The overall goal of this procedure is to prepare thin tissue slices from aquatic tiger salamander retina, and use them for simultaneous whole cell voltage clamp recordings from photoreceptors and second order horizontal and bipolar cells. This is accomplished by first dissecting a salamander eye and isolating the retina on a piece of nitrocellulose membrane. The second step is to cut thin slices of retina and position them in the recording chamber.
Next, the chamber is placed on an upright fixed stage Microscope and patch pipettes are positioned above the retinal slices. The final step is to obtain whole cell recordings from a presynaptic, photoreceptor, and postsynaptic horizontal or bipolar cell. Ultimately, paired whole cell recordings can be used to manipulate synaptic release and study the interactions among cells in the retina.
The retinal slice preparation was originally developed in the laboratories of Frank Werblin and Sam Wu.Unlike single cell preparations slice preparations retained intact ntic contact, and unlike whole mount retina preparations, slicing the retina exposes various retinal layers, allowing ready access to all of the different cell types. In the retina Slice. Preparations can be used for electrophysiological recordings, including simultaneous recording for multiple cells and for imaging experiments.
This preparation has proven extremely valuable for studying synaptic interactions among retinal neurons. In this video, we describe preparation of slices using tiger salamander retina, and emphasize some of the small details that can make the difference between success and failure. Begin this procedure by assembling the chamber.
Place two beads of vacuum grease spaced eight to 10 millimeters apart across the recording chamber to form a channel for super fase and to embed the retinol slices at a second beat of grease, a few millimeters beyond each of these two beads of grease to act as a levy to limit spillover. Then place a small triangular piece of Kim wipe at the end of the chamber to ensure fluid contact with the reference electrode. After that, press a piece of nitrocellulose membrane flat against the glass microscope.
Slide onto two small beads of vacuum grease. To prepare the tissue slicer. Break a double-edged razor blade into four pieces.
Then attach one of them to the slicing arm. Ensure the cutting edge of the razor blade lays flat against the recording chamber by cutting a thin slice of nitrocellulose filter paper. Next, after euthanizing a salamander by decapitation nucleate the eye using a pair of small Venice scissors under the microscope, cut the skin that connects the eye to the surrounding orbit.
Then pull the eye forward and slide the scissors under it to cut through the eye muscles and optic nerve. Afterward, place the enucleated eye on a bed of cotton on a linoleum block. Now make a small incision in the center of the cornea with a sharp surgical blade.
Remove the cornea by sliding the fine Venice scissors into the incision and extending the cut radially out toward the aura Serrata. Then cut circumferentially around the aura serrata by rotating the linoleum block or the cotton between the cuts. After cutting all the way around the eye, remove the cornea and lens by pulling them out of the side of the eye cup.
Transfer the remaining eye cup onto a hard surface of the linoleum block moistened with amphibian saline solution. Cut it into thirds with a sharp razor blade in a fine sawing motion to ensure that it cuts all the way through the sclera. Then place one or two pieces of IUP on the nitrocellulose membrane with the retinal surface facing down.
Submerge the remaining pieces with additional saline. After that, place them in a refrigerator in case additional slices are cut later. Now, gently press the piece of IUP against the nitrocellulose membrane with a pair of fine forceps.
Submerge the nitrocellulose membrane and IUP piece with several drops of cold amphibian saline. Then drain away the saline solution by touching a piece of Kim wipe at the edges of the nitrocellulose membrane and iup. After that, submerge the IUP into nitrocellulose membrane with several drops of cold amphibian saline solution.
Again, subsequently peel away the sclera choroid retinal pigment epithelium to isolate the retina. If the retina has not adhered tightly, drain the saline away with a kim wipe. In order to pull the retina more firmly onto the nitrocellulose membrane, replace the saline and repeat the procedure if necessary.
Next, fill the chamber with cold amphibian saline. Transfer it to the stage of the tissue slicer. Then slice the retina and nitrocellulose membrane into 125 micron thin strips by pressing the razor blade gently but firmly through them.
After cutting the entire retina into slices, transfer the retinal slices one by one to the main channel of the recording chamber. To transfer a slice, hold a strip of membrane in place while sliding the chamber beneath it. Be sure to keep the slice submerged when it is above the center part of the chamber.
Embed the edges of the nitrocellulose membrane in the strips of vacuum grease and rotate it 90 degrees. To view the retinal layers, press the nitrocellulose membrane flat against the glass surface, even if there is no retina on every piece. Place strips of nitrocellulose membrane at about millimeter regular intervals to help break up the surface tension and thereby improve fluid flow.
Continue to transfer the slices one by one until the slices have been placed along the entire length of the perfusion channel. After all of the slices have been transferred, move the recording chamber to the stage of an upright fixed stage microscope. Connect the reference electrode lead to a ground wire on the amplifier head stage.
Focus on the slices using a long working distance water immersion 40 to 60 x objective. Next super. Fuse the slices at a rate of one millimeter per minute with amphibian saline solution bubbled with oxygen.
Connect the suction line to the output line of the chamber. Then adjust the outflow to balance the inflow by rotating the beveled end of the suction needle or by moving the kim wipe at the end of the chamber closer or farther away from the opening at the tip of the outflow needle. Examine the slices under dim or infrared light.
Next, identify a pair of cells, a photoreceptor and a nearby horizontal or bipolar cell for wholesale. Recording Philip pipette prepared before the experiments with the intracellular solution, mount it on the electrode holder. Next, elevate the microscope objective, slightly.
Position the photoreceptor pipette beneath the objective. Then lower it so that the tip is positioned just above the slice and adjust the focus simultaneously. To avoid going too far.
Repeat this procedure with the second pipette. Next, check the pipette resistance with a five to 10 millivolt depolarizing pulse. Adjust any offset in the baseline current level on the amplifier.
After that, apply slight positive pressure, and in the meantime, position the postsynaptic pipette so that it contacts the horizontal or bipolar cell body. Then position the presynaptic pipette so that it contacts the cell body of a rod or cone photoreceptor. While monitoring the resistance release, the positive pressure on the postsynaptic pipette, sometimes the release of positive pressure is sufficient to form a giga ohm seal.
After the tip resistance has increased to more than 100 mega ohms, apply a holding potential of minus 60 millivolts. Then when a giga om seal is obtained, cancel the pipette capacitance transient. Using the capacitance compensation circuitry of the patch clamp amplifier, repeat the ceiling procedure with the photoreceptor pipette, applying a holding potential of minus 70 millivolts and cancel the pipette capacitance.
Transient, then rupture the patch by applying suction to each cell. Rupture of the patch is evident by the appearance of whole cell capacitance transient. Now, confirm the identity the postsynaptic cell physiologically by applying a light flash and delivering a series of voltage steps from minus 120 to plus 40 millivolts in 20 millivolt increments.
To assess if the cells are synaptically connected, deliver a 25 to 100 millisecond 60 millivolt. Step depolarization to the photoreceptor and look for post-synaptic currents. In the second order neuron, here are the representative traces of light responses from the neurons in vertical slices of salamander retina.
The cone horizontal cell and off bipolar cell all display an outward current in response to the light onset. The prominent inward current following the light flash in the horizontal and bipolar cell recordings here is caused by the increased release of glutamate from the photoreceptors as they depolarize at light offset. The on bipolar cell responds with an inward current at the light onset, which results from a sign inverting metabotropic glutamate receptor signaling cascade, and the activation of tripm one channels.
Horizontal cells and bipolar cells can be distinguished from one another by their current voltage relationships. Horizontal cells typically have linear or inwardly rectifying current voltage relationships with an input resistance less than 500 mega ohms bipolar cells have an outwardly rectifying current voltage relationship and higher input resistance between 0.5 to two giga ohms. This figure shows the representative results from the recordings of a cone horizontal cell pair and a cone off bipolar cell pair.
In each case, depolarizing the cone to minus 10 millivolts from a holding potential of minus 70 millivolts evoked a voltage gated calcium current in the cone and fast inward postsynaptic. Current in the horizontal or bipolar cell Retinal slice. Preparations have been used in hundreds of studies on the anatomy and physiology of the retina.
They're particularly advantageous for examining responses of multiple cells simultaneously since they facilitate recording and imaging from multiple cells at the same time. After watching this video, you should have a good understanding of how to dissect the retina, cut thin retinal slices, position them in the recording chamber, and obtain wholesale recordings from pairs of synaptically connected selves.
