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320 VOLUME 34 | NUMBER 3 | JULY 2003 NATURE GENETICS

In rodents, the electroencephalogram (EEG) during paradoxicalsleep and exploratory behavior is characterized by thetaoscillations. Here we show that a deficiency in short-chain acyl-coenzyme A dehydrogenase (encoded by Acads) in mice causesa marked slowing in theta frequency during paradoxical sleeponly. We found Acads expression in brain regions involved intheta generation, notably the hippocampus. Microarray analysisof gene expression in mice with mutations in Acads indicatesoverexpression of Glo1 (encoding glyoxylase 1), a gene involvedin the detoxification of metabolic by-products. Administrationof acetyl-L-carnitine (ALCAR) to mutant mice significantlyrecovers slow theta and Glo1 overexpression. Thus, anunderappreciated metabolic pathway involving fatty acid β-oxidation also regulates theta oscillations during sleep.

The EEGs of slow-wave sleep and paradoxical sleep differ considerably.Slow-wave sleep is characterized by large-amplitude slow oscillations,whereas paradoxical sleep is characterized by the absence of slow waves

and, in rodents, the presence of theta oscillations (5–9 Hz). Theta oscilla-tions also accompany exploratory behavior1, and long-term potentiation(LTP) in the hippocampus is optimally induced with theta stimulations2.The implication of theta in learning and memory is further indicated bythe finding that its elimination blocks and facilitation enhances LTPinduction and memory3,4. Although a role for sleep in learning andmemory has been repeatedly advocated, this issue is controversial5,6.Because there is evidence that theta is directly involved in memory func-tions of the hippocampus7, an important point is whether theta servesthe same function during wakefulness and paradoxical sleep6.

Previously, we have reported that different genes regulate thetaoscillations during sleep and exploratory behavior8. Visual inspec-tion of the EEG during paradoxical sleep indicated differences in the

1Biochemistry and Genetics Unit, Department of Psychiatry, University of Geneva and Geneva University Hospitals, Chemin du Petit-Bel-Air 2, 1225 Chêne-Bourg,Switzerland. 2Neuropsychiatry Division, Department of Psychiatry, Geneva University Hospitals, 1225 Chêne-Bourg, Switzerland. 3Department of Morphology, CentreMedical Universitaire, 1211 Geneva, Switzerland. 4Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. 5Departmentof Biological Sciences, Stanford University, Stanford, California 94305, USA. Correspondence should be addressed to M.T. (mehdi.tafti@medecine.unige.ch).

Deficiency in short-chain fatty acid β-oxidation affectstheta oscillations during sleepMehdi Tafti1, Brice Petit1, Didier Chollet1, Elisabeth Neidhart1, Fabienne de Bilbao2, Jozsef Z Kiss3,Philip A Wood4 & Paul Franken5

Figure 1 Mapping of Tpf. (a) 8-s EEG samples for paradoxical sleep fromrepresentative BALB/cByJ (C) and C57BL/6J (B) mice. (b) EEG spectra (± 1 s.d.) indicate that TPF is 1 Hz faster in B mice (n = 10 per strain). (c) Chromosome 5 haplotype in 132 C × B backcross mice (blue, allelesfrom C mice; brown, C × B heterozygous alleles). Number of mice perhaplotype is indicated below bars. The TPF segregation allowed genotypeassignment in all mice. Map indicates loci distances in cM (± s.e.) and lodscores for linkage between MIT markers and Tpf28. The difference betweenhaplotype (left) and distances (right) is due to the inclusion of 31 micerecombinant between D5Mit155 and D5Mit139. (d) Left, Haplotype of C × B recombinant-inbred (RI) strains (blue, alleles from BALB/cBy (C)mice; brown, alleles from C57BL/6ByJ (B) mice). MIT marker numbers areindicated above bars. Asterisk denotes the Acads position. Note thediscrepancy between the marker order in c and d. Our map positioned Tpfand Acads between D5Mit240 and D5Mit188, whereas the Mouse GenomeDatabase map positioned Acads distal to D5Mit188. Right, CorrespondingTPF during paradoxical sleep (mean ± s.d.). C, BALB/cBy; B, C57BL/6ByJ.TPF in all C × B recombinant-inbred strains and their parental was fasterthan in mutant BALB/cByJ mice (ANOVA, F6,45 = 31.55, P < 2 × 10–12;Scheffe’s test, P < 0.01).

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frequency of theta among inbred mouse strains (Fig. 1a,b).Quantitative EEG analysis showed that theta-peak frequency (TPF)varied greatly with genotype8. Among 11 strains (Table 1) analyzedso far, four had significantly slower theta.

We crossed a slow-theta strain (BALB/cByJ or C) with a fast-thetastrain (C57BL/6J or B). TPF in reciprocal F1 generations was similar tothat of B mice but significantly faster than that of C mice (Table 1),indicating that the allele of C mice is recessive. Linkage analysis in 47 C× B F2 mice identified a single location on chromosome 5, which wenamed Tpf (lod score = 11.5 between D5Mit240 and D5Mit188; P < 4 ×10–12), strongly suggesting that TPF is an autosomal recessive pheno-type under the control of a single gene. We then generated a C × B back-cross panel (C × B F1 mice × C mice; n = 101) and, on the basis of ourfindings in F2 and inbred mice, classified them as slow TPF (<6.5 Hz) orfast TPF (>6.5 Hz). Tpf in these mice was linked to D5Mit240 (lod score= 8.8; P < 2 × 10–10). From 200 additional backcross mice, we selectivelyphenotyped 31 recombinants between D5Mit155 and D5Mit139 flank-ing D5Mit240. Haplotype analysis placed Tpf in a 2.4-cM interval delim-ited by D5Mit177 and D5Mit406 (Fig. 1c).

The Tpf region contains the gene Nos1 encoding neuronal nitric-oxide synthase. Nitric oxide is important in theta-induced LTP9 andsleep10. But TPF in Nos1-null mice does not differ from TPF in fast Bmice (P > 0.9; Table 1). The same region is also linked to abnormalnon-shivering thermogenesis, a recessive condition caused by a loss-of-function mutation in Acads11. We therefore genotyped all F2 andbackcross mice for the 278-bp deletion in Acads12. Among 226 meiosesscreened, we found no recombination between Acads and Tpf, linkingTpf to Acads.

The Acads mutation appeared spontaneously in BALB/cByJ after itwas separated from BALB/cBy in 1982 (ref. 12) and is responsible forthe absence of a functional ACADS enzyme13–15. ACADS catalyses thefirst step in the β-oxidation of C4–C6 fatty acids. EEG analysis showedthat TPF in wild-type BALB/cBy mice was significantly faster than inmutant mice (P < 5 × 10–6; Table 1 and Fig. 1d). We also determinedTPF in C × B recombinant-inbred lines derived from BALB/cBy andC57BL/6ByJ before the Acads mutation appeared. TPF in C57BL/6ByJmice and in four C × B recombinant-inbred lines recombinant in theTpf region was significantly faster than in mutant BALB/cByJ mice(Table 1 and Fig. 1d). Because significant differences were stillobserved among these recombinant-inbred strains, we generated 42 F2mice from a cross between C × B recombinant-inbred line 7 and C × Brecombinant-inbred line 5, but we found no linkage between TPF andany marker on any recombinant chromosomes including chromo-some 5. This confirms that apart from the Acads mutation, no otherTPF-associated genes are segregating. We sequenced full-length AcadscDNAs from other inbred strains and found a G→T substitution atposition 281 (changing Gly94 to aspartic acid) in some, but not all,fast-theta strains, indicating that Acads mutations affect theta inBALB/cByJ mice only.

We also analyzed TPF during waking episodes with clear theta activ-ity in all our mice, but waking TPF did not correlate with paradoxicalsleep TPF and was not linked to any markers on chromosome 5 (datanot shown). Furthermore, waking TPF did not differ between wild-type mice (7.67 ± 0.36 Hz) and mice with the mutation in Acads (7.58± 0.27 Hz; P > 0.9), indicating that the mutation affects theta oscilla-tions specifically during sleep.

ACADS deficiency causes organic aciduria13,14, suggesting thatalthough these mice seem asymptomatic, some toxic effects mightoccur. In humans, ACADS deficiency is differentiated from otherdefects in fatty acid oxidation by abnormalities of the central nervoussystem, including mental retardation, microcephaly and seizures16. Wetherefore carried out a brain histopathological examination, which didnot identify any gross structural abnormalities, focal lesions or evidentneurodegeneration. But brain weight was lower in mutant mice (397 ±19 mg in BALB/cByJ versus 444 ± 13 mg in BALB/cBy; P < 10–6)although body weight was similar (27.4 ± 1.8 g versus 27.2 ± 2.2 g; P >0.9; n = 24 per strain).

Using western-blot analysis, immunohistochemistry and in situhybridization, we verified that ACADS protein and mRNA areexpressed in brain and liver of wild-type but not mutant mice (Fig. 2a).In wild-type mice, ACADS protein (Fig. 2b–e) and mRNA (Fig. 2f–k)are expressed exclusively in neurons of discrete brain regions, includingthose involved in theta generation.

To identify eventual changes in gene expression caused by theACADS deficiency, we used microarray analysis with total brain RNAsfrom mutant and wild-type BALB/cBy mice. Among 12,488 genes andexpressed sequence tags (ESTs) analyzed, 2 ESTs showed a consistentlyhigher levels of expression in mutant mice (AI852001: 2.9 times higher,P < 2 × 10–4; AI848952: 3.2 times higher, P < 3 × 10–3; Fig. 3a). BLASTanalysis indicated that both ESTs were derived from Glo1, which

Table 1 TPF during paradoxical sleep

Genotype TPF (Hz) n

A/J 6.12 ± 0.14 6

BALB/cByJ 6.25 ± 0.22 10

AKR/J 6.25 ± 0.25 7

C3H/HeJ 6.40 ± 0.20 7

DBA/2J 7.03 ± 0.19 9

LP/J 7.21 ± 0.22 7

PL/J 7.21 ± 0.17 7

C57BL/6J 7.25 ± 0.23 10

SPRET/Ei 7.25 ± 0.18 5

129/Ola 7.44 ± 0.18 8

C57BR/cdJ 7.61 ± 0.20 7

C × B F1 6.92 ± 0.13 6

B × C F1 7.08 ± 0.13 6

C × B F2 6.83 ± 0.33 47

C × B BC 6.66 ± 0.33 132

C57BL/6ByJ 7.36 ± 0.23 6

BALB/cBy 7.10 ± 0.22 8

C × B RI1 7.21 ± 0.19 6

C × B RI4 7.33 ± 0.20 6

C × B RI5 7.59 ± 0.23 8

C × B RI7 6.78 ± 0.23 8

(C × B RI7) × (C × B RI5) F2 7.21 ± 0.25 42

Nos1–/– 7.19 ± 0.24 4

Acadl–/– 7.70 ± 0.24 5

Acadl+/+ 7.62 ± 0.28 4

BALB/cByJ + ALCAR 6.71 ± 0.27 7

TPF values are expressed as mean ± s.d. Comparisons among the first 11 inbredstrains indicated two groups: slow (6.12–6.40 Hz; strains A/J, C3H/HeJ, AKR/Jand BALB/cByJ) and fast TPF (7.03–7.61 Hz; DBA/2J, SPRET/Ei, PL/J, LP/J,C57BL/6J, 129/Ola and C57BR/cdJ; ANOVA, F10,72 = 50.18, P < 10–12; post-hoc Scheffe test between slow and fast strains, P < 0.01). Parental strains for C × B F1, B × C F1, C × B F2 and C × B backcross (BC) are C57BL/6J (B) andBALB/cByJ (C). Comparisons among C × B F1, B × C F1 and their parental strainsC and B indicated no difference between F1 and B mice, but F1 mice weresignificantly different from C mice (ANOVA, F3,28 = 54.92, P < 8 × 10–12; post-hoc Scheffe test between F1 and C, P < 0.01). Parental strains for C × B RI1 toRI7 (RI, recombinant inbred) are C57BL/6ByJ and BALB/cBy.

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encodes an enzyme involved in metabolic detoxification. For confirma-tion, we used real-time RT–PCR and added Hagh (encoding glyoxylase2, the second enzyme in the glyoxylase system) and Bdh (encoding β-hydroxybutyrate dehydrogenase, the primary ketone-body convertingenzyme; Fig. 4), which were not present on our microarrays. Glo1expression was upregulated in brain (by a factor of 2.0) and liver (by afactor of 3.5) of Acads mutant mice (Fig. 3b). Unexpectedly, Bdh wasdownregulated in brain and upregulated in liver, indicating that theAcads mutation has tissue-specific effects (Fig. 3b). In situ hybridiza-tion showed overlapping expression patterns for Glo1 and Acads withmaximal expression in hippocampus (Fig. 2l).

Although no Acads mutations were found in the three other slow-theta strains, defects in related metabolic pathways cannot beexcluded. We found that Glo1 expression in the brain was signifi-cantly greater in all four slow-theta strains (Fig. 3c), strongly indicat-ing that changes in Glo1 expression constitute a molecular markerfor theta frequency in mice.

ACADS is one of four acyl-coenzyme A (CoA) dehydrogenases thatparticipate in fatty acid β-oxidation. Mutations in other ACAD mem-bers might also affect TPF. But TPF does not differ between mice withmutations in Acadl (encoding long-chain acyl-CoA dehydrogenase;ref. 17) and wild-type mice (Table 1). Also, Glo1 expression in brain(1.37 ± 0.22 in Acadl mutants versus 1.45 ± 0.32 in wild-type mice)and liver (3.20 ± 0.10 in Acadl mutants versus 3.47 ± 0.23 in wild-typemice) is unaffected (n = 4, in triplicates; P > 0.7).

BALB/cByJ mice seem ‘clinically’ normal, even with severeaciduria and secondary carnitine deficiency as in humans18.Experimental riboflavin deficiency aggravates their aciduria andcarnitine deficiency, but both could be partially restored byALCAR18. L-carnitine is involved in transporting long-chain fattyacids into mitochondria, producing ATP and removing excessshort- and medium-chain fatty acids19. ALCAR treatment has alsobeen shown to partially restore changes in mitochondrial functionassociated with memory loss and DNA/RNA oxidation in aged

Figure 2 Expression pattern of Acads and Glo1.(a) Western blot of ACADS protein in brain (50 µg)and liver (10 µg total protein) of wild-type (lanes1–3; n = 3) and Acads mutant (lanes 4–6; n = 3)mice. Lane 7, 0.1 µg purified human ACADS.(b–e) Immunocytochemical localization of ACADSin the brain of C57BL/6J mice using anti-ratACADS polyclonal antibody. ACADS issubstantially expressed in the hypothalamicparaventricular (PVN) and suprachiasmatic nuclei(b), cingulate gyrus (CG; c), medial habenula(MH; d) and hippocampal dentate gyrus (DG; e).(f–k) In situ hybridization of Acads mRNA. Acadsis expressed in suprachiasmatic nuclei (f),piriform (PIR) and entorhinal cortices (ENT; g),granular cells in the cerebellum (h),paraventricular nuclei (PVN; i), medial habenula(j) and hippocampal dentate gyrus (DG; k). Aweaker signal is apparent in pyramidal cells ofhippocampal CA regions (CA1). White matterstructures including the optic chiasm (OC), stria medullaris (SM) and ependymal cells of the plexus choroid, the third ventricle (3V) and dorsal third ventricle(D3V) are all negative. Expression pattern of Glo1 in the hippocampus (l) and other brain regions is similar to that of Acads. Scale bar corresponds to 100 µm(f,i,j), 150 µm (b–e) or 200 µm (g,h,k,l).

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Figure 3 Changes in gene expression in Acads mutant, wild-type and inbred mice. (a) Microarray analysis of brains from wild-type and Acads mutant mice.Circles indicate the expression of 4,447 genes and ESTs that could be reliably detected (that is, they were present on all six chips ‘n = 4,422’ or present onthree chips of either strain ‘n = 25’). Green line indicates a similar signal level; red lines delineate relative change by a factor of 2 between strains. Only 2EST clones (arrows), corresponding to Glo1, were significantly upregulated in the brain of Acads mutant mice. (b) Real-time RT–PCR expression of selectedgenes (Glo1, Hagh, Bdh and Acads) in brain and liver of wild-type and Acads mutant mice. Data are mean ± s.d. from nine mice per strain analyzed intriplicate. Significance in t-test is indicated by asterisks (***P < 10–6, **P < 10–3, *P < 0.05). (c) Real-time RT–PCR expression of Glo1 in brain of inbredmouse strains (n = 3, each analyzed in triplicate). Glo1 expression (mean ± s.d.) is upregulated in all slow theta strains (<6.5Hz; AKR/J, BALB/cByJ,C3H/HeJ and A/J) as compared with fast theta strains (>6.5Hz; C57BL/6J, SPRET/Ei, 129/Ola, BALB/cBy and C57BR/cdJ; ANOVA F8,30 = 28, P < 10–6;Bonferroni test, P < 0.05–10–3).

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rats20. The increased Glo1 expression in Acads mutants suggestschanges in mitochondrial oxidative function. We therefore treatedBALB/cByJ mice with ALCAR and partially recovered TPF (P <0.003), although not to the wild-type level (P < 0.02; Table 1). Eightweeks of treatment with ALCAR also recovered the increased brainGlo1 expression in BALB/cByJ mice (real-time RT–PCR: 1.97 ± 0.30versus 2.79 ± 0.32; P < 0.002), confirming a causal relationshipbetween Glo1 expression level and TPF.

Although we could not identify brain abnormalities in BALB/cByJmice, the partial rescue of our phenotype by ALCAR suggests thatsome toxicity might accompany the ACADS deficiency. The natureof this toxicity is unknown, but accumulated butyryl-CoA in theabsence of ACADS, which can be partly detoxified by ALCAR prob-ably activates the glyoxylase system (Fig. 4). The glyoxylase pathwayis a metabolic detoxification system including two enzymes, glyoxy-lase 1 and 2, with reduced glutathione as cofactor21. We proposethat accumulated butyryl-CoA can be transformed into succinateand malate through propionyl-CoA22 and activate the glyoxylasesystem (Fig. 4). The finding that all four slow-theta strains haveupregulated expression of Glo1 in the brain indicates that the gly-oxylase pathway is the final common step underlying theta fre-quency differences in mice.

Liver fatty acid β-oxidation produces ketone bodies that are used asan alternative energy source in the adult when circulating glucose con-centration is low. We found that Bdh expression is downregulated inthe brain but upregulated in the liver of Acads mutants. Thus, ACADSdeficiency might be compensated in the liver by a mechanism notfunctional in the brain, such as increased use of long-chain fatty acids.

This is consistent with the fact that long-chain fatty acids are less effi-ciently transported across the blood-brain barrier than are short- andmedium-chain fatty acids23,24 and with our findings that ACADL defi-ciency does not affect TPF or the glyoxylase system.

Our present and previous findings8 indicate that theta oscillations areregulated differently during sleep and waking. Given the expression ofAcads in the hippocampus, the poor memory performance of the fourslow-theta strains (see URL below) and the suggested role of sleep inmemory processes5, theta oscillations might have a role in cognitivefunction during sleep. Whether changes in TPF during sleep can affectwaking cognitive functions needs further investigation. Our mostnotable finding is that a deficiency in the fatty acid metabolism pathwayaffects hippocampal theta oscillations. Brain fatty acid metabolism isnot thought to be important in the adult, although β-oxidation enzymeactivity is observed in the adult brain25. Our results suggest that becauseACADS deficiency affects theta oscillations only during sleep, sleepmight represent a condition favoring or requiring β-oxidation.

METHODSEEG recording and analysis. The methods concerning the recording and analysisof the EEG in mice are described in detail elsewhere8,26. All mice were adult male(10–14 weeks old) individually housed in an experimental room under a 12-hlight/dark cycle (lights on at 8:00). Food and water were available ad libitum. Weimplanted EEG and electromyogram (EMG) electrodes under deep pentobarbi-tal anesthesia. We allowed mice 10–14 d of recovery from surgery and habituationbefore the experiments. We recorded the EEG and EMG signals continuously forat least 24 h. Both signals were amplified, filtered and converted from analog todigital. We subjected the EEG signal to fast-Fourier transform analysis yieldingpower spectra between 0 and 25 Hz (0.25-Hz resolution) using a 4-s window. Weclassified the behavior in each of these 4-s epochs as slow-wave sleep, paradoxicalsleep or wakefulness by visual inspection of the EEG and EMG signals. Individualvalues for TPF were based on EEG power spectra from all artifact-free 4-s epochsscored as paradoxical sleep during a 12-h light period. Spectra were averaged andTPF was assigned to the frequency bin with the highest EEG power. Waking TPFwas based on the EEG spectra derived from 4-s epoch in which clear theta oscilla-tions were present during the same 12-h light period and was calculated in thesame way as for paradoxical sleep. All P values listed in the text refer to t-testsunless indicated otherwise. The experimental protocols were approved by thelocal veterinary office (Office Vétérinaire Cantonal de Genève) and the ethicalcommittee of the Geneva School of Medicine.

α-oxoaldehydes:Glyoxal

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Figure 4 Metabolic pathways involved in the activation of the glyoxylasesystem. Glyoxyl formed by lipid peroxidation and slow oxidativedegradation of glucose and glycated proteins (red arrows) is converted into glycolate. Methylglyoxyl from glyceraldehydes-3-phosphate anddihydroxyacetone-phosphate (DHA-P) degradation, from acetone producedby ketone-body metabolism and from amino-acid catabolism (blackarrows) is converted into D-lactate. 4,5-dioxovalerate formed by thereduction of α-ketoglutarate and oxidative degradation of 5-aminolevulinate (heme biosynthesis, not shown) is converted to 2-hydroxyglutarate through glutathione-dependent glyoxylase 1 (Glo1) and 2 (Hagh) action. The Acads mutation induces a decreased ketone-bodymetabolism (green arrows) in brain as evidenced by reduced β-hydroxybutyrate dehydrogenase (Bdh) expression. This pathway isupregulated in liver through overproduction of acetyl-CoA formed from β-oxidation of long- and medium-chain fatty acids. Acads deficiency is accompanied by organic aciduria evident from overproduction ofbutyrylglycine, butyrylcarnitine, ethylmalonate and methylsuccinate.Accumulated butyryl-CoA can be transformed into succinate and malatethrough propionyl-CoA and explains the urinary excretion of excessethylmalonate and methylsuccinate, hallmarks of ACADS deficiency.Transformation of succinate/malate into α-ketoglutarate (directly orthrough the tricarboxylic-acid (TCA) cycle) could ultimately activate theglyoxylase system and also compensate for the reduced acetyl-CoA supplyto the TCA cycle.

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Linkage and sequence analysis. We extracted DNA from the liver in all C × B F2

and backcross mice and used it for genotyping 89 Mit SSLP markers (ResearchGenetics) at intervals of approximately 20 cM throughout the genome. We car-ried out linkage analysis with MapManagerQT. Total RNA was extracted frombrains and livers, and full-length Acads was reverse-transcribed, amplified byPCR, subcloned (pGEM-T vector; Promega) and sequenced on both strands ininbred strains BALB/cByJ, C57BL/6J, AKR/J, A/J and C57BR/cdJ.

cRNA preparation, cDNA microarray hybridization and real time RT–PCR.Nine mutant BALB/cByJ and nine wild-type BALB/cBy mice were killed by cer-vical elongation between 10:00 and 11:00, and brains and livers were quicklyremoved, frozen on dry ice and stored at –80 °C. We isolated total RNA fromwhole brain and liver by a commercial RNA extraction kit (RNAXEL, Eurobio),treated it with DNase and cleaned it using RNeasy columns (Qiagen). Wepooled together equal quantities of total brain RNA from three individual miceof each strain. We generated a hybridization mixture containing 15 µg ofbiotinylated c-RNA according to the protocols provided by Affymetrix andhybridized it to mouse Affymetrix MG-U74A-v2 chips. The chips were washed,scanned, and analyzed with Affymetrix software MAS 5.0 and DMT 3.0. Wecarried out pairwise analyses between the data from three BALB/cByJ and thethree BALB/cBy cDNA microarray chips, resulting in nine comparison sets,each providing a relative change in expression for each gene.

As templates in real-time RT–PCR, we used total RNA from each of thesame nine pooled RNA samples, three from each strain that were hybridized tothe microarrays, their corresponding liver RNAs and RNAs extracted from thebrain and liver of inbred strains (all killed between 10:00 and 12:00). Briefly,we carried out reverse transcription on 500 ng of total RNA (in 20 µl) usingrandom priming and SuperScript II (Invitrogen). We carried out quantitativePCR reactions using Syber Green PCR Master Mix and Prism SDS 7900 HT(Applied Biosystems) according to the manufacturer’s protocols. All primerswere designed with Primer Express Software (Applied Biosystems) and allamplified fragments corresponded to exon-exon junctions. We analyzed allsamples in triplicates and normalized the threshold cycle values for targetgenes to the threshold cycle of a reference gene (Actb encoding β-actin) basedon each PCR amplification efficiency (calculated based on four cDNA concen-trations run in triplicates) as previously described27. The Glo1, Hagh, Bdh andAcads primer sequences, as well as the primer sequences of the reference geneActb, are available on request.

Histopathology, immunohistochemistry and in situ hybridization. We anes-thetized adult (12-week-old) BALB/cBy, C57BL/6J and Acads mutantBALB/cByJ mice with sodium pentobarbital and perfused them through theascending aorta with physiological saline followed by a solution of 4%paraformaldehyde in 0.1 M phosphate buffered saline (PBS; pH 7.35). We thenremoved brains, post-fixed them for 2 d in the same fresh fixative and thenprocessed them for paraffin embedding. For each mouse, we cut four alternateseries of coronal serial sections (7 µm) of the whole brain with a microtomeand collected them on slides pretreated with 3-aminopropyltriethoxy-silane(Sigma). We removed the paraffin from the sections in xylol, rehydrated them,heated them in a microwave oven for 3 × 5 min in 0.01 M citrate buffer, pH 6,and cooled them for 20 min. We stained one series of alternate sections withcresyl violet and mounted them in Eukitt. We processed other alternate sectionsfor immunohistochemistry using a polyclonal antibody to ACADS (1:750).Briefly, we incubated sections in 10% normal goat serum (Dako) for 20 min toblock nonspecific staining and in the primary antibody overnight at 4 °C. Afterwashing them in PBS, we incubated them for 1 h in a peroxidase-conjugatedgoat secondary antibody to rabbit (1%, Dako). All solutions were diluted inPBS (pH 7.4) with 0.2% Triton X-100, 3% bovine serum albumin (Sigma).Tissue-bound peroxidase was visualized by diaminobenzidine (0.05%, in 0.6%H2O2 in 0.1 M PBS, pH 7.4).

For in situ hybridization, we quickly removed the brains, froze them inisopentane at –40 °C and stored them at –80 °C. We cut 12-µm sections in acryostat at –20 °C and thaw-mounted them on poly-L-lysine glass slides. Wefixed sections in 4% paraformaldehyde and 0.05% glutaraldehyde for 30 min,washed them in 5× standard sodium citrate (SSC) buffer for 15 min and cov-ered them with hybridization buffer containing the digoxigenin-dUTP-labeled

antisense probes. We cloned the full-length Acads cDNA from the inbredstrain A/J and Glo1 cDNA from the inbred strain BALB/cByJ into pGEM-Tvector (Promega) and verified correct cloning by sequencing on both strands.The plasmids were linearized by HindIII (Acads) or NcoI (Acads and Glo1),and the digoxigenin-dUTP-labeled antisense probes (688–994 bp) were gener-ated by in vitro transcription with SP6 RNA polymerase using DIG RNA label-ing Kit (Roche) and following the manufacturer’s instructions. We shortenedthe probes to approximately 200 bp by alkaline hydrolysis, purified them andresuspended them in the hybridization buffer at 400 ng ml–1. We incubatedsections with the hybridization buffer at 59 °C overnight and then washed theslides under high stringency and treated them with RNase A at 37 °C for 45 min. After incubation in a blocking buffer containing normal goat serumfor 1 h at room temperature, the slides were incubated overnight at 4 °C withalkaline phosphatase-conjugated antiserum to digoxigenin. We detectedbound probes using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate. No in situ hybridization signal was detected in sections pre-treatedwith RNase A for 30 min.

Immunoblot assays. We subjected 10–50 µg of protein extracts to SDS–PAGEon 15-well (6-cm × 8-cm minigels, 1.0 mm thick) 8–12% polyacrylamide gels.Proteins were electrophoretically transferred to nitrocellulose membranes thatwere incubated in PBS containing 5% non-fat dry milk, 0.2% Tween 20 and0.5% bovine serum albumin (blocking solution) for 1 h at room temperature.We then incubated the membranes either overnight at 4 °C or for 1.5 h atroom temperature in the blocking solution containing the antibody toACADS (1:2,000 dilution). The secondary antibody, a horseradish peroxi-dase–linked donkey antibody to rabbit IgG, was incubated at 1:7,000 dilutionin the blocking solution at room temperature for 2 h. We detectedimmunoreactivity with an enhanced chemiluminescence (ECL) western-blotdetection system (Amersham), followed by exposure to AmershamHyperfilm ECL for 1–10 min.

ALCAR treatment. We gave ALCAR (0.5% w/v in drinking water) to mice forfour (n = 3) or eight (n = 4) weeks before sleep recordings. We compared TPFin BALB/cByJ mice treated with ALCAR versus BALB/cByJ mice providedwith tap water.

URLs. Information on behavioral and (neuro-) physiological differencesamong inbred strains of mice is available at http://www.informatics.jax.org/external/festing/mouse/STRAINS.shtml. The program MapManagerQT can beobtained at http://mapmgr.roswellpark.org/mmQTX.html. Map positions ofMIT markers and gene symbols are based on the Mouse Genome Database,http://www.informatics.jax.org.

Enzyme nomenclature and Enzyme Classification numbers. Short-chain acyl-CoA dehydrogenase (also butyryl-CoA dehydrogenase), EC 1.3.99.2; glyoxylase1 (also lactoylglutathione lyase), EC 4.4.1.5; glyoxylase 2 (also hydroxyacyl glu-tathione hydrolase), EC 3.1.2.6; β-hydroxybutyrate dehydrogenase (also 3-hydroxybutyrate dehydrogenase), EC 1.1.1.30. See the Enzyme StructuresDatabase at http://www.biochem.ucl.ac.uk/bsm/enzymes/ for EC numbers.

GenBank accession numbers. Acads, L11163; Glo1, AK005055; Hagh,BC019817; Bdh, BC027063. Microarray accession numbers in GEO are93268_at and 93269_at.

ACKNOWLEDGMENTSWe thank J. Vockley for providing the ACADS antibody; J. Garcia-Sevilla, M. FerrerAlcon, C. Walzer and B. Pastori for help in immunoblot assays; P. Descombes, M. Docquier and O. Schaad for assistance in microarray and real-time RT–PCRanalyses; A. Malafosse, N. Gregersen and S. Ghisla for discussions; and J. Flint andU. Schibler for critical reading of our first drafts. M.T. was supported by the SwissNational Science Foundation and the Geneva University Hospitals. P.A.W. and P.F. were supported by grants from the National Institutes of Health (the Center for Research Resources and the Heart, Lung, and Blood Institute, respectively). M.T. and P.F. were also supported by a grant from Roche Research Foundation.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

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NATURE GENETICS VOLUME 34 | NUMBER 3 | JULY 2003 325

Received 9 January; accepted 5 May 2003Published online 8 June 2003; doi:10.1038/ng1174

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