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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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. Furth....

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 full.......

Representative 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 parameter.......

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 vivo11,12,13. Since then, refined methods have allowed more.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
2 mm socketWPI2026-10materials to prepare electrode
Ag/AgCl ElectrodeWorld Precision InstrumentsEP1materials to prepare electrode
Ames' mediumSigma AldrichA1420perfusion media
barium chlorideSigma AldrichB0750potassium channel blocker
DL-AP4Tocris0101broad spectrum glutamatergic antagonist
OcuScience Ex Vivo ERG AdapterOcuSciencen/aex vivo ERG specimen holder
Threaded luer connectorMcMaster-Carr51525K222 or 51525K223materials to prepare electrode

References

  1. Perlman, I., Kolb, H., Fernandez, E., Nelson, R. . Webvision: The Organization of the Retina and Visual System. , (1995).
  2. Bonezzi, P. J., Tarchick, M. J., Renna, J. M. Ex vivo electroretinograms made easy: performing ERGs using 3D printed components.

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