유전적으로 망막 신경절 라벨 셀에서 라이트 응답을 기록하기 위해 절연 망막 준비

The overall goal of this procedure is to record light evoked responses from genetically labeled ganglion cells in the isolated mouse retina. This is accomplished by first dissecting the retina from the mouse eye. The second step of the procedure is to treat the retina with enzyme to remove the remaining vitreous.

The third step of the procedure is to mount the retina ganglion cell side up in the recording chamber, mount the chamber on the microscope stage, and identify the genetically labeled ganglion cells. The final step of the procedure is to obtain a whole cell recording of the identified ganglion cell and to record light evoked responses. Ultimately, results can be obtained that show the functional properties of a defined ganglion cell, type in the mouse retina, and define the signals that it conveys to the brain through single cell patch clamp recordings.

Hi, my name is Tiffany Schmidt and I'm a neuroscience graduate student in the lab of Dr.Paulo Ka Fuji at the University of Minnesota. This technique allows identification of and recording from a defined, genetically labeled ganglion cell population and subsequent morphological analysis. This technique can help answer key questions in the visual field, such as how different ganglion cell subtypes process key features of visual stimuli Prior to performing the surgery.

To remove the retina, the first task is to mix up the eight solutions needed for the experiment. The ingredients for all solutions used in this experiment are given in the accompanying text. Transfer 200 milliliters of the extracellular solution to a 250 milliliter beaker for retina storage.

Place the remaining extracellular solution in a separatory funnel for gravity profusion during recording. Both of these solutions should be kept saturated with 95%oxygen, 5%carbon dioxide at all times. To visualize the dendrites during the experiment, add a fluorescent tracer such as LOR 5 94 to the intracellular solution.

If you plan to do immunohistochemistry after the whole cell recording, add 0.3%neuro biotin to the intracellular solution. Once the necessary solutions are prepared, euthanize a mouse by carbon dioxide asphyxiation, nucleate the eyeball by gently inserting an angled number two forceps behind the eye and lifting out the eyeball. Use ophthalmologic scissors to clean off the extraocular muscles and to cut the optic nerve.

Place the eyeball in a 35 millimeter Petri dish filled with extracellular solution. Cut away the cornea with ophthalmologic scissors and pull out the lens using a number five forceps. Use number five forceps to tear open the sclera and sever the remaining optic nerve where the retina and sclera meet.

Gently pull the retina away from the eye cup by running the forceps in the space between the retina and sclera. To save time, do each step on both retinas Before moving on to the next step, a crucial step is to remove the vitreous attached to the retina. Start by using forceps to grab the transparent vitreous above the retina.

If the vitreous is removed successfully, you'll see a small, clear gelatinous substance on the tip of the forceps. Slice each retina in half to maximize the amount of usable tissue and to make it easier to mount the retina in the recording chamber. Place the retinas in the oxygenated extracellular solution at room temperature and minimize light exposure by allowing only minimal ambient light, such as light from computer screens, the retinas remain viable for approximately six hours.

Following dissection, take 500 microliters of the extracellular solution from the solution in which the extra retinas are bubbling. Dilute into it. An aliquot of the collagenase hyaluronidase enzyme solution.

Put the enzyme solution in a 35 millimeter Petri dish and transfer one of the dissected retinas into the Petri. Rotate the Petri dish for 15 minutes all the time gently super fusing 95%oxygen, 5%carbon dioxide in the solution. The enzyme solution removes any remaining vitreous and allows for easier access to the ganglion cell layer For retinas from younger animals with less vitreous, or if adverse effects are observed, the digestion can be shortened to five minutes.

Using a plastic transfer pipette with a tip cut off. Remove the retina from the enzyme containing medium. Wash the retina extensively in extracellular solution.

Transfer the retina into the recording chamber. With the photoreceptor layer facing down, wick away any remaining fluid with either a pipette or tissue and roll out the sides of the retina to flatten the tissue. Being careful to only touch the edges of the retina and not the ganglion cell layer.

To anchor the retina, place a platinum ring with nylon mesh over the retina, and then immediately fill the chamber with extracellular solution. Mount the recording chamber onto the microscope stage and then attach the tube for gravity perfusion of the extracellular solution, the vacuum suction tube and the ground wire. Adjust the gravity so that it drips at one milliliter per minute or faster.

If the perfusion rate is too slow, the tissue will not be sufficiently oxygenated and synaptic signaling may be disrupted. The next step is to set up the electrode for whole cell patch recording. Select a pipette with a resistance in the range of three to five mega ohms.

Fill the recording pipette with a defrosted aliquot of intracellular solution and insert it into the pipette holder using infrared illumination and differential interference contrast or DIC optics under a 10 x objective. Visualize the retina and the recording pipette. The infrared light is used to minimize the retina's exposure to visible light and to minimize light adaptation, switch to a 40 x objective and bring the electrode into view just above the tissue.

The mouse line used in this study expresses EGFP in its ganglion cells, and thus the ganglion cells are visible under epi fluorescence. Microscopy illuminate the retina with epi fluorescence at 480 nanometers. If the retina is healthy, individual ganglion cells should be visible.

Unhealthy retinas will have large granulated nuclei and should be discarded. Select one of the fluorescent ganglion cells for recording and use lab tape to mark the selected cell on the computer monitor. Switch to infrared optics so that the selected ganglion cell body is visible.

Position the recording electrode near the targeted ganglion cell. Clean the area immediately covering the cell by applying gentle positive pressure to the recording. Electrode pressure is applied via a 12 cc plastic syringe connected to the pipette holder with polyethylene tubing on the amplifier zero.

Any voltage offsets. Place the electrode onto the selected ganglion cell until a dimple appears on the cell surface. With the amplifier in voltage clamp mode, apply a test pulse and monitor the current passing through the pipette.

Apply negative pressure to create a seal between the electrode and cell. Release the negative pressure when the seal begins to form and wait until the holding current is indicative of a giga electrical seal between the cell's membrane and the pipette. Apply gentle pulses of negative pressure until capacitive transients appear indicating that whole cell recording mode has been obtained, and then release any application of pressure.

If the access resistance is greater than 30 mega ohms, this can often be ameliorated by additional very gentle pulses of negative pressure. After whole cell mode is obtained, turn off the infrared light and cover the microscope with a black cloth. Allow the cell to dark adapt for five minutes.

After five minutes, the preparation is ready for recording. In current clamp mode, turn on full field white light stimulation and record the response of the ganglion cell. If the ganglion cell is to be stimulated multiple times, the retina should be allowed to dark adapt as necessary between stimulations.

The light stimulus may also be modified at this point to include patterned or chromatic stimuli in order to assess the ganglion cells response properties following recording, the fluorescent tracer in the intracellular solution will have diffused to the dendrites by switching between epi fluorescence and DIC infrared optics. The tracer can be visualized under epi fluorescence at 594 nanometers, and you can examine the dendrites to determine whether the ganglion cell is on off or by stratified. The neuro biotin that was added to the intracellular solution has diffused into the ganglion cell, and so the next step is to prepare the retina for immunohistochemistry.

Retract the pipette gently so as to minimize disruption of the cell membrane. Note that if you record and fill only one retinal ganglion cell per preparation, you can then make specific correlations between the recorded ganglion cell and its detailed morphology. Next, place the retina in the Paraform aldehyde solution and fix overnight at four degrees Celsius after fixation overnight.

Rinse the fixed retina in PBS making at least six changes of PBS solution over the course of two or more hours. After rinsing, transfer the retina to a plate containing blocking solution, put the plate on a shaker and let it sit for two hours. At room temperature, replace the blocking solution with the strep evidence solution and put the retina back on the shaker and let it incubate for two days at four degrees Celsius.

After two days, rinse the retina in PBS doing six rinses of 20 minutes each to mount the retina. Place it ganglion cell side up on a glass slide using vector shield. Mounting media cover with a glass cover, slip and seal with nail polish, the labeled ganglion cell can now be looked at under a confocal microscope.

This figure shows GFP positive retinal ganglion cells visualized under epi fluorescent illumination at 40 times magnification on the left and under DIC illumination on the right. If the retina is healthy and the dissection was successful, individual ganglion cells are visible under DIC at high magnification. Unhealthy retinas will have large granulated nuclei and should be discarded.

This figure shows the type of light response that is typical of an M two on stratifying intrinsically photos sensitive retinal ganglion cell. Depending on the type of retinal ganglion cell genetically labeled in a given preparation, the response properties will differ. If the synaptic response of a given ganglion cell is intact, then the cell should respond at light onset or offset with action potentials.

And in the case of an intrinsically photo sensitive ganglion cell, a sustained depolarization, this figure of a neuro biotin filled ganglion cell shows the morphology of a typical M1 off stratifying, intrinsically photosensitive retinal ganglion cell. Following whole cell recording immunohistochemistry allows a correlation of the dendritic morphology and the retinal ganglion cell's electrophysiological response. While attempting this procedure, it's important to remember to oxygenate the retinal well and to perfuse the tissue rapidly in order to preserve synaptic responses.

Thanks for watching this demonstration.