Determining a Test Growth Hormone Effect on Kisspeptin Neurons using a Whole-Cell Patch Clamp
筆記録
Transfer a brain slice of interest, one at a time, to the recording chamber using an acrylic transfer pipette. Hold the slice with a slice anchor so it does not move during the aCSF perfusion. Position the slice to the center of the recording chamber using the immersion microscope's low-power objective lens 10x or 20x.
The slice position is critical to allow a good view of the desired region under the microscope and for a perfect reach for the recording micropipette. After locating the region of interest, switch the objective lens to the high power lens, 63x, and focus on the tissue level, observing the endogenous fluorescent protein and shapes of the cells in the target region to locate the kisspeptin cells on the surface of the brain slice.
When a possible target cell is located, mark it on the computer screen with the mouse cursor or by drawing a format like a square over the area of interest. The computer screen marked helps guide the recording micropipette's position to the cell. After determining the exact location of the target cell, lift the objective, and introduce the recording micropipette filled with the internal solution in the electrode holder, ensuring the internal solution is in contact with the silver electrode.
Apply a positive pressure before submerging the micropipette in the aCSF solution using a 1- to 3-milliliter air-filled syringe connected to the micropipette holder through a polyethylene tubing to prevent debris from entering the micropipette. Using the micro-manipulator, guide the micropipette below the center of the objective. Move the micro-manipulator buttons to guide the x, y, z-axis of the micropipette toward the cell of interest.
Adjust the focus to see the micropipette's tip and bring the focus closer, but not too close to the slice. Reduce the speed of the micro-manipulator, and slowly lower the micropipette to the plane of focus, ensuring that the micropipette tip does not abruptly penetrate the slice, but slowly descends until it touches the cell's surface or the target region.
Applying light positive pressure with the 1- to 3-milliliter air-filled syringe to clear any debris from the approach path, slowly Move the micro-manipulator on the x, y, z-axis to bring the micropipette tip closer and touch the target cell, which causes a dimple by the applied pressure. After establishing the dimple, with the help of voltage clamp mode on the software, apply weak brief suction by mouth for 1 to 2 seconds through the tube connected to the micropipette holder to generate the seal between the micropipette to the cell.
If the seal remains mechanically stable without noise interference for about a minute, set the holding voltage at the closest physiological resting potential of the cell of interest. For kisspeptin hypothalamic neurons, minus 50 millivolts is recommended. Next, apply a brief suction by mouth to break the plasma membrane at the micropipette tip sealed placed to the cell so that the ruptured membrane does not clog the micropipette, or attract a sizable membrane portion, or the cell.
An adequate whole-cell configuration can be achieved by performing suction with sufficient force. Check the system settings manual used. After breaking the cell membrane, enable the Whole Cell option on voltage clamp mode, and click on the auto command referring to the Whole Cell tab.
Next, check the cell viability parameters in the software as described in the text manuscript. Monitor the series' resistance and the cells' steady-state capacitance during the experiments. Once the whole-cell configuration is properly achieved, measure synaptic currents in voltage-clamp mode. Record the changes in resting membrane potential and induced resting membrane potential variations in the current clamp mode.
