Figure 4. Mechanism of activity-induced mitochondrial arrest.

(A, B) Perfusing layer II/III pyramidal neurons (postnatal day [P] 5 + 3–7 DIV) with high-potassium medium [50 mM] triggered a massive influx of calcium and significantly reduced mitochondrial motility within 2 min (n = 9 cells; 107 mitochondria, paired t-test, p = 0.04). (C-E) Stimulating synaptic vesicle release with latrotoxin (LTX) interrupted mitochondrial motility entirely. (C) Example kymographs from recordings during baseline, in the presence of tetrodotoxin (TTX), TTX and LTX, and after washout of LTX. Basal calcium levels were elevated and mitochondrial motility was absent during the presence of LTX. (D) Averaged time course of mitochondrial motility and GCaMP6 ΔF/F₀ for the duration of the experiments. Shaded areas and horizontal bars indicate SEMs of values and time points, respectively. (E) Percentage of moving mitochondria across all conditions (p = 0.0058, repeated measures ANOVA, *p = 0.028 (baseline vs. LTX + TTX), *p = 0.022 (TTX vs. TTX + LTX), post hoc t-test with Bonferroni multi-measures correction, n = 5 cells, 92 mitochondria). (F-G) Triggering calcium transients with focal application of glutamate (100 µM) in the presence of TTX did not affect mitochondrial motility significantly (P5 + 3–7 DIV, n = 74 transients from 13 cells, 146 mitochondria, paired t-test, before vs. after, 10.32 ± 2.09 vs. 7.12 ± 1.12, p = 0.1).