Ablation of Unilateral Hippocampal GABAergic Neurons: A Novel Mouse Model of Mesial Temporal Lobe Epilepsy With Hippocampal Sclerosis methods
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AAV and Reagents
AAV vectors were packaged with titers of 1-10 × 10 12 viral genomes (v.g.)/ml, and 200 nL of these vectors were injected into a single site within the target area of the mouse brain for modeling. Specifically, the rAAV2/9-mCherry-flex-DTA (5.5 × 10 12 v.g./mL, expressing mCherry in all infected cells, simplified as AAV-flex-DTA) was obtained from Addgene plasmid (#58536). The rAAV2/9-DIO-taCasp3-TEVp-mCherry (2.0 × 10 12 v.g./mL, expressing mCherry only in cre-positive cells, simplified as AAV-DIO-taCasp3-TEVp) and the rAAV2/9-DIO-mCherry (2.0 × 10 12 v.g./mL) as negative control (NC) were kind gifts from the Chinese Institute for Brain Research. KA (ab144490) was purchased from Abcam, and 100 nL diluted as 2...
vEEG Recordings and Analyses
Following the insertion of skull electrodes, mice were connected to the NeuSen H signal acquisition system (Neuracle, Changzhou) for 24-h continuous monitoring, commencing on the 8th day post-surgery and continuing for the subsequent 3 weeks. To assess seizure severity, the revised Racine (r-Racine) scale (-1 to 7) was employed, where a tonic-clonic seizure with continuous spike-wave discharges was scored as 5 (when the mouse lay on its belly) or 6 (when it lay on its side or exhibited wild jumping) and was assigned a score of 7 if animal death happened several minutes after seizure onset [ ]. The onset and termination of seizures were precisely determined based on the EEG signal, with the EEG filter settings adjusted to 1-70 Hz and the notch filter activated.
Ablation of Hippocampal GABAergic Neurons by AAV Expressing DTA or taCasp3-TEVp Induced SRS
SRS characteristics induced by hippocampal GABAergic neuron ablation. (A) Timeline of the vEEG recording during 1- and 4-week after modeling. (B) Schematic of the positions where electrodes were inserted in the mouse brains. (C) Representative EEG tracings depicting the interictal period and SRS onset in hippocampal GABAergic neuron ablation models (AAV-mCherry-flex-DTA and AAV-DIO-taCasp3-TEVp-mCherry) and KA injection models. (D) The epileptogenic time course of each group. n = 10 for each group. There is a significant difference in comparison of DTA-CA1 versus DTA-DG ( p < 0.05), DTA-DG versus Casp3-CA1 ( p < 0.001), DTA-DG versus KA- central ( p < 0.01), Casp3-CA1 versus Casp3-DG ( p < 0.01), and Casp3-DG versu...
Ablation of Hippocampal GABAergic Neurons by AAV Expressing DTA or taCasp3-TEVp Induced SRS
The overall effects of the DTA model and the taCasp3 model were comparable, with the latter group showing a seemingly weaker SRS severity, although the difference was not statistically significant ( p > 0.05, Figure ). Regarding the differences among ablation regions, the SRS severity of the CA1 group was generally weaker, characterized by a lower seizure frequency (Figure ), a lower Grade 6 seizure ratio (Figure ), no lethal seizures (Figure ), and a slightly shorter duration (Figure ). Notably, the CA1 model stood out with approximately 50% of mice exhibiting no SRS (Figure ). The overall SRS pattern in the KA model was intermediate compared to each group of hippocampal GABAergic neuron ablation (Figure ). In a word, it was found that GABAergic neuron ablation led to chronic SRS, comparable to KA models.
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AAV vectors were packaged with titers of 1-10 × 10 12 viral genomes (v.g.)/ml, and 200 nL of these vectors were injected into a single site within the target area of the mouse brain for modeling. Specifically, the rAAV2/9-mCherry-flex-DTA (5.5 × 10 12 v.g./mL, expressing mCherry in all infected cells, simplified as AAV-flex-DTA) was obtained from Addgene plasmid (#58536). The rAAV2/9-DIO-taCasp3-TEVp-mCherry (2.0 × 10 12 v.g./mL, expressing mCherry only in cre-positive cells, simplified as AAV-DIO-taCasp3-TEVp) and the rAAV2/9-DIO-mCherry (2.0 × 10 12 v.g./mL) as negative control (NC) were kind gifts from the Chinese Institute for Brain Research. KA (ab144490) was purchased from Abcam, and 100 nL diluted as 2 mg/mL in saline was injected per mouse brain.
Following the insertion of skull electrodes, mice were connected to the NeuSen H signal acquisition system (Neuracle, Changzhou) for 24-h continuous monitoring, commencing on the 8th day post-surgery and continuing for the subsequent 3 weeks. To assess seizure severity, the revised Racine (r-Racine) scale (-1 to 7) was employed, where a tonic-clonic seizure with continuous spike-wave discharges was scored as 5 (when the mouse lay on its belly) or 6 (when it lay on its side or exhibited wild jumping) and was assigned a score of 7 if animal death happened several minutes after seizure onset [ ]. The onset and termination of seizures were precisely determined based on the EEG signal, with the EEG filter settings adjusted to 1-70 Hz and the notch filter activated.
SRS characteristics induced by hippocampal GABAergic neuron ablation. (A) Timeline of the vEEG recording during 1- and 4-week after modeling. (B) Schematic of the positions where electrodes were inserted in the mouse brains. (C) Representative EEG tracings depicting the interictal period and SRS onset in hippocampal GABAergic neuron ablation models (AAV-mCherry-flex-DTA and AAV-DIO-taCasp3-TEVp-mCherry) and KA injection models. (D) The epileptogenic time course of each group. n = 10 for each group. There is a significant difference in comparison of DTA-CA1 versus DTA-DG ( p < 0.05), DTA-DG versus Casp3-CA1 ( p < 0.001), DTA-DG versus KA- central ( p < 0.01), Casp3-CA1 versus Casp3-DG ( p < 0.01), and Casp3-DG versus KA- central ( p < 0.01), with no significant difference between the other two groups. (E) Summary of the weekly SRS frequency for each group highlighting the mean SRS rate and differences between groups. n = 6 for NC group, n = 10 for each other group,...
The overall effects of the DTA model and the taCasp3 model were comparable, with the latter group showing a seemingly weaker SRS severity, although the difference was not statistically significant ( p > 0.05, Figure ). Regarding the differences among ablation regions, the SRS severity of the CA1 group was generally weaker, characterized by a lower seizure frequency (Figure ), a lower Grade 6 seizure ratio (Figure ), no lethal seizures (Figure ), and a slightly shorter duration (Figure ). Notably, the CA1 model stood out with approximately 50% of mice exhibiting no SRS (Figure ). The overall SRS pattern in the KA model was intermediate compared to each group of hippocampal GABAergic neuron ablation (Figure ). In a word, it was found that GABAergic neuron ablation led to chronic SRS, comparable to KA models.
Machine-readable layer
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