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Cell, Volume 138
Supplemental Data
Synaptic PRG-1 Modulates
Excitatory Transmission
via Lipid Phosphate-Mediated Signaling Thorsten Trimbuch, Prateep Beed, Johannes Vogt, Sebastian Schuchmann, Nikolaus Maier, Michael Kintscher, Jörg Breustedt, Markus Schuelke, Nora Streu, Olga Kieselmann, Irene Brunk, Gregor Laube, Ulf Strauss, Arne Battefeld, Hagen Wende, Carmen Birchmeier, Stefan Wiese, Michael Sendtner, Hiroshi Kawabe, Mika Kishimoto-Suga, Nils Brose, Jan Baumgart, Beate Geist, Junken Aoki, Nic E. Savaskan, Anja U. Bräuer, Jerold Chun, Olaf Ninnemann, Dietmar Schmitz, and Robert Nitsch
2
A
B-/-+/-+/+ -/-+/-+/+
~4.1 kb
~6 kb~9.8 kb
~6.7 kb
5‘Probe 3‘Probe
-/-+/-+/+
305 bp
706bp
C
Targeting construct
5‘ arm (4.6kb) 3‘ arm (1.3kb)
Wild type locus
1 2 4 5 6 7
3
Not I BamH I BamH I Sal I
Targeted locus
3
1 2 73
Xba I Xba I~9.8kb
Xba I BamH I~6.7kb
5‘ProbeEcoR I EcoR I
EcoR I EcoR I
~6kb
~4.1kb
3‘Probe
7MC1-neoIRES LacZ3xstop MC1-neoIRES LacZ3xstop
MC1-neoIRES LacZ3xstop MC1-neoIRES LacZ3xstop
11 2
33 2
D-/-+/+
175
25
47.5
6283
kDa
Anti-PRG1
Anti-ß-Actin
E-/-+/+
Anti-PRG1
CA1CA1
Figure S1. Generation of constitutive PRG-1 KO mice.
(A) Diagram of the targeting strategy. Numbered exons of the Prg-1 gene are represented as grey
boxes and their relative positions are indicated. The black box represents the first 77 bp of exon 4 that
are present in the targeting vector. Red boxes indicate the relative position of the putative
phosphatase domains on exons 4 to 6. The 5’ (4.6 kb) and 3’ (1.3 kb) homology arms were inserted
via Not I / BamH I and BamH I / Sal I restriction digest from PCR products in the targeting construct.
For homologous recombination, an IRES-LacZ-MC1-neo cassette was inserted, leading to a
translational stop of PRG-1 in exon 4 due to the 3 stop codons in each frame, which preceded the
IRES sequence. Assessment of correct recombination was performed by southern blot analysis. The
corresponding restriction enzymes, fragment lengths and relative position of the external radioactive-
3
labeled probes (hatched boxes) are indicated. Relative position of oligonucleotides for genotyping of
tail tip DNA is shown as black arrows numbered from 1 to 3. 1: 5’-GGG CTG ACC GCT TCC TCG
TGC TTT AC-3’, 2: 5’- CGG GGA TGT GCA CCA ATT GGG AAG AG-3’, 3: 5’-CCG TGA TTG CTT
GTT CCT TCT AGT GTG-3’. (B) Southern Blot analysis from wildtype (WT) (+/+), heterozygous (+/-),
and homozygous mutant (-/-) mouse tail DNA hybridized with the 32P-labeled 5’ and 3’ probes. WT
and mutant-specific bands are indicated. (C) Genotyping analysis of WT, heterozygous and PRG-1
KO mice using primers, shown in (A), results in the amplification of a 706 bp fragment for the WT allele
and 305 bp fragment for the Prg-1 KO allele. (D) Western Blots of brain homogenate of WT and PRG-
1 KO mice using a PRG-1-specific antibody revealed absence of the PRG-1 protein in PRG-1-deficient
animals. ß-Actin antibody serves as loading control. (E) Immunohistochemistry of WT and PRG-1 KO
hippocampal CA1 pyramidal cells revealed absence of PRG-1 signal in PRG-1-deficient animals
(Scale bar 100 μm).
4
0
5
10
15
20
Bod
y w
eigh
t [g]
*** ***
******
***
P5-6
P9-11
P10-
14
P20-22
P30-3
5P4
2-56
P25-
27
A
n = 155, status at the third week of life
Het WT KO dead KO0
10
20
30
40
50
Perc
enta
geof
tota
l num
bero
f mic
eC
WTP40-60
KoP40-60
0
200
400
Bra
inw
eigh
t[m
g]
*B
WT-littersPRG-1-KO
1
34
2
6
78
5
9
Bod
y le
ngth
[cm
]
WT PRG-1-KO
P21
Figure S2. General observations concerning the PRG-1 KO mouse line.
(A) Body weight measurement of the offspring revealed that the PRG-1 KO mice had a significantly
lower body weight up to day P35 (ANOVA with post hoc Bonferroni for selected groups, p < 0.001 (WT
n = 43, KO n = 44)). The image shows a WT and a PRG-1 KO mouse at P21. (B) Brain weight of WT
and PRG-1 KO mice revealed a significantly lower brain weight in the PRG-1 KO mice (unpaired t test,
p < 0.05, WT n = 21, KO n = 22). The image illustrates the morphologic difference between the brains
from a WT and a PRG-1 KO mouse. (C) Mating of PRG-1-heterozygous mice results in genotypes,
which correspond to mendelian expectations. While all genotypes were viable at birth, approximately
50% of the PRG-1 KO mice died around the third week of life. Data are represented as mean +/- SEM.
5
P21 WT P21 PRG-1 -/-
Synaptoporin Synaptoporin
CA3CA3
CA3
CA1
CA3
CA1
VGlut1 punctae/50 µm
WT PRG1-KO0
25
50
75
100
VG
lut1
pun
ctae
/50
µm
150 µm
50 µm
50 µm
A
E
DC
B
F
P21 WT
P21 PRG-1 -/-
VGlut1 5 µm
VGlut1
H
G
I
Figure S3. No changes in synaptic innervation at P21.
Synaptoporin staining revealed similar mossy fiber (MF) innervation pattern of the CA3 pyramids in the
PRG-1 KO mice at P21 (A,B). Higher magnification of the synaptoporin expression in the
infrapyramidal MF (C,D) and in the terminal part of the suprapyramidal MF (E,F) at the CA3/CA1-
border displayed similar innervation pattern. VGlut1 staining reveals the density of the glutamatergic
innervation of the apical CA1 dendrites in the stratum radiatum in the WT and PRG-1 KO animals
(G,H). Note the negative contours of the unstained apical CA1-dendrites. (I) Quantitative analysis of
VGlut1-punctae revealed no significant changes (t-test) in the glutamatergic innervation (n = 5 animals
per group; 4 slices per animal). Data are represented as mean +/- SEM.
6
5‘ probe 3‘ probe 3‘ probe (dNeo)
~18.5kb
~11kb
~18.5kb
~9.4kb
~18.5kb
~7.4kb
+/+ +/+ +/fl +/fl +/fl(dNeo)
~9.4kb
A
B
C
+/fl
1kb
0.8kb
0.6kb
0.4kb
0.2kb
D
175
83
47.5
25
Anti-PRG-1+/+ +/fl fl/fl +/- -/-
Anti-ß-Actin
+/+ +/fl fl/fl +/-
kDa
Targetingconstruct
5‘ arm (10kb) 3‘ arm (3.5kb)
Wild typelocus
1 2 4 5 6
Not I
Targetedlocus
3
Spe I~18.5kb
5‘Probe 3‘Probe
2 4 5 6 73
Neo
Spe I Not I
Spe I
1 2 4 5 63
Spe I Spe ISpe I
Neo
~11kb ~9.4kb (~7.4kb after Neo deletion)
7
7
1 2 43
Figure S4. Generation of conditional PRG-1 KO mice.
(A) Diagram of targeting strategy. Numbered exons of the Prg-1 gene are represented as grey boxes
and their relative positions are indicated. The red boxes indicate the relative position of the putative
phosphatase domains on exons 4 to 6. In the targeting vector two green triangles flanking exons 4 to 6
represent LoxP sites, while the yellow triangles flanking the neomycin selection cassette (Neo)
represent frt sites. Correct homologous recombination and deletion of Neo by FLP recombinase
expression within the ES cells was detected by southern blot analysis utilizing Spe I as restriction
enzyme and two external 32P-labeled DNA probes (hatched boxes). Relative locations of
oligonucleotides for genotyping by PCR analysis are indicated as black arrows numbered 1 to 4. 1: 5’-
CCC TGT TAA GAA TTG ACC-3’, 2: 5’- GAC ATG ACA CAA ATA TCA GGG-3’, 3: 5’-GTT ATG TTG
CAC TTA CAT ATC AGT GTA G-3’, 4: 5’-CAC ACA CAG TGC TTA CTT TAA GAC TG-3’. (B) Results
7
of the recombination events are shown. Corresponding fragments sizes of WT allele (+/+, 18.5 kb),
heterozygous floxed allele (+/fl, 11 kb 5’ probe, 9.4 kb 3’ probe) and heterozygous floxed allele after
Neo deletion (+/fl(ΔNeo), 7.4 kb 3’ probe) are indicated. (C) Genotyping by PCR analysis of mouse tail
DNA. By using the primer 1 and 2 (shown in (A)), PCR of WT DNA produced a DNA product of 698 bp
(Lane 2); heterozygous floxed DNA, two products (698 bp and 771 bp (Lane 3)); homozygous floxed
DNA, a PCR product of 771 bp (Lane 4). For functional tests we bred homozygous floxed mice with
Cre-deleter mice, expressing Cre recombinase within the germ line, which resulted in deletion of
exons 4 to 6. Lane 4 shows the PCR product of a heterozygous PRG-1 KO mouse using primers 3
and 4 (180bp). (D) Western blot of WT, heterozygous and homozygous floxed, as well as
heterozygous and homozygous PRG-1-deficient brain homogenates, using PRG-1 antibody and anti-
ß-Actin as loading control are shown.
8
Figure S5. Subcellular fractionation of mouse cerebral cortex from PRG-1 KO and WT litter
mice.
Western blot analysis was performed on subcellular fractionations of the PRG-1 KO and WT mouse
cerebral cortices using anti-PSD-95 and anti-RabGDI antibodies (#1, #2, #5, #6, and #7, Prg-1 KO; #3,
#4, #8, #9, and #10, WT) to control the PSD preparations. Note that PSD-95 is enriched in the PSD
fraction while RabGDI is abundant in the SC+CSV fraction, but not in the PSD fraction.
9
A
B
C
H253KGFP
YZ XY
XZ
XYYZ
XZ
PRG-1GFP
PRG-1 GFP merge
YZ
H253K GFP
YZ
merge
PRG-1H253K/Homer1
PRG-1/Homer1
Figure S6. 3D reconstruction of in utero electroporated Prg-1 constructs.
(A) Prg-1 was electroporated at E14 and analyzed at postnatal day (P) 21. Images of the PRG-1
staining (red) and the co-electroporated GFP signal (green) are shown in the xy-, xz- and yz-planes in
the section view overlay mode. Xz- and yz-planes are located as displayed by the white crosshair.
Note the membrane localization of the reconstituted PRG-1 in the yz-plane (arrows). PRG-1 staining in
the xz-plane at the intersection with the yz-plane, which is located at the dendritic membrane, confirms
membrane localization. Sections were scanned with a Leica confocal laser-scanning microscope
equipped with Argon and HeNe lasers and the excitation lines 488 nm and 543 nm. Imaging of serial
stacks was performed with a 63x oil objective, 4.8x zoom and a z-separation of 0.1 μm. 3D
reconstructions were performed with Volocity software (Improvision, Tubingen, Germany). Further
immune stainings showed colocalization of PRG-1 with the postsynaptic marker Homer1 (indicated by
the purple staining, arrows) confirming localization of the reconstituted PRG-1 at the postsynaptic
membrane. (B) Prg-1-H253K was electroporated and analyzed under the same conditions as
described above. PRG-1 staining revealed similar distribution and membrane localization (arrows) of
the mutated PRG-1 molecule. Immune staining confirmed colocalization of the H253K mutated PRG-1
with Homer1 pointing to localization of the mutated PRG-1 at the postsynaptic membrane (purple
staining, arrowheads). (C) Higher magnification of a cross section of a dendrite in the yz-plane
revealed similar distribution and membrane localization of PRG-1 (left), as well as of the mutated
PRG-1 (H253K) protein (right).
10
PRG-1-KO mEPSCs
50 pA0.5 s
mIPSCsWT
+ LPA
mIP
SC
freq
uenc
yra
tio
0
1
0-5-10 5 10
LPA
LPA
0
1
0-5-10 5 10
mE
PS
Cfr
eque
ncy
ratio
+ LPA
30 pA1 s
0
1
PRG-1-KO
mE
PSC
freq
uenc
yra
tio[L
PA
/ co
ntro
l]
**
0
1
WTmIP
SCfr
eque
ncy
ratio
[LP
A / c
ontr
ol]
A
B
Figure S7. Effect of exogenously applied LPA on mIPSCs in C57/B6 and on mEPSCs in PRG-1 -/- mice. (A) Application of 10 µM LPA did not alter the mIPSCs in C57/B6 (wild-type) hippocampal CA1
neurons. For the analysis the mIPSC frequency of each analyzed neuron was averaged for the last 4
minutes under control conditions and in steady state after LPA application, and these values then
compared. This revealed a mean ratio of 1.02 ± 0.04 (n = 7, p = 0.61, paired t-test). (B) In PRG-1 -/-
mice the mEPSC frequency increased after the application of 10 µM LPA. The analysis was performed
as in (A) and disclosed a mean ratio of 1.34 ± 0.08 (n = 7, p < 0.01). Note that this increase was
smaller compared to wild-type animals (p < 0.001, independent t-test). Data are represented as mean
+/- SEM.
11
LPA2-R WT 1:1000 LPA2-R KO 1:1000A
B
LPA2 Munc
VGlut-1 merge Figure S8. LPA2 receptor localization.
(A) Rat monoclonal antibody (Clone 10D5, against the c-terminus of LPA2 (Shida et al., 2004))
specifically recognizes LPA2 receptors. Immune staining using the 10D5 antibody in a dilution of
1:1000 displayed a clear and distinct signal in WT mice but no signal in LPA2-KO mice. Images were
obtained using a Leica TCS-SL (Leica Microsystems, Heidelberg, Germany) confocal scanning
microscope equipped with an Ar laser (488) using same settings (excitation laser intensity, detector
gain and offset) and oil immersion objective lens 63x NA 1.4. (zoom 5) (Scale bar 5 μm). (B) Triple
immunolabeling detected the LPA2–R in excitatory terminals in the hippocampus. The rat monoclonal
antibody (Clone 10D5, 1:1000) revealed colocalization with the presynaptic active zone marker
Munc13-1 in glutamatergic presynaptic terminals containing VGluT1 (as shown at higher magnification
in the insert) (Scale bar 1 μm).
12
Bod
y w
eigh
tat P
20-2
2 [g
]
0
2
4
6
8
10
******
WT PRG-1-KO PRG-1/LPA2-KO
Figure S9. Normal bodyweight in PRG-1/LPA2-R double-deficient animals at P20-22.
We observed in PRG-1/LPA2 -/- mice no signs of seizures or increased mortality at around P21. We
measured normal body weight in these animals, which were not significantly different to WT mice, but
differed significantly to PRG-1 -/- mice at this age. (ANOVA with post hoc Bonferroni for selected
groups, p < 0.001 (WT n = 26, PRG-1 -/- n = 27, PRG-1/LPA2 -/- n = 5). Data are represented as mean
+/- SEM.
13
LPA2
PRG-1
LPA2
GluGlu
GluGlu
GluGluGlu
Glu
Glu
synaptic cleft
PRG-1PSD
LP
LPLP LP
LP
LPLP
presynapticterminal
postsynapticglutamatergic
neuron
GliaLPGlia
L/LP
LP LP
LPA 2
G
G
LPLP
Figure S10. Proposed model of PRG-1 action at the excitatory synapse.
This study provides evidence for the modulatory action of the lipid phosphate phosphatases-related
protein PRG-1 in synaptic transmission of hippocampal glutamatergic neurons. PRG-1 is located at
the postsynaptic side at the PSD and controls bioactive lipid content in the synaptic cleft by uptake into
the intracellular compartment. Thereby, bioactive lipids, able to signal via presynaptic LPA2-receptors
and delivered by glial cells (Savaskan et al., 2007) to the synaptic cleft, are controlled by postsynaptic
PRG-1.
14
Supplemental Experimental Procedures Generation of PRG-1-deficient mice. We generated constitutive KO mice by targeted disruption of
the prg-1 gene via homologous recombination in E14-TG2a-derived embryonic stem (ES) cells. The
recombination was designed to result in the replacement of the last 119 bp of exon 4, full replacement
of exons 5 and 6 and replacement of the first 494 bp of exon 7 with a cassette containing at the 5’end
3 stop codons in every frame followed by an internal ribosome entry site, the lacZ reporter coding
sequence, an independent promoter (MC1)-controlled neomycin-resistance gene, and a
polyadenylation sequence (Figure S1A). The homology arms were PCR-amplified from C57Bl/6
genomic DNA. The 5’ homology arm was an approximately 4.6 kb PCR fragment corresponding to a
region between exon 2 and exon 4, whereas the 3’ homology arm, a 1.3 kb PCR fragment,
corresponded to a region within exon 7. One positive recombined ES-cell clone tested using southern
blot analysis was microinjected into C57BL/6 blastocysts. The resulting chimeric males were mated
with C57Bl/6 females. Transgenic progeny was genotyped by southern blot and PCR analysis of tail
tip DNA (Figure S1B and S1C). Protein expression was analyzed by western blotting and by
immunohistochemistry using PRG-1 antibody (1:500; custom-made antibody against aa 624-637,
NP_808332) and ß-Actin antibody (1:5000, Sigma-Aldrich, Munich, Germany) (Figure S1D and S1E).
To delete the prg-1 gene in a cell-specific fashion we generated a conditional knockout mouse line for
PRG-1. For the vector construct we utilized a fast recombination method mediated by the lambda
phage Red proteins described by Liu et. al. (Liu et al., 2003). Using this method we introduced two
LoxP sites in the genomic locus of the prg-1 gene by homologous recombination in E14-TG2a-derived
ES cells. This allowed us to delete the genomic sequence between intron 3 and intron 6 by Cre-
mediated recombination (Figure S4A) in an in utero electroporation approach. The homology DNA was
subcloned from a BAC containing mouse genomic DNA of chromosome 3 (RP23-340J18). The 5’
homology arm comprised sequences reaching from intron 2 to intron 3 and was 10 kb long. The 3’
homology arm was 3.5 kb long and comprised sequences from intron 6 to exon 7. Furthermore, a
neomycin-resistance cassette within the targeting vector was flanked by two frt-recombination sites,
which made removing the cassette possible in targeted ES cells by transfecting the CAGGS-FLPe
expression construct (Schaft et al., 2001). Positive recombination within the targeting locus was
characterized by southern blot analysis (Figure S4B). Three (3) positive recombined ES-cell clones
were then microinjected into C57BL/6 blastocysts. Chimeric males were mated with C57Bl/6 females;
its progeny were genotyped by PCR analysis of tail tip DNA (Figure S4C). The conditional KO mice
were tested for function through breeding with Cre-deleter mice (Schwenk et al., 1998) Protein
expression was analyzed by western blotting as described for the constitutive KO mice (Figure S4D).
Immunohistochemistry. The following primary antibodies were used: PRG-1 (1:1500; custom-made
antibody against aa 624-637, NP_808332), NeuN, VGlut2, GAD67, GluR1, GluR2/3, GluR2, NR2a&b,
PSA-NCAM (1:1000), Homer1 (1:500) (Chemicon, Temecula USA), synaptophysin, synaptoporin,
VGlut1, VGat, NR1 (1:1000), Gephyrin (1:500) (Synaptic Systems, Göttingen, Germany), GluR4
(1:1000 Upstate Biotechnology, Lake Placid, USA), GFAP (1:1000 DAKO, Glostrup, Denmark) and
Parvalbumin, Calbindin and Calretinin (1:5000; Swant, Bellinzona, Switzerland), ProSAP2/Shank3
15
(1:750 provided by T. Böckers), LPA2-R (1:1000; Clone 10D5, (Shida et al., 2004)), Munc13-1 (1:3500;
kindly provided by F. Varoqueaux).
Quantitative analysis of GluR2 and VGlut1. Analysis of synaptically localized GluR2 and VGlut1
was performed on immunofluorescent stainings using an antibody against an extracellular GluR2
epitope (1:300 Chemicon (Rouach et al., 2005)) and an antibody against VGluT1 (1:1000; Synaptic
Systems). Permeabilization was performed using a freeze-thaw protocol with liquid nitrogen followed
by 48 h incubation of the primary antibodies at 4°C. Quantitative analysis of colocalized GluR2 and
VGluT1 along the dendrites of CA1 pyramids was performed using unbiased stereological methods as
described (Mokin and Keifer, 2006). VGlut1 innervation along the CA1 dendrites was automated
quantified by using the image software MethaMorph (Version 6.2r6, Molecular Devices Corporation,
PA, USA). All images were acquired with 63x oil objective (zoom 5) with a Leica confocal laser-
scanning microscope (SL). To avoid bias all quantifications were performed using the same predefined
journals (Mokin and Keifer, 2006). Punctae were defined as an area of > 0.04 µm² to exclude
unspecific background signal.
Subcellular fractionation. Subcellular fractionation of the cerebral cortex was performed based on a
protocol described previously (Cotman and Taylor, 1972; Mizoguchi et al., 1989). The cerebrum from
one P19 mouse was homogenized in 3 ml of 0.32 M sucrose solution containing 1 mM NaHCO3 and
layered onto the discontinuous sucrose gradient composed of 2.85 ml of 0.85 M, 1.0 M, and 1.2 M
sucrose layers. After centrifugation at 82,500 g for 2 h at 4°C, the layer of 0.32 M sucrose solution,
bands between 0.32 M and 0.85 M sucrose solutions, between 0.85 M and 1.0 M sucrose solutions,
and between 1.0 M and 1.2 M sucrose solutions, and the pellet were collected as the cytosol, P2A
(myelin enriched fraction), P2B (the endoplasm reticulum and Golgi complex enriched fraction), P2C
(synaptosome), and P2D (mitochondria enriched fraction) fractions respectively (Carlin et al., 1980).
P2C fraction was centrifuged at 32,800 g for 20 min and the pellet was resuspended with 2.5 ml of 6
mM Tris/Cl pH 8.0 for the hypo-osmotic shock. This suspension was separated by centrifugation at
32,800 g for 20 min and the supernatant was collected as the synaptic cytoplasm fraction and the
crude synaptic vesicle fraction (SC+CSV). The pellet (crude synaptic plasma membrane fraction;
CSM) was extracted with 6 mM Tris/Cl pH 8.0 containing 1% Triton and further fractionated by
centrifugation at 32,800 g for 20 min. The pellet was used as the postsynaptic density fraction (PSD).
2.5 microgram of proteins from each fraction were separated by SDS-PAGE and transferred to the
PVDF membrane followed by western blotting. As it has been shown that Anti-RabGDI is enriched in
the SC fraction (Nonaka et al., 1991), we used this antibody and anti-PSD95 as subcellular
fractionation control. Quantification of the protein amount was performed by secondary antibodies
coupled with Alexa Fluor 680 (Molecular Probes) using the Odyssay Infrared Imaging System (LI-
COR, Lincoln, USA).
Expression plasmids and in utero Electroporation. The plasmids pCAG-mPRG1 and pCAG-Cre
were constructed by cloning the corresponding coding frame into the MCS of a pCAG-IRES-EGFP
vector (Kawauchi et al., 2003). For generation of the mutated plasmid pCAG–PRG1H253K the triplet
CAG was exchanged to AAG by site-specific mutagenesis (stratagene). For in utero electroporation
16
plasmids were prepared at 4 µg/μl by using the EndoFree Plasmid Kit (Qiagen). We used time-
pregnant mice at E15-E16 (post coitum). After having anesthetized the mice with a Ketamin-Xylazin-
mix (Ketamine 10mg/ml; Xylazine 1mg/ml), the uterine horns were exposed. The DNA solution (1-
1.5µl/embryo) was injected through the uterine wall into the lateral ventricle of two of the embryos by
pulled glass capillaries (World Precision Instruments, Sarasota, USA). Electric pulses were delivered
to embryos by holding the injected brain through the uterine wall with forcep-type electrodes
(CUY650P5) connected to a square–pulse generator (CUY 21 Edit, Unique Medical Imada, Miyagi,
Japan). Five (5) 38-V pulses of 50 ms were applied at 950 ms intervals. The uterine horns were
carefully put back into the abdominal cavity before the muscle wall and skin were stitched.
Electrophysiology. For investigating the electrographic seizure activity in vivo, craniectomies were
performed under deep halothane anesthesia without damaging the underlying dura. A standard
miniature drill equipped with a 0.3 mm diameter carbide dental burr was used. The electrodes were
placed at the following coordinates of the left hemisphere: 2.0 mm posterior from bregma and 3.0 mm
lateral from midline with a subdural reference electrode above the cerebellum. In a number of
experiments electrodes were placed at both hemispheres. The implanted electrodes were fixed using
microconnectors (GM-4; Microtech, Boothwyn, USA) and dental acryl (RR; Dentsply, Germany). The
incision for electrode implantation was sutured using 6-0 monofilament nylon (Ethicon, Johnson &
Johnson, Dilbeek, Belgium). After recovery from anesthesia, the pups were returned into their original
litter. First recordings were taken 24 hours after electrode implantation. Four channel recordings were
obtained with epidural electrodes at P20 in the Laplacian derivation, which re-references the signals to
the local average signal at two sites on each side of the head. Recordings were taken at waking state
at midday with the BrainStar® EEG System (Schwind, Erlangen, Germany) at a 256 Hz sampling rate,
70 Hz high pass and time constant of 0.3 sec filter setting.
For SPW-R detection, our event identification method was based on voltage thresholds in the
non-filtered LFP signal. We estimated the baseline noise by fitting a normal distribution to all samples
smaller than the mode, and their reflection with respect to the mode. A threshold was set at 3.5 STD,
and all samples larger than this threshold were considered as parts of possible events. As events
typically last 50-150 ms, we introduced an additional length criterion. From the above candidates, only
events in which a continuous deflection of over 1 STD from baseline lasted for more than 20 ms were
considered SPW-R events.
For gamma oscillation PSD, two minutes of unprocessed data stretches were analyzed. Specific
gamma band power was assessed by integration of the resulting PSD functions over frequency
(integration limits: 25 to 45 Hz). Ripple PSD was determined on otherwise unprocessed data of 50
SPW-ripple complexes randomly picked from the total number of events detected in the given
experiment. Power line hum (50 Hz) and its harmonics were removed. Further analyses included
approximation of 1/f in the given PSD function, i.e. the theoretical baseline PSD level, by linearly fitting
between onset and offset of the ripple-PSD peak. Specific ripple power was computed – within 120 to
300 Hz integration limits – by subtracting the area below the fitted line from the total area below ripple
17
peak. Spectral frequencies of ripple and gamma oscillations were derived from the peak values in the
respective frequency bands.
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