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.
Solutions
Prepare recording chambers
Our recording chamber consists of a microscope slide with an adhesive well in which small silicone blocks cut out of another adhesive well are placed to provide stability for the tissue slices.
Preparation of retinal slices
To avoid bleaching of the photoreceptors, the dissection is carried out in IR or dim red light light. If one is interested in recording responses from the rod pathway, only IR light should be used (IR dissecting microscope and no room light. Additional IR goggles should be used for procedures not done under the microscope). If one is mainly interested in recording cone light responses, dim red room light or a red flash light can be used. The microscope, however, should still have IR oculars.
If photoreceptor input is irrelevant for the experiment, procedures can be carried out in normal room light using a regular microscope.
Placing the tissue in the recording rig
Visual Stimulation
Visual stimuli are programmed in Matlab using the Psychtoolbox and are projected onto the retina through the top port of the microscope using a microscopy image injector (Mbf bioscience). A Bits++ Digital Video Processor (Cambridge Research Systems) is used to obtain a 14bit luminance scale and to synchronize stimulus presentation with data acquisition. The visual stimuli used in this video are bright or dark bars (width = 460µm) of 100% contrast, which were presented for two seconds on a steady uniform background (luminance = 8 * 104 photons/µm/s, size: 1.84 x 1.38mm).
Electrophysiology
Benefits:
Disadvantages:
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Multiclamp 700A | Patch Clamp Amplifier | Molecular Devices | ||
SZX9 | Dissection Micropscope | Olympus | ||
BX50WI | Upright Microscope | Olympus | Equipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp | |
P-97 | Micropipette Puller | Sutter Instruments | ||
Lucivid | Microscope image injector | Mbf bioscience | ||
8-channel perfusion system | Microperfusion | Parker Hannfin corporation | ||
Bits++ | Digital Video Processor | Cambridge Research Systems | ||
Infrared Oculars | Andere | ITT | ||
Adhesive silicone wells | Andere | Molecular Probes | 20mm diameter, 1.0mm deep | |
Membrane Filters | Filter | Milipore | HAWPO1300 | .45µm HA |
Borosilicate Glass Capillaries | Electrode glass | World Precision Instruments | 1B150-4 | |
Syringe Filters | Filter | Whatman | 6789-0402 | 4mm filters, .2um Nylon Membrane, Polypropylene housing |
IR camera | Micropscope mounted camera | Sony | SPT-M324 | Extrawave HAD, B7W video camera |
Picrotoxin | Reagent | Sigma | P1675 | |
Strychnine | Reagent | Sigma | S0532 | |
Imidazole-4-acetic acid sodium salt | Reagent | Sigma | I7013 | |
Larval tiger salamanders | Animal | Charles E. Sullivan (Nashville, TN) |