Method Article

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

DOI:

10.3791/771

⸱

July 2nd, 2008

 ,  , 
1Program in Neuroscience, Boston University, 2Department of Biology, Boston University, 3Department of Biomedical Engineering, Boston University

In This Article

Summary

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This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

Abstract

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We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents. The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

Protocol

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 Solutions

  • Intracellular solution for ganglion cells (in mM): 100 K-gluconate, 8 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, 4 ATP, .5 GTP, 90 µM Sulforhodamine B (for staining), adjusted to pH = 7.4 with KOH
  • Intracellular solution for bipolar cells (in mM): 90 K-gluconate, 8 KCl , 1 MgCl2, 10 BAPTA, 10 HEPES, 4 ATP, .5 GTP, 90 µM Sulforhodamine  B (for staining), adjusted to pH = 7.4 with KOH
  • Extracellular Ringer’s solution (in mM): 112 NaCl, 5 Glucose, 5 HEPES, 2 KCl, 2 CaCl2, 1 MgCl2, adjusted to pH = 7.75 with NaOH. Oxygenation of the solution is not necessary.
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Discussion

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Benefits:

  1. All cell types are accessible
  2. Easy identification of cell types, in particular if a fluorescent dye is added to the electrode solution
  3. Pharmacological agents can easily reach target cells
  4. The patch clamp technique allows to investigate the role of different ion channels in retinal computations. The same information can not be obtained through extracelluar spike recordings or recordings with sharp electrodes.
  5. This technique can b.......

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Multiclamp 700APatch Clamp AmplifierMolecular Devices
SZX9Dissection MicropscopeOlympus Corporation
BX50WIUpright MicroscopeOlympus CorporationEquipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp
P-97Micropipette PullerSutter Instrument Co.
LucividMicroscope image injectorMBF Bioscience
8-channel perfusion systemMicroperfusionParker Hannifin Corporation
Bits++Digital Video ProcessorCambridge Research Systems
Infrared OcularsOtherITT Visual Information Solutions
Adhesive silicone wellsOtherMolecular Probes, Life Technologies20mm diameter, 1.0mm deep
Membrane FiltersFilterEMD MilliporeHAWPO1300.45ÎĽm HA
Borosilicate Glass CapillariesElectrode glassWorld Precision Instruments, Inc.1B150-4
Syringe FiltersFilterWhatman, GE Healthcare6789-04024mm filters, .2um Nylon Membrane, Polypropylene housing
IR cameraMicropscope mounted cameraSony CorporationSPT-M324Extrawave HAD, B7W video camera
PicrotoxinReagentSigma-AldrichP1675
StrychnineReagentSigma-AldrichS0532
Imidazole-4-acetic acid sodium saltReagentSigma-AldrichI7013
Larval tiger salamandersAnimalCharles E. Sullivan

References

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  1. Cook, P. B., Lukasiewicz, P. D., McReynnolds, J. S. GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina. Journal of Neuroscience. 20, 806-812 (2000).
  2. Heflin, S. J., Cook, P. B.

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Tags

Whole cell Patch ClampRetinal Slice PreparationLight Evoked CurrentsElectrophysiological RecordingsTiger Salamander ModelGanglion Cell RecordingVisual Stimuli ProjectionPharmacological Perfusion SystemElectrode Resistance MeasurementSynaptic Circuitry Analysis

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