A library of synthetic cannabinoids (SCs) was screened for their ability to reduce the high velocity seizure-like swim behavior of 5 day old zebrafish larvae. Compounds were screened at (A) 250 µM and (B) 10 µM. Each data point represents the mean velocity change in swim behavior of six fish treated with an individual compound. The red data points represent compounds that failed to go into solution or identified as toxic after 90-min exposure. (C) Summary of compound effects after screening 370 SCs at 250 and 10 µM. The threshold for inhibition of seizure activity was determined as a reduction in mean swim velocity of ≥40%. (D) The total number of compounds identified as positive from the 250 and 10 µM library screen. (E) Heat map representing the 20 compounds which successfully reduced the mean swim velocity by >40% in the 250 and 10 µM screening.

Behavioral screening of 20 compounds which were identified as positive from the library screens. (A) Heat map representing the change of mean swim velocity of the 20 hit compounds. The 20 SCs which were identified from the blind screens were retested at 1, 10, and 100 µM to confirm a dose response effect. Each box represents the percent change in mean velocity from a locomotion assay on at least 6 scn1lab larvae. Compounds marked with a cross were identified as toxic; threshold for positive hits shown by solid line. Scale at right represents the range of % change in mean velocity values. (B) Representative swim behavior traces obtained during a 10 min recording epoch for the top two compounds, #9001523 and #147666.

Electrophysiological assay for compounds identified in the locomotion-based screening assay. (A) Local field potential (LFP) recordings were obtained with an glass micro-electrode placed under visual guidance in the midbrain of agar-immobilized scn1lab larvae that had previously showed reduced seizure-like behavior in the locomotion assay. Representative examples of events classified as interictal- or ictal-like are shown. Scale bars: 1 mV, 0.5 s, (B) Bar graphs show the frequency of epileptiform events in a 10 min recording epoch for scn1lab larvae exposed to DMSO vehicle (scn1lab mutants; n = 17), JWH 018 N-(5-chloropentyl) analog (compound #10521; n = 10), JWH 018 N-(2-methylbutyl) isomer (compound #10690; n = 12), 5-fluoro PB-22 5-hydroxyisoquinoline isomer (compound #14550; n = 11), 5-fluoro ADBICA (compound #14766; n = 13), JWH 018 adamantyl analog (compound #9000799; n = 13), and AB-FUBINACA 3-fluorobenzyl isomer (compound #90001523; n = 11). Mean ± SEM and individual data points are shown. One-way analysis of variance with Dunnet's multiple comparisons was used to test for significance. **p < 0.001; *** p < 0.0001. (C) Representative electrophysiology traces (10 min) are shown for SC compounds 10521, 10690, 14550, 14766, 9000799, and 900015323 compared to an scn1lab mutant zebrafish (red). Scale bars: 1 mV, 10 s.

Structural comparison of SC identified to reduce spontaneous seizures in the scn1lab Dravet syndrome zebrafish mutant. (A) 14550; 5-fluoro PB-22 5-hydroxyisoquinoline isomer, (B) 10521; JWH 018 N-(5-chloropentyl) analog, (C) 14766; 5-fluoro ADBICA, (D) 10690; JWH 018 N-(2-methylbutyl) isomer, and (E) 9001523; AB-FUBINACA 3-fluorobenzyl isomer.