The overall goal of this horizontal slice preparation of the mouse retina is to preserve the morphological and physiological integrity of horizontally oriented neurons in order to study their functional role in synaptic circuits of the retina. This method can help answering key questions in the visual information processing field such as how sensory signals are encoded and integrated in the ribbon synapses of the repeated retina. The main advantage of this technique is that horizontally oriented interneurons like horizontal cells and amacrine cells are functionally intact and accessible to electronic recordings.
Demonstrating the procedure will be Andrea Nerz a technician in my laboratory. To begin this procedure remove the eyeball by placing a pair of curved forceps under the eye. Gently lift up the eyeball to expose the optic nerve and cut it with a spring scissor.
Then transfer the eyeball to a 35 millimeter Petri dish containing two milliliters of AMS medium. Under the stereo microscope adjust the magnification to a value that allows optimal visual control. Next, steady the eyeball with a pair of forceps and puncture the cornea at the transition to sclera with another pointed forceps or a 20 gauge needle.
Be careful not to damage the retina while puncturing the cornea. Subsequently, insert the spring scissors into the small hole. Steady the eyeball with forceps while cutting a circle around the cornea and keep the incision very close to the surface in order not to cut into the retina.
Carefully remove the cornea lens and vitreous body. The vitreous body comprises only a thin transparent layer in the back of the eye. However, it is essential for the vitreous body to be fully removed for vibratome sectioning.
In the Petri dish position the eye cup to show a clear hole down to the retina. The rim of the opening should consist of scleral tissue only. Next, grab the sclera with forceps and carefully tear the tissue apart.
It is critical not to rupture the retina during this step. At this step the retina and the rest of the eye cup should be connected only at the optic nerve head. Cut the attaching point at the optic nerve head with spring scissors.
The retina should now be freely floating as a single piece of tissue in AMS medium. Next, transfer the retina to a cover slip with a large diameter Pasteur pipette and cut the retina into four to six small rectangles of approximately two-by-three square millimeters using a curved scalpel blade. Avoid pinching the retinal tissue within the rectangles.
In this procedure, carefully transfer two to three retinal pieces into a glass Petri dish together with some of the AMS medium with a large diameter plastic Pasteur pipette. Remove as much of the remaining AMS medium as possible with a glass Pasteur pipette. Then immediately cover the retinal pieces with two milliliters of 37 degree Celsius dissolved agarose.
Gently push the retinal pieces to the bottom of the Petri dish. Position the ganglion cell layer to be facing down as fast and precise as possible since the agarose solidifies quickly. At the same time arrange the retinal pieces to be parallel to the bottom of the glass dish to prevent the sections from getting tangential or oblique.
After that, transfer the embedded retinal pieces to the refrigerator to harden the agarose before further processing. Now carefully detach the agarose block containing the retinal pieces from the Petri dish. Use a scalpel blade to lift the agarose block from the glass carefully.
Afterward, place the agarose block on a glass cover slip. Adjust its size with a scalpel blade and leave one to two millimeters of agarose on each side of the retinal pieces. Next, glue the agarose block to the vibratome specimen holding using an instant adhesive.
Cover the block immediately by pouring the previously bubbled AMS medium into the buffer tray of the vibratome. In this figure a horizontal cell located near the surface of the slice was visualized with dote contrast optics. This image shows a horizontal cell filled with Oregon Green 488 BAPTA-1 via a patch electrode which was still attached to the cell.
Shown here is the overlay of a dye injected horizontal cell and an array of cone pedicles labeled with flouisine coupled peanut agglutinin, which reveals the putative synaptic contacts. In this figure, whole cell voltage clamp recording at a holding potential of minus 60 millivolts shows high frequency tonic synaptic activity. At higher temporal resolution synaptic events display monophasic waveforms or more complex current trajectories.
In this figure a current response of a horizontal cell to a light stimulus is shown. Light induces a closure of ion channels and a block of synaptic activity. And in this figure cone photo receptors were electrically stimulated with an extracellular electrode.
Post synaptic excitatory currents are graded with stimulation strength. Once mastered, this procedure can be done in less than one hour if it is performed properly. Following this procedure other methods like calcium imaging can be performed in order to answer additional questions like the processing of visual information in retinal networks.
After watching this video you should have a good understanding of how to cut horizontal slices of the mammalian retina with functional synaptic inputs to horizontally extended cell types.
