Figure 3. Simulation of AP-induced synaptic Ca²⁺ profiles.

(A) Estimation of the simulation volume and Ca²⁺ simulations. The left hand image shows a confocal scan of a 3^rd instar larval NMJ stained against the AZ marker Bruchpilot (BRP) (genotype: w[1118]; P{w[+mC]=Mhc-SynapGCaMP6f}3–5 (Bloomington Stock No. 67739). The right hand image shows a higher magnification of the indicated region. To determine the dimensions of the simulation volume, the average distance of each AZ to its closest four neighboring AZs (k-NND = k^th nearest neighbor distance) was determined. The average inter-AZ distance to each of the closest four neighboring AZs (1- through 4-NND) is depicted on the left. Average and SEM of inter-AZ distances (1-4-NND) are depicted on the right. White scale bars: Left: 5 µm; right: 1 µm. (B) Example illustration of the Ca²⁺ simulation. The simulation volume is a cylinder whose base area (radius 624 nm) is the same as a square with side length of the mean 1–4-NND. The local Ca²⁺ concentration is shown at different time points following an AP-induced Gaussian Ca²⁺ current (the area/height is a free parameter, see Table 2, the FWHM is 0.36 ms). The simulation started at t=0 ms, Ca²⁺ influx was initiated at t=0.5 ms and peaked at t=2 ms. The Ca²⁺ (point) source is located in the AZ center (black dot) and the Ca²⁺ concentration is determined at 10 nm height from the plasma membrane. (C) Example simulation of the local Ca²⁺ concentration profile in response to stimulation with a pair of APs (current was initiated at 0.5 and 10.5 ms and peaked at 2 and 12 ms). Simulations were performed using the best fit parameters of the single sensor model described below (see Figure 4, Table 2). Top left: Ca²⁺ transients in response to the first AP at two distances: 95.9 nm and 122.1 nm (the mean of Rayleigh/integrated Rayleigh). Top right: AP-induced Ca²⁺ transient at 122.1 nm for all experimental extracellular Ca²⁺ concentrations. Bottom left: Semi-logarithmic plot of Ca²⁺ decays toward baseline after the 2^nd transient (residual Ca²⁺) at different extracellular Ca²⁺ concentrations ([Ca²⁺]_ext). Time constant of decay is τ = 111 ms. Bottom right: Residual Ca²⁺ levels at 122.1 nm after 10.5 ms of simulation as a function of extracellular Ca²⁺ concentrations. Data depicted in panel A were collected from 17 different animals. Used genotype: w[1118]; P{w[+mC]=Mhc-SynapGCaMP6f}3–5 (Bloomington Stock No. 67739, panel A). Materials and methods section ‘Fly husbandry, genotypes and handling’ lists all exact genotypes. Values used for graphs can be found in the accompanying source data file (Figure 3—source data 1). GCaMP6m experiment is summarized in Figure 3—figure supplement 1. Ca²⁺ signals for all optimised models (below) are summarised in Figure 3—figure supplement 2.

Figure 3—figure supplement 1. Experiment to determine the dependence of AP-induced Ca²⁺ influx on the extracellular Ca²⁺ concentration.

(A) Example frame of maximum fluorescence during recording of presynaptic GCaMP6m at 6 mM extracellular Ca²⁺. Red dotted line indicates the ROI used for read-out of the fluorescence signal. (B) 10 s fluorescence trace of experiment shown in A. At the 5 s mark, 20 APs are given over 1 s. Subtracting the fluorescence at 4.75 s from the maximum fluorescence gives the value dF plotted in panel D. (C) Baseline-subtracted fluorescence (see Materials and methods for details) traces of 5 different animals over the whole range of extracellular Ca²⁺ concentrations. 8.3 mM EGTA was added at the end to quench Ca²⁺ influx. (D) Quantification of dF (see panel B) per cell and Ca²⁺ concentration. The nonlinear fit with hill coefficient, m, of 2.43 (as previously determined for GCaMP6m Barnett et al., 2017) is indicated as a dashed black line, see Materials and methods for details. Mean is shown as black bars ± SEM. Used genotype: w[1118]; P{y[+t7.7] w[+mC]=20XUAS-IVS-GCaMP6m}attP40 crossed to Ok6-GAL4. Materials and methods section ‘Fly husbandry, genotypes and handling’ lists all exact genotypes. Data summary as well as best fit Hill curve corresponding to the depicted graph can be found in the accompanying source data file (Figure 3—source data 1).

Figure 3—figure supplement 2. Ca²⁺ profiles of all models using best fit parameters (reported in Table 1; Table 2).

Left: Both transients at 1.5 mM extracellular Ca²⁺ concentration at 95.9 nm and 122.1 nm. Middle: For all experimental extracellular Ca²⁺ concentrations an AP induced Ca²⁺ transient at a distance of 122.1 nm distance from the Ca²⁺ source is shown. Right: Semilogarithmic plot of Ca²⁺ decays at the different extracellular Ca²⁺ concentration. Time constant of decay is τ = 111 ms in all models. Plots of Ca²⁺ in the single-sensor model are the same as Figure 3C. Parameters can be found in Tables 1,2.