Gene expression in the rat brain during sleep deprivation and recovery sleep: an Affymetrix GeneChip study.

Previous studies have demonstrated that macromolecular synthesis in the brain is modulated in association with the occurrence of sleep and wakefulness. Similarly, the spectral composition of electroencephalographic activity that occurs during sleep is dependent on the duration of prior wakefulness. Since this homeostatic relationship between wake and sleep is highly conserved across mammalian species, genes that are truly involved in the electroencephalographic response to sleep deprivation might be expected to be conserved across mammalian species. Therefore, in the rat cerebral cortex, we have studied the effects of sleep deprivation on the expression of immediate early gene and heat shock protein mRNAs previously shown to be upregulated in the mouse brain in sleep deprivation and in recovery sleep after sleep deprivation. We find that the molecular response to sleep deprivation and recovery sleep in the brain is highly conserved between these two mammalian species, at least in terms of expression of immediate early gene and heat shock protein family members. Using Affymetrix Neurobiology U34 GeneChips , we also screened the rat cerebral cortex, basal forebrain, and hypothalamus for other genes whose expression may be modulated by sleep deprivation or recovery sleep. We find that the response of the basal forebrain to sleep deprivation is more similar to that of the cerebral cortex than to the hypothalamus. Together, these results suggest that sleep-dependent changes in gene expression in the cerebral cortex are similar across rodent species and therefore may underlie sleep history-dependent changes in sleep electroencephalographic activity.

Altered rapid eye movement sleep timing in serotonin transporter knockout mice.

The monoamine neurotransmitter serotonin has long been implicated in development and maintenance of sleep patterns, yet the role of the serotonin transporter (SERT) in these processes has not been evaluated in detail. We report that genetically engineered SERT knockout mice exhibit more REM sleep (REMS) than wild type littermates (11 vs 7% of recording time under baseline conditions) and display more frequent REMS bouts that last longer. This phenotype resembles the previously reported long-term effect of repeated treatment with SERT inhibitor compounds rather than the acute REMS suppressing effect of treatment with such compounds, and is thus likely to reflect neuroadaptations to the absence of SERT, rather than an acute effect of its absence in the adult. While electroencephalographic (EEG) spectra did not differ between SERT knockout and wild type mice during non-REM sleep (NREMS) or REMS, the dynamics of the EEG during the transition from NREMS to REMS differed between the genotypes. The surge in EEG power in both the 6-9 Hz and 10-16 Hz ranges that occurs just prior to the onset of REMS (pre-REMS power surge) is of greater magnitude in SERT knockout mice than in wild type littermate controls. This observation contrasts with the reduced magnitude pre-REMS power surge observed in rats subjected to REMS deprivation relative to yoked controls. These results indicate that the pre-REMS power surge is influenced by REMS history and by monoaminergic transmission. Genetic differences in serotonin systems and developmental exposure to SERT blockers are likely to exert effects on REMS.

Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background.

The hypocretins (1 and 2) have emerged as key regulators of sleep and wakefulness. We developed a high-throughput enzyme immunoassay (EIA) to measure total brain hypocretin levels from large numbers of mice. Hypocretin levels were not altered by circadian time or age. However, significant differences in one or both hypocretin peptides were observed between different mouse strains. We studied hypocretin levels in knockout and transgenic mouse models with obesity, circadian gene mutations or monoaminergic defects. Compared to controls, only histamine receptor knockouts had lower hypocretin levels. This was most pronounced in H1 receptor knockouts suggesting the existence of a positive feedback loop between hypocretin and histaminergic neurons.

Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice.

The hypocretin/orexin ligand-receptor system has recently been implicated in the sleep disorder narcolepsy. During the dark (active) period, null mutants of the prepro-orexin (prepro-hypocretin) gene have cataplectic attacks and increased levels of both rapid eye movement (REM) and non-REM (NREM) sleep. Intracerebroventricular injection of one of the encoded neuropeptides, orexin-A, early in the light period increases wakefulness and reduces REM sleep in the rat, suggesting that this system may be involved in the normal regulation of sleep and wakefulness. To further test this hypothesis, we measured hypocretin (hcrt) mRNA levels by both Northern hybridization and Taqman analysis in mouse and rat hypothalamus after short-term (6 h) sleep deprivation (SD) and 2-4 hours after recovery from SD. Although our SD procedures effectively induced a sleep debt and increased c-fos mRNA expression in the cortex and hypothalamus as described by other investigators, we found that hcrt mRNA levels were not significantly changed in either species either after SD or after recovery from SD. If the hcrt system is involved in normal regulation of sleep and wakefulness, longer periods of SD may be necessary to affect hcrt mRNA levels or changes may occur at the protein rather than mRNA level. Alternatively, this system may also be involved in another function that counterbalances any SD-induced changes in hcrt mRNA levels.

Circadian and homeostatic control of rapid eye movement (REM) sleep: promotion of REM tendency by the suprachiasmatic nucleus.

The daily timing of rapid eye movement (REM) sleep reflects an interaction between the circadian pacemaker located in the suprachiasmatic nucleus of the hypothalamus (SCN) and a homeostatic process that induces compensatory REM sleep in response to REM sleep loss. Whether the circadian variation in REM sleep propensity is caused by active promotion, inhibition, or passive gating of REM sleep homeostasis by the SCN is unknown. To investigate these possibilities, compensatory responses to 24 hr REM sleep deprivation (RSD) were compared between SCN-lesioned (SCNx) and sham-lesioned rats at different times of day in constant dark. The attempts to enter REM sleep (REM tendency) increased during RSD in all rats and were modulated by circadian phase in sham-lesioned, but not SCNx rats. REM sleep homeostasis interacted with circadian time, such that REM tendency doubled during the rest phase in sham-lesioned rats relative to SCNx rats (F((6,93)) = 17.9; p = 0.0001). However, REM tendency was indistinguishable between SCNx and sham-lesioned rats during the activity phase, suggesting the SCN does not inhibit REM tendency at this time. By contrast, the amount of compensatory REM sleep examined 2, 6, 12, or 24 hr after RSD did not depend on circadian phase. Thus, transitions into REM sleep are facilitated by the SCN during the rest phase, but the amount of REM sleep, once initiated, is determined primarily by homeostatic mechanisms. This work supports a role for the SCN in the active promotion of REM sleep at specific times of day.

Caffeine during sleep deprivation: sleep tendency and dynamics of recovery sleep in rats.

The adenosine antagonist caffeine disrupts sleep, but whether caffeine promotes wakefulness by interfering with the expression of sleep or by attenuating sleepiness is unknown. The ability of caffeine to reduce sleep tendency in rats was directly tested by quantifying the number of stimuli needed to maintain wakefulness during sleep deprivation for 6 h after systemic caffeine treatment. In addition, the influence of caffeine on the dynamics between nonrapid-eye-movement (NREM) and rapid-eye-movement (REM) sleep was investigated by comparing the magnitude and time course of the compensatory sleep responses for 42 h postsleep deprivation. Caffeine significantly reduced the attempts to sleep during sleep deprivation, F(1,9) 8.83, p = 0.0157; 44.9% of vehicle), but did not change compensatory slow-wave activity during recovery sleep. During the initial recovery phase, caffeine suppressed compensatory REM sleep and reduced, but did not block, compensatory NREM sleep duration and continuity. By 42 h postsleep deprivation, the amount of NREM recovered (70.0% of deficit) did not differ from vehicle. In contrast, the REM sleep deficit recovered after caffeine (100%) was more than after vehicle (43.9%). Thus, caffeine slowed the rate of compensatory sleep after sleep deprivation, as indexed by the duration of sleep states and sleep continuity.