Name: preparation of horizontal slices of adult mouse retina for electrophysiological studies
Uploaded: 2017-01-27T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies at JoVE.com

This work was supported by the Deutsche Forschungsgemeinschaft DFG FOR701 and FE464/11-1 to A.F. The authors want to acknowledge the excellent technical assistance of Andrea Nerz.

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 ...

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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...

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 cell1,2,3,4.
However, vertical sectioning always causes severe truncation of the dendritic fields of many retinal neurons, leading to considerable loss of synaptic contacts. Especially in the outer retina, horizontal cells are affected due to their laterally spread extended dendritic fields and axon terminal systems5. In a vertical slice preparation, the synaptic input of hundreds of cone photoreceptor terminals to the dendrites of a horizontal cell is thus reduced to a few sparse contacts. This might suffice to study the properties of individual synapses, but it does by no means represent the signal processing capabilities underlying the synchronized activation of photoreceptor ribbon synapses.
We therefore developed a horizontal slice preparation, which leaves almost all of the cone photoreceptor synaptic input to horizontal cell dendrites intact and functional. The plane of sectioning runs parallel to the surface of the retina, ideally cutting through the inner nuclear layer between horizontal cells and amacrine cells. Thus, horizontal slices of the outer retina are created containing, on the presynaptic site, photoreceptors with inner and outer segments as well as synaptic terminals, and horizontal cells and bipolar cells on the postsynaptic site. This preparation is well suited to investigate coordinated synaptic inputs into horizontal cell dendrites by all cone photoreceptors within its dendritic field. It thus might prove useful to better understand the workings of the photoreceptor ribbon synapse, which is crucial for the transfer of visual information from the matrix of photoreceptor activation into a postsynaptic response.

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preparation of horizontal slices of adult mouse retina for electrophysiological studies

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&#160;(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.

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...

Watch this Scientific Journal Video about Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies at JoVE.com

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

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.

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

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