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.
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.
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 limitations2,3. 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 retina2,3,5,6. 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 holder3,5. 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.
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
2. Animal preparation
3. Equipment preparation
4. Tissue preparation
5. Mounting the tissue on the specimen holder
6. Recording retinal neuronal cell function
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.
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 to obtain large responses include a careful dissection technique, high perfusion rate, and perfusion temperature close to physiological values (35-38 °C in the mammalian retina). Under these conditions, response amplitudes and kinetics in both cell types were relatively stable over time but slowly declined approximately 40-45 min after retinas were mounted on the specimen holder (Figure 3).
Compared to photoreceptors, ON-bipolar cell function is more easily disrupted, for example, by damage to the retina during dissection and mounting or by a drop in the temperature and/or perfusion speed. While reduced temperature in the specimen holder greatly slowed the kinetics of both photoreceptors and ON-bipolar cells, it decreased the amplitude of the b-wave but not the a-wave (Figure 4A). Conversely, slowing the perfusion rate from 2.1 mL/min to 0.6 mL/min reduced the amplitudes of both photoreceptor and ON-bipolar cell responses but did not affect the implicit time (time from stimulus onset to response peak) of either the a- or the b-wave (Figure 4B). Cessation of perfusion for 10 min followed by reperfusion resulted in a complete loss of ON-bipolar cell function with preserved photoreceptor responses (Figure 5).
Figure 1: Ex vivo electroretinogram specimen holder and recording setup. (A,B) The ex vivo ERG specimen holder comprises a dome to mount the isolated retina, which is connected to a perfusion line to continuously deliver Ames' medium. Electrodes are connected through narrow channels to both the photoreceptor side of the retina via the perfusion line and the inner retina through the filter paper glued to the dome. These electrodes are connected to a differential amplifier, which enables measurement of potential differences in the retina in response to light stimuli. (C) The specimen holder is placed onto the stage of a microscope, which has been modified to deliver light flashes and connected to the perfusion line, which delivers heated, oxygenated Ames' medium by gravity. The entire recording setup is shielded by a Faraday cage to minimize electrical noise. This figure has been modified from 9. Abbreviation: ERG = electroretinogram. Please click here to view a larger version of this figure.
Figure 2: Example traces of ex vivo photoreceptor and ON-bipolar cell responses. Addition of pharmacological agents to the perfusate allows quantification of contributions from individual retinal cell types to the ex vivo electroretinogram. Photoreceptor (PR) light responses are isolated in the presence of 100 µM barium chloride (BaCl2), a blocker of K+ channels expressed by Müller glial cells, as well as 40 µM DL-AP4, a glutamate receptor blocker, which inhibits signal transmission from photoreceptors to ON-bipolar cells (B). ON-bipolar cell (ON-BPC) function (C) is determined by subtracting the photoreceptor component (B) from the combined photoreceptor and ON-bipolar cell response in the presence of barium chloride alone (A). Photoreceptor light responses can be obtained from human donor retinas with a death to enucleation delay of <5 h (D), whereas retinas enucleated within 20 min of death frequently also give ON-bipolar cell responses (E) (see 8 for further information). Figure 2A–C is modified from 9. Abbreviations: PR = photoreceptor; ON-BPC = ON-bipolar cell. Please click here to view a larger version of this figure.
Figure 3: Stability of photoreceptor and ON-bipolar cell function in the ex vivo electroretinogram over time. (A) Light responses were recorded every minute from photoreceptors alone in the presence of both 100 µM barium chloride and 40 µM DL-AP4. (B) Dim light flashes in the presence of 100 µM barium chloride alone are heavily dominated by ON-bipolar cell function, although they contain a small photoreceptor component. Light responses from isolated retinas typically stabilize after perfusing the ex vivo specimen holder for 15-20 min, and were stable for at least 20-25 min before starting to decline. Please click here to view a larger version of this figure.
Figure 4: Combined photoreceptor and ON-bipolar cell responses at different temperatures and perfusion speeds. (A) Lowering of the temperature inside the specimen holder from 37 °C to room temperature greatly slowed photoreceptor and ON-bipolar cell kinetics but only reduced the ON-bipolar cell amplitude in the mixed photoreceptor and ON-bipolar cell response. (B) Reduction of the perfusion rate from 2.1 mL/min to 0.6 mL/min resulted in decreased photoreceptor and ON-bipolar cell amplitudes but no change in the response kinetics. Please click here to view a larger version of this figure.
Figure 5: ON-bipolar cell responses are more sensitive to cessation of perfusion. (A) Large photoreceptor and ON-bipolar cell responses were recorded following perfusion with 2.1 mL/min for 20 min. (B) After cessation of perfusion for 10 min followed by reperfusion for 10 min at 2.1 mL/min, photoreceptor responses were present, whereas ON-bipolar cells responses were completely lost. Please click here to view a larger version of this figure.
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 vivo11,12,13. Since then, refined methods have allowed more widespread use of ex vivo ERG recordings14,15, although response amplitudes, particularly those from ON-bipolar cells, remained comparatively small16. To overcome these limitations, retinal function is today more commonly measured with in vivo ERG, despite experimental constraints and more complicated interpretation of the recorded waveforms. Although the ex vivo ERG attempts to replicate physiological conditions as closely as possible, it nevertheless measures retinal function in an artificial environment and in the absence of systemic factors and pathological changes. However, when combined with the in vivo ERG, this limitation can be used to answer important questions, such as whether changes in retinal function in disease are intrinsic to retinal cells or caused by systemic changes17.
Recently, newly designed ex vivo ERG specimen holders have simplified the methodology, making the ex vivo ERG accessible to a broader research community2,3,5,18. Modifications to existing patch clamp or multielectrode array equipment described in this protocol will enable more laboratories to perform ex vivo ERG with minimal financial investments and space requirements. In particular, recent developments in ex vivo ERG technology have amplified the responsiveness of mammalian isolated retinas and resulted in a superior signal-to-noise ratio, which remains good despite additional amplification steps described in this protocol. Nevertheless, the decline of the light responses, mainly from ON-bipolar cells19, has perhaps hampered the more widespread use of the ex vivo ERG. The use of Ames' or Locke's medium results in larger photoreceptor and ON-bipolar cell responses, making them preferable to, for example, HEPES-buffered Ringer solution for ex vivo ERG3. Other laboratories stabilized ex vivo ON-bipolar cell function by supplementing with glutamine or glutamate19. This report demonstrates how to obtain large and stable light responses from isolated retinas, including from human donor eyes, as previously reported8.
Important experimental parameters include the temperature of the retina, which should be kept within the physiological range, and a rapid perfusion rate. The most noticeable effects of lowering the temperature in the ex vivo ERG specimen holder are slower response kinetics in both photoreceptors and ON-bipolar cells and an attenuated ON-bipolar cell amplitude. Although lowering the temperature appears to have little effect on the photoreceptor amplitude in the combined response from photoreceptors and ON-bipolar cells, this may be an artifact due to differential changes in the response kinetics from both cell types.
A sufficient perfusion rate appears to be especially critical for retinal function, most likely to supply oxygen and nutrients and remove waste products. While both photoreceptor and ON-bipolar cell amplitudes are somewhat diminished by a moderate reduction in the perfusion rate, even a short cessation of perfusion abolished ON-bipolar but not photoreceptor function. This implies that photoreceptor responses are more robust and may be more easily preserved in eyes that undergo experimentation with a significant delay, such as human donor eyes8. In this context, it is notable that ON-bipolar cell function is not diminished by storing eye cups in oxygenated Ames' medium for several hours. We therefore hypothesize that the loss of ON-bipolar cell function when the perfusion in the ex vivo specimen holder is stopped may be due to rapid depletion of oxygen and other nutrients in the small volume surrounding the retina in the specimen holder. This is supported by reports that a short delay from death to enucleation is critical to record photoreceptor and, particularly, ON-bipolar cell responses from human retinas, and that postmortem hypoxia is the most likely candidate for irreversible damage to retinal neuronal function8. While experimental parameters for ex vivo ERG were optimized in the mouse retina, they nevertheless successfully improved recording conditions for retinal specimens from large animals and human donor eyes.
The authors have nothing to disclose.
This work was supported by National Eye Institute grants EY02665 and EY031706 and International Retinal Research Foundation to Dr. Vinberg, National Institutes of Health Core Grant (EY014800), and an Unrestricted Grant from Research to Prevent Blindness, New York, NY, to the Department of Ophthalmology & Visual Sciences, University of Utah. Dr. Frans Vinberg is also a recipient of a Research to Prevent Blindness/Dr. H. James and Carole Free Career Development Award, and Dr. Silke Becker of an ARVO EyeFind grant. We thank Dr. Anne Hanneken from The Scripps Research Institute for providing the donor eye used for recordings shown in Figure 2E.
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 |