Fig. 2

Measurement of axonal ATP levels before and after injury and ischemia. A, B Schematic (A) and color-coded heatmap index (B) of the GO-ATeam2 ATP probe. GO-ATeam2 is a FRET-based and red-shifted ATP sensor (Nakano et al. 2011) that emits at 560 nm (OFP) when it is bound to ATP, and 510 nm (GFP) when it is free from ATP, and thus higher 560 nm/510 nm intensity ratios indicate relatively higher intracellular ATP levels. C-E Schematic (C), representative images (D), and quantifications (E) showing the dynamic changes in axonal ATP levels within axon bundles and growing tips after vacuum aspiration-based axotomy. WT or snph KO cortical neurons in microfluidic chambers were infected with lentiviruses encoding GO-ATeam2 and imaged at DIV14 before or at different time points post injury. Scale bars, 50 μm (bundles) and 10 μm (tips). F-H Schematic (F), representative images (G), and quantifications (H) showing the dynamic changes of cytosolic ATP levels within cell bodies and axons after the OGD-R treatment of cortical neurons. Cortical neurons were infected with lentiviruses encoding GO-ATeam2 and imaged at DIV14 before or at different time points after OGD-R. Scale bars, 10 μm. Data were quantified from n = 30 (E) or n = 22–43 images (H) from more than 3 chambers per condition in three independent experiments. Data were displayed as the mean ± SEM by one-way ANOVA with Tukey’s multiple comparisons test