Supplementary Subjects and Method
Animals
Nat8l KO, wild type (WT) mice and C57BL6/J (Japan SLC Inc., Shizuoka, Japan) were housed
in plastic cages and were maintained in a regulated environment (25°C ± 1°C, 50% ± 5% humidity),
with a 12-h light/dark cycle (lights on at 8:00a.m, off at 8:00p.m). Food (CE2; Clea Japan Inc.) and
tap water were available ad libitum.
Constructs
For the glutathione S-transferase (GST) pull-down assay, a GST-His construct was made by the
insertion of a histidine repeat sequence, which was synthesized by annealing double-stranded
oligonucleotides, into the XhoI site of pGEX-4T-2 (GE Healthcare). Mouse Shati cDNA was
amplified by Advantage GC cDNA PCR kit (Clontech) from a full-length mouse cDNA library
constructed from mouse adult brain, and cloned between the BamHI and XhoI sites of the pGEX-His
vector (GST-SHATI-His). For immunoprecipitation and immunocytochemistry, Shati cDNA,
followed by an initiation codon and a FLAG sequence (FLAG-SHATI), was inserted into the NheI
and XhoI sites of pcDNA3.1 (+) (Life technologies).
GST pull-down assays and MASS spectrometry
GST-His and GST-SHATI-His constructs were introduced into E. coli. The E. coli were grown
to an OD600 of 1.0, and expression was induced by the addition of 0.1mM IPTG at 37°C for 3 h. The
-1-
cultures were centrifuged (3000 × g, 15min), and the pellet was placed on ice and re-suspended in
lysis buffer A (50mM phosphate, pH 7.4, 300mM NaCl, 0.5% Triton X-100) containing 1mM
phenylmethylsulfonyl fluoride (PMSF) and complete protease inhibitor cocktail, EDTA free
The resulting lysates were clarified by centrifugation (16000 × g, 15min). The supernatants were
incubated for 4 h at 4°C with TALON metal affinity resin (TAKARA) that had been washed three
times and re-suspended in lysis buffer. Then, the resin was washed three times, and lysis buffer B
(40mM Tris-HCl pH 7.4, 100mM NaCl, 0.5% Triton X-100, 2mM EDTA) containing 150mM
imidazole
was
added.
The
eluted
solution
was
incubated
overnight
at
4°C
with
glutathione-sepharose beads (Amersham Bioscience). The beads were then washed three times with
lysis buffer B, followed by elution with lysis buffer B containing 50mM reduced glutathione.
Further purification was performed using a centrifugal filter device (Amicon Ultra-50k, Millipore),
with degraded protein fragments of less than 50 kDa being removed.
Mice were killed by cervical dislocation, and the whole brain was removed, minced in ice-cold
PBS, and homogenized on ice in lysis buffer B using a potter homogenizer. Centrifugation and all
subsequent purification steps were carried out at 4°C. Cellular debris was removed from the
homogenate by ultracentrifugation (100 000 × g, 1 h). The resulting supernatant was pre-cleared by
incubation with glutathione-sepharose beads overnight at 4°C, and the cleared supernatant were
used for the pull-down assay. Beads that had been incubated with GST-His or GST-SHATI-His
were added to the supernatant, and the sample was incubated with rotation for 4 h at 4°C. The beads
were then washed with wash buffer (40mM Tris-HCl pH 7.4, 100mM NaCl, 2mM EDTA) and
-2-
eluted with elution buffer (40mM Tris-HCl pH 7.4, 500mM NaCl, 2mM EDTA). A 20%
trichloroacetic acid (TCA)/acetone solution was added to the eluate and chilled for 2 h at -20 °C,
and then centrifuged (12 000 × g, 10min). The pellet was washed twice with acetone, dried at room
temperature, and boiled in sodium dodecyl sulphate (SDS) sample buffer.
Proteins obtained from the GST pull-down assay were separated using a 12.5%
polyacrylamide gel and stained with silver nitrate. Protein bands were excised from the gel and
incubated with decolourizing solution (6mM K3Fe(CN)6, 1.5mM Na2SO3) for 15min at room
temperature. After the solution was removed, the gels were washed four times with water for 10
min. After the gel pieces were dehydrated for 5 min using acetonitrile, the solvent was removed,
and the gels were dried in a vacuum centrifuge. A volume of 10mM dithiothreitol (DTT) in 100mM
NH4HCO3 sufficient to cover the gel pieces was added, and the proteins were reduced for 30 min at
56°C. After the samples had cooled to room temperature, the DTT solution was replaced with
acetonitrile. After incubation for 5 min, the solvent was removed, the gels were dried in a vacuum
centrifuge and 100mM iodoacetamide in 100mM NH4HCO3 was added. After a 30-min incubation
at room temperature in the dark, the gel pieces were dehydrated twice by the addition of acetonitrile.
The liquid phase was removed, and the gel pieces were completely dried in a vacuum centrifuge.
The gel pieces were rehydrated in digestion buffer containing 100mM NH4HCO3 and 20ng/μL
trypsin (mass spectrometry grade; Promega) in an ice-cold bath. After 30 min, the gel pieces were
covered with trypsin solution and incubated at 37°C for 16 h. The supernatant was collected in a
low absorption tube. The gel pieces were added to 50μL of solution A (2% acetonitrile, 0.1 %
-3-
trifluoroacetic acid (TFA)) followed by mixing for 10 min, and further 33 μL of solution B was
added, followed by mixing for 20 min. Moreover, the gels were added 42 μL of solution B
following by mixing for 20 min, and then the supernatant was collected the tube and centrifuged
(13,000 g, 10 min, 4°C) to remove residual gels. The supernatant was transferred to a new tube and
evaporated by a vacuum centrifuge until the volume is about 5μL. The remaining solution was
collected for MASS analysis. A 10-μL volume of solution A was added to the empty tube and also
collected with adherent peptides to the tube wall. All mass spectra were obtained with an LC/MS
spectrometer. Searches were performed by Mascot (Matrix Science).
Immunoprecipitation and Immunocytochemistry
For immunoprecipitation, COS7 cells were transfected with the FLAG-SHATI construct and
cultured for 48 h. Then, the cells were scraped into RIPA buffer (50 mM HEPES pH 7.5, 1 % Triton
X-100, 0.1 % SDS, 150 mM NaCl, 1 % deoxycholate sodium, 1 mM DTT, 0.2 mM
phenylmethylsulfonyl fluoride and complete protease inhibitor cocktail [Roche]) and lysed on ice
with a 27-G needle. After centrifugation, the resulting supernatant was collected and incubated with
either anti-FLAG M2 (1:1000; Sigma), anti-AP2 (1:1000; Abcam), or anti-AP2 (1:1000; BD
Transduction Laboratories), together with Protein G beads (GE Healthcare) at 4 °C for 3 h,
followed by boiling in sample buffer for SDS-PAGE. In western blotting, anti-FLAG M2 (1:1000)
and anti-AP2 (1:1000) were used as primary antibodies.
For immunocytochemical analysis, the transfected COS7 cells were fixed with PBS
-4-
containing 4% (w/v) paraformaldehyde 48 h after transfection, and permeabilized with 0.3% (w/v)
Triton X-100 in 100 mM Tris-HCl (pH 7.6). The cells were incubated with a primary antibody,
such as anti-FLAG M2 (1:1000; Sigma), anti-AP2 (1:1000; Abcam) in PBS containing 2% BSA
overnight at 4°C, and were subsequently incubated with the Alexa 488- or Alexa 546-labeled
secondary antibody (Molecular Probes) in PBS containing 2% BSA, for 3 h at room temperature.
Cell images were acquired on a Zeiss LSM510 Meta laser scanning confocal microscope (Carl
Zeiss).
Locomotor activity test
Locomotor activity was measured using an infrared detector (Scanet SV-10; Melquest Co.
Ltd, Japan), with mice placed in a plastic box (45 × 26 × 40 cm high), and determined as described
previously (Niwa, 2007). For measurement of basal locomotor activity, mice were habituated for 1
h in the box, and after 24 h, locomotor activity was measured over a period of 1 h without any
treatment. For measurement of sensitivity to the D1 agonist, mice were habituated for 1 h in the box,
and 24 h later, locomotor activity was measured over a period of 1 h after treatment with SKF81927
at a dose of 0.5mg/kg, s.c. For measurement of methamphetamine (METH)-induced sensitization,
mice were habituated for 2 h in the box for 2 d and then administered METH (1mg/kg, s.c) or saline
(SAL) once a day for 5 d and then challenged with METH (0.3mg/kg, s.c) on day ten. Locomotor
activity was measured over a period of 2 h immediately after the METH or saline administration on
d 1, 3, 5 and 10.
-5-
Conditioned place preference test
Conditioned place preference test was carried out as described previously (Niwa, 2007). The
apparatus used for the place-conditioning task consisted of two compartments: a transparent
Plexiglas box and a black Plexiglas box (both 15 × 15 × 15 cm high). To enable mice to easily
distinguish the two compartments, the floors of the transparent and black boxes were covered with
white plastic mesh and black frosted Plexiglas, respectively. The two boxes could be divided with a
sliding door (10 × 15 cm high). For habituation, the sliding door was opened, and the mouse was
allowed to move freely between the boxes for 15 min once a day for 3 d (day 1 to 3). In the
preconditioning test on the third day, we measured the time that the mouse spent in the black and
transparent boxes using a Scanet SV-20LD (Melquest, Toyama, Japan). The box in which the
mouse spent the most time was referred to as the “preferred side” and the other box as the
“non-preferred side.” Conditioning was performed over six consecutive days (from day 4 to 9).
Mice were given METH or saline in the apparatus with the sliding door closed. That is, a mouse
was subcutaneously given METH and put in its non-preferred side for 20 min. On the next day, the
mouse was given saline and placed opposite the drug-conditioning site for 20 min. These treatments
were repeated for three cycles. In the post-conditioning test on the tenth day, the sliding door was
opened, and we measured the time that the mouse spent in the black and transparent boxes for 15
min, using the Scanet SV-20 LD. Place-conditioning behaviour was expressed as a CPP value,
which was calculated as: [(post-value) − (pre-value)], where post-value and pre-value represent the
-6-
differences calculated by the time spent in the saline conditioning site from that in the METH
conditioning site in the post-conditioning test on the tenth day and pre-conditioning tests on the
third day, respectively.
HPLC
The quantification of N-acetyl aspartate (NAA) in brain was carried out using HPLC after
pre-column derivatization with
4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED) as
described previously (Song, 2012; Fukushima, 2008; Arai, 2008).
The mice were administered METH (1 or 2 mg/kg, SC) for 6 days and decapitated without
anasthaesia 24 h after the final treatment, and the brains were removed and separated into the
required parts. All dissections were conducted according to the atlas of Franklin and Paxinos (1997).
For example, “frontal c
(anteroposterior [AP]: +1.98 to +1.10mm; mediolateral (ML); ±1.0mm from Bregma; dorsoventral
[DV]: from the dura to 2.5m
al
sections (AP: +1.10 to +0.38mm; ML: ±1.0mm to ±2.0mm; DV: -2.5mm to -3.5mm). The isolated
brain regions were immediately homogenized using an Astrason Ultrasonic Processor XL (Misonix)
with 10 vols of phosphate-buffered saline (PBS) under ice-cold conditions, and then centrifuged at
-7-
3000 × g for 10 min at 4 °C. The resulting supernatant was stored at −80 °C until use. To a 20μL
aliquot of each sample, 40μL CH3CN and 40μL CH3OH were added. The aliquot was then
centrifuged at 600 × g for 10 min at 4°C. A 20μL sample of this supernatant was then added to a
transparent tube. Subsequently, 10μL of 200µM (±)-methylsuccinic acid (MSA) in CH3CN was
added and mixed vigorously. After reacting at 60°C for 30 min, the reaction mixtures were diluted
10-fold using 0.1% HCOOH in H2O–CH3CN (50:50). Subsequently, a 50μL aliquot of each
solution was loaded onto a solid phase extraction (SPE) cartridge bearing a membrane coated with
sulfonated poly(styrene-divinylbenzene) polymer (SCX, MonoSpin®; GL Sciences, Tokyo, Japan)
and initialized with 50μL of 0.1 % HCO2H in H2O–CH3CN (50:50). Finally, 10μL of each solution
was injected into the HPLC system. A Shimadzu LC 2010C HPLC system (Shimadzu Corporation,
Kyoto, Japan) was used with a Hitachi L-7485 fluorescence detector (Hitachi Ltd, Tokyo, Japan)
and a Shimadzu C-R8A recorder (Shimadzu Corporation, Kyoto, Japan). A Cadenza CD-C18
separation column (250 × 4.6mm; i.d., 3µm; Imtakt Co. Ltd, Kyoto, Japan) was used along with a
TSK guard gel ODS-80Ts guard column (3.2 × 1.5mm; i.d., 5mm; Tosoh Co. Ltd). The mobile
phases A and B used in the study were 0.1% HCO2H in H2O and 0.1% HCO2H in CH3CN,
respectively. The mobile phase was eluted according to the following program: 0–25 min, B% = 34
(isocratic); 25–35 min, B% = 34–42 (linear gradient). The flow rate was maintained at a constant
0.8mL/min. The column temperature was set at 40°C, and the detection wavelength was set at
562nm, with excitation at 438nm, as reported previously (Fukushima, 2008; Arai, 2008). The
-8-
protein concentration was determined using the Bradford method, and the NAA concentration in the
mouse brain was expressed as nmol/mg protein.
Magnetic Resonance Imaging
Magnetic resonance imaging (MRI) was performed using a 1.5T GE Sigma EXCITE system. A
three-dimensional volumetric acquisition of a T1-weighted gradient echo sequence produced a
gapless series of 124 sagittal sections using a spoiled gradient recalled acquisition in the steady state
(SPGR) sequence (TE/TR, 4.2/12.6 ms; flip angle, 15°; acquisition matrix, 256 × 256; 1NEX, FOV,
24 × 24cm; slice thickness, 1.4mm). MR images were processed using optimized voxel-based
morphometry (VBM) in Statistical Parametric Mapping 5 (SPM5) running on MATLAB R2010b
(MathWorks,
Natick,
MA)
according
to
the
VBM5.1-Manual
(http://dbm.neuro.uni-jena.de/vbm/vbm5-for-spm5/manual/) and as previously described (Good,
2001; Ashburner, 2000). We screened all scans and found no gross abnormalities, such as infarcts,
haemorrhages or brain tumorus, in any of the subjects. Each image was visually examined to
eliminate images with motion or metal artifacts, and then the anterior commissure-posterior
commissure line was adjusted. The normalized segmented images were modulated by multiplication
with Jacobian determinants of the spatial normalization function to encode the deformation field for
each subject as tissue volume changes in normal space. Finally, images were smoothed with a
12-mm full-width, half-maximum isotropic Gaussian kernel.
-9-
Statistical
analyses
were
performed
with
SPM8
software
(http://www.fil.ion.ucl.ac.uk/spm/software/spm8/). We performed hypothesis-driven regions of
interest
(ROIs)
analysis
on
the
caudate
nucleus
defined
using
PickAtlas
(http://fmri.wfubme.edu/software/PickAtlas) to explore the effects of the SHATI genotype on
caudate nucleus grey matter volume in healthy subjects. The effect of genotype on the brain region
was assessed statistically using a two-sample t-test model in SPM8. We contrasted caudate nucleus
volume between groups with differing genotype (individuals with C/C genotype versus A-carriers).
Age, sex and years of education were included as covariates of no interest into the analysis to
control for confounding variables. Non-sphericity was estimated. The analysis yielded statistical
parametric maps {SPM (t)} based on a voxel-level height threshold of p < 0.001 (uncorrected for
multiple comparisons). Clusters of more than 100 contiguous voxels were considered in the analysis.
Family-wise error (FWE) correction was applied to multiple testing to avoid type I errors. The
significance level was set at p<0.05 (FWE corrected) for caudate nucleus regions of interest defined
using PickAtlas. Anatomic localization was performed according to both MNI and Talairach
coordinates;
the
latter
were
obtained
from
M.
(http://www.mrccbu.cam.ac.uk/Imaging/Common/mnispace.shtml).
- 10 -
Brett’s
transformations
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