Elsevier

Brain Research

Volume 885, Issue 2, 8 December 2000, Pages 303-321
Brain Research

Interactive report
Gene expression in the brain across the sleep–waking cycle1

https://doi.org/10.1016/S0006-8993(00)03008-0Get rights and content

Abstract

Sleep and waking differ significantly in terms of behavior, metabolism, and neuronal activity. Recent evidence indicates that sleep and waking also differ with respect to the expression of certain genes. To systematically investigate such changes, we used mRNA differential display and cDNA microarrays to screen ∼10 000 transcripts expressed in the cerebral cortex of rats after 8 h of sleep, spontaneous waking, or sleep deprivation. We found that 44 genes had higher mRNA levels after waking and/or sleep deprivation relative to sleep, while 10 were upregulated after sleep. Known genes that were upregulated in waking and sleep deprivation can be grouped into the following categories: immediate early genes/transcription factors (Arc, CHOP, IER5, NGFI-A, NGFI-B, N-Ras, Stat3), genes related to energy metabolism (glucose type I transporter Glut1, Vgf), growth factors/adhesion molecules (BDNF, TrkB, F3 adhesion molecule), chaperones/heat shock proteins (BiP, ERP72, GRP75, HSP60, HSP70), vesicle- and synapse-related genes (chromogranin C, synaptotagmin IV), neurotransmitter/hormone receptors (adrenergic receptor α1A and β2, GABAA receptor β3, glutamate NMDA receptor 2A, glutamate AMPA receptor GluR2 and GluR3, nicotinic acetylcholine receptor β2, thyroid hormone receptor TRβ), neurotransmitter transporters (glutamate/aspartate transporter GLAST, Na+/Cl transporter NTT4/Rxt1), enzymes (aryl sulfotransferase, c-jun N-terminal kinase 1, serum/glucocorticoid-induced serine/threonine kinase), and a miscellaneous group (calmodulin, cyclin D2, LMO-4, metallothionein 3). Several other genes that were upregulated in waking and all the genes upregulated in sleep, with the exception of the one coding for membrane protein E25, did not match any known sequence. Thus, significant changes in gene expression occur across behavioral states, which are likely to affect basic cellular functions such as RNA and protein synthesis, neural plasticity, neurotransmission, and metabolism.

Introduction

The functions of sleep remain obscure despite its ubiquitous occurrence [75] and its exquisite homeostatic regulation [6]: in virtually all species studied so far, prolonged waking is followed by a compensatory increase in the duration and/or the intensity of sleep [88]. Such homeostatic regulation of sleep suggests that a distinct physiological, biochemical, or molecular process may build up beyond its usual level if sleep initiation is postponed, and that uncovering such processes may provide critical clues as to the functions of sleep.

Over the past few years, evidence has been collected indicating that several compounds accumulate in the brain during short periods of spontaneous waking or sleep deprivation, potentially leading to a need for restoration. For example, the neurotransmitter and metabolic by-product adenosine has been shown to increase in proportion to the amount of waking in the basal forebrain region [74]. Recent studies have provided evidence that the expression of certain genes changes as a function of behavioral state. In the rat cerebral cortex, the expression of tumor necrosis factor[94], interleukin 1[48], [86], cortistatin[19] and BDNF[70] increases after prolonged waking or sleep deprivation relative to sleep, while the expression of neurogranin and dendrin decreases after 24 h of sleep deprivation [61], [62], [76]. For a few other genes, such as tyrosine hydroxylase[4], [73], growth hormone-releasing hormone, somatostatin[91], and galanin[90], localized changes have been demonstrated after total or selective REM deprivation in certain hypothalamic or brainstem nuclei. Finally, several laboratories have shown that the transition from sleep to waking is accompanied by the activation of the expression of immediate-early genes, such as c-fos and NGFI-A, in many brain regions (see Refs. [13], [14], [16]). The finding of a significant change in the expression of these transcription factors between sleep and waking suggests that other ‘late’ genes, in addition to those already identified, may in turn be activated or deactivated in relation to the behavioral state of the animal.

While targeted experiments aimed at studying specific genes have proved very useful, it is unlikely that they will ever offer an exhaustive picture of the regulation of gene expression by sleep and waking. An alternative to candidate gene approaches is the systematic investigation of all the genes whose expression in the brain changes in relation to different behavioral states. As a first step in this direction, in a recent study we used mRNA differential display to screen for changes in gene expression in the cerebral cortex of rats after short periods (3 h) of sleep, spontaneous waking, and sleep deprivation [12]. It was found that several immediate early genes, as well as mitochondrial genes encoded by the mitochondrial genome, are differentially expressed across the sleep–waking cycle.

The present paper continues and extends previous work, with the goal of systematically establishing the differences in gene expression that occur between sleep and waking. To this end, we analyzed the expression of ∼10 000 genes in the cerebral cortex of rats after sustained periods (8 h) of sleep, spontaneous waking, and sleep deprivation. We have used two complementary approaches: mRNA differential display, which allowed us an unbiased screen for random mRNAs without prior assumptions as to which transcripts might change, and cDNA microarray technology, which allowed us to screen 1176 known mRNAs, most of which are specifically expressed in the rat brain. We focused on the cerebral cortex as it appears to be the structure most significantly affected by sleep deprivation in humans [34] and is the main target of the restorative effects of sleep according to several influential hypotheses about the functions and the local mechanisms of sleep [41], [50].

Section snippets

Recordings

Male Wistar WKY rats (Charles River, 300–350 g) were anesthetized with pentobarbital (65–75 mg/kg) and implanted with stainless steel, round-tipped miniature screw electrodes in the skull to record the electroencephalogram (EEG), and with silver electrodes in the neck muscles of both sides to record the electromyogram. EEG electrodes were located over frontal cortex (2 mm anterior to the bregma and 2 mm lateral to the midline) and over occipital cortex (4 mm posterior to the bregma and 3.8 mm

Sleep percentages

Rats kept in a 12:12 light/dark cycle are asleep for most of the light period and awake for most of the dark period. We selected rats that had been asleep for the first 8 h of the light period, rats that had been spontaneously awake for the first 8 h of the dark period, and rats that had been sleep deprived during the light period for 8 h (see Section 2.1 for details). The three experimental conditions were chosen to distinguish between changes in gene expression related to sleep and waking per

General findings

The present study provides a comprehensive evaluation of changes in gene expression in the cerebral cortex as a function of behavioral state. The results obtained after sustained periods of sleep, sleep deprivation, and spontaneous waking (8 h) complement and extend previous findings from a screening of gene expression after shorter periods of sleep and waking (3 h [12], [13], [14]). Several general conclusions can be drawn from these experiments. First, only a small minority of the genes

Conclusions

The results of the systematic screening of brain gene expression during sleep and waking lead to several conclusions. As shown here, sleep and waking differ not only in terms of behavior, metabolism, and neuronal activity, but they are characterized by the up- or down-regulation of distinctive categories of genes. The few genes that are specifically upregulated during sleep are presently still unidentified and await further characterization. The genes that are upregulated after periods of

Acknowledgements

This work was carried out as part of the experimental neurobiology program at The Neurosciences Institute, which is supported by Neurosciences Research Foundation. The Foundation receives major support for this program from Novartis Pharmaceutical Corporation. We thank Glen A. Davis, Marijo C. Gallina, and Donald F. Robinson for their expert contribution.

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