Abstracts

Rationale: We present a detection method applied to electrophysiological signals recorded with steel electrodes, positioned on immobilized zebrafish larvae that undergo pentylenetetrazole (PTZ)-induced seizures. Our long-term objective is to investigate the time course of seizure development in zebrafish larvae in regards to electrophysiological, cardiac, and behavioral variables. Methods: Wild-type zebrafish larvae at 4, 5, and 7 days post fertilization (dpf) were immobilized in low-melting agarose (1.2%) prepared with E3 solution after receiving a solution of tricaine  (0.04%) and paralyzing agent d-tubocurarine (10 uM). Heartbeat was monitored visually under a stereoscope. Seizures were induced with application of 15 mM (for 5 and 7 dpf) to 60 mM (for 4 dpf) of PTZ on the outside of the agarose gel, following a previously published protocol (Baraban 2005). We record signals using a medical grade steel electrode (316M) bent in order to touch the dorsal side of the head without impaling it. A second electrode is immersed in the agarose (ground). Signals were recorded at 20k samples per second and 800x amplification with an Intan RHD2000.  We implemented a graphical user interface (GUI) for automated detection of seizure onset and seizure latency; the GUI optimizes signal visualization, filtering, and power spectral density. Figure 1 shows one of its panels. Data can be sub-sampled at a selectable rate. Artifacts are identified by large peaks (see yellow arrows) and replaced with zeroes.  Then the user selects the most appropriate signal for the filters and for the power spectral density (shown as a spectrogram in figure 2). Seizure latency is measured by the detection of a peak in the power spectral density until the signal returns to baseline levels. Results: Data from 14 files containing more than 10 hours of measurements from a total of 5 larvae were analyzed. Signals were band-pass filtered from 50 to 500 Hz. Seizure detection was performed on a sample of the files and validation was done manually against notes taken during the experimental sessions. The times of seizures or myoclonic episodes noted matched the times the program detected those physiological activities, within a window of 30 seconds. The analyzed data showed that myoclonic and seizure episodes lasted from 0.2 seconds up to 4.9 seconds. The validated seizure episodes last longer compared to the myoclonic episodes. Conclusions: The graphical user interface can be leveraged for any measurements (electrical, video, chemical) and expanded to detect in particular electrophysiological variables of interest. We intend to further develop this system to propose a low-cost electrode, acquisition, and data analysis integrated setup for multichannel electrophysiology and chemical analyses for zebrafish studies.  Reference: Baraban S et al. Pentylenetetrazole-Induced Changes in Zebrafish Behavior, Neural Activity, and c-fos Expression. Neuroscience 2005; 131:759-768. Funding: Funding Sources: CEPID-BRAINN 2013/07559-3 and FAPESP 2014/15640-8, Brazil.