Behavioral characteristics as potential biomarkers of the development and phenotype of epilepsy in a rat model of temporal lobe epilepsy

The present study performed a detailed analysis of behavior in a rat model of epilepsy using both established and novel methodologies to identify behavioral impairments that may differentiate between animals with a short versus long latency to spontaneous seizures and animals with a low versus high number of seizures. Temporal lobe epilepsy was induced by electrical stimulation of the amygdala. Rats were stimulated for 25 min with 100-ms trains of 1-ms biphasic square-wave pluses that were delivered every 0.5 s. Electroencephalographic recordings were performed to classify rats into groups with a short latency (< 20 days, n = 7) and long latency (> 20 days, n = 8) to the first spontaneous seizure and into groups with a low number of seizures (62 ± 64.5, n = 8) and high number of seizures (456 ± 185, n = 7). To examine behavioral impairments, we applied the following behavioral tests during early and late stages of epilepsy: behavioral hyperexcitability, open field, novel object exploration, elevated plus maze, and Morris water maze. No differences in stress levels (e.g., touch response in the behavioral hyperexcitability test), activity (e.g., number of entries into the open arms of the elevated plus maze), or learning (e.g., latency to find the platform in the Morris water maze test during training days) were observed between animals with a short versus long latency to develop spontaneous seizures or between animals with a low versus high number of seizures. However, we found a higher motor activity measured by higher number of entries into the closed arms of the elevated plus maze at week 26 post-stimulation in animals with a high number of seizures compared with animals with a low number of seizures. The analysis of the Morris water maze data categorized the strategies that the animals used to locate the platform showing that the intensity of epilepsy and duration of epileptogenesis influenced swimming strategies. These findings indicate that behavioral impairments were relatively mild in the present model, but some learning strategies may be useful biomarkers in preclinical studies.


Supplementary
Comparison of RODA analysis of Morris water maze test day between the sham and stimulated animals at weeks 9 and 27 after stimulation.
The probabilities (%) of transitioning between strategies were checked in the test trials (weeks 9 and 27) in the sham animals and stimulated (SE) groups. Rows and columns indicate the starting and ending strategies respectively. Row values (for the same starting strategy) were normalized (sum of each row equals 100%). Low strategies: thigmotaxis, incursion. Medium strategies: scanning, focused search, chaining reaction, self-orienting. High strategies: scanning surrounding, scanning target. Thigmotaxis and incursion were assigned to low-level strategies because the animals are staying mostly in the areas close to the walls of the arena. Scanning, focused search, chaining response and self-orienting were assigned to medium-level strategies because the animals explored inner parts of the arena. Scanning surroundings and scanning target were assigned to high-level strategies because the animals passed or focused on areas of the arena contained the platform. Sham animals at both weeks 9 and 27 transitioned more often from a medium-level to a high-level strategy and less often from a high-level to a low-level strategy than the SE group. Based on the strategy transitions from low to medium levels and low to high levels, little difference was seen in SE animals between weeks 9 and 27, whereas the sham animals were more likely to end with a medium-level strategy at week 27.  Figure S7 Comparison of RODA analysis of the Morris water maze test day between the sham, short-latency and long-latency groups at weeks 9 and 27 after stimulation.

Supplementary
The probabilities (%) of transitioning between strategies were checked in test trials (weeks 9 and 27) in the sham, long-latency, and short-latency groups. Rows and columns indicate the starting and ending strategies, respectively. Row values (for the same starting strategy) were normalized (sum of each row equals 100%). Low strategies: thigmotaxis, incursion. Medium strategies: scanning, focused search, chaining reaction, self-orienting. High strategies: scanning surrounding, scanning target. Thigmotaxis and incursion were assigned to low-level strategies because the animals mostly stayed in areas close to the walls of the arena. Scanning, focused search, chaining response, and self-orienting were assigned to medium-level strategies because the animals explored inner parts of the arena. Scanning surroundings and scanning target were assigned to high-level strategies because the animals passed or focused on areas of the arena that contained the platform. Sham animals at week 9 transitioned more often from medium-to high-level strategies than the other two groups; similar conclusion is obtained for week 27 but only between sham and long latency groups. The sham and long-latency groups at both weeks 9 and 27 transitioned more often from high-to medium-level strategies. The short-latency group at week 9 transitioned much more often from high-to low-level strategies and during week 27 the difference on the transition probabilities between high and low and high and medium level strategies are more equalized. For the transition probabilities between low and medium and low and high strategies transitions the sham groups increases the transitions of the former between weeks 9 and 27 while the short latency group does the opposite, it starts from more equalized transitions between low and medium and low and high strategies on week 9 but on week 27 it increases the latter. The long-latency group did not exhibit such differences between weeks 9 and 27, and the transitions from low-level strategies to medium-and high-level strategies were more equalized.  Figure S10 Comparison of RODA analysis of Morris water maze test day between the sham, non-epileptic and epileptic animals at week 9 after stimulation.

Supplementary
The probabilities (%) of transitioning between strategies were checked in test trials at week 9 in the sham, non-epileptic, and epileptic animals. Rows and columns indicate the starting and ending strategies, respectively. Row values (for the same starting strategy) were normalized (sum of each row equals 100%). TT, thigmotaxis; IC, incursion; SC, scanning; FS, focused search; CR, chaining reaction; SO, self-orienting; SS, scanning surrounding; ST, scanning target. Thigmotaxis and incursion were assigned to low-level strategies because the animals mostly stayed in areas close to the walls of the arena. Scanning, focused search, chaining response, and self-orienting were assigned to intermediate-level strategies because the animals explored inner parts of the arena. Scanning surroundings and scanning target were assigned to high-level strategies because the animals passed or focused on areas of the arena that contained the platform. Epileptic animals had a higher probability of transitioning between low-and high-level strategies. The sham and non-epileptic groups had nearly equal probabilities of transitioning between low-and medium-level strategies and between low-and high-level strategies. Sham animals had more equalized probabilities of transitioning between medium-and low-level strategies and medium-and high-level strategies. The non-epileptic and epileptic groups had a higher probability of transitioning between medium-and low-level strategies. The sham and non-epileptic groups had a higher probability of transitioning between high-and medium-level strategies than the epileptic group, which had a higher probability of transitioning between high-and low-level strategies.
Supplementary Figure S13 Comparison of RODA analysis of Morris water maze test day between the sham, low seizure number and high seizure number groups at weeks 9 and 27 after stimulation The probabilities (%) of transitioning between strategies were checked in test trials (weeks 9 and 27) in the sham, low-seizure-number, and high-seizure-number groups. Rows and columns indicate the starting and ending strategies, respectively. Row values (for the same starting strategy) were normalized (sum of each row equals 100%). TT, thigmotaxis; IC, incursion; SC, scanning; FS, focused search; CR, chaining reaction; SO, self-orienting; SS, scanning surrounding; ST, scanning target. Thigmotaxis and incursion were assigned to low-level strategies because