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antibody (ab30868; 1 mg/ml; Abcam), rabbit polyclonal anti-LGI1

antibody (sc-28238 H56; 1 mg/ml; Santa Cruz) and monoclonal anti

-tubulin antibody (1/2000, Sigma Aldrich).

Animal surgery

Under deep peritoneal anaesthesia (ketamine 100 mg/kg and xylazine

10 mg/kg) homozygous, heterozygous and wild-type postnatal day 8

mice (4–5 g) and heterozygous and wild-type postnatal day 21 mice

(10–12 g) were implanted with two nickel–chromium epidural

electrodes placed symmetrically in the somatosensory cortex (2.5 mm

posterior to bregma, 1.84 mm lateral to midline). Two electrodes were

placed along the median line, the anterior one as neutral and

the posterior one as reference. A nickel–chromium bipolar electrode

was placed in the dorsal hippocampus in postnatal day 8 mice

(1.7 mm posterior to bregma, 1.8 mm lateral to midline and 1.6 mm

ventral).

Intracranial video-EEG recordings

Animals were placed in a round transparent cage in which they

were free to move, and were connected to a recording system.

EEG signals were ampliﬁed with a band-pass ﬁlter setting of

0.5–100 Hz with a 24-channel system (Medelec, Oxford Instruments)

and digitized at 1024 Hz with a 22-bit resolution. Since postnatal

day 9 newborn pups are not weaned, recordings were limited to

4–5 h per day. Animal behaviour and EEG signal were visually

inspected.

Audiogenic stimuli

After 1 min of habituation in a Plexiglas box, mice were exposed to a

loud acoustic stimulus (11 kHz, 93 dB) generated by a function gener-

ator (Wavetek 131A) connected to four loudspeakers. The sound was

terminated either when a seizure was triggered or after 80 s (four

times 20 s with a 2 s interval between each exposure) as previously

(Yagi et al., 2005). Mice were subjected to a single auditory stimula-

tion, and responses were studied by an investigator blind to the

genotype of the animal.

Histochemistry

Mice aged postnatal day 8 and postnatal day 14 were deeply anaes-

thetized with sodium pentobarbital (50 mg/kg by intraperitoneal injec-

tion) and then perfused with 4% paraformaldehyde in a 0.1 mol/l

phosphate buffer, pH 7.4. Brains were removed, postﬁxed in the

same ﬁxative for 2 h at 4



C, cryoprotected for 24 h in a 30% sucrose

solution, frozen in isopentane (30



C) and stored at 80



Immunohistochemistry was performed using 20 mm free-ﬂoating sec-

tions. For all experiments, a series of three littermate mice correspond-

ing to each genotype were processed simultaneously. Antibodies used

were rabbit polyclonal antibody against ZnT3 (1:500; kindly provided

by R. Palmiter), rabbit polyclonal antibody against glial ﬁbrillary acidic

protein (1:4000; Dako) and biotinylated secondary antibody (Vector

Laboratories). A Nissl counterstaining (0.8% cresyl violet) was done

to reveal neuronal cytoarchitecture. Brain slices were labelled

with Fluoro-Jade C according to manufacturer’s instructions (Histo-

Chem Inc.).

Statistical analysis

Mendelian and sex ratios were assessed by using the 

-test. For the

body-weight plot, we have compared weight ratios between two

timepoints rather than absolute values in order to avoid variability of

body weight at birth between independent litters. A Kruskal–Wallis

test was used to compare body weight evolution of the three geno-

types (68 pups from 7 litters). Subsequently, we performed a Mann–

Whitney test to compare LGI1

/

mice to LGI1

+/

and wild-type mice.

The thickness of the granule cell layer was measured in three brain

sections from three mice of each genotype. Means were compared

using a Mann–Whitney test.

Results

Generation of LGI1-deﬁcient mice

We targeted the LGI1 gene in murine embryonic stem cells by

homologous recombination with a conditional Cre-LoxP approach.

LGI1

loxP/+

mice with a ﬂoxed LGI1 conditional allele were pro-

duced in a 75% C57BL/6, 25% 129S2Sv/pas hybrid line.

LGI1

loxP/+

males were crossed with PGK-Cre females (C57BL/6),

which express the Cre recombinase early and ubiquitously under

the control of the phosphoglycerate kinase 1 (PGK) promoter.

Recombination was observed in all organs due to maternal trans-

mission of active Cre recombinase in the oocyte (Lallemand et al.,

1998), leading to the deletion of exons 6 and 7 with a frameshift

generating a premature stop codon at residue 179 of the protein

(Fig. 1A).

Breeding pairs of adult heterozygous LGI1

+/

yielded litters with

wild-type (

+/+

), heterozygous (

+/

) and homozygous (

/

) geno-

types born in Mendelian ratios. Of 472 mice born, 120 were

LGI1

+/+

, 248 were LGI1

+/

and 104 were LGI1

/

as predicted

by Mendelian transmission (

= 2.3, nonsigniﬁcant), suggesting

that loss of both LGI1 alleles during embryogenesis is not lethal.

Sex ratios in LGI1

/

mice were approximately 1 : 1 (107 females,

123 males; 

= 1.1, nonsigniﬁcant) as expected.

LGI1 protein expression was examined by Western blot

of whole brain lysates from LGI1

/

, LGI1

+/

and wild-type

littermate mice. Immunoblot with an antibody against residues

200–300 of LGI1 (ab30868) revealed a single band of 65 kDa.

The intensity of the band was reduced by about half in LGI1

+/

lysate and the band was absent in LGI1

/

, conﬁrming that the

full-length LGI1 protein was completely ablated (Fig. 1B). A

second LGI1 antibody (sc-28238) directed against the

N-terminus (amino acids 35–90) detected the full-length protein,

but not a lower band, suggesting that a putative truncated protein

(179 amino acids) was absent (Fig. 1C). Neither antibody

cross-reacted with other LGI subfamily proteins, whereas a com-

mercial anti-LGI1 antibody (sc-9581, N18) directed against the

N-terminal region may do so (S. Baulac, personal communication).

We also examined the developmental and tissue expression pat-

tern of LGI1 using the speciﬁc ab30868 antibody. LGI1 expression

could be detected at low levels as early as embryonic day 16 and

increased with age, reaching plateau levels in the adult (Fig. 1D).

LGI1 expression was only detected in mouse brain and spinal cord

extracts (Fig. 1E).

LGI1 knockout mice Brain 2010: 133; 2749–2762 | 2751

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