1. No category

Supporting Information
Sagi et al. 10.1073/pnas.1420162111
SI Materials and Methods
Materials. Most generic reagents were obtained from SigmaAldrich Chemical Co.; ketamine HCl was from Fort Dodge;
[32P]γATP 3000 Ci·mmol−1. For physiological experiments, stock
solutions of 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline
(NBQX; Tocris Bioscience) and (S)-4-carboxyphenylglycine
(Tocris) were dissolved in dimethyl sulfoxide (DMSO, <0.1%
final concentration) whereas L-NAME (Tocris) and APV (Tocris) were dissolved in water. CREB1 recombinant protein
(H1385P01) was from Abnova. Antibodies were as follows:
β-actin (4967, 1:1,000, Cell Signaling Technology); GFP
([Western blot (WB)] MAB2510, 1:1,000, Millipore; [immunohistochemistry (IHC)] 1218, 1:500, Abcam); sGCβ1 [(WB)
G4405, 1:5,000, Sigma; (IHC) 1:200]; VGAT [(WB) AC5062P,
1:500, Millipore; (IHC) 131003, 1:500, SYSY]; synaptophysin
(ab52636, 1:200000, Abcam); α-synuclein (ab1903, 1:5,000, Abcam);
DARPP-32 (1) [(WB, IHC), 1:2,000]; T34-DARPP-32 (2)
(1:5,000); p44/42 MAPK (4696, 1:2,000, Cell Signaling); p44/
42 ERK1/2 (T-202/Y-204) (9101, 1:1,000, Cell Signaling);
CREB (9104, 1:1,000, Cell Signaling); pCREB (S-133) [9198,
1:1,000 (WB), 2 μg (ChIP, blocking peptide 1090s, 10 μg), Cell
Signaling]; RNA Polymerase II N-20 [Sc899, 4 μg (ChIP,
blocking peptide Sc899p, 10 μg), Santa Cruz Biotechnologies].
Animals. In behavioral experiments, L-NAME, 10 mg/kg, was
injected intraperitoneally 10 min before the test. For lesion experiments, 26–28 gram C57/Bl6 mice (WT or Drd1-EGFP) were
anesthetized with ketamine/xylazine. For lesion experiments, 26–
28 gram C57/Bl6 mice (WT or Drd1-EGFP) were anesthetized
with ketamine/xylazine. 6-Hydroxydopamine HCl (1 μL/ 3.6 μg)
was dissolved in 0.9% sterile NaCl with 0.02% ascorbic acid
immediately before it was stereotaxically injected at a constant rate
(100 nL·min−1) using a calibrated glass micropipette (Drummond
Scientific), to the right medial forebrain bundle (MFB) (coordinates
were −1.2 mm, −0.7 mm, −5.00 mm lateral, anterior, and ventral
from bregma). Control animals were injected with the vehicle.
For all biochemical studies including Western blot, transporter
activities, and binding, mice were killed by cervical dislocation,
and striata were quickly removed. For RNA isolation studies and
for synaptic preparation studies, brains were dissected immediately without prior freezing. For immunohistochemical studies,
mice were perfused with PBS followed by 4% paraformaldehyde.
For chromatin i.p., mice were perfused under 3% isoflurane
with 20 mL of cold PBS containing heparin (10 u·mL−1), sodium
phosphate (20 mM), sodium vanadate (1 mM), and glycerophosphate (5 mM), followed by 50 mL of 1.42% formaldehyde
and 20 mL of cold PBS containing 125 mM glycine. For electrophysiology studies, mice were deeply anesthetized with ketamine
and xylazine, transcardially perfused with oxygenated, ice-cold,
artificial cerebral spinal fluid (ACSF), and decapitated. For
analysis of cyclic nucleotide levels and protein phosphorylation
level, mice were killed by focused microwave irradiation. For
primary cortical culture, cortical cells from embryonic day 17 rat
embryos were isolated, triturated, and plated.
Characterization of sGCβ1 KD in Vitro. The sequence of the mouse
soluble guanylyl cyclase sGCβ1 gene (Gucy1b3) was confirmed
from IMAGE clone 6822142 (Invitrogen) by sequencing, and the
gene was subcloned into pEGFP-N3 (Clontech). An shRNA
construct against sGCβ1 (sequence ACCACAGATCCCCGCACTGAGATAGACATGAACTTCCTGTCATTCATGTCTATCTCAGTGCTTTTTTGGAATCTAGAACTATAGCTAGAGSagi et al. www.pnas.org/cgi/content/short/1420162111
CATGGCTACGTAGATAAGTAGCATGGCGGGTTA) was
inserted into pAAV.H1 as previously described (3). AAV2
particles were packaged with either pAAV.siGucy or control
plasmids (pAAV.siLuc), and viruses were concentrated to 5 ×
1011 viral particle number (VPN) using heparin columns (GE
Healthcare). To study the effect of AAV.siGucy on endogenous
sGCβ1 level and activity, 25 × 108 VPN were transduced into
2 × 106 cortical neurons on day 2 in vitro (DIV), after which
the medium was replaced every 3 d.
Translatome Profiling. For global transcript analysis, 15 ng of
RNA was amplified with a Two-Cycle Target Labeling kit and
hybridized to Mouse Genome 430 2.0 arrays, which were
washed and scanned according to the manufacturer’s instructions
(Affymetrix). CEL files of three biological replicates were imported into Genespring GX (Agilent Technologies) and processed
with the GC-RMA algorithm. Changes in gene expression were
determined using the Student t test followed by the false discovery
rate (FDR) with the Benjamini–Hochberg procedure to adjust for
multiple comparisons.
For quantitative PCR (qPCR) analysis, 15 ng of RNA was
reverse transcribed using a WT-Ovation kit (Nugen Technologies), and 20 ng of cDNA was used as a template for analysis,
using TaqMan Gene Expression Assays. Fluorescence was
detected using ABI 7900HT (Applied Biosystems).
Immunohistochemistry. Brains were postfixed for 1 h in 4% PFA
and cryopreserved in 30% sucrose overnight, after which they
were frozen and cut on a cryostat. Sections (30 μm) were washed
twice in PBS, blocked in 1% normal goat serum (Invitrogen),
and incubated overnight with primary antibodies. For immunofluorescent detection, Alexa secondary antibodies (Invitrogen)
and the nucleic acid dye, DRAQ5 (Alexis), were used according
to the manufacturers’ instructions. Fluorescent images were
detected using a confocal LSM 710 microscope (Carl Zeiss). For
positive cell number and immunolabeling quantification analyses, X20 air and ×40 oil (N.A. = 0.75) objectives were used,
respectively. Brain sections were scanned at 0.45-μm depth intervals (total depth of 10 μm). For labeling quantification of
VGAT and DARPP-32, serial sections throughout the dorsal
striatum and the globus pallidus and substantia nigra were used.
Mean pixel values from the grayscale images were determined
using ImageJ software (Universal Imaging), in fields of 1940 ±
84 μm2 (three to six fields per section, four to five striatal
midbrain/ globus pallidus sections per animal), followed by
subtraction of the mean pixel value of the nonspecific background from within the field. The ratio between VGAT and
DARPP mean pixel values was determined for each field.
To quantify the level of VGAT in dSPN and iSPN, Drd1-TRAP
mice were injected with either AAV.Gucy or control AAV.
Striatal sections were immunostained for GFP, nuclear marker,
and VGAT. Regions of interest were defined around somas
(30–40 somas per slice, five slices per brain), and mean pixel
value of VGAT immunolabeling was quantified (71 ± 18
GFP-positive cells from control; 66 ± 12 GFP-positive cells
from KD; 102 ± 27 GFP-negative cells from control; 115 ± 33
GFP-negative cells from KD). The value of somatic VGAT
was identified as either dSPN or iSPN according to the presence
of GFP labeling. Similar analysis was performed for VGAT using
antibodies for parvalbumin or neuropeptide Y.
1 of 11
Electrophysiology. Tissue processing, mIPSC recording protocols,
and data analysis were performed as described (4). The striatonigral afferents were stimulated by placing the poles of the
stimulating electrode in the striatum on either side of the injection site. Synaptic potentials were evoked by just suprathreshold pulses (1.5–2 × threshold) to minimize activation of
the neighboring globus pallidus. The paired pulse protocol
consisted of two stimulating pulses with an interstimulus interval of 100 ms (10 Hz). This protocol allowed for the initial
IPSC to recover to baseline before the second pulse. Clampfit
software, ver. 10.3.0.2 (Molecular Devices) was used to analyze
the paired-pulse ratio, amplitude of pulse 2/amplitude of pulse 1.
Pooled data are presented as means ± SE or cumulative probability plots using IGOR 5.00 (WaveMetrics). Whole-cell voltageclamp recordings were performed using standard techniques.
Individual slices were transferred to a submersion-style recording
chamber on an Olympus Optical BX50WI microscope and continuously superfused with an ACSF at a rate of 2–3 mL·min−1 at
22–23 °C. Whole-cell voltage-clamp recordings were performed
on either striatal medium spiny neurons or SNr neurons and were
detected in the slice with the help of an infrared-differential interference contrast video microscopy with a Photometrics coolSNAP HQ2 camera/controller system. The following were added
to the superfusion medium for all experiments to isolate mIPSCs:
50 μM 2-amino-5-phosphonopentanoic acid (AP-5; Tocris
Cookson) to block NMDA glutamate receptors, 5 μM 1,2,3,4tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide
(NBQX) to block AMPA/kainite glutamate receptors, and 1 μM
tetrodotoxin (TTX; Alomone Labs) to block sodium channels.
Patch electrodes were made by pulling Sutter BF150-86-10 glass
on a P-97 Flaming/Brown micropipette puller (Sutter Instrument
Co.) and fire polished before recording. Pipette resistance was
typically 2.5–4 MΩ after filling with an internal solution containing the following: 140 mM CsCl, 1.5 mM MgCl2, 10 mM
Hepes, 0.1 mM BAPTA-Cs, 5 mM QX-314, 2 mM ATP-Na2,
0.4 mM GTP-Na2, pH 7.25–7.3 adjusted with CsOH, 270–280
mOsm·L−1. IPSCs were recorded with a Multiclamp 700A
amplifier, a Digidata 1322A 16-bit data acquisition system, and
pClamp software, ver. 9.2 (Molecular Devices). For mIPSCs,
neurons were voltage-clamped at −80 mV and allowed to reach
a stable baseline (∼5 min) before mIPSCs were recorded for 7
min. Mini Analysis (Synaptosoft Inc.) was used to analyze
mIPSC amplitude, frequency, 10–90% rise time, and decay time.
A threshold of five times the root-mean-square baseline noise
level (commonly∼20–25 pA) was set for event detection. Records were then visually inspected, and all events triggered by
noise were discarded. Frequency and amplitude analysis was
carried out on all mIPSCs that met threshold criteria. Events
were then selected for 10–90% rise time and decay time analysis
on the following criteria: (i) Events with 10–90% rise times faster
than 1.5 ms were selected to minimize space clamp errors and
electrotonic filtering; (ii) Events with decay times faster than 45
ms were selected to minimize events in which multiple events
precluded accurate decay-time measurement. For stimulated
IPSCs, SNr neurons were voltage-clamped at −50 mV and allowed to stabilize for ∼5 min. Stimulation (200 μs) was performed using steel bipolar electrodes (Frederick Haer & Co.)
delivered by a constant current isolated stimulator model Ds3
(Digitimer Ltd.). The striatonigral afferents were stimulated by
placing the poles of the stimulating electrode in the striatum on
either side of the injection site. Synaptic potentials were evoked
by just suprathreshold pulses (1.5–2 × threshold) to minimize
activation of the neighboring globus pallidus. The paired-pulse
protocol consisted of two stimulating pulses with an interstimulus interval of 100 ms (10 Hz). This protocol allowed for
the initial IPSC to recover to baseline before the second pulse.
Clampfit software, ver. 10.3.0.2 (Molecular Devices) was used to
analyze the paired-pulse ratio, amplitude of pulse 2/amplitude of
Sagi et al. www.pnas.org/cgi/content/short/1420162111
pulse 1. Pooled data are presented as means ± SE or cumulative
probability plots using IGOR 5.00 (WaveMetrics).
Optogenetic Stimulation and Recording. Slices were generated from
animals that were genetically engineered to have the Cre protein
expressed under the A2a promoter. Floxed channelrhodopsin
(hChR) was introduced into these animals either through injection of a modified channelrhodopsin virus [AAV2/9.EF1a.
DIO.hCHR2(H134R)-EYFP.WPRE.hGH; Addgene 20298;
University of Pennsylvania Vector Core, Philadelphia) into the
dorsal lateral striatum or through genetic crossing into an Ai32
line that expresses floxed channelrhodopsin in all cells. Presynaptic GABA release was induced in hChR2-expressing cells
using a 200-μs duration pulse of 470 nm wavelength light delivered via an LED light source (CoolLED EE-100) through the
objective. The intensity of the LED used varied from cell to cell
and was determined by the value that yielded a middle range of
responses from an evoked I/O curve. After induction of deep
anesthesia (ketamine and xylazine injected intraperitoneally at
50 mg/kg and 4.5 mg/kg, respectively, with additional isoflurane
administration by inhalation), 2- to 4-mo-old A2a-Cre transgenic
mice (blk6) were perfused transcardially with 5–10 mL of icecold artificial CSF (aCSF) containing 124 mM NaCl, 3 mM KCl,
1 mM CaCl2, 1.5 mM MgCl2, 26 mM NaHCO3, 1 mM NaH2PO4,
and 16.66 mM glucose, continuously bubbled with carbogen
(95% O2 and 5% CO2). The animals were subsequently killed by
decapitation. The brain was removed from the skull and dissected on ice, and sagittal brain slices (300 μm) were prepared
from the isolated hemispheres using a Leica VT1200 vibrating
blade microtome (Leica Microsystems). The slices were then
transferred to a holding chamber where they were incubated in
aCSF containing 2 mM CaCl2 and 1 mM MgCl2, pH7.4, at 35 °C
for 60 min, after which they were stored at room temperature.
Slices were transferred to a submersion-style recording chamber
mounted on an Olympus BX51 upright, fixed-stage microscope
(Olympus Corporation) and were continuously perfused (2–3
mL/min) with carbogen-bubbled aCSF at room temperature,
22–24 °C. Neurons were visualized through infrared-differential
interference contrast video microscopy using a CoolSNAP HQ2
CCD camera (Photometrics) operated by in-house software.
Patch pipettes were pulled from thick-walled borosilicate glass
on a Sutter P-97 puller (Sutter Instrument Co.) to resistances of
4–6 MΩ when filled with recording solution. The internal recording solution contained 120 mM cesium chloride, 10 mM
tetraethylammonium chloride, 10 mM Hepes, 2 mM QX314
chloride (Tocris Biosciences), 0.2 mM EGTA, 3 mM MgATP,
and 0.3 mM NaGTP; pH was adjusted to 7.3 with CsOH and
osmolarity to 270–280 mOsM. Electrophysiological recordings
were obtained with a Multiclamp 700B amplifier (Molecular
Devices). The amplifier bridge circuit was adjusted to compensate for series resistance and was continuously monitored during
recording. Current signals at the headstage of the patch-clamp
amplifier were filtered at 2 kHz and digitized at 10 kHz using a
Digidata 1440A data acquisition board and pCLAMP10 software
(both from Molecular Devices). The amplitudes were obtained
from the peak current resulting either from a single pulse or from
the first pulse of a double-pulse stimulus protocol. The intertrial
interval in both cases was 30 s. The intensity of the LED used
varied from cell to cell and was determined by the value that
yielded a middle range of postsynaptic responses from an evoked
I/O curve. Off-line data analysis and figure preparation were
performed with Clampfit 10 (Molecular Devices) software, Prism
statistical software (GraphPad Software), and Adobe Photoshop
and Illustrator (Adobe Systems).
Western-Blot Analysis. Proteins were extracted from dissected
tissues using 1% SDS buffer. For analysis of nuclear and cytoplasmic fraction contents, the NE-PER extraction kit (Thermo
2 of 11
Scientific) was used according to the manufacturer’s instructions.
Protein concentrations were determined by the bicinchoninic
acid (BCA) method, and 10 μg of total protein from total lysates,
100 μg from cytoplasmic extracts, or 20 μg from nuclear extracts
were loaded and separated by SDS/PAGE and transferred to
nitrocellulose membranes, which were incubated with primary
antibodies followed by goat Alexa 680-linked IgG (Molecular
Probes) or goat IRDye800-linked IgG (LI-COR). Fluorescence
was detected using the Odyssey infrared imaging system (LICOR) and quantified using the Odyssey software (LI-COR).
Cyclic Nucleotide Analysis. Frozen striata were weighed and homogenized in 120 μL of 75% ethanol. Samples were dried and
lysed in 0.1 normal HCl. cGMP and cAMP levels were determined using an enzyme-linked immunoassay kit (Assay Designs) according to the manufacturer’s instructions.
Electromobility-Shift Assay. Probes for the CRE sequence in the
VGAT (5′ VGAT WT, CACTCAGATTCTGCGTCAGGGCCTCTT) gene were purchased from Invitrogen, labeled with [γ-32P]
ATP, filtered, and annealed. Labeled probe duplexes (17 nM)
were incubated with 20 ng of recombinant CREB1 in a buffer
containing 20 mM Tris (pH 7.9), 4 mM MgCl2, 5 mM DTT,
0.5 mg·mL−1 BSA, 13 ng·μL−1 poly dGdC, 0.5% PEG 8000,
and 12% glycerol for 40 min at 30 °C. Excess of the WT probes
(3.4 μM) or CRE-mutated probes (5′ VGAT dT: CACTCAGATTCTGTTTTTGGGCCTCTT) were added to some samples.
Samples were loaded on a 6% DNA Retardation gel (Invitrogen)
and separated by electrophoresis at 125 V in the presence of 0.5×
TBE for 40 min. Dried gels were exposed to Kodak Biomax MR
film (Eastman Kodak Company) overnight at −80 °C, and images
were developed and analyzed.
Chromatin Immunoprecipitation. Dissected brain areas from individual animals were lysed in cell lysis buffer containing 10 mM
Hepes, 5 mM MgCl2, 150 mM KCl, 0.5 mM DTT, 1× protease
inhibitor mixture (Roche), sodium phosphate (20 mM), sodium
vanadate (1 mM), and glycerophosphate (5 mM), and chromatin
was isolated using a SimpleCHIP Enzymatic Chromatin IP kit
(Cell Signaling Technology) according to the manufacturer’s
instructions with the following modifications: DNA was sheared
using sonication (9 cycles of 15× 1 s, power level 2 on Microson
XL sonicator; Misonix). Although chromatin input samples
1. Hemmings HC, Jr, Greengard P (1986) DARPP-32, a dopamine- and adenosine 3′:5′monophosphate-regulated phosphoprotein: Regional, tissue, and phylogenetic distribution. J Neurosci 6(5):1469–1481.
2. Valjent E, et al. (2005) Regulation of a protein phosphatase cascade allows convergent
dopamine and glutamate signals to activate ERK in the striatum. Proc Natl Acad Sci
USA 102(2):491–496.
Sagi et al. www.pnas.org/cgi/content/short/1420162111
contained 10 μL of sheared chromatin, each i.p. sample contained 150 μL of sheared chromatin, 30 μL of magnetic beads,
650 μL of ChIP dilution buffer containing 3% BSA, and antibody. Specific activities of the RNA polymerase II (4 μg per i.p.
sample) or S133 pCREB (2 μg per i.p. sample) antibodies, were
confirmed by comparing them to IgG (20 μg per i.p. sample),
and by using blocking peptides. DNA custom probes and
primer sets were designed using the Primer Express software
(Applied Biosystems). Primers for the proximal promoter region
of GAPDH and VGAT were F-GGGCCACGCTAATCTCATTTT, R-GTTCACACCGACCTTCACCAT, Probe-CTCCTGCAGCCTCGTCCCGTAGAC; and F-TTCCCTCAGCCTCCTCCAT, RCCTGACGCAGAATCTGAGTGTTA, Probe-CTCCCAGGCACCGGGCTTC, respectively. Primers and probe sets for these
genes were tested using sheared genomic DNA and showed
similar efficiencies. Gene levels from chromatin elutes were measured by qPCR using ABI 7900HT (Applied Biosystems).
Open Field. Each mouse underwent a 1-h trial, which was analyzed
in 5-min bins to quantify distance traveled and time in motion.
Data were collected automatically using Fusion 3.2 software
(AccuScan).
Three-Day Accelerating Rotarod. The mouse was placed on a cylinder that can rotate at constant or accelerating speeds (4–40
rpm) (Med-Associates). The latency to fall off the rod was measured for each of three trials per day. The average latency was
used as the primary variable.
Statistical Analysis. Changes in global gene expression were determined using Student t test followed by the false discovery rate
with the Benjamini–Hochberg procedure to adjust for multiple
comparisons. Differences in expression of a gene with a P value
lower than 0.05, FDR less than 0.1, and fold change over 1.5-fold
were considered significant. Cyclic nucleotide levels, protein
levels, and phosphorylation levels (determined by Western blot)
and bound protein levels after ChIP (qPCR) were analyzed using
a one-way ANOVA test followed by a post hoc Student t test.
mRNA levels (qPCR) were statistically confirmed using a onetailed Student t test whereas statistical analysis of the changes in
mean values of mIPSC and IPSC were analyzed using unpaired
Student’s t tests. Statistical analysis of the averages of immunolabeling ratios was performed using a two-tailed Mann–Whitney U test.
3. Musatov S, Chen W, Pfaff DW, Kaplitt MG, Ogawa S (2006) RNAi-mediated silencing of
estrogen receptor alpha in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors. Proc Natl Acad Sci USA 103(27):10456–10460.
4. Heiman M, et al. (2008) A translational profiling approach for the molecular characterization of CNS cell types. Cell 135(4):738–748.
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A.
GFP
DARPP-32
B.
250
N.S
200
GFP+ cells
Merge
AAV.siLuc
AAV.siGucy
150
100
50
0
DARPP-32 +
DARPP-32 -
Fig. S1. Mice were injected with either AAV.Luc or AAV.siGucy, and striatal sections were coimmunolabeled for GFP and DARPP-32. (A) Representative images
from AAV.siGucy-injected mice showing colocalization of GFP in cells expressing DARPP-32, indicating that the targeting area was the striatum. (B). After
transfection with either AAV.Luc or AAV.siGucy, 252 ± 15.5 and 242 ± 21.1 cells positive for GFP were inspected, respectively, for DARPP-32 labeling; 232 ± 25.3
and 220 ± 29.4 of the respective cells were also positive for DARPP-32 (n = 3 mice per group).
A
B
VGAT
Control
Striatum
sGCβ1 KD
Striatum
Merge
control
sGCβ1KD
9
VGAT level (A.U)
GFP (Drd1)
10
8
7
6
*
*
5
4
3
2
1
0
GFP +
GFP -
Fig. S2. VGAT immunolabeling in dSPN and iSPNs. (A) Representative images showing colocalization of VGAT and GFP from Drd1-BacTRAP mice transfected
with either AAV.Luc or AAV.siGucy. Mean pixel value of VGAT labeling was determined in somas from GFP-positive (indicated by open arrows; 131 ± 33 cells
from control, 157 ± 42 cells from KD) and GFP-negative cells (indicated by closed arrowheads; 210 ± 56 cells from control, 229 ± 38 cells from KD), from
Drd1-BacTRAP control (n = 5 mice) and sGCβ1 KD (n = 6 mice). (Scale bar: 50 μm.) (B) Analysis of VGAT intensity indicates that VGAT level in sGCβ1 KD mice is
reduced in both GFP positive (*P = 0.02 vs. control) and negative neurons (**P = 0.04 vs. control).
Sagi et al. www.pnas.org/cgi/content/short/1420162111
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A.
NPY
PV
B.
Merge
VGAT
Merge
VGAT
12
control
sGCβ1 KD
10
8
6
4
2
0
NPY
PV
Fig. S3. No effect of sGCβ1-KD on VGAT immunolabeling in striatal GABAergic interneurons. (A) Representative images from striatal sections of sGCβ1-KD
illustrating prominent VGAT immunoreactivity in either Neuropeptide-Y (NPY, Top)-expressing or parvalbumin (PV, Middle)-expressing neurons. (Scale bars:
Top, 50 μm, Bottom, 10 μm.) (B) Mean pixel values were determined for VGAT in somas of NPY-expressing neurons (18 ± 3.2, 23 ± 3.1 cells from control and KD,
respectively) or PV neurons (26 ± 4.7, 31 ± 6.2 cells from control and KD, respectively). Bars represent mean ratios of mean pixel values as percent of that in
control ± SD.
Sagi et al. www.pnas.org/cgi/content/short/1420162111
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A Control
B
sGCβ1 KD
Paired pulse ratio (P2/P1)
2.0
1.5
50 ms
200 pA
200 pA
1.0
0.5
0.0
Control sGCβ1 KD
50 ms
Fig. S4. Extrastriatal transmission is unchanged in sGCβ1 KD. (A) Representative traces of paired-pulse IPSCs in substantia nigra neurons from control (n = 12
mice) and sGCβ1 KD (n = 9 mice) elicited by striatal stimulation. (B) Bar graph summary of the data presented in (B). Bars represent means ± SEM.
Cytoplasm
PKG1
Striatum
Hippocampus
control sGC-KD control sGC-KD
PKA
Synaptophysin
Nucleus
PKG1
PKA
CREB
B
140
Protein level ( % of control )
A
120
Nucleus
Cytoplasm
100
80
*
*
60
40
20
0
PKA
PKG1
C
D
2401 attctgCGTC Agggcctctt tcacgaggat tcacccccat tctctcgtcc tgtggcttag
CRE-like cite
2461 gcacaagtct gtctccatct agcgccccct agcgatagtt ttgggctata ccccagggtc
5'- Start
2521 ccttttcaag aggatcagct gagctcctgg gtctggttgc ctctttgcac cacagggcat
2581 gttcgtgctg
3'-End
Exon 2
Lane
1
2
3
4
VGAT- CRE
CREB
WT X 200
+
-
+
+
-
+
+
+
-
+
+
+
dT X 200
Fig. S5. PKG1 and CREB involved in the transcriptional regulation of vgat. (A) Representative immunoblot images of proteins in the nuclear and cytoplasmic
fractions from brain lysates of control (Luc, n = 4 mice) and sGCβ1 KD (Gucy, n = 4 mice). (B) Quantification of the data presented in A, indicating that the levels
of PKG1 in both subcellular fractions are reduced in sGCβ1 KD mice (*P < 0.05). Bars represent averages of protein levels from striatal sGCβ1 KD normalized to
synaptophysin (cytoplasm) or CREB (nucleus) as percentage of control ± SD. (C) cAMP regulatory element in Slc32a1. Sequence analysis of mouse genomic
VGAT DNA (slc32a1, GenBank accession nos. NM_009508.2, NT_039207.7) identified a conserved cAMP response element (CRE)-like motif (in bold capitals) in
the intron of the gene [(−) strand]. Forward and reverse primers (in bold) were designed for qPCR analysis of the levels of genomic DNA bound to Ser 133
phospho-CREB or RNA polymerase II. Reverse primer overlaps the site of exon 2 initiation (right angle arrow). (D) CREB interacts with vgat. Electromobility shift
assay showing the migration of the vgat gene on a native gel. Radiolabeled probes of the CRE-like motif in the vgat gene (17 nM, lane 1) were incubated with
recombinant CREB1 (20 ng, lanes 2–4), in the presence of excess unlabeled WT (3.4 μM, lane 3) or CRE-mutated probes (3.4 μM, lane 4).
Sagi et al. www.pnas.org/cgi/content/short/1420162111
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A.
DARPP-32
VGAT
Merge
Cont.
Substantia
Nigra
Ipsi.
Cont.
Globus
Pallidus
Ipsi.
B.
125
VGAT/ DARPP-32
( % of contralateral)
100
75
50
25
0
Globus Pallidus
Substantia Nigra
Fig. S6. Effect of dopaminergic lesion on VGAT labeling in extrastriatal terminals of SPNs. (A) Representative images from a 6-OHDA–treated mouse show full
colocalization of VGAT in terminals of SPNs (identified by DARPP-32 staining) both in the substantia nigra (Top row) as well as in the globus pallidus (Third
row). No change in VGAT labeling was detected in either area after the lesion. (Scale bar: 20 μm.) (B) Mean pixel values were determined concomitantly for
DARPP-32 and VGAT. Bars represent mean ratios of mean pixel values as a percentage of the contralateral ± SD.
Sagi et al. www.pnas.org/cgi/content/short/1420162111
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Table S1. Effect of sGCβ1 KD on mRNA expression of synaptic vesicle-related genes in Drd1expressing SPNs
Gene level, A.U.
Protein\mice
SNARES
VAMP1
VAMP2
VAMP3 cellubrevin
VAMP4
VAMP7
Sec22-like1
SNAP-23
SNAP-25
SNAP-29
SNAP-47
Syntaxin 1A
Syntaxin 1B2
Syntaxin 2
Syntaxin 3
Syntaxin 6
Syntaxin 7
Syntaxin 13
Syntaxin 16b
vti1a
Tomosyn (b, m)
Amisyn
Small GTPases and related proteins
Rab1
Rab2
Rab2b
Rab3A
Rab3B
Rab3C
Rab4A
Rab5C
Rab6b
Rab7
Rab8A
Rab8B
Rab9B
Rab10
Rab11B
Rab12
Rab14
Rab15
Rab18
Rab21
Rab24
Rab25
Rab26
Rab27B
Rab30
Rab31
Transporter and channel proteins
VGLUT1
VGLUT2
VGLUT3
VGAT
VAChT
VMAT2
V-ATPase E2 subunit
ATPase, H+ transporting, V1 subunit A1
ATPase, aminophospholipid transporter
(APLT), class I, type 8A, member 1
Sagi et al. www.pnas.org/cgi/content/short/1420162111
Control
sGCβ1 KD
1,153
19,923
1,026
269
480
6,395
65
3,023
281
—
3,956
21
—
293
13,165
5,614
—
—
296
627
42
843
20,515
1,290
533
1,022
6,222
187
3,417
331
—
4,951
29
—
107
13,734
8,270
—
—
344
864
51
16,892
9,759
13,734
13,459
3,022
888
836
280
17,336
13,878
998
1,644
574
8,120
10,787
1,701
27,255
14,909
3,317
1,947
6,579
1,474
708
236
701
1,537
19,029
13,654
16,092
12,498
3,052
886
860
217
18,065
16,454
938
2,474
956
8,028
10,101
2,145
31,424
13,180
4,063
2,474
6,989
1,596
794
283
1,041
1,771
408
180
—
10,070
16
8
57
7,563
3,564
591
185
—
6,226
4
9
79
9,303
4,949
8 of 11
Table S1. Cont.
Gene level, A.U.
Protein\mice
Proline transporter
GLT1 solute carrier family 1 (glial high-affinity
glutamate transporter), member 2
NTT4
VAT-1 homolog
Na/K-ATPase
F1-ATPase a1
F1-ATPase b1
Cytoskeletal proteins
Tubulin a4
Actin beta cytoplasmatic
Actin gama cytoplasmatic
Actin alpha smooth muscle
ARP3
Dynein Cytoplasmatic, light chain1
Internexin-alpha
Kinesin family 3C
Kinesin family 21A
Kinesin family 2C
Kinesin family 5C
Kinesin family 5B
Kinesin family 2A
Kinesin family 3A
Kinesin family 1B
Kinesin family 17
Kinesin family 3B
Kinesin family 13A
Septin 5
Septin 3
Septin 7
Septin 4
Myosin Va
Myosin VIIb
Cell-surface proteins
Basigin
Cadherin 13
Contactin 1
Contactin-associated protein 1
Brain link protein 2 hyaluronan and
proteoglycan link protein 4
LSAMP
N-CAM
Rab33
Rab35
Arl10
RalA
Rabphilin3A
c-K Ras2
Di-Ras2
Arfaptin
Arhgap1
Rac1B
RhoB
Other trafficking and SV proteins
Synapsin1
Synapsin2
Synapsin3
Synaptophysin
Synaptogyrin 1
Synaptogyrin 2
Synaptogyrin 3
Sagi et al. www.pnas.org/cgi/content/short/1420162111
Control
sGCβ1 KD
—
64
—
72
2,679
396
40,749
36,539
37,173
3,190
396
37,947
40,734
38,627
42,096
46,048
40,703
596
14,328
44,737
4,131
3,917
3,411
3,115
2,636
1,671
1,298
975
907
345
306
277
22,879
8,062
6,410
2,986
7,127
80
42,295
46,190
40,703
336
17,537
43,585
3,981
6,762
5,784
1,940
2,644
2,508
2,166
972
693
612
657
404
17,461
8,660
10,830
3,320
8,923
52
1,111
1,596
9,508
12
333
706
1,472
7,154
12
321
—
—
3,022
1,682
6,578
1,962
3,341
2,782
14,940
1,360
35
51
237
—
—
3,052
1,930
7,083
2,393
3,415
2,552
16,912
1,976
38
42
255
2,363
12,230
47
18,718
215
130
3,650
2,472
18,878
34
18,010
192
103
3,061
9 of 11
Table S1. Cont.
Gene level, A.U.
Protein\mice
Control
sGCβ1 KD
Synaptogyrin 4
Scamp1
Synaptotagmin XI
Synaptotagmin I
Synaptotagmin VII
Synaptotagmin XVI
Synaptotagmin IV
Synaptotagmin V
Synaptotagmin II
Synaptotagmin XII
MUNC-18
VPS33a
VPS45
NSF
VAP-33
CSP
TRAPPC1
TRAPPC3
TRAPPC5
Bassoon
Doc2b
Lamp-1
Piccolo
Similar to SNAP25
b-SNAP
Reticulon 1
Reticulon 2
Reticulon 3
Reticulon 4
Snap25bp
Pantophysin
OBCAM
Neuronal growth
Neurotrimin
Stromal cell-derived factor 2-like 1
Thymus cell antigen 1, theta
Ig superfamily, member 4B
Intercellular adhesion molecule 4
Myelin basic protein
Myelin-associated glycoprotein
Myelin proteolipid
PLP
Signaling proteins
CaMK II a
CaMK II b
CaMK II g
CaMK II d
CaMK II 4
Casein kinase 1a1
Casein kinase 1d2
Casein kinase 1g3
Casein kinase 1g2
Casein kinase 1e
Casein kinase 2a
Casein kinase 2b
PKCa
PKCb1
PKCc
PKCd
PKCe
PKCz
4,574
12,658
14,605
9,261
2,885
2,610
1,623
1,490
868
736
337
1,517
747
19,395
9,314
8,619
1,840
2,948
822
1,087
—
7,177
935
43,127
8,546
17,837
1,331
22,574
44,016
1,558
—
546
4,940
1,524
496
18,014
1,913
4,075
31,075
801
17,919
28,644
4,220
14,385
19,904
10,799
2,532
3,589
1,505
1,150
907
363
242
1,592
1,011
24,283
11,490
9,401
1,551
2,861
997
1,077
—
5,950
1,103
52,455
10,089
17,507
1,160
22,857
44,016
1,151
—
797
6,511
1,612
480
12,953
1,596
3,072
33,496
489
17,969
23,096
3,741
18,243
1,371
331
6,739
12,703
12,135
4,027
2,543
2,433
1,956
1,451
13,257
27,224
257
236
4,149
10,964
2,664
23,675
2,046
729
9,330
18,205
12,995
5,653
2,208
2,729
2,236
1,760
13,904
33,181
349
238
4,275
10,783
Sagi et al. www.pnas.org/cgi/content/short/1420162111
10 of 11
Table S1. Cont.
Gene level, A.U.
Protein\mice
Control
sGCβ1 KD
ITPR1
PI4K2 a
PI3Kd
PI3Kp85a
PI3Kp110a
PLD3
S100A16
16,737
3,421
38
1,095
617
7,444
4,998
19,732
3,309
40
2,100
1,067
5,736
4,740
The gene list is composed of synaptic vesicle-related proteins (1). Averaged expression values from Affymetrix
Mouse Genome 430 2.0 arrays of control and sGCβ1-KD Drd1-TRAP mice (n = 3 samples per group; each RNA
sample was pooled from striata of six mice). Bold values indicate a statistically significant difference (Materials
and Methods) in expression level between conditions, showing that the expression level of 4.5% of the synaptic
vesicle-related genes is down-regulated in sGCβ1 KD mice. None of the other seven genes down-regulated
by sGCβ1 KD is functionally related to VGAT, suggesting that the transcriptional regulation of VGAT by NO
is specific.
1. Takamori S, et al. (2006) Molecular anatomy of a trafficking organelle. Cell 127(4):831–846.
Sagi et al. www.pnas.org/cgi/content/short/1420162111
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