The overall goal of this demonstration is to describe a method for detecting high threshold and subthreshold oscillations in pedunculopontine neurons. This method can help answer questions about, for example, how cells maintain their firing frequency. How do populations maintain their firing frequency?
We know that circuits cannot maintain gamma band or 40 to 60 per second oscillations. Therefore, you require intrinsic membrane properties to maintain that kind of firing frequency. Demonstrating the procedure will be Susan Mahaffey and Dr.Stasia D'Onofrio.
To prepare standard aCSF, first prepare stock solutions A and B and keep them refrigerated at four degrees Celsius for up to two weeks. Before each experiment, mix 50 milliliters of stock A with 50 milliliters of stock B.Then bring the mixture to one liter with distilled water and oxygenate it with carbogen at room temperature for at least 30 minutes. Transfer to a beaker for stirring and oxygenate.
After bubbling, add the calcium chloride. Next, prepare sucrose aCSF by preparing stock solution C.Before each experiment, mix 300 milliliters of stock C with 600 milliliters of distilled water and oxygenate it with carbogen at room temperature for at least 30 minutes. After that, transfer 100 milliliters of sucrose aCSF solution to a clean beaker, place it on ice, and keep it oxygenated with carbogen.
Now, fill up a vibratome cutting chamber with sucrose aCSF and keep it oxygenated. Turn on the glycerol-based refrigerating system coupled to the cutting chamber and wait 15 minutes to allow it to cool down to about four degrees Celsius. Next, remove the pup's brain by placing a spatula under the olfactory bulb and gently pushing it out from the most rostral area towards the most caudal area.
Subsequently, place the brain into the ice-cold oxygenated sucrose aCSF. Make a parasagittal cut on the right hemisphere to remove approximately 1/3 of the hemisphere. Then, glue the trimmed side of the brain onto the metal disk.
Subsequently, fix the metal disk to the vibratome cutting chamber and slice 400 micron sagittal sections containing the PPN. Keep the PPN slices at room temperature for 45 minutes prior to whole-cell patch clamp recording. 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 property. After that, fill the recording patch pipette with intracellular high potassium solution.
Insert the pipette in the headstage holder. Next, apply a small positive pressure using a one 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 micromanipulater. 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 the 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 50 millivolts and release the negative pressure.
Apply negative pressure continuously until the cell membrane is ruptured and electrical access is achieved in the wholesale 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 wholesale access.
Continuously monitor the resting membrane potential of the PPN neuron being recording. If the resting membrane potential shifts towards depolarizing or hyperpolarizing values, apply a small amount of direct current to keep the optimal potential at 50 millivolts. The reason why we use current ramps is so that we can slowly depolarize the membrane without activating potassium channels that keep you from depolarizing the membrane sufficiently high to reach the threshold of these high-threshold calcium channels that we're studying.
Shown here are the representative membrane potentials responding to the depolarizing two-second-long square steps of increasing current injected intracellularly through the recording pipette in the presence of synaptic blockers and TTX. This figure shows a power spectrum of the membrane oscillations induced by the indicated current steps in three recordings. The frequencies induced were all low in power and low in frequency.
This figure shows the current ramps of 200, 400, and 700 picoamps and the membrane responses. Note that the membrane is sufficiently depolarized to reach 30 millivolts, at which high-threshold calcium channels begin to open, as in the blue recording, and the 10 millivolt level at which their oscillation amplitude is maximal, as in the red recording. This graph shows a power spectrum of the membrane oscillations induced by the current ramps in the corresponding three recordings.
The frequencies induced by the 400 picoamp ramp were in the beta range, and by the 700 picoamp ramp were in the gamma range. Once mastered, this technique can be done in about an hour or less. When you're doing your recording, it's really important to make sure that you have a tight seal and that you have capacity compensation and series resistance well-monitored to make sure that there are no major changes during the recording procedure.
Once you've isolated these oscillations, what you can do is test to determine if they're really mediated by N-type or P/Q-type calcium channels. You can use conotoxin to block N-type channel oscillations and agatoxin to block P/Q-type channel oscillations. As we developed this technique, it became much easier for you to demonstrate that indeed, high threshold calcium channels were dependent for intrinsic membrane oscillations, and it became a breakthrough in the field of sleep research to determine that the reticular activating system could actually generate gamma band oscillations by itself.
