Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Summary

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Abstract

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

Introduction

As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural....

Protocol

Ethical approval – procedures involving animal subjects were conducted in accordance with the rules and regulations of the National Institutes of Health guidelines for research animals, as approved by the institutional animal care and use committee of Baylor College of Medicine.

1. External and Internal Solutions

1. Use mammalian Ringer's solution during retina dissection and as the external solution in subsequent electrophysiological recording. Prepare the mammalian Ringer's solution from 10x stock solution (without calcium) on the day of recording, and add CaCl₂ drop-wise after 15 min of carbogenation (95% O₂ and 5....

Results

Representative recordings of ON- and OFF-type SACs from a deafferentated mouse retina are shown in Figure 1. Cholinergic cells in both GCL and INL can be reliably identified by tdTomato fluorescence and targeted for whole-cell patch clamp recording under DIC (Figure 1A) to reveal oscillation of their membrane potentials (top traces) and the synaptic currents that drive it (bottom traces, Figure 1B). Inhibitory and excitatory synaptic curr.......

Discussion

Many labs have recorded from GCL neurons in the flat-mount preparation^15-18, but our procedures allow recording from INL neurons. We hereby emphasize several steps that are critical for successful routine recordings.

The freshness and flatness of the retina are important for penetrating it with a recording pipette. In this regard, the firm attachment of the retina to the punched nitrocellulose membrane is paramount and is achieved by transient absorption of solution followed by time.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fixed-stage fluorescent microscope with DIC	Olympus	BX51-WI
Micromanipulators	Sutter	MP-225
Patch clamp amplifier	A-M System	AM2400
AD converter	National Instrument	NI-USB-6221
Heater controller	Warner Instrument	TC-324B
Inline heater	Warner Instrument	SC-20
Peristaltic pump	Rainin	Dynamax
pipette puller	Sutter Instrument	P-1000
Glass tube with filament	King Precision Glass	Customized
Stimulator	A.M.P.I.	Master-8
Biocytin	Sigma	B4261
NaCl	Sigma	S6191
KCl	Sigma	P5405
NaHCO3	Fisher	BP328-1
Na2HPO4	Sigma	S0876
Na2HPO4	Sigma	S5011
CaCl2	Sigma	C5670
MgSO4	Sigma	M1880
D-glucose	Sigma	G6152
K-gluconate	Sigma	G4500
ATP-Mg	Sigma	A9187
Li-GTP	Sigma	G5884
EGTA	Sigma	E0396
HEPES	Sigma	H4034
KOH	Sigma	P5958
Cs-methanesulfonate	Sigma	C1426
CsOH	Sigma	232041
Syringer filter	Nalgene	171
1 ml syring	Rainin	17013002
10 μl pipette tip	Genesee Scientific	24-130RL
Streptavidin-488	ThermoFisher	S-11223
10x PBS	Lonza	17-517Q