The overall goal of this procedure is to prepare the Drosophila flight motor neuron five for in C two patch clamp recordings. This is accomplished by first visualizing the drosophila ventral nerve cord with the GFP tagged flight motor neurons one to five through a 40 x water dipping lens. The second step is to use a broken protease filled patch pipette to apply protease across the GFP tagged motor neurons in order to loosen the debris glia and trachea that obstruct the access to motor neuron five.
Next debris glia and trachea are removed by gently sucking the loosened debris and glia into the pipette and pulling the trachea out of the way with the application of positive and negative pressure. Alternately, at this point, bright light is used to remove the remaining debris that cannot be seen with fluorescent light through a GFP filter. Ultimately, the cleaned motor neuron five can be seen faintly in bright light, but seen well using fluorescent light with a GFP filter.
After rinsing the cells with saline for 15 to 20 minutes, they're prepared for subsequent situ two patch clamp recordings. The procedure I'm going to show you today will help answer key questions in the field of neurophysiology, such as which ionic channels underlie, which ionic currents, and in turn, which ionic currents underlie behavior. Generally, individuals new to this technique will struggle because it is hard to see the subtle changes of the appearance of the neurons during the cleaning procedure, which is necessary prior to the pet clamp experiment.
A clean cell membrane is a crucial prerequisite for subsequent situ pet lamp recordings, and since it is so hard to judge on the cleanliness of the cell membrane, I am gonna show you a visual demonstration of the procedure. How to do that today? Begin this procedure by mounting the Petri dish on an upright fixed stage epi fluorescent microscope.
Next, position the dish with the fly in it so that the anterior part faces the side where the electrode holder is mounted. Then view the ventral nerve cord with a high magnification, high NA water dipping lens and A GFP filter. The motor neurons five are located bilaterally on the dorsal surface of the ganglion in the meso thoracic neuro mirror, close to the midline of the ganglion between prominent trachea motor neurons five with their dendrites appear as a diffuse green area.
The somato will become more distinct after cleaning. However, their dendrites will remain blurry due to the tissue covering them. Here we use neutral density filters to avoid overexposure of the tissue by too much fluorescent light, especially blue light.
If bleaching occurs, most likely the light output is too strong and the cell is photo damaged. Make sure that the suction is steady and the saline outlet is positioned in a way that the solution level in the recording chamber does not fluctuate up and down. A steady and stable perfusion is reflected by the constant never interrupted gurgling sound that is produced by the constant suction of saline out of the bath.
Now use a cock or specific diameter tubing to regulate the perfusion rate of the recording chamber at approximately two milliliters per minute. The volume of the chamber should be as small as possible to guarantee fast exchange of the solutions in the chamber, which will assure fast removal of protease and blockers. From the recording chamber, bend and attach the hypodermic needles to the narrow tubings as the saline inlet and outlet.
Then place them near the recording chamber with plasticine. Next, pull a patch pipette with a pipette puller. Break the tip a little under the microscope with a clean pair of forceps.
Then fill the pipette with 2%protease and PBS buffer without clogging it. Insert the pipette into the electrode holder. To ensure proper control of the applied pressure, the holder needs to be tightly sealed.
Then attach a tubing to the small outlet on the pipette holder. Secure it in a way that tugging at the free end of the tubing does not affect the inserted pipette Check for the movement of the pipette under the microscope. Next, insert a plastic pipette tip into the free end of the tubing for the application of positive or negative pressure to the pipette.
Now, visualize the enzyme filled pipette under the 40 x water dipping lens with bright light. Focus on the pipette tip. If it is clogged, try to get the dirt out by applying positive or negative pressure.
If the dirt does not come out, prepare a new enzyme filled pipette. Next, switch to the fluorescent light. Lower the pipette with enzyme carefully and refocus until the cell body can be seen.
When the pipette is close to the cell body, switch the perfusion off. Apply positive pressure in short bursts towards the cell body so that everything that is hampering access to the cell will be loosened. Then move the dorsally to catch the loose debris that may be left over from the ganglion sheath surrounding the ventral nerve cord with alternating application of positive and negative pressure.
Use the pipette like a vacuum cleaner to clean around the cell body. This cleaning procedure is done until the membrane looks free of debris. Next, use high intensity blue fluorescent light to quickly view the membrane and make sure that it is clean in order to visualize the motor neurons switch to bright light and remove the cells and debris that could not be seen with fluorescent light.
After the cleaning procedure is finished, switch the perfusion system back on and the light off. Then rinse the cells for about 15 to 20 minutes with saline or the solution that will be used. Pull the patch pipettes from boro silicic glass capillaries with a vertical pipette puller.
Then conduct conventional situ to whole cell patch clamp recordings in voltage clamp and current clamp mode shown here is an example of the calcium currents recorded from motor neuron five. There are at least two different calcium currents, a low voltage activated transient calcium current and a sustained high voltage calcium current. And this is an example of the potassium currents recorded from motor neuron five.
The total potassium current is shown in this figure, and this figure shows only the sustained potassium current pure transient potassium current is obtained by subtracting the sustained potassium currents from the total potassium current. Here is an example of the firing patterns elicited by somatic current injections in motor neuron five. The resting membrane potential is negative 59 millivolts Once mastered, the procedure can be done in five minutes, excluding the prior section of the animal and subsequent rinsing of the preparation.
After watching this video, you should have a good understanding of how to clean identified neurons in the TRALA ventral nerve cord to prepare them for subsequent inside your patch lamp recordings.
