Optogenetic disruption of sleep continuity impairs memory consolidation

Abstract

Memory consolidation has been proposed as a function of sleep. However, sleep is a complex phenomenon characterized by several features including duration, intensity, and continuity. Sleep continuity is disrupted in different neurological and psychiatric conditions, many of which are accompanied by memory deficits. This finding has raised the question of whether the continuity of sleep is important for memory consolidation. However, current techniques used in sleep research cannot manipulate a single sleep feature while maintaining the others constant. Here, we introduce the use of optogenetics to investigate the role of sleep continuity in memory consolidation. We optogenetically targeted hypocretin/orexin neurons, which play a key role in arousal processes. We used optogenetics to activate these neurons at different intervals in behaving mice and were able to fragment sleep without affecting its overall amount or intensity. Fragmenting sleep after the learning phase of the novel object recognition (NOR) task significantly decreased the performance of mice on the subsequent day, but memory was unaffected if the average duration of sleep episodes was maintained at 62-73% of normal. These findings demonstrate the use of optogenetic activation of arousal-related nuclei as a way to systematically manipulate a specific feature of sleep. We conclude that regardless of the total amount of sleep or sleep intensity, a minimal unit of uninterrupted sleep is crucial for memory consolidation.

Fig. 1.

Optogenetic stimulation of Hcrt cells every 60 s disrupts sleep integrity but not total sleep time or the overall composition of sleep. Mice expressing ChR2 under the Hcrt promoter were stimulated with a blue laser diode (477 nm; 20 mW) through an optical fiber aimed at the lateral hypothalamus. Trains of 10 s (20 Hz, 15-ms light pulse) with 30- or 60-s intervals between the stimuli were used. Control mice expressing only the fluorescent marker were stimulated in 60-s intervals. (A) Schematic representation of the cannula placement and the placing of the EEG/EMG recording setup are also shown. Sleep was recorded during the time of stimulation (10:00 AM–2:00 PM). Intervals of 4 s were visually scored for wake, NREM, and REM sleep. Data are presented as total over the 4 h of stimulation from artifact-free intervals. (B) Number of transitions from sleep (NREM and REM) to wake. (C–E) The average duration in seconds of a single NREM (C), REM (D), or wake episode (E). (F–H) The percentage of total time spent in wake (F), NREM (G), or REM (H). (I) The percentage of time spent in wake during the first hour immediately after the stimulation session ended (2:00–3:00 PM). Values are represented as means ± SEM; n = 8–9 in each group. One-way ANOVA (factor “stimulation”) was followed by Tukey's multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.0001.

Fig. 2.

EEG power density during NREM and REM sleep is not affected by Hcrt stimulations every 60 s. We performed fast Fourier transformation (FFT) analysis of sleep for two groups: control (expressing only the fluorescent marker) and ChR2 mice, both stimulated every 60 s. The relative EEG power of each frequency bin was normalized to the total power (0.4–20 Hz). (A and B) The curves represent relative power densities (mean ± SEM) for NREM (A) and REM (B) sleep. (C) Distribution of arousal events by their duration. The duration of each arousal event was determined by specific scoring in 2-s intervals. These events were divided into three categories: events shorter than 2 s, events between 2 and 10 s, and events longer than 10 s. The percentage of each category out of the total events was determined and shown here as a comparison between control and ChR2-expressing mice, both stimulated at the 60-s paradigm. Two-way ANOVA (factors “stimulation” and “wake duration”) with Bonferroni correction was used to determine the significance of the data. (D) Relative power densities (mean ± SEM) for transitional stages between NREM and REM sleep (12 s of NREM before the presence of stable REM sleep). Two-way ANOVA (factors “stimulation” and “frequency”) with Bonferroni correction (n = 6–7 mice per group; *P < 0.05, **P < 0.01, ***P < 0.0001).

Fig. 3.

Hcrt stimulation every 60 s impairs performance in the novel object recognition task via a sleep-dependent mechanism. (A) Schematic representation of the behavioral paradigm. At the training session on the first day (9:30 AM) mice (ChR2 mice and the controls) were given 10 min to adapt to an open field arena followed by 5 min to explore two similar objects placed in the same open arena. At the end of the session, the optic fibers were inserted and mice were stimulated during their resting phase for 4 h every 60 s. At the testing session 24 h later, mice were reexamined in the same arenas with one of the previously encountered objects replaced with a novel one. (B) Representative videographs of the overall pathway of a mouse in a testing session determined automatically by the Viewpoint Videotrack system. Red lines represent movements and green dots indicate pauses in movement. Differences in exploration of the novel (N) and the familiar (F) object are evident (the intensity of green dots around the circled areas) in the control but not in the ChR2 mice (stimulated every 60 s). (C–E) Percentage of time spent exploring each object (novel or familiar) was determined. Student's t test was used to determine whether they are significantly different (for each group of mice; n = 8–10 per group). Significance is indicated by asterisks above the statistically significant groups. Difference in novel object explorations between groups is indicated above the lines connecting statistically significant groups (Student's t test in C and E and one-way ANOVA in D). (C) Novel object exploration in the control and ChR2 mice stimulated every 60 s (mean ± SEM). (D) Exploration of novel object in three groups of mice (control or ChR2 mice) that were injected immediately after the acquisition session with either the Hcrt receptor 1 antagonist (SB334867, 10 mg/kg i.p.) or vehicle alone as indicated in the figure. All mice were stimulated in the same paradigm of 60-s intervals (n = 7–8 per group). (E) Dark phase; control and ChR2 mice were exposed to the training session with dark onset (9:30 PM) instead of the onset of light as shown in C. The testing session took place again, 24 h later (n = 6–7 per group). ***P < 0.001.

Fig. 4.

Hcrt stimulation at different intervals differentially affects performance in the novel object recognition task. (A) Schematic representation of the behavioral paradigm. The same paradigm as described in Fig. 4 was used but Hcrt stimulations were introduced in four different intervals (schematic view). (B) Percentage of novel object exploration (out of the total time) for each mouse is shown (n = 6–9 per group). The red lines represent means ± SEM; one-way ANOVA (**P < 0.01).

Fig. 5.

Intact sleep is crucial for memory consolidation in the novel object recognition task in mice. Quantification of the total EEG power in the delta (0.4–4 Hz) and theta (4–9 Hz) frequency bands for control and ChR2 mice stimulated at 60-s intervals and ChR2 mice stimulated every 120 s during NREM (A), REM (B), and transitional stages (C) between NREM and REM (12 s of NREM before the presence of stable REM). Two-way ANOVA (factors “stimulation” and “frequency”) with Bonferroni correction was used to determine the significance of the data. Correlation between the sleep duration (D) or fragmentation of sleep (E) (represented by the number of sleep interruptions) and performance on the test phase is represented by percentage of novel object exploration for the pooled control and ChR2 mice stimulated every 30, 60, and 120 s (n = 29).