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.

Behavior analysis of scn1lab^s552 homozygous mutant larvae during long-term exposure of AEDs. To examine the effect of long-term exposure of AEDs on scn1lab^s552 swim behavior was analyzed using the DanioVision running EthoVision XT software (DanioVision, Noldus Information Technology). As previously described (Sourbron et al., 2017b), 6 dpf scn1lab^s552 homozygous mutants were identified based on pigmentation and arrayed in a 96-well plate. Larvae were treated with 2% DMSO (control) or compound using drug concentrations previously published (Zhang et al., 2015; Sourbron et al., 2016, 2017a). After 22-h incubation at 28°C with a 14:10 h light/dark photoperiod, and 30 min of chamber habituation, the swim behavior of 7 dpf larvae was analyzed for 10 min under dark conditions. Both the (A) total distance moved and (B) velocity were analyzed by normalizing the activity of AED treated larvae to vehicle treated controls (previously described as method A, Sourbron et al., 2017b). Graphs represents mean (±SD) normalized to control treated larvae from three independent experiments using the average from 12 larvae per treatment each time. Statistical significance was determined by one-way ANOVA followed by Dunnet's multiple comparison test. No statistical significance changes in swim behavior of scn1lab^s552 homozygous mutants when exposed to known AEDs or putative AEDs. Recommended treatments (blue), contraindicated treatments (red) and recent experimental treatments (yellow) for DS are shown. Acetaminophen is also shown (gray). These behavioral results, which also fail to distinguish between any of the experimental situations tested, are entirely consistent with the data independently obtained using the iZAP system and fail to support the validation of this low micromolar, 22-h exposure assay as an effective means to identify drugs with antiepileptic activity in scn1lab^s552 homozygous mutants.

Summary of the compound screening results using the scn1lab^s552 homozygous mutant larvae to identify drugs for DS. Using our pharmacologically validated dual-stage assay screening protocol, 2,863 compounds have been blind tested for anti-seizure activity in the scn1lab^s552 homozygous zebrafish larvae. (A) Seven commercially sourced drug libraries were screened including the MicroSource Discovery Systems' International Drug Collection (Baraban et al., 2013) and Pharmakon Collection (Dinday and Baraban, 2015) and Selleckchem's, ion channel library, GPCR compound library, a serotonin modulating compound library (Griffin et al., 2017), a natural product library and a FDA-approved compound library. Compounds highlighted in this plot were reported by other groups as being effective anti-seizure compounds, but could not be confirmed as such in our hands. Specifically, these compounds failed to induce an antiepileptic response when screened blinded as part of these commercial libraries. Blind screening of libraries allows for unbiased testing of compounds regardless of their mechanism of action. (B) A summary of identified mechanism of actions of all compounds screened highlighting the broad range of mechanisms covered by these libraries. Clemizole, trazodone and lorcaserin effectively suppressed seizure activity in the scn1lab^s552 homozygous zebrafish larvae. These compounds have known activity at serotonin 2 receptors. 4.3% of all drugs tested are known to modulate serotonin signaling, however, only these drugs were effective. The serotonin reuptake inhibitor fenfluramine is also effective in suppressing scn1lab^s552 homozygous larvae seizure activity and is currently in clinical trial for DS. (C) The FDA approved compound library, and (D) natural product library were also screened for compounds inhibiting seizure activity. Plots show the locomotor seizure behavior for 5 dpf scn1lab^s552 mutants during the first stage screening. The threshold for inhibition of seizure activity (positive hits) was determined as a reduction in mean swim velocity of 40% (red line). Red data points represent compounds that were classified as toxic as treated larvae have no visible heartbeat or movement in response to touch after 90 min exposure. Green data points represent known AEDs. The natural product huperzine A which has been shown to be effective against hyperthermia induced seizures is labeled. No additional lead compounds were identified in these libraries.

Summary of behavioral screening results for anti-seizure compounds in DS zebrafish larvae. In total, seven commercially available libraries have undergone blind screening for compounds which suppress the seizure activity in scn1lab^s552 homozygous larvae. These include the MicroSource Discovery Systems' International Drug Collection (Baraban et al., 2013) and Pharmakon Collection (Dinday and Baraban, 2015) and Selleckchem's, ion channel library, GPCR compound library, a serotonin modulating compound library (Griffin et al., 2017), a natural product library and a FDA-approved compound library. Plots represent the results from the first blinded screening of each library. A hit is recognized as a compound which reduces the high-speed seizure like swim behavior by more than 40% (>2 S.D.). Once a hit is retested it is then screened by an independent laboratory member. If identified as a hit again, the compound is unblinded and independently sourced for further testing including the second stage electrophysiology assay. The serotonin library exhibited the greatest percentage of positive hits in the first-pass behavioral assay consistent with subsequent identification of four serotonin modulating drugs for the potential treatment of DS e.g., clemizole, trazodone, lorcaserin and fenfluramine. It is also interesting to note that the percentage of toxic drugs is greatest in the ion channel library cohort. Approximately 20% of compounds are identified as toxic when screened at 250 μM as they result in decreased or absent heart beat and/or an absent touch-evoked escape response after 90 min of exposure. When screening at 667 μM, 48% of compounds were identified as toxic. The majority of compounds fail to elicit any significant change in the swim velocity of the larvae.