Mitochondrial content.

<p>Mitochondrial content was scored by immunofluorescent staining of larvae expressing mito-GFP in motoneurons. D42>w¹¹¹⁸ or D42>khc^wt larvae were used as controls for the two mutant phenotypes D42>khc^N262S and D42>khc^wt+N262S. (A) Confocal images of immunofluorescent staining of the ventral nerve cord (VNC) of mid-third-instar larvae. Staining for Even-skipped (eve, mangenta) was used to visualize the RP2 and aCC motoneuron (white arrow heads). The medially located interneurons (cyan arrowheads) are negative for mito-GFP (green). No reduction on mitochondrial content was observed in the two mutant phenotypes. Scale bar: 20 µm, and 5 µm in right panels. The star indicates the segment in the left panel that was used for the right panels. (B) Confocal images of immunofluorescent staining showing NMJ 6/7, segment A2 of mid-third-instar <i>Drosophila</i> larvae. In mutant larvae the mitochondrial content at the NMJ was lowered compared to the control larvae. An anti-HRP immunofluorescent staining was used to visualize neuronal membranes. Scale bar: 10 µm. The arrowhead indicates the section in the upper panel that was used for the enlargement shown in the lower panel. (C–E) Quantification of the mitochondrial number (C), size (D) and density (E) at NMJ 6/7, segment A2 of mid-third-instar <i>Drosophila</i> larvae. Mitochondrial Density is the mitochondrial Area Fraction relative to the NMJ size as quantified by the HRP staining. Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. ** p<0.01, *** p<0.001. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line.