Figure 3

Axonal projection from TL to tectum is glutamatergic. (A) Dorsal view confocal image of EGFP and DsRed fluorescence in the brain of a Tg(hspGGFF23C,uas:egfp,vglut2b:rfp) larva. (B) Magnified views of region indicated by box in (A) showing the EGFP (B), DsRed (B′), and merged (B′′) fluorescence channels. Arrows indicate neurons with EGFP and DsRed colocalization. (C) Van Steensel's cross-correlation functions of EGFP and DsRed overlays. Local maxima of Pearson's Coefficient (PC) near ΔX = 0 indicates colocalization. N = 10 larvae. (D) Dorsal view confocal image of the tectum in a 7 dpf Tg(hspGGFF23C,uas:egfp) larva fixed and immunofluorescently stained with anti-EGFP antibody. Note dense plexus of EGFP-positive axons in SM. (E) Side-view of a single tectal lobe from tectum in (D), including immunofluorescent staining with anti-VGluT1 antibody in red channel. Note superficial EGFP labeling in SM layer and punctate VGluT1 immunofluorescence. Note increased density of puncta in SM layer. (F) High-resolution dorsal view of a SM subregion within tectum in (D). Note overlap between VGluT1 puncta and EGFP-positive plexus and lack of VGluT1 labeling in EGFP-negative regions. (G) Van Steensel's cross-correlation functions of EGFP and DsRed overlays for image in (F′). Local maxima of Pearson's Coefficient (PC) near ΔX = 0 indicates colocalization. N = 4 larvae. Scale bar: 150 μm in (A), 30 μm in (B,E), 75 μm in (D), and 15 μm in (F).