Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice
Transkript
In this procedure, transfer a slice to the submersion chamber, place a screen on top of it to hold the slice down, and perfuse it at 1.5 milliliters per minute, with oxygenated aCSF containing the selected receptor antagonist.
In order to isolate intrinsic membrane properties, what you need to do is block the fast synaptic transmission around the cell, and also block the ability to generate action potentials. So you have to use synaptic blockers and tetrodotoxin or TTX in order to block action potentials. That way, the cell that you are patching is the one that generates those properties, and no synaptic input is responsible for those, and that's what we're studying here — intrinsic membrane properties.
After that, fill the recording patch pipette with intracellular high potassium solution. Insert the pipette in the head stage holder. Next, apply a small positive pressure using a 1-milliliter syringe connected to the pipette holder. Using a 4x objective together with the near-infrared differential interference contrast optics, locate the PPN nucleus, which is dorsal to the superior cerebellar peduncle.
Slowly, lower the recording pipette to the PPN nucleus using a mechanical micromanipulator. Then, position the recording pipette in the PPN pars compacta, which is located immediately dorsal to the posterior end of the peduncle. Using a 40x water immersion lens, bring the recording pipette in contact with a PPN neuron, and rapidly apply negative suction to form a seal with the cell.
Use voltage-clamp seal software to monitor the pipette resistance during negative suction. Then, slowly increase the negative pressure. When the resistance value of the pipette reaches 80 to 100 megaohms, rapidly change the holding potential to minus 50 millivolts and release the negative pressure.
Apply negative pressure continuously until the cell membrane is ruptured and electrical access is achieved in the whole-cell configuration. Before suction, the resistance is low. In this case, 2.5 megaohms. As suction is applied, step amplitude decreases towards holding. When the seal is formed, note the resistance increases to 167 megaohms.
With additional suction, a gigaseal is formed. Resistance briefly increases to 1.2 gigaohms before acquiring whole-cell access. Continuously monitor the resting membrane potential of the PPN neuron being recorded. If the resting membrane potential shifts towards depolarizing or hyperpolarizing values, apply a small amount of direct current to keep the optimal potential at minus 50 millivolts.
