Seizure threshold was determined in individual male FVB mice by the timed intravenous pentylenetetrazole (PTZ) test, as described previously (60), in a total of 57 naive and 55 epileptic mice. Epilepsy was induced by pilocarpine (61), and seizure thresholds were determined 7 to 9 weeks after SE, when all mice had developed epilepsy. Groups of 8 to 20 mice were used per threshold determination. PTZ infusion was stopped at the first seizure, and seizure threshold was calculated in milligrams of PTZ per kilogram of body weight on the basis of the infusion time needed to induce this seizure end point (60). PTZ thresholds were determined every 4 hours during the light and dark phase. The maximum number of threshold determinations was limited to three per animal with 7 days in between (repeated seizure threshold tests do not alter the threshold if an interval of at least 48 hours lies within the determinations) (60, 62, 63). In addition, we performed two PTZ seizure threshold determinations at the same time point (ZT4:30) to compare PTZ thresholds at the beginning and at the end of the experiment, demonstrating the reproducibility of the method.

Data were analyzed by using the Prism 5 software (GraphPad, La Jolla, CA, USA). Thresholds of the control and SE animals were separately analyzed using one-way ANOVA and Bonferroni post hoc tests and combined by using a two-way ANOVA with Student’s t tests as post hoc analysis. All tests that were used were two sided; a P < 0.05 was considered significant.

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