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: [email protected] (A.S.); [email protected] (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
30
Embed
Supplementary Materials for - Sciencescience.sciencemag.org/highwire/filestream/594177/field_highwire... · Supplementary Materials for ... standardize the conditions of both behavioral
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
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