Feline Lindhout

134 4 for 10 min with Syt antibodies targeting the luminal side of the synaptic vesicle protein, which were diluted (1:200) in the same original NB medium supplemented with 50 μM bicuculline at 37°C in 5% CO2. Next, cells were fixed, stained with secondary antibodies, and subjected to image quantifications and analysis. Image quantification and analysis Quantifications of fluorescent Syt intensity at presynaptic boutons: Presynaptic boutons were identified by swellings along the axon using expressed RFP as fill, similar as described previously (Bamji et al, 2003; Leal-Ortiz et al, 2008; Spangler et al, 2013). Fluorescent intensity of internalized Syt at each bouton was measured using a circular region of interest with a fixed size of ø 1.39 μm/ø 7 pix. SCRN1 knockdown quantifications: SCRN1 knockdown efficiency was analyzed in cortical neurons (DIV4) co-transfected with RFP and a single SCRN1 shRNA. The average fluorescence intensity was measured of the somatic region without nucleus. Quantifications of SCRN1 recruitment and ERmorphology: For analyzing SCRN1 recruitment to ER in COS7 cells, the number of cells showing obvious enriched SCRN1 localization at ER structures was scored. For analyzing reticular ER structures in COS7 cells, the number of cells containing less than ~ 30% detecTable ER tubules in cytoplasm was scored as “non- reticular ER localization”. Live-cell imaging Live-cell imaging (other than electric field stimulation experiments) was conducted on an inverted microscope Nikon Eclipse Ti-E (Nikon), equipped with a Plan Apo VC 100x NA 1.40 oil objective (Nikon), Plan Apo VC 60x N.A. 1.40 oil objective (Nikon), a Plan Apo VC 40x NA 1.40 oil objective (Nikon), a Yokogawa CSU-X1-A1 spinning disk confocal unit (Roper Scientific), a Photometrics Evolve 512 EMCCD camera (Roper Scientific), and an incubation chamber (Tokai Hit) mounted on a motorized XYZ stage (Applied Scientific Instrumentation), all controlled by MetaMorph (Molecular Devices) software. Cells were imaged in their original medium. During acquisition, the objective was kept at 37°C and the imaging chamber was kept at 37°C in 5% CO2. Relative basal Ca 2+ levels’ measurements Hippocampal neurons were transfected with Ca 2+ indicators GCaMP6f or R-GECO1, and mRFP or GFP to identify transfected neurons and presynaptic boutons. Field of views with axonal structures for all conditions were selected based on similar expression levels, while remaining blind for the expression levels of Ca 2+ indicators. Duo-color time-lapses were acquired of 21 frames with 30-s time interval, with a Z-stack of three planes with 0.5- μm interval for each frame, and cells were treated with 1–10 μM ionomycin (Santa Cruz, SC3592) prior to frame 13. Fluorescent intensities of GCaMPf or R-GECO1 at single boutons were measured for the maximum intensity projections of each frame using a fixed ROI. Fluorescent values were corrected for background fluorescence and normalized to the maximum fluorescent intensity within seven frames after ionomycin treatment (F/F