Sleep/wake movement velocities, trajectories and micro-arousals during maturation in rats

BACKGROUND Sleep is regulated by two main processes. The circadian process provides a 24-h rhythm and the homeostatic process reflects sleep pressure, which increases in the course of wakefulness and decreases during sleep. Both of these processes undergo major changes during development. For example, sleep homeostasis, measured by means of electroencephalogram (EEG) slow-wave activity (SWA, EEG power between 0.5 and 4.5 Hz), peaks around puberty and decreases during adolescence. In humans and rats these changes have been related to cortical maturation. We aimed to explore whether additional parameters as state dynamic (dynamic of sleep/wake behavior) parameters of movement velocity, trajectories and micro-arousals provide markers of rat maturation. The state dynamics reflect the stability of sleep within a specific sleep stage. We applied a state space technique (SST), a quantitative and unbiased method, based on frequency band ratios of the EEG to analyze the development of different sleep/wake states and state dynamics between vigilance states. EEG of recording electrodes at the frontal and parietal lobe were analyzed using conventional scoring criteria and SST. RESULTS We found that movement velocity, trajectories between sleep states and micro-arousals changed as an inverse U-shaped curve across maturation. At all ages, movement velocity over the frontal lobe is higher compared to the parietal lobe, while the number of trajectories and micro-arousals are reduced. Furthermore, we showed that SWA correlates negatively with movement velocity and the number of micro-arousals. The velocity in the parietal lobe correlates positively with the number of micro-arousals. As for SWA, trajectories seem primarily to depend on sleep homeostasis regulatory mechanisms while the movement velocity seems to be modulated by other sleep regulators like the circadian rhythms. CONCLUSIONS New insights in sleep/wake state dynamics are established with the SST, because trajectories, micro-arousals and velocities are not evident by traditional scoring methods. These dynamic measures may represent new indicators for changes in sleep regulatory processes across maturation

Diurnal changes in electrocorticogram sleep slow-wave activity during development in rats

According to the homeostatic regulation of sleep, sleep pressure accumulates during wakefulness, further increases during sleep deprivation and dissipates during subsequent sleep. Sleep pressure is electrophysiologically reflected by electroencephalogram slow-wave activity during non-rapid eye movement sleep, and is thought to be stable across time. During childhood and adolescence the brain undergoes massive reorganization processes. Slow-wave activity during these developmental periods has been shown in humans to follow an inverted U-shaped trajectory, which recently was replicated in rats. The goal of this study was to investigate in rats the diurnal changes of slow-wave activity during the inverted U-shaped developmental trajectory of slow-wave activity. To do so, we performed longitudinal electrocorticogram recordings, and compared the level of slow-wave activity at the beginning with the slow-wave activity level at the end of 24-h baselines in two sets of Sprague-Dawley rats. In younger animals (n = 17) we investigated specific postnatal days when overall slow-wave activity increases (postnatal day 26), peaks (postnatal day 28) and decreases (>postnatal day 28). The same analysis was performed in older animals (postnatal day 48, n = 6). Our results show a gain of slow-wave activity across 24 h on postnatal day 26, followed by no net changes on postnatal day 28, which was then followed by a loss of slow-wave activity during subsequent days (>postnatal day 28). Older animals did not show any net changes in slow-wave activity across 24 h. These results cannot be explained by differences in vigilance states. Thus, slow-wave activity during this developmental period may not only reflect the trajectory of sleep pressure but may additionally reflect maturational processes

MOESM1 of Sleep/wake movement velocities, trajectories and micro-arousals during maturation in rats

Additional file 1: Fig. S1. a Point densities of 2D state space plots of an “average” animal with distinct clusters at the frontal lobe across 4 selected days. Each plot shows 6 h of EEG activity, and each point represents 1 s of EEG activity. Warm colors indicate regions where the average density is high and cool colors indicate low average density. The numbers in the color bar are arbitrary. b “Average” state space densities on 4 selected days, projected into ratio 2. Each of the vigilance state space point densities (not shown) was projected separately into ratio 2. Blue—Wake; Green—REM; Red – NREM and Black—summation of all vigilance sleep state

SWA trajectory across age in sham and caffeine treated animals.

<p>(<b>A</b>) Sample ECoG traces of a sham and caffeine treated animal on P30 and P38, respectively. (<b>B</b>) Trajectory of sleep slow wave activity (ECoG power between 1 and 4 Hz, averaged over the first 3 hours after light onset) between postnatal day 25 (P25) and P45 for sham (n = 17) and caffeine (n = 11) treated animals. The grey shaded background illustrates the period of caffeine administration. A two-way repeated measures ANOVA with factor age (P25–P45) and condition (caffeine and sham) was significant for age and condition (p<0.05). Crosses indicate increased SWA in caffeine compared to sham treated animals (black, p<0.05, gray, p<0.08), unpaired Student's t-test).</p

Vigilance states across age.

<p>Wakefulness and non rapid eye movement (NREM) sleep in sham (sh, n = 15) and caffeine (caf, n = 11) treated animals, expressed as a percentage of 12 hours recording time (rec. time) for the light and dark period before (P29), during (P31) and after (P38) caffeine treatment. A two-way repeated measures ANOVA with factor age (P29, P31 and P38) and condition (caffeine and sham) performed for NREM sleep and wakefulness during the light period was significant for age. The same analyses for NREM sleep and wakefulness during the dark period revealed an effect of age and an interaction between condition and age (all, p<0.05). The group comparison during caffeine application (P31) showed increased wakefulness and decreased NREM sleep during the dark period, respectively (#p<0.05, unpaired Student's t-test).</p

Behavioral changes across age.

<p>(<b>A, B, C</b>) The amount of grooming, quiet waking and exploration expressed as a percentage of total behavior are shown for P28 and P42. Significant changes across age are illustrated by an asterisk (p<0.05, paired Student's t-test). (<b>D</b>) The increase in object exploration time from P28 to P42 was reduced in caffeine (n = 9) compared to sham (n = 8) treated animals (#p<0.05, Mann-Whitney U-test).</p

Structural changes across age.

<p>(<b>A</b>) Representative example image of a coronal section stained for vesicular acetylcholine transporter protein (VAChT), a specific marker for cholinergic presynaptic terminals. The dotted black box indicates the location of the randomly selected images for further analyses. One example, indicated by the solid black box is enlarged for postnatal day 30 (P30, right image). Below representative images for P42 after either sham or caffeine treatment are shown. (<b>B</b>) Reduction of the VAChT stained area, assumed to reflect cholinergic presynaptic terminals from P30 to P42 (n = 6 per group, *p<0.05, Mann-Whitney U-test). Caffeine treated animals show a diminished reduction of presynaptic cholinergic terminals at P42.</p

Caffeine reduces the build up of slow wave energy (SWE) during early caffeine treatment.

<p>Filled circles represent accumulated SWE (see Materials and Methods for details) in 3 hour intervals during the period when caffeine was initiated on P30 until the end of the second day of treatment (P31). The period of caffeine administration is illustrated by the grey shaded background. The white and black bars at the top of each graph indicate the 12 hour light and the 12 hour dark period, respectively. Crosses indicate reduced SWE in caffeine (n = 11) compared to sham (n = 17) treated animals (p<0.05, unpaired t-test). Error bars indicate SEM.</p