Time course of Run/no-Run bouts and sleep episodes.

<p>Time course of the duration of Run and no-Run bouts during waking, and of NREM and REM sleep episodes starting from the first consolidated period of sleep during the dark period until the end of the light phase (all data are means of Days 1 and 3). Since the duration of waking and the timing of sleep onset differed between individuals, the entire time period (waking: Day 1 - 355–677 minutes, Day 3∶404–741 minutes; sleep: the remaining time until the end of 24 h) was subdivided into 5 time intervals, each comprising 1/5th of all waking bouts or sleep episodes. Mean values+SEM (n = 8); one-way ANOVA, factor ‘time’.</p

The fine structure of waking and sleep is related to the slow homeostatic process.

<p>(<b>A</b>) Relationship between the average duration of Run bouts (left) with the duration of waking (starting from dark onset until the occurrence of the first consolidated sleep period), and between the average duration of Run - no-Run cycle and the duration of NREM sleep episodes (right). (<b>B</b>) Ratios of average duration of Run - no-Run cycles during waking and NREM - REM sleep cycles (’Architecture’), the total amount of waking and sleep in 24 h (‘Amount’), and the time constants of the Process S, Ti and Td (after Huber et al., 2000; ‘Dynamics’). For all three bar plots wake-related variables (red bars (W): duration of Run - no-Run cycle, total 24-h waking and Ti) are expressed relative to sleep-related variables (blue bars (S): duration of NREM-REM sleep cycle, total 24-h sleep and Td). (<b>C</b>) The relationship between the ratio of waking cycle (duration of Run – no-Run cycle) and sleep cycle (duration of NREM – REM sleep cycle) and the ratio of total waking and sleep during 24 h (left) or the capacity to sustain continuous waking (right). R- and P-values resulting from Pearson correlations. Day 3, n = 8 mice for all analyses.</p

Temporal structure of behaviour and brain activity.

<p>A periodic structure is manifested on all three days: in brain EEG activity on the day with access to the running wheel (<b>A</b>), during waking without access to the running wheel on Day 2 (<b>B</b>) and during sleep on Day 1 (<b>C</b>). Each panel depicts a 3-h interval (panels <b>A</b> and <b>B</b>: beginning 1.5 hours after dark onset on Day 1 and 2 respectively; panel <b>C</b>: 5 h after light onset on Day 1), for which the spectrogram of the occipital EEG was computed (top in each panel), along with the IR- and RW-activity (middle in each panel). The three vigilance states, waking (W), NREM sleep (N) and REM sleep (R) are shown in the hypnograms below each panel. Note that the overall amplitude of changes during spontaneous waking on Day 2 was lower than on Days 1 or 3 (the scaling on the color plots in Fig. 5 is adjusted to the maximal values).</p

Intra individual stability and inter individual variability of waking and sleep.

<p>Correlation of four variables between Day 1 and 3: Waking duration starting from dark onset until the occurrence of the first consolidated sleep period, running wheel intensity (RW counts/min within Run bouts), total 24-h NREM sleep amount and the mean duration of NREM sleep episodes (n = 8 mice). r- and p-values correspond to Pearson correlations between the variables. Note the remarkable intra individual stability of all four variables between the two days.</p

Power spectra analysis following ceftriaxone administration.

<p><b>A.</b> Reduction in theta power at day 10. Waking mean absolute power spectra of Day 0 and Day 10 for frontal (above) and parietal (below) EEG channel. <b>B.</b> Power spectra analysis relative to the baseline illustrating a reduction of theta power at Day 10 (shown in detail for single animals in the small inset) and a return to the baseline eight days after CEF withdrawal (Day 16) in frontal (above) and parietal (below) EEG channels. Statistical significance (p<0.05) is represented by black dots. Values are mean ± sem. <b>C.</b> Time course analysis of relative spectra (7–9 Hz frequency band) showing a significant reduction of theta power for frontal and parietal channels two days (Day 10) and four days (Day 12) after CEF withdrawal compared to the baseline (Day 0). *p<0.05. Values are mean ± sem. <b>D.</b> Example of power spectrum relative to the baseline for a representative animal during the entire length of the experiment. <b>E.</b> Frontal and parietal relative spectra showing a broad band (7–13 Hz) reduction in power during NREM sleep at day 10. Statistical significance (p<0.05) is represented by black dots. Values are mean ± sem. <b>F.</b> Frontal and parietal relative spectra showing a reduction in theta power during REM sleep at day 10. Statistical significance (p<0.05) is represented by black dots. Values are mean ± sem.</p

Lack of pathological elements after ceftriaxone treatment.

<p><b>A.</b> Schematic description of electrodes location. <b>B.</b> Saline and CEF Treatment schedule. Day 0 represents the baseline. <b>C.</b> Waking absolute spectra, raw EEG and EMG signals of baseline, day 10 and day 16. Signals appeared stable across the entire length of the experiment and the signal quality was not affected by CEF treatment.</p

Effects of ceftriaxone treatment on motor activity.

<p><b>A.</b> Time-course of the amount of time expressed in 4s epochs spent in waking, NREM and REM sleep. Values are expressed as mean ± sem. <b>B.</b> Example of EMG activity during the entire length of the experiments. Note the intense activity after the end of CEF treatment. The grey line represents the threshold above and below which the motor activity is identified as active waking or quiet waking, respectively. <b>C–D.</b> Quantitative analysis of EMG activity (C) and Motion activity (D) during active and quiet waking for light and dark periods. Values are relative to the baseline (day 0) and expressed as mean ± sem. * (p<0.05), ** (p<0.01). <b>E–F.</b> Negative correlation between the time-course of relative theta power and the EMG (E) or Motion activity (F).</p

The arousal-promoting and sleep-promoting effects of nocturnal light exposure in mice depend upon different neural pathways.

<p>In response to blue light, M1 pRGC projections to the SCN result in activation of the adrenal gland via the ANS. This alertness promoting pathway is associated with corticosterone release and increased waking. This pathway is strongly activated by blue light due to the spectral sensitivity of melanopsin which peaks ~480 nm. Loss of melanopsin results in reduced activation of this pathway, resulting in enhanced sleep induction. Additional arousal-promoting pathways undoubtedly contribute to this response. By contrast, green light results in activation of the sleep-promoting VLPO pathway, most likely via non-M1 melanopsin pRGCs that are more dependent upon rod and cone input. As melanopsin plays a critical role in rod and cone adaptation, under bright light conditions, loss of melanopsin results in attenuated sleep induction via this pathway. Moreover, the resultant saturation of rod and cone pathways also results in a loss of chromatic responses.</p

Molecular responses to light in SCN and VLPO are wavelength-dependent.

<p>A <i>Fos</i> induction in response to a 30 min light pulse was studied in wildtype mice exposed to blue or green light in both the SCN and VLPO (two-way ANOVA for wavelength x brain region interaction F_(2,31) = 10.968 <i>p</i> ≤ 0.001). Mice exposed to blue light show greater <i>Fos</i> induction in the SCN compared with green light (posthoc Tukey dark versus blue <i>p</i> = 0.001, dark versus green <i>p</i> = 0.035, blue versus green <i>p</i> = 0.008). By contrast, in the VLPO, a greater response to green light than blue light was observed (dark versus blue <i>p</i> = 0.880, dark versus green <i>p</i> = 0.015). As a control, <i>Gal</i> induction was also studied, showing no induction in the SCN but significant induction in the VLPO (two-way ANOVA for wavelength and x brain region interaction F_(2,30) = 3.774 <i>p</i> = 0.035). This response was only evident in response to green light (posthoc Tukey dark versus blue <i>p</i> = 0.742, dark versus green <i>p</i> = 0.011, blue versus green <i>p</i> = 0.003). Histograms show mean ± SEM, <i>n</i> = 5–8/group. Significant differences indicated by *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤0.01, * <i>p</i> ≤0.05, NS = not significant. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002482#pbio.1002482.s006" target="_blank">S6 Data</a>.</p

Wavelength-dependent effects on light on sleep induction and sleep duration.

<p>(<b>A</b>) Mice exposed to different wavelengths (405 nm violet, 470 nm blue, or 530 nm green) for 1 hr at ZT14 exhibited differences in sleep onset and sleep duration. (<b>B</b>) Sleep induction is delayed in response to blue light exposure. One-way ANOVA for wavelength, F_(2.23) = 18.791, <i>p</i> ≤ 0.001. Posthoc Tukey violet versus blue <i>p</i> = 0.003, violet versus green <i>p</i> = 0.041, blue versus green <i>p</i> ≤ 0.001. (<b>C</b>) Total sleep duration during the 1 h light pulse is reduced in response to blue light. Data plotted as mean percentage ± SEM (<i>n</i> = 8–10/group). Horizontal black-white-black bar illustrates the light pulse condition from ZT14 until ZT15. One-way ANOVA for wavelength, F_(2.23) = 4.391, <i>p</i> = 0.024. Posthoc Tukey violet versus blue <i>p</i> = 0.046, violet versus green <i>p</i> = 0.517, blue versus green <i>p</i> = 0.008. Statistical differences indicated by *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤ 0.01, * <i>p</i> ≤ 0.05, NS = not significant. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002482#pbio.1002482.s001" target="_blank">S1 Data</a>.</p