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Supplementary Materials and Methods
To verify brain-specific overexpression of CRH in CRH-COE-Nes and CRH-COE-Cam mice,
ten-week-old mice were sacrificed in the morning (ZT4), under an overdose of isoflurane.
Brains were carefully removed and immediately shock frozen on dry ice. Frozen brains were
cut on a cryostat in 20 µm-thick sections. For in situ hybridization cryostat sections of CRHCOEwt, con, het, hom-Nes brains as well as of CRH-COEcon,het,hom-Cam brains were mounted on
SuperFrost Plus slides (Menzel GmbH, Braunschweig, Germany). All sections were
processed for in situ hybridization according to a modified version of the procedure described
by Dagerlind et al.1 The riboprobe nucleotides 1306-1661 of GenBank accession no.
AY128673 was used to detect CRH gene. Specific riboprobes were generated by PCR
applying T7 and T3 primers using a plasmid containing above mentioned cDNA as templates.
Antisense and sense cRNA probes were transcribed from 200 ng of respective PCR product
and directly used as a template for the synthesis of radiolabeled transcripts by in vitro
transcription with 35S-UTP (Amersham Biosciences, Piscataway, NJ, USA) using T7 and T3
RNA polymerase (Roche, Penzberg, Germany), respectively. After 20 min of DNase I
(Roche) treatment, the probes were purified by the RNeasy Clean up protocol (Qiagen,
Hilden, Germany) and measured in a scintillation counter. For hybridization, sections were
pretreated, and prehybridized as described previously.1 Subsequently, they were hybridized
overnight with a probe concentration of 7106 cpm/ml at 57°C and washed at 64°C in 0.1
SSC and 0.1 mM DTT.
Reference List
1. Dagerlind A, Friberg K, Bean AJ, Hokfelt T (1992): Sensitive mRNA detection using
unfixed tissue: combined radioactive and non-radioactive in situ hybridization
histochemistry. Histochemistry 98:39-49.
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Supplementary Figure 1 Conditional overexpression of CRH in the mouse brain. (A)
Schematic representation of the R26 locus, which was engineered to harbour a Creinducible CRH-LacZ expression unit (R26flopCRH). Nes-Cre-mediated removal of the
transcriptional terminator results in a R26 promoter-driven overexpression of CRH and
LacZ within the entire CNS whereas Camk2a-Cre is restricting CRH overexpression to
the anterior forebrain. CNS- and forebrain-restricted overexpression of CRH in CRHCOE-Nes and CRH-COE-Cam mice respectively was demonstrated by in situ
hybridization. Representative autoradiographs of sagittal brain sections of (B) wild-type
(wt), (C) control (con), (F) heterozygous (het) and (G) homozygous (hom) CRH-COENes mice as well as of (D) heterozygous (het) and (E) homozygous (hom) CRH-COENes are depicted. (H) X-Gal staining demonstrates CNS-restricted LacZ reporter gene
expression on a sagittal brain section of a homozygous (hom) CRH-COE-Nes animal.
SA: splice acceptor; STOP: transcriptional terminator; loxP sites are indicated as black
arrowheads; R26 exons are represented by green boxes. PVN: paraventricular nucleus of
the hypothalamus; BST: bed nucleus of stria terminalis; OB: olfactory bulb; CX: cortex;
HIP: hippocampus.
Supplementary Figure 2 Differences in time course power densities of slow-wave
activity (SWA) during non-REM sleep in three genotypes of CRH-COE-Nes mice. Data
are plotted with SEM representing mean values calculated 30 min each. Since time spent
in non-REM sleep is minimum in the first half of the dark period, data are shown only
for the latter half period. Hatched area, CRH-COEcon-Nes (Nes con), n = 9; gray circles,
CRH-COEhet-Nes (Nes het), n = 9; black circles, CRH-COEhom-Nes (Nes hom), n = 8.
Data sets were selected from the animals shown in Figure 2, in which EEG noises
(artifacts) did not interfere with automatic FFT analyses. Horizontal open bar, light
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period; horizontal filled bar, dark period; horizontal gray bar, sleep deprivation (SD).
Two-way ANOVA among 3 genotypes; during the 12-h light period on the baseline day
F[2,536] = 14.44, P < 0.01, during ZT20 – 22.5 in the dark period on the baseline day
F[2,174] = 4.37, P < 0.05. Heterozygous and/or homozygous mice showed significant
differences during baseline compared with con mice, indicated by its respective line and
*. No statistical significances were found in SWA on the day of sleep deprivation.