Fig. 1: SF-iGluSnFR fluorescence imaging of quantal synchronous and asynchronous release in tens of individual presynaptic boutons supplied by a single axon.

A detailed illustration of the experimental paradigm in a representative wild-type neuron (total N = 16 neurons from 5 culture preparations were recorded). a Reconstructed mosaic image of a pyramidal neuron expressing the SF-iGluSnFR probe. b A region of interest (ROI) in the axonal arbour selected for imaging of presynaptic SF-iGluSnFR responses, corresponding to the yellow box in (a). c Heat maps of SF-iGluSnFR responses to the 1st, 21st, and 41st action potentials (APs) during a 5 Hz train of 51 action potentials. The images are averages of 3 frames from a band-pass filtered image stack immediately after each action potential (see Methods and Supplementary Movie 1). d Maximal projection of the band-pass filtered image stack, revealing locations of all presynaptic boutons that released at least one vesicle during the stimulation train. e Analysis of quantal SF-iGluSnFR responses in two representative boutons (Boutons 1 and 2 in d). Left, somatic action potential escape currents time-aligned with band-pass filtered and deconvolved SF-iGluSnFR signals. Quantal release events were identified as local maxima on the deconvolved traces located above the threshold θ (horizontal dashed lines) corresponding to 4 standard deviations of the background noise (see also Supplementary Figs. 2–6 and Methods). Blue circles mark synchronous and red circles mark asynchronous release events (see Fig. 2 for the definition of the synchronous/asynchronous release time threshold). Right, quantal analysis. To determine the amplitude of SF-iGluSnFR signal corresponding to release of a single vesicle (q, green lines on deconvolved traces) the positions of peaks on the amplitude histograms were fitted with a sum of 4 Gaussian functions (black lines) (Methods). This was then used to calculate in each bouton the total number of vesicles released per action potential (release efficacy n_T) and the fraction of asynchronous release events (n_A/n_T) (see Supplementary Figs. 5 and 6 for more examples).