JoVE Encyclopedie van Experimenten
Neurowetenschap
0 weergaven • 3:18 min. • June 17th, 2025
Take primary rodent cortical neurons cultured on a coated glass-bottom dish containing media.
These cells express a genetically encoded ALS-associated protein and a calcium biosensor.
Replace the media with a low-potassium chloride buffer.
Incubate to maintain resting neuronal activity and baseline calcium levels.
Mount the dish on a confocal microscope stage and position the perfusion tubing.
Using bright-field illumination, focus on the neurons, then switch to fluorescence imaging.
Using the microscope’s laser, excite the biosensor and record the fluorescence emission to determine baseline intracellular calcium levels.
Next, perfuse the dish with high-potassium chloride buffer.
The high potassium concentration depolarizes the neurons, opening voltage-gated calcium channels at the presynaptic terminals. This allows calcium influx, which initiates neuronal synaptic activity.
The increased intracellular calcium binds to the biosensor, causing a conformational change that enhances fluorescence.
Continue recording fluorescence to measure real-time changes in intracellular calcium levels, revealing neuronal synaptic activity.
48 hours post-transfection with GCaMP6m, incubate the primary rodent c
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