For imaging, cultured neurons were placed in a stimulation chamber between parallel platinum wires
(RC-49MFSH, Warner Instruments). Stimulation (300 bipolar pulses of 10 V/cm, each of a duration of 1 µs, at 20 Hz for 15 s), was delivered using a high-power stimulus-isolation unit (SIU-102B, Warner
Instruments) driven by an isolated pulse-stimulator (2100, A-M Systems). 50 images were obtained (43 at 0.2 Hz and then 7 images at 0.125 Hz) per experiment. At least 30 synaptic regions of
interest (ROIs) were analyzed in each case. The baseline sypHy fluorescence (F0)
in each synapse was the average value measured in 6 pre-stimulation images. The
fluorescence increment at time t [DF(t)=F(t)-F0] was normalized by the baseline value for each synapse. Synaptic DF(t)/F0 values were averaged across synapses in
each experiment (shown as symbols in bar-chart graphs). These were averaged to
obtain mean values for each experimental condition. Non-responding synaptic
puncta were excluded. Experiments were performed using at least three
independent cultures on different days. To confirm the presence of h-α-syn-mCherry and tagBFP-synapsins, fluorescent-tagged proteins were imaged before each experiment. All pHluorin assays were performed at room temperature. Fluorescence measurements were performed on a Nikon TiE
inverted microscope driven by the NIS-elements software package (Nikon). The
microscope was equipped with an Andor Neo 5.5 sCMOS camera (Oxford
Instruments), a 40X 0.75 NA Plan Fluor objective, EGFP and Cy3 filter cubes
(TE-series, Chroma), BFP, mCherry and Cy5 filter cubes (Semrock), and a
perfect-focus mechanism (Nikon).