Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Although there have been attempts to test retinal implantable devices using this viable method, I thought of a detailed protocol from sample attention to data analysis has been lacking. With this work, we aim to fill this gap, providing researchers from different backgrounds with the necessary tools to confidently embark on neural retinal stimulation experiments. Calcium imaging is a popular technique for studying neural activity that offers several advantages over nanoparticle methods.

It provides cellular resolution and can target the specific cell types. It is particularly useful for testing new images, as it allows discrimination between active and inactive cells upon electrical stimulation. We are introducing a robust methodology for studying the responses of the retinal neurons by the use of calcium imaging.

This approach could offer insights into selective excitation of the cells. A critical step in developing better stimulation protocols, improving implant performance, and advancing the state of the art. Our laboratory focus on developing advanced microscopy techniques.

Currently, we are working in recording calcium dynamics in 3D to gain a better understanding of the interaction in the whole tissue. 2 to 3 weeks before imaging, the vitreous of an anesthetized rats eye is injected with the viral particles carrying the genetically encoded calcium indicator. Using fundus copy and CT, the retinal structure is examined for adverse reactions from the calcium indicator injection.

Two weeks after the injection, the ganglion cells layer or GCL, exhibited fluorescence emission. Then, the retina excision from the euthanized rat is initiated. Take the eyes expressing AAV two kg camp 5G and remove all tissue surrounding the eyeball using small curved forceps and fine spring scissors.

Next, take a three by three centimeter piece of filter paper. Position it on the cover of a 3.5cm dish and drench the filter paper with Ames medium. Position the eyeball on the paper with the anterior segment oriented towards the operator.

Use a pair of straight forceps to hold the eyeball at approximately a 45 degree angle from the dish surface. Use the gap between the straight forceps to guide an incision with a blade, then immerse the eyeball into Ames medium. Use straight forceps and fine spring scissors to separate the anterior and posterior segments of the eye.

Remove the lens carefully using two pairs of straight forceps, and detach the retina from the sclera. Using fine spring scissors, make an incision in the sclera towards the optic nerve and cut until the retina has been successfully separated from the eye.Cup. Transfer the excised piece of the retina onto the mounting membrane using a cut tip plastic pipette.

Then use a pair of straight forceps to flat mount the retina, ensuring the ganglion cell layer is oriented upwards. Next, use a plastic pipette fitted with a 100 microliter pipette tip to remove the media, facilitating adherence of the retina piece to the porous membrane. Next, flip the assembly onto the micro electrode array or meaa, positioning the gxl on top of the electrodes and then fill the sample bath with oxygenated Ames medium.

Place the G camp 5G expressing rat retina mounted on the meaa, with the GCL facing the electrodes on the microscope stage. Connect the perfusion system and set the parameters to constantly perfused the sample bath with the oxygenated Ames medium. Next, inspect the retina using an inverted fluorescence microscope fitted with a fluorescent lamp, an citc filter and a CMOs camera.

Look for an area where stimulating electrodes and the fluorescence from G.Camp 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 soma by navigating to more and clicking multi Measure. Enable to measure all 600 slices and one row precise options to obtain a single table in which columns correspond to ROI, size and rows.

Correspond to time frames. Then extract the centroid from the ROI size 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 cells soma upon five bursts of pulse trains every 10s during a 60s 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.