Fig. 4.

Post-traumatic seizures and unresponsive zebrafish behaviours increase with more severe injury. To assess decreased locomotor activity following higher injury and pressure intensities, seizures and loss of consciousness were measured manually. (A) Zebrafish larvae were recorded after injury using either the foam block or clamp syringe holder. Larvae were manually scored (blinded) for the presence of seizure behaviour using three categories: no seizure, stage I seizure, or stage II/III seizure. The proportion of larvae displaying seizure trended with increased maximal pressure levels (N=48 no TBI, N=24,100 g clamp, N=24,200 g clamp, N=24,300 g clamp, N=24,100 g foam block, N=19,200 g foam block, and N=22,300 g foam block injured larvae. Each experimental group was repeated twice). (B) A threshold of 1 min or more of inactivity was set to quantify larval inactivity after injury in the videos used for manual seizure scoring. The total duration of these inactive bouts was quantified. Inactivity was significantly higher than no TBI controls for the highest pressure clamp injury group (300 g) and all foam block injury groups (P<0.05-0.001). (C) Following TBI larval zebrafish display a stunned phenotype where they do not respond to stimuli in the form of a fin poke, whereas non-injured fish respond vigorously. The proportion of larvae that exhibit this stunned phenotype increased for higher injury intensity groups versus lower injury groups (purple=highest intensity, N=20; blue=middle intensity, N=13; green=lowest intensity, N=11. Each group had two replicates). Across experiments, dead larvae were removed from quantification. Larvae were verified as dead by a lack of heartbeat in the fin poke response assay and zero movements while floating during video recording. A one-way ANOVA with Dunnett's multiple comparisons of means was used to test for statistical significance between injured larva inactivity and no TBI inactivity (*P<0.05, ** P<0.01, ***P<0.001).