The Fmr1 KO mice exhibit reduced and fragmented sleep during the light phase.

(A) Waveforms of daily rhythms in sleep behavior under standard 12:12 h light-dark (LD) cycles in WT (blue circle) and Fmr1 KO (yellow triangle) mice. Zeitgeber time (ZT) 0 corresponds to lights on, and ZT 12 to lights off. Sleep was defined by immobility and binned in 1-hr intervals. Both genotypes exhibited clear diurnal rhythms, but the Fmr1 KO mice showed significantly less sleep during the light phase and at several times in the dark phase. Asterisks indicate significant differences between genotypes at individual time points (two-way ANOVA with genotype and time as factors, followed by the Holm-Sidak’s multiple comparisons test, *P < 0.05). The white/black bar on the top indicates the LD cycle; gray shading indicates the dark phase time-period. (B–D) Immobility-defined sleep metrics during the light-phase. Compared to WT, Fmr1 KO mice showed a greater number of sleep bouts, indicating sleep fragmentation, and of shorter duration. Histograms show the means ± SEM with the values from individual animals overlaid. Genotypic differences were analyzed by t-test (*P < 0.05). See Table 1.

Altered behavioral sleep parameters in the Fmr1 KO mice.

Comparisons of sleep behavior in age-matched male WT and Fmr1 KO mice (n = 6/group). Values are shown as the averages ± SEM. For the 24-hr data set, values were analyzed using a t-test. Possible day/night differences were analyzed with two-way ANOVA using genotype (WT vs. Fmr1 KO) and time (day vs. night) as factors, followed by the Holm- Sidak’s multiple comparisons test. Asterisks indicate significant differences between genotypes, while crosshatch those between day and night. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant differences.

The Fmr1 KO mice show unstable locomotor rhythms.

(A) Representative actograms showing daily rhythms in wheel-running activity under LD followed by constant darkness (DD) in WT (left) and Fmr1 KO (right) mice. Activity levels were normalized to 85% of the most active mouse. Each row represents two consecutive days, and the second day is repeated at the beginning of the next row. (B) Waveforms of daily rhythms in cage activity in WT (blue circle) and Fmr1 KO (yellow triangle) mice under the LD cycles. Activity under LD (1-hr bins) was analyzed by two-way ANOVA with genotype and time as factors followed by the Holm-Sidak’s multiple comparisons test (*P < 0.05). There were significant effects of both time (F = 8.84; P = 0.003) and genotype (F = 39.75; P < 0.001) on the temporal pattern of the locomotor activity rhythms. Note that significant genotypic differences were found before and after dawn. Activity metrics in LD (C) and DD (D). Rhythmic power under both LD and DD conditions was significantly reduced in the mutants, which also presented higher imprecision in the activity onset in DD. Histograms show the means ± SEM with the values from individual animals overlaid. Genotypic differences were analyzed by t-test (*P < 0.05). See Table 2.

Activity rhythms were altered in the Fmr1 KO mutants.

The locomotor activity rhythms of adult male WT and Fmr1 KO mice in the standard 12 h:12 hr LD cycles and constant darkness (DD) were monitored using wheel running activity (n=6/group). Values are shown as the averages ± SEM. If the assumptions of normality and equal variance were met, a t-test was used to analyze the data, otherwise the Mann-Whitney Rank sum test was used. Asterisks indicate significant differences between genotypes. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant differences.

The Fmr1 KO mice display deficits in light-regulated circadian behaviors.

(A, B) Photic- suppression (masking) of activity in response to a 1-hr light (300 lx, 4500K) pulse at ZT 14 (lights off; n=10/genotype). The activity level during the light exposure was compared to the activity level during the equivalent hour (ZT 14-15) on the day before the treatment (baseline activity). (A) The genotypic difference in the fold change was determined by t-test, with the mutants showing a significantly reduced suppression of activity as compared to the WT (*P = 0.05). (B) Changes in the activity levels of each individual mice during the baseline window and the light masking were analyzed using a paired t-test. Within-genotype comparisons showed significant suppression in WT (P < 0.001), but not in KO mice (P = 0.12). (C, D) Entrainment induced by a 6-hr-phase advanced LD cycle. Representative actograms of light-induced phase shifts of wheel-running activity rhythms (C). The white/black bars on the top of actograms indicate the LD cycle before (upper) and after (lower) the 6-hr-phase advance. The gray shading in the waveforms indicates the dark phase time-period. The arrows next to the actograms indicate the day when the 6 hours-phase advance was applied. (D) Quantification of the days to re-entrain show that the KO mice required more time to adjust (two-way ANOVA: genotype effect F(1,285) = 130.157, P < 0.001; followed by the Holm-Sidak’s multiple comparisons test *P < 0.001). The entrainment shifting in the WT (blue circle) and the Fmr1 KO (yellow triangle) was quantified by the difference between the activity onset and the new ZT12 on each day. The yellow and blue arrow heads in the graph indicate the day when the activity rhythms are considered well entrained. See Table 3.

The Fmr1 KO mice exhibit difficulty in adapting to the skeleton photic period (SPP).

(A) Representative actograms of daily rhythms in cage activity under standard LD cycles (2 weeks) followed by the SPP (1h light:11h dark:1h light:11h dark) in WT (left) and Fmr1 KO (right) mice. The white/black bars on the top of actograms indicate the baseline LD cycle (upper) and the SPP LD cycles (lower). The gray shading in the waveforms indicates the time of the dark phases. (B) Measures of locomotor activity rhythms under SPP. Many of the parameters measured were significantly different between the genotypes with the mutants being more impacted and showing lower rhythmic power and increased variability of the activity onset. In addition, the mutants displayed higher activity during their day. Histograms show the means ± SEM with the values from each individual animal overlaid. Significant differences (P < 0.05), determined by t-test or Mann-Whitney test, are indicated with an asterisk. (C, D) Light-induced phase delay of free-running activity rhythms in mice exposed to light (300 lx, 4500K) for 15 min at circadian time (CT) 16. Mice were held in constant darkness, by definition, CT 12 is the beginning of the activity cycle in DD for a nocturnal organism. Examples of light-induced phase shifts of wheel-running activity rhythms (C) of WT (left) and Fmr1 KO (right) and quantified phase delay (D). In the representative actograms, the yellow lines indicate the best-fit line of the activity onset across the 10 days before and after the light pulse. The amount of phase delay is determined by the difference between the two lines on the day after the light pulse. The sunny-shape symbols indicate when the mice were exposed to light (CT16). Compared to WT, the Fmr1 KO showed reduced phase shift of their activity rhythms (Mann Whitney U *P = 0.011). See Table 3.

Deficits in circadian light response in the Fmr1 KO mice.

The circadian light response of male adult WT and Fmr1 KO mice was evaluated using four behavioral assays and wheel-running activity. First, masking or suppression of activity that occurs when mice are exposed to 1-hour of light during the night at ZT 14 (n=10 per group). Second, the number of days required for the activity rhythms to re-synchronize to a 6 hours phase advance of the LD cycle (n=11 per group). Third, the mice were held on a skeleton photoperiod (1hr:11hr:1h:11h LD) and basic locomotor activity parameters were measured. Fourth, to measure the magnitude of a light-evoked phase shift of the circadian system, mice were held in constant dark (DD) and exposed to light for 15 min at CT 16 (n=8 per group). Values are shown as the averages ± SEM. If the assumptions of normality and equal variance were met, a t-test was used to analyze the data; otherwise, the Mann-Whitney Rank sum test was used. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant differences.

Abnormal Retinal-Suprachiasmatic Nucleus connectivity in the Fmr1 KO mice.

To trace the projections from the retina to the suprachiasmatic nucleus (SCN) via the Retino-Hypothalamic tract (RHT), WT and Fmr1 KO mice received a bilateral intravitreal injection of Cholera Toxin (β-subunit) conjugated to Alexa Fluor555 and were perfused 72 hours later. (A) Lower intensity of the fluorescently labelled RHT projections can be observed both laterally and medially to the ventral part of the SCN in the Fmr1 KO mice (white arrows) as compared to WT, suggesting a loss of afferent projections to the SCN. (B, C) Densitometric analysis of the distribution of the cholera toxin fluorescence intensity in the ventral SCN (Suppl. Fig. 2) of WT and Fmr1 KO mice. The intensity peaks of the profile plot of 4 to 5 consecutive coronal sections containing the middle SCN were aligned and then averaged to obtain a single curve per animal. Results are shown as the mean ± standard deviation (SD) for the left (B) and the right (C) SCN of each genotype. (D, E) Light-induction of cFos was greatly reduced in the SCN of the Fmr1 KO mice compared to WT. Mice held in DD, were exposed to light (300 lx, 4500K) for 15 min at CT 16, and perfused 45 minutes later (CT 17). (D) Representative serial images of light-evoked cFos expression in the SCN. The inset in the lower left panel shows the lack of cFos immunopositive cells in the SCN of mice held in DD but not exposed to light. Dotted magenta lines delineate the SCN. OC=Optic Chiasm, V=third Ventricle, (E) The number of immune-positive cells in the left and right SCN from 3-5 consecutive coronal sections per animal were averaged to obtain one number per animal and are presented as the mean ± SD per genotype. One-way ANOVA followed by Bonferroni’s multiple comparisons test, *P=0.0201. See Table 4

Compromised retinal-SCN connectivity.

Subtle decrease in the relative intensity of Cholera Toxin (β subunit) in the retinal afferents to the suprachiasmatic nucleus (SCN) of Fmr1 KO mice. There was a stronger impact of the loss of FMRP on the induction of light-evoked cFos expression in the SCN. Control no pulse = WT mice held in DD but not exposed to the light pulse at CT16. Histomorphometrical analysis of the SCN revealed no differences between WT and Fmr1 male mice. All measurements were performed by two independent observers masked to the experimental groups. Results are shown as the mean ± SD. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant

The deficits in social recognition and repetitive behaviors of the Fmr1 KO mice correlate with altered sleep behavior.

Tests of Social behavior: (A) In the first stage of the 3-chambers social test, when the testing mouse is given a choice between a stranger mouse and an inanimate object, the Fmr1 KO mice spent less time with the stranger mouse and had a lower social preference index (SPI) than the WT. (B) In the second stage, the testing mouse is given the choice between a chamber with a novel mouse and the one with the familiar mouse. The mutants spent less time with the novel mouse compared to the familiar one, and also in this phase exhibited lower social novelty preference (SNPI) as compared to the WT. (C) The possibility of reduced social recognition was further tested with the 5-trial social test. In this test, the stranger mouse becomes a familiar mouse after 4 exposures to the testing mouse, then a novel mouse is introduced in the 5th trial. The WT mice showed a higher interest for the novel mouse compared to the Fmr1 KO mice. Tests of repetitive behaviors: The amount of digging in the bedding (D) and the percentage of marbles buried (E) were measured with the marble bury test. The Fmr1 KO mice spent longer time digging and buried more marbles compared to WT. (F) Grooming behavior, assessed in a novel arena, was significantly higher in the Fmr1 KO mice as compared to WT. Histograms show the means ± SEM with the values from the individual animals overlaid. Significant differences (*P < 0.05) were determined by t-test or Mann-Whitney test. See also Table 5. Sleep duration (min, G, H) correlated with impaired social recognition and abnormal grooming behaviors. Sleep fragmentation, measured by number of sleep bouts, correlated only with grooming (I,J) (Pearson Correlation test). See Table 6.

Fmr1 KO mice present with deficits in social discrimination.

Comparisons of social discrimination behavior in age-matched WT and Fmr1 KO male mice (n = 8 per group) were assessed using the 3-chamber and the 5-trial social interaction test. Social Preference Index (SPI) = difference in the time spent with the novel mouse and object divided by the sum of the time spent with the novel mouse and the object. Social Novelty Preference Index (SNPI) = difference in the time spent with the novel and familiar mouse divided by the sum of the time spent with both the novel and familiar mice. The repetitive behavior in WT and Fmr1 KO mice was (n=14/genotype) assessed using the marble bury and grooming tests. Values are shown as the averages ± SEM. If the assumptions of normality and equal variance were met, a t-test was used to analyze the data, otherwise, the Mann-Whitney test. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant differences.

Correlation between sleep disturbances and the severity of impaired behaviors in the Fmr1 KO mice.

Data obtained from age- matched WT and Fmr1 KO mice housed under standard LD cycles were tested for associations with the Pearson Correlation test. The most prominent sleep phenotypes were usually observed during the animals’ light-phase sleep, hence, only measures between ZT 0-12 were used for these analyses. The correlation coefficients are reported, those significant are shown in bold and labeled with an asterisk. Alpha = 0.05.

Amelioration of sleep/wake rhythms in the Fmr1 KO mutants by Time Restricted Feeding (TRF).

(A, B) Waveforms of daily rhythms in cage activity using IR detection in the WT (circle) and Fmr1 KO (triangle) mice under ad libitum feeding (ALF) or TRF. The activity waveforms (1-hr bins) were analyzed using a three- way ANOVA with genotype, feeding regimen, and time as factors followed by Holm-Sidak’s multiple comparisons test. There were significant effects of genotype (F(1, 767) = 13.301; P < 0.001) and time (F(23,767) = 94.188; P < 0.001), as well as significant interactions between genotype and time (P < 0.001) and feeding regimen and time (P < 0.001) on the locomotor activity rhythms of both WT and Fmr1 KO mice. The green area indicates the time-period when the food hoppers were opened for 6 hours between ZT 15 and ZT 21. (C- E) Measures of locomotor activity rhythms: Both genotypes exhibited an increase in the power of the rhythms under TRF compared to ALF controls. The increase in early-day and late-night activity as well as the onset variability seen in the Fmr1 KO mice was corrected by the TRF. Data are shown as the means ± SEM; two- way ANOVA followed by Holm-Sidak’s multiple comparisons test with genotype and feeding regimen as factors, *P < 0.05 significant differences within genotypes (different feeding regimen); #P < 0.05 significant differences between genotypes (same feeding regimen). (F, G) Waveforms of daily rhythms of immobility- defined sleep. The sleep waveforms (1-hr bins) were analyzed by two-way ANOVA with time and feeding regimen as factors followed by the Holm-Sidak’s multiple comparisons test. There were significant effects of time for both WT (F(23, 351) = 9.828, P <0.001) and Fmr1 KO (F(23, 351) = 1.806, P = 0.014) mice, but not of feeding regimens. Missing data points precluded the use of three-way ANOVA for these measures. (H-J) Measures of immobility-defined sleep in the light phase. Both genotypes held on TRF exhibited an increase in sleep duration and in sleep bout length as well as a reduction in sleep fragmentation, measured by the number of sleep bouts, compared to their ALF counterparts. Data are shown as the means ± SEM; two-way ANOVA followed by Holm-Sidak’s multiple comparisons test with genotype and diet as factors, *P < 0.05 significant differences within genotype - between diet regimens; #P < 0.05 significant differences between genotypes - same feeding regimen. See Table 7.

Scheduled Feeding improved sleep/wake rhythms in the Fmr1 KO mutants.

Locomotor activity rhythms and immobility-defined sleep were recorded from WT and Fmr1 KO mice on ad libitum feeding (ALF) or time-restricted feeding (TRF; n=8 per group). As the running wheels interfere with the feeders, we used IR to measure the activity rhythms in these experiments. Since the most prominent sleep phenotypes were observed during the light-phase sleep and sleep recordings were paused during the dark phase for adding (ZT15) and removing (ZT21) food, the analyses below only focused on the effects of TRF on sleep during the light-phase sleep (ZT 0-12). Values are shown as the averages ± SEM. Data were analyzed by two-way ANOVA with genotype and treatment as factors, followed by the Holm-Sidak’s multiple comparisons test. Asterisks indicate significant differences within genotype - different feeding regimen, while crosshatch significant differences between genotypes – same feeding regimen. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant differences.

Improved social memory and stereotypic grooming behavior in the Fmr1 KO mice after 2 weeks of TRF.

(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, perhaps, able to distinguish the novel mouse from the familiar mouse. The time spent in social interactions with the novel mouse in the 5th-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 4th (familiar mouse) and 5th (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.

Scheduled Feeding improved social recognition memory and reduced grooming behavior in the Fmr1 KO mice.

Adult male WT and Fmr1 KO mice on ALF or TRF (n=8 per group) were exposed to the 5-trial social test and the grooming test. Data are shown as the averages ± SEM and were analyzed by two-way ANOVA with genotype and treatment as factors followed by the Holm-Sidak’s multiple comparisons test. Asterisks indicate significant differences within genotype – different feeding regimen, while crosshatch those between genotypes – same feeding regimen. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant differences.

TRF repristinates the levels IL-12 and IFNƳ in the plasma of Fmr1 KO mice to WT levels.

(A) The levels of selected plasma pro-inflammatory markers are shown. The increase in IL-12 and IFNƳ in the mutants is lowered by TRF to WT levels. The full list of the assayed makers is reported in Table 9. Data were analyzed with 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 feeding regimen, and #P < 0.05 between genotypes – same feeding regimen. (B-G) Correlations between IL-12 or IFN-γ levels and social recognition or grooming behavior. IL-12 levels correlated with both behaviors. Data were analyzed using the Pearson Correlation, and the coefficients are reported in Table 10.

Scheduled feeding affects the levels of plasma cytokines in WT and Fmr1 KO mice.

The levels of several plasma cytokines were measured in WT and mutants held on ALF or TRF regimen (n=8 per group). Values are shown as the averages ± SEM. Data were analyzed by two- way ANOVA with genotype and treatment as factors followed by the Holm-Sidak’s multiple comparisons test. Asterisks indicate significant differences within genotype – different feeding regimen, while crosshatch those between genotypes – same feeding regimen. Alpha = 0.05. Degrees of freedom are reported between parentheses. Bold values indicate statistically significant differences.

Correlation of the plasma levels of selected inflammatory markers with the severity of sleep disturbances and behavioral deficits.

Data from all 4 groups (WT ALF, WT TRF, Fmr1 KO ALF, Fmr1 KO TRF) were pooled and the Pearson Correlation was applied. The correlation coefficients are reported, those significant are shown in bold and labeled with an asterisk. Alpha = 0.05.