Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Summary

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Abstract

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades^1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome^3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Protocol

1. Preparing Electrodes

1. Prepare the recording and reference electrodes by dipping them both in 1M NaCl and driving 15V between them using a 1 Hz sine wave for ~ 20 s.
2. Make the recording electrodes using a micropipette puller (Sutter Instruments P-97). For patch-pipettes use a borosilicate glass with an outer diameter of 1.2 mm and an inner diameter of 0.69 mm (Sutter Instruments, Cat#B120-69-10). Measure the series resistance through the tip of the electrode. Based on the size of the target cells choose an pipette tip diameter; for cell bodies ~ 20 μm in diameter use ~ 5 MΩ for cell bodies ~ 5 μm in diameter use ~ 12 MΩ.
3. Prepar....

Discussion

Slice recording from vertebrate retinas have been made for decades^1,2, and have been quite successful in providing a deeper understanding of how the retinal circuitry encodes incident light. The advantages of cutting with a vibrating microtome rather than directly with a razor blade is that the retinal slices incur less damage at the surface. With a suction pipette it is easy to remove this damage and target healthy cells just below the surface that retain their connectivity in the retinal circuit, where mos.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Micropipette Puller	Sutter Instrument Co.	P-97
Borosilicate glass	Sutter Instrument Co.	B120-69-10
Polyethylene tubing	Intramedic	PE205
Agar	Sigma-Aldrich	A7002
Low gelling temp agarose	Sigma-Aldrich	A0701
Ames’ Medium	Sigma-Aldrich	A1420
Penicillin-Streptomycin	Sigma-Aldrich	P0781