The overall goal of this procedure is to study the light responses and the morphology of single neurons in the intact retina by using a mouse line that expresses a fluorescent marker exclusively in specific cell types. This is accomplished by first dissecting the eye to get an isolated retina. The second step is to put the retina under a laser scanning microscope and visualize a cell by two photon infrared excitation.
A micro pipette is then used to obtain a whole cell patch clamp configuration for recording and dye filling. Ultimately, this preparation allows recordings of cellular responses to photo stimulation and reveals the morphology of the recorded cell by confocal microscopy. The main advantage of this technique over existing methods like unguided patch clamp recording from the retina is that recording can be visually directed towards a fluorescently labeled cell without stimulating the retina with the excitation light.
This method can help to answer key questions in retinal research, such as what is the contribution of a given cell population to the processing of visual information? Even if the cell population has low density and does not carry any distinct morphological features that may facilitate identification. Begin this experiment by adapting the mouse in the dark for at least three hours before the experiment.
In the meantime, prepare the extracellular solution as described in the accompanied manuscript and bubble it with carbogen at room temperature. Next, sacrifice the mouse and nucleate the eyes with a pair of curved iris scissors. Transfer them to a dish of extracellular solution.
Then place it under a dissecting microscope with a pair of spring scissors. Remove the cornea and ciliary body by opening the eye bulb along the aura serrata. Take out the lens and carefully separate the retina from the pigment epithelium.
After that, cut the optical nerve between retina and pigment epithelium. Remove the retina from the eye cup. Note that the inside of the curled up retina is the ganglion cell side.
The outside is the photoreceptor side. Subsequently, remove the vitreous from the inner retinal surface by gently pulling it off with the aid of a wooden toothpick. Then apply short incisions along the retinal perimeter to facilitate flattening of the tissue.
Next, transfer the retina to the recording chamber with the photoreceptor side down. Spread it out on the glass bottom with a fine brush and immobilize it with a nylon strung frame of stainless steel. Place the recording chamber in darkness under an upright laser scanning microscope.
Super fuse the retinal preparation continuously at not less than five milliliters per minute. With car oxygenated extracellular solution heated to 35 degrees Celsius, the microscope is situated on a shock absorbing air table inside a faraday cage. For electronic shielding, cover the cage with a non-transparent curtain to keep the preparation and darkness.
We also screen off light from computer monitors by red transparent films. Tune the infrared laser to 850 to 870 nanometers or longer wavelengths. Switch to mode locked condition and use two photon excitation to visualize GFP expressing cells.
For patch clamp recording, fill boro silicate glass micro pipettes with the intracellular solution containing a fluorescent probe as described in the accompanied manuscript. Then insert the micro pipette into the holder. Make sure that the reference electrode is in contact with the extracellular solution in the recording chamber.
Next, apply pressure to the micro pipette target A GFP expressing cell using a 40 fold water immersion Objective, Omicron cell bodies are situated in the ganglion cell layer as well as in the proximal part of the interclear layer before the micro pipette contacts with the targeted cell penetrate the inner limiting membrane at the retinal surface. Successful penetration is recognized by the intracellular solution that is driven out from the micro pipette tip and the detachment of the inner limiting membrane from the underlying retinal tissue. The fluorescent dye in the intracellular solution can reveal the micropipets position in the two photon image while approaching the desired cell.
Then release the pressure from the micro pipette and obtain a whole cell patch clamp configuration. In current clamp mode, a usable recording should give a membrane potential of minus 50 to minus 55 millivolts and last for at least 20 minutes. After that, present the visual stimuli created on a computer monitor.
Tune the stimulus intensity by inserting neutral density filters into the beam path and adjust the spatial position of the stimulation to the center of the recorded cell. Next, start the stimulation protocol and record the light responses. Use the stimulus generator to trigger the recording software.
Include a photo diode within the beam path to keep record of the stimulus timing. Be careful not to pull out the cell body when retracting the micro pipette. When it is done for dye filling, let the fluorescent agent diffuse into the recorded cell for 30 to 45 minutes.
Here is an example of the GFP expressing cells in a retinal flat mount of a under their promoter For tyrosine hydroxylase, two populations can be distinguished by the brightness of the GFP signal. Type one dopaminergic cells located in the interclear layer, express weak fluorescence as shown by the arrowheads. In figure A.Type two cells were intensely labeled and have cell bodies either in the layer as shown by arrows in figure A or displaced in the ganglion cell layer as shown in figure B, figure C shows a dendritic stratification of type two cells in stratum three of the inner plexiform layer shown here is an example of the morphology of a type two cell injected with the tracer neuro biotin.
The tracer was later on visualized by binding to fluorescently labeled streptavidin, which is shown here in magenta. Here are different response patterns of a type two cell located in the ganglion cell layer to white light. A prolonged stimulus of three seconds was used for better distinction of the response components at stimulus, onset, and offset Following this procedure.
Other methods like pharmacological experiments, calcium imaging or immunochemistry can be performed in order to answer additional questions like the functional role of a given cell type in the retina circuitry.
