Akira Terao

A role for cryptochromes in sleep regulation

BackgroundThe cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes.ResultsUnder all three conditions, cry1,2-/- mice exhibit the hallmarks of high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS consolidation, and EEG delta power during NREMS). This unexpected phenotype was associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and albumin d-binding protein (dbp), which are known to be transcriptionally inhibited by CRY1,2. To further examine the relationship between circadian genes and sleep homeostasis, we examined wild type mice and rats following sleep deprivation and found increased levels of per1,2 mRNA and decreased levels of dbp mRNA specifically in the cerebral cortex; these changes subsided with recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and casein-kinase-1ε did not change with sleep deprivation.ConclusionsThese results indicate that mice lacking cryptochromes are not simply a genetic model of circadian arrhythmicity in rodents and functionally implicate cryptochromes in the homeostatic regulation of sleep.

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.

Immune response gene expression increases in the aging murine hippocampus.

Using GeneChips, basal and lipopolysaccharide (LPS)-induced gene expression was examined in the hippocampus of 3-, 12-, 18- and 24-month-old male C57BL/6 mice to identify genes whose altered expression could influence hippocampal function in advanced age. Gene elements that changed with age were selected with a t-statistic and specific expression patterns were confirmed with real-time quantitative PCR. Basal expression of 128 gene elements clearly changed with age in the hippocampus. Fourteen gene elements showed increased expression with age and these increases were validated after LPS stimulation. Major histocompatibility complex (MHC) TL region and thymic shared antigen (TSA-1) gene expression increased, suggesting T cell activation in the hippocampus with age. Cytokine (interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha) and chemokine (macrophage chemotactic protein-1) expression increased sharply in 24-month-old mice. These findings are in contrast to a decrease in the peripheral immune response, documented by decreased T cell proliferation and decreased ratios of naive to memory T cells. Age-related increases in inflammatory potential in the brain may contribute to neurodegenerative diseases of the aged.

Age-related decline in hypocretin (orexin) receptor 2 messenger RNA levels in the mouse brain

The hypocretin (Hcrt; also known as orexin) system has been implicated in arousal state regulation and energy metabolism. We hypothesize that age-related sleep problems can result from dysfunction of this system and thus measured messenger RNA (mRNA) levels of preprohcrt in the hypothalamus, and hcrt receptor 1 (hcrtr1) and hcrt receptor 2 (hcrtr2) in eight brain regions of 3, 12, 18 and 24 months old C57BL/6 mice. Expression of preprohcrt and the colocalized prodynorphin did not change with age. Whereas an age-related change in hcrtr1 mRNA expression was observed only in the hippocampus, hcrtr2 mRNA levels declined in the hippocampus, thalamus, pons, and medulla; these reductions ranged from 33 to 44%. Declining trends (P < 0.1) in hcrtr2 mRNA levels were also observed in the cortex, basal forebrain and hypothalamus. These results are consistent with the hypothesis that an age-related deterioration occurs in the Hcrt system that may contribute to age-related sleep disorders.

Age-related changes in histamine receptor mRNA levels in the mouse brain.

Several lines of evidence indicate that the histaminergic (HA) system is important for wakefulness and behavioral state regulation. We investigated the hypothesis that age-related changes in HA system occur which may be related to decreased alertness in aging. Although histidine decarboxylase mRNA levels did not change with age in C57BL/6 mice, significant differences were found in histamine H1 receptor (H1R), histamine H2 receptor (H2R), and histamine H3 receptor (H3R) mRNA levels in several brain regions. The most widespread changes were observed in H1R mRNA, which were significantly lower (27-38%) in the cortex, hypothalamus, hippocampus and medulla of 24-month-old mice relative to 3-month-old animals. Age-related changes in H2R mRNA levels were restricted to the pons and cerebellum and decreased H3R mRNA was found only in the medulla. In conjunction with the age-related decrease in hypocretin receptor 2 mRNA levels we have previously reported, decreased HA receptor mRNA levels may contribute to diminished alertness, sleep continuity, and diurnal rhythms of sleep and wakefulness in the aged.

Modulation of the promoter region of prepro-hypocretin by α-interferon

Abstract Hypocretins 1 and 2 (also called orexins A and B, respectively) are hypothalamic neuropeptides that have recently been shown to be involved in the sleep disorder narcolepsy and possibly in the normal regulation of sleep and wake functions. These two peptides are derived from a single precursor molecule called prepro-hypocretin, also known as prepro-orexin. We have cloned a 450 bp fragment from the 5′-flanking region of the human prepro-hypocretin gene and demonstrated that this fragment has promoter activity in vitro. Deletions at the 5′ end from −450 to −188 reduced the promoter activity by ∼50%. Further deletion from the 5′-end to −69 almost completely abolished promoter activity. The 450 bp fragment contains a number of potential transcription factor binding sites, including an interferon (IFN) response element. Our studies demonstrate that α-IFN strongly inhibits the promoter activity of both 450 and 188 bp fragments in a dose-dependent manner. The inhibitory effect of α-IFN is consistent with recent studies which suggest that hypocretin 1/orexin A may be involved in modulating arousal states and with the literature indicating involvement of immune-related molecules in sleep regulation.

Cyclooxygenase in the vagal afferents: is it involved in the brain prostaglandin response evoked by lipopolysaccharide?

The vagal afferents are proposed to transmit abdominal immune signals to the brain. In this immune-brain communication, prostaglandins might play a mediator role. In fact, prostaglandin receptors are abundant in the vagal afferents. We examined here the presence of cyclooxygenase, an enzyme necessary for prostaglandin biosynthesis, in the vagal afferents of rats. We also tested whether the vagal afferents contribute to the elevation of prostaglandin E2 in the brain after intraperitoneal injection of lipopolysaccharide. Under normal conditions, cyclooxygenase-1-like immunoreactivity was constitutively expressed in the vagal afferents at their central terminals and in their cell bodies. Cyclooxygenase-2-like immunoreactivity was absent in the vagal afferents under normal as well as lipopolysaccharide-challenged conditions. Instead, cyclooxygenase-2-like immunoreactivity was induced in brain endothelial cells by the lipopolysaccharide challenge. The elevation of prostaglandin E2 in the cerebrospinal fluid after lipopolysaccharide challenge was not inhibited, but was rather enhanced, by the bilateral vagotomy. These results suggest that the vagal afferents potentially generate prostaglandins, which may locally modulate the vagal signal transmission, but that the vagal afferents are not essential to the elevation of prostaglandin E2 in the brain after intraperitoneal challenge with LPS.

Gene expression in the rat brain during prostaglandin D2 and adenosinergically-induced sleep.

Previous studies have supported the hypothesis that macromolecular synthesis occurs in the brain during sleep as a response to prior waking activities and that prostaglandin D2 (PGD2) is an endogenous sleep substance whose effects are dependent on adenosine A2a receptor‐mediated signaling. We compared gene expression in the cerebral cortex, basal forebrain, and hypothalamus during PGD2‐induced and adenosinergically‐induced sleep to results from our previously published study of recovery sleep (RS) after sleep deprivation (SD). Immediate early gene expression in the cortex during sleep induced by PGD2‐ or by the selective adenosine A2a agonist CGS21680 showed limited similarity to that observed during RS while, in the basal forebrain and hypothalamus, widespread activation of immediate early genes not seen during RS occurred. In all three brain regions, PGD2 and CGS21680 reduced the expression of arc, a transcript whose expression is elevated during SD. Using GeneChips®, the majority of genes induced by either PGD2 or CGS21680 were induced by both, suggesting activation of the same pathways. However, gene expression induced in the brain after PGD2 or CGS21680 treatment was distinct from that described during RS after SD and apparently involves glial cell gene activation and signaling pathways in neural‐immune interactions.