마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

This video demonstrates the dissection of mouse retina and subsequent acquisition of single cell suction recordings for cones. The eyeballs of a euthanized dark adapted mouse are cauterized at the dorsal part and nucleated under dim red light. Eyeballs are then hemis sectored.

Along the equator, the cornea and ventral parts of the eyeballs are then removed. The dorsal retina is isolated from the pigment epithelium layer, and sliced into small, thin pieces using a razor blade. The slices of retina are then placed in the recording chamber that fits to a microscope stage.

Several photoreceptor cell bodies are drawn into the recording electrode. When a cone photoreceptor is drawn in the electrode, a bright test flash will induce photo response with amplitude above four picoamps. Hi.Hi.I am Jin from Dr.Mir Kops Laboratory in the Department of Ophthalmology and the visual census at Washington University in St.Louis.

Today we will show you a procedure for recording photo responses from single Moscon photoreceptors. We use this procedure in our laboratory to study the adaptation properties of mammalian cons, the photoreceptors that mediate our daytime vision. So let's get started.

One day prior to the recording, experiment dark. Adapt a mouse by placing it in a light tight cage overnight. Use a micro pipette puller to fabricate recording electrodes from Borrow silicate glass tubing under a compound microscope.

Use a heating filament to fire. Polish the tip of each electrode to smoothen it and reduce its opening to the desired diameter. For a mouse cone single cell suction, recording the inner diameter on the electrode tip should be about six to seven micrometers.

Prepare a 1%agri solution in distilled water. Place the solution in boiling water to melt the agar. Once the agar has melted, transfer it to a syringe and fill the glass pipettes with the solution.

To prepare reference electrodes allow the to solidify for 10 minutes at room temperature. After the agar has solidified, use a diamond's knife to cut each pipette in half to make two reference electrodes. Place the reference electrodes in a scintillation vial filled with reference solution and soak them for at least 24 hours at four degrees Celsius to allow the solution to penetrate the agar gel.

Reference electrodes can be used for up to three months from the date of preparation. Used electrodes are discarded following the experiment. The recording system is composed of an inverted microscope mounted on an air suspension table to minimize vibration noise shielded by a verity cage to minimize electromagnetic noise.

A motorized micro manipulator is used for positioning the recording electrode. The optical stimulator consists of tungsten filament, computer-driven shutters and optical filters to control wavelength and light intensity. A patch clamp amplifier is used to detect membrane currents from individual photoreceptor cells.

Throughout the recordings, the tissue will be perfused. Place one liter of prepared perfusion buffer in a 40 degree Celsius water bath near the recording system. Then using tubing, connect the perfusion buffer to 95%oxygen, 5%carbon dioxide so that it bubbles continuously.

Using a photometer measure, the un attenuated light power of un attenuated light from the optical stimulator, the detector of the photometer is placed on the microscope stage in the plane where the preparation will be placed. Subsequently, convert the reading given in micro watts into photons per square, micrometer per second to estimate the number of photons per test. Flash taking into account the attenuation of the light by neutral density filters.

Once the un attenuated light power has been measured, remove the photometer from the stage and mount the recording chamber to be used for the experiments on the stage of the inverted microscope. Using tubing, connect the profusion solution bottle through a regulator valve to the recording chamber. Routing the tubing through a heating resistor.

Ensure that the heating resistor is placed immediately before the recording chamber. Then connect the bypass tubing from the regulator valve to the recording chamber. Initiate profusion by opening and adjusting the flow regulator valve to a flow rate of one to 1.5 milliliters per minute.

The profusion solution drains out of the recording chamber and into a collection flask by gravity. Use a syringe with a blunt needle to fill the electrode holders with reference solution. Using a microfill syringe needle fill and flush the recording electrode with reference solution, ensuring that there are no bubbles in the electrode.

Insert the reference electrode in an electrode holder and connect to the reference pole of the amplifier head stage. Use a rubber ring to bind the thermal couple to the reference electrode. Making sure to place the electrode and thermal couple tips close together to ensure that the temperature reading is taken as close to the cell as possible.

Then insert the recording electrode in an electrode holder and mount the holder to the head stage of the amplifier. Using tubing, connect a small reservoir of mineral oil to the side port of the electrode holder. Gentle suction by mouth through tubing connected to the reservoir will draw the cell in the electrode.

Using the computer software, open one of the shutters of the optical stimulator. To visualize the beam area of the test flash on the microscope stage, focus the microscope to the bottom of the recording chamber. Using the micro manipulator, move the electrode to the center of the test flash beam area.

Position the reference electrode close to the tip of the recording electrode. Adjust the voltage for the heater so that the temperature of the solution in the recording chamber is about 36 to 38 degrees Celsius. When a seal test, which measures voltage to preset current amplitude, to calculate resistance to check the resistance of the recording electrode normally about 900 kilo ohms.

The seal test enables detection of bubbles in the recording or reference electrodes or inappropriate tip size of the recording electrode. The seal test is software controlled by the acquisition program clamp X.The resistance reading is shown on the computer screen. If the resistance is too low, less than 500 kilo ohms, the tip of the electrode is probably too large and a new electrode will need to be installed.

If the resistance is higher than one milli ohm, a bubble might be present in the electrode or its tip is too narrow and the electrode will need to be flushed or replaced. Once the recording apparatus is ready to go, prepare the retinal slices for the experiment under a dim red light, use the tip of the hot dissecting needle to cauterize the sclera on the most dorsal point of each eyeball of a euthanized dark adapted mouse. Then nucleate the eyeballs using scissors.

Use blunt forceps to transfer the eyeballs to a dissecting microscope equipped with infrared illumination and infrared image converters. All further dissection manipulations are done under infrared light. Then using micro scissors, cut the eyeball open along the equator except for the cautery point.

Use sharp forceps to remove the lens. Then remove as much of the vitreous as possible with the cautery point as reference. Use a razor blade to remove the ventral part of the eye cup.

Then remove the cornea. Next, use forceps to peel the dorsal retina from the pigment epithelium layer. Then place the retina vo receptor side up on the bottom of a new Petri dish filled with one to two milliliters of a 10 millimolar glucose reference solution with 0.1%BSA gently pressed down on the retina to flatten it using a razor blade.

Slice the retina into small thin pieces. Then incubate the preparation in a light type box saturated continuously with pure oxygen before adding the retina slices onto the recording chamber. Shut off the heater temporarily.

Switch the profusion flow to the bypass tubing. Then using a kim wipe, drain most of the solution from the chamber. Turn on the infrared illumination.

Remove the retina suspension from the incubator and transfer the slices to the recording chamber. Using a wide opening transfer pipette, allow the slices to settle down for two minutes. After the retina slices have settled, switch the direct perfusion and heater back on.

Identify a piece of retina with its photoreceptor outer segments sticking out. Position the tip of the recording electrode above the outer nuclear layer of the retina using mouth suction. Draw several photoreceptor cell bodies gently into the electrode with the cells drawn into the electrode.

Give a 20 millisecond 500 nanometer test flash. The light induced change in the current flowing through the photoreceptor represents the test flash response. A healthy cone will produce a maximum response amplitude of four to seven pico, MPIs or pa.

When stimulated with un attenuated test flash. Once a good cell is found lower gently the mineral oil reservoir by hand to apply slight negative pressure to the tip of the electrode to help hold the cell inner segment in the electrode. If the cell is responsive, record a series of responses to test flashes with intensity ranging from threshold to saturating.

Using this technique, we can obtain cone photo responses from mouse retinal slices with the help of a suction electrode. Here, an overlay of several recordings from the same cell is shown. Photo responses of a single cone were induced by test flashes of increasing intensity from dim, which generates threshold response of about one pico am here to bright intensity, which generates saturated response of about 5.5 pico amper.

As flash intensity is increased, the amplitude of photo response increases until it reaches saturation. The flashes are given at time equals zero. We'll just showing you how to record photo responses from single moscon photoreceptors using a suction electrode.

When doing this procedure, it's important to remember to obtain the do do part of the retina, cut the retina into thin slices, keep the electrode clean or bubbles and minimize both mechanical and electro electromagnetic noises. So that's it. Thanks for watching and good luck with your experiments.