Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes
June 27th, 2022
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.
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.
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Overview
This study presents a protocol for enhancing ex vivo electroretinogram (ERG) recording methods to study retinal neuron function. The approach focuses on ON-bipolar cells and photoreceptor functionality using various setups, providing insights into pharmacological effects on the retina in healthy and diseased states.
Key Study Components
Area of Science
- Electrophysiology
- Neuroscience
- Retinal biology
Background
- Challenges in recording retinal function effectively in ex vivo settings.
- Importance of ON-bipolar cells and photoreceptors in retinal health and disease.
- Need for reliable methods to quantify pharmacological interventions.
- Existing techniques often lack accessibility and precision.
Purpose of Study
- To improve the recording of retinal light responses using modified equipment.
- To facilitate the analysis of photoreceptor and ON-bipolar cell functionalities.
- To evaluate pharmacological responses in retinal cells.
Methods Used
- The protocol employs ex vivo ERG with either multi-electrode arrays or patch clamp systems.
- Mouse retina and human donor retinas are utilized as biological models.
- Each setup includes critical parameters for amplification and filtering to ensure accurate recordings.
- The importance of maintaining temperature and perfusion rates during the experiment is emphasized.
- Calibration of light outputs using photodiodes is crucial for controlled stimulation.
Main Results
- Enhanced methods allow stable recordings from both photoreceptors and ON-bipolar cells.
- Responses remain consistent, with slight declines observed after extended mounting times.
- Temperature and perfusion rate significantly affect the amplitude of responses, particularly the B wave.
- Findings clarify the differences in photoreceptor function across human retina regions.
Conclusions
- This study demonstrates a reliable method for exploring retinal neuron functions.
- Improved accessibility allows for advanced pharmacological studies relevant to retinal health.
- The findings enhance understanding of neuronal mechanisms and potential interventions for retinal diseases.
Frequently Asked Questions
Executive Industry Relevance
Optimizing ex vivo ERG conditions enables stable, high-fidelity recording of retinal neuronal responses, particularly from ON-bipolar cells and photoreceptors, supporting mechanistic de-risking in retinal target validation. The method allows quantitative assessment of pharmacological interventions in isolated tissue, reducing systemic variability and improving predictive confidence in preclinical models. This positions the technique as a translational bridge for evaluating retinal therapeutics across species, including human donor tissue.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of retinal neuronal cell-type-specific contributions to light responses, supporting target hypothesis testing and pathway clarification.
- Operational Value: Facilitates pharmacological agent introduction to quantify efficacy on retinal function, improving target de-risking.
- Predictive Value: Stabilized ON-bipolar cell B-wave recordings allow assessment of compound effects over time, supporting lead identification and portfolio triage.
Screening & Assay Development
- Assay Readiness: Optimized perfusion speed and physiological temperature maximize response amplitude and stability, enabling reliable compound screening.
- Quantitative Output: Provides high signal-to-noise measurements of photoreceptor and ON-bipolar cell responses, supporting dose-response and kinetic analyses.
- Scalability: Adaptable to retina samples from small and large eyes, including human donor tissue, supporting cross-species assay standardization.
Translational & Preclinical Research
- Translational Continuity: Enables comparison of photoreceptor function between human macular and peripheral retina, supporting biomarker alignment.
- Disease-Relevant System: Maintains retinal function ex vivo for up to five hours postmortem, allowing evaluation of pathological changes and therapeutic interventions.
- Mechanistic De-risking: Isolates tissue-intrinsic retinal responses, minimizing confounding systemic influences in preclinical evaluation.
Pipeline & Workflow Integration
The optimized ex vivo ERG method integrates into the discovery continuum from early target validation through preclinical evaluation, supporting data-driven go/no-go decisions in retinal therapeutic development.
- Discovery Biology: Supports hypothesis testing of retinal neuronal mechanisms and pathway-specific contributions to visual function.
- Screening: Delivers reproducible, quantitative light response readouts enabling reliable compound evaluation in retinal assays.
- Analytics: Generates kinetic and amplitude data (A and B waves) that allow comparison of treatment effects across conditions and timepoints.
- Translational Research: Connects findings from model systems to human donor retina, supporting preclinical-to-clinical continuity.
- Enterprise Reuse: Establishes a standardized, reusable platform for retinal functional assessment across discovery and preclinical teams.
Operational & Enterprise Impact
- Scientific Value: Provides mechanistic clarity on retinal cell-type-specific responses, reducing ambiguity in target validation.
- Operational Value: Standardized perfusion and temperature controls improve reproducibility and reduce variability in retinal function measurements.
- Strategic Value: Enables earlier, more confident go/no-go decisions by improving predictive confidence in retinal target engagement.
- Portfolio Impact: Supports risk-adjusted advancement of retinal therapeutics by de-risking mechanistic uncertainty in preclinical models.
Implementation Considerations
- Requires expertise in retinal tissue dissection, electrophysiology, and pharmacological agent handling.
- Dependent on specialized equipment including differential amplifiers, LED drivers, photodiodes, and perfusion systems.
- Necessitates standardized protocols for tissue mounting, perfusion speed (at least 1 mL/min), and physiological temperature maintenance.
- Adaptation across model systems requires optimization of biopsy punch size and dissection techniques for varying eye dimensions.
- Practical limitations include gradual decline in response stability after 40–45 minutes post-mounting, necessitating timely experimental execution.
