Method Article

Horizontal Slice Preparation of the Retina

DOI:

10.3791/108

⸱

November 20th, 2006

 ,  , 
1Dpt of Physiology and Biophysics, Dalhousie University, 2Massachusetts General Hospital, Harvard Medical School

In This Article

Summary

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Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Abstract

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Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Protocol

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  1. Prepare an agar block (30 mg/ml, ie 3%, in distilled water). Dissolve and microwave it first and pour it into a glass dish. Keep the dish in a refrigerator at 4 oC.
    Tip: The agar block should be prepared in advance.

  2. Prepare low-temperature-melting agarose gel (25 mg/ml, ie 2.5%, in medium). Dissolve and microwave it. Keep it in the hot water bath at 35 oC.
    Tip: Keep the agarose gel NOT too hot. If it is too hot, it takes a long time to be solidified, and the gel may kill the retina

    .
  3. Euthanize the animal, enucleate an eyeball and open it as an eyecup. To liquefy vitreous humor, the opened eyec....

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Discussion

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There are several important advantages of the horizontal slice preparation of the retina.

First, the morphology of the cells that expand dendrites horizontally, such as amacrine cells and horizontal cells, is preserved. It has been shown that there are over 20 types of amacrine cells in the retina (MacNeil and Masland, 1998), so that it is quite important to compare the morphology of the cells with the physiological property.

Second, in the whole-mount retina, th.......

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Disclosures

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All experimental protocols were conducted according to the National Institutes of Health guidelines for animal use.

Acknowledgements

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We thank Drs. Richard H. Masland and Akimichi Kaneko for giving us valuable advices and suggestions to establish the procedure of the horizontal slice preparation of the retina.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AgarSigma-AldrichA-1296
Agarose LWako Pure Chemical Industries, Ltd.317-01182Low-temperature melting agarose
HyaluronidaseSigma-AldrichType I-S, 300 U/mg
Mediumin mM: 102 NaCl, 3.1 KCl, 2.0 CaCl2, 1.0 MgCl2, 23 NaHCO3, and 10 glucose, maintained at pH 7.8, or, Ames’ Solution (Sigma)
Microwave
Hot water bath35 °C
Vibratome slicerLeica MicrosystemsVT1000S
MF-Membrane filtersEMD MilliporeHAWP01300Filter paper , pore size 0.45 µm
Dissection toolsforceps, blade, pinch, twizzer, etc.
Instant gluee.g. Alonalpha, Krazy Glu
50 mL Syringe
Syringe filterEMD MilliporeSX0001300

References

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  1. Azuma, T., Enoki, R., Iwamuro, K., Kaneko, A., Koizumi, A. Multiple spatiotemporal patterns of dendritic Ca2+ signals in goldfish retinal amacrine cells. Brain Res. , 64-73 (2004).
  2. Koizumi, A., TC, J. akobs, Masland, R. H.

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Tags

Horizontal Slice PreparationRetina SlicingVibratome SlicerPatch Clamp RecordingCalcium ImagingImmunocytochemistrySingle Cell RT PCRDendritic MorphologyRetinal NeuronsAgarose Gel Embedding

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