The overall goal of this procedure is to perform trans retinal electroretinogram recordings from isolated intact mouse retina to obtain rod and cone driven photo responses. First glass capillaries and a perfusion chamber are prepared for recordings. Both eye cups from dark adapted mouse are hemis effected.
Under infrared illumination and cornea lens and vitreous are removed. The retina is peeled from the pigment epithelium and placed on the filter paper photoreceptor side. Up.The retina on the filter paper is transferred to the perfusion chamber on the microscope stage and placed between two electrodes connected to the head stage of a differential amplifier.
Finally, rod and cone mediated photo responses are recorded from the flat mounted mouse retina. The main advantage of this technique over existing methods like classic single cell recordings, is that it allows drug accessible longtime recordings from rod and con fatter receptors from valve type and genetically modified mice. It is becoming particularly useful for obtaining stable, fatter response from the scarce and fragile mouse.
Cons Begin by preparing the glass capillaries. Make 10 milliliters of 1.2%agar in a hot water bath. Fill glass capillaries with the agar solution using a plastic syringe where the agar quickly solidifies.
Cut the capillaries into halves using a diamond knife. Soak the resulting glass electrodes in mouse electrode solution for at least 24 hours at four degrees Celsius. Now prepare the microscope generally to minimize mechanical and electromagnetic noise.
Mount the microscope on an anti vibration table and shield it within a Faraday cage. However, these features are not essential. Calibrate the light source using a photometer placed at the plane of the retina.
In the case of LED Light Source, use an optical diffuser to achieve light uniformity using plastic tubing and a glass capillary. Make a connection between the lower electrode and the electrode holder. Then fill the lower electrode space of the perfusion chamber with electrode solution.
Make sure there are no bubbles in the electrode, the connecting tubing or the electrode holder. Remove any bubbles by purging the solution with a syringe. After placing the recording chamber under the microscope, connect the lower electrode holder to the positive head stage pole of the differential amplifier.
Then center and align the chamber's lower electrode opening with the stimulus light spot bubble, the bicarbonate buffered perfusion solution with 95%oxygen, 5%carbon dioxide while incubating it in a 40 degree Celsius water bath. The solution pH will become 7.2. Connect the perfusion line to the recording chamber and turn the perfusion on.
The solution flows from the bottle to the recording chamber by gravity. Passing through a ceramic resistor, reheating it to 36 or 37 degrees Celsius. To reduce heat loss, the heater should be located as close to the recording chamber as possible.
Ideally, on the stage of the microscope, set a flow regulator in the solutions path and adjust it to about one milliliter per minute. Now, connect the upper electrode to the second negative pole of the head stage using an electrode holder using an O-ring, bind the thermocouple probe to this electrode with their tips as close as possible so that the temperature reading is taken near the center of the retina. Next, immerse the tip of the upper electrode in the perfusion solution and place it in the center of the chamber.
Allow 10 to 15 minutes for baseline stabilization. Finally, adjust the DC voltage for the heater so that the temperature of the solution is 36 to 37 degrees Celsius. Prepare the mouse by dark, adapting it overnight.
Use wild type mice like C 57 black six for rod driven trans retinal recordings or rod transducing alpha knockout mice, lacking rod signaling for cone trans retinal recordings Before working in the dark, prepare a piece of square filter paper by making a two to 2.5 millimeter hole at its center and greasing its corners. All of the following steps must be performed under infrared light. The dissecting microscope is outfitted with infrared image converters from the hemis sectored eyeballs.
Remove both corneas and lenses. Remove as much of the vitreous as possible during these processes. Proceed by working with one eye cup at a time.
The second eye cup can be saved for several hours in a dish of incubation solution in a light type box saturated with pure oxygen and a room temperature to record from whole retinas. Peel the first retina from the pigment epithelium layer, and if necessary, thoroughly clean it with forceps to remove remaining granules of pigment epithelium. To record from the dorsal region of the retina, remove the ventral part of the eye cup before peeling the retina using a razor blade or micro scissors.
And with the corry markers reference, remove the ventral eye cup. Use a pipette to transfer the isolated retina into a drop of perfusion solution on the lid of a Petri dish. Then add five to six small drops of electrode solution containing barium chloride.
Place the prepared filter with the hole in a drop of perfusion solution with the retina using forceps. Position the retina on top of the filter paper with the photoreceptor side up. Then press it edges on the periphery to flat mount it.
The retina should be centered over the opening in the filter paper. Now transfer the filter paper with the retina to the perfusion chamber and place it above the stimulus spot aligned lower electrode space. Slightly press the grease edges of the filter paper to bond it with the chamber.
Lastly, place the upper electrode over the retina center so the electrode slightly touches the photoreceptor layer. Now proceed with the experiment where making trans retinal recordings. For our analysis, we use peak lamp 10 data acquisition and analysis Software from molecular devices rotten ate our accordance.
We made using a 505 nanometers LED light from this LED source During long recording sessions. To maintain efficient suppression of the glial component of the photo response, the lower electrode solution is changed. Prior to testing, each retina should suffice.
The flash intensity of the LED light source was controlled by a combination of computerized control of the LED voltage switchable resistors and a set of neutral density filters placed between the LED and the retina. The duration of the LED test flash typically 20 milliseconds, was controlled by a computer. These C 57 black six mouse rod driven trans retinal recordings, show traces that represent averages of five to six responses at dim light intensities, and two to three responses at saturating light intensities.
Each trace of this cone driven trans retinal recoding data from the rod transducing alpha knockout mouse represents an average of five to 10 responses at dim light intensities, and three to five responses at saturating light intensities. So after watching this video, we should have a good understanding of how to perform. And eight, our electrogram coatings from isolated mouse retina to obtain rot and con driven photo responses.
