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James A Kuchenbecker, Sara Patterson, Michael B Manookin, Ethan D Buhr, Maureen Neitz, Jay Neitz; An ex vivo electroretinogram to study spectral mechanisms and cone pathways in the retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
The electroretinogram (ERG) has been used to study mouse models of human disorders of the cone pathways and to test possible treatments. By varying temporal and spectral characteristics of light stimuli it is possible to analyze different spectral mechanisms and to partially separate responses from different cell types in the outer retina. We explored the possibility of further dissecting the function of cone pathways by combining techniques of spectral and temporal manipulation of light in an ex vivo ERG that allows efficient pharmacological manipulations. The gradient of S-opsin co-expression from dorsal to ventral retina complicates the use of mice as a model for human cone function, but with the ex vivo ERG preparation it is possible to separately isolate and record from ventral and dorsal regions.
Eyes were enucleated, the retina dissected, and small pieces of retina from defined locations were placed in a closed ex vivo ERG chamber and perfused with Ames' solution at 37°C. ERG responses were recorded transretinally to single-flash, flickering and on-off stimuli. Light arriving from the photoreceptor side was from a Gooch & Housego OL 490 Agile Light Source that has an incorporated diffraction grating and Digital Light Processor; it was controlled by custom ERG hardware and software to deliver programmable spectral output capable of producing cone isolating stimuli and narrowband lights for measuring spectral sensitivity functions.
Stable responses with a high signal-to-noise ratio could be recorded for several hours from a single preparation. It was possible to record and compare responses before and after application of pharmacological agents including L-AP4 and GABA antagonists gabazine and TPMPA; finally recordings before and after washout of the drugs were reproducible. ERG spectral sensitivity functions recorded from the ventral retina showed distinctly higher sensitivity to short wavelength lights compared to their counterparts recorded from dorsally derived explants.
An ex vivo ERG system was capable of recording cone mediated, chromatic responses from specific locations in the retina. S-opsin mediated sensitivity was greater in ventral than dorsal retina consistent with the observed gradient of S vs. M opsin expression. The methods describe here enable the study of models of human disorders of the cone pathways and to test possible treatments.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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