This protocol demonstrates the conditions needed to maintain retinal function in ex vivo ERG, particularly the extremely sensitive B wave produced by ON-bipolar cells. With ex vivo ERG, we can measure the function of different types of retinal neurons with a high signal to noise ratio while permitting the easy introduction of pharmacological agents. This method is particularly amenable to quantifying the efficacy of pharmacological agents on retinal function and disease.
To begin, convert a multi-electrode array setup by connecting the ex vivo ERG specimen holder to a differential amplifier via a headstage. The amplifier must be plugged into the analog input of the interface board of the multi-electrode array system. Use the recording software for the multi-electrode array to record and store the input data from the ex vivo ERG.
Set the gain of the differential amplifier to 100 and add an additional 10X voltage amplification, depending on the digitizer specifications. Set the low pass filter to 100 hertz. Alternatively, to convert a patch clamp setup, connect the ex vivo ERG specimen holder via a headstage to a differential amplifier, which must be connected to the headstage of the patch clamp amplifier.
Use the patch clamp system software and the digitizer to record and store the input data from the ex vivo ERG. Set the parameters as shown earlier. Connect LEDs with the appropriate wavelengths to the microscope.
To control light stimuli, use an LED driver controlled by analog outputs from a digitizer. Control the LEDs with recording software that will enable the triggering of light stimuli. Then, calibrate the light output of the LEDs at the position of the retina in the specimen holder using a photodiode.
If necessary, insert neutral density filters to dim the light intensity in the light path. To prepare the electrode, insert a silver silver chloride pellet electrode into a threaded lure connector. Fill the inside of the lure connector with hot glue, and insert a two millimeter socket into the hot glue from the non-threaded side.
Solder a silver wire of the EP-1 electrode to the two millimeter socket. Screw the finished electrode with an O-ring on the thread into the electrode ports of the ex vivo ERG specimen holder. For large eyes, including human donor eyes, clean the globe of the remaining connective tissue and remove the anterior segment and lens.
Use a scalpel to make a cut approximately three millimeters from the limbus. Insert curved dissection scissors into the incision and cut along the limbus to remove the anterior portion of the eye with the lens. Obtain retinal specimens for ex vivo electroretinography with a three to six millimeter disposable biopsy punch.
To mount the tissue on the specimen holder, place the lower half of the specimen holder into a large Petri dish, and fill it with oxygenated Ames'medium such that the dome of the specimen holder is just submerged. Carefully grasp the edge of the retina with fine forceps and transfer the retina onto the dome of the ex vivo specimen holder, photoreceptor side facing up. Lift the specimen holder from the Ames'solution, taking care that the retina stays in place.
Completely dry the plate of the specimen holder to minimize noise, electrical crosstalk, and signal shunting. Then assemble both halves of the specimen holder with four screws and connect the perfusion line. Drive the electrode in the lower half of the specimen holder and connect the anode cable to the inner retinal side and the cathode cable to the photoreceptor side.
Perfuse the specimen holder with at least one milliliter per minute of oxygenated Ames'medium for 10 to 20 minutes to allow light responses to stabilize. Ex vivo ERG enabled the recording of photoreceptor and ON-bipolar cell light responses from the mouse retina. Recording of photoreceptor responses from human donor retinas was possible with up to five hours postmortem delay of enucleation and less than 20 minutes delay for ON-bipolar cell responses.
Under ideal conditions, response amplitudes and kinetics in both cell types were relatively stable over time, but showed a slow decline 40 to 45 minutes after retinas were mounted on the specimen holder. Reduced temperature in the specimen holder greatly slowed the kinetics of both photo receptors and ON-bipolar cells. However, it decreased the amplitude of the B wave but not the A wave.
Conversely, slowing the perfusion rate reduced the amplitudes of both photo receptor and ON-bipolar cell responses but did not affect the implicit time of either the A or the B wave. Cessation of perfusion for 10 minutes followed by reperfusion resulted in a complete loss of ON-bipolar cell function with preserved photoreceptor responses. Sufficient perfusion speed and physiological temperature are critical to obtaining stable responses, in particular, from ON-bipolar cells in the ex vivo ERG.
This protocol allows the in-depth exploration of the differences in photoreceptor function in the human macular and periphery, answering questions that could previously only be carried out in non-human primates.
