Multichannel EEG Recordings on PTZ-induced Zebrafish Epilepsy Model

Abstract

Despite recent interests in using zebrafish for human disease studies, sparked by their economics, fecundity, easy handling, and homologies to humans, the electrophysiological tools or methods for zebrafish are still inaccessible. Although zebrafish exhibit more significant larval-adult duality than any other animals1, most electrophysiological studies using zebrafish are biased in using larvae these days. The results of larval studies not only differ from those conducted with adults, but also are unable to delicately manage electroencephalographic montages due to their small size. Hence, we developed a methodology of non-invasive long-term multichannel electroencephalographic recording on adult zebrafish using a custom-designed electrode array and perfusion system. The multichannel EEG array was prepared as a flexible printed circuit board (FPCB) based on polymide film. The array had a thickness of 80 μm and was flexible enough to adhere to the curved head surface of zebrafish. Zebrafish were anesthetized by 15 ppm of eugenol, until they were anesthetized in stage 3. When they did not show reflex responses, their head skin was gently dried using cotton swabs. Then, the electrode array was attached onto zebrafish heads. During the recordings, 7.5 ppm of eugenol (2~3ml/min) was orally injected continuously through an intubator. By providing 7.5 ppm of eugenol, animals were successfully maintained in the anesthesia state. After 10 minutes of baseline recording, 15 mM of pentylenetetrazole (PTZ) was orally injected to evoke seizure activities. All recording sessions were performed for 60 minutes using 17 zebrafish, and all the animals survived through the sessions. During the one hour of recording, animals exhibited 4.93 seizures per hour. The seizure activities showed two patterns, high amplitude theta activities and absence-like seizure activities. The high amplitude theta activity events were comparable to seizure pattern in all other species2,3. Our techniques with multichannel recording capability allowed for studying seizure dynamics on epileptic zebrafish for the first time. 71% of seizure activities were onset from telencephalon as expected4. In addition, our results suggested that there is a possibility that some seizures started from the cerebellum, which rarely happens in human5. Our results provide a new pathway for future neuroscience research using zebrafish by overcoming the challenges for aquatic organisms such as precision, serviceability, and continuous water seepage.