Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function.

Detailed experimental procedures and materials can be found in the Supplemental Information . For ratiometric imaging, coverslips with neurons were fixed, permeabilized, and labeled for immunocytochemistry using antibodies conjugated to two different fluorophores. For optogenetic stimulation, primary neuronal cultures were sparsely transfected with ChR2-YFP and stimulated for 48 hr. For quantification of synapse-specific correlation, coverslips were processed three-color immunocytochemistry, and recruitment of presynaptic machinery to the individual Bsn-positive puncta was correlated with the local R A/D values. For live imaging of presynaptic function, neurons were sparsely transfected with the vesicle cycling sensor CMV::SypHy or Ca 2+ sensor SyGCaMP6F and subjected to field stimulation while imaging. Images were analyzed using MATLAB codes (MathWorks). For correlative live-fixed imaging of presynaptic structure and function, live images of SypHy responses were aligned with the fixed ratiometric images using a MATLAB routine ( Figure S6 ). For STORM imaging, samples were fixed, permeabilized, stained for the proteins of interest, and imaged using either a commercially available N-STORM Nikon system or a custom-built setup as described before ( Winterflood et al., 2015 ). Imaging was performed in objective-type near-total internal reflection fluorescence (TIRF) mode. An image-correlation-based drift correction was employed. All data analysis was performed in ImageJ and MATLAB. Statistical analysis was carried out using GraphPad Prism 6.0. Sample distribution was assessed using D’Agostino and Pearson’s omnibus normality test; to assess the significance of differences between datasets, Mann-Whitney test was used unless noted otherwise. Error bars indicate 10–90 percentile range. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Supplemental Information Document S1. Supplemental Experimental Procedures and Figures S1–S6 Document S2. Article plus Supplemental Information