Figure 1.
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).
Figure 2.
SWA trajectory across age in sham and caffeine treated animals.
(A) Sample ECoG traces of a sham and caffeine treated animal on P30 and P38, respectively. (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).
Figure 3.
EEG NREM sleep spectrum across age.
Average NREM sleep EEG power between 0.5 and 25 Hz across 24 hours for sham treated animals is shown for P26 (n = 17), P28 (n = 17) and P38 (n = 15). Crosses indicate power differences across age (grey, p<0.05, black, p<0.01), paired Student's t-test).
Figure 4.
Behavioral changes across age.
(A, B, C) 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). (D) 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).
Figure 5.
Structural changes across age.
(A) 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) 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.
Figure 6.
Caffeine reduces the build up of slow wave energy (SWE) during early caffeine treatment.
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.