Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Summary

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Abstract

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Introduction

Electroretinography measures retinal function in response to light¹. It is integral to studying retinal physiology and pathophysiology, and measuring the success of therapies for retinal diseases. The in vivo ERG is widely used to assess retinal function in intact organisms, but it has significant limitations^2,3. Amongst these, the quantitative analysis of individual retinal cell types in the in vivo ERG is hampered, since it records the sum of potential changes, and therefore overlaying responses, from all retinal cells to light stimuli⁴. Furthermore, it does not readily allow addition of drugs to the retina, is vulnerable to systemic influences, and has a relatively low signal-to-noise ratio. These disadvantages are eliminated in the ex vivo ERG that investigates the function of the isolated retina^2,3,5,6. The ex vivo ERG allows the recording of large and stable responses from specific retinal cell types by addition of pharmacological inhibitors and easy evaluation of therapeutic agents, which can be added to the superfusate. At the same time, it removes influences of systemic effects and eliminates physiological noise (e.g., heartbeat or breathing).

In the ex vivo ERG, retinas or retinal samples are isolated and mounted photoreceptor-side up on the dome of the specimen holder^3,5. The specimen holder is assembled, connected to a perfusion system that supplies the retina with heated, oxygenated media, and placed onto the stage of a microscope, which has been modified to deliver computer-controlled light stimuli. To record the responses elicited by light, the specimen holder is connected to an amplifier, digitizer, and recording system (Figure 1). This technique allows isolation of responses from rod and cone photoreceptors, ON-bipolar cells, and Müller glia by changing the parameters of the light stimuli and adding pharmacological agents.

An existing patch clamp or multi-electrode array (MEA) setup can be converted to record ex vivo ERG, either in conjunction with a commercially available ex vivo ERG adapter or a custom polycarbonate computer numerical control (CNC)-machined specimen holder, to measure light responses in retinas from small animal models, such as mice. This modification increases the accessibility of ex vivo ERG while minimizing the need for specialized equipment. The design of the specimen holder simplifies the mounting technique and integrates electrodes, eliminating the need for manipulation of microelectrodes compared to previously reported transretinal ex vivo ERG methods⁷. The perfusion rate and temperature inside the specimen holder are important factors that affect the response properties from photoreceptors and ON-bipolar cells. By adjusting these conditions, the ex vivo ERG can be reliably recorded from the isolated mouse retina over prolonged periods of time. Optimized experimental conditions allow ex vivo ERG recordings in retinal punches from larger retinas, including large animal eyes and human donor eyes⁸.

Protocol

All experiments using mice were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Studies Committee at the University of Utah. Pig eyes for demonstration of this video were obtained postmortem from the slaughterhouse (Sustainable Swine Resources, Johnsonville). Eyes were obtained from human donors after brain or cardiac death with consent for research use through the Utah Lions Eye Bank, the San Diego Eye Bank or Lifesharing, which are fully accredited by the FDA, the Association of Organ Procurement Organizations (AOPO) and the Eye Banks of America Association. The use of human donor eyes had exempt status at the University of Utah (IRB no. 00106658) and ScrippsHealth IRB (IRB no. 16-6781).

1. Setting up the ex vivo ERG

1. To convert a multielectrode array setup, connect the ex vivo ERG specimen holder via a head stage to a differential amplifier, which is plugged into the analog input of the interface board of the multielectrode array system. Use the recording software for the multielectrode 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 Hz.
2. To convert a patch clamp setup, connect the ex vivo ERG specimen holder via a head stage to a differential amplifier, which is connected to the head stage of the patch clamp amplifier. Use the patch clamp system software and digitizer to record and store the input data from the ex vivo ERG. Set the gain of the differential amplifier to 100 and apply an additional 10x voltage amplification via the patch clamp head stage. Set the low pass filter to 100 Hz.
3. Connect LEDs with the appropriate wavelengths (e.g., approximately 530 nm to elicit rod photoreceptor and ON-bipolar cell responses) to the microscope. Control the LEDs with recording software that enables the triggering of light stimuli. To control light stimuli, use an LED driver controlled by analog outputs from a digitizer.
4. 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 into the light path to dim the light intensity.
5. Use a commercially available or custom built ex vivo ERG specimen holder.
   NOTE: Drawings for CNC machining from polycarbonate can be obtained from the authors upon request.
6. To prepare the electrode, insert an Ag/AgCl pellet electrode into a threaded luer connector. Fill the inside of the luer connector with hot glue and insert a 2 mm socket into the hot glue from the non-threaded side. Solder a silver wire of the EP1 electrode to the 2 mm socket. Screw the finished electrode, with an o-ring on the thread, into the electrode ports of the ex vivo ERG specimen holder.
   ​NOTE: Electrodes can be used for a long time. If the pellet surface accumulates dirt and/or gets dark (this can cause high offset voltage and/or electrical drift), it can be "polished" using fine sandpaper.
7. At least 1 day prior to experimentation, glue filter papers onto the domes of the ex vivo ERG specimen holder using epoxy glue, ensuring that the glue does not obstruct the electrode channel (see ³ for video).

2.  Animal preparation

1. Dark adapt animals for at least 6 h or overnight.

3. Equipment preparation

1. Fill the perfusion line of the ex vivo ERG with Ames' medium bubbled with 5% carbon dioxide and 95% oxygen. Connect the perfusion line to a heating element with a DC current source or heat controller near the specimen holder to warm the Ames' medium, so that the retina is kept at approximately 35-38 °C.
2. Fill both halves of the ex vivo ERG specimen holder with electrode solution, seal the perfusion line with luer stoppers, connect the electrodes, and assemble the specimen holder with four screws (see ³ for video).
3. Check the resistance and offset voltage between the electrodes in the assembled specimen holder by inserting the probes of the multimeter into the electrodes.
   NOTE: If there are no blockages, and electrodes are in good condition, the resistance should be below 100 kΩ and offset voltage below 5 mV.
4. Connect the assembled specimen holder to the perfusion line and place onto the stage of a microscope set up to deliver light flashes. Ensure that the specimen holder and perfusion line do not contain any air bubbles.

4. Tissue preparation

1. Sacrifice the animal with CO₂ and immediately enucleate the eyes, or obtain large animal or human donor eyes.
2. Clean the globe of the remaining connective tissue and extraocular muscles. Trim off the optic nerve.
3. On filter paper, carefully place a small incision with a razor blade along the ora serrata, approximately 1 mm from the limbus in the mouse eye. Insert fine vannas scissors into the incision and cut along the limbus to remove the anterior portion of the eye with the lens.
4. Move the eye cup into a dish containing Ames' medium. Grasp the sclera with fine forceps, taking care to not touch the retina. Insert vannas scissors between the retina and the sclera and cut the sclera from the peripheral toward the central part. Take care not to touch or damage the retina.
5. Immobilize the sclera by holding one side of the incision with vannas scissors against the bottom of the dissection dish. Grasp the sclera with forceps on the other side of the incision. Remove the sclera without touching or damaging the retina by pulling apart the sclera on either side of the incision to allow isolation of the retina with minimal damage to the tissue.
6. Remove the anterior segment with the lens from the fellow eye and store the eye cup at room temperature in Ames' solution bubbled with 5% carbon dioxide and 95% oxygen.
   NOTE: Functional light responses from eyes stored in this way can be obtained for several hours.
7. In large eyes, including human donor eyes, clean the globe of the remaining connective tissue and remove the anterior segment and lens, similarly to the procedure described for the mouse eye. Use a scalpel to make a cut approximately 3 mm 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 3-6 mm disposable biopsy punch.

5. Mounting the tissue on the specimen holder

1. Place the lower half of the specimen holder into a large Petri dish and fill with oxygenated Ames' medium so the dome of the specimen holder is just submerged.
2. 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.
3. Lift the specimen holder from the Ames' solution, taking care that the retina stays in place.
4. Completely dry the plate of the specimen holder to minimize noise, electrical crosstalk, and signal shunting.
5. Assemble both halves of the specimen holder with four screws and connect the perfusion line. Dry 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.
6. Perfuse the specimen holder with at least 1 mL/min of oxygenated Ames' medium for 10-20 min to allow time for light responses to stabilize.

6. Recording retinal neuronal cell function

1. Record response families by exposing the retina to light flashes of increasing intensity. For example, record mouse rod photoreceptor response families with light intensities ranging from approximately 10 to 1,000 photons/µm² and those from mouse ON-bipolar cells with light intensities ranging from approximately 0.6 to 20 photons/µm².
2. Measure photoreceptor light responses (a-wave) in the presence of 100 µM barium chloride, which blocks potassium channels in Müller glial cells, and 40 µM DL-AP4, which blocks glutamatergic signal transmission to ON-bipolar cells (Figure 2B).
3. To isolate the b-wave, which originates from the function of the ON-bipolar cells, first record combined light responses from both photoreceptor and ON-bipolar cells in the presence of barium chloride alone (Figure 2A). Then, perfuse for 5-10 min with Ames' medium containing barium chloride, as well as DL-AP4, and record photoreceptor responses to the same light stimuli as before (Figure 2B). Subtract photoreceptor responses from combined photoreceptor and ON-bipolar cell responses, thus calculating ON-bipolar cell light responses alone (Figure 2C).

7. Optimizing ON-bipolar cell function

NOTE: The b-wave, which originates from the ON-bipolar cells, is highly sensitive to the temperature in the specimen holder and the perfusion rate.

1. Maintain a perfusion rate of at least 0.5 mL/min to obtain the b-wave.
   NOTE: A higher perfusion rate of 1-2 mL/min is preferable to maintain a large and stable response from ON-bipolar cells.
2. Ensure that for a given perfusion rate, the temperature in the specimen holder near the retina is close to body temperature (i.e., approximately 35-38 °C).
   NOTE: It is important adjust the temperature of the perfusate, which is heated before it reaches the ex vivo ERG specimen holder, so that it is within the optimal temperature range at the retina.
3. Store eye cups for later experimentation protected from light and in oxygenated Ames' medium at room temperature to maintain normal a- and b-waves for several hours.

Results

Ex vivo ERG enables recording of reproducible and stable photoreceptor and ON-bipolar cell light responses, for example, from the mouse retina (Figure 2A-C). Recording of photoreceptor responses from human donor retinas is possible with up to 5 h postmortem delay of enucleation (Figure 2D) and of ON-bipolar cell responses with a <20 min enucleation delay (Figure 2E). Important parameters...

Discussion

Originally developed in 1865 by Holmgren to measure retinal light responses from the amphibian retina¹⁰, technical constraints initially prevented the ERG from being widely used. Nevertheless, seminal studies by Ragnar Granit and others identified the cellular origins of the ERG and measured photoreceptor and ON-bipolar cell responses ex vivo^11,12,13. Since then, refined methods have allowed more...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 mm socket	WPI	2026-10	materials to prepare electrode
Ag/AgCl Electrode	World Precision Instruments	EP1	materials to prepare electrode
Ames' medium	Sigma Aldrich	A1420	perfusion media
barium chloride	Sigma Aldrich	B0750	potassium channel blocker
DL-AP4	Tocris	0101	broad spectrum glutamatergic antagonist
OcuScience Ex Vivo ERG Adapter	OcuScience	n/a	ex vivo ERG specimen holder
Threaded luer connector	McMaster-Carr	51525K222 or 51525K223	materials to prepare electrode