Series (time in seconds) of live-cell images of cytosolic (A) mCherry and (B) GCaMP6s in DD postsynaptic spines reveals (C) dynamic GCaMP6s vs stable mCherry signals, n = 11 movies, 31 spines. (D) GCaMP6s transients occur in neighboring spines more frequently (>50%) than predicted by a random distribution (KS test, p<0.0001). Scale bars = 500 nm. (E–H) VA motor neuron activation is correlated with Ca++ transients in DD1 spines. GCaMP6s fluorescence imaged (at 0.5 s intervals) with periodic optogenetic activation (at 2.5 s intervals) of ceChrimson, detects Ca++ transients with (E) ATR (n = 14) but not with carrier (F) (EtOH) (n = 12). Circles at the top right corner of each panel correspond to red light on (red) for ceChrimson activation vs off (black). Scale bars = 500 nm. (G) GCaMP6s fluorescence throughout the 15 s recording period plotted for ATR (green) (n = 14) vs carrier (EtOH) (black) (n = 12). (H) Plot of the standard deviation (SD) of GCaMP6s signal at each time-point shows that fluctuations in the ATR-treated samples (green boxes) are significantly greater than in EtOH controls (black circles), F-test, *=p < 0.0001. Additionally, SDs are significantly different between timepoints before and after light activation (T6 vs T7 and T11 vs. T12). F-test, ^=p < 0.05. ND = not determined. Purple bars denote interval with red-light illumination (e.g., ceChrimson activation). (I–K) Ca++ propagation to neighboring spines depends on intracellular Ca++ stores. (I) Graphical representation of the experimental paradigm: 561 nm laser excitation at a single spine (excitation area, pink) with subsequent GCaMP6s changes recorded from three different regions of interest at: (1) the excited spine, (2) an adjacent spine and (3) a distant spine. (J) In the wild type, significant Ca++ changes are detected at the excited spine (p=0.0182), adjacent spine (p=0.0319) and distant spine (p=0.0402), n = 15 videos. (K) In ryanodine-treated worms, significant Ca++ changes are not detected at the excited spine (p>0.999), at an adjacent spine (p=0.924) or at a distant spine (p=0.552), n = 16 videos.