Timelines for the experimental procedures for (A) Cohort 1 and (B) Cohort 2.

(A) Representative images of cerebral cortex and hippocampus stained for 82E1-positive Aβ deposits in WT and (A’) 22-month-old AppNL-G-F mice. A” compares the percent area impacted by Aβ deposition in five brain regions between the two strains. (B) Iba1 immunostaining of the cerebral cortex and hippocampus of WT and (B’) 22-month-old AppNL-G-F mice. (B”) Proportion of 5 brains regions showing severe microgliosis in AppNL-G-F compared to WT mice. Abbreviations: CTX, cortex; RSC, retrosplenial cortex; PPC, posterior parietal cortex; SSc, somatosensory cortex; HPC, hippocampus. Scale bar: 700 µm. Two-way ANOVA revealed significant effect of genotype for both Aβ and Iba1. **p < 0.01 and ***p < 0.001 based on Šidák’s multiple comparisons test.

EEG spectrograms (0-25 Hz) across the 24-h period for a representative individual of each sex and each genotype.

The light and dark phases are indicated in the bar at the top of the spectrogram. Note lower spectral power (indicated by the presence of cooler colors in the 0-5Hz delta range) during the dark phase in the female AppNL-G-F mouse, suggesting that female mice of this strain sleep less during the dark phase. In contrast, the more consolidated periods with higher power (indicated by the presence of warmer colors in the delta/theta range(?)) in the AppNL-G-F/NL-G-F males and females during the light period may indicate more consolidated sleep periods during this phase.

Hourly percent time spent in (A) Wake, (B) NREM sleep and (C) REM sleep as well as activity counts (D) and subcutaneous body temperature (E) across a 24-h recording period in Cohort 1 WT and AppNL-G-F mice recorded at 14-18 months and at 18-22 months of age.

Panels A’-E’ present the corresponding total amounts of each state or physiological parameter for the same 24-h period at each age. **p < 0.01 and ***p < 0.001 based on between group post hoc comparisons using Fisher’s LSD test.

Effects of Age, Genotype, Time of Day and their interactions on vigilance state amounts and activity during baseline for Cohort 1 determined by a mixed-effects

Amount of time in (A) Wake, (B) NREM sleep and (C) REM sleep as well as total activity counts (D) and subcutaneous body temperature (E) during the 12-h light phase in WT and AppNL-G-F mice at 14-18 months and at 18-22 months of age.

Panels A’-E’ present the corresponding results for the 12-h dark phase at each age. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 based on between group post hoc comparisons using Fisher’s LSD test.

Effects of Age, Genotype, Age, Time of Day and their interactions on the number of bouts of each state during baseline for Cohort 1 determined by a mixed-effects

Mean bout duration during (A) Wake, (B) NREM and (C) REM sleep during the 12-h light phase in WT and AppNL-G-F mice at 14-18 months and at 18-22 months of age.

Panels A’-C’ present the corresponding results for the 12-h dark phase at each age. *p < 0.05 and **p < 0.01 based on between group post hoc comparisons using Fisher’s LSD test.

Effects of Age, Genotype, Time of Day and their interactions on bout durations during baseline for Cohort 1 mice determined by a mixed-effects

(A) Distribution of Wake Bouts binned in durations varying from <60 sec to >260 sec in 18-22 month old WT and AppNL-G-F mice. (B) Activity counts per 24-h period in WT and AppNL-G-F mice during the light phase at both ages. Lines connect the activity of levels of each individual studied at the two ages. (C) Activity counts per 24-h period in WT and AppNL-G-F mice during the dark phase at both ages. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 based on between group post hoc comparisons using Fisher’s LSD test.

Comparison of homeostatic response to 6-h sleep deprivation in 18-22 month old WT and AppNL-G-F mice.

(A) Hourly amounts of NREM sleep from ZT0 to ZT12 in AppWT/WT mice on a baseline day (gray line) vs. a day on which 6-h sleep deprivation (SD) occurred (black line) from ZT0-6 (shaded area) followed by a 5-h recovery sleep (RS) opportunity from ZT6-11. (B) Same as A but for AppNL-G-F mice. (C, D) Same as A and B but for REM sleep. (E, F) EEG delta power in NREM sleep during the RS period (ZT6-11) after the 6-h SD (ZT0-6) compared to the baseline day in (E) WT and (F) AppNL-G-F mice. Values are mean ± SEM. * p < 0.05 based on between condition post hoc comparisons using Fisher’s LSD test.

EEG power density during Wake, NREM and REM sleep in WT (black) and AppNL-G-F (red) mice at 14-18 months (A, A’ and A”) and at 18-22 months (B, B’ and B”). Panels C-H present the EEG Power Density during Wake at both ages in the delta (C), theta (D), alpha (E), beta (F), low gamma (G) ranges and the gamma/delta ratio (H). Panels C’-H’ provide comparable information for NREM sleep. Panels C’’-H’’ provide similar information for REM sleep as well as the theta/delta ratio (I”). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 based on Fisher’s LSD test after mixed-effects ANOVA.

Mean percentage of time spent in Wake (A), NREM (B), and REM (C) sleep on 14 consecutive days for the 31 mice recorded when they ranged from 18.3-24.8 months of age.

Percent time in sleep/wake states for male and female WT and AppNL-G-F mice at 18.3-24.8 months of age.

Percent time for Wake (A-C), NREM (A’-C’), and REM sleep (A’’-C’’) averaged across the 14 24-h periods recorded (A-A’’) and for the corresponding light (B-B’’) and dark phases (C-C’’). Across the 24-h periods, AppNL-G-F mice of both sexes exhibited a greater amount of Wake (A) and decreased amounts of REM sleep (A’’) in comparison to their age-matched WT littermate controls. While this effect is most pronounced in the dark phase in both sexes (C-C’’), it is also evident during the light phase in female mice (B-B’’). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 based on between group post hoc comparisons using Fisher’s LSD test.

Effects of Genotype, Sex, and Genotype x Sex interaction on vigilance state amounts during baseline and during the light and dark phases.

Mean bout durations of sleep/wake states for male and female WT and AppNL-G-F mice at 18.3-24.8 months of age.

Wake (A-C), NREM (A’-C’), and REM sleep (A’’-C’’) bout durations were averaged for the 14 24-h periods recorded (A-A’’) and for the corresponding light (B-B’’) and dark phases (C-C’’). AppNL-G-F mice exhibit longer Wake (A), NREM (A’) and REM sleep (A’’) bouts in comparison to age-matched WT littermate controls. This effect is largely consistent across both the light (B-B’’) and dark (C-C’’) phases. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 based on between group post hoc comparisons using Fisher’s LSD test.

Effects of Genotype, Sex, and Genotype x Sex interaction on bout duration of each vigilance state during baseline and the light and dark phases.

Effects of Genotype, Sex, and Genotype x Sex interaction on the number of bouts of each vigilance state during baseline and the light and dark phases.

The number of sleep/wake state bouts for male and female AppNL-G-F and WT mice at 18.3-24.8 months of age.

The number of Wake (A-C), NREM (A’-C’), and REM sleep (A’’-C’’) bouts were summed across the 14 24-h periods (A-A’’) recorded as well as the light (B-B’’) and dark (C-C’’) phases. Male AppNL-G-F mice exhibit fewer Wake (A), NREM (A’) and REM sleep (A’’) bouts in comparison to their age-matched wildtype (WT) littermate controls. This effect is evident in both the light (B-B’’) and dark (C-C’’) phases as well. Female AppNL-G-F mice exhibit fewer REM sleep bouts than their age-matched wildtype (WT) littermates in all 3 time comparisons. *p < 0.05, **p < 0.01 and ***p < 0.001, ****p < 0.0001 based on between group post hoc comparisons using Fisher’s LSD test.

EEG spectral power (0.5-15 Hz) during (A) NREM and (B) REM sleep for male and female WT and AppNL-G-F mice at 18.3-24.8 months of age.

Red bars indicate the frequency range over which female WT and AppNL-G-F mice are significantly different; blue bars in B indicate the frequency range over which male WT and AppNL-G-F mice differ. Bars indicate p < 0.05 using unpaired t-test.

Multiple Sleep Latency Test (MSLT) results from 18-24 month old AppWT/WT (WT) and AppNL-G-F/NL-G-F (KI) mice.

A. Percent time in NREM sleep during each 20 min nap opportunity in WT (black) and KI (red) mice. The five 20 min SD periods are indicated by horizontal bars below the abscissa. B. Percent time in REM sleep during each 20 min nap opportunity. C. Mean percent NREM time during the 5 nap opportunities in WT and KI mice. D. Mean NREM sleep latency in the WT and KI mice. E. Mean percent REM time during the 5 nap opportunities in WT and KI mice. F. Mean REM sleep latency between WT and KI mice. Values are mean ± SEM. **, p < 0.01.

Arousal state amounts during baseline (BL) and during recovery sleep (RS) during the 6-h of the light phase after cessation of 6-h sleep deprivation (left) and the subsequent 12-h dark phase (right) in AppWT/WT (WT) and AppNL-G-F/NL-G-F (KI) mice.

A and A’. Mean hourly amounts of Wakefulness. B and B’. Mean hourly amounts of NREM sleep. C and C’. Mean hourly amounts of REM sleep. Values are mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.