Electroclinical characterization of epileptic seizures in leucine-rich, glioma-inactivated 1-deficient mice

Abstract

Mutations of the LGI1 (leucine-rich, glioma-inactivated 1) gene underlie autosomal dominant lateral temporal lobe epilepsy, a focal idiopathic inherited epilepsy syndrome. The LGI1 gene encodes a protein secreted by neurons, one of the only non-ion channel genes implicated in idiopathic familial epilepsy. While mutations probably result in a loss of function, the role of LGI1 in the pathophysiology of epilepsy remains unclear. Here we generated a germline knockout mouse for LGI1 and examined spontaneous seizure characteristics, changes in threshold for induced seizures and hippocampal pathology. Frequent spontaneous seizures emerged in homozygous LGI1(-/-) mice during the second postnatal week. Properties of these spontaneous events were examined in a simultaneous video and intracranial electroencephalographic recording. Their mean duration was 120 +/- 12 s, and behavioural correlates consisted of an initial immobility, automatisms, sometimes followed by wild running and tonic and/or clonic movements. Electroencephalographic monitoring indicated that seizures originated earlier in the hippocampus than in the cortex. LGI1(-/-) mice did not survive beyond postnatal day 20, probably due to seizures and failure to feed. While no major developmental abnormalities were observed, after recurrent seizures we detected neuronal loss, mossy fibre sprouting, astrocyte reactivity and granule cell dispersion in the hippocampus of LGI1(-/-) mice. In contrast, heterozygous LGI1(+/-) littermates displayed no spontaneous behavioural epileptic seizures, but auditory stimuli induced seizures at a lower threshold, reflecting the human pathology of sound-triggered seizures in some patients. We conclude that LGI1(+/-) and LGI1(-/-) mice may provide useful models for lateral temporal lobe epilepsy, and more generally idiopathic focal epilepsy.

Figure 1

Generation of the LGI1 knockout mouse. (A) Diagram showing LoxP and LoxP-Neo-LoxP sequences indicated by triangles (floxed allele). Exons 6 and 7 were removed by crossing the LGI1^loxP mice with a PGK-Cre strain (knockout allele). (B–C) Western blots demonstrating the absence of native LGI1 protein in whole brain lysate of LGI1^−/− mouse (at postnatal day 13) using the ab30868 antibody (B), and spinal cord lysate of LGI1^−/− mouse using the sc-28238 antibody (C). Equal amounts of total protein were loaded as demonstrated by α-tubulin levels. (D) Western blot showing LGI1 expression during embryonic and postnatal development on C57BL/6 mouse brain lysate (ab30868) and α-tubulin used for internal control. (E) Western blot showing restricted expression of LGI1 in the brain and spinal cord of a postnatal day 15 wild-type mouse (ab30868). Equal amounts of total protein were loaded and Ponceau S staining was used as internal loading control.

Figure 2

Spontaneous seizures in homozygous LGI1^−/− mice. Frames from a video recording of a spontaneous seizure in a postnatal day 16 LGI1^−/− mouse. (A) Onset of the seizure with forelimb and hind limb flexion and loss of postural equilibrium, (B) asymmetrical tonic extension (arrow) with rigidity of the tail, (C) chewing mechanism (arrow), (D) beginning of the hypertonic phase, (E) hypertonic phase with characteristic rigid hind limb extension and (F) postictal immobility. The behavioural seizure lasted for 100 s.

Figure 3

Video–EEG recordings of LGI1^−/− , LGI1^+/− and wild-type mice. (A) Epidural EEG recording in a postnatal day 10 LGI1^−/− mouse showing the onset of an electroclinical seizure (upper trace corresponds to the right cortex and bottom trace to the left cortex). Behavioural modifications were correlated with EEG changes: 1 = immobility; 2 = repeated clonies of the four limbs, agitation; 3 = myoclonic jerks of the trunk. In the lower panel, expanded EEG traces show low voltage fast activities (LVFA) following spikes, and bursts of polyspikes (PS) with increasing duration. No EEG abnormality was seen in age-matched LGI1^+/− (B) or wild-type (C) mice. (D) Epidural EEG recording in a postnatal day 10 LGI1^−/− mouse. In the lower panel, expanded EEG traces show initial spikes discharge with increasing amplitude, and then pseudo-periodic slow potentials with over-imposed polyspike activity. Note the asymmetry at the end of the seizure.

Figure 4

Simultaneous video–EEG recordings of the cortex and the hippocampus in homozygous LGI1^−/− mice. (A) Two electrodes located in the dorsal hippocampus (Hp) and one in the adjacent cortex (Cx) record an electroclinical seizure. Behavioural modifications are correlated with EEG event: 1 = immobility; 2 = myoclonic jerks; 3 = agitation and wild running; 4 = four-limb hypertonia. In the lower panel, an expanded EEG trace shows initial spike discharges of increasing amplitude, which begin earlier in the hippocampus than in the cortex and then pseudo-periodic slow potentials with an imposed polyspike activity. (B) Brief electrical seizure, with no clinical correlate. In this example, which was limited to the hippocampus, polyspikes, resembling interictal activity followed a low voltage fast activity of duration 2 s (see the lower panel for magnification). (C) Interictal activity, restricted to the hippocampus, showing high-amplitude spikes and polyspikes. This activity is increased after electroclinical seizures. (D) Postictal delta slow waves and depression of the activity at the end of an electroclinical seizure. (E) Physiological theta rhythm, with maximum amplitude in the hippocampus. (F and G) EEG analysis of 52 spontaneous electroclinical seizures recorded in LGI1^−/− mice (n = 6). (F) Percentage of mice showing seizures from postnatal days 9–15 during recordings (curve; axis on the right) and seizure frequency (histogram; axis on the left). (G) Seizure duration. Means and SEM are indicated.

Figure 5

Premature death and reduced body weight in homozygous LGI1^−/− mice. (A) Kaplan–Meier survival curves of LGI1^−/− (n = 25), LGI1^+/− (n = 52) and wild-type (n = 23) mice from postnatal day 0 until postnatal day 20. Of the LGI1^−/− mice 50% had died at postnatal day 17. (B) Body weight was comparable for LGI1^−/− (n = 21), LGI1^+/− (n = 27) and wild-type (n = 20) animals between postnatal days 8 and 10. Between postnatal days 8 and 14, the body-weight ratio of LGI1^−/− mice was reduced with respect to LGI1^+/− and wild-type mice. *P < 0.05. (C) LGI1^−/− mice were smaller than LGI1^+/− and wild-type littermates at postnatal day 14. (D) Stomach content of LGI1^−/− mice (empty) and LGI1^+/− mice (full) at postnatal day 14. At P9, stomach sizes and contents of LGI1^−/− and LGI1^+/− mice were similar.

Figure 6

Lower threshold for audiogenic seizures in heterozygous LGI1^+/− mice. (A) Variation with age in susceptibility to audiogenic seizures of LGI1^+/− mice exposed to a sound stimulus (11 kHz, 95 dB) compared with wild-type littermates. At postnatal Day 21, susceptibility was low, while at postnatal day 28, LGI1^+/− mice exhibit a significant susceptibility to audiogenic seizures compared with wild-type. *P < 0.05. LGI1^+/− (n = 25), wild-type (n = 17). (B) Epidural EEG recording in a P28 LGI1^+/− mouse under auditory stimulation. During the auditory stimulation (dashed arrow), the mouse was immobile and the EEG showed a normal background activity. Later, the mouse exhibited wild running (associated with movement artifacts), followed by a tonic phase and death (associated with suppression of brain activity).

Figure 7

Brain morphology of LGI1^−/− mice. (A–C) at postnatal day 8 and (D–F) at postnatal day 14 (P14). Nissl-stained coronal brain sections show similar brain morphology in (A and D) wild-type (n = 3), (B and E) LGI1^+/− (n = 3) and (C and F) LGI1^−/− mice (n = 3). Scale bars: 650 μm. Cx = cortex; Hipp = hippocampus; Th = thalamus; Ag = amygdala.

Figure 8

Seizure-induced hippocampal damage in homozygous LGI1^−/− mice aged postnatal day 14. (A–C) Nissl-staining shows dentate granule cell dispersion in LGI1^−/− mice (C). (D–I) Glial fibrillary acidic protein immunostaining with cresyl violet counterstaining of the hippocampus of (D) wild-type (n = 3), (E) LGI1^+/− (n = 3) and (F) LGI1^−/− (n = 3) mice. Enlarged images of the dentate gyrus in (G) wild-type, (H) LGI1^+/− and (I) LGI1^−/− mice. Reactive astrocytes are observed in LGI1^−/− mice (F and I). (J–O) Zinc transporter 3 (ZnT3) immunostaining with cresyl violet counterstaining of the hippocampus of (J) wild-type (n = 5), (K) LGI1^+/− (n = 4) and (L) LGI1^−/− (n = 5) mice. Enlarged images of the dentate gyrus from (M) wild-type, (N) LGI1^+/− and (O) LGI1^−/− mice showing mossy fibre sprouting in the inner molecular layer of the dentate gyrus of LGI1^−/− mice (L, O). (P–R) Fluoro-Jade C positive neurons in the hippocampus (P), CA3 region (Q) and CA1 region (R) of LGI1^−/− mice. Scale bars: 160 μm (D–F, J–L); 60 μm (A–C, G–I, M–R). Hipp = hippocampus.

Figure 9

Absence of morphological alterations in LGI1^−/− mice aged postnatal day 8 before seizures. (A–C) Nissl-staining of dentate gyrus. (A) Wild-type (n = 3), (B) LGI1^+/− (n = 3) and (C) LGI1^−/− (n = 3) mice. (D–F) Glial fibrillary acidic protein immunostaining with cresyl violet counterstaining of the hippocampus of (D) wild-type (n = 3), (E) LGI1^+/− (n = 3) and (F) LGI1^−/− (n = 3) mice. (G–I) Zinc transporter 3 (ZnT3) immunostaining with cresyl violet counterstaining of the hippocampus of (G) wild-type (n = 3), (H) LGI1^+/− (n = 3) and (I) LGI1^−/− (n = 3) mice. Scale bars: 300 µm (A–B); 40 μm (D–I).