Homeostatic changes in presynaptic calcium influx detected with SyGCaMP2. A, Fluorescence image of cultured hippocampal neurons expressing SyGCaMP2 (left) and pseudocolored images showing the relative change in fluorescence 0.3 s from the beginning of a train of 3 APs delivered at 20 Hz. B, The response to 3 APs was larger in a culture treated with TTX for 2 d. Note the variability in the amplitude of the presynaptic calcium signal between different boutons. Scale bar, 10 μm. C, Averaged SyGCaMP2 response to 1 AP (left) and 3 APs (right). Control cultures are black (n = 805 synapses), TTX-treated cultures are red (n = 1200), and gabazine-treated cultures are green (n = 386). Error bars are SEM. D, Peak amplitude of the SyGCaMP2 signal plotted as a function of the number of APs delivered at 20 Hz. Line fits have the following slopes: control (black), 0.078 ± 0.003; TTX (red), 0.104 ± 0.003; gabazine (green), 0.063 ± 0.004. Error bars are SEM.