The overall goal of this procedure is to perform whole-cell patch clamp recording from inner retinal neurons in the flat-mount mouse retina preparation. This method can help answer key questions in the retinal neurobiology field such as the diversity of neurons, their synaptic connections under the normal and pathological conditions. The main advantage of this technique is that both the vertical and lateral connections are preserved allowing the retina circuits with large lateral components to be studied.
Generally, individuals new to this method will struggle because retina is a thin, delicate tissue which must be handled with great care and patience. Practice, however, is the best way to master it. To begin this procedure, roll the mouse eyeballs in a clean paper towel to remove any blood.
Then, immerse them in the carbogenated mammalian Ringer solution. Next, punch a hole on the limbus with a 23-gauge needle and bisect the eyeballs by cutting along the limbus with micro scissors. Then, remove the cornea and lens using fine forceps.
For retinal whole-mount, gently peel the entire retina off the pigmented epithelium with fine forceps and make four 1.5 to two millimeter orthogonal cuts from the edge toward the optic nerve head. After that, immerse a punched nitrocellulose membrane into the dish and gently drag the retina over it with the GCL side up. Then, place the optic nerve head into the center hole when preparing a whole-mount retina.
Subsequently, transfer the membrane with the retina into another clean dish. Gently flatten the retina as a Maltese cross with a fine paint brush and lay all four edges over the holes. Blot the nitrocellulose membrane with a piece of dry filter paper and remove the vitreous and inner limiting membrane with forceps and a paint brush.
Make sure that all the retinal edges are fully attached to the membrane before transferring the assembly into the recording chamber with a sealed glass cover slip at the bottom. Secure the membrane to the cover slip with vacuum grease and rehydrate the retina with the external solution. Be careful not to trap any air bubbles underneath the assembly.
Next, set the chamber onto the stage of an upright microscope. Profuse the chamber with 34 degrees Celsius carbogenated external solution at a rate of about three milliliters per minute. Examine the retina under a 10x subjective lens first.
Then, use a 60x water immersion lens to see GCL and INL neurons under DIC and the epifluorescent setting. To visualize tdTomato expressing SACs, use a white LED light source in combination with an excitation filter of 554 nanometers and an emission filter of 581 nanometers. In this procedure, filter the internal solution through a syringe filter into the custom backfill filament.
Then, insert the filament into a freshly pulled micropipette and dispense the internal solution near the tip until the solution covers the silver electrode wire for more than five millimeters. Afterward, fasten the micropipette onto an electrode holder with a suction pull through which pressure inside the electrode can be adjusted by pushing or pulling the plunger of a tube connected, 10 milliliter plastic syringe. Next, locate the pipette under the objective and bring it down to about 100 micrometers above the retina.
Under the current follower mode, use DC offset to zero the standing DC voltage signal. Measure the pipette resistance under the current clamp mode by injecting fixed amplitude square wave currents through the pipette while it is in the bath. Neutralize the difference by turning the Raccess knob and use the reading on it to calculate pipette resistance.
After that, slowly bring the electrode to about 10 micrometers above the retina. Apply positive pressure to the electrode. Then, watch the reflection change near the pipette tip as it approaches the retina.
Quickly but gently, force the pipette into the GCL and reduce the positive pressure immediately. Move the pipette toward a labeled neuron and avoid contacting other neurons, blood vessels and endfeet of Muller cells. Apply more positive pressure, if needed, to prevent electrode clogging.
Next, position the pipette tip near the labeled neuron until a dimple is visible. Then, release the positive pressure and allow the plasma membrane to bounce back onto the pipette tip. Apply 20 to 120 picoamps negative currents to the pipette to help the formation of a gigaohm seal.
Allowing cell membrane to bounce back after releasing the positive pressure is important because an excellent seal formed naturally between the membrane and the pipette opening is important to preserve the cell morphology after recording. If necessary, apply a gentle suction to pull the plasma membrane into the pipette. Wait five minutes after the seal formation to rupture the cell membrane in order to allow the clearance of the spilled internal solution by superfusion.
After the membrane has been ruptured and while in the current clamp mode, switch on the bridge balance and adjust it using the Raccess knob. Record the excitatory and inhibitory postsynaptic currents in the voltage clamp mode by holding the cell at reversal potentials. After recording, gently remove the pipette from the soma.
Transfer the assembly from the chamber to a clean dish. Then, detach and reattach the retina onto another flattened nitrocellulose membrane without punching holes to ensure retina flatness during fixation. The tissue is now ready for staining.
These images show that cholinergic cells in both GCL and INL can be reliably identified by tdTomato fluorescence and targeted for whole-cell patch clamp recording under DIC. The oscillation of their membrane potentials can be revealed by current clamp recording, and the oscillation driven by the inhibitory and excitatory synaptic currents are revealed by holding the cells at zero millivolts and negative 75 millivolts respectively under the voltage clamp conditions. Here, post hoke histological characterization of recorded cells indicates the typical SAC dendritic morphology and stratification levels in the IPL.
While attempting this procedure it is important to remember to secure the retina membrane assembly in the recording chamber and rehydrate without trapping air bubbles underneath in a tightly and delicated manner to provide a recordable sample. Following this procedure, other methods like dual-patch clamp recordings can be performed in order to answer additional questions like whether the oscillations of the target multiple cells are driven by the same synaptic mechanisms. After its development, this technique paves the way for researchers in the field of retinal neurobiology to explore the synchronicity of oscillation among neighboring retinal neurons in photoreceptor degenerated retinas.
After watching this video, you should have a good understanding of how to remove vitreous humor and prepare a retinal flat-mount for targeted whole-scale patch clamp recording from inner retinal neurons.
