Loss of αsyn does not affect mitochondrial-derived ATP levels at the nerve terminal. Loss of αsyn does not affect mitochondrial-derived ATP levels at the nerve terminal. A–C, ATP levels of hippocampal neurons were assessed using an ATP YEMK FRET sensor, and synaptic boutons were identified with mCherry-synaptophysin. Basal ATP levels in Tyrodes buffer containing glucose and pyruvate were identical in neurons isolated from control and αsyn KO mice (A; n = 14–15 coverslips, not significant (NS) by unpaired two-tailed t test). Electrical field stimulation (10 Hz*60 s, blue lines) in pyruvate buffer without (B) and with (C) 2-deoxyglucose (2DG, 5 mM) and iodoacetate (IAA, 1 mM) to completely block glycolysis reduced ATP levels similarly in neurons in control and αsyn KO mice (compilation of two experiments, n = 6–7 coverslips/group with 15–20 boutons/coverslip). NS for ATP level of αsyn KO versus control groups at corresponding time points. Note that overall ATP levels (control and αsyn KO) decreased after the first electrical stimulation (B and C, p < 0.01 for ATP at 10 min versus 9 min pre-stimulation time points), while the acute drop in ATP levels after the second stimulations did not reach significance. D–F, Synaptic transmission at individual boutons was assessed using a pH-sensitive GFP targeted to synaptic vesicles (VGLUT1-pHluorin), again in pyruvate buffer, as well as 2DG and IAA to force reliance on glycolysis. Neither αsyn KO (D) or shRNA against αsyn (E, F) affected synaptic vesicle cycling after repeated stimulation (10 Hz*60 s, blue lines). Bar graph confirms that shRNA decreased αsyn levels by immunofluorescence (E) (compilation of three experiments, n = 10–12 coverslips/group with 10–15 cells/coverslip). NS for extent of endocytosis [(amplitude endocytosis)/(amplitude exocytosis)] versus respective control by two-way ANOVA and Sidak’s posthoc test. All graphs show mean ± SEM. Divya Pathak et al. eNeuro 2017;4:ENEURO.0216-16.2017 ©2017 by Society for Neuroscience