SV exo-endocytosis assays were performed as previously described (21, 42). Neurons were stimulated with an electric field generated in custom-made chambers housing 18-mm coverslips with platinum electrodes (8-mm distance between electrodes). For the generation of the electrical impulses, we used a Stimulus Isolator combined with an A310 Accupulser Stimulator (both from World Precision Instruments, USA) with an initial nominal output of 100 mA. Stimulation was performed either on neurons expressing the bidirectional series of pBI-CMV1-pHluorin plasmids (VAMP-2 based) or coexpressing the orange mOr2-Synaptophysin (mOr2-SypHy) indicator of SV recycling (21) together with the Biosettia plasmids for down-regulation. Coexpression was always confirmed by imaging both the enchanced GFP and the mOr2 channel. To test the efficiency of the pBI-CMV1-pHluorin plasmids, we included, in the initial screening, a version encoding the TetTxLC, which cleaves VAMP-2 and blocks neurotransmitter release. Unless otherwise stated, live experiments were performed in Tyrode’s solution [124 mM NaCl, 5 mM KCl, 30 mM glucose, 25 mM Hepes, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.4)] supplemented with 10 μM CNQX (Tocris Bioscience, Cambridge, UK) and 50 μM 2-amino-5-phosphonopentanoic acid (D-AP5; Tocris Bioscience, Cambridge, UK) to avoid spontaneous network activation. At the end of each experiment, 50 mM NH4Cl was applied to evaluate total SV content. Live imaging was performed with an inverted Nikon Ti epifluorescence microscope (Nikon, Tokyo, Japan) equipped with a Plan Apochromat 60× 1.4 numerical aperture oil immersion objective, an HBO-100W lamp, an IXON X3897 Andor camera (Northern Ireland, UK), and an Okolab cage incubator system (Okolab, Ottaviano, Italy) to maintain a constant temperature of 37°C. The system was operated through the NIS-Elements AR software (version 4.20; Nikon). ND2 Images were imported using the Bio-Formats (Open Microscopy Environment) plug-in. For quantifications, the difference in fluorescence intensity before and at the peak of the electrical stimulation (ΔF) was normalized to the maximum of fluorescence (Fmax) measured upon NH4Cl application. For simplicity, all ΔF/Fmax values are expressed as a percentage of the signal measured in control cells (either expressing pBI-CMV1-pHluorin or the scramble shRNA vector from Biosettia). Each experiment (N) corresponds to a different neuronal preparation. For each neuronal preparation, the ΔF/Fmax values were collected and averaged from ~500 synapses from three or more coverslips. Image analysis was performed by identifying the boutons in the exocytotic hotspots with a custom-made National Institutes of Health ImageJ macro based on the “Time Series Analyzer” ImageJ plugin (available at For visualization of calcium dynamics, neurons were loaded in their own medium for 30 min with the acetoxymethyl ester version of Cal-590 (Cal-590-AM; AAT Bioquest), a fluorescein-based Ca2+ indicator with single-photon excitation and emission peak wavelengths of 570 and 590 nm, respectively. After a 10-min reequilibration in their culture medium without dye, coverslips were quickly washed in Tyrode’s solution. Neurons were imaged and stimulated in the same settings as for the SV exo-endocytosis assay. Image analysis measuring signal intensities was performed using custom ImageJ macros. Briefly, circular regions of interest were placed on transfected cell bodies (identified by the GFP signal), and fluorescence was background-corrected. For each condition more than eight fields from three different preparations were measured. For the analysis of spontaneous activity, cells were imaged in the absence of CNQX and D-AP5, peaks surpassing baseline noise by at least twofold were recorded, and the frequency was determined. For stimulated activity (in the presence of CNQX and D-AP5), ΔF was calculated in a 10-s time frame around the stimulation maximum. ΔF was then normalized to the baseline signal measured just before the beginning of the stimulation (F0). For all experiments with neuroLNC overexpression, we also performed the analysis of the fluorescence at the peak maximum (which, depending on the timing of the imaging and of the stimulation, might be slightly shifted in different experiments).

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