Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.
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 decades1,2. 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 microtome3,4. 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.
1. Preparing Electrodes
2. Preparing Solutions
3. Day of Experiment
4. Begin Experiment
5. Slicing
6. Recording
7. Representative Results
Figure 1. Here, light-evoked potentials of a dark-adapted retinal neuron to flashes of light of increasing strength are shown.
Figure 2. Fluorescence picture from a retinal slice where cells have been filled with the fluorophore, Lucifer Yellow. Evoked fluorescence shows the morphology of cells from which patch recordings were made.
Slice recording from vertebrate retinas have been made for decades1,2, 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 most of the cell types have been identified5,6, and that display robust responses to light. Thus patch clamp recordings from dark-adapted retinal slices can allow an investigator to determine the roles played by identified cell classes in neural computations.
The authors have nothing to disclose.
This work was supported by NIH Grant EY17606 (APS).
Material Name | Tip | Company | Catalogue Number | Comment |
---|---|---|---|---|
Micropipette Puller | Sutter Instruments | P-97 | ||
Borosilicate glass | Sutter Instruments | B120-69-10 | ||
Polyethylene tubing | Intramedic | PE205 | ||
Agar | Sigma | A7002 | ||
Low gelling temp agarose | Sigma | A0701 | ||
Ames’ Medium | Sigma | A1420 | ||
Penicillin-Streptomycin | Sigma | P0781 |