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Fig. 4. NCS-1 knockdown affects the action potential-induced fura-2 Ca²⁺ signal in growth cones in an activity-dependent manner. (A) Representative epifluorescent images of Ca²⁺ signals in L. stagnalis neurites treated with the control dsRNA or NCS-1 dsRNA. The Ca²⁺ ratiometric signals excited at 340 and 380 nm (F_340/380) were obtained at rest (basal) or elicited by two, four or six depolarization-induced action potentials. Action potentials were evoked by 1 second depolarization using an intracellular sharp electrode impaled into the soma. (B) Representative examples showing that the Ca²⁺ signal (F_340/380) was as a linear function of the number of action potentials. The Ca²⁺ signal from a single growth cone region was plotted against the number of action potentials used to evoke the response. The line represents a linear best fit of the data, which shows no apparent saturation of the signal within the range of action potentials used. The slopes of the fits are 0.037±0.004 (control), 0.031±0.003 (control dsRNA) or 0.011±0.001 (NCS-1 dsRNA). The slope was used to quantify the dynamics of Ca²⁺ change resulting from action potential stimulation. (C) Summary of the slope of the fit described in B from three to five cells with treatments as indicated. The mean slope dependencies are 0.037±0.006 (control, n=4), 0.027±0.003 (control dsRNA, n=3), and 0.010±0.004 (NCS-1 dsRNA, n=5). The data are presented as mean±s.e.m. Asterisk indicates significant difference (P<0.05) from the controls. AP, action potentials.

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