Supplementary Information
Astroglial Glutamate Transporter Deficiency Increases Synaptic Excitability and
Leads to Pathological Repetitive Behaviors in Mice
Tomomi Aida, Junichi Yoshida, Masatoshi Nomura, Asami Tanimura, Yusuke Iino,
Miho Soma, Ning Bai, Yukiko Ito, Wanpeng Cui, Hidenori Aizawa, Michiko
Yanagisawa, Terumi Nagai, Norio Takata, Kenji F. Tanaka, Ryoichi Takayanagi,
Masanobu Kano, Magdalena Götz, Hajime Hirase, Kohichi Tanaka
1
Supplementary Materials and Methods
Mice
Mice were housed by genotype in groups of 3-5 animals per cage and maintained on a
regular 12 hours light/dark cycle (8:00-20:00 light period) at a constant 25 °C. Food and
water were available ad libitum. The ages of the mice used in each experiment were
indicated below. Both sexes were used unless otherwise noted.
Histology
Mice (n = 3 for each conditions or genotypes) were deeply anesthetized with
pentobarbital (100 mg/kg, i.p.) and fixed by perfusion with 4% paraformaldehyde
The ages of the mice are indicated in the text. Brains were removed, post-fixed
in 4% PFA, transferred to 30% sucrose for cryoprotection, embedded in OCT
(Sakura, Tokyo, Japan) and cryosectioned at 30 μm (for GLT1 immunohistochemistry
and stainings of ROSA26-CAG-TdTomato reporter mice) or 50 μm (for neuronal
count, GFAP and IbaI immunohistochemistry). Facial skin was also removed,
overnight in 4% PFA, dehydrated, embedded in paraffin and sectioned at 4 μm. Nissl
hematoxylin-eosin staining was performed following standard protocols. For
immunohistochemistry, cryosections were permeabilized and blocked with 0.3% Triton
X-100, 1% BSA and 10% normal goat serum in PBS and were incubated with primary
antibodies overnight at 4 °C. The following antibodies were used: polyclonal anti-GLT1
(1:10,000, a gift from M. Watanabe, Hokkaido University) (Tanaka et al, 1997),
polyclonal anti- glial fibrillary acidic protein (GFAP, 1:1,000, Z0334, Dako Carpinteria,
CA, USA), monoclonal anti-S100 β (1:200, S2532, Sigma), polyclonal anti-IbaI
(1:1,000, 019-19741, Wako Pure Chemicals, Osaka, Japan) and monoclonal anti-NeuN
(1:1,000,
MAB377,
Millipore,
Bedford,
MA,
USA),
goat
anti-mouse
IgG-AlexaFluor488
(1:1000,
A-11001,
Life
Technologies),
goat
anti-rabbit
IgG-AlexaFluor488
(1:1000,
A-11034,
Life
Technologies),
goat
anti-mouse
IgG-AlexaFluor594 (1:1000, A-11005, Life Technologies), and goat anti-rabbit
IgG-AlexaFluor594
(1:1000,
A-11012,
Life
Technologies).
For
GLT1
immunohistochemistry, signal was visualized by using the Envision-Plus Rabbit HRP
2
System (Dako) and the DAB Peroxidase Substrate Kit (Vector Laboratories,
Burlingame, CA, USA). Images were acquired under a constant exposure condition on a
BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan) with 4× objective lens using
attached software. For fluorescent immunohistochemistry of GFAP, S100β, IbaI, and
NeuN, signals were visualized by Alexa Fluor 488- or 594-conjugated goat anti-rabbit
or mouse IgG. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI,
D1306, Life Technologies). Sections were mounted with Fluoromount (Diagnostic
BioSystems, Pleasanton, CA, USA). Images were acquired under a constant exposure
condition between control and mutant sections on an LSM 510 META laser-scanning
confocal microscope (Carl Zeiss, Oberkochen, Germany) with 40× objective lens using
attached Zeiss LSM Image Browser software.
Western blot analysis
Control and mutant mice (n = 3, the ages of the mice are indicated in below) were
sacrificed with pentobarbital (100 mg/kg, i.p.). Brains were dissected, separated into the
cerebral cortex, thalamus and striatum and homogenized in lysis buffer [20 mM
Tris-HCl, pH 7.4; 10% sucrose and Complete Protease Inhibitor Cocktail tablet (Roche
Diagnostics)]. After sonication, protein concentrations were determined by BCA Assay
(Sigma, St. Louis, MO, USA), and the samples were diluted with equal amounts of 2×
sample buffer (250 mM Tris-HCl, pH 6.8; 4% SDS; 10% glycerol and 1% β
-mercaptoethanol) as previously described (Regan et al, 2007). After denaturation by
heating at 95 °C for 10 min, 10 μg of each sample was separated by 4–20% SDS-PAGE
(Mini-PROTEAN TGX Precast Gel, Bio-Rad, Hercules, CA, USA) along with a protein
standard ladder (Blue Star, Nippon Genetics, Tokyo, Japan). The samples were then
transferred to PVDF membranes and blocked in TBS with 0.1% Tween 20 (TBS-T) and
5% skim milk for 1 hour at room temperature. Membranes were then incubated with
primary antibodies in TBS-T containing 1% skim milk at 4 °C overnight. Next, the
membranes were washed, incubated with HRP-conjugated secondary antibodies at room
temperature for 1 hour, washed, and visualized with Luminata Forte Western HRP
substrate (Millipore). Gel images were taken every 30 sec for 10 min using Image Lab
software (Bio-Rad). The following antibodies were used: polyclonal anti-GLAST
3
(1:2,500, a gift from M. Watanabe, Hokkaido University) (Watase et al, 1998),
polyclonal anti-GLT1 (1:5,000, a gift from M. Watanabe, Hokkaido University)
(Tanaka et al, 1997), polyclonal anti-EAAC1 (1:500, Santa Cruz Biotechnology, Santa
Cruz, CA, USA, sc-25658), monoclonal anti- β -Actin (1:5000, Santa Cruz
Biotechnology, sc-47778), HRP-conjugated anti-rabbit IgG (1:10,000, Jackson
ImmunoResearch
Laboratories,
West
Grove,
PA,
USA,
711-035-152)
and
HRP-conjugated anti-mouse IgG (1:10,000, Jackson ImmunoResearch Laboratories,
715-035-151). For EAAC1 quantification, membranes were stripped with stripping
buffer (0.2 M glycine, 0.1% SDS and 1% Tween 20, pH 2.2) twice for 5 min and
washed with PBS twice for 10 min and TBS-T twice for 5 min. Band intensities of gel
images within linear signal range were quantified using Image Lab software and
normalized with band intensities of β-Actin. For GLT1, total band intensities of both
monomer and dimer were quantified.
Experiment 3: Effects of GLT1 deletion on behaviors.
Behavior experiments were performed as previously described (Nakatani et al, 2009).
Elevated plus maze test. The elevated plus-maze test apparatus (O’Hara) consisted of
two open and two closed arms (25 × 5 cm). The apparatus was placed at a height of 55
cm. Mice were placed in the center, and the behaviors were recorded during a 6-min test
period. Time spent in the open arms was recorded. Data acquisition and analysis were
performed automatically using Image EP software (O’Hara).
Light-dark box test. The light-dark box test apparatus (O’Hara) consisted of a cage (21 ×
42 × 25 cm) divided into two sections of equal size by a partition with a door. The light
chamber was brightly illuminated (390 lx), while the dark chamber was dark (2 lx).
Mice were placed into the dark chamber and allowed to move freely between the two
chambers with the door open for 10 min. The latency to enter the light chamber was
recorded automatically using Image LD software (O’Hara).
Open field test. Mice were placed in the center of the open field test apparatus (50 × 50
× 40 cm, O’Hara). Time spent in the center was recorded automatically using Image OF
software (O’Hara). Data were collected for 30 min.
4
Reciprocal social interaction test. The resident mice and intruder (C57BL/6J) male mice
were housed in different cages. Resident mice were individually housed for 24 h. Then,
an intruder was introduced into the home cage of a resident mouse, and behaviors were
video recorded for 10 min. Social behaviors (time spent in contact) were manually
measured.
Three-chamber social interaction test. The three-chamber social interaction test
apparatus (O’Hara) consisted of a box divided with clear partitions into 3 chambers (20
× 40 × 22 cm). The partitions have small square openings (5 × 3 × 3 cm) that allow
mice to freely explore all of the chambers. An unfamiliar C57BL/6J male mouse
(stranger) was introduced into a small, round wire cage that was placed in one of the
side chambers, and an empty wire cage was placed in the other side chamber. The side
chamber containing the stranger was systematically alternated between trials. Test mice
were first placed in the middle chamber and allowed to explore all of the chambers for a
10 min session. Time spent in the quadrant around the wire cage was automatically
analyzed using Image CSI software (O’Hara).
Experiment
4:
Effects
of
GLT1
deletion
on
seizure
susceptibility
and
electroencephalogram.
Susceptibility to kainate-induced seizures. Seizure severity was graded as follows: 0, no
abnormality; 1, immobility, cessation of normal behavior; 2, rigid posture with extended
tail or forelimbs; 3, repetitive behaviors, including head nodding, head bobbing,
twitching or scratching; 4, forelimb clonus with partial or intermittent rearing; 5,
continuous forelimb clonus/rearing or repeated rearing and falling; 6, loss of posture,
generalized tonic-clonic whole body convulsions or hyperactivity/jumping behavior;
and 7, mortality.
Experiment 5: Effects of GLT1 deletion on corticostriatal synaptic transmission.
c-Fos mappings. Cryosections (20 μ m) were prepared as described above and
mounted on MAS-coated glass slides (Matsunami, Osaka, Japan). The sections were
5
fixed with 4% PFA, treated with proteinase K (Roche Diagnostics, Basel, Switzerland),
fixed again with 4% PFA and hybridized with digoxigenin (DIG)-labeled riboprobes
(Roche Diagnostics) against mouse c-Fos cDNA (NM_010234; base pairs 800-1296) at
70 ℃ overnight. Then, the sections were washed, blocked with lamb serum and
incubated with an alkaline phosphatase–conjugated anti-DIG antibody (Roche
Diagnostics). Color was developed with the chromagens nitroblue tetrazolium and
5-bromo-4-chloro-3’-indolylphosphate (Nacalai Tesque, Kyoto, Japan). Images were
acquired under a constant exposure condition using a BIOREVO BZ-9000 microscope
from the striatum (from Bregma; AP +0.98 to -0.82 mm, 5-6 sections per each mouse),
medial prefrontal cortex (AP +1.54 to +1.18 mm, 1-2 sections) and thalamus (AP -0.70
to -1.58 mm, 1-3 sections). The number of c-Fos positive cells for each brain region,
indicated in the figure, was automatically quantified using ImageJ software (NIH) under
a constant threshold level for all sections of the same staining.
Western blotting of synaptic molecules. Striata were homogenized in 10 volumes of
buffered sucrose [0.32 M sucrose, 4 mM HEPES/NaOH (pH 7.4), 1 mM EDTA and
protease and phosphatase inhibitor cocktails (Roche)] and then centrifuged at 800 g for
15 min at 4 °C. The supernatants were collected and centrifuged at 9,000 g for 15 min,
and pellets were collected as crude synaptoneurosomal (P2) fractions. The P2 fractions
were resuspended in lysis buffer and subjected to western blot analysis as described
above. The following antibodies were used: monoclonal anti-NR1 (1:250, BD
Biosciences, San Jose, CA, USA, 556308), polyclonal anti-NR2A (1:200, Covance,
Princeton, NJ, USA, PRB-513P), polyclonal anti-NR2B (1:500, a gift from M.
Watanabe, Hokkaido University), polyclonal anti-GluR1 (1:500, a gift from M.
Watanabe, Hokkaido University), monoclonal anti-GluR2 (1:1,000, Millipore,
MAB397), polyclonal anti-PSD95 (1:1,000, Abcam, Cambridge, MA, USA, 18258) and
monoclonal anti-synaptophysin (1:1,000, Millipore, MAB5258). For synaptophysin, the
membrane was stripped with stripping buffer (0.2 M glycine, 0.1% SDS and 1% Tween
20, pH 2.2) twice for 5 min and washed with PBS twice for 10 min and TBS-T twice for
5 min. Western blot analyses were performed as described above.
Electrophysiology. Mice were decapitated under anesthesia with 100% CO2, and the
brains were cooled in ice-cold, modified external solution (120 mM choline-Cl, 2 mM
KCl, 8 mM MgCl2, 28 mM NaHCO3, 1.25 mM NaHPO4 and 20 mM glucose bubbling
6
with 95% O2 and 5% CO2). Slices were cut using a Leica VT1200 slicer. For recovery,
slices were incubated for at least 1 hour in the normal bath solution (125 mM NaCl, 2.5
mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3 and 20
mM glucose [pH 7.4] bubbling with 95% O2 and 5% CO2). Medium spiny neurons were
identified visually through their medium-sized, spherical somata and their
electrophysiological properties. Resistance of the patch pipette was 2–3 MΩ when filled
with the intracellular solution (140 mM CsCl, 10 mM HEPES, 10 mM BAPTA-K4, 4.6
mM MgCl2, 4 mM Na2-ATP, 0.4 mM Na2-GTP [pH 7.3], adjusted with CsOH). The
pipette access resistance was compensated by 80%. The PULSE software (HEKA
Electronik) was used for stimulation and data acquisition. The signals were filtered at 3
kHz and digitized at 20 kHz. Stimulus pulses (duration: 0.1 ms; intensity: 0-80 V) were
applied between the pipettes to evoke EPSCs in medium spiny neurons.
Microdialysis. A straight cellulose dialysis probe (1.0 mm in length, 350 μm outer
diameter, 50,000 Da cutoff, A-I-4-01, Eicom, Kyoto, Japan) was slowly inserted into
the left striatum (stereotaxic coordinates in mm: AP 0.0-0.5, ML 2.0-2.5, DV 3.0) of
urethane-anesthetized (dosage: 1.65 g/kg) adult Ctrl and iKO mice (n = 6 and 5,
respectively). The probe was equilibrated for at least 120 min while perfusing with 2 μ
l/min HEPES-ACSF into the striatum. Dialysate samples were collected every 10 min,
including the recovery period for equilibration. Each dialysate sample was frozen at -30
°C when it was collected and stored at -80 °C at the end of the experiment. The
location of the dialysis probe was verified by observing Nissl-stained 60 μm-thick serial
coronal brain sections as described above. OPA (o-phthalaldehyde) HPLC
(high-performance liquid chromatography) analysis was performed using a GL-7453A
(GL Science, Tokyo, Japan) for glutamate detection. The data were acquired using
Power Chrom software (Eicom). Spectral peak quantification and analysis were
performed using custom Matlab (Mathworks, Natick, MA, USA) programs. Probe
stabilization was first checked by analyzing the 120 min recovery period. Once the
equilibration was confirmed, the subsequent dialysate sample was collected to
determine the basal concentration of glutamate. In vitro recovery of the dialysis probes
was determined by placing the probes in HEPES-ACSF that contained a known
concentration of glutamate at a flow rate of 2 μl/min. Consecutive 10 min samples were
collected, yielding a recovery rate of 2.4 % for glutamate.
7
Figure S1 A schematic diagram showing the generation of GLASTCreERT2/+/GLT1flox/flox,
Ctrl, and iKO mice.
8
a
Tmx or Oil
0
1
Analysis
2
3
4
Age (weeks)
Ctrl
b
Tmx or Oil
iKO
Analysis
12 13 14 15 16 17 18 19 20
Ctrl
Age (weeks)
iKO
Figure S2 The extent of GLT1 deletion after injection of tamoxifen in neonatal and
adult GLASTCreERT2/+/GLT1flox/flox mice. (a) GLASTCreERT2/+/GLT1flox/flox mice at P1
were injected with tamoxifen or corn oil for 1 day. Immunoreactivity for GLT1 was
almost completely eliminated in the brains of the neonatal-iKO mice. (b)
GLASTCreERT2/+/GLT1flox/flox mice at 12 weeks old were injected with tamoxifen or corn
oil for 5 days. Immunoreactivity for GLT1 was mildly reduced in the brains of the
adult-iKO mice. The scale bars represent 1 mm.
9
a
GFAP
b
TdTomato
c
Merge
d
S100β
e
TdTomato
f
Merge
g
NeuN
h
TdTomato
i
Merge
Figure S3 The expression pattern of Cre recombinase in GLASTCreERT2 mice treated
with tamoxifen from P19 to P23. Astrocytes [labeled with GFAP (a, c) or S100β (d, f),
green] but not neurons [labeled with NeuN (g, i), green] were targeted for
recombination as demonstrated by the Cre-sensitive expression of the reporter signal
tdTomato (red, b, c, e, f, h, i) throughout the brain. Photos were taken of the
hippocampus (a-c) and prefrontal cortex (d-i). The scale bars represent 20 μm.
10
Figure S4 The weights of brain rostral to the medulla were measured in Ctrl (n = 6) and
iKO (n = 8) mice (8-9 weeks old female). Student’s t-test was used to compare wet
brain weight between Ctrl and iKO mice.
11
Figure S5 Neuronal quantification. (a-h) NeuN positive cells were quantified for Ctrl
and iKO mice (3-4 sections per mice, n = 3). Layer 2/3 in somatosensory cortex (a, b),
thalamus (c, d), dorsal striatum (e, f), and CA1 in hippocampus (g, h) are shown.
Student’s t-tests were used to compare number of NeuN positive cells per square
millimeters between Ctrl and iKO mice. The scale bars represent 50 μm.
12
Figure S6 No gliosis and scarring in the iKO brain. GFAP (a, red) and IbaI (b, green)
immunohistochemistry of layer 2/3 in somatosensory cortex, thalamus, dorsal striatum,
and CA1 in hippocampus of Ctrl and iKO mice (3-4 sections per mice, n = 3) are
shown. Blue: DAPI. The scale bars represent 50 μm.
13
Figure S7 The ablation kinetics of GLT1 and the time-course of overgrooming in iKO
mice are shown. (a) Western blot analysis of GLT1 in the cerebral cortex, thalamus and
striatum of Ctrl and iKO mice at different time points (wpi, weeks post injection) after
the first tamoxifen injection (n = 3). Student’s t-tests were used to compare Ctrl and
iKO mice for each brain regions at each time point. A significant reduction in GLT1
expression was detected at 3 weeks after the first injection. Data for wpi 6 are the same
as in Figure 1b. (b) A significant increase in grooming in a new environment was
detected at 5 weeks after the first tamoxifen injection (n = 10 and 12 for Ctrl and iKO,
respectively). Statistical significance was calculated by two-way repeated measures
ANOVA with post hoc t-test. *P < 0.05, ***P < 0.005. All data are presented as the
mean ± SEM.
14
a
b
40
Tic-like movements
(Times/10min)
Grooming (s/10min)
300
250
200
150
100
50
0
30
20
10
0
WT
KO
WT
KO
Figure S8. EAAC1 knockout mice (C57BL/6 genetic background) did not show (a)
excessive grooming or (b) tic-like movements (8 months old, n = 10 for wild-type and
n= 13 for EAAC1 KO mice). Student’s t-tests were used to compare duration of
grooming and number of tic-like movements between WT and EAAC1 KO mice.
15
Figure S9. c-fos mapping in the medial prefrontal cortex (mPFC) and thalamus (n = 8
for Ctrl and 9 for iKO mice). c-Fos positive cells within each boxed region (red) were
counted. Statistical significance was calculated by Student’s t-test. Scale bars: 500 μm.
16
GLAST
EAAC1
β-actin
150
Normalized Protein (%)
Ctrl iKO
125
Ctrl
iKO
100
75
50
25
0
GLAST
EAAC1
Figure S10 Expression of GLAST and EAAC1 in the striatum of iKO and Ctrl mice.
Western blot analysis confirmed the absence of upregulation of the other non-targeted
glutamate transporters GLAST and EAAC1 in iKO mice. Student’s t-tests were used to
compare levels of GLAST and GLT1 proteins between Ctrl and iKO mice (n = 3).
17
1400
Glu Conc (nM)
1200
1000
800
600
400
200
0
Ctrl
iKO
Figure S11. Microdialysis in the striatum of Ctrl mice (n = 11, white bar) and iKO (n =
5, black bar). Extracellular glutamate levels were not altered in iKO mice. Student’s
t-tests were used to compare glutamate concentrations between Ctrl and iKO mice.
18
Video S1 Excessive grooming observed in iKO mice.
Video S2 Tic-like movements observed in iKO mice.
19
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behavior in a chromosome-engineered mouse model for human 15q11-13
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Regan MR, Huang YH, Kim YS, Dykes-Hoberg MI, Jin L, Watkins AM, et al (2007).
Variations in promoter activity reveal a differential expression and physiology of
glutamate transporters by glia in the developing and mature CNS. J Neurosci 27:
6607–6619.
Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, et al (1997).
Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter
GLT-1. Science 276: 1699–1702.
Watase K, Hashimoto K, Kano M, Yamada K, Watanabe M, Inoue Y, et al (1998).
Motor discoordination and increased susceptibility to cerebellar injury in GLAST
mutant mice. Eur J Neurosci 10: 976–988.
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