Brainstem and hypothalamic regulation of sleep pressure and rebound in newborn rats

Abstract

Sleep pressure and rebound comprise the two compensatory or "homeostatic" responses to sleep deprivation. Although sleep pressure is expressed by infant rats as early as postnatal day (P)5, sleep rebound does not appear to emerge until after P11. We reexamined the developmental expression of these sleep-regulatory processes in P2 and P8 rats by depriving them of sleep for 30 min using a cold, arousing stimulus delivered to a cold-sensitive region of the snout. This method effectively increased sleep pressure over the 30-min period (i.e., increases in the number of arousing stimuli presented over time). Moreover, sleep rebound (i.e., increased sleep during the recovery period) is demonstrated for the first time at these ages. Next, we showed that precollicular transections in P2 rats prevent sleep rebound without affecting sleep pressure, suggesting that the brainstem is sufficient to support sleep pressure, but sleep rebound depends on neural mechanisms that lie rostral to the transection. Finally, again in P2 rats, we used c-fos immunohistochemistry to examine neural activation throughout the neuraxis during sleep deprivation and recovery. Sleep deprivation and rebound were accompanied by significant increases in neural activation in both brainstem and hypothalamic nuclei, including the ventrolateral preoptic area and median preoptic nucleus. This early developmental expression of sleep pressure and rebound and the apparent involvement of brainstem and hypothalamic structures in their expression further solidify the notion that sleep-wake processes in newborns-defined at these ages without reference to state-dependent EEG activity-provide the foundation on which the more familiar processes of adults are built.

Figure 1

(A) Location of thermal receptive fields (denoted by circles) responsive to cold stimulation on the face in rats. Adapted from Dickenson et al., 1979. (B) Timeline depicting the procedure for Experiment 3. After a 30-min baseline period, the 30-min deprivation period began. To allow for c-fos induction so as to detect changes in neural activation in response to sleep deprivation, pups in the Deprivation group were sacrificed 90 min after the end of the deprivation period. Similarly, pups in the Recovery group were sacrificed 90 min after they were allowed 60 min of recovery sleep. Sham pups were also sacrificed at these times but were never deprived of sleep.

Figure 2

Representative data from a P2 rat at the beginning and end of the deprivation period in Experiment 1. Top: Nuchal EMG from the first 5 min of the deprivation period. Bottom: Nuchal EMG from the last 5 min of the deprivation period. Arrows denote manual presentation of the arousing orofacial stimulus.

Figure 3

(A) Mean number of presentations of the arousing stimulus for each 5-min interval of the deprivation period in Experiment 1 for P2 (filled circles) and P8 (open squares) rats. At both ages, the number of presentations increased significantly over the 30-min deprivation period, indicative of sleep pressure. * significant difference from the first 5-min interval. Mean sleep bout durations of (B) P2 and (C) P8 rats for each 30-min period of the experiment for Control (open circles) and Deprived (filled squares) groups. Mean bout durations were significantly reduced in Deprived subjects during the deprivation period, but significantly increased during the recovery periods, indicative of sleep rebound. * significant difference from the Control group. All means are presented with standard errors.

Figure 4

(A) Location of precollicular decerebrations in the P2 rats in Experiment 2. Black lines indicate the anterior-to-posterior range of the transections across all subjects. (B) Mean number of presentations of the arousing stimulus for each 5-min interval during the deprivation period for Sham+Deprived (filled squares) and Transected+Deprived (open circles) groups. In both groups, the number of presentations increased significantly over the 30-min deprivation period, indicative of sleep pressure. * significant difference from the first 5-min interval. (C) Mean sleep bout durations for the Sham+Deprived (filled squares), Transected+Deprived (open circles), and Transected+Undeprived (filled triangles) groups. Mean bout duration was significantly reduced in both Deprived groups during the deprivation period, but only the Sham+Deprived group exhibited a significant increase in bout duration during the recovery periods. † significant difference from Transected+Undeprived. * significant difference from Sham+Deprived and Transected+Undeprived. All means are presented with standard errors.

Figure 5

Mean number of Fos-ir positive cells per mm² for each area sampled in the P2 rats in Experiment 3. Data are clustered into 3 groups reflecting nuclei that exhibited wake-active (left), sleep-active (middle), and state-indifferent (right) responses to sleep deprivation and recovery. See Figure 1B for the timeline for this experiment and the designation of experimental groups. * significant difference from Sham groups. † significant difference from Sham and Deprivation groups. All means are presented with standard errors. LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; DMH, dorsomedial hypothalamus; PnO, nucleus pontis oralis; MnPO, median preoptic nucleus; VLPO, ventrolateral preoptic nucleus; SCN, suprachiasmatic nucleus; MPA, median preoptic area; BF, basal forebrain; PVN, paraventricular nucleus.

Figure 6

Representative examples of Fos labeling in (A) hypothalamic and (B) brainstem sections from P2 rats in Experiment 3. (A) Left: coronal section depicting median preoptic nucleus (MnPO; green box) and ventrolateral preoptic nucleus (VLPO; red box) for a Recovery P2 subject. Middle: Enlarged sections illustrating Fos labeling in the MnPO (top) and VLPO (bottom). Right: Corresponding sections from the MnPO (top) and VLPO (bottom) in a Sham subject. (B) Representative coronal brainstem sections from Recovery (left) and Sham (right) pups. AC, anterior commissure; 3V, third ventricle; PnO, nucleus pontis oralis; MnR, median raphe nucleus.