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. 2010 May 1;19(9):1702-11.
doi: 10.1093/hmg/ddq047. Epub 2010 Feb 3.

Lgi1 null mutant mice exhibit myoclonic seizures and CA1 neuronal hyperexcitability

Y Eugene Yu  1 , Lei Wen, Jeane Silva, Zhongyou Li, Karen Head, Khalid Sossey-Alaoui, Annie Pao, Lin Mei, John K Cowell
Affiliations

Lgi1 null mutant mice exhibit myoclonic seizures and CA1 neuronal hyperexcitability

Y Eugene Yu et al. Hum Mol Genet. .

Abstract

LGI1 in humans is responsible for a predisposition to autosomal dominant partial epilepsy with auditory features (ADPEAF). However, mechanisms of how LGI1 mutations cause epilepsy remain unclear. We have used a mouse chromosome engineering strategy to create a null mutation for the gene ortholog encoding LGI1. The Lgi1 null mutant mice show no gross overall developmental abnormalities from routine histopathological analysis. After 12-18 days of age, the homozygous mutant mice all exhibit myoclonic seizures accompanied by rapid jumping and running and die shortly thereafter. The heterozygous mutant mice do not develop seizures. Electrophysiological analysis demonstrates an enhanced excitatory synaptic transmission by increasing the release of the excitatory neurotransmitter glutamate, suggesting a basis for the seizure phenotype. This mouse model, therefore, provides novel insights into the mechanism behind ADPEAF and offers a new opportunity to study the mechanism behind the role of LGI1 in susceptibility to myoclonic seizures.

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Figures

Figure 1.
Figure 1.
Generation of the Lgi1 mutation. (A) Strategy to generate the deletion at the Lgi1 locus based on Cre/loxP-mediated recombination. H, HpaI; N, NdeI; S, SmaI; 3′ 3′-HPRT; 5′, 5′-HPRT; Ag, K14-Aguoti gene; Ty, tyrosinase minigene; P, puromycin-resistance gene; N, neomycin-resistance gene; arrowhead, loxP site. Integration sites for MICER clones are shown adjacent to intron 2 and downstream of exon 8. Following Cre-loxP recombination, the HPRT gene is reconstituted from the two fragments located in the vector arms and the two coat color markers are inserted. (B and C) Southern blot analysis of samples from ES cell DNA that were digested with NdeI. DNA isolated from the parental AB2.2 ES cells and an ES cell clone targeted with MHPN-127h1 (B, lanes 1 and 2) was hybridized with Probe A. Cellular DNA isolated from an ES cell clone targeted with MHPN-127h1 (C, lane 1) and two ES cell clones carrying the Lgi1 deletion (C, lanes 2 and 3) hybridized with Probe B.
Figure 2.
Figure 2.
FISH analysis of ES cells carrying the heterozygous deletion of the Lgi1 gene. An ES cell metaphase is shown (left). The two copies of chromosome 19 are identified in the box. Individual copies of chromosome 19 (right) were identified by BAC clone RP23-359A49 located close to the centromeric region (red). When co-hybridized with DNA probes from within the deleted region (see text), only one of these chromosomes (arrow) shows the presence of the Lgi1 locus (green).
Figure 3.
Figure 3.
PCR strategy for identification of the homozygous deletion of the Lgi1 gene. (A) Six pups were analyzed, of which four (2, 3, 5 and 6) were positive for the HPRT gene, indicating the presence of the Cre/loxP-mediated recombination product, although this PCR analysis could not distinguish between heterozygous and homozygous deletions. (B) All four of these pups were positive for proximal genomic region 2 (P2) and distal genomic regions 7 and 8 (P7 and P8) which lie outside the deletion (see text). When analyzed for genomic regions within the deletion, pups 3 and 6 were negative, demonstrating that they carry the homozygous deletion and the other two (2 and 5) carry a hemizygous deletion. RT–PCR analysis of wild-type (lanes 1, 4, 5 and 8) and mutant null (lanes 2, 3, 6 and 7) mice demonstrates the absence of PCR products from the hippocampus of the null mice (C). RT–PCR analysis of RNA from hippocampus of the wild-type mice (D) at postnatal days 2–26 demonstrate expression of Lgi1 during early weeks of postnatal life. When wild-type and heterozygous littermates were analyzed using semi-quantitative RT–PCR, evidence for reduced LGI1 expression was seen in the hippocampus, although expression is clearly present at all time points in the heterozygotes (E).
Figure 4.
Figure 4.
Spontaneous epileptiform-discharge activity in the CA1 regions of hippocampal slices from wild-type (+/ + ) and Lgi1 null (−/ − ) mice. Field responses were recorded extracellularly in a modified ACSF (0 mm MgSO4, 5 mm KCl, 1.6 mm CaCl2). (A) Representative traces of the spontaneous epileptiform burst discharges from mutant and wild-type mice were shown. Quantitative analysis of the burst discharge incidence (B) showed increased frequency in the mutant compared with the wild-type mice (both n = 7, **P < 0.01).
Figure 5.
Figure 5.
The intrinsic properties of CA1 pyramidal neurons. (A) Representative response of a CA1 pyramidal neuron to 200 ms depolarizing and hyperpolarizing somatic current pulses. Quantitative analysis showed that the evoked spike frequencies of CA1 pyramidal neurons produced by a 200 pA suprathreshold somatic current injection (B) were similar in wild-type and Lgi1-mutant neurons. The resting membrane potential (RMP) (C) and input resistance (D) of CA1 pyramidal neurons did not vary significantly between wild-type and Lgi1-mutant mice (both n = 9, P > 0.05).
Figure 6.
Figure 6.
Analysis of miniature and evoked EPSCs and IPSCs in hippocampal CA1 pyramidal neurons from wild-type (+/ + ) and Lgi1 null (−/ − ) mice. (A) Representative traces of the mEPSCs are shown. Quantitative analysis of mEPSC frequency (B) showed significantly higher frequencies in the mutant compared with the wild-type mouse (both n = 12, **P < 0.01), and cumulative distribution of mEPSC amplitudes (C) showed no significant difference between mutant and wild-type control mice. (D) Representative traces of the mIPSC in CA1 neurons for mutant and wild-type mice are shown. Quantitative analysis of the mIPSC frequency (E) and cumulative distribution of mIPSC amplitudes (F) showed no significant difference between Lgi1-mutant mice and wild-type controls (both n = 8). (G) Representative traces showed that the evoked AMPA (lower) and NMDA (upper) receptor-mediated EPSCs were increased in Lgi1-mutant CA1 pyramidal neurons. (H) The quantification of eEPSC amplitude is shown (n = 7 for wild-type mice and n = 8 for Lgi1-mutant mice, respectively; **P < 0.01, *P < 0.05, compared with wild-type controls). Representative traces (I) and quantification (J) showed that the eIPSC amplitudes were similar in CA1 pyramidal neurons from the wild-type and Lgi1-mutant mice (both n = 7, P > 0.05). The amplitudes for AMPA-eEPSCs, NMDA-eEPSCs and eIPSCs in wild-type mice were 270.6±15.3 pA, 278.0±18.7 pA and 712.9±44.6 pA, respectively.
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