On-demand seizures facilitate rapid screening of therapeutics for epilepsy
- Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, United States
- Center for Neurotrauma, Neurodegeneration, and Restoration, Corporal Michael J. Crescenz Veterans Affairs Medical Center, United States
- Department of Bioengineering, University of Pennsylvania, United States
- Department of Neurology, Perelman School of Medicine, University of Pennsylvania, United States
- Center for Neuroengineering and Therapeutics, University of Pennsylvania, United States
- Division of Neurology, Department of Pediatrics, The Children’s Hospital of Philadelphia, United States
Figures
Figure 1
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.
Figure 2
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 naive animals (bottom left). Fiber was implanted above cells expressing Thy1-ChR2 in the CA1 (bottom right). (B) Two thirds (Dalic and Cook, 2016) of CA1-IHK animals did not have a visible CA1 cell layer, while the rest (Fiest et al., 2017) had a thinned cell layer. In contrast, all CA3 IHK (Asadi-Pooya et al., 2017) 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.
Figure 3 with 2 supplements
Induced seizures resembled naturally occurring spontaneous seizures in chronically epileptic animals.
(A) Example of electrographic signal from spontaneous seizure in epileptic animal, with three 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 afterdischarges exceeding 5 s. (E) Compiled table of SVM accuracies across epileptic animals (n=8). Induced activity was classified as similar to spontaneous seizures in 88.1% of all successful activations. Failed activations were classified as baseline 100% of the time.
Figure 3—figure supplement 1
Threshold determination for inducing consistent afterdischarges.
(A) Flowchart for determining threshold stimulus conditions (laser power and total duration) that induce consistent afterdischarges (>5 s). Stimulation frequency (10 Hz) and pulse duration (25 ms) were kept constant (B) Threshold power for n=10 epileptic (9.14±4.75 mW) and n=6 naive animals (6.17±1.58 mW; Wilcoxon rank sum test, p=0.137). (C) Threshold duration was comparable between the same epileptic animals (6.30±1.64 s) and naive animals (5.67±1.03 s; Wilcoxon rank sum test, p=0.7133). (D) Epileptic animals had comparable success rate of induction (88% ± 8.8%) at threshold to naive animals (86 ± 6.0 %; Wilcoxon rank sum test, p=0.8506). One naive animal never reached consistent induction above 66.7%. (E) With the threshold stimulus, induced activity mean duration was longer in epileptic animals (30.98±4.69 s) than in naive animals (20.87±2.19 s; Wilcoxon rank sum test, p=0.0005).
Figure 3—figure supplement 2
Examples of behavioral and electrographic seizures induced in epileptic animals.
(A) Seizure depicted in (B), from animal #100. Key frames depict stiffening of the tail (yellow), forelimb clonus with backpedaling (blue), rearing (pink), and loss of righting reflex (purple), with approximate locations of the frame marked by lines on the waveform. (B) Example of induced seizure from another epileptic animal (#107). In this seizure, onset was characterized by freezing (orange) before progressing to clonus, tail stiffening, rearing (pink) and – eventually – uncontrolled jumps (green) and loss of righting reflex (purple). (C) Example of Racine 2 behavioral and electrographic seizure induced in epileptic animal #107. Approximate locations of key frames marked in color. Initially, there was no obvious seizure behavior (black). The animal froze (orange) and the tail stiffened (pink) towards the end of the event, signaling a seizure has occurred. (D) Example of failed induction in epileptic animal (#105). Stimulus elicited no electrographic discharges. In this trial, stimulus power was under that of the threshold stimulus for consistent induction in this animal.
Figure 4 with 1 supplement
Inducing seizures in epileptic animals differs from optical kindling in naive animals.
(A) Initial optogenetic activations in naive animals induced low frequency afterdischarges. Representative EEG trace with three segments of 10 s at the start, middle, and end of activity 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 naive and epileptic animals. Percent of behavioral electrographic events significantly differed between naive and epileptic animals on stimulation day 1 through 3. Average Racine score of electrographic inductions significantly differed between naive 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 naive (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; naive – 58 inductions, n=7). Linear Mixed Effect Model: * p<0.05, ** p<0.01, *** p<0.001.
Figure 4—figure supplement 1
Examples of inductions in naive animal #110.
(A) Afterdischarges depicted in Figure 4A. Animal displayed independent movement (black) and grooming (gray) during spiking. Behavioral Score: No seizure. Electrographic Score: Afterdischarges induced. (B) Seizure depicted in Figure 4B Animal begins shaking (blue) before embarking on a wild run with a stiff tail (pink). Finally, the animal ended up rearing and performing an uncontrolled jump (purple). Behavioral Score: Racine 5. Electrographic Score: Seizure induced.
Figure 5 with 1 supplement
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 hr washout occurred between subsequent ASM applications. (B) Post ASM application, rates of inducing electrographic afterdischarges (any electrographic activity lasting more than five seconds) and behavioral seizures (generalized seizures with seizure behaviors) 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.
Figure 5—figure supplement 1
Racine level of seizures, measured as percentage of all inductions in epileptic animals before and after drug application.
(A) 5 mg/kg Diazepam reduces the Racine level of behavioral seizures in all animals, including animal 107, which was a diazepam non-responder on one of two testing days. (B) Levetiracetam also reduces the Racine level of behavioral seizures in all animals. In both graphs, data between multiple testing days was aggregated by animal.
Tables
Table 1
Behavioral scoring and Racine Scale assignments.
| Racine | Behaviors |
|---|---|
| 0 | Normal behavior, grooming, free movement (even with slight limp) |
| 1 | Clear freezing/flattening of the body |
| 2 | Tail stiffening/forelimb clonus/uncontrolled shaking |
| 3 | Rearing |
| 4 | Wild run/backpedaling |
| 5 | Uncontrolled jumping/loss of righting reflex |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic Reagent (Mus musculus) | Thy1-ChR2-YFP | Jackson Laboratory (JAX) | IMSR_JAX:007612 | |
| Chemical compound | Kainic Acid | Hello Bio | HB0355 | |
| Chemical compound, drug | Diazepam | Dash Pharmaceuticals | 69339013634 | 5 mg/ml |
| Chemical compound, drug | Levetiracetam | Sigma Aldrich | PHR-1447 | |
| Other | Optic Fiber Cannula | RWD | 907-03011-00 | 1.25 mm ferrule, 400 micron core, 0.39 NA |
| Other | Optic Fiber (FC-PC) | RWD | 807-00059-00 | 200 micron core, 0.22 NA |
| Other | Optic Fiber (FC-FC) | Doric | D202-2075 | 200 micron core, 0.22 NA |
| Other | Laser Power Source | Laserglow | LRS-0473 | |
| Chemical compound, drug | Cresyl Violet Acetate | Sigma Aldrich | C5042 | For Staining |
| Software, algorithm | MATLAB | MATLAB | RRID:SCR_001622 | 2019b |
| Software, algorithm | R Project for Statistical Computing | R | RRID:SCR_001905 | 4.4.0 |
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On-demand seizures facilitate rapid screening of therapeutics for epilepsy
eLife 13:RP101859.
https://doi.org/10.7554/eLife.101859.3
