Interactive report
Differences in gene expression between sleep and waking as revealed by mRNA differential display1

https://doi.org/10.1016/S0169-328X(98)00057-6Get rights and content

Abstract

In order to systematically investigate differences in gene expression between sleep and waking, mRNA differential display was used to examine mRNAs from the cerebral cortex of rats who had been spontaneously asleep, spontaneously awake, or sleep deprived for a period of 3 h. It was found that, while the vast majority of transcripts were expressed at the same level among these three conditions, the expression of a subset of mRNAs was modulated by sleep and waking. Half of these transcripts had known sequences in databases. RNAs expressed at higher levels during waking included those for the transcription factors c-fos, NGFI-A, and rlf, as well as three transcripts encoded by the mitochondrial genome, those for subunit I of cytochrome c oxidase, subunit 2 of NADH dehydrogenase, and 12S rRNA. As shown by in situ hybridization, the level of RNAs encoded by the mitochondrial genome was uniformly higher during waking in many cortical regions and in several extracortical structures. By contrast, mRNA levels corresponding to two mitochondrial protein subunits encoded by the nuclear genome were unchanged. This finding suggests the hypothesis that an increase in the level of mitochondrial RNAs may represent a rapid regulatory response of neural tissue to adapt to the increased metabolic demand of waking with respect to sleep.

Introduction

The molecular correlates of sleep and waking are still largely unknown. It was recently found, however, that in many brain regions mRNAs and protein products of several immediate early genes (IEGs) are present at higher levels after a few hours of spontaneous or forced waking than after corresponding periods of sleep 5, 10, 11, 20, 27, 39, 41, 42, 43. IEGs such as c-fos and NGFI-A are transcription factors that can regulate the expression of other genes [24], raising the question of whether the level of other mRNAs differs between periods of spontaneous sleep and waking. According to previous studies, sleep deprivation of up to 48 h influences the overall level of transcription and translation in the brain 7, 40. Other studies have shown that RNA levels in the cerebral cortex are modulated by EEG synchronization [19]. Subtractive cDNA cloning has been used to isolate a few forebrain transcripts whose levels are modulated by 24 h of sleep deprivation [46]. However, it is unknown whether the levels of specific mRNAs vary between spontaneous sleep and waking.

In the present study, we used a modified version of mRNA differential display 29, 44to systematically compare mRNA levels in the cerebral cortex of rats between the following 3 conditions: 3 h of spontaneous sleep, 3 h of spontaneous waking, and 3 h of forced waking induced by gentle handling. These 3 conditions were chosen in order to restrict the search for differentially expressed mRNAs to those that are related to sleep and waking per se, as opposed to those related to circadian factors or handling (see Table 1). A period of 3 h was chosen because IEG mRNA and protein levels differ markedly depending whether an animal spent such an interval awake or asleep 11, 43. Furthermore, sleep propensity is at its highest 3 h after light onset and at its lowest 3 h after light offset [8]. It is also known that 3 h of sleep deprivation are sufficient to trigger the homeostatic regulation of sleep, as indicated by subsequent episodes of sleep rebound [49].

To highlight the functional consequences of sleep and waking, our analysis focused on the cerebral cortex. Many of the leading restorative hypotheses about the functions of sleep 23, 34and its local mechanisms [26]consider the cerebral cortex as a prime target. Furthermore, the cerebral cortex is the structure that shows the greatest functional impairment after sleep deprivation in humans [23]. Finally, previous studies have demonstrated that significant differences in the levels of IEGs between sleep and waking are found in most cortical regions 5, 10, 11, 20, 27, 39, 41, 42, 43.

Section snippets

Recordings

Male Wistar WKY rats (Charles River, 300–350 g) were anesthetized with pentobarbital (60–75 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

All rats were recorded continuously for several days after adaptation to the recording environment to establish baseline percentages and distributions of waking, non-REM sleep, and REM (rapid eye movement) sleep. Baseline data, reported in Table 2, were in agreement with standard values [8]. Table 3 shows percentages of waking, non-REM sleep, and REM sleep corresponding to the last 3 recording hours for the 3 groups of rats used in this study. The first group comprised rats sacrificed after

Discussion

A systematic comparison of mRNA levels in the cerebral cortex of rats after 3 h of sleep, 3 h of sleep deprivation, and 3 h of spontaneous waking was performed using mRNA differential display. Six transcripts that were present at higher levels in waking than in sleep were identified. Three were mitochondrial transcripts encoded by the mitochondrial genome and three were transcription factors. Their increased expression in waking was confirmed using in situ hybridization and RPA. Several other

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. C.C. is a Joseph Drown Foundation Fellow. We thank Dr. Melvena Teasdale and Glen A. Davis for their expert contribution and Dr. Maria Pompeiano and Dr. J. Michael Salbaum for their help in the initial stages of this project.

References (53)

  • M.T.T Wong-Riley et al.

    Brain cytochrome oxidase subunit complementary DNAs: isolation, subcloning, sequencing, light and electron microscopic in situ hybridization of transcripts, and regulation by neuronal activity

    Neuroscience

    (1997)
  • P. Arrighi, C. Cirelli, G. Tononi, M. Pompeiano, Jun B expression during sleep-waking states and after sleep...
  • G Attardi et al.

    Biogenesis of mitochondria

    Ann. Rev. Cell Biol.

    (1988)
  • R Basheer et al.

    Effects of sleep on wake-induced c-fos expression

    J. Neurosci.

    (1997)
  • D Bauer et al.

    Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR)

    Nucl. Acid. Res.

    (1993)
  • P Bobillier et al.

    The effect of sleep deprivation upon the in vivo incorporation of tritiated amino acids into brain proteins in the rat at three different age levels

    J. Neurochem.

    (1974)
  • A.A Borbély et al.

    Daily patterns of sleep, motor activity and feeding in the rat: effects of regular and gradually extended photoperiods

    J. Comp. Physiol.

    (1978)
  • C Cirelli et al.

    Fos-like immunoreactivity in the rat brain in spontaneous waking and sleep

    Arch. Ital. Biol.

    (1993)
  • C Cirelli et al.

    Sleep deprivation and c-fos expression in the rat brain

    J. Sleep Res.

    (1995)
  • C Cirelli et al.

    Sleep-waking changes after c-fos antisense injections in the medial preoptic area

    Neuroreport

    (1995)
  • C Cirelli et al.

    Neuronal gene expression in the waking state: a role for the locus coeruleus

    Science

    (1996)
  • C. Cirelli, M. Pompeiano, G. Tononi, Immediate early genes as a tool to understand the regulation of the sleep-waking...
  • T Curran et al.

    Isolation and characterization of the c-fos (rat) cDNA and analysis of posttranscriptional modification in vitro

    Oncogene

    (1987)
  • J Douglass et al.

    PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine

    J. Neurosci.

    (1995)
  • M Ereciñska et al.

    ATP and brain function

    J. Cereb. Blood Flow Metab.

    (1989)
  • R Gelfand et al.

    Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable

    Mol. Cell Biol.

    (1981)
  • Cited by (90)

    • Mechanisms of sleep and circadian ontogeny through the lens of neurodevelopmental disorders

      2019, Neurobiology of Learning and Memory
      Citation Excerpt :

      For example, chronic sleep deprivation kills rodents and flies but does not result in brain injury (Cirelli, Shaw, Rechtschaffen, & Tononi, 1999). To study the molecular events that differentiate sleep from wake and accompany sleep need almost all have utilized forced sleep deprivation (SD) during a period when an animal would habitually be asleep (Cirelli, Faraguna, & Tononi, 2006; Cirelli, Gutierrez, & Tononi, 2004; Cirelli & Tononi, 1998, 1999, 2000; Thompson et al., 2010; Wang et al., 2018). These studies have summarily demonstrated that wakefulness promotes the expression of genes associated with synaptic plasticity.

    • Neuroscience-driven discovery and development of sleep therapeutics

      2014, Pharmacology and Therapeutics
      Citation Excerpt :

      Prior to genome-wide association studies applied to humans, searches for specific “sleep genes” had been conducted in animal studies (Valatx et al., 1972). Such genetic approaches allow to reveal state-dependent gene expression or associated traits by means of polymerase chain reaction (PCR) (Cirelli & Tononi, 1998), microarray (Terao et al., 2003; Cirelli & Tononi, 2004), quantitative trait loci (QTL) analyses (Toth & Williams, 1999; Tafti et al., 2003) among others. Using advanced cDNA microarray technology with a verification by real-time quantitative PCR and ribonuclease protection assay, many wake- or sleep-dependent transcripts in the rat cortex have been demonstrated, and the results were interpreted to suggest function of sleep, e.g. its involvement in protein synthesis and neural plasticity (Cirelli et al., 2004, 2006).

    View all citing articles on Scopus
    1

    Published on the World Wide Web on 30 March 1998.

    View full text