Epileptic behaviors in mice measured by inducted current in magnetic fieldHsiang-Chin Lu1, Andrew Chih-Wei Huang2, Tien-Tzu Yang3, Bai Chuang Shyu1*

1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC2 Department of Psychology, Fo Guang University, Yilan, Taiwan, ROC

3 Taipei Municipal Heping High School, Taipei, Taiwan, ROC

Introduction Epilepsy is a common neurological disorder. Approximately 1% of population suffers from this disease. Many methods were used to assess the seizures of epilepsy in animal model. For example, the video recording of the home cage behavior were measured by an observer for scoring. However, some deficits have been concerned, including the manual analysis of mouse seizures requires the monitoring over long time, and the result will likely to be affected by personal subjective factors. In contrast, the magnetic field assessment is a new method to automate measuring spontaneous pain behavior in an animal model with a non-subjective measurement. Accordingly, we proposed that the magnetic field assessment could be an effective and objective method to evaluate the seizure responses. The present study was designed to develop a novel method for assessing seizure behavior in a mice model.

Conclusion In this study, we found the magnetic field assessment is an alternative approach to replace the home cage behavior recording and transducer recording techniques for high resolution recording of seizure activity. The present findings might provide some implications in clinical evaluations.

Material and MethodPreparation of animals Male C57BL/6 mice (8 weeks old about 25~30 g) were housed in an air-conditioned room under a 12-h light/dark cycle at constant room temperature (23±2°C) and humidity (50%) with free access to food and water. All experiments were carried out in accordance with theguidelines of the Academia Sinica Institutional Animal Care and Utilization Committee.

Surgery C57BL/6 mice were initially anesthetized with 4% isoflurane (in pure O2), and then placed in a stereotaxic apparatus. Animals were maintained under anesthesia with 1.6% isoflurane and the body temperature were maintained at 36.5~37.5°C during the surgery with a homeothermic blanket system. To record the electroencephalography (EEG) of the cortex, a craniotomy centered 1~2 mm anterior to Bregma and 1.5~2.5 mm lateral of the midline wasperformed, and the Omnetics connector was placed on the scalp.

Drugs Ethosuximide (ETS) and valproic acid (VPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The doses ETS used in this study were 200~600 mg/kg, and VPA used in this study were 200~400mg/kg. All drugs were dissolved in distilled water were administered intraperitoneal (i.p.) 30 min before treatment with PTZ. The doses of each drug wereselected based on the findings of previous reports.

Acute Pentylenetetrazole (PTZ)-induced seizures protocol and behavior recording After the surgery of placing Omnetics connector for one week, A coil magnetic sensor was used to couple with a body-mounted magnet to mice, and placed in a box (15 × 15 × 30 cm). Five minutes later, the mice behavior was observed before and after PTZ i.p. injection (St. Louis, MO, USA).The experimental mice were acutely treated with convulsive doses (50 mg/kg), and the control mice received 0.1 ml/25 g saline injections. After each drug injection, the convulsive behaviors of the mice were observed for 30 min and resultant convulsions were classified and scored according to the criteria of a previous report (Itoh et al., 2004; Itoh and Watanabe, 2009) as follows: 0: normal; 1: immobilization; 2: short myoclonic jerk; 3: myoclonic jerk; 4: generalized clonic seizures (GCS) with kangaroo posture; 5: generalizedtonic–clonic seizures (GTCS) with loss of posture tone.ResultThe experiment protocol and recording system

A

B

CFree moving

PTZ-induced seizure

Evaluation the behavior recording of the PTZ injection before and after

The influence of anti-epilepsy drugs on behavior activity

The behaviors were corresponded to behavior score

EthosuximideA B Valproic acid

B EEG recording

behavior recording

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Behavioral recording-1

Anti-Epilepsy Drug / saline injection

Behavioral recording-2

PTZ / saline injection

5 min 5 min30 min 30 min

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Fig1. A. Timeline for Anti-Epilepsy Drug, and PTZ injection in the experiment. B. Overview of the proposed system. C. An exemplification of the EEG and the behavior (Coil and transducer) recording. D. The video recording of the home cage behavior, which using the self-developed software were measured by an observer for scoring.

Fig2. A. Non-PTZ injection, the mice was free moving in the observation box, and the Fast Fourier transform (FFT) of the free moving recording wave displayed a curve from upper left (low frequency) to lower right (high frequency). B. A specific peak was find by the FFT analysis after the PTZ-induced seizure C. A 19~21 Hz frequency band was a prominentfeature of the group of mice after the treatment of PTZ.

Fig3. The behavior frequency band was blocked by anti-epilepsy drugs: ethosuximide (600 mg/kg) and valproic acid (400 mg/kg). A. The different doses of the ethosuximide from200~600 mg/kg. B. The different doses of the valproic acid from 200~400 mg/kg.

Fig4. The frequency band distinguished the epileptic behavioral from that of spontaneous body movement. Further analysis of the behavior occurring the different score, we found that the behavior occurring 19~21 Hz were corresponded to behavior score No.4 (generalizedclonic seizures with kangaroo posture).