Ageing-related modification of sleep and breathing in orexin-knockout narcoleptic mice

Abstract

Narcolepsy type-1 (NT1) is a lifelong sleep disease, characterised by impairment of the orexinergic system, with a typical onset during adolescence and young adulthood. Since the wake-sleep cycle physiologically changes with ageing, this study aims to compare sleep patterns between orexin-knockout (KO) and wild type (WT) control mice at different ages. Four groups of age-matched female KO and WT mice (16 weeks of age: 8 KO-YO and 9 WT-YO mice; 87 weeks of age: 13 KO-OLD and 12 WT-OLD mice) were implanted with electrodes for discriminating wakefulness, rapid-eye-movement sleep (REMS), and non-REMS (NREMS). Mice were recorded for 48 h in their home cages and for 7 more hours into a plethysmographic chamber to characterise their sleep-breathing pattern. Regardless of orexin deficiency, OLD mice spent less time awake and had fragmentation of this behavioural state showing more bouts of shorter length than YO mice. OLD mice also had more NREMS bouts and less frequent NREMS apneas than YO mice. Regardless of age, KO mice showed cataplexy-like episodes and shorter REMS latency than WT controls and had a faster breathing rate and an increased minute ventilation during REMS. KO mice also had more wakefulness, NREMS and REMS bouts, and a shorter mean length of wakefulness bouts than WT controls. Our experiment indicated that the lack of orexins as well as ageing importantly modulate the sleep and breathing phenotype in mice. The narcoleptic phenotype caused by orexin deficiency in female mice was substantially preserved with ageing.

Keywords: apnea; cataplexy; elderly; hypnic; rodent.

FIGURE 1

Wake–sleep structure during home‐cage recordings. (a–c) Percentage of time spent in wakefulness, non‐rapid‐eye‐movement sleep (NREMS), and rapid‐eye‐movement sleep (REMS), respectively, by OLD and young (YO) orexin‐knockout (KO) and wild‐type (WT) control mice. (d–f) Mean episode length during wakefulness, NREMS, and REMS, respectively. Data are reported as mean ± SEM, with symbols representing data in individual mice. *, **, ***, and **** p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively, for the main effect of genotype or age (2‐way ANOVA)

FIGURE 2

Wake–sleep structure as a function of the light–dark period in home‐cage recordings. (a, b) mean length and number of wakefulness episodes. (c) Number of non‐rapid‐eye‐movement sleep (NREMS) episodes, as a function of the light (resting) and the dark (active) period in OLD and young (YO) orexin‐knockout (KO) and wild‐type (WT) control mice. Data are reported as mean ± SEM, with symbols representing data in individual mice. ^### and ^@@@ p < 0.001, main effect of genotype or age (3‐way ANOVA), respectively. ***p < 0.001, post‐hoc corrected comparison between KO and WT in the dark period, with significant photoperiod × genotype interaction at 3‐way ANOVA. ^$$$ p < 0.001 for the post‐hoc corrected comparison between KO‐YO and WT‐YO, with significant age × genotype × photoperiod interaction or age × genotype interaction at 3‐way ANOVA. (d–g) Representative 12 h hypnograms (dark period, from Zeitgeber 12 to 24) of WT‐YO, KO‐YO, WT‐OLD and KO‐OLD, respectively. N, NREMS; R, REMS; W, Wakefulness. Red arrows highlight cataplexy‐like episodes in KO mice. KO, orexin‐knockout; WT, wild‐type; YO, young.

FIGURE 3

Electroencephalogram spectral analysis during wake–sleep states in home‐cage recordings. Electroencephalographic (EEG) spectral power (expressed as mean ± SEM of the percentage of total EEG spectral power) during wakefulness, non‐rapid‐eye‐movement sleep (NREMS), and rapid‐eye‐movement sleep (REMS) expressed as a percentage of total EEG spectral power (a, c, e, respectively) exhibited by OLD and young (YO) orexin‐knockout (KO) and wild‐type (WT) control mice. (b, d, f) Median (with the 95% confidence interval) values of the EEG peak frequency during wakefulness, NREMS, and REMS, with symbols indicating values in individual mice. ^# p < 0.05 for the post‐hoc corrected comparison between KO‐YO and KO‐OLD after significant Kruskal‐Wallis test. * and ^## genotype (p < 0.05) and age (p < 0.01) global effects, respectively, with Mann–Whitney test with the false discovery rate correction after significant Kruskal‐Wallis test. The black bar indicates p < 0.05 for the post‐hoc corrected comparison between OLD and YO mice, with significant age × EEG frequency interaction. KO, orexin‐knockout; WT, wild‐type; YO, young.

FIGURE 4

Electroencephalogram spectral analysis during cataplexy‐like episodes. Electroencephalographic (EEG) spectral power (expressed as mean ± SEM of the percentage of total EEG spectral power) and peak (median with the 95% confidence interval) during cataplexy‐like episodes (CLE) are shown for OLD and young (YO) orexin‐knockout (KO). *p < 0.05 for Mann–Whitney test with the false discovery rate correction, with significant Kruskal‐Wallis test. Black bars indicate p < 0.05 for the post‐hoc corrected comparison between KO‐OLD and KO‐YO mice with significant age × EEG frequency interaction.

FIGURE 5

Sleep breathing phenotype. (a, b) Ventilatory period (VP) during non‐rapid‐eye‐movement sleep (NREMS) and rapid‐eye‐movement sleep (REMS), respectively, exhibited by OLD and young (YO) orexin‐knockout (KO) and wild‐type (WT) control mice. (c, d) Tidal volume (TV, corrected per body weight) during NREMS and REMS, respectively. (e, f) The minute ventilation (MV, corrected per body weight) during NREMS and REMS. Data are reported as median values (with the 95% confidence interval), with symbols indicating values in individual animals. ** and *** p < 0.01 and p < 0.001, respectively, for the post‐hoc corrected comparison between KO‐OLD and WT‐OLD and between KO‐YO and WT‐YO with significant Kruskal‐Wallis test. ^# p < 0.05 genotype or age global effect, Mann–Whitney test with the false discovery rate correction after significant Kruskal‐Wallis test. The significant age main effect is not reported in (b) to increase readability. KO, orexin‐knockout; WT, wild‐type; YO, young.

FIGURE 6

Sighs and sleep apneas. (a) Sighs (augmented breath) occurrence rate during non‐rapid‐eye‐movement sleep (NREMS) exhibited by OLD and young (YO) orexin‐knockout (KO) and wild‐type (WT) control mice. (b, c) Sleep apnea (breath pause) occurrence during NREMS and rapid‐eye‐movement sleep (REMS), respectively. Data are reported as median (with the 95% confidence interval) values, with symbols indicating results in individual mice. * p < 0.05, for the post‐hoc corrected comparison between WT‐YO and WT‐OLD, and between KO‐YO and KO‐OLD with significant Kruskal‐Wallis test.

FIGURE 7

Principal components analysis. A principal components analysis (PCA) was run on 31 electroencephalographic, sleep and breathing variables of the present experiment. Scores of principal component (PC) 1 and PC2 (i.e. the two PCs explaining most of the variance in the dataset) are shown for OLD and young (YO) orexin‐knockout (KO) and wild‐type (WT) control mice. (a) Segregation of the present experimental population using PC1 and PC2 as, respectively, the x‐ and the y‐axis of a bidimensional graph. (b, c) Results of statistical comparisons between groups for PC1 and PC2, respectively. * and *** p < 0.05 and p < 0.001, respectively, for the main genotype and age effect (2‐way ANOVA).