By measuring the electrophysiological properties of cell membranes, the question of the main proves that modulate the electrical activity of a cell can be solved. Whole-cell patch clamp recording is a peripheral technique to study ionic currents in individual cells and interrogated the cellular response to different stimuli. The best performancy on whole-cell patch clamp pre-recordings will be achieved after practicing and studying a lot.
To begin brain dissection make an incision using surgical scissors in the skin at the top of the decapitated animal's skull from caudle to rostral and remove the scalp. Next, cut the inter parietal plate along the sagittal suture with the iris scissors and remove the occipital bone. Slide the osteotome under the parietal bone and gently pull it out until the brain is exposed.
After exposing the brain, turn the head upside down. Gently lower the brain to visualize the trigeminal nerve on each side. Then cut the trigeminal nerve using Castroviejo curved scissors.
After visualizing the hypothalamus identify the optic nerve and cut it gently. Next, cut the far anterior portion of the frontal lobe. Remove the brain and immediately immerse it in the slicing solution until acquiring the slices.
To slice the brain samples, place the brain on a filter paper to dry the excess solution. Then with a sharp cutting razor blade perform a coronal cut separating the brainstem with the cerebellum from the rest of the tissue. Next, glue the caudle portion of the brain to the base of a vibratome and fill the slicing device chamber with the slicing solution or aCSF solution cooled to zero to two degrees Celsius.
Pack dry ice around the vibratome chamber to keep the slicing solution cold during the procedure. Insert the razor blade into the vibratome and set the device's cutting parameters, such as speed to three, frequency to nine, and feed to 250 micrometers. During the tissue slicing procedure transfer the slices using an acrylic transfer pipette to the recovery chamber and wait 60 minutes for tissue recovery after slice acquisition.
Before starting cell sealing and recording, turn on the microscope, amplifier, digitizer and micro manipulator. Build the recording chamber attached to the microscope with the aCSF solution. Use a perfusion pump to constantly perfuse the aCSF at two milliliters per minute.
Check the software settings and create specific protocols for the recordings according to the type of experiment. 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 mark helps guide the recording pipette 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 one to three 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 XYZ 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 plain 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 a one to three milliliter air-filled syringe to clear any debris from the approach path.
Slowly move the micro manipulator on the XYZ 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 one to two 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. The cell's series resistance and the whole-cell capacitance are automatically calculated and instantly displayed by the software.
Next, check the cell viability parameters in the software as described in the text manuscript. Monitor the series resistance and the cell 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. The possible effects of human recombinant growth hormone on the activity of hypothalamic kisspeptin neurons were studied with the help of whole-cell patch clamp recordings. The administration of human recombinant growth hormone, HGH at 20 micrograms per gram induced a significant hyperpolarization of the resting membrane potential in 5 out of 12 AVPV/PeN kisspeptin neurons and 9 out of 14 recorded ARH kisspeptin neurons.
The AVPV/PeN kisspeptin and ARH kisspeptin hyperpolarized neurons significantly changed the resting membrane potential compared to the unresponsive cells. The effects on resting membrane potential during HGH application followed a significant reduction of the whole-cell input resistance of approximately 0.9 to 0.7 gigaohms on AVPV/PeN kisspeptin neurons and 1.7 to 1.0 gigaohms on ARH kisspeptin neurons. During the perfect brain lines performed via careful gigaseal and the area thing at the cell via beach parameters are required to achieve the best whole-cell recordings.
The micropipette solution of the recorded cell can be collected and used for single cell RTPCR allowing the molecular characterization of an isolated cell. Whole-cell patch clamp technique combined with the use of genetically modified animals represents a breakthrough in the understanding of reproduction allowing the definition of kisspeptin neurons increased properties, and they are major modulators.
