Separating the contribution of glucocorticoids and wakefulness to the molecular and electrophysiological correlates of sleep homeostasis.

STUDY OBJECTIVES: The sleep-deprivation-induced changes in delta power, an electroencephalographical correlate of sleep need, and brain transcriptome profiles have importantly contributed to current hypotheses on sleep function. Because sleep deprivation also induces stress, we here determined the contribution of the corticosterone component of the stress response to the electrophysiological and molecular markers of sleep need in mice. DESIGN: N/A SETTINGS: Mouse sleep facility. PARTICIPANTS: C57BL/6J, AKR/J, DBA/2J mice. INTERVENTIONS: Sleep deprivation, adrenalectomy (ADX). MEASUREMENTS AND RESULTS: Sleep deprivation elevated corticosterone levels in 3 inbred strains, but this increase was larger in DBA/2J mice; i.e., the strain for which the rebound in delta power after sleep deprivation failed to reach significance. Elimination of the sleep-deprivation-associated corticosterone surge through ADX in DBA/2J mice did not, however, rescue the delta power rebound but did greatly reduce the number of transcripts affected by sleep deprivation. Genes no longer affected by sleep deprivation cover pathways previously implicated in sleep homeostasis, such as lipid, cholesterol (e.g., Ldlr, Hmgcs1, Dhcr7, -24, Fkbp5), energy and carbohydrate metabolism (e.g., Eno3, G6pc3, Mpdu1, Ugdh, Man1b1), protein biosynthesis (e.g., Sgk1, Alad, Fads3, Eif2c2, -3, Mat2a), and some circadian genes (Per1, -3), whereas others, such as Homer1a, remained unchanged. Moreover, several microRNAs were affected both by sleep deprivation and ADX. CONCLUSIONS: Our findings indicate that corticosterone contributes to the sleep-deprivation-induced changes in brain transcriptome that have been attributed to wakefulness per se. The study identified 78 transcripts that respond to sleep loss independent of corticosterone and time of day, among which genes involved in neuroprotection prominently feature, pointing to a molecular pathway directly relevant for sleep function.

Melanopsin as a sleep modulator: circadian gating of the direct effects of light on sleep and altered sleep homeostasis in Opn4(-/-) mice.

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4(-/-)) mice under various light-dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7-10 Hz) and gamma (40-70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4(-/-) mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-hratio1-h schedule revealed that the failure to respond to light in Opn4(-/-) mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4(-/-) mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4(-/-) mice slept 1 h less during the 12-h light period of a LD 12ratio12 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4(-/-) mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4(-/-) mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.

Glycogen content in the cerebral cortex increases with sleep loss in C57BL/6J mice.

We hypothesized that a function of sleep is to replenish brain glycogen stores that become depleted while awake. We have previously tested this hypothesis in three inbred strains of mice by measuring brain glycogen after a 6h sleep deprivation (SD). Unexpectedly, glycogen content in the cerebral cortex did not decrease with SD in two of the strains and was even found to increase in mice of the C57BL/6J (B6) strain. Manipulations that initially induce glycogenolysis can also induce subsequent glycogen synthesis thereby elevating glycogen content beyond baseline. It is thus possible that in B6 mice, cortical glycogen content decreased early during SD and became elevated later in SD. In the present study, we therefore measured changes in brain glycogen over the course of a 6 h SD and during recovery sleep in B6 mice. We found no evidence of a decrease at any time during the SD, instead, cortical glycogen content monotonically increased with time-spent-awake and, when sleep was allowed, started to revert to control levels. Such a time-course is opposite to the one predicted by our initial hypothesis. These results demonstrate that glycogen synthesis can be achieved during prolonged wakefulness to the extent that it outweighs glycogenolysis. Maintaining this energy store seems thus not to be functionally related to sleep in this strain.

Glucocorticoids influence brain glycogen levels during sleep deprivation.

We investigated whether glucocorticoids [i.e., corticosterone (Cort) in rats] released during sleep deprivation (SD) affect regional brain glycogen stores in 34-day-old Long-Evans rats. Adrenalectomized (with Cort replacement; Adx+) and intact animals were sleep deprived for 6 h beginning at lights on and then immediately killed by microwave irradiation. Brain and liver glycogen and glucose and plasma glucose levels were measured. After SD in intact animals, glycogen levels decreased in the cerebellum and hippocampus but not in the cortex or brain stem. By contrast, glycogen levels in the cortex of Adx+ rats increased by 43% (P < 0.001) after SD, while other regions were unaffected. Also in Adx+ animals, glucose levels were decreased by an average of 28% throughout the brain after SD. Intact sleep-deprived rats had elevations of circulating Cort, blood, and liver glucose that were absent in intact control and Adx+ animals. Different responses between brain structures after SD may be due to regional variability in metabolic rate or glycogen metabolism. Our findings suggest that the elevated glucocorticoid secretion during SD causes brain glycogenolysis in response to energy demands.

Changes in brain glycogen after sleep deprivation vary with genotype.

Sleep has been functionally implicated in brain energy homeostasis in that it could serve to replenish brain energy stores that become depleted while awake. Sleep deprivation (SD) should therefore lower brain glycogen content. We tested this hypothesis by sleep depriving mice of three inbred strains, i.e., AKR/J (AK), DBA/2J (D2), and C57BL/6J (B6), that differ greatly in their sleep regulation. After a 6-h SD, these mice and their controls were killed by microwave irradiation, and glycogen and glucose were quantified in the cerebral cortex, brain stem, and cerebellum. After SD, both measures significantly increased by approximately 40% in the cortex of B6 mice, while glycogen significantly decreased by 20-38% in brain stem and cerebellum of AK and D2 mice. In contrast, after SD, glucose content increased in all three structures in AK mice and did not change in D2 mice. The increase in glycogen after SD in B6 mice persisted under conditions of food deprivation that, by itself, lowered cortical glycogen. Furthermore, the strains that differ most in their compensatory response to sleep loss, i.e., AK and D2, did not differ in their glycogen response. Thus glycogen content per se is an unlikely end point of sleep's functional role in brain energy homeostasis.

Sleep deprivation decreases glycogen in the cerebellum but not in the cortex of young rats.

We tested whether brain glycogen reserves were depleted by sleep deprivation (SD) in Long-Evans rats 20-59 days old. Animals were sleep deprived beginning at lights on and then immediately killed by microwave irradiation. Glycogen and glucose levels were measured by a fluorescence enzymatic assay. In all age groups, SD reduced cerebellar glycogen levels by an average of 26% after 6 h of SD. No changes were observed in the cortex after 6 h of SD, but in the oldest animals, 12 h of SD increased cortical glycogen levels. There was a developmental increase in basal glycogen levels in both the cortex and cerebellum that peaked at 34 days and declined thereafter. Robust differences in cortical and cerebellar glycogen levels in response to enforced waking may reflect regional differences in energy utilization and regulation during wakefulness. These results show that brain glycogen reserves are sensitive to SD.