Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 135 4 max). Relative basal Ca 2+ levels were determined by the F 0/F max ratio, where baseline values (F 0) were obtained by averaging the fluorescent intensities of the five frames prior to ionomycin treatment. Fluorescent recovery after photo-bleaching Fluorescent recovery after photo-bleaching experiments were conducted on the characteristic dense ER clusters in TagRFP-ER expressing hippocampal neurons showing this phenotype, or in regular dense ER structures for control conditions, using the ILas system (Roper Scientific; Fig 4E). Fluorescence recovery of TagRFP-ER in bleached regions can be interpreted as the result from two processes: (i) diffusion of TagRFP-ER within existing ER tubules (Yalcin et al, 2017) and (ii) local ER remodeling within the photo-bleached region. The FRAP area size and imaging settings were kept the same for all conditions. For analysis, fluorescence intensity of the bleached region was corrected for background noise and for overall bleaching occurring during acquisition. Next, the post-bleaching fluorescent recovery values were normalized to the baseline fluorescence, which was defined by the average fluorescent intensity of five initial frames prior to onset of photo-bleaching. Electric field stimulation and real-time Ca 2+ dynamics All experiments were carried out in modified Tyrode’s solution (pH 7.4, 25 mM HEPES, 119 mM NaCl, 2.4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose). Objective was prewarmed to 37°C with objective heater (Tokai Hit). Hippocampal neurons were placed in a stimulation chamber (World Precision Instruments) and stimulated (50 Aps, 20 Hz) by electric field stimulation (platinum electrodes, 10 mm spacing, 1 ms pulses of 50 mA, alternating polarity) applied by constant current stimulus isolator (WPI A385, World Precision Instruments) in the presence of 10 μM 6-cyano-7 nitroquinoxaline-2,3-dione and 50 μM D,L- 2-amino-5-phosphonovaleric acid (CNQX/AP5; Tocris Bioscience). Imaging was performed on an inverted Nikon Eclipse TE2000 microscope equipped with mercury lamp (Nikon). Fluorescence emission was detected using a 40× oil-immersion objective [Nikon Apo, numerical aperture (NA) 1.3] and ET-GFP filter (GCaMP) or ET-mCherry (mCherry), together with a EMCCD camera (Evolve 512, Photometrics) controlled by MetaMorph 7.7 software (Molecular Devices). Images were acquired every 650 ms with exposure times between 50 and 100 ms in 1 × 1 binning mode. Quantitative analyses of GCaMP experiments were performed with custom macros in Igor Pro (WaveMetrics) using an automated detection algorithm as described previously (Wienisch & Klingauf, 2006). Statistical analysis Statistical details are included in corresponding Figure legends. P-values are annotated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. Data processing and statistical analysis were conducted in Prism GraphPad (version 7.0) software.