Abnormal sleep/wake dynamics in orexin knockout mice

Abstract

Study objectives: Narcolepsy with cataplexy is caused by a loss of orexin (hypocretin) signaling, but the physiologic mechanisms that result in poor maintenance of wakefulness and fragmented sleep remain unknown. Conventional scoring of sleep cannot reveal much about the process of transitioning between states or the variations within states. We developed an EEG spectral analysis technique to determine whether the state instability in a mouse model of narcolepsy reflects abnormal sleep or wake states, faster movements between states, or abnormal transitions between states.

Design: We analyzed sleep recordings in orexin knockout (OXKO) mice and wild type (WT) littermates using a state space analysis technique. This non-categorical approach allows quantitative and unbiased examination of sleep/wake states and state transitions.

Measurements and results: OXKO mice spent less time in deep, delta-rich NREM sleep and in active, theta-rich wake and instead spent more time near the transition zones between states. In addition, while in the midst of what should be stable wake, OXKO mice initiated rapid changes into NREM sleep with high velocities normally seen only in transition regions. Consequently, state transitions were much more frequent and rapid even though the EEG progressions during state transitions were normal.

Conclusions: State space analysis enables visualization of the boundaries between sleep and wake and shows that narcoleptic mice have less distinct and more labile states of sleep and wakefulness. These observations provide new perspectives on the abnormal state dynamics resulting from disrupted orexin signaling and highlight the usefulness of state space analysis in understanding narcolepsy and other sleep disorders.

Figure 1

Spectral ratios of EEG activity define a 2-dimensional state space with distinct clusters. Each plot shows 24 hours of EEG activity, and each point represents 1 second of EEG activity. In these graphs, the color of each state space point is determined from conventional scoring: Wake (blue), NREM sleep (red), REM sleep (green), and Cataplexy (magenta). Centroids of each cluster are denoted by white dots. A. State space analysis of a typical WT mouse. B. Same for a typical OXKO mouse. The relative locations of state clusters are conserved between WT and OXKO mice, but OXKO mice have cataplexy and show less cluster separation than WT mice.

Figure 2

OXKO mice have abnormal distributions of EEG activity during wake and sleep. A, B—Point densities were averaged across mice of each genotype to create average WT and OXKO mouse density plots. WT mice (n = 6) have a Wake cluster that includes a region corresponding to active wakefulness with higher theta activity (high ratio 1), a highly concentrated NREM sleep cluster, and a relatively sparse REM sleep cluster, reflecting much less time spent in REM sleep compared to other states. In contrast, OXKO mice (n = 7) have a smaller Wake cluster concentrated closer to the transition region between Wake and NREM sleep. The color scale represents point densities with warm colors indicating higher densities. C, D—State space densities for individual WT and OXKO mice projected into the ratio 2 dimension. Each color represents data for an individual mouse. The projection yields 2 peaks: the left peak is associated with Wake and the right peak is associated with NREM sleep. The horizontal peak-to-peak distances are shorter in OXKO mice (one-way ANOVA, P < 0.05). Trough height is higher in OXKO mice, reflecting more time spent in the transition region between Wake and NREM sleep (one-way ANOVA, P < 0.01). E—Difference plot showing the average density pattern of OXKO mice subtracted from that of WT mice. Here the color scale highlights differences between genotypes: warm colors indicate regions where the average density is greater in WT mice, and cool colors indicate higher density in OXKO mice. WT mice spend more time in deep (delta-rich) NREM sleep and active (theta-rich) Wake, whereas OXKO mice spend more time in light NREM sleep and the Wake/NREM sleep transition region.

Figure 3

OXKO mice have faster movements in certain regions of the state space. Plot colors represent the directionless (A, B), vertical (C, D), or horizontal (E, F) velocities of EEG activity originating in that region of the state space. Different color maps were used for each pair of panels to emphasize differences between genotypes. A, B—Average directionless velocity plots in WT and OXKO mice distinguish between stable states where velocities are slow (cool colors) and transition regions where velocities are fast (warm colors). C, D—The vertical (ratio 2) components of velocity vectors highlight the fast transitions between Wake and NREM sleep. In OXKO mice, this region of fast vertical velocities extends far into the middle of the Wake cluster. E, F—The horizontal (ratio 1) components of the velocity vectors highlight the fast transitions between Wake and the REM sleep/Cataplexy clusters. For both horizontal and vertical velocities, the regions of fast transitions are larger in OXKO mice (one-way ANOVA, P < 0.01). This shows that regions of sleep/wake behavior that are normally stable are susceptible to rapid changes in OXKO mice, possibly contributing to the frequent state transitions in these animals.

Figure 4

Using a contour algorithm, we delineated cluster core boundaries for each state. A—Cluster core boundaries for Wake, NREM sleep, and REM sleep are drawn over a density plot for a representative WT mouse. B—Cluster core areas provide a measure of EEG homogeneity with smaller clusters corresponding to less variable activity. The Wake cluster is smaller and the REM sleep cluster is larger in OXKO mice compared to WT mice (one-way ANOVA, P < 0.05). C—Boundaries defined using the contour algorithm have high positive predictive value. In both genotypes, over 95% of data encompassed by the Wake boundary were independently scored as Wake using conventional scoring. The NREM sleep and REM sleep/Cataplexy cluster cores also have high positive predictive values. D—The cluster core boundaries are moderately sensitive: 50% to 80% of all Wake and NREM sleep fall within these boundaries. The boundaries for REM sleep and Cataplexy are less sensitive, probably because these states are relatively uncommon, and data were sparse.

Figure 5

The trajectories of individual transitions are normal in OXKO mice with the exception of transitions from Wake into Cataplexy. In all panels, the cluster core boundaries for each state for WT or for OXKO mice are shown (Wake - Blue; NREM - Red; REM-green; Cataplexy - magenta). Each panel also includes information about transition trajectories as described below. A—Representative transition trajectories from Wake to NREM sleep (red lines) and from NREM sleep to Wake (blue lines) in an individual WT mouse. These transitions tend to arc right and left, respectively, suggesting that falling asleep and waking up are not simply inverse processes. B—Representative transition trajectories from NREM sleep to REM sleep (green lines) and from REM sleep to Wake (blue lines) in an individual WT mouse. C, D—Origins of transition trajectories in representative WT and OXKO mice are localized to specific regions of the state space. Origins are color-coded according to the terminating state: Wake (blue dots), NREM sleep (red dots), REM sleep (green dots). Origination regions are generally conserved between genotypes, although transitions from Wake to REM sleep/Cataplexy are common in OXKO mice (green dots on the right side of the Wake cluster core boundary). True Wake-to-REM sleep transitions almost never occur in WT mice, but due to the proximity of the Wake and REM sleep clusters, the boundary crossing approach occasionally identifies a few false positive Wake-to-REM sleep transitions. E—Representative transition trajectories from Wake to Cataplexy (magenta lines) in an individual OXKO mouse. The direct movement from the Wake cluster to the Cataplexy/REM sleep region distinguishes these transitions from normal episodes of REM sleep that follow NREM sleep. F—Representative transition trajectories from Cataplexy to Wake (blue lines) in an individual OXKO mouse. These transitions follow paths similar to transitions from REM sleep to Wake suggesting similar processes for terminating both Cataplexy and REM sleep. Furthermore, they constitute a reversal of the trajectories from Wake to Cataplexy indicating symmetry in EEG dynamics during transitions in and out of Cataplexy not observed with other transitions.