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

Molecular Brain Research 56 Ž1998. 293–305

Interactive report

Differences in gene expression between sleep and waking as revealed by mRNA differential display 1 Chiara Cirelli, Giulio Tononi

)

The Neurosciences Institute, 10640 John J. Hopkins DriÕe, San Diego, CA 92121, USA Accepted 18 February 1998

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. q 1998 Elsevier Science B.V. Keywords: Sleep; Sleep deprivation; Wakefulness; Cerebral cortex; Mitochondrion; Fos; Zifr268; Differential PCR display; Rat

1. 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 ŽIEGs. are present at higher levels after a few hours of spontaneous or forced waking th an after co rresp o n d in g p erio d s o f sleep w5,10,11,20,27,39,41–43x. IEGs such as c-fos and NGFI-A are transcription factors that can regulate the expression of other genes w24x, 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 w7,40x. Other studies have shown that RNA levels in the cerebral cortex are modulated by EEG synchronization w19x. Subtractive

) Corresponding author: Tel.: q1 Ž619. 626 2100; Fax: q1 Ž619. 626 2199; E-mail: [email protected] 1 Published on the World Wide Web on 30 March 1998.

0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 0 5 7 - 6

cDNA cloning has been used to isolate a few forebrain transcripts whose levels are modulated by 24 h of sleep deprivation w46x. 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 w29,44x to 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 Žsee 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 w11,43x. Furthermore, sleep propensity is at its highest 3 h after light onset and at its lowest 3 h after light offset w8x. 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 w49x.

C. Cirelli, G. Tononir Molecular Brain Research 56 (1998) 293–305

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Table 1 Differential expression of bands identified by mRNA differential display, subdivided by factor Experimental condition S–L Factor

Spontaneous sleep, light period, 3 h

Sleep Waking Circadian, light Circadian, dark Handling

≠ ≠

W–L

W–D

Sleep deprivation, light period, 3 h

Spontaneous waking, dark period, 3 h

≠ ≠

≠ ≠

≠

The 3 experimental conditions permit to distinguish between a differential change associated with the factors sleep ŽS–L., waking ŽW–L, W–D., circadian Žlight period: S–L and W–L; dark period: W–D., and handling ŽW–L.. For example, a band that is expressed at higher levels in conditions W–L and W–D Žarrowheads. is associated with the factor waking, while a band that is expressed at higher or lower levels in condition W–L is associated with the factor handling.

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 w23,34x and its local mechanisms w26x 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 w23x. Finally, previous studies have demonstrated that significant differences in the levels of IEGs between sleep and waking are found in most cortical regions w5,10,11,20,27,39,41–43x.

2. Materials and methods 2.1. Recordings Male Wistar WKY rats ŽCharles River, 300–350 g. were anesthetized with pentobarbital Ž60–75 mgrkg. and implanted with stainless steel, round-tipped miniature screw electrodes in the skull to record the electroencephalogram ŽEEG., and with silver electrodes in the neck muscles of both sides to record the electromyogram. EEG electrodes were located over frontal cortex Ž2 mm anterior to the bregma and 2 mm lateral to the midline. and over occipital cortex Ž4 mm posterior to the bregma and 3.8 mm lateral to the midline.. After surgery rats were housed individually in sound-proofed recording cages where lighting and temperature were kept constant ŽLD 12:12, light on at 10:00, 24 " 18C, food and drink ad libitum.. One week after surgery the rats were connected by means of a flexible cable and a commutator ŽAirflyte, Bayonne, NJ. to a Grass electroencephalograph ŽQuincy, MA; mod. 78., and recorded continuously until the percentages and distributions of sleep states were normal and within published values w8x. Each day from 09:30 to 10:00 the rats were gently stroked using a small painter’s brush to familiarize them with the sleep deprivation procedure. After adaptation, the rats were recorded for as many days as required to satisfy the criteria for any of three experimental groups. Sleep–Light ŽS–L, n s 7. rats were sacrificed during the

light hours Žaround 13:00. at the end of a long period of sleep Žmean " SEM s 53.0 " 12.7 min, interrupted by periods of waking not longer than 2 min. after spending at least 80% of the previous 3 h asleep. Waking–light ŽW–L, n s 7. rats were sacrificed during the light period Žaround 13:00. after 3 h of total sleep deprivation by gentle handling. Sleep deprivation was achieved by eliciting an orienting reaction whenever a slowing of the EEG was noted. Waking–Dark ŽW–D, n s 6. rats were sacrificed during the dark period Žaround 1:00. after a long period of continuous waking Žmean " SEM s 62.8 " 11.4 min, interrupted by periods of sleep not longer than 1 min., and after spending at least 80% of the previous 3 h awake. Animal care was in accordance with institutional guidelines. 2.2. Tissue and RNA preparation After sacrifice by decapitation, the head was cooled in liquid nitrogen and the whole brain was removed and dissected into various brain regions. Samples were immediately frozen on dry ice and then stored at y808C. Total RNA was isolated from the left cerebral cortex of each animal by using RNA-zol ŽBiotecx, Houston, TX. according to the manufacturer’s instructions. Final RNA concentrations were determined spectrophotometrically. 2.3. mRNA differential display mRNA differential display was performed as in w44x. Briefly, total RNA Ž2 m g. from each animal was individually reverse transcribed using superscript II reverse transcriptase ŽGIBCO-BRL, Gaithersburg, MD. and one of three 3X composite anchor primers E1T12 M ŽE1 s 5X CGGAATTCGG, M s A, C, or G.. Each reverse transcription reaction was then amplified by PCR in the presence of w a- 33 PxdATP ŽNew England Nuclear-Du Pont, Natick, MA.. The primers used were one out of the three 3X anchor primers used for the reverse transcription and one out of twenty E2-AP 5X arbitrary primers, where

C. Cirelli, G. Tononir Molecular Brain Research 56 (1998) 293–305 Table 2 Baseline percentages of waking, non-REM sleep and REM sleep during the light and dark periods for the rats used for the differential display study

Light period Dark period

Waking

Non-REM sleep

REM sleep

35.9"2.3 70.3"3.0

48.0"1.9 24.5"2.5

16.2"0.7 5.2"0.5

Values are expressed as percentages of the recording time Žmean "SEM; ns 20..

E2 s CGTGAATTCG and AP is a sequence of 10 bp with a presence of 50% G q C and A q T and an absence of uninterrupted self-complementarity of more than two nucleotides, as in w6x. To specifically detect c-fos and NGFI-A mRNAs, the following 5X primers were used: c-fos: CTG ACT CAC TGA GCT CGC CC w15x; NGFI-A: TAG GTC AGA TGG AAG ATC TC w37x. For cytochrome c oxidase subunit IV, the following primers were used: 5X-ATC CCT CAT ACC TTT GAT CG-3X Žsense.; 5XTGA CCT GAA CTT GAC TCC CA-3X Žantisense; w50x.. PCR conditions were as follows: 4 cycles of denaturation at 948C for 1 min, annealing at 458C for 5 min and extension at 728C for 1.5 min, followed by 30 cycles of denaturation at 948C for 30 s, annealing at 588C for 2 min and extension at 728C for 1 min, and an additional period of 5 min at 728C. Radiolabeled PCR products were separated on denaturating polyacrylamide gels ŽGenomyx, Foster City, CA. and visualized by autoradiography. To avoid problems with false positives and to compare intra- with inter-group variance, samples from individual animals were not pooled but run in parallel. To minimize variability due to technical inconsistencies, PCR reactions were performed in duplicate for each animal. Differentially expressed bands were quantified by using a phosphorimager ŽMolecular Dynamics, Sunnyvale, CA., recovered from dried gels, reamplified by PCR using the same primers and subcloned into pCRTMII vectors using the TA cloning kit ŽInvitrogen, San Diego, CA.. Plasmid DNA sequencing of cloned cDNAs was carried out using the Cy5TM AutoRead sequencing kit ŽPharmacia Biotech, Sweden.. Sequences were screened using the BLAST program and the EMBL and GenBank databases ŽDec 1997 release.. 2.4. Ribonuclease protection assays and in situ hybridization Confirmation of the results was performed for each differentially expressed band by using ribonuclease protection assays ŽRPA; on the same rats used for mRNA differential display. andror in situ hybridization experiments Žon different rats.. RPA was preferred to Northern blot analysis due to its higher sensitivity w28x. Antisense RNA probes were synthesized by run-off transcription from a linearized DNA template using the MAXIscript in vitro transcription kit ŽAmbion, Austin, TX. and w a-

295

32

PxUTP ŽNew England Nuclear-Du Pont.. RPA was performed using the RPAIITM kit ŽAmbion.. Total RNA from the left cerebral cortex Ž2 m g from each rat. was hybridized with an excess of w a- 32 PxUTP-labeled riboprobe. To normalize the amount of sample RNA, a b-actin riboprobe was used to measure b-actin mRNA. For in situ hybridization, rats implanted and recorded as described above were sacrificed after 3 h of sleep Ž n s 7., 3 h of sleep deprivation by gentle handling Ž n s 5., or 3 h of spontaneous waking Ž n s 2.. In situ hybridization was performed on frontal sections as in w14x. Antisense RNA probes were the same used for RPA. Pretreatment of tissue sections with RNAse eliminated true hybridization signals. Hybridization with sense RNA probes showed no specific hybridization signal. In some cases, different riboprobes complementary to different regions of the target mRNA were used separately in consecutive tissue sections and yielded similar hybridization patterns. Slides were scanned with a phosphorimager ŽMolecular Dynamics. and then exposed to Biomax film ŽEastman Kodak, New Haven, CT.. To detect F1-ATPase subunit alpha mRNA, a riboprobe was used corresponding to nucleotides 645-836 of the rat F1-ATPase subunit alpha gene ŽGenbank acc. no. X56133.. 2.5. Statistical analysis The signal intensity of each differentially expressed band was calculated after scanning the gel with a phosphorimager by measuring the average volume of each band for each lane and subtracting the background. The mean density within and among conditions was calculated and significant differences among conditions were evaluated using ANOVA followed by Bonferroni correction. RPA and in situ hybridization signals were also quantified densitometrically with a phosphorimager. For in situ hybridization, the entire cortex was outlined bilaterally at 8 different rostro-caudal levels. The relative optical density for each region of every subject was calculated by subtracting the optical density of the white matter from the same section. Since corresponding sections from a sleeping rat and from a waking rat were always hybridized together, they were

Table 3 Percentages of waking, non-REM sleep and REM sleep for the last 3 recording hours before sacrifice in the 3 groups of rats

S–L W–L W–D

Waking

Non-REM sleep

REM sleep

21.0"2.7 95.4"0.9 88.8"1.5

63.4"2.7 4.6"0.9 9.9"1.8

15.6"0.5 0.0 0.5"0.3

S–L: rats sacrificed after spontaneous sleep during the light period Ž ns 7.. W–L: rats sacrificed after being kept awake by gentle handling during the light period Ž ns 7.. W–D: rats sacrificed after spontaneous waking during the dark period Ž ns6.. Values are expressed as percentages of the recording time Žmean "SEM..

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paired for statistical analysis. The percent increase in density between sleep and waking was calculated for each section. The mean percent increase in density between each pair of sleeping and waking animals were compared using Student’s t-test for paired samples.

3. Results 3.1. Sleep percentages All rats were recorded continuously for several days after adaptation to the recording environment to establish

Fig. 1. mRNA differential display analysis of rat cerebral cortex RNA in Sleep–Light animals ŽS–L, 1 to 7., Waking–Light animals ŽW–L, 8 to 14., and X Waking–Dark animals ŽW–D, 15 to 20. using a 5 primer specific for rat c-fos mRNA ŽA. and for rat NGFI-A mRNA ŽB.. For each rat, 2 PCR reactions were performed from the same reverse transcription reaction. Each lane on the gel represents one PCR reaction from one rat. On each gel the band corresponding to c-fos ŽA. and NGFI-A ŽB. is indicated by an arrowhead. The graph on the right shows the densitometric analysis performed by scanning the gel with a phosphorimager. For both c-fos and NGFI-A, the difference in signal intensity between the three groups was statistically significant, with S-L rats differing from both W–L and W–D rats Ž P - 0.05, ANOVA with post hoc pairwise comparisons using Bonferroni correction.. For NGFI-A, the difference in signal intensity between W–L and W–D rats was also significant. The signal intensity units on the ordinate depend on the amount of exposure of each gel in the phosphorimager and are meaningful only for comparisons within the same gel.

C. Cirelli, G. Tononir Molecular Brain Research 56 (1998) 293–305

baseline percentages and distributions of waking, non-REM sleep, and REM Žrapid eye movement. sleep. Baseline data, reported in Table 2, were in agreement with standard values w8x. 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 spontaneous sleep during the light period ŽS–L., the second group comprised rats sacrificed during the light period that had been kept awake by gentle handling ŽW–L., and the third group comprised rats sacrificed after spontaneous waking during the dark period ŽW–D.. 3.2. Validation of the screening procedure We first examined whether mRNA differential display was able to detect differences between sleep and waking in the expression levels of c-fos and NGFI-A that had previously been reported w5,10,11,20,27,39,41–43x. Specific 5X primers were used to generate PCR products from the transcripts of rat c-fos and NGFI-A. In agreement with a previous report w16x, more than 120 PCR products were generated with the c-fos specific 5X primer and with the NGFI-A specific 5X primer, as observed on the respective gels. This confirms that multiple species of mRNAs within the cerebral cortex can serve as hybridization targets for such primers. In both gels, all but one of the PCR products were present at the same level in all sleeping, spontaneously awake, or sleep-deprived rats. The single band that was expressed at higher levels after sleep deprivation and spontaneous waking than after sleep was excised from each gel, cloned, and sequenced. It was found to correspond to a c-fos transcript in the gel obtained with c-fos specific primers ŽFig. 1A. and to a NGFI-A transcript in

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the second gel ŽFig. 1B.. The relative levels of c-fos and NGFI-A PCR products were determined by densitometric analysis ŽFig. 1.. As shown in the figure, the level of the corresponding transcripts was higher in the two waking conditions than in the sleep condition, in agreement with previous results obtained with other methodologies w5,10,11,20,27,39,41–43x. 3.3. General results of mRNA differential display Twenty arbitrary primers were screened against all 3 anchor E1T12M primers, giving a total of 60 gels. The mean number of PCR products visualized per lane in each gel was 150–200, thus theoretically allowing the evaluation of over 9000–12 000 RNA species. The band patterns obtained in separate experiments utilizing the same primers were reproducible, producing ) 90% equivalent bands, while patterns were markedly different when different primer pairs were used. In this study, PCR products from each animal were run in different lanes of the gel. This allowed us to examine differences among individual animals Žintra-group comparisons. as well as differences among the experimental conditions Žinter-group comparisons.. The overwhelming majority of PCR products were present at similar levels in all rats Žsee Fig. 1.. This indicates that in these inbred animals the inter-individual variability in mRNA levels is not large. Furthermore, this indicates that the vast majority of the transcripts are present at similar levels after 3 h of sleep and of waking. In several cases, visual inspection identified bands that showed inter-group differences. The level of signal intensity of these bands on each gel was quantified by densitometric analysis. Nineteen bands showed changes between

Table 4 Characterization and sequence analysis of 15 bands differentially expressed in sleep and waking as revealed by mRNA differential display Band name Žbased on the primers used.

Factor Žwaking or sleep.

%Change Žphosphorimager quantification.

Sequence analysis

E1T12Grap9- A E1T12Grap9- B E1T12Grap9- C E1T12Grap9- D E1T12Grap13 E1T12CrFOS1 E1T12Crap3 E1T12CrNGFI-A E1T12Crap15 E1T12ArAP1 E1T12Arap3 E1T12Grap5 E1T12Arap5 E1T12Arap2 E1T12Crap12

Waking Waking Waking Waking Waking Waking Waking Waking Waking Waking Waking Waking Waking Waking Sleep

34% 54% 45% 97% 79% 83% 82% 174% 82% 61% 60% 50% 94% 47% y55%

12S rRNA 12S rRNA 12S rRNA NADH dehydrogenase subunit 2 Unknown c-fos Unknown NGFI-A NGFI-A cytochrome Coxidase subunit I zn-15 related zinc finger Žrlf. gene Unknown Unknown Unknown Unknown

Only bands showing a change of at least 30% between sleep and waking, as measured by scanning the gels with a phosphorimager, are listed. No significant differences were found between W–L and W–D rats, except for the bands corresponding to NGFI-A. Changes are expressed as mean percent increase or decrease in waking conditions Žaverage of W–L and W–D. with respect to sleep. For the 2 bands corresponding to NGFI-A, the increase was X twice as large with the specific primer than with the 5 arbitrary primer.

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Fig. 2. mRNA differential display analysis of rat cerebral cortex RNA in Sleep–Light animals ŽS–L, 1 to 7., Waking–Light animals ŽW–L, 8 to 14., and X X X X Waking–Dark animals ŽW–D, 15 to 20. using the 5 primer E2-AP9 ŽAP9s 5 -TCGGTCATAG-3 . and the 3 anchored primer E1T12G. PCR reactions were performed and run in duplicate for each animal as described in Figure 1. Sequence analysis showed that bands A–C were 380–350bp cDNAs complementary to the rat mitochondrial 12S rRNA, while band D was a 340bp cDNA complementary to the rat subunit 2 of NADH dehydrogenase. The graphs below the gel show the densitometric analysis performed by scanning the gel with a phosphorimager. Values for 12S rRNA correspond to band C. For both 12S rRNA and subunit 2 of NADH dehydrogenase, the difference in signal intensity between S–L and W–L rats, and between S–L and W–D rats was statistically significant Ž P - 0.05, ANOVA with Bonferroni correction..

Fig. 3. RPA showing mRNA levels of cytochrome c oxidase subunit I in S–L, W–L, and W–D animals. A ß-actin antisense riboprobe was used to normalize the amount of sample RNA. Lane 1 Žfrom left.: molecular weight markers. Lane 2: cytochrome c oxidase subunit I riboprobe hybridized with 10 mg yeast RNA, incubated without RNase mixture. Most of the signal is the full-length probe of 280 bp. Lanes 3–8: cytochrome c oxidase subunit I and ß-actin probes hybridized under conditions of excess probe with 2 mg RNA pooled from the cerebral cortex of S–L Žlanes 3–4., W–L Žlanes 5–6., and W–D animals Žlanes 7–8.. The protected fragments are 170 bp for cytochrome c oxidase subunit I and 250 bp for ß-actin. The gel was exposed for 2 days for cytochrome c oxidase subunit I and for 7 days for ß-actin. The graph on the right shows the densitometric analysis performed by scanning the gel with a phosphorimager. The ordinate values refer to signal intensity.

C. Cirelli, G. Tononir Molecular Brain Research 56 (1998) 293–305

25% and 174% among the 3 experimental conditions. Of these, 14 bands were higher in waking than in sleep and 4 were higher in sleep than in waking. One band was found to be differentially expressed between the light and the dark period Žcircadian band.. No bands were found to be specifically expressed in the sleep-deprived rats with respect to the spontaneously awake or sleeping groups. 3.4. Sequencing of cDNAs Fifteen transcripts that showed differences between sleep and waking of at least 30% were excised from the gel, reamplified, cloned, and sequenced ŽTable 4.. They in-

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cluded one transcript that was present at higher levels in sleep, and 14 transcripts that were present at higher levels in waking. Partial DNA sequence analysis revealed flanking sites complementary to the PCR primers in all cases. All species were 300–800 bp in size. Comparison of these sequences with DNA data bases revealed identical sequences with 6 known genes. Six sequences, including the one with higher levels in sleep, could not be assigned to any previously reported gene. Three of the identified transcripts with higher expression levels in waking were RNAs coded by the mitochondrial genome: subunit I of cytochrome c oxidase, subunit 2 of NADH dehydrogenase, and 12S rRNA. Fig. 2 shows four bands ŽA–D. corre-

Fig. 4. In situ hybridization for cytochrome c oxidase subunit I mRNA in brain sections of a rat sacrificed at 13:00 after 3 h of spontaneous sleep Žsections A–D. and of a rat sacrificed at the same circadian time after 3 h of waking induced by gentle handling Žsections A’–D’.. A’–D’ show an increase in the hybridization signal for cytochrome c oxidase subunit I mRNA in many brain regions including cerebral cortex, olfactory nuclei, caudate-putamen, thalamus, and hippocampal formation. AON, anterior olfactory nucleus; CA1, field CA1 of Ammon’s horn; Cg, cingulate cortex; CPu, caudate putamen; Ent, entorhinal cortex; Fr, frontal cortex; HL, hindlimb area of the cortex; Par, parietal cortex; RS, retrosplenial cortex. Scale bar s 1mm.

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C. Cirelli, G. Tononir Molecular Brain Research 56 (1998) 293–305

sponding to the mitochondrial 12S rRNA ŽA–C. and to subunit 2 of NADH dehydrogenase ŽD.. The other 3 identified transcripts that were expressed more in waking corresponded to the transcription factors c-fos, NGFI-A, and to the rat homologue of the human Zn-15 related zinc finger Žrlf. gene w33x. NGFI-A was isolated twice, the first time when using its specific 5X primer, and the second time when using a 5X arbitrary primer. 3.5. RNAse protection assays and in situ hybridization analysis of transcripts encoded by the mitochondrial genome The increase of c-fos and NGFI-A mRNA levels during waking confirms previous results w5,10,11,20,27,39,41–43x. To confirm the increase in the level of 3 mitochondrial transcripts during waking, purified DNA fragments cut from the gel were used to prepare antisense RNA probes for RPA andror in situ hybridization experiments. In the case of subunit I of cytochrome c oxidase a second riboprobe complementary to a different region of the target mRNA was also employed as an additional control. RPA performed on pooled RNA from the 3 experimental groups

confirmed the differential expression between sleep and waking of cytochrome c oxidase subunit I mRNA ŽFig. 3.. Higher levels of expression during waking for 12S rRNA were also tested and confirmed with RPA Ždata not shown.. A higher level of mitochondrial transcripts during waking was also demonstrated with in situ hybridization in a set of rats different from those used for mRNA differential display. Fig. 4 shows the results for cytochrome c oxidase subunit I. Two different riboprobes gave similar results. Autoradiograms of consecutive sections hybridized with riboprobes for 12S rRNA, subunit 2 of NADH dehydrogenase, and subunit I of cytochrome c oxidase, are shown in Fig. 5. Table 5 shows the percent change in signal intensity as quantified by densitometry with a phosphorimager. In all 3 cases, the higher levels of mitochondrial RNAs determined with mRNA differential display were confirmed by in situ hybridization. The level of mitochondrial RNAs after waking appeared to be uniformly higher in many cortical regions and to extend to extracortical structures ŽFigs. 4 and 5.. Due to the limited number of sections at the appropriate anatomical levels, a quantitative analysis was performed only for 12S rRNA levels in the thalamus and in the hippocampal

Fig. 5. In situ hybridization for 3 mitochondrial genes Ž12S rRNA, NADH dehydrogenase subunit 2, and cytochrome c oxidase subunit I. in brain sections of a rat sacrificed at 13:00 after 3 h of spontaneous sleep Žsections A–C. and of a rat sacrificed at the same circadian time after 3 h of waking induced by gentle handling Žsections A’–C’.. An increase in mitochondrial RNA levels during waking is noticeable in cerebral cortex, hippocampal formation, and some thalamic nuclei. MHb, medial habenular nucleus; Par, parietal cortex, Te, temporal cortex. Scale bar s 1 mm.

C. Cirelli, G. Tononir Molecular Brain Research 56 (1998) 293–305 Table 5 Differences in transcript levels of mitochondrial and nuclear origin between sleep and waking as revealed by in situ hybridization Gene

% increase

12S rRNA NADH dehydrogenase subunit 2 Cytochrome c oxidase subunit I Cytochrome c oxidase subunit IV F1-ATPase subunit alpha

37.5 "11.6 ) 12.2 "3.4 ) 15.8 "4.6 ) 4.4 "4.4 6.6 "6.2

Values represent the percent increase Žmean"SEM. of the average hybridization signal in the cerebral cortex for waking rats Ž ns 7. with respect to sleeping rats Ž ns 7.. ) p- 0.05, Student’s t-test for paired comparisons.

formation. It was found that 12S rRNA levels were significantly higher in waking than in sleep both in the thalamus Žpercent increase 25.2 " 8.9%, p - 0.03. and in the hippocampal formation Žpercent increase 29.1 " 7.8%, p 0.01.. 3.6. Expression of transcripts for mitochondrial protein subunits encoded by the nuclear genome The 3 mitochondrial transcripts isolated through mRNA differential display are encoded by the mitochondrial genome. To function in the respiratory chain, the protein products of these transcripts need to be assembled together with mitochondrial proteins encoded by nuclear genes. For instance, cytochrome c oxidase contains 13 subunits, of which subunits I–III are of mitochondrial origin, and the others are of nuclear origin w4,9x. To determine whether the

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nuclear-encoded subunits were also present at higher levels in waking than in sleep, 2 primers were designed to generate PCR products from the rat cytochrome c oxidase subunit IV mRNA. Experiments with both mRNA differential display Ždata not shown. and in situ hybridization ŽFig. 6, Table 5. showed no change in the level of subunit IV mRNA between waking and sleep. In situ hybridization experiments using a riboprobe specific for the subunit alpha of F1-ATPase, another mitochondrial enzyme of nuclear origin which is a key component of the respiratory chain, also showed no change in mRNA levels between sleep and waking ŽFig. 6, Table 5..

4. 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 transcripts whose sequence did not match database entries were present at higher levels after 3 h of waking and a few were present at higher levels after 3 h of sleep. The overwhelming majority of transcripts, however, were expressed at similar levels in all three conditions.

Fig. 6. In situ hybridization shows that levels of 2 nuclear-encoded mitochondrial transcripts are similar after 3 h of waking and after 3 h of sleep. Two sets of frozen sections from rat brain were processed for in situ hybridization using w a- 32 PxUTP-labeled riboprobes specific for cytochrome c oxidase subunit IV mRNA ŽA,A’. and F1-ATPase subunit alpha ŽB,B’.. A and B are consecutive sections from a rat that was sacrificed at 13:00 after 3 h of spontaneous sleep Žsame rat as in Figure 5.. Sections A’ and B’ are from a rat that was sacrificed at the same circadian time after 3 h of waking induced by gentle handling Žsame animal as in Figure 5.. Scale bar s 1 mm.

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4.1. Differential gene expression between sleep and waking: general considerations The systematic comparison of cortical mRNA levels between multiple individuals and experimental conditions performed in the present study indicates that only a small fraction of the genes expressed in the cerebral cortex of rats is differentially expressed between 3 h of sleep, 3 h of waking, and 3 h of sleep deprivation. Of 19 differentially expressed transcripts, 18 were present at different levels in the two waking conditions vs. spontaneous sleep. This confirms, in agreement with previous work using in situ hybridization and immunocytochemistry, that 3 h spent predominantly in waking or sleep are a sufficient period of time for detecting state-dependent differences in mRNA levels. We did not find any transcript that was specifically increased in forced waking with respect to spontaneous waking. This supports previous findings that changes in the level of IEGs after short periods of sleep deprivation by gentle handling are comparable to those observed after spontaneous waking w11,42,43x. Finally, we isolated only one transcript whose level was higher during the light than during the dark period, but we did not proceed to its further characterization. The limited number of differentially expressed bands that were isolated out of thousands of bands that were screened suggests that the genes whose expression is modulated by short periods of sleep and waking represent a very specific subset of the genes expressed in the cerebral cortex. While it is possible that further differences in mRNA expression levels could emerge by comparing periods of sleep and waking shorter or longer than 3 h, it should be kept in mind that the mRNAs isolated in this study represent an incomplete sample of the transcripts that are differentially expressed between sleep and waking. The number of different mRNAs contained in a given tissue is estimated to vary between 12 000 and 30 000 w3x. If each PCR product visualized in a gel corresponded to a different gene, a total of 60 gels with an average of 175 bands per gel should have screened up to ; 10 000 mRNAs. In a pilot experiment in which 14 nearby bands were cut from the same gel and sequenced, 4 Ž28%. turned out to correspond to the same gene, suggesting that the actual number of genes screened was less than 7000. Depending on the number of different transcripts present in the cerebral cortex, we estimate that between 20 and 60% of the cortical transcripts have been screened in this study. This can account for the fact that some transcripts known to be modulated by sleep and waking, such as jun B w1,36,39x were not isolated, although differences in the experimental protocols, such as the length of sleep deprivation, may also be responsible. Other factors could contribute to an underestimation of the number of differentially expressed transcripts. Since the overwhelming majority of mRNAs are rare w3x, it is important to know whether mRNA differential display can detect low prevalence mRNAs. Liang et al.

w30x showed that rare mRNAs could be detected, and a recent report showed that mRNA differential display was at least as sensitive as subtractive hybridization in detecting rare mRNAs w51x. Finally, it is possible that small changes in mRNA levels may have remained undetected, although in several cases we were able to detect and confirm with in situ hybridization differences as small as 10–20%. In the present study, when differences in mRNA levels between sleep and waking were detected with differential display, they were always confirmed with RPA and in situ hybridization andror they were consistent with previous findings, thus validating the power and reliability of this technique. In early experiments with mRNA differential display, a significant number of differentially expressed bands could not be confirmed by Northern blot analysis. We substantially reduced this problem by performing, for each combination of primers, duplicate PCR reactions for each individual subject and by selecting only those bands that were consistently increased or decreased within a group. This allowed us to distinguish between inter-individual variability and state-dependent effects. RPA and radioactive in situ hybridization were chosen to confirm the results because of their sensitivity. In all cases, the latter techniques confirmed the differences detected by mRNA differential display, although the magnitude of such differences was less pronounced. 4.2. Differential gene expression between sleep and waking: mitochondrial RNAs and mRNAs for transcription factors Of the 15 bands differentially expressed between sleep and waking that were cloned and sequenced, the band that was higher in sleep and 5 of the bands that were higher in waking correspond to unknown genes. The identification of these genes is currently in progress. The remaining 9 bands that were higher in waking correspond to 6 known genes. Of these, 3 were the IEGs c-fos, NGFI-A, and the rat homologue of the human Zn-15 related zinc finger Žrlf. gene. The latter gene, which has been implicated in transcriptional regulation w33x, was not known to be induced by waking. The detection of changes in c-fos and NGFI-A expression between sleep and waking is consistent with previous results w5,10,11,20,27,39,41–43x. Changes in IEG mRNA and protein expression in the cortex have been related to behavioral and anatomical plasticity w24x. Fos protein expression in the preoptic area can alter the regulation of sleep w12x. The rapid modulation in the expression of such genes with behavioral state change, which is mediated in part by the locus coeruleus, has been linked to the inability to acquire new information during sleep w13x. These observations suggest that changes in the expression of IEGs with behavioral state may have significant functional consequences.

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The other 3 identified transcripts that were present at higher levels in waking were the mitochondrial genes for subunit I of cytochrome c oxidase, for subunit 2 of NADH dehydrogenase, and for 12S rRNA. This represents the first report indicating that sleep and waking are accompanied by changes in the levels of mitochondrial RNAs. Furthermore, the occurrence of a change in mitochondrial transcript levels in as rapidly as 3 h had not previously been described, although it is in agreement with the rapid turnover of these RNAs w18x. In the cerebral cortex, mRNA and protein levels of several cytochrome c oxidase subunits were found to decrease in a matter of a few days as a consequence of sensory deafferentation w21,22,52x. An increase in cytochrome c oxidase activity has been described in enteric neurons stimulated for 8 h with veratridine w35x and in the suprachiasmatic nucleus during the light phase w31x. The parallel increase of 3 different mitochondrial transcripts is consistent with the transcription of the mitochondrial H-strand as a polycistronic unit w2x. Changes in mRNA levels between sleep and waking were demonstrated for the mitochondrial subunit I, but not for the nuclear-encoded subunit IV of cytochrome c oxidase. This dissociation is consistent with previous studies showing that the transcription of mitochondrially encoded subunits responds more quickly and more significantly than that of nuclear subunits to changes in neuronal activity and energy demand w22,52x. Recent evidence suggests that mitochondria contain larger amounts of nuclear-encoded cytochrome c oxidase subunits, and that it is the synthesis of mitochondrial subunits, followed by the holoenzyme assembly, that is governed by dynamic local energy needs w38,52x. Cytochrome c oxidase holoenzyme can still be assembled for 5 h in hepatic cells in the absence of cytoplasmic protein synthesis w25x. Moreover, protein levels of mitochondrial, but not of nuclear subunits of cytochrome c oxidase correlate with cytochrome c oxidase activity w38x. In fact, mRNA levels of mitochondrial subunits are a more rapid indicator of changes in cellular energy requirements than is enzyme activity itself w22x. The mean difference in mitochondrial mRNA levels between waking and sleep conditions as evaluated by in situ hybridization ranged between 12 and 37%. Such a difference after just 3 h of behavioral state change is remarkable if one considers the functional role and the abundant and ubiquitous expression of these genes. By way of comparison, after 1–2 weeks from monocular enucleation, cytochrome c oxidase subunit III mRNA levels in the lateral geniculate nucleus decreased by 35–45% w52x. The mean increase of c-fos mRNA levels in cortex after 3 h of waking as assessed by in situ hybridization with oligonucleotides w11x and riboprobes Žthis study, data not shown. also ranged between 20 and 30%. According to in situ hybridization, the increase in the level of mitochondrial RNAs after 3 h of waking was distributed diffusely in most cortical regions and it extended to subcortical struc-

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tures. It remains to be determined whether such increase was more prominent in certain cortical layers, whether it was limited to specific cellular populations Že.g. neurons vs. glia. andror to certain cellular compartments Že.g. cell bodies vs. neuropil.. 4.3. Mitochondrial mRNAs and brain metabolism in waking and sleep The finding of higher levels of mitochondrial RNAs after short periods of waking with respect to equal periods of sleep raises the question of its functional relevance. About 95% of cerebral energy metabolism is dependent on mitochondrial oxidative phosphorylation w47x. Brain energy expenditure is tightly controlled by brain activity w32x, mainly because of the high metabolic cost of ion pumping by Na q rK q ATPase to counteract membrane depolarization w17x. Cerebral glucose metabolism is 20–30% higher in waking than in non-REM sleep in several species, including the rat w34,45x. The higher metabolic cost of waking is probably related to the relative depolarization of cortical and thalamic cells and to the facilitation of synaptic transmission in this behavioral state w48x. Mitochondria must be able to respond efficiently to both short-term and long-term changes in energy demand dictated by synaptic depolarization. Sustained changes in respiration rate require changes in the amount of respiratory chain enzymes andror changes in the number of mitochondria w22x. The results of the present study indicate that a few hours of spontaneous waking result in an increase in the level of mitochondrial RNAs in cortical tissue. This finding suggests the hypothesis that an increased transcription of mitochondrial subunits may represent a regulatory response of neurons or glia to adapt to the increased metabolic demand of waking with respect to sleep. A recent report indicating that persistent depolarization increases cytochrome c oxidase subunit mRNA levels in neuronal culture of rat cortex is consistent with this hypothesis Žw53x.. In future work, it will be important to determine whether the increase in mitochondrial RNA levels extends beyond a few hours of waking, whether it is accompanied at a later stage by an upregulation of nuclear-encoded mitochondrial subunits, and whether this response can be sustained in conditions of prolonged sleep deprivation.

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

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and Dr. Maria Pompeiano and Dr. J. Michael Salbaum for their help in the initial stages of this project. w21x

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