www.sciencemag.org/cgi/content/full/339/6117/335/DC1

Supplementary Materials for

Adolescent Stress–Induced Epigenetic Control of Dopaminergic Neurons via

Glucocorticoids

Minae Niwa, Hanna Jaaro-Peled, Stephanie Tankou, Saurav Seshadri, Takatoshi Hikida,

Yurie Matsumoto, Nicola G. Cascella, Shin-ichi Kano, Norio Ozaki, Toshitaka Nabeshima,*

Akira Sawa*

*To whom correspondence should be addressed. E-mail: asawa1@jhmi.edu (A.S.); tnabeshi@meijo-u.ac.jp (T.N.)

Published 18 January 2013, Science 339, 335 (2013)

DOI: 10.1126/science.1226931

This PDF file includes:

Materials and Methods

Figs. S1 to S9

Tables S1 and S2

References

2

Materials and Methods

Animals

DISC1 dominant-negative transgenic mice under control of the prion protein promoter

(DISC1-DN-Tg-PrP) were generated at the Transgenic Core Laboratory of the Johns Hopkins

University. Heterozygous transgenic line 51 mice and wild-type littermates established by mating

with C57BL6 mice maintaining the purity of the genetic background (24) were compared in

further experiments. Considering the variability due to the insertion site of the transgene, we also

used line 5 as a reference to confirm consistent phenotypes between lines 51 and 5 (Fig. 2B; Fig.

S7). Mice were group-housed in wire-topped clear plastic cages (21×32×13 cm) or isolated in

wire-topped opaque polypropylene cages (12.5×20×11 cm) (10) from 5 weeks of age

continuously until sampling after behavioral tests, under a controlled environment (23 ± 1˚C; 50

± 5% humidity; light and dark cycles started at 8 am and 8 pm, respectively) with free access to

food and water. All animal care and use was in accordance with guidelines for the care and use of

laboratory animals issued by the National Institutes of Health, Japanese Pharmacological Society,

Johns Hopkins, and Meijo University. Primers for the RT-PCR to test the expression of DISC1

constructs were as follows:

sense, PrP exon 1 5'-TGCGTCGCATCGGTGGCA-3';

antisense, human DISC1 5'-TCTCACAGAGGTCACAGTAGGGGCTGCTGCAC-3'.

Protein expression of exogenous mutant DISC1 was tested by immunoprecipitation-coupled

Western blotting (IP-Western), which was equivalent or even lower than that of overall

endogenous DISC1: a rabbit anti-exon 2 DISC1 antibody (25) for precipitation and a mouse

anti-DISC1 antibody 3D4 provided by Dr. Korth (26) for Western detection were used. In situ

hybridization was performed as previously described (27) with minor modifications, by using two

distinct transgenic models expressing the human dominant-negative DISC1, DISC1-DN-Tg-PrP

3

or under control of the Ca2+/calmodulin-dependent protein kinase II promoter

(DISC1-DN-Tg-CaMKII), respectively. The probe sequences were as follows:

hmDISC1 (reacting with both human and mouse DISC1) sense,

5’-TCGAACAGCCAGGGCTGCAGCTGTTGCTACTCAACTCTGCTGATGGCAGAGAGCCT

GCTGCCCGGACACGC-3';

hmDISC1 antisense,

5'-GGCCGGTGTCCGGGCAGCAGGCTCTCTGCCATCAGCAGAGTTGAGTAGCAACAGC

TGCAGCCCTGGCTGT-3';

human-specific DISC1 (huDISC1) sense,

5'-TCGACGGGACCTCAGGGCCACAGCCAGGAGCTGTTGCTACTGC-3';

huDISC1 antisense,

5'-GCCGCAGTAGCAACAGCTGCAGCTTGTGGCCCTGAGGTCCCG-3'.

Drug treatment

RU38486 [mifepristone: 17-hydroxy-11-(4-dimethylamino-phenyl)-17-(prop-1-ynyl)-estra-4,9-

dien-3-one] (12, 28) or methamphetamine (METH; 1 mg/kg, i.p.) was dissolved in saline with 2%

ethanol or saline, respectively. To examine whether the behavioral, dopaminergic, and epigenetic

changes in the GXE model are mediated by the glucocorticoid receptor (GR) signaling, mice

were administered RU38486 (20 mg/kg, s.c., once per day) from 5 weeks to decapitation after the

behavioral experiments (in this study, we observed blockade of biological cascades downstream

of GR, regardless of the levels of corticosterone). During the 2-day behavioral analysis for

prepulse inhibition (PPI) and forced swim test or the 1-day analysis for locomotion, daily

administration of RU38486 (20 mg/kg, s.c.) was made continuously to avoid washout periods. To

standardize the conditions of both behavioral studies and in vivo microdialysis, such

4

administration included one injection 30 min prior to these experiments according to the

published protocols (10, 12). We did not observe any significant differences among CTL, E, and

G groups in behavioral tests and neurochemical analyses (Figs. 1, 2, and 3A). Thus, we used the

CTL+Veh group as a control in the following experiments.

Behavioral analyses

Two cohorts (the first cohort was used for PPI test followed by forced swim test, and the second

cohort for testing locomotor activity) were used.

Prepulse inhibition test

The PPI test was carried out as described previously (29) with minor modifications. The mouse

was subjected to 50 startle trials, each trial consisting of one of five conditions: (i) a 40-ms,

120-dB noise burst presented alone; (ii-iv) a pre-pulse (20-ms noise burst) that was 4, 8, or 16 dB

above background noise (i.e., 74, 78, or 86 dB) followed, 100 ms later, by a 40-ms, 120-dB noise

burst; or (v) no stimulus (background noise alone), which was used to measure baseline

movement in the startle chamber. Each of these five trial types (i-v) was repeated 10 times in a

pseudorandom order to give 50 trials. Percent PPI of a startle response was calculated as: 100 -

[(startle response on prepulse + startle pulse) / (startle response on startle pulse)] × 100.

Forced swim test

Each mouse was placed in a transparent glass cylinder (8 cm in diameter x 20 cm high),

containing water at 22-23 oC to a depth of 15 cm, and forced to swim for 10 min. The duration of

immobility was measured using digital counters with infrared sensors (30). Immobility time was

calculated as follows: 600 (sec) - swimming time (sec) = immobility time (sec).

5

Locomotor activity test

To measure novel environment-, saline-, and METH (1 mg/kg, i.p.)-induced locomotor activity,

mice were placed in a transparent acrylic cage, and locomotion was measured every 5 min for 6 h

(habituation session for 2 h; saline session for 2 h; METH session for 2 h) by using digital

counters with infrared sensors as described previously (31), with minor modifications. Figs. 1C,

3F, S5E, and S5F show column graphs of accumulating counts for each session. The respective

locomotions over time graphs (Fig. S3) show that the mice were habituated at the end of the

habituation session and recovered from the saline injection before the METH injection.

Neurochemical and biochemical analyses

Histology

Histological procedures were performed as previously described (32) with minor modifications.

Brains were fixed with 4% paraformaldehyde, and coronal sections, including frontal cortex (Fc)

or ventral tegmental area (VTA), were obtained with a cryostat at 20 µm. For

immunohistochemistry, the following primary antibodies were used; anti-tyrosine hydroxylase

(TH) (1:100, Millipore) and anti-glial fibrillary acidic protein (GFAP) (1:200, Millipore).

Fluorescent secondary antibodies conjugated to Alexa 488 and Alexa 647 (Molecular Probes) as

well as biotin-conjugated secondaries were used for chromogen detection. Nissl staining with

cresyl violet was carried out as published (33).

Contents of monoamines

Fc, nucleus accumbens (NAc), and caudate putamen (CPu) were rapidly dissected out according

to the atlas of Franklin and Paxinos (34). The total levels of dopamine (DA), norepinephrine (NE),

6

and 5-hydroxytryptamine (5-HT) were determined using a high-performance liquid

chromatography (HPLC) system, as previously described (35); Briefly, each frozen brain sample

was weighed and homogenized with an ultrasonic processor in 350 μl of 0.2 M perchloric acid

containing isoproterenol as an internal standard. The homogenate was placed on ice for 30 min

and centrifuged at 20,000 × g for 15 min at 4˚C. The supernatant was mixed with 1 M sodium

acetate to adjust the pH to 3.0 and injected into an HPLC system equipped with a reversed-phase

ODS column and an electrochemical detector.

In vivo microdialysis

Microdialysis was carried out as previously described (29, 31), with a minor modification. A

guide cannula was implanted into Fc (15o angle from anteroposterior (AP): +1.7 mm,

mediolateral (ML): -1.0 mm from Bregma, dorsoventral (DV): -2.0 mm from the dura) or NAc

(AP: +1.7 mm, ML: -0.8 mm from Bregma, DV: -4.0 mm from the dura) according to the atlas of

Franklin and Paxinos (34). Ringer’s solution (147 mM NaCl, 4 mM KCl, and 2.3 mM CaCl2) was

perfused at a flow rate of 1.0 μl/min. The dialysates were collected every 10 min and analyzed by

an HPLC system. Six samples were taken to establish baseline levels of extracellular DA. The

extracellular levels of DA upon METH challenge (1 mg/kg, i.p.) were measured in Fc and NAc,

to clarify tonic and phasic responses in mesocortical and mesolimbic dopaminergic neurons,

respectively (36). Of note, administration of RU38486 did not change the basal levels of

extracellular DA in Fc (shown in Fig. 3B as the comparison of CTL+Veh and CTL+RU).

Western blotting

Western blotting was performed as previously described (29) with minor modifications. Fc and

NAc were dissected out according to the atlas of Franklin and Paxinos (34). Mouse anti-TH

7

monoclonal (1:1,000, Millipore), rat anti-dopamine D1 receptor (D1R) monoclonal (1:250,

Sigma-Aldrich), rabbit anti-dopamine D2 receptor (D2R) polyclonal (1:500, Millipore), mouse

anti-GFAP monoclonal (1:1,000, Millipore), and goat anti-β-actin polyclonal antibody (1:500,

Santa Cruz Biotechnology) antibodies were used as primary antibodies. Horseradish

peroxidase-conjugated anti-mouse, anti-rat, anti-rabbit, and anti-goat IgG (1:2,000, Kierkegaard

& Perry Laboratories) were used as secondary antibodies. These biochemical assessments were

made from the brains of mice 24 h after the last RU38486 treatment.

Levels of plasma corticosterone

Blood was collected as described previously (10) with minor modifications. One day after the

behavioral tests, blood was collected from cohort 1 between 9 am to 11 am. Plasma

corticosterone levels were determined using a commercially available enzyme immune assay kit.

Retrograde tracing and fluorescence-activated cell sorting (FACS)

Retrograde tracing was performed as described previously (16) with minor modifications. In

retrograde tracer analysis, male DISC1-DN-Tg-PrP and wild-type littermates at 5 weeks of age

were used. The location of the injection site in Fc (15o angle from AP: +1.7 mm, ML: ±1.0 mm

from Bregma, DV: -2.0 mm from the dura) and NAc (AP: +1.42 mm, ML: ±1.6 mm from

Bregma, DV: -4.75 mm from the dura) was standardized among animals by using stereotaxic

coordinates (34). Mice were bilaterally injected with 100 nl of the green and red retrograde beads

into Fc and NAc of mice, respectively. For sufficient labeling, a survival period for retrograde

tracer transport was 3 weeks after injection of the beads. According to the mouse brain atlas of

Franklin and Paxinos (34), the VTA regions (AP: -2.92 to -3.88 mm; ML: ±0.75 to ±0.25 mm

from Bregma; DV: +4.1 to +4.6 mm from the dura) labeled with green and red retrograde beads

8

were dissected from coronal sections with tweezers by using Fluorescent Protein Flashlight (37),

which includes both blue and green high intensity light-emitting diodes (LEDs) so that we could

screen for green and red fluorescence. The samples from 5 mice of each group were pooled for

FACS. After obtaining single cell suspensions from dissected cells, the only cells with these latex

beads were sorted by FACSAria at the flow cytometry core facility of the Johns Hopkins

University by a standard protocol (25).

Bisulfite sequencing

Genomic DNA from FACS sorted cells labeled with retrograde beads was isolated and bisulfite

conversion was performed with Epi Tect Plus LyseAll Bisulfite Lit. The sequences of the bisulfite

primers used for the amplification of the mouse Th gene, with the nucleotide position of the first

base indicated in parentheses (GenBank Accession No. X53503), were as follows (38):

(3301) 5'-TTTTGGTTTGATTAGAGAGTTTTAGATG-3',

(3608) 5'-AATTCTATCTCCACAACCCTTACCA-3',

(3382) 5'-GAGGGTGATTTAGAGGTAGGTGTTG-3',

(3605) 5' -TCTATCTCCACAACCCTTACCAAAC-3'.

The PCR amplification of Th with primers F3301-R3608 was followed by a nested PCR with

primers F3382-R3605, yielding an amplicon of 225 bp encompassing 11 of 20 CpGs in the Th

promoter. PCRs were performed with the AccuPrime Taq DNA Polymerase System according to

the manufacturer’s protocol. The following cycling conditions were applied: 94 oC for 10 min

followed by 40 cycles of 94 oC for 45 sec, 55 oC for 45 sec, 72 oC for 1 min, and a 10-min

extension step at 72 oC. After the first round of DNA amplification, a 1-μl aliquot of the PCR

solution was used for the nested PCR. The amplified DNA was cloned into the pGEM-T Easy

T/A vector system and sequenced using a 3730xl DNA Analyzer at the synthesis and sequencing

9

core facility of the Johns Hopkins University in a standard protocol. These biochemical

assessments were made from the brains of mice 24 h after the last RU38486 treatment.

MRI

In vivo MRI scans were performed in the 11.7T Bruker Biospec small animal imaging system. A

three-dimensional, fast-spin echo, diffusion-weighted (DW) imaging sequence with twin

navigation echoes was used (39, 40).

Statistical analysis

Numbers of animals are summarized in Table S1. All data are expressed as the mean ± SE.

Statistical differences between two groups were determined with Student’s t test. Statistical

differences among three groups or more were determined using a one-way analysis of variance

(ANOVA), two-way ANOVA, three-way ANOVA, and an ANOVA with repeated measures,

followed by the Bonferroni multiple comparison test (Table S2). Statistically significance: ** p <

0.01, * p < 0.05.

Fig. S1 (1)

Construct

RT-PCR IP with DISC1 antibodies

Input

Wt Mut

IPEx2 IgG

Wt Mut Mut

A

B C

(kDa)

Truncated DISC1 GAPDH

- + OB Fc Hc Cb

98

64

OB Fc Hc Cb

10

In situ hybridization (Low magnification)

D

Endogenous DISC1 DISC1-DN-Tg-PrP (G)

DISC1-DN-Tg-CaMKII

E15

E18

P7

P28

Adult

E15

E18

P7

P28

Adult

E15

E18

P7

P28

Adult

Fig. S1 (2) 11

In situ hybridization, adult (40x magnification)F

Cortex Hippocampus Hypothalamus Thalamus

DISC1-DN-Tg-CaMKII

DISC1-DN-Tg-PrP (G)

EndogenousDISC1

E In situ hybridization, E15 (40x magnification)

Cortex Hippocampus Pituitary gland

DISC1-DN-Tg-CaMKII

DISC1-DN-Tg-PrP (G)

EndogenousDISC1

Fig. S1 (3) 12

Fig. S1. Generation of DISC1-DN-Tg-PrP mice. (A) Design of the transgenic construct. Sequences of a C-terminal truncated dominant-negative human DISC1 (corresponding to amino acids 1-597) were inserted under control of the prion protein promoter in a modified pMM403 vector. (B) Dominant-negative transgene mRNA expression assayed by RT-PCR. Expression was widely observed in the brain at 2 months of age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control of expression, the levels of which were similar among several different brain regions. OB, olfactory bulbs; Fc, frontal cortex; Hc, hippocampus; Cb, cerebellum. (C) Expression of the dominant-negative DISC1 was confirmed at the protein level by IP-Western with protein extracts prepared from the cerebellum of DISC1-DN-Tg-PrP mice. (D) Developmental expression of the dominant-negative DISC1 in DISC1-DN-Tg-PrP mice was compared with exogenously expressed DISC1 in DISC1-DN-Tg-CaMKII mice (S5) and endogenous DISC1. In situ hybridization on sagittal sections at embryonic ages 15 (E15), E18, postnatal ages 7 (P7), P28, and adult (approximately 4 months) at low magnification. Scale bar, 1 mm. (E) High magnification images of in situ hybridization at E15. Scale bar, 40 μm. Dominant-negative DISC1 was developmentally expressed in cortex, hippocampus, and anterior pituitary gland in the DISC1-DN-Tg-PrP model, compatible to the endogenous DISC1 expression, in sharp contrast to negligible expression of the dominant-negative DISC1 in these brain regions in the DISC1-DN-Tg-CaMKII mice. (F) High magnification images of in situ hybridization in the adult brain. Scale bar, 30 μm. Endogenous DISC1 was expressed in cortex, hippocampus, hypothalamus, and thalamus. The PrP promoter expressed the mutant DISC1 widely in the brain, including hypothalamus and thalamus, whereas the CaMKII promoter led to the expression of the mutant in more restricted brain regions, especially cortex and hippocampus. High magnification images in Fig. S1E and F were prepared from the areas indicated by red squares in Fig. S1D. Representative figures from experiments repeated more than 3 times are presented.

13

Birth 5 (weeks)

Wild-type

DISC1-DN-Tg-PrP

IsolatedGXE

(2 months)

C 8 20

CTL (5 months)

Group-housed Group-housed

Group-housed Group-housedG

(5 months)

Isolated Group-housedGXE-G

(5 months)

Group-housed

Group-housed

Birth 5 (weeks)

Group-housed

Wild-type

DISC1-DN-Tg-PrP

CTL+VehGroup-housed + Vehicle

CTL+RUGroup-housed + RU38486

GXE+VehIsolated + Vehicle

Isolated + RU38486 GXE+RU

B 8

Group-housed

Birth 5 (weeks)

Group-housed

Wild-type

DISC1-DN-Tg-PrP

CTLGroup-housed

EIsolated

GGroup-housed

Isolated GXE

A 8

Group-housed

Fig. S2. Schemes of experimental schedules.

14

15

CTLEGGXE

B C

0

2000

4000

0 120 240 360Time (min)

Lo

com

oto

r ac

tivi

ty (

cou

nts

/ 5

min

)

0

2000

4000

0 120 240 360Time (min)

Lo

com

oto

r ac

tivi

ty (

cou

nts

/ 5

min

) CTLEGGXE

Male Female

Saline

METH

Saline

METH

** * **

** **

D

0

2000

4000

0 120 240Time (min)

Lo

com

oto

r ac

tivi

ty (

cou

nts

/ 5

min

)

360

Saline

METH

CTL+VehCTL+RUGXE+VehGXE+RU **

****

ACTLEGGXE

Saline

METH

******

0 120 240Time (min)

3600

2000

4000

Lo

com

oto

r ac

tivi

ty (

cou

nts

/ 5

min

)

Fig. S3. Five-min binning of the data for locomotor activity. (A) Aberrant locomotor activity in the GXE model. n=18 to 23 (n=9 or 10, male; n=9 to 13, female). (B and C) Gender influence on locomotor activity: n=9 or 10, male for (B); n=9 to 13, female for (C). Values are means ± SE. ** p < 0.01, * p < 0.05. (D) Effects of RU38486 on locomotor activity. n=12 or 13 (n=6 or 7, male; n=5 to 7, female).

16

0

15

30

Bo

dy

wei

gh

t (g

)

CTLEGGXE

Fig. S4. No difference in the body weight was observed among CTL, E, G, and GXE groups at 2 months of age. Values are means ± SE. n = 13 to 17 (n = 6 to 8, male; n = 7 to 9, female).

17

***

PP

I (%

)

74 78 86Prepulse (dB)

0

50

100Male

PP

I (%

)

074 78 86

50

100Female

Prepulse (dB)

0

250

500 Male

0

250

500

Imm

ob

ilit

y ti

me

(sec

)

Female

74 78 860

50

100

PP

I (%

)

Male

Prepulse (dB)74 78 86

0

50

100

PP

I (%

)

Female

Prepulse (dB)

CTL+VehCTL+RUGXE+VehGXE+RU

CTLEGGXE

CTL+VehCTL+RUGXE+VehGXE+RU

A B

C D

E F

G H

CTLEGGXE

CTLEGGXE

CTLEGGXE

*

Imm

ob

ilit

y ti

me

(sec

)

*

**

*** *

***

18

Lo

com

oto

r ac

tivi

ty(x

103

cou

nts

/ 2h

)

0

25

50 Male

Habituation Saline METH

Lo

com

oto

r ac

tivi

ty(x

103

cou

nts

/ 2

h)

Female

0

25

50

Habituation Saline METH

CTLEGGXE

CTLEGGXE

**

****

**

Fig. S5. Gender influence on the behavioral abnormalities in the GXE model. (A to F)Impaired performance of PPI [(A) n = 6 to 8, male; (B) n = 7 to 9, female], forced swim [(C) n = 6 to 8, male; (D) n = 7 to 9, female], and locomotor activity [(E) n = 9 or 10, male; (F) n = 9 to 13, female] at 2 months of age. (G and H) Effects of a GR antagonist RU38486 during the isolation period on the impaired performances of PPI [(G) n = 8 to 10, male; (H) n = 4 to 10, female] in the GXE model at 2 months. The trend in the behavioral changes in GXE mice was preserved in both genders. CTL, wild-type without isolation; E, wild-type with isolation; G, DISC1-DN-Tg-PrP without isolation; GXE, DISC1-DN-Tg-PrP with isolation; CTL+Veh, CTL treated with vehicle; CTL+RU: CTL treated with RU38486; GXE+Veh, GXE treated with vehicle; GXE+RU, GXE treated with RU38486. Values are means ± SE. ** p < 0.01, * p < 0.05.

19

A Nissl

0

50

100

150

GF

AP

/ β

-act

in (

% o

f co

ntr

ol)

GFAP

β-actin

C D GFAPFc Fc

Fc

CTLEGGXE

CTL E

G GXE

I

II/III

IV

V

VI

I

II/III

IV

V

VI

CTL E

G GXE

I

II/III

IV

V

VI

I

II/III

IV

V

VI

BIn vivo MRI

0

3

6L

V v

olu

me

(mm

3 )CTL

G

20

Fig. S6. No robust changes at the gross anatomical and neurochemical levels. (A) Anatomical analyses of the lateral ventricles of DISC1-DN-Tg-PrP mice detected by in vivo MRI scan. n = 6 or 7 (n = 5, male; n = 1 or 2, female). (B) Nissl staining in Fc at 2 months of age. Scale bar, 200 μm. (C) Expression levels of GFAP in Fc at 2 months of age detected by Western blotting. (D) Expression levels of GFAP in Fc at 2 months of age assessed by immunostaining. Values are means ± SE. n = 6, male. Scale bar, 200 μm. Representative figures from experiments repeated more than 3 times are presented.

** CTLEGGXE

Fc Line 5

0.0

0.1

0.2

Bas

al e

xtra

cellu

lar

DA

(n

M)

21

Fig. S7. Representative demonstration of the consistent phenotypes between lines 5 and 51 of the DISC1-DN-Tg-PrP mice. Basal levels of extracellular DA in Fc of line 5 GXE model. There was no difference in the variability due to the insertion site of the transgene between lines 5 and 51 (also see Fig. 2B). Values are means ± SE. n = 6-7, male. ** p < 0.01.

***

0

50

100

150

D1R

/ β

-act

in (

% o

f co

ntr

ol)

D1Rβ-actin

D2Rβ-actin

A Fc FcB

0

50

100

150

D2R

/ β

-act

in (

% o

f co

ntr

ol) CTL

EGGXE

CTLEGGXE

C NAc

D1R

β-actin

0

50

100

150

D1R

/ β

-act

in (

% o

f co

ntr

ol)

0

50

100

150

D2R

/ β

-act

in (

% o

f co

ntr

ol)

NAc

D2R

β-actin

D

CTLEGGXE

CTLEGGXE

***

22

Fig. S8. Differences in expression of dopamine receptors in Fc and NAc of the GXE model.(A to D) Levels of D1R and D2R in Fc (A and B) and NAc (C and D) at 2 months of age, respectively. Expression of D2R in Fc was elevated in the GXE model. Values are means ±SE. n = 6, male. ** p < 0.01, * p < 0.05.

Fc

D2R

β-actin

0

50

100

150D

2R /

β-a

ctin

(%

of

con

tro

l)

BFc

TH

β-actin

0

50

100

150

TH

/ β

-act

in (

% o

f co

ntr

ol)

A

CTL+VehCTL+RUGXE+VehGXE+RU

CTL+VehCTL+RUGXE+VehGXE+RU

**** *

**** *

Fig. S9 23

Fig. S9. Influence of glucocorticoids on neurochemical abnormalities in the GXE model. (A and B) Normalization effect of RU38486 during the isolation period on the decreased levels of TH (A) and increased levels of D2R (B) in Fc of the GXE model at 2 months of age. Values are means ± SE. n = 6, male. ** p < 0.01, * p < 0.05.

Table S1

Fig.1 A B C (S3A)

Numberof

animals

per group 13 to 17 13 to 17 18 to 23

male 6 to 8 6 to 8 9 or 10

female 7 to 9 7 to 9 9 to 13

Fig.2 A B C D E F G

Numberof

animals

per group 7 6 7 6 6 6 6

male 7 6 7 6 6 6 6

female 0 0 0 0 0 0 0

Fig.3 A B C D E F (S3B)

Numberof

animals

per group 7 or 8 7 7 13 to 19 13 to 19 12 or 13

male 7 or 8 7 7 8 to 10 8 to 10 6 or 7

female 0 0 0 4 to 10 4 to 10 5 to 7

Fig.4 C D E F

Numberof

animals

per group 5 (pooled) 5 (pooled) 5 (pooled) 5 (pooled)

male 5 (pooled) 5 (pooled) 5 (pooled) 5 (pooled)

female 0 0 0 0

Fig.S4

Numberof

animals

per group 13 to 17

male 6 to 8

female 7 to 9

Fig.S5 A B C D E (S3C) F (S3D) G H

Numberof

animals

per group 6 to 8 7 to 9 6 to 8 7 to 9 9 or 10 9 to 13 8 to 10 4 to 10

male 6 to 8 0 6 to 8 0 9 or 10 0 8 to 10 0

female 0 7 to 9 0 7 to 9 0 9 to 13 0 4 to 10

Fig. S6A S6C S7 S8A S8B S8C S8D S9A S9B

Numberof

animals

per group 6 or 7 6 6 or 7 6 6 6 6 6 6

male 5 6 6 or 7 6 6 6 6 6 6

female 1 or 2 0 0 0 0 0 0 0 0

Table S1. Numbers of animals per study.

24

Fig. 1A 1B 1C (S3A) 2A 2B 2C 2D 2E 2F 2G

ANOVA three-way

two-way repeated two-way two-way two-way two-way two-way repeated repeated

genotypeF (1, 162)

= 37.57F (1, 54)

= 6.73-

F (1, 24)

= 5.79F (1, 20)

= 6.69F (1, 24)

= 1.86F (1, 20)

= 26.46F (1, 20)

= 0.99- -

environmentF (1, 162)

= 0.22F (1, 54)

= 8.03-

F (1, 24)

= 3.55F (1, 20)

= 5.38F (1, 24)

= 0.64F (1, 20)

= 4.07F (1, 20)

= 0.32- -

group - -F (3, 75)

= 4.21- - - - -

F (3, 20)

= 0.13F (3, 20)

= 1.64

prepulseF (2, 162)

= 45.02- - - - - - - - -

time - -F (24, 1800)

= 12.60- - - - -

F (10, 200)

= 59.65F (10, 200)

= 15.73genotype

x environmentF (1, 162)

= 6.07F (1, 54)

= 4.38-

F (1, 24)

= 0.54F (1, 20)

= 0.02F (1, 24)

= 1.20F (1, 20)

= 2.15F (1, 20)

= 0.07- -

gentype x prepulse

F (2, 162)

= 0.14- - - - - - - - -

environment x prepulse

F (2, 162)

= 0.28- - - - - - - - -

group x time

- -F (72, 1800)

= 1.82- - - - -

F (30, 200)

= 0.28F (30, 200)

= 0.83genotype

x environment x prepulse

F (2, 162)

= 0.29- - - - - - - - -

Fig. 3A 3B 3C 3D 3E 3F (S3B) 4C 4D 4E 4F

ANOVA two-way two-way repeatedthree-way

two-way repeated two-way two-way two-way one-way

genotypeF (1, 36)

= 2.08- - - - -

F (1, 36)

= 54.76F (1, 68)

= 0.01- -

environmentF (1, 36)

= 9.28- - - - -

F (1, 36)

= 14.77F (1, 68)

= 0.54- -

group -F (1, 27)

= 14.06F (3, 24)

= 3.85F (1, 180)

= 28.07F (1, 60)

= 20.36F (3, 47)

= 20.57- -

F (1, 38)

= 94.65F (3, 42)

= 76.17

drug -F (1, 27)

= 6.30-

F (1, 180)

= 2.64F (1, 60)

= 1.44- - -

F (1, 38)

= 22.96-

prepulse - - -F (2, 180)

= 46.34- - - - - -

time - -F (11, 264)

= 41.39- -

F (23, 1081)

= 31.11- - - -

genotype x environment

F (1, 36)

= 1.68- - - - -

F (1, 36)

= 1.19F (1, 68)

= 1.24- -

group x drug

-F (1, 27)

= 2.41-

F (1, 180)

= 10.70F (1, 60)

= 6.41- - -

F (1, 38)

= 26.30-

group x time

- -F (33, 264)

= 3.69- -

F (69, 1081)

= 1.67- - - -

group x prepulse

- - -F (2, 180)

= 0.72- - - - - -

drug x prepulse

- - -F (2, 180)

= 0.10- - - - - -

group x drug

x prepulse- - -

F (2, 180)

= 1.02- - - - - -

Table S2. Summary of statistical analyses with F values.

Table S2 (1) 25

Fig. S4D S5A S5B S5C S5DS5E

(S3C)S5F

(S3D)S5G S5H

ANOVA two-way three-waythree-way two-way two-way repeated repeated three-waythree-way

genotypeF (1, 54)

= 0.01F (1, 69)

= 25.43F (1, 81)

= 14.68F (1, 23)

= 3.44F (1, 27)

= 2.95- - - -

environmentF (1, 54)

= 1.18F (1, 69)

= 0.00F (1, 81)

= 0.33F (1, 23)

= 2.55F (1, 27)

= 5.29- - - -

group - - - - -F (3, 33)

= 0.76F (3, 38)

= 4.24F (1, 96)

= 9.71F (1, 83)

= 25.39

drug - - - - - - -F (1, 96)

= 14.16F (1, 83)

= 2.55

prepulse -F (2, 69)

= 29.10F (2, 81)

= 18.75- - - -

F (2, 96)

= 36.33F (2, 83)

= 13.99

time - - - - -F (24, 792)

= 8.05F (24, 912)

= 5.68- -

genotype x environment

F (1, 54)

= 2.26F (1, 69)

= 4.88F (1, 81)

= 2.01F (1, 23)

= 1.83F (1, 27)

= 2.30- - - -

gentype x prepulse

-F (2, 69)

= 0.20F (2, 81)

= 0.29- - - - - -

environment x prepulse

-F (2, 69)

= 0.08F (2, 81)

= 0.29- - - - - -

group x drug

- - - - - - -F (1, 96)

= 3.75F (1, 83)

= 2.41group x time

- - - - -F (72, 792)

= 1.46F (72, 912)

= 1.37- -

group x prepulse

- - - - - - -F (2, 96)

= 0.61F (2, 83)

= 0.64drug

x prepulse- - - - - - -

F (2, 96)

= 0.04F (2, 83)

= 0.11genotype

x environment x prepulse

-F (2, 69)

= 0.03F (2, 81)

= 0.32- - - - - -

group x drug

x prepulse- - - - - - -

F (2, 96)

= 0.66F (2, 83)

= 0.68

Fig. S6C S7 S8A S8B S8C S8D S9A S9BANOVA two-way two-way two-way two-way two-way two-way two-way two-way

genotypeF (1, 20)

= 0.05F (1, 21)

= 8.85F (1, 20)

= 0.92F (1, 20)

= 31.09F (1, 20)

= 0.06F (1, 20) =

2.28- -

environmentF (1, 20)

= 2.83F (1, 21)

= 5.43F (1, 20)

= 4.36F (1, 20)

= 2.37F (1, 20)

= 0.77F (1, 20) =

2.08- -

group - - - - - -F (1, 20)

= 14.41F (1, 20)

= 15.17

drug - - - - - -F (1, 20)

= 3.60F (1, 20)

= 6.54genotype

x environmentF (1, 20)

= 0.65F (1, 21)

= 0.60F (1, 20)

= 0.38F (1, 20)

= 1.97F (1, 20)

= 0.23F (1, 20) =

0.00- -

group x drug

- - - - - -F (1, 20)

= 8.71F (1, 20)

= 3.54

Table S2 (2) 26

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