The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.
Cells: Rat (or mouse) primary hippocampal neuronal cultures (14-28 days in vitro).
Stimulation: Electrical, delivered via two platinum electrodes; 70-90 mV
Microscopy: 60x oil lens on an inverted CCD fluorescent microscope.
Software: Slidebook (Intelligent Imaging Innovations, Santa Monica, CA)
See supplemental methods page for further details
As 300 action potentials (APs) is sufficient to induce at least one round of vesicle recycling, a loading stimulus of 300 APs or more is often given to label the recycling pool of vesicles. Though more intense loading stimuli, such as 900 APs, may allow a nominal number of additional vesicles to be labeled, this also increases the amount of time the dye can embed in extracellular membranes, leading to greater non-specific staining. I have found the wash step to be critical to ensure proper destaining. It is important to perfuse with flow rate 1 to 1.5 mL per minute, usually for 10 + 2 minutes. A single image can be taken ~7 minutes into the wash to monitor the extent of dye removal. If necessary, the flow rate and or wash time can be slightly increased. For our experiments it was undesirable for the wash to proceed greater than 10-12 minutes as this increases the likelihood of losing vesicle-labeling through spontaneous exocytosis events which may occur even when the cell is at rest. For the destaining step, 900 to 1200 APs were administered to release all the labeled vesicles. Additionally another 1200 to 2000 APs were often given to obtain a baseline value of “releasable fluorescence”, used to normalize the destaining data.
The authors have nothing to disclose.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
APV | Sigma | A5282 | ||
FM 4-64 | Molecular Probes | T-3166 | ||
CNQX disodium salt | Sigma | C-239 |