Place the GCaMP 5G expressing rat retina mounted on the MEA with the GCL facing the electrodes on the microscope stage. Connect the perfusion system and set the parameters to constantly perfuse the sample bath with the oxygenated AMS medium. Next, inspect the retina using an inverted fluorescence microscope fitted with a fluorescent lamp, an FITC filter, and a CMOS camera.
Look for an area where stimulating electrodes and the fluorescence from GCaMP expressing cells are visible. In the pulse generator devices software, configure the electrical stimulation parameters like the shape, amplitude, duration, phase delay, and frequency of pulses for application. Connect the camera to the pulse generator using the output trigger signal and set the camera software's capture mode to external start trigger.
Press the start button in the camera software so that it awaits an external trigger to start. Initiate the image acquisition process using the pulse generator software and save the captured images, ensuring the file name includes all the applied electrical stimulation parameters. Now open image J and segment the region of interest using the area selection tools, add it to the ROI manager, and save it as a zip folder using the ROI manager menu.
Extract the mean gray value from the cell somas by navigating to more and clicking multi measure. Enable the measure all 600 slices and one row per slice options to obtain a single table in which columns correspond to ROIs and rows correspond to timeframes. Then extract the centroid from the ROIs using the measure command.
To correct the photo bleaching effect and minimize the background, select approximately 15 to 20 frames from the non-stimulating intervals before each burst and fit these frames to a linear curve to ensure optimal data analysis. The calcium traces of cell somas upon five bursts of pulse trains every 10 seconds during a 60 second image acquisition are shown. Frames from the non-stimulating periods are used to correct the photo bleaching effect.
The relationship between the current required to activate the cells and the distance from the stimulating electrode showed that cells located closer to the stimulating electrode required lower current values to evoke a response.
