Release probability distributions for mathematical modelling were obtained by measurements of readily releasable pool using the styryl dye FM1-43 (Invitrogen, Fisher-Scientific) in rat primary hippocampal neurons co-cultured with astrocytes as described previously (Goda and Stevens, 1998 (link); Letellier et al., 2019 (link)). Images were captured on an inverted Olympus IX71 microscope equipped with an EMCCD camera (Andor Technology, Oxford Instruments) controlled by Metamorph software (Molecular Devices). The extracellular solution consisted of (in mM) 137 NaCl, 5 KCl, 10 D-Glucose, 5 HEPES pH 7.3, 2 CaCl2, 2 MgCl2, 0.01 CNQX, 0.1 picrotoxin at 300 mOsm. Briefly, neurons (used at DIV11-14) were stimulated by a pair of field electrodes (positioned ~10 mm apart) using 40 action potentials at 20 Hz in the presence of 10 μM FM1-43 and for an additional minute in FM1-43 but without stimulation to allow for completion of endocytosis. Subsequently, cells were washed in extracellular solution containing 1 mM Advasep-7 (Biotium) for 1 min to facilitate dye removal, and then the wash was continued for a total period of 10 min. Images were acquired before and after the unloading stimulation, which was 600 action potentials at 20 Hz. The signal remaining was taken as background. The images were analyzed by OpenView software (Kaufman et al., 2012 (link)) kindly provided by Dr. Noam Ziv.
Advasep 7
Manufactured by Biotium
Sourced in United States
Advasep-7 is a non-ionic detergent used for solubilizing and purifying proteins. It is a mild and effective detergent that can help maintain protein structure and function during extraction and purification processes.
Automatically generated - may contain errors
Lab products found in correlation
Fm 1 43fx, by Thermo Fisher Scientific (2 mentions)
Fm1 43, by Thermo Fisher Scientific (1 mentions)
Ix 71 microscope, by Oxford Instruments (1 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Synaptored c2, by Biotium (1 mentions)
Ruthenium red, by Merck Group (1 mentions)
Baclofen, by Bio-Techne (1 mentions)
Fm1 43, by Biotium (1 mentions)
6 protocols using advasep 7
1
Measuring Synaptic Vesicle Release Probability
2
FM Dye Labeling of Active Synaptic Vesicles
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For FM-dye labeling of active synaptic vesicle cycling, cells were exposed to 10 μM SynaptoRed™ C2 (Equivalent to FM®4–64; Biotium; 70027) in Stimulation Buffer consisting of 31.5 mM NaCl, 90 mM KCl, 5 mM Hepes, 1 mM MgCl2, 2 mM CaCl2, 30 mM glucose, and 50 μM D-AP5 (Thomas Scientific; 14539–5) for 2 min. Cells were then incubated with 10 μM SynaptoRed™ C2 in Wash Buffer consisting of 50 μM D-AP5 and 10 μM CNQX in Hibernate A for 5 min. Cells were washed with Wash Buffer alone, Wash Buffer with 1 mM ADVASEP-7 (Biotium; 70029), and finally Wash Buffer alone. Cells were then fixed in 4% paraformaldehyde/4% sucrose in PBS for 10 min at room temperature and immunostained for synaptic markers (details below).
3
FM 1-43 Labeling and Inhibition Assay
4
Fluorescent Dyes and Neurotoxin Protocol
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5
Presynaptic Vesicle Dynamics via FM1-43FX
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Presynaptic terminals were labeled with 10 μM FM1-43FX (Life Technologies) in modified Tyrode buffer using a stimulus train of 40 pulses at 20 Hz or 900 pulses at 10 Hz. FM dye was applied to cells 5 s before stimulation and was removed 30 s after the stimulus train ended. Neurons were washed with 500 μM Advasep-7 (Biotium) in modified Tyrode buffer for 30 s then washed by perfusion of modified Tyrode buffer for 3 min before imaging. Destaining was performed by stimulation with 900 pulses at 10 Hz. After one load and unload cycle, neurons were treated for 20 min with 1 μM cPAF or vehicle. FM dye was then loaded and unloaded two more times. Background correction was performed in ImageJ with the rolling ball algorithm (20 pixel radius), which is designed to correct for unevenly illuminated background. Image stacks were analyzed in Volocity. A maximally loaded image (after 900 pulse load) was used to identify FM positive synaptic puncta using the Find Spot function. This function identifies local intensity maxima above a set threshold within a radius of 0.75 μm. A circular ROI, 1 μm in diameter, was placed around each identified spot to track fluorescent changes for that puncta over time. Only puncta whose fluorescence after the 900 pulse load decreased by 50% or more after an unload cycle were included in the data set.
6
FM1-43 Uptake Assay for Synaptic Vesicle Dynamics
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FM1-43 uptake experiments were performed according to our previously published protocol (Hiesinger et al., 2005 (link)). In brief, embryo fillets were exposed to 10 µM FM1-43FX with or without 30 mM K+ for 5 min and washed with ice-cold zero Ca2+ saline for 5 min containing 0.5 mM EGTA and 1 mM Advasep-7 (Biotium). Embryo fillets were fixed and stained for 5 min with concentrated HRP antibody (1:10) in the absence of detergent and subsequently with highly concentrated Cy5-conjugated secondary antibody. 3D confocal high-resolution datasets were obtained immediately after staining because, in our hands, the fixed FM1-43FX loses >50% fluorescence within 24 h. The surface of HRP-demarcated boutons was reconstructed and the mean voxel intensity in the FM1-43 channel calculated for the total volumes inside (boutons) and outside (muscle). Finally, inside/outside ratios were calculated and are presented in Fig. 4 C . At least 10 embryos were analyzed per genotype.
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