Figure 1

Figure 1

Electrophysiology analysis of scn1lab^s552 homozygous mutant larvae during long-term exposure of AEDs. For non-invasive long-term monitoring of electrographic seizure activity in larval zebrafish, we used an integrated zebrafish activity platform (iZAP) microfluidic recording system previously described by our laboratory (Hong et al., 2016). The iZAP system consists of three primary components: (i) microfluidic unit, (ii) multi-electrode array, and (iii) integrated electronic unit with multichannel amplifiers for simultaneous monitoring of surface EEG activity on five independent electrodes for up to 12 larvae at an acquisition rate of 1 kHz. For these experiment, scn1lab^s552 homozygous mutants screened from larval clutches based on pigmentation are loaded into the iZAP at 5 dpf and continuously monitored for 24-h in embryo medium (or test drug) supplemented with 300 μM pancuronium. A custom algorithm based on distinct features of the zebrafish electrographic seizure signal and spatial correlation between the 5 surface electrodes was used to detect and score seizure events, as described (Hong et al., 2016). For each test compound, we first obtained a 2-h baseline recording, followed by a 22-h drug exposure; at least seven larvae were tested per drug/per experiment and all experiments included at least one biological replicate. Recordings were normalized to the baseline seizure activity and presented as an EEG index where a ratio of 1.0 would represent no change in activity at 22-h vs. baseline and 0.0 would represent a complete suppression of seizure activity. (A) Plot of all drug treatments and controls (embryo media) tested in the iZAP device using the low micromolar concentrations published in Sourbron et al. (2016), Sourbron et al. (2017b), or Zhang et al. (2015). Recommended treatments (blue), contra-indicated treatments (red), and recent experimental treatments (yellow) for DS are shown; acetaminophen is also shown (gray). Note that electrographic seizure activity diminishes by approximately 60% from baseline in all scn1lab^s552 homozygous mutants larvae with no significant differences noted between putative antiepileptic drugs, control and acetaminophen exposures using these low micromolar, 22-h drug exposures (Sourbron et al., 2016, 2017a). Graph represents mean (±SD). Statistical significance was determined by one-way ANOVA followed by Dunnet's multiple comparison test. (B) Representative raw EEG recordings from one surface electrode channel (of five) are shown for the entire 24-h recording period. Baseline recordings are indicated in black; drug exposures are shown in blue. These raw traces highlight the reduction in electrographic seizure activity seen over time under any recording condition at this stage of scn1lab^s552 larval development. In contrast to these data, but consistent with our acute LFP recording protocol, we previously used the iZAP recording device to demonstrate a significant and reversible suppression of electrographic activity monitored in scn1lab^s552 homozygous mutants (2-h drug exposure epochs) for valproate (1 mM), topiramate (1 mM), lorcaserin (250 μM) and trazodone (250 μM) (Hong et al., 2016; Griffin et al., 2017) further highlighting the discrepancy between acute and chronic drug assays.