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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