This study investigates the real-time measurement of intracellular calcium levels in primary rodent cortical neurons using a genetically encoded calcium biosensor. The methodology involves imaging techniques to assess neuronal synaptic activity through changes in fluorescence.
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 cortical neurons with low potassium chloride ACSF for 15 minutes. Then mount the dish on the imaging platform and visualize GCaMP6m fluorescence using a FITC filter and a 20 times or 40 times objective. After selecting the imaging field and engaging perfect focus, take a single still image with bright field FITC and fluorescence marker channels to mark neuronal boundaries.
Next, initiate "Run now" in the acquisition software and carry out the basal recording for three to five minutes. Then perfuse ACSF containing 50 millimolar potassium chloride to the neurons as demonstrated previously, and record for five minutes. When the image acquisition stops, save the experiment and proceed to data analysis using the confocal software.
For image analysis, open the time-lapse images and the confocal software, and align the images by clicking on Image, followed by Processing, followed by Align Current Document. Then select Align to the First Frame. Select regions of interest along the neurites using the ROI selection tool, and also mark an ROI representing background fluorescence intensity. Next, initiate the measure function from the time measurement panel to measure raw fluorescence over time for the selected ROIs. After measurement, export the raw fluorescence intensities to the spreadsheet software.
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