supplemental information - The Huguenard Lab

Neuron, Volume 78
Supplemental Information
Endogenous Positive Allosteric
Modulation of GABAA Receptors
by Diazepam binding inhibitor
Catherine A. Christian, Anne G. Herbert, Rebecca L. Holt, Kathy Peng, Kyla D. Sherwood,
Susanne Pangratz-Fuehrer, Uwe Rudolph, and John R. Huguenard
Inventory of Supplemental Information
Figure S1. Effects of FLZ and the α3(H126R) Mutation on nRT sIPSC Duration Persist into
Adulthood, Related to Figure 1.
Figure S2. FLZ Preincubation Does Not Alter sIPSC Kinetics or Frequency in VB, Related to
Figure 2.
Figure S3. Map of AAV Genome Packaged Into AAV-DJ-CMV-DBI-T2A-hrGFP Virus,
Related to Figure 5.
Figure S4. Clonazepam Occludes nRT-Dependent Potentiation, and Combined GAT Blockade
and FLZ Eliminates all nRT-Dependent Potentiation of Sniffer Responses, Related to Figure 6.
Figure S5. α3(H126R) Mutants Exhibit More Severe Experimental Absence Seizures Than
Wild-Types, Related to Figure 7.
Table S1. Summary of nRT sIPSC parameter values (mean + SEM) for cells from WT and
α3(H126R) mice with and without FLZ treatment.
Table S2. Summary of nRT sIPSC parameter values (mean + SEM) for cells from WT and
nm1054 mice.
Experimental Procedures
Supplemental References
Christian et al.
Supplemental Figures and Legends
Figure S1. Effects of FLZ and the α3(H126R) Mutation on nRT sIPSC Duration Persist
into Adulthood, Related to Figure 1. (A) sIPSC half width in individual wild-type nRT cells
(n=9) in slices from P48-54 mice before (Con) and during FLZ treatment. Open boxes indicate
mean + SEM during control and FLZ conditions. (B) Mean + SEM for sIPSC half width in WT
(black bar, n=11 cells) and α3(H126R) mutant mice (blue bar, n=8 cells). *p<0.05 vs. WT;
***p<0.001 vs. Con.
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Figure S2. FLZ Preincubation Does Not Alter sIPSC Kinetics or Frequency in VB, Related
to Figure 2. (A-D) Probability distributions constructed from 100 randomly selected events per
cell comparing half width (A), 90% width (B), weighted decay time constant (τd,w; C), and
interevent interval (D) in VB cells under control (n=8, black lines) and FLZ-preincubated (n=4,
red lines) conditions.
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Christian et al.
Figure S3. Map of AAV Genome Packaged Into AAV-DJ-CMV-DBI-T2A-hrGFP Virus,
Related to Figure 5. Control AAV-DJ-CMV-hrGFP virus did not contain DBI and T2A
sequences. Courtesy of Michael Lochrie, Stanford Neuroscience Gene Vector and Virus Core.
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Christian et al.
Figure S4. Clonazepam Occludes nRT-Dependent Potentiation, and Combined GAT
Blockade and FLZ Eliminates all nRT-Dependent Potentiation of Sniffer Responses,
Related to Figure 6. (A) Responses averaged across all WT patches in the presence of CZP,
normalized to peak amplitude. (B) Mean + SEM for 90-10% decay time of responses (p>0.6)
obtained in patches placed in VB (n=6) or nRT (n=6) in the presence of 100 nM – 1 µM CZP.
(C) Responses averaged across all WT patches in the presence of GAT antagonists (4 µM NNC711 and 10 µM SNAP-5114), normalized to peak amplitude. (D) Responses averaged across all
WT patches in the presence of GAT antagonists and FLZ, normalized to peak amplitude. (E)
Mean + SEM for 90-10% decay time of responses obtained in the presence of GAT antagonists
alone (turquoise bars, VB n=7, nRT n=6; p<0.001) or GAT antagonists and FLZ (magenta bars,
VB n=6, nRT n=8; p>0.9). Patches placed in VB are not significantly different between the two
groups (p>0.1). hν symbol – 1 ms UV laser stimulus.
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Figure S5. α3(H126R) Mutants Exhibit More Severe Experimental Absence Seizures Than
Wild-Types, Related to Figure 7. (A) Continuous EEG recordings showing 4-6 Hz activity in
response to PTZ injection. (B) Mean + SEM for SWD rate following PTZ injection (Time 0) in
WT (n=7) and α3(H126R) mice (n=4). Gray bars indicate times at which seizure parameters
differed between mutants and controls (p<0.05).
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Supplemental Tables
Amplitude
(pA)
1090%
Rise
time
(ms)
Half
width
(ms)
90%
width
(ms)
τd,w
(ms)
sIPSC
Charge
(fC)
τfast
(ms)
τslow
(ms)
%
slow
Freq.
(Hz)
WT
control
-20.58 +
1.11
1.31 +
0.05
122.14
+ 6.24
255.61
+
16.63
105.74
+ 3.05
-2033 +
134
39.59
+
2.15
177.02
+
11.50
65.14
+
3.09
1.02
+
0.18
WT+FLZ
-25.64 +
2.84
** vs. WT
con
1.14 +
0.04
97.62
+ 6.48
***vs.
WT
con
229.93
+
16.81
97.91
+ 5.78
*vs.
WT
con
-2461 +
337
p=0.06 vs.
WT con
29.49
+
2.89
**vs.
WT
con
152.09
+ 9.95
*vs.
WT
con
64.26
+
3.64
1.15
+
0.32
α3(H126R)
control
-17.16 +
2.17
1.42 +
0.06
102.15
+ 4.72
* vs.
WT
con
186.21
+
11.34
** vs.
WT
con
93.70
+ 4.66
* vs.
WT
con
-1320 +
189
* vs. WT
con
31.93
+
2.40
* vs.
WT
con
144.40
+ 8.85
* vs.
WT
con
62.44
+
4.49
1.10
+
0.20
α3(H126R)
+FLZ
-18.69 +
1.94
p=0.069 vs.
α3(H126R)
con
1.35 +
0.06
107.24
+ 9.72
181.09
+
16.30
91.42
+ 7.10
-1548 +
169
p=0.06 vs.
α3(H126R)
con
41.38
+
4.19
158.70
+
19.82
49.94
+
4.22
1.48
+
0.36
Table S1. Summary of nRT sIPSC parameter values (mean + SEM) for cells from WT and
α3(H126R) mice with and without FLZ treatment. Values calculated from P4-14 WT cells
(n=23, 13 treated with FLZ) and α3(H126R) cells (n=20, 10 treated with FLZ). Comparisons
between WT and α3(H126R) control groups performed using independent two-tailed t-tests.
Effects of FLZ within each genotype assessed using paired t-tests. Note that 4 of 10 α3(H126R)
cells exhibited an increase in sIPSC amplitude in response to FLZ, indicating that the increase in
amplitude represents a non-specific effect, which is also reflected in the slight increase in charge
transfer with FLZ treatment in both genotypes. Con, control; τd,w, weighted decay time constant.
*p<0.05, **p<0.01, ***p<0.001. Related to Figures 1 and 2.
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Christian et al.
Amplitude
(pA)
1090%
Rise
time
(ms)
Half
width
(ms)
90%
width
(ms)
τd,w
(ms)
sIPSC
Charge
(fC)
τfast
(ms)
τslow
(ms)
%
slow
Freq.
(Hz)
WT
-11.78 +
0.27
1.53 +
0.04
71.19 +
3.09
98.27 +
4.23
55.48
+ 1.80
-644.06
+ 16.30
25.73
+ 1.64
95.44
+ 6.70
57.43
+
2.71
2.28 +
0.19
nm1054
-11.48 +
0.15
1.60 +
0.04
61.75 +
2.27
*
80.99 +
2.55
**
49.74
+ 1.85
*
-566.51
+ 16.64
**
18.48
+ 1.37
**
74.98
+ 5.28
*
64.77
+
3.72
3.43 +
0.28
**
Table S2. Summary of nRT sIPSC parameter values (mean + SEM) for cells from WT and
nm1054 mice. Values calculated from P22-29 WT cells (n=14) and nm1054 cells (n=13).
Comparisons between genotypes performed using independent two-tailed t-tests. Note that
events are shorter than those in Supplementary Table 1 due to older age of animals. τd,w,
weighted decay time constant. *p<0.05, **p <0.01 vs. WT. Related to Figure 5.
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Extended Experimental Procedures
Animals
All procedures were approved by the Administrative Panel on Laboratory Animal Care at
Stanford University. Mice were bred and housed on a 12:12 light-dark photoperiod with food
and water available ad libitum.
α3(H126R) mice:
Wild-type (WT) C57BL/6 mice (Charles River Laboratories, Hollister, CA) were
compared with α3(H126R) mice bred on a C57BL/6 background. Where indicated, some EEG
experiments were performed using α3(H126R) mice generated on the 129X1/SvJ background
(Löw et al., 2000) and compared to WT 129X1/SvJ mice (Jackson Laboratory, Bar Harbor, ME).
The α3 subunit gene is located on the X-chromosome; mutant animals were thus either
hemizygote male or homozygote female.
nm1054 mice:
Following an established breeding scheme (Ohgami et al., 2005), an initial cross of
nm1054 heterozygote (Het) males with WT 129S6/SvEvTac female mice (Taconic Farms,
Oxnard, CA) yielded Het male and female progeny, which were then bred to each other to
produce WT, Het, and homozygous mutant littermates. Here we refer to the homozygous mutant
mice as nm1054. No heterozygotes were used in this study. The 129S6/SvEvTac background
was chosen because this mutation has been shown to demonstrate a high degree of prenatal and
early postnatal lethality on the C57BL/6J background (Ohgami et al., 2005).
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EEG recordings
In this study we use “EEG” to refer to electrocorticography. For EEG apparatus
implantation, mice older than P30 of either sex were anesthetized with ketamine (80 mg/kg i.p.)
and xylazine (16 mg/kg i.p.). EEG recordings were obtained from either metal skull screws or
silver wires implanted above the left and right frontal and parietal cerebral cortices. Animals
were housed individually or in pairs following EEG apparatus implantation. At least 1 week
later, mice were placed in the recording area and EEG activity and simultaneous video were
recorded.
Experimental absence seizures were induced in WT, α3(H126R), and nm1054 mutant
mice via s.c. injection of pentylenetetrazol (PTZ, Tocris Bioscience, Ellisville, MO). EEG
activity and simultaneous video were recorded for up to 90 min post-PTZ injection. Recordings
in WT and α3(H126R) mutants on the 129X1/SvJ background strain, and in WT and nm1054
mice, used a 40 mg/kg dose of PTZ. In C57BL/6 mice, this dose was consistently observed to
generate myoclonic jerks that often progressed to brief episodes of tonic-clonic convulsions (n=3
out of 4 mice, compared to 1 out of 12 129X1/SvJ mice tested), indicating a narrower therapeutic
window for PTZ induction of SWDs in this strain. This is consistent with previous reports of
induction of partial and generalized clonic seizures at this PTZ dose in C57BL/6 mice and
related strains (Wong et al., 2003; Heurteaux et al., 2004; Hentschke et al., 2006; Cheung et al.,
2010). Furthermore, SWD activity was disrupted in all animals following these convulsions.
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Virus generation and injections
The AAV-DJ serotype was chosen for its high degree of infection efficacy (Grimm et al.,
2008; Xu et al., 2012). The AAV-DJ-CMV-DBI-T2A-hrGFP virus was generated in the Stanford
Neuroscience Gene Vector and Virus Core (supported in part by NINDS grant P30 NS06937501A1) by calcium-phosphate-mediated transfection of AAV-293 cells (Agilent Technologies,
Santa Clara, CA) and purified using an iodixanol step gradient (Hermens et al., 1999). For
transfection 27 µg of pAAV CMV DBI-T2A-hrGFP vector, 27 µg of adenovirus helper plasmid
(pHELPER, Agilent), and 27 µg AAV rep-cap helper plasmid (pRC-DJ, Mark Kay, Stanford)
were used per T-225 flask of cells transfected. After ultracentrifugation, the iodixanol was
diluted with TBS and the AAV was concentrated using 100 kDal molecular weight cutoff
ultrafiltration devices (Millipore, Billerica, MA). The genomic titer (6.1 x 1012 vector
genomes/ml) was determined by Q-PCR using primers that amplify and a probe that detects the
hGH polyA region. Control AAV-DJ-CMV-hrGFP virus was also obtained from the Virus Core.
The infectious titer was determined by infecting 3.0 x 105 HEK 293T cells on a 24-well plate in a
volume of 0.3 ml in the presence of 4 µM etoposide. GFP+ cells were counted by fluorescence
microscopy 2 days post-infection. Both viruses were injected at a concentration of 6.4 x 109
infectious units/ml.
Bilateral stereotaxic injections of either control or DBI-expressing AAVs were performed
under isoflurane anesthesia between P48-60. Injections were made using a 10-µl syringe, 34gauge needle, and a syringe pump (World Precision Instruments, Sarasota, FL) to control the
injected volume (1 µl) and flow rate (120 nl/min). To target nRT, the stereotaxic coordinates
were 1.25 mm posterior to Bregma, 1.9 mm lateral to the midline and 2.8 mm ventral to the dural
surface. For injections in nm1054 mice, which typically have smaller brains (Lee et al., 2008),
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Christian et al.
the Bregma-Lambda distance was divided by 4.21 mm, the published standard distance (Paxinos
and Franklin, 2001), and the coordinates were scaled accordingly for each mouse. Brain slices
were prepared for electrophysiology as described below at 2-3 weeks post-injection. Infected
cells expressing GFP were visualized using epifluorescence microscopy. No differences in
recording properties within a group were observed between GFP-positive and –negative cells.
Slices in which the injection targeting failed and fluorescence was only observed in VB were not
included for experimentation.
Brain slice preparation
Mice of either sex at ages P4-81 were anesthetized with i.p. pentobarbital sodium (55
mg/kg) and killed via decapitation and the brain was quickly removed and placed in ice-cold (~4
o
C) oxygenated (95% O2/5% CO2) sucrose slicing solution containing (in mM): 234 sucrose, 11
glucose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, and 0.5 CaCl2 (310 mOsm).
Horizontal thalamic slices (250 µm thickness) containing nRT and VB were prepared as
previously described (Huguenard and Prince, 1994) using a Leica VT1200 microtome (Leica
Microsystems, Bannockburn, IL). Slices were incubated and continuously oxygenated in warm
(~32oC) artificial cerebrospinal fluid (ACSF) containing (in mM): 10 glucose, 26 NaHCO3, 2.5
KCl, 1.25 NaHPO4, 1 MgSO4, 2 CaCl2, and 126 NaCl (298 mOsm) for 1 hour and then
transferred to room temperature (~21-23 oC) for at least 15 min prior to recording. In some cases
slices were preincubated by being placed in ACSF containing 100 nM clonazepam (CZP, Sigma,
St. Louis, MO), 1 µM flumazenil (Hunkeler et al., 1981) (FLZ; also known as Ro15-1788,
Sigma), the GAT-1 and GAT-3 antagonists 1,2,5,6-Tetrahydro-1-[2[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711, 4
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µM, Tocris Bioscience, Minneapolis, MN) and 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)3-piperidinecarboxylic acid (SNAP-5114, 10 µM, Tocris) and/or 1 µM finasteride (FIN, Sigma)
[45 min for FIN, a treatment duration demonstrated to remove endogenous neurosteroid effects
(Tokuda et al., 2010)] when transferred to room temperature and maintained for >30 min prior to
recording.
Patch-clamp electrophysiology
For recording, individual slices were placed in a recording chamber continuously
superfused at 2 ml/min with oxygenated ACSF at room temperature. Slices were stabilized in the
recording chamber >5 min before experimentation. Neurons in nRT or VB were visualized using
a Zeiss Axioskop fixed-stage upright microscope (Carl Zeiss Inc., Thornwood, NY). Patchclamp recordings were made using a MultiClamp 700A amplifier with Clampex 9.2 software,
and signals digitized using a Digidata 1322A (Molecular Devices, Sunnyvale, CA). Borosilicate
glass recording pipettes were prepared using a Model P-97 Flaming/Brown micropipette puller
(Sutter Instrument Co., Novato, CA) to 2-5 MΩ tip resistance when filled with intracellular
pipette solution. Access resistance (Rs), measured from the peak of the averaged current response
to 65 40-ms 5 mV depolarizing steps from a holding potential of -70 mV, was <20 MΩ in all
whole-cell recordings. For within-cell drug treatments, data were discarded if access resistance
increased >20% during recording. Appropriate vehicle controls (<0.2% DMSO) were included in
the control ACSF solution. Only one cell per slice was recorded for within-cell drug treatment
comparisons, no more than two cells from the same nucleus (nRT or VB) were recorded per
slice, and no more than six cells in the same group were recorded per mouse.
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IPSCs:
Patch pipettes were filled with a near-isotonic chloride solution containing (in mM): 135
CsCl, 10 HEPES, 10 EGTA, 2 MgCl2, 5 QX-314, and pH adjusted to 7.3 with CsOH (290
mOsm). Recordings were not corrected for an estimated -5 mV liquid junction potential. To
isolate GABAergic IPSCs, ionotropic glutamate receptors were blocked with either kynurenic
acid (1 mM, Ascent Scientific, Princeton, NJ) or a combination of D-(-)-2-amino-5phosphonovaleric acid (APV, 100 µM, Ascent) plus 6,7-dinitroquinoxaline-2,3-dione (DNQX,
20 µM, Ascent). Cells were recorded in voltage-clamp mode with membrane potential clamped
at -60 mV. For recordings of spontaneous IPSCs, signals were recorded in gap-free mode and
low-pass filtered at 2 kHz or 4 kHz in nRT and VB, respectively, with gain set at 20 mV/pA. For
evoked intra-nRT IPSCs, a bipolar tungsten stimulating electrode was placed in nRT ~100-200
µm from the recording electrode. Threshold intensity (10-40 V, 80-100 µs pulse width) was
defined as that which produced 50% successes and 50% failures, and evoked currents were
recorded at 1.5X threshold at 10 s stimulation intervals.
GABA uncaging:
Outside-out patches of membrane from nRT or VB cells were obtained by slowly pulling
away the recording pipette shortly after breaking into the whole-cell configuration. Stable
patches were maintained in voltage-clamp mode at a membrane holding potential of -30 mV. In
all cases the recording pipette was pulled completely out of the slice to ensure total patch
excision. Sniffer patches were placed ~25-50 µm into the slice, a depth at which cell bodies
could be visualized easily. CNB-caged GABA (Invitrogen, Carlsbad, CA) was added to a
recirculating 10-20 ml bath solution containing APV and DNQX. 0.1 mM caged GABA was
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Christian et al.
used for VB patch recordings, and 1 mM was used for nRT patch recordings, to account for the
lower GABA affinity of nRT patches (Schofield and Huguenard, 2007). An ultraviolet laser
beam (355 nm wavelength; DPSS Lasers, Santa Clara, CA) was directed into the epifluorescence
port of the microscope and through the back aperture of a 60X water immersion objective. The
tip of the recording pipette was positioned in the center of the laser spot to ensure maximal
photolysis of caged GABA focused near the membrane patch. 1 ms-duration laser pulses were
delivered at 10 s intervals. Recordings were low-pass filtered at 2 kHz with gain set at 20
mV/pA. For experiments in which patches pulled from VB were moved to nRT, final
localization of the patch electrode tip in nRT was confirmed by capturing a video frame from the
microscope using a 2.5X or 10X objective. Placement of patches in VB or nRT was alternated to
ensure that conditions of slice incubation time and health were equivalent across groups.
Histology and immunocytochemistry
For DBI staining in Figures 5A-B, mice were anesthetized with Beuthanasia-D
(110mg/kg) and perfused transcardially with saline followed by 4% paraformaldehyde (PFA,
Sigma) in 0.1 M phosphate buffer at pH 7.4. The brains were removed and post-fixed in 4% PFA
at 4oC overnight, then cryoprotected in 30% sucrose buffer and frozen on dry ice. Horizontal 50
µm slices were cut with a sliding microtome (Microm; HM 400). Free-floating sections were
incubated for 1 hr in 10% Normal Donkey Serum followed by incubation with primary
antibodies against DBI (rabbit polyclonal, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA) at
4oC for 48 hrs. Sections were then rinsed in PBS and incubated for 2 hrs with corresponding
fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA).
Sections were mounted on slides and coverslipped with Vectashield mounting media (Vector
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Christian et al.
Laboratories, Burlingame, CA). Z-stacks of images with an optical distance of 0.5 microns were
captured with a laser scanning confocal microscope (Zeiss LSM 510) using 40X and 63X oilimmersion objectives. Secondary antibodies tagged to Fluorescein 488 and Cy3 were excited
with 488 and 594 nm lasers and observed through 510-530 and 560-615 emission filters,
respectively. A pinhole of 1 airy unit and identical settings for the detector gain and amplifier
offset were used to capture all confocal images.
Virus-infected slices were fixed in 4% PFA overnight after recording, washed in PBS,
and resectioned to 50 µm thickness. Free-floating sections were then treated as described above
using primary antibodies for DBI. The hrGFP signal produced following viral infection was
strong enough to be imaged without the use of antibody amplification.
Data analysis and statistics
sIPSCs were analyzed using the custom software programs wDetecta and WinScanSelect
(J.R.H., http://huguenardlab.stanford.edu/apps/wdetecta). Event detection threshold was
confirmed for each cell and was typically set at 4-8 pA above baseline. sIPSCs were sorted into
type-1 (those that decayed completely to baseline before the initiation of a subsequent event),
type-2 (those whose decay was interrupted by a subsequent event), and type-3 (those whose
initiation occurred during the decay phase of a previous event). The weighted decay time
constant (τd,w) was calculated by dividing the total charge transfer (in fC) by the peak amplitude
(in pA). To calculate τfast and τslow for a given cell, averaged type-1 events were obtained for each
cell and fit with a double exponential function using Clampfit with DC offset set to 0. The decay
of the averaged IPSC was fit to the following equation: I=A1e−t/τ1+A2e−t/τ2, in which τ1
represented τfast. The percentage of the decay represented by the slow component (% slow) was
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Christian et al.
calculated by the function A1/(A1+A2). For nonstationary variance analysis (Sigworth, 1980; De
Koninck and Mody, 1994; Schofield and Huguenard, 2007), only type-1 events with rise times
<2 ms were used. A mean current response was obtained by averaging all such events recorded
in control conditions for each cell, and the mean current was normalized to the peak amplitude of
each individual event. The variance from the cell mean trace was calculated for each individual
sIPSC from the peak-to-baseline return portion of the event, and the mean variance and mean
amplitude for each cell was divided into 100 equal bins. These data were plotted and fit to the
parabolic function σ2-σ2Noise = iIm − I2m/N where σ2 is the variance, Im is the mean current, i is the
unitary current, N is the number of channels, and σ2Noise is the variance in current baseline noise
for the 30 ms preceding event onset. Evoked IPSC and uncaging recordings were analyzed using
Clampfit.
EEG recordings were analyzed using a continuous wavelet transform method in
MATLAB (MathWorks, Natick, MA) to isolate SWD events (Schofield et al., 2009). Detection
parameters were set to identify SWD events as periods in which the scale-averaged wavelet
power was above a 99% confidence level threshold. Wavelet power ranges for positive detection
were set in the 160 to 333 ms band (3 to 6.25 Hz) for screw recordings, or the 160 to 250 ms
band (4 to 6.25 Hz) for wire recordings. Events with duration less than 800 ms were rejected and
subsequent events separated by a gap less than 800 ms long were merged. Identification of
spontaneous SWD events was confirmed via manual analysis of the power spectrum of each
event in Clampfit 9.2 (Molecular Devices).
Data were transferred to Excel (Microsoft, Redmond, WA), Origin 7 (Microcal Software,
Northampton, MA), and SigmaStat (Aspire Software, Ashburn, VA) for statistical analysis.
Comparisons between groups were made using two-tailed independent t-tests, nonparametric
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Christian et al.
Mann-Whitney Rank Sum Tests, or one-way ANOVA with Tukey’s post hoc means comparison
tests. Within-cell comparisons were performed using two-tailed paired t-tests. Cumulative
probability distributions were constructed using up to 100 randomly selected sIPSCs (events) per
cell and compared using two-sample Kolmogorov-Smirnov (KS) goodness of fit tests. For
amplitude and kinetics analyses, only type-1 events were used; all three types of events were
used in analysis of frequency and interevent interval. Differences within each genotype for EEG
parameters across different time points after PTZ injection were assessed using one-way repeated
measures ANOVA. Data are presented as means + SEM. Statistical significance was set at
p<0.05 for means comparisons, and p<0.001 for KS tests.
Supplemental References
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Chang, N., Li, L., et al. (2010). Identification of BERP (brain-expressed RING finger protein) as
a p53 target gene that modulates seizure susceptibility through interacting with GABAA
receptors. Proc Natl Acad Sci USA 107, 11883–11888.
Grimm, D., Lee, J.S., Wang, L., Desai, T., Akache, B., Storm, T.A., and Kay, M.A. (2008). In
vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of
adeno-associated viruses. J Virol 82, 5887–5911.
Hentschke, M., Wiemann, M., Hentschke, S., Kurth, I., Hermans-Borgmeyer, I., Seidenbecher,
T., Jentsch, T.J., Gal, A., and Hübner, C.A. (2006). Mice with a targeted disruption of the Cl/HCO3- exchanger AE3 display a reduced seizure threshold. Mol Cell Biol 26, 182–191.
Hermens, W.T., Ter Brake, O., Dijkhuizen, P.A., Sonnemans, M.A., Grimm, D., Kleinschmidt,
J.A., and Verhaagen, J. (1999). Purification of recombinant adeno-associated virus by iodixanol
gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene
transfer in the nervous system. Hum Gene Ther 10, 1885–1891.
Heurteaux, C., Guy, N., Laigle, C., Blondeau, N., Duprat, F., Mazzuca, M., Lang-Lazdunski, L.,
Widmann, C., Zanzouri, M., Romey, G., et al. (2004). TREK-1, a K+ channel involved in
neuroprotection and general anesthesia. EMBO J 23, 2684–2695.
Lee, L., Campagna, D.R., Pinkus, J.L., Mulhern, H., Wyatt, T.A., Sisson, J.H., Pavlik, J.A.,
Pinkus, G.S., and Fleming, M.D. (2008). Primary ciliary diskinesia in mice lacking the novel
ciliary protein Pcdp1. Mol Cell Biol 28, 949–957.
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Ohgami, R.S., Campagna, D.R., Antiochos, B., Wood, E.B., Sharp, J.J., Barker, J.E., and
Fleming, M.D. (2005). nm1054: a spontaneous, recessive, hypochromic, microcytic anemia
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