Interactive reportDifferences in gene expression between sleep and waking as revealed by mRNA differential display1
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.
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Published on the World Wide Web on 30 March 1998.