regulation of Homer1a. In conclusion, converging evidence strongly
implicates Homer1a as a brain-coping marker against stressors, and
our findings suggest that Homer1a might represent the molecular
link between sleep, cognition, and neuropsychiatric disorders.
Materials and Methods
Animal Handling. A ll ex periments were perfor med in ac c or-
dance with the protoc ols approved by the Ethical Committee
of the State of Vaud Veterinary Office, Switzerland. Sleep-
deprivation and sleep-recording procedures are described in SI
Mater ials and Methods.
cRNA Preparation, cDNA Microarray Hybridization, and Real-Time
For the first experiment, we isolated total RNA from whole
brain and liver by using the RNAXEL kit (Eurobio), treated the
RNA with DNase, and cleaned it using RNeasy columns (Qiagen).
Equal quantities of total RNA from three individual mice of each
strain were pooled in triplicate (nine mice of each strain in each
condition). A hybridization mixture containing 15
g of biotin-
ylated cRNA was hybridized to GeneChip Mouse Expression Set
430. Chips were washed, scanned, and analyzed with Affymetrix
For the around-the-clock microarray experiment, RNA from
whole brain was isolated and purified with the RNeasy Lipid Tissue
Midi kit (Quiagen) and DNase-treated. All RNA quantities were
assessed with a NanoDrop ND-1000 spectrophotometer, and the
quality of RNA was controlled on Agilent 2100 bioanalyzer chips.
Equal amounts of total RNA were pooled from three mice within
each of the 24 experimental groups (three strains, two conditions,
4ZT⫽ 24; in triplicate: 24 䡠 3 ⫽ 72 chips). Three micrograms of
each of these pools were used to perform the chip array experiment,
according to the Affymetrix Gene Expression procedure. Twelve
micrograms of biotinylated cRNA from each sample were frag-
mented and hybridized to GeneChip Mouse 430 2.0 arrays, accord-
ing to standard procedures. Microarray analyses and qPCR verifi-
cations were performed as reported in SI Materials and Methods.
Hybridization. Total protein extract was
prepared with RIPA lysis buffer. Protein concentration was calcu-
lated by using the bicinchoninic acid assay (Pierce) with BSA as a
standard. Eighty micrograms of each fraction were analyzed by
SDS/PAGE, followed by We stern blotting using antibodies as
follows: mouse anti-tubulin 1/1,000 (Santa Cruz), goat anti-
Homer1a 1/200 (Santa Cruz), mouse anti-Flag M2 1/300 (affinity-
purified; Sigma), and rabbit anti-GFP 1/2,500 (AbCam). Secondary
antibodies were all coupled with HRP, except for the anti-goat
antibody, which was IRDye800-conjugated for Lycor analysis.
In situ hybridizations with coronal cryosections of 12
performed according to Allen Brain Atlas protocols (enzymatic
BCIP/NBT revelation) (37). All reagents and solutions were pur-
chased and prepared based on Eurexpress II in situ hybridization
consortium instructions. GFP and Homer1a riboprobes were syn-
thesized by in vitro transcription on a linearized pGEM-Easy vector
(Promega) containing the corresponding sequences. The cDNA
insert of this plasmid was generated by RT-PCR from mouse brain
RNA, using the following primers: Homer1a for ward, 5⬘-
GCTGTCAGAAGCTTAGGATGTG-3⬘; Homer1a reverse,
5⬘-AAAGTGCAGAAAGTCCAGCAGC-3⬘; GFP forward, 5⬘-
GAGCTGGACGGCGACGTAAACG-3⬘; and GFP reverse, 5⬘-
FISH was perfor med as described in ref. 38, using anti-DIG-
POD 1/600 (Roche), anti-FLU-AP 1/100 (Roche), and SA-A lexa
488 (Molecular Probes) and counterstained with DAPI (Sigma).
Transgenic and mRNA Tagging. Transgenic mice were generated as
described in Fig. 3. See SI Materials and Methods for details.
We thank K. Harshman, A. Paillusson, and M. Bueno for assistance in
microarray and real-time RT-PCR analyses at the Lausanne DNA Array
Facility; P. Descombes, M. Docquier, D. Chollet, and C. Delucinge for
assistance in microarray and real-time RT-PCR analyses at the Geneva
Genomics Platform, National Center for Competence in Research Frontiers
in Genetics; S. Excoffier for help in the transgenic construction; P. Seeburg,
M. Schwarz (Max Planck Institute, Heidelberg, Germany), and P. Worley
(Johns Hopkins School of Medicine, Baltimore, MD) for providing Homer1
mutant mice; and A. Vassali for constructive discussions. This work was
supported by the Swiss National Science Foundation and the State of Vaud
(M.T.) and in part by National Institutes of Mental Health Grant MH67752
1. Dijk DJ, Franken P (2005) in Principles and Practice of Sleep Medicine, eds
Kryeger TH, Roth TH, Dement W (Saunders, Philadelphia), pp 418 –434.
2. Krueger JM, Obal F (1993) J Sleep Res 2:63–69.
3. Tononi G, Cirelli C (2006) Sleep Med Rev 10:49– 62.
4. Huber R, Ghilardi MF, Massimini M, Ferrarelli F, Riedner BA, Peterson MJ,
Tononi G (2006) Nat Neurosci 9:1169–1176.
5. Marshall L, Helgadottir H, Molle M, Born J (2006) Nature 444:610–613.
6. Molle M, Marshall L, Gais S, Born J (2004) Proc Natl Acad Sci USA
7. Franken P, Chollet D, Tafti M (2001) J Neurosci 21:2610–2621.
8. Van Dongen HP, Baynard MD, Maislin G, Dinges DF (2004) Sleep 27:423–433.
9. Tucker AM, Dinges DF, Van Dongen HP (2007) J Sleep Res 16:170–180.
10. Shifman S, Bell JT, Copley RR, Taylor MS, Williams RW, Mott R, Flint J
(2006) PLoS Biol 4:e395.
11. Cirelli C, Gutierrez CM, Tononi G (2004) Neuron 41:35–43.
12. Mackiewicz M, Shockley KR, Romer MA, Galante RJ, Zimmerman JE,
Naidoo N, Baldwin DA, Jensen ST, Churchill GA, Pack A (2007) Physiol
13. Nelson SE, Duricka DL, Campbell K, Churchill L, Krueger JM (2004) Neurosci
14. Huber R, Tononi G, Cirelli C (2007) Sleep 30:129–139.
15. Terao A, Steininger TL, Hyder K, Apte-Deshpande A, Ding J, Rishipathak D,
Dav is RW, Heller HC, Kilduff TS (2003) Neuroscience 116:187–200.
16. Wisor JP, Morairty SR, Huynh NT, Steininger TL, Kilduff TS (2006) Neuroscience
17. Franken P, Thomason R, Heller HC, O’Hara BF (2007) BMC Neurosci 8:87.
18. Wisor JP, O’Hara BF, Terao A, Selby CP, Kilduff TS, Sancar A, Edgar DM,
Franken P (2002) BMC Neurosci 3:20.
19. Cirelli C, Faraguna U, Tononi G (2006) J Neurochem 98:1632–1645.
20. Terao A, Grec o MA, Davis RW, Heller HC, Kilduff TS (2003) Neuroscience
21. Roy PJ, Stuart JM, Lund J, Kim SK (2002) Nature 418:975–979.
22. Kunitomo H, Uesugi H, Kohara Y, Iino Y (2005) Genome Biol 6:R17.
23. Yang Z, Edenberg HJ, Davis RL (2005) Nucleic Acids Res 33:e148.
24. Satoh K, Takeuchi M, Oda Y, Deguchi-Tawarada M, Sakamoto Y, Matsubara
K, Nagasu T, Takai Y (2002) Genes Cells 7:187–197.
25. Bottai D, Guzowski JF, Schwarz MK, Kang SH, Xiao B, Lanahan A, Worley
PF, Seeburg PH (2002) J Neurosci 22:167–175.
26. Worley PF, Zeng W, Huang G, Kim JY, Shin DM, K im MS, Yuan JP, Kiselyov
K, Muallem S (2007) Cell Calcium 42:363–371.
27. Xiao B, Tu JC, Worley PF (2000) Curr Opin Neurobiol 10:370 –374.
28. Kakizawa S, Kishimoto Y, Hashimoto K, Miyazaki T, Furutani K, Shimizu H,
Fukaya M, Nishi M, Sakagami H, Ikeda A, et al . (2007) EMBO J 26:1924–1933.
29. Yamamoto K, Sak agami Y, Sugiura S, Inokuchi K, Shimohama S, Kato N
(2005) Eur J Neurosci 22:1338–1348.
30. Moriguchi S, Nishi M, Komazak i S, Sakagami H, Miyazaki T, Masumiya H,
Saito SY, Watanabe M, Kondo H, Yawo H, et al. (2006) Proc Natl Acad Sci USA
31. Blethyn KL, Hughes SW, Toth TI, Cope DW, Crunelli V (2006) J Neurosci
32. Tappe A, Klugmann M, Luo C, Hirlinger D, Agarwal N, Benrath J, Ehren-
gruber MU, During MJ, Kuner R (2006) Nat Med 12:677–681.
33. Kato A, Ozawa F, Saitoh Y, Fukazawa Y, Sugiyama H, Inokuchi K (1998) J Biol
34. Southw ick SM, Vythilingam M, Charney DS (2005) Annu Rev Clin Psychol
35. Szumlinski KK, Kalivas PW, Worley PF (2006) Curr Opin Neurobiol 16:251–
36. Giedke H, Schwarzler F (2002) Sleep Med Rev 6:361–377.
37. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF,
Boguski MS, Brockway KS, Byrnes EJ, et al. (2007) Nature 445:168–176.
38. Ishii T, Omura M, Mombaerts P (2004) J Neurocytol 33:657–669.
Maret et al. PNAS
December 11, 2007