Fig. 1

Identifying AS1, a novel compound that reverses sensitized thermal aversion, from a small molecule screen. A Left: photograph of a 5dpf zebrafish larva in a single arena of a 32-well temperature choice assay plate. Middle: representative traces/motion tracking of a zebrafish larva when both sides of the plate are set to 28.5 °C. No preference is demonstrated. Bottom: representative traces of a zebrafish larva given the choice between 28.5 and 31.5 °C after incubation in AITC. Clear preference for the 28.5 °C side is shown. B Results from screening Plate 43 in the sensitized temperature preference assay. Fish in all conditions except Pool 3 significantly favored the 28.5 °C side of the arena, whereas fish incubated in Pool 3 significantly chose the 31.5 °C side. N = 50 larvae for the control condition, N = 49 for the AITC only condition, N = 64, 52, 57, 63, 63, 61, 59, 63, 54, 58 larvae for Pools 2–11, respectively. C Demultiplexing Pool 45–3. Fish in all conditions except Pool F significantly preferred the 28.5 °C side, whereas fish in Pool F preferred the 37.5 °C side. N = 52, 63, 63, 63, 64, 58, 61, 64, 62, 57 larva for control, AITC, and groups A–H. *** denotes significant difference between the control and Pool F groups and the AITC only group. D The chemical structure of AS1 (4-propan-2-yl-N-pyridin-4-ylbenzamide). E Comparison of AS1 with two other potential novel analgesics, AS2 and AS3. N = 29, 58, 64, 49, 63 larvae for control, AITC, and AS1-3 conditions, respectively. * denotes significant difference from DMSO-treated control fish; # denotes significant difference from AITC-treated fish. *^/# p < 0.05, **^/## p < 0.01, ***^/### p < 0.001. One-way ANOVA with Dunnett’s multiple comparison tests was performed in B and C. One-way ANOVA with Tukey’s multiple comparisons test was performed in E. For all choice experiments (B, C, E), a one-sample t test was performed with a hypothetical mean of 50% to determine if fish were significantly choosing one side of the arena