Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Summary

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Abstract

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.

To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows (1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.

Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

Introduction

Visual information is coded as a dynamic spatio-temporal pattern of photoreceptor activation, and the ribbon synapse between photoreceptors and second-order neurons determines the downstream transfer of visual information. The vertical slice preparation of the mammalian retina is a highly valuable tool to study signal processing in vertical pathways, i.e. between photoreceptors and bipolar cells, and between bipolar cells and some types of amacrine cell^1,2,3,4.

However, vertical sectioning always causes s....

Protocol

All procedures were carried out in accordance with the guidelines for animal experiments issued by the Federal Republic of Germany.

NOTE: Because the retinal tissue will be devoid of oxygen during prolonged parts of this procedure, all steps should be carried out as fast as possible. Mice should be dark-adapted at least 3 hr before dissection. To avoid light adaptation of the retina, preparation and slicing should be carried out under dim red light.

1. Preparation of Media and Other Chemicals

1. Prepare Ames' Medium⁶. After dissection, immerse retinal tissue in Ames....

Results

In horizontal slices of the mouse retina, horizontal cell bodies could be easily identified according to their unique morphology with several primary dendrites emerging from a polygonal cell body (Figure 1A). Horizontal cell bodies located near the surface of the slice were routinely accessible for patch-clamp electrodes. During recordings, cells were filled with a fluorescent dye, revealing their intricate dendritic arborization, which closely resembled the morphology of.......

Discussion

In contrast to vertical slices, the dendritic morphology of radially oriented neurons like horizontal cells and amacrine cells is well preserved. Compared to whole retina preparations, these cell types are easily accessible for microelectrodes and can be studied by both electrophysiological and imaging techniques. Here, we focused on the outer retina; however, a similar approach has been reported especially for amacrine cells of the inner retina¹⁰.

The properties of the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ames' Medium	Sigma	A1420
Sodium Bicarbonate	Sigma	S5761
Hepes	Sigma	H3375
Carbogen	Air Liquide
Agarose	Sigma	A9045
35 mm Petri dish (plastic)	Nunc Thermofisher	150318
Glass Petri dish	Chemoline	50-1470-40
Isoflurane	Dispensary university hospital
Vibratome injector blade	Biosystems	39053250
Vibratome	Leica Microsystems	VT1200
Vibrocheck	Leica Microsystems
Microwave
Water bath
Forceps
20 G needles
Spring scissors
Curved scalpel blades
Stereo microscope
Plastic Pasteur pipette
Glass Pasteur pipette