An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

Summary

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Abstract

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina ¹. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.

The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes ^{2 3 4} and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

Protocol

Animal Use Statement: Animals were cared for in accordance with guidelines described in Guide for the Care and Use of Laboratory Animals, using protocols approved by the University of Minnesota Institutional Animal Care and Use Committee.

1) Preparation of solutions

1. Before performing electrophysiological recording, intracellular, extracellular, and enzyme solutions need to be prepared.
2. Extracellular solution: mix one bottle (8.8g) of powdered Ames' medium (Sigma) with 1 l of H₂O and 1.9 g of sodium bicarbonate (23 mM). At all points the extracellular solution is saturate....

Discussion

This technique is applicable to any retinal ganglion cell recordings from an isolated retina. Though in the mouse line used above intrinsically photosensitive, melanopsin-expressing RGCs are labeled with EGFP^{2, 5}, this protocol is readily transferable to other fluorescently labeled RGC lines or can be generalized to record from randomly selected RGCs as well. This technique is particularly valuable because the dendritic arbor of each RGC and its respective input neurons are left entirely intact. Recordings ca.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ames’ Medium	Sigma-Aldrich	A1420
Collagenase	Worthington Biochemical	LS005273
Hyaluronidase	Worthington Biochemical	LS002592
Sodium Bicarbonate	Sigma-Aldrich	S5761
AlexaFluor 594 Hydrazyde	Invitrogen	A10442
Neurobiotin	Vector Laboratories	SP1120
Electrodes	Sutter Instrument Co.	BF120-69-10
Forceps	Fine Science Tools	11252-30
Ophthalmologic Scissor	Fine Science Tools	15000-00
Streptavidin 594	Invitrogen	S32356
Vectashield	Vector Laboratories	H-1000
Goat Serum	Jackson ImmunoResearch	005-000-001
Triton X-100	Sigma-Aldrich	T8787
Paraformaldehyde	Electron Microscopy Sciences	19210