Figure 3

DFP exposure caused neuronal hyperexcitation. (a) As experimental set-up, 5 dpf Tg[Huc:GCaMP5G] larvae were exposed to either 15 µM DFP or vehicle (1% DMSO), and calcium transients were recorded in brain neurons during the next 6 h. (b) Scheme of a 5 dpf larva head with the red box showing the region of interest in the brain, uncovering the optic tectum (OT). (c,d) Snapshot views of calcium imaging in a 5 dpf Tg[Huc:GCaMP5G] larva brain showing baseline calcium transients (c in Fig. 2e) and seizure-like hyperactivity seen 3 h after exposure to 15 µM DFP (d in Fig. 2f). (e,f) Baseline calcium transients detected in 5 dpf Tg[Huc:GCaMP5G] control larvae (n = 3) (e) and massive calcium transients detected in 5 dpf Tg[Huc:GCaMP5G] larvae exposed for 6 h to 15 µM DFP (n = 4) (f). (g) Amplitude of calcium transients detected in 5 dpf Tg[Huc:GCaMP5G] larvae at different time points following exposure to either 15 µM DFP (n = 4) or vehicle (n = 3) (two-way ANOVA with Sidak’s multiple comparisons test: n.s., non-significant; **, p < 0.01; ***, p < 0.001). (h) Number of calcium transients showing ΔF/F₀ > 0.04 in 5 dpf Tg[Huc:GCaMP5G] larvae at different time points following exposure to either 15 µM DFP (n = 4) or vehicle (n = 3) (two-way ANOVA with Sidak’s multiple comparisons test: n.s., non-significant; **, p < 0.01; ****, p < 0.0001). (i) Pattern of calcium transients seen in 5 dpf Tg[Huc:GCaMP5G] larvae exposed for 5 h to 15 µM DFP and then to 15 µM DFP + 40 µM diazepam (DZP) for an additional hour. (j) Number of calcium transients showing ΔF/F₀ > 0.04 in 5 dpf Tg[Huc:GCaMP5G] larvae exposed for 5 h to 15 µM DFP and then to 15 µM DFP + 40 µM diazepam (DZP) for an additional hour (n = 6) (Student’s unpaired t-test: **, p < 0.01).