전 세포 전압 클램프 기록을위한 전기 생리 학적 방법

The overall goal of this procedure is to obtain whole-cell voltage clamp recordings from a single photoreceptor cell to measure light-induced currents arising from single photon absorption and from the repeated responses to intense light. This method can help address key topics of the sensory transduction field such as visual transduction for both physical and ionic mechanisms that underlie phototransduction and the properties of TRP channels. The advantages of using this technique includes full control of the membrane voltage and extremely high signal-to-noise ratio and the ability to introduce specific indicators for optical measurements.

Generally, individuals new to this method will struggle with the minute size of the preparation and the need to perform the dissection quickly due to a time metabolic vulnerability. For this procedure, raise flies at a density of about 20 per vial. At the time of the experiment, have a few vials with pupae that are about to be closed and have been under constant darkness for at least 24 hours.

Two hours prior to collecting adults, remove any eclosed adults. Always handle the dark adjusted flies in tissues under red light illumination. While waiting for the freshly eclosed flies, prepare the recording chamber.

Onto the bottom of the chamber, fix the coverslip using melted paraffin or high vacuum silicon grease to complete the bath. Then mount the bath to the stage of the inverted microscope. Into the bath, place the perfusion system tube, the suction system tube, and the ground wire for the electrophysiology setup.

Before starting the dissection, prepare some solutions on the backside of a 60 millimeter Petri dish. Deposit four separate drops of calcium-free ES and one drop of TS.Now, using rough tweezers, catch a fly that has recently eclosed by its wings or body. Then using fine tweezers, detach the head and quickly transfer the head to the first drop of calcium-free ES.Under a stereoscope, dissect the head in half along the sagittal plane using a second pair of fine tweezers.

Make sure that both eyes remain intact. Now transfer 1/2 of the head to the second calcium-free ES drop and the other half of the head to the third calcium-free ES drop. Then proceed with dissecting one of the two eyes.

Using fine tweezers, remove as much of the tissue surrounding the eye as possible to ensure that no harm is caused to the retina. Then firmly grasp the edge of the cornea with fine tweezers and scoop out the retina. The result should be an empty intact cornea separated from an intact retina.

The pipettes are kept in ethanol, therefore, when using a new trituration pipette, always rinse it with water first. Now fill the rinsed pipette with a small volume of calcium-free ES from the fourth drop. Then, using gentle mouth aspiration, draw the isolated retina into the pipette.

Avoid trapping air bubbles with extreme caution. Then deposit the retina into the drop of TS.Next, repeat the retina isolation on the second eye so that both are now in the TS drop. Then delicately wipe away only the calcium-free ES from the Petri dish and add six more drops of TS to the dish surface.

Transfer both retinas into new drops of TS.Then replace the trituration pipette with one that has a small diameter opening. After rinsing it out, fill the pipette with a drop of TS.Using this pipette, rapidly and repeatedly aspirate and expirate both retinas to separate some ommatidia from the pigment cells. The TS will cloud up as this happens.

Next, transfer the remains of both retinas to another drop of TS.Then, load the pipette with the cloudy TS drop containing the isolated ommatidia. Expirate the solution into the bath chamber. Repeat this procedure with decreasing smaller diameter trituration pipettes until the retina has dissolved completely.

Now, wait a minute. During this pause, the isolated ommatidia will bind to the bottom of the chamber. After a minute, start flowing ES containing 1.5 millimolar calcium using the perfusion system.

As the chamber is filled, make sure that the ground wire and the suction tube are submerged. Let the solution flow for 60 to 90 seconds before proceeding. To begin, use the 40x objective to carefully inspect the ommatidia in the bath.

Notice that the elongated photoreceptor cell bodies forming the ommatidium are detached from their axons but remain viable. Now choose a suitable ommatidium for the experiment. Select when it meets the following criteria.

The outer membrane must be smooth and intact. The long axis must be nearly at a right angle relative to the electrode's approach from the distal side. And the ommatidium should not be surrounded by any excess tissue.

Once selected, center the ommatidium in the field of vision. Now fill a pipette with the appropriate intracellular solution for the experiment. And round the pipette to the electrode holder.

Then blow into the pipette's tube to create some positive pressure and lock the pressure in by closing the tube valve. Next, insert the pipette into the bath chamber and guide it close to the distal section of the ommatidium until a small dimple is observed in the membrane, which is due to the positive pressure from the patched pipette. Now open the membrane test module in the recording software to apply continuous square voltage pulses of two millivolts at a rate of 100 hertz.

Next, using the patch clamp amplifier controls, set the junction potential to zero to set the base of the square pulse to zero current. Then open the valve to release the pressure in the pipette and gently aspirate to create negative pressure. The pipette should then attach to the cell membrane.

Once this occurs, close the valve to lock in the pressure. Now check the electrode resistance. It should be elevated to between 100 and 150 megaohms.

Then release the negative pressure in the pipette by opening the valve again. The resistance should now leap up to between one and two gigaohms which indicate a C has been formed between the pipette and the cell. Next, using the patch clamp amplifier, offset the capacitive current of the pipette.

To make the whole-cell configuration, break the cell membrane using mouth aspiration. Create rapid short forceful bouts of negative pressure in the electrode. Upon obtaining the whole-cell configuration, a sudden increase in the capacitance will occur.

Then using the patch clamp amplifier, set the holding potential to the required voltage and offset the capacitive currents in series resistance. Now close the black French curtain of the Faraday cage and begin the experiment. After obtaining the whole-cell configuration with a patch pipette containing the fluorescent dye, Lucifer yellow, it can be seen that just one of the photoreceptors forming the ommatidium has been loaded with the dye.

This photoreceptor will also be electrically isolated from its neighbors in the ommatidium. Whole-cell voltage clamped ommatidia from wild type flies and flies with mutations in the genes InaC or InaD showed quantum bumps in response to continuous dim light. A slow termination of the bumps was observed in both mutants relative to wild type flies.

Normalized responses to 500 millisecond light pulses delivered at 150, 000 photons per second, the altered physiologies are neatly illustrate. Using the preparation, it was even possible to measure whole-cell voltage clamped quantum bump responses to one millisecond brief dim light. Such single photon responses were compared in wild type arrestin mutant and ninaC mutant flies.

Both mutants responded with a train of bumps. Normalized responses to a 500 millisecond light pulse delivered at 15, 000 photons per second. So the slow termination of the macroscopic response is observed in the arrestin-2 and ninaC mutants.

After watching this video, you should have a good understanding of how to make whole-cell recordings from Drosophila photoreceptor cells. Once mastered, this technique can be done in one hour if it is performed properly. This technique has paved the way for researchers to better understand TRP channels and inositol lipid signaling, as well as the role of intrinsically photosensitive retinal ganglion cells found in mammals which are involved in non-image forming light dependent activities.