Experimental schematic for on-demand seizure induction in epileptic animals.

(A) Chronic epilepsy was induced in Thy1-ChR2 mice via intrahippocampal kainic acid injection into the CA3. (B) Mice were implanted with EEG recording apparatus consisting of two cortical screws, one set of insulated braided wire targeting the hippocampus, a ground screw, and a reference screw. A fiber was positioned so that the tip illuminated the CA1. (C) 10 Hz of 473 nm light delivered into the CA1 activated Thy1-ChR2 neurons and induced seizures on demand in vivo.

Intrahippocampal kainic acid (IHK) injection into the CA3 reduced CA1 damage without impacting average spontaneous seizure count.

(A) Cresyl violet staining of hippocampal slices from IHK injected animals visualized IHK induced damage to the hippocampus. The CA1 layer was eliminated in CA1 IHK animals (top). In the CA3 IHK animals, the CA1 layer was present but thinned (middle). Both CA1 IHK and CA3 IHK animals displayed expansion of the dentate gyrus structure and extensive CA3 damage when compared to naïve animals (bottom). (B) Two thirds (4) of CA1-IHK animals did not have a visible CA1 cell layer, while the rest (2) had a thinned cell layer. In contrast, all CA3 IHK (10) animals had a visible CA1 cell layer. All 10 CA3 IHK and 6 CA1 IHK animals had extensive damage to the CA3 coupled with expansion of the dentate gyrus (DG). (C) Two tailed Mann-Whitney test comparing the average number of spontaneous seizures per day showed no significant difference (p = 0.635) between CA1 IHK and CA3 IHK animals.

Induced seizures resembled naturally occurring spontaneous seizures in chronically epileptic animals.

(A) Example of electrographic signal from spontaneous seizure in epileptic animal, with 3 segments of 10 s enlarged for clarity. (B) Electrographic signal from first induction in the same animal. (C) Change in features from baseline (gray) to computationally defined segments (first tercile – beginning, second tercile – middle, and final tercile – end). In the first tercile, the extent of increase in 1 – 30 Hz band power, 300 + Hz band power, line length, and area significantly differed between 138 induced (n = 10) and 337 spontaneous behavioral seizures (n = 8). Differences become less significant by the middle and the final third. Linear Mixed Effect Model: * p < 0.05, ** p < 0.01, *** p < 0.001 (D) Linear Support Vector Machine (SVM) classified inductions from the animal in (A) and (B). Successfully induced seizures were more closely associated with spontaneous seizures than with either baseline activity or optogenetic activations that failed to induce seizures. (E) Compiled table of SVM accuracies across epileptic animals (n = 8). Induced seizures were classified as similar to spontaneous seizures in 88.1% of all successful activations. Failed activations were classified as baseline 100% of the time.

Inducing seizures in epileptic animals differs from optical kindling in naïve animals.

(A) Initial optogenetic activations in naïve animals induced low frequency afterdischarges. Representative EEG trace with 3 segments of 10 s at the start, middle, and end of seizure enlarged. (B) Following multiple days of stimulation, application of activation stimulus in the same animal induced Racine 5 seizures. (C) Rate of electrographic afterdischarges from optogenetic activation did not significantly differ between naïve and epileptic animals. Percent of behavioral electrographic events significantly differed between naïve and epileptic animals on stimulation day 1 through 3. Average Racine score of electrographic inductions significantly differed between naïve and epileptic animals on stimulation day 1 through 4. Pairwise T test: * p < 0.05, ** p < 0.01, *** p < 0.001. (D) In the first 4 days of stimulation, inductions in naïve (100 inductions, n = 7) and epileptic animals (87 inductions, n = 10) significantly differed in the feature space. Differences were reduced, but still existed, on stimulation day 5 or later (epileptic – 75 inductions, n = 7; naïve – 58 inductions, n = 7). Linear Mixed Effect Model: * p < 0.05, ** p < 0.01, *** p < 0.001.

Induced seizures in epileptic animals responded to both diazepam and levetiracetam.

(A) Experimental timeline for testing ASM efficacy in on-demand seizure model. Stimulus for seizure induction was tested for consistency before ASM application. A 48-hour washout occurred between subsequent ASM applications. (B) Post ASM application, rates of inducing electrographic afterdischarges and behavioral seizures were significantly reduced. Paired One Tailed Wilcoxon Signed Rank Test, * p < 0.05, ** p < 0.01 (C) Probability of successful induction of activity (averaged across all epileptic animals) increased the more time has passed since ASM injection into the mouse.