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A Method of Horizontally Sliced Preparation of the Retina

  • Ryosuke EnokiEmail author
  • Amane Koizumi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 935)

Abstract

Various types of retinal neurons, including amacrine, ganglion, and horizontal cells, expand neurites (dendrites or axons) in horizontal direction and make synaptic or electrical contacts with other cells to integrate the visual information. Many types of ion-channels and receptors are located along these neurites, and these horizontal connections critically contribute to the information processing in the retinal circuits. However, many of previous electrophysiological and immunocytochemical studies employed slice preparations cut by vertical direction in which most of these cells and their neurites were severely damaged and removed. This might lead to the underestimation of active and passive conductance in horizontally expanding neurites, and also missing of morphological information of horizontal structures. Here, we describe an alternative slicing method of horizontally cut preparation of the retina. The slice is made horizontally at the inner layer of the retina using a vibratome slicer after the retina is embedded in the low-temperature melting agarose gel. This horizontal slice preparation enables us to directly access cells in the inner retina by patch-clamp recording, calcium imaging, single RT-PCR, and immunocytochemistry. The method described here would offer an alternative strategy for studying the functions of neurons and neural circuits in the retina.

Key words

Retina Amacrine cell Ganglion cell Horizontal cells Patch-clamp recording RT-PCR Immunocytochemistry Slice 

Notes

Acknowledgement

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.

References

  1. 1.
    Masland RH (2001) The fundamental plan of the retina. Nat Neurosci 4(9):877–886PubMedCrossRefGoogle Scholar
  2. 2.
    Koizumi A, Jakobs TC, Masland RH (2004) Inward rectifying currents stabilize the membrane potential in dendrites of mouse amacrine cells: patch-clamp recordings and single-cell RT-PCR. Mol Vis 10:328–340PubMedGoogle Scholar
  3. 3.
    Briggman KL, Helmstaedter M, Denk W (2011) Wiring specificity in the direction-­selectivity circuit of the retina. Nature 471(7337):183–188PubMedCrossRefGoogle Scholar
  4. 4.
    MacNeil MA, Masland RH (1998) Extreme diversity among amacrine cells: implications for function. Neuron 20(5):971–982PubMedCrossRefGoogle Scholar
  5. 5.
    Azuma T, Enoki R, Iwamuro K, Kaneko A, Koizumi A (2004) Multiple spatiotemporal patterns of dendritic Ca2+ signals in goldfish retinal amacrine cells. Brain Res 1023(1):64–73PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Photonic Bioimaging CenterHokkaido University Graduate School of MedicineSapporoJapan

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