The overall goal of this puff technique is to conduct the pharmacological administration in whole-cell patch-clamp recording. This method can help answer key questions in the electrophysiology field, such as pharmacological administration. The main advantage of this technique is that the drug concentration around the recording site increases rapidly, thus preventing the membrane receptors from desensitization.
Generally, individual new to this method will struggle because the preparation of acute prefrontal cortex slice and the position of puff micropipette are relatively difficult. To begin this procedure, crush the frozen cutting solution to obtain a slush. Keep the slushy solution on ice, and bubble it with 95%oxygen, 5%carbon dioxide.
Next, place the dissected head into a beaker filled with ice-cold cutting solution, and take it out after a few seconds. Cut the skull along the midline in a caudal-rostral direction with fine scissors, and remove the lateral skull portions. Then, remove the brain with a fine spatula, and drop it into the ice-cold cutting solution.
Afterward, place the brain on the lid of a nine-centimeter Petri dish filled with ice. Rinse the brain with ice-cold cutting solution. Then, cut off the cerebellum and olfactory bulb with a razor blade.
Subsequently, apply cyanoacrylate glue to the Vibratome specimen holder. Carefully place the brain block on the drop of glue, rostral side up, and immediately immerse it in the ice-cold cutting solution. Adjust the blade holder to an angle of 15 degrees with reference to the horizontal plane.
Section the brain tissues into 350 micron coronal slices. Then, transfer the slides into the recovery chamber filled with ACSF, and incubate them for one hour with constant bubbling of 95%oxygen, 5%carbon dioxide. In our experiment, the preparation of acute prefrontal cortex slice is also critical in the procedure.
A healthy brain slice means active neurons that can be patched tightly. In this procedure, make glass borosilicate micropipettes for use as recording electrodes or puff micropipettes. For recording electrodes, tip resistances are in the range of four to six megaohm, while puff micropipettes have tip diameters in the range of two to five micrometers.
Add sodion channel blocker to ACSF to block fast sodion channel and thus action potentials, and maintain a constant flow rate of this solution of two to three milliliter per minute in the recording chamber. Bubble the ACSF with 95%oxygen, 5%carbon dioxide constantly to ensure the viability of the slices. Transfer a brain slice to the recording chamber, using a Pasteur pipette with its fine tip cut to fit the size of the slice.
Next, place a platinum slice anchor above the slice to hold it on the platform. After that, fill the recording micropipettes with internal solution, and fill the puff micropipettes with GABA at 10 micromolar, 50 micromolar, and 100 micromolar, or ACSF as vehicle control. To prevent blockage with debris, apply slight positive pressure using a one-milliliter syringe before immersing the recording electrode in the ACSF.
Under a microscope, position the recording electrode and puff micropipette so that the tips appear in the center of the monitor. Then, identify a target neuron. Adjust the microscope focus while gradually lowering the puff micropipette, and place it above the recording neuron at an angle of 45 degrees.
Keep the distance between the tip of the puff micropipette and the target neuron in the range of 20 to 40 micrometers. Slowly and carefully approach the neuron with the recording electrode, and then release the positive pressure. Apply a weak and brief suction through the tubing connected to the electrode holder to create a gigaohm seal.
Maintain the voltage at zero millivolts. After the formation of the gigaohm seal, compensate the fast and slow capacitance manually or automatically. Then, apply a brief and strong suction through the tubing to break into the whole-cell mode.
Subsequently, record evoked IPSC currents in voltage clamp mode. Deliver single or paired GABA puffs through the puff micropipette controlled by a Master-8 voltage-step generator. Change the puff duration to obtain the best evoked IPSC currents results, and measure the evoked IPSC currents in the LPS-induced depression model.
Shown here, are the sample IPSCs induced by 10, 50, and 100 micromolar GABA. And here are the puff duration response curves. The repetitive currents evoked by GABA at 50 micromolar puffs at interpuff intervals of one, two, or three seconds are shown here.
One mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it's important to remember to obtain healthy brain slices. After its development, this technique paved the way for researchers in the field of pharmacological administration to explore the effect of drug in electrophysiology.
After watching this video, you should have a good understanding of how to conduct the pharmacological administration in whole-cell patch-clamp recording. Don't forget that working with TTX can be extremely hazardous and precautions, such as wearing gloves, should always be taken while performing this procedure.
