The effect of Neuroligin-2 absence on sleep architecture and electroencephalographic activity in mice

Abstract

Sleep disorders are comorbid with most psychiatric disorders, but the link between these is not well understood. Neuroligin-2 (NLGN2) is a cell adhesion molecule that plays roles in synapse formation and neurotransmission. Moreover, NLGN2 has been associated with psychiatric disorders, but its implication in sleep remains underexplored. In the present study, the effect of Nlgn2 knockout (Nlgn2^-/-) on sleep architecture and electroencephalographic (EEG) activity in mice has been investigated. The EEG and electromyogram (EMG) were recorded in Nlgn2^-/- mice and littermates for 24 h from which three vigilance states (i.e., wakefulness, rapid eye movement [REM] sleep, non-REM [NREM] sleep) were visually identified. Spectral analysis of the EEG was performed for the three states. Nlgn2^-/- mice showed more wakefulness and less NREM and REM sleep compared to wild-type (Nlgn2^+/+) mice, especially during the dark period. This was accompanied by changes in the number and duration of individual episodes of wakefulness and sleep, indexing changes in state consolidation, as well as widespread changes in EEG spectral activity in all states. Abnormal 'hypersynchronized' EEG events have also been observed predominantly in Nlgn2^-/- mice. These events were mainly observed during wakefulness and REM sleep. In addition, Nlgn2^-/- mice showed alterations in the daily time course of NREM sleep delta (1-4 Hz) activity, pointing to modifications in the dynamics of sleep homeostasis. These data suggest that NLGN2 participates in the regulation of sleep duration as well as EEG activity during wakefulness and sleep.

Keywords: Cell adhesion molecule; Delta activity; Electroencephalography; Knockout mice; Neuroligin; Sleep regulation; Wakefulness.

Fig. 1

Time spent (min ± SEM) in wakefulness, NREM sleep and REM sleep in Nlgn2^+/+, Nlgn2^+/− and Nlgn2^−/− mice measured using EEG and EMG recordings. a) Total time spent in wake, NREM sleep and REM sleep for the total 24 h recording, the 12 h Light and the 12 h Dark periods. Significant genotype effects were found for 24 h wake (F_2,37 = 4.8, p = 0.014), 24 h REM sleep (F_2,37 = 16.9, p < 0.0001), 12 h Light REM sleep (F_2,37 = 8.5, p < 0.001), 12 h Dark wake (F_2,37 = 6.8, p = 0.003), 12 h Dark NREM sleep (F_2,37 = 6.4, p = 0.004) and 12 h Dark REM sleep (F_2,37 = 6.4, p = 0.004). No significant genotype effect was found for 24 h NREM sleep (F_2,37 = 2.7, p = 0.08), 12 h Light wake (F_2,37 = 1.3, p = 0.3) and 12 h Light NREM sleep (F_2,37 = 1.1, p = 0.3). Stars show significant post-hoc Tukey HSD comparisons between indicated genotypes (*: p < 0.05; **: p < 0.01). b) Hourly distribution of wake, NREM sleep and REM sleep. For wakefulness, significant genotype and time effects were found (respectively, F_2,37 = 4.8, p = 0.014 and F_23,851 = 51.3, p < 0.0001), but no significant interaction was observed (F_46,851 = 1.2, p = 0.14). For NREM sleep, a significant time effect was found (F_23,851 = 47.0, p < 0.0001), but no significant genotype effect or interaction was observed (respectively, F_2,37 = 2.7, p = 0.08 and F_46,851 = 1.3, p = 0.07). For REM sleep, significant genotype and time effects were found (respectively, F_2,37 = 16.9, p < 0.0001 and F_23,851 = 52.3, p < 0.0001), but no significant interaction was observed (F_46,851 = 0.8, p = 0.7). Grey backgrounds indicate the 12 h dark period

Fig. 2

Parameters of vigilance state consolidation/fragmentation in Nlgn2^+/+, Nlgn2^+/− and Nlgn2^−/− mice quantified for the 12 h Light and the 12 h Dark periods using EEG and EMG recordings. a) Mean duration of individual episodes of vigilance states. Significant genotype effect was found for 12 h Light NREM sleep (F_2,37 = 4.3, p = 0.02) and 12 h Dark wake (F_2,37 = 4.9, p = 0.01). No significant genotype effect was found for 12 h Light wake (F_2,37 = 3.2, p = 0.052), 12 h Light REM sleep (F_2,37 = 0.2, p = 0.8), 12 h Dark NREM sleep (F_2,37 = 1.1, p = 0.3), and 12 h Dark REM sleep (F_2,37 = 0.1, p = 0.9). b) Number of individual episodes of vigilance states. Significant genotype effects were observed for all states for both the 12 h Light and 12 h Dark (12 h Light wake F_2,37 = 3.6, p = 0.04; 12 h Light NREM sleep F_2,37 = 3.5, p = 0.04; 12 h Light REM sleep F_2,37 = 3.3, p = 0.048; 12 h Dark wake F_2,37 = 4.3, p = 0.02; 12 h Dark NREM sleep F_2,37 = 4.2, p = 0.02; 12 h Dark REM sleep F_2,37 = 3.5, p = 0.04). Stars show significant post-hoc Tukey HSD comparisons between indicated genotypes (*: p < 0.05; **: p < 0.01). Grey backgrounds indicate the 12 h dark period

Fig. 3

Abnormal EEG events in Nlgn2^+/+, Nlgn2^+/− and Nlgn2^−/− mice. a) Representative 12-s EEG traces (black), and corresponding EMG traces (blue), showing abnormal EEG events for mice of the three genotypes during wakefulness (upper traces) and REM sleep (lower traces). Scale bars are the same for the two states of all mice. b) Absolute spectral power of events calculated between 1 and 50 Hz and averaged for each genotype for mice showing events (n = 3 Nlgn2^+/+, n = 5 Nlgn2^+/−, n = 12 Nlgn2^−/−). c) Total number of events (upper panel) observed for the 24 h recording in the three genotypes, and their mean duration for mice showing events (lower panel). The number of events was significantly affected by genotype (F_2,37 = 13.7, p < 0.0001). **: p < 0.01 between indicated points (planned comparisons). For mice showing events, the duration of events was significantly affected by genotype (F_2,17 = 3.7, p < 0.05). *: p < 0.05 between indicated points (planned comparisons). d) Number of events calculated separately for the 12 h Light and the 12 h Dark periods in Nlgn2^−/− mice only. The number of events did not significantly differ between the 12 h Light and the 12 h Dark periods (F_1,11 = 3.5, p = 0.09). e) Number of events calculated separately for the three vigilance states in Nlgn2^−/− mice only. The number of events was significantly different between vigilance states (F_2,22 = 9.9, p < 0.001). **: p < 0.01 compared to the other two states; *: p < 0.05 compared to wakefulness

Fig. 4

Power spectra of the 24 h EEG recording computed between 1 and 50 Hz with a 1-Hz resolution separately for the three vigilance states in Nlgn2^+/+, Nlgn2^+/− and Nlgn2^−/− mice. a) Absolute spectral power for wakefulness, NREM sleep and REM sleep. For wakefulness, significant genotype effects were found for frequencies between 1 and 6 Hz and between 9 and 22 Hz (F_2,37 > 3.9, p < 0.03). For NREM sleep, significant genotype effects were found for frequencies between 1 and 37 Hz (F_2,37 > 3.4, p < 0.05). For REM sleep, significant genotype effects were found for frequencies between 1 and 6 Hz and between 9 and 27 Hz (F_2,37 > 3.2, p < 0.05). b) Relative spectral power for wakefulness, NREM sleep and REM sleep. For wakefulness, significant genotype effects were found for frequencies 2 to 3 Hz, 6 to 8 Hz, and 14 to 50 Hz (F_2,37 > 3.6, p < 0.04). For NREM sleep, significant genotype effects were found for frequencies 2 to 3 Hz, 4 to 6 Hz, 9 to 19 Hz, and 27 to 50 Hz (F_2,37 > 3.3, p < 0.05). For REM sleep, significant genotype effects were found for frequencies 1 to 3 Hz, 4 to 9 Hz, 11 to 13 Hz, 15 to 16 Hz, 20 to 21 Hz, and 22 to 50 Hz (F_2,37 > 3.3, p < 0.05). Red symbols indicate Hz-bins for which Nlgn2^−/− mice are significantly different from Nlgn2^+/+ mice (simple effect analysis; p < 0.05). For clarity, significant differences between Nlgn2^+/− and Nlgn2^+/+ mice have not been represented

Fig. 5

Twenty-four hour time course of delta activity (1–4 Hz) during NREM sleep and theta activity (6–9 Hz) during wakefulness in Nlgn2^+/+, Nlgn2^+/− and Nlgn2^−/− mice measured using EEG. a) 24 h dynamics of NREM sleep absolute delta activity (upper panel) and relative delta activity (lower panel). For absolute activity, significant genotype and interval effects have been found (respectively, F_2,37 = 11.7, p < 0.001 and F_17,629 = 38.0, p < 0.0001), as well as a tendency for significant interaction (F_34,629 = 2.3, p = 0.05). For relative activity, a significant interaction has been observed (F_34,629 = 3.4, p < 0.0001). Red symbols indicate intervals for which Nlgn2^−/− mice are significantly different from Nlgn2^+/+ mice, and dark red symbols indicate intervals for which Nlgn2^+/− mice are significantly different from Nlgn2^+/+ mice (simple effect analysis; p < 0.05). b) 24 h dynamics of waking absolute theta activity (upper panel) and relative theta activity (lower panel). For both absolute and relative activity, a significant interval effect was found (respectively, F_17,612 = 3.6, p < 0.01 and F_17,612 = 5.3, p < 0.0001), but no significant genotype effect (respectively, F_2,36 = 2.2, p = 0.13 and F_2,36 = 0.7, p = 0.5) or interaction (respectively, F_34,612 = 1.2, p = 0.2 and F_34,612 = 1.3, p = 0.13) was observed. Grey backgrounds indicate the 12 h dark period