr1 KO+ALF groups did not exhibit differences in Figures 7I and 7J, especially considering that Fmr1 KO mice displayed more sleep bouts but shorter bout lengths in Figures 1C and 1D.

We appreciate the reviewer’s attention to the subtle details of the behavioral measurement of sleep and believe the reviewer to be referring to differences in the behavioral measurements of sleep with data shown in Table 1 and Table 7. The first set of experiments described in this study was carried out between 2016 and 2017 and involves the comparison between WT and Fmr1 KO mice. The WT and mutants were obtained from JAX. In this initial set of experiments (Table 1), the total amount of sleep in 24 hrs was reduced in the KO, albeit not significantly, and these also exhibited sleep bouts of significantly reduced duration. The pandemic forced us to greatly slow down the research and reduce our mouse colonies. Post-pandemic, we used new cohorts of Fmr1 KO ordered again from JAX for the TRF experiment presented in this study. In these cohorts, the KO mice exhibited a significant reduction in total sleep (Table 7) and the sleep bouts were still shorter but not significantly. We have added to our text to explain that the description of the mutants and TRF interventions were carried out at different times (2017 vs 2022). We would like to emphasize that we always run contemporaneously controls and experimental groups to be used for the statistical analyses. We believe that the data are remarkably consistent over these years, even with different students doing the measurements.

Furthermore, it is not specified whether the results in Figure 7 were collected after two weeks of scheduled feeding (for how many days?) or if they represent the average data from the two-week treatment period.

This is another good point raised by the reviewer. The activity measurements are collected during the 2 weeks (14 days) then the TRF was extended for a 3 more days to allow the behavioral sleep measurements.

We have added a supplementary figure (Supp Fig 3) depicting the different experimental designs.

The rationale behind analyzing "ZT 0-3 activity" in Figure 7D instead of the parameters shown in Figures 2C and 2D is also unclear.

We have added to our explanation. In prior work, we found that the TRF protocol has a big impact on the beginning of the sleep time, hence, we specifically targeted this 3-hours interval in the analysis.

In Figure 7F, some data points appear to be incorrectly plotted. For instance, the dark blue circle at ZT13 connects to the light blue circle at ZT14 and the dark blue circle at ZT17. This is inconsistent, as the dark blue circle at ZT13 should link to the dark blue circle at ZT14. Similarly, it is perplexing that the dark blue circle at ZT16 connects to both the light blue and dark blue circles at ZT17. Such errors undermine confidence in the data. The authors need to provide a clear explanation of how these data were processed.

Thank you for bringing this to our attention. The data were plotted correctly, however, those data points completely overlapped with those behind, masking them. We have now offset a bit them for clarity.

Lastly, in the Figure 7 legend, Table 6 is cited; however, this appears to be incorrect. It seems the authors intended to refer to Table 7.

We have corrected this error, thank you.

(6) Similar to the issue in Figure 7F, the data for day 12 in Supplemental Figure 2 includes two yellow triangles but lacks a green triangle. It is unclear how the authors constructed this chart, and clarification is needed.

We have corrected this error. As the reviewer pointed out, we filled the triangle on day 12 with yellow instead of green.

(7) In Figure 8, a 5-trial test was used to assess the effect of scheduled feeding on social behaviors. It is essential to present the results for all trials (1 to 4). Additionally, it is unclear whether the results for familial mice in Figure 8A correspond to trials 1, 2, 3, or 4.

The legend for Figure 8 also appears to be incorrect: "The left panels show the time spent in social interactions when the second novel stranger mouse was introduced to the testing mouse in the 5-trial social interaction test. The significant differences were analyzed by two-way ANOVA followed by Holm-Sidak's multiple comparisons test with feeding treatment and genotype as factors." This description does not align with the content of the left panels. Moreover, two-way ANOVA is not the appropriate statistical analysis for Figure 8A. The authors need to provide accurate details about the analysis and revise the figure legend accordingly.

We apologies for the confusing Figure legend which has been revised:

“Fig. 8: TRF improved social memory and stereotypic grooming behavior in the Fmr1 KO mice. (A) Social memory was evaluated with the 5-trial social interaction test as described above. The social memory recognition was significantly augmented in the Fmr1 KO by the intervention, suggesting that the treated mutants were able to distinguish the novel mouse from the familiar mouse. The time spent in social interactions with the novel mouse in the 5^th-trial was increased to WT-like levels in the mutants on TRF. Paired t-tests were used to evaluate significant differences in the time spent interacting with the test mouse in the 4^th (familiar mouse) and 5^th (novel mouse) trials. *P < 0.05 indicates the significant time spent with the novel mouse compared to the familiar mouse. (B) Grooming was assessed in a novel arena in mice of each genotype (WT, Fmr1 KO) under each feeding condition and the resulting data analyzed by two-way ANOVA followed by the Holm-Sidak’s multiple comparisons test with feeding regimen and genotype as factors. *P < 0.05 indicates the significant difference within genotype - between diet regimens , and #P < 0.05 those between genotypes - same feeding regimen. (C) TRF did not alter the overall locomotion in the treated mice. See Table 8.”

To assess social recognition memory, mice underwent a five-trial social interaction paradigm in a neutral open-field arena. Each trial lasted 5 minutes and was separated by a 1-minute inter-trial interval. During trials 1–4, the test mouse was exposed to the same conspecific (Stimulus A) enclosed within a wire cup to permit olfactory and limited tactile interaction. In trial 5, a novel conspecific (Stimulus B) was introduced. Time spent investigating the stimulus B mouse (defined as sniffing or directing the nose toward the enclosure within close proximity) was scored using AnyMaze software. A progressive decrease in investigation time across trials 1–4 reflects habituation, while a significant increase in trial 5 indicates dishabituation and intact social recognition memory. In our data, there was not a lot of habituation in both genotypes, but clear differences can be appreciated between trial 4 with the now familiar mouse and trial 5 with novel mouse. Fig. 8A plots the results from individual animals in Trial 4 with a familiar mouse and in Trial 5 with a novel mouse, we have well specified this in the legends. As such, these data were analyzed with a pair t-test.

We used Tow-Way ANOVA to analyse the data reported in Panel 8B and as well as the results in Table 8. This has been clarified in the legend.

(8) The circadian activity and sleep behaviors of Fmr1 KO mice have been reported previously, with some findings consistent with the current manuscript, while others contradict it. Although the authors acknowledge this discrepancy, it seems insufficiently thorough to simply state that the reasons for the conflicts are unknown. Did the studies use the same equipment for behavior recording? Were the same parameters used to define locomotor activity and sleep behaviors? The authors are encouraged to investigate these details further, as doing so may uncover something interesting or significant.

We agree with the reviewers, and believe that the main differences were likely in the experimental design and possibly interpretation.

(9) Some subtitles in the Results section and the figure legends do not align well with the presented data. For example, in the section titled "Reduced rhythmic strength and nocturnality in the Fmr1 KOs," it is unclear how the authors justify the claim of altered nocturnality in Fmr1 KO mice. How do the authors define changes in nocturnality? Additionally, the tense used in the subtitles and figure legends is incorrect. The authors are encouraged to carefully review all subtitles and figure legends to correct these errors and enhance readability.

Nocturnality is defined as the % of total activity within a 24-h cycle that occurred in the night, since this can be confusing and we agree that it was not well explained we have removed it from the subtitle/figure legends.

We have adjusted the subtitles as recommended; however, the tense of the verbs might be a matter of writing style.

Reviewer #2 (Public review):

Summary:

In the present study, the authors, using a mouse model of Fragile X syndrome, explore the very interesting hypothesis that restricting food access over a daily schedule will improve sleep patterns and, subsequently, behavioral capacities. By restricting food access from 12h to 6h over the nocturnal period (active period for mice), they show, in these KO mice, an improvement of the sleep pattern accompanied by reduced systemic levels of inflammatory markers and improved behavior. Using a classical mouse model of neurodevelopmental disorder (NDD), these data suggest that eating patterns might improve sleep quality, reduce inflammation and improve cognitive/behavioral capacities in children with NDD.

Strengths:

Overall, the paper is very well-written and easy to follow. The rationale of the study is generally well-introduced. The data are globally sound. The provided data support the interpretation overall.

Thank you for the positive comments.

Weaknesses:

(1) The introduction part is quite long in the Abstract, leaving limited space for the data provided by the present study.

We have revised the Abstract to better focus on the most impactful findings as suggested.

(2) A couple of points are not totally clear for a non-expert reader: - The Fmr1/Fxr2 double KO mice are not well described. What is the rationale for performing both LD and DD measures?

We did not use the Fmr1/Fxr2 double KO mice in this study.

While measurement of day/night differences in activity rhythms are standardly done in a light/dark (LD) cycle, the organisms must be under constant conditions (DD) to measure their endogenous circadian rhythms (free running activity); this is often needed to uncover a compromised clock as entrainment to the LD cycle can mask deficits in the endogenous circadian rhythms.

(3) The data on cytokines and chemokines are interesting. However, the rationale for the selection of these molecules is not given. In addition, these measures have been performed in the systemic blood. Measures in the brain could be very informative.

The panel that we used had 16 cytokines/chemokines which are reported in Table 9. The experiment included WT and mutants held under 2 different feeding conditions with an n=8 per group. If we are able to obtain more resources, we would like to also carry out a comprehensive investigation of immunomediator levels as well as RNA-seq or Nanostring in selected brain regions associated with ASD aberrant behavioural phenotypes, for instance the prefrontal cortex.

(4) An important question is the potential impact of fasting vs the impact of the food availability restriction. Indeed, fasting has several effects on brain functioning including cognitive functions.

We did not address this issue in the present study. Briefly, the distinction between caloric restriction (CR) and TRF, in which no calories are restricted, has important mechanistic implications in mouse models. While both interventions can impact metabolism, circadian rhythms, and aging, they operate via overlapping but distinct molecular pathways. These have been the topic of recent reviews and investigations. Importantly, the fast-feed cycle can also act as a circadian entrainer (Zeitgeber)

Ribas-Latre A, Fernández-Veledo S, Vendrell J. Time-restricted eating, the clock ticking behind the scenes. Front Pharmacol. 2024 Aug 8;15:1428601. doi: 10.3389/fphar.2024.1428601. PMID: 39175542; PMCID: PMC11338815.

Wang R, Liao Y, Deng Y, Shuang R. Unraveling the Health Benefits and Mechanisms of Time-Restricted Feeding: Beyond Caloric Restriction. Nutr Rev. 2025 Mar 1;83(3):e1209-e1224. doi: 10.1093/nutrit/nuae074.

(5) How do the authors envision the potential translation of the present study to human patients? How to translate the 12 to 6 hours of food access in mice to children with Fragile X syndrome?

Time-restricted feeding (TRF) is a type of intermittent fasting that limits food intake to a specific window of time each day (usually 8–12 hours in humans), is being actively studied in adults for benefits on metabolic health, sleep, and circadian rhythms. However, applying TRF to children is not currently recommended as a general intervention, and there are important developmental, medical, and ethical considerations to take into account.

On the other hand, we believe that the Fmr1 KO mouse is a good preclinical model for FXS because it closely recapitulates key molecular, cellular, and behavioral phenotypes observed in humans with the disorder. A number of the behavioral phenotypes seen in the mouse mirror those seen in patients including increased anxiety-like behavior, sensory hypersensitivity, social interaction deficits and repetitive behaviors so there is strong face validity.

As we show in this study, Fmr1 KO mice present with disrupted sleep/wake cycles and reduced amplitude of circadian rhythms, consistent with findings in individuals with FXS. This makes the Fmr1 KO an excellent model to test out circadian based interventions such as scheduled feeding.

We believe that pre-clinical research in Fmr1 KO mice bridges the gap between basic discovery and human clinical application. It provides a controlled, cost-effective, and biologically relevant platform for understanding disease mechanisms and testing interventions. These types of experiments need to be done before jumping to humans to ensure that the human trials are scientifically justified and ethically sound.

Reviewer #1 (Recommendations for the authors):

The authors should:

(1) Revise the Methods section for clarity and completeness.

We have re-worked the methods for clarity and completeness.

(2) Provide consistent and precise definitions for all parameters and terms.

We believe that we have provided definitions for all terms.

(3) Clarify the rationale for experimental designs, such as the feeding schedule.

We have added to the rationale for the feeding schedule. This feeding schedule has been used in a number of prior studies including our own. All this work is cited in the manuscript.

(4) Reanalyze and transparently present data, including individual trial results.

We have added to the figure showing the individual trail results for the 5-trial tests as requested (Supplementary Fig. 2).

(5) Conduct appropriate statistical tests and correct figure legends.

We believe that we have carried out appropriate statistical tests and have carefully rechecked the figure legends.

(6) Investigate discrepancies with prior studies to enhance the discussion.

We have added to our discussion of prior work.

(7) Improve language quality and ensure consistency in terminology and grammar.

We have edited the manuscript to improve language quality.

Reviewer #2 (Recommendations for the authors):

(1) The Abstract should be rewritten to provide more room for the obtained data.

We have re-written the Abstract to focus on the most impactful findings.

(2) An additional sentence describing the double KO mice should be added.

We did not use double KO mice in this study.

(3) The rationale for studying LD and DD should be provided.

Measurement of day/night differences are standardly done in a light/dark cycle. To measure the endogenous circadian rhythms, the organisms must be under constant conditions (Dark/Dark).

(4) The data on cytokines/chemokines should be strengthened by performing a larger panel of measures both in blood and the brain.

The panel that we used had 16 cytokines/chemokines which we report in Table 9. This was a large experiment with 2 genotypes being held under 2 feeding conditions with n=8 mice per group. If we are able to obtain more resources, we would like to also carry out RNA-seq in different brain regions.

(5) The authors should discuss in more detail the potential role of fastening vs restriction of food access.

We did not address this issue in the present study. Briefly, the distinction between caloric restriction (CR) and TRF when no calories are restricted has important mechanistic implications in mouse models. While both interventions can impact metabolism, circadian rhythms, and aging, they operate via overlapping but distinct molecular pathways.

(6) The authors should also provide some insight into their view on the potential translation of their experimental studies.

We believe that the Fmr1 KO mouse is considered a good preclinical model for FXS because it closely recapitulates key molecular, cellular, and behavioral phenotypes observed in humans with the disorder. A number of the behavioral phenotypes seen in the mouse mirror those seen in patients including increased anxiety-like behavior, sensory hypersensitivity, social interaction deficits and repetitive behaviors so there is strong face validity. As we demonstrate in this study, Fmr1 KO mice exibit disrupted sleep/wake cycles and reduced amplitude of circadian rhythms, consistent with findings in individuals with FXS. This makes the Fmr1 KO an excellent model to test out circadian based interventions such as scheduled feeding.

Still we are mindful that the translation of therapeutic findings from mouse to human has proven challenging e.g., mGluR5 antagonists failed in clinical trials despite strong preclinical data (Berry-Kravis et al., 2016). Therefore, we are cautious in overreaching in our translational interpretations.

Berry-Kravis, E., Des Portes, V., Hagerman, R., Jacquemont, S., Charles, P., Visootsak, J., Brinkman, M., Rerat, K., Koumaras, B., Zhu, L., Barth, G. M., Jaecklin, T., Apostol, G., & von Raison, F. (2016). Mavoglurant in fragile X syndrome: Results of two randomized, double-blind, placebo-controlled trials. Science translational medicine, 8(321), 321ra5. https://doi.org/10.1126/scitranslmed.aab4109).