Fig 1. Rotenone inhibition of RC complex I in zebrafish.

Rotenone treatment effects were tested on larval development, brain physiology, and mitochondrial membrane potential. Rotenone inhibits development in a concentration-dependent manner when larvae are treated from 6–36 hours post fertilization (hpf). (A) While control treated larvae developed normally, (B) Rotenone (100 nM) treatment drastically inhibited development, such that embryos did not develop past the tail bud stage. (C) Developmental effects were concentration dependent, as scored both by the larvae arresting before the tail bud stage and their greater size of yolk extension. (D–E) Rotenone induced brain cell death occurred when larvae were treated for 4 hours on 7 days post fertilization (dpf), as indicated by arrowhead in control (D) and rotenone-treated (E) animals. (F) The severe brain phenotype induced by rotenone on 7 dpf was significantly prevented by pre-treating larvae with 50 uM probucol (***, p < 0.001). N=3 biological replicate experiments. (G–I) Rotenone treatment decreased the mitochondrial membrane potential of 7 dpf larval trunk muscle. Relative fluorescence quantitation in a flank skeletal muscle region of interest was performed with TMRE to assess mitochondrial membrane potential (G) compared to Mitotracker green FM (MTG, H) as loading control and indicator of mitochondrial content. (I) Rotenone treatment for 4 hours on 7 dpf induced a significant reduction of mean TMRE brightness normalized to MTG (I). n=6 replicates evaluated. *, p=0.026. Error bars represent (± SD).