Hi, my name is Ken Paradiso. I work for Ling Gang Wu at the National Institute of Neurological Disorders and Stroke Intramural program, which is located at the NIH in the Bethesda campus. I'm gonna be showing you the techniques for the caly have held presynaptic recording Patch clamping.
So to prepare the Brain tissue, we remove the brain from the rat. We put the brain inside of this solution, which would be ice cold, low calcium solution, and it would be bubbling with carbogen to make sure that there's an oxygen supply to the brain at all times. And it also has to be ice cold to minimize DA to minimize the amount of damage.
So the brain would now be in this solution. We would take it out, cut it appropriately so that it can be put onto the block, mounted on the block with Sano correlate or crazy glue, but fill the chamber then with ice cold, low calcium solution, bubble it, place it onto the vibrator and then cut slices at 180 to 190 microns in thickness at a frequency blade frequency of 70 hertz and a forward advanced speed of 0.01 to 0.02 millimeters per second. So that would be 10 to 20 microns per second.
Once each slice is made, we transfer the slices from the ice cold saline over to this chamber, which contains normal calcium solution. This is the same solution that we record in and it's at 37 degrees physiological temperature. We then put the slices in here for recovery.
It stays in here for half an hour to an hour before the physiology Experiments are run. So here I have the brain and I'm going To pull it out of the solution. So this is a a larger brain for demonstration purposes.
This is from a P 20 rat. We'll turn it over. It's the brain stem that we're interested in.
So this area down here and the calyx have held is right about in that area right there. So what we're going to do is cut off the rest of the brain, isolate the brainstem, and then prepare the brainstem for the slicing chamber. Okay, so we're gonna slice off the rest of the brain just isolating out the brainstem.
I'm gonna turn it over to demonstrate where we are. Normally we wouldn't do it this way, and again, we can just get rid of that whole portion. Turning it back over, we've isolated the brainstem and we're just do another cut right there.
Okay, that's it. That's the brainstem. Calyx of held is right about there and there.
This is the brainstem. Again, we'll push it back a little bit. We're gonna cut off one side of the cerebellum and that's gonna allow the brain stem to sit sideways like this.
We put in a little bit of Sano acrylic glue and keeping it laid sideways quickly fill it up with cold ice, cold low calcium solution. Get the oxygen line in there as soon as possible. We now pick it up and bring it over to the vibrato.
We bring it in fast to get to the beginning of the brain, and then once we get to the beginning of the brain, we drop the speed down to 0.01 millimeters per second. So it's 10 micros per second. So it's moving at an incredibly slow rate.
The first portion of the brain is, is the part that has the mtb, which is the medial Niles of the trapezoid body. And that's the part that contains the, okay, so once I have it in the transfer pipette, I try to bring it as close to the tip of the pipette as possible. And there's the slice of the brain and I rather quickly transfer it into this chamber, which contains a standard recording solution that is normal levels of calcium and also a higher temperature.
And that will allow the slices to recover after the slicing procedures that they're ready from physiology experiments. Okay, so from here on in, it's gonna be the same thing over and over. The Following equipment is what I'll be using to do the patch clamping.
We have a fixed stage upright microscope. The stage is fixed, as I said, so the microscope moves underneath it from on a manipulator. We use a wigs and Newman micro manipulator to hold the stage, the head stage of the amplifier, we use an EPC 10 patch clamp amplifier from heca.
And what I also should have mentioned is that we use DIC with infrared light. So we have a IR camera here, and so everything that we see will be seen on the screen when we're looking for CAEs. The patch clamp amplifier is controlled by this software, which is called Pulse, which is also, which is also from Heca, and will control the amplifier and all of the physiology experiment.
Results will come out on this screen. The caly have held is a large presynaptic terminal, and because it's large, it allows us to physically patch onto the terminal. So we can take presynaptic recordings and that's kind of a unique thing, especially in the CNS.
So this allows us to measure calcium channels activated presynaptically. It also, we also can measure action potential activity if we wanted to stimulate the nerve that leads to the calyx. What we do in this lab is measure a process called exocytosis, which is the fusion of vesicles that contain neurotransmitter with the membrane.
Now in those vesicles fuse with the membrane in the process called exocytosis, they add membrane to the synaptic terminal, and we can measure that as a change in capacitance. We'll get an increase in capacitance as membrane is added to the terminal so that, so the experiments I'll be showing today are capacitance measurements, presynaptically when the membrane is removed. So you can't just keep on adding membrane, you of course have to remove some.
That process is called endocytosis or membrane uptake. We can also measure that and that will be measured as a decrease in the capacitance level and we'll show examples of that later on. Now what this allows us to do is to monitor calcium channel activity and also monitor some of the proteins that are involved in exocytosis and endocytosis and study them.
We can put in things like peptides to block proteins that are involved in exo or endocytosis. We can also use toxins that do the same thing. We can also adjust the amounts of calcium that's coming into the terminal, so we can really study the exocytosis process, the release of neurotransmitter, as well as the uptake of membrane that follows that event.
This is the tube containing our intracellular recording solution. We prepare it ahead of time and then we freeze it and we can usually use it for several weeks to a month before having to make new solution. Again, we thought immediately before doing our recordings, and you're always going to get a certain amount of dust particles or other type of debris that's going to get into your tube.
You can do one of two things. You can either take the solution out and filter it, or you can spin it for a minute or two and then pull off the solution, leaving a bottom portion that would have debris and other material inside of it. So I prefer to spin it and I'll do that right now.
So this is the tube containing our intracellular solution. It's thawed. We're going to spin it down for about two to three minutes in a little tiny centrifuge, and that should be enough to get any large dust particles out of the solution.
So now I just pull off the top, the solution that we just spun, being careful not to agitate it, transferring it to a clean tube, and I pull off about 90%of it. I try not to again, get that last little bit. So I'll stop right about there.
This I put on ice immediately so that it stays nice and cold during the experiment. That's very important. This is the, this is one of our pipettes.
We use this for intracellular recordings. It's thick walled bore silica glass, two millimeters outer diameter, 1.16 millimeters inner diameter. First, what we do is we backfill the pipette.
So we apply suction, we put the pipette into the recording solution and apply suction to try and fill the very, very tip of the pipette, which otherwise won't fill any other way. Once we've done that, we then take a capillary tubing and fill the rest of the electrode just taking the back of your pipette. This is a standard physiology technique and literally flicking it.
You run the danger of breaking the end of the pipette, but you also will have, if you don't do that, you'll often have air bubbles. Okay, we now put it onto our pipette holder, which is connected to our head stage of the patch plant amplifier. There's a silver chloride wire there.
The silver chloride wire comes into contact with the intracellular solution, and that allows us to measure the electrical Activity in the presynaptic terminal. So what we do is we Find the slice that has a lot of CAEs in it. We want a high density of CAEs.
So this is, we're looking for the medial nucleus of the trapezoid body, which is the MNTB, and that's where the terminal, which forms the caly upheld, or which is the calyx app, how is found. And so what we do is start scanning through the slice. So for example, there we have a calyx that's a little bit deep off of the surface and there's not much to patch onto, so we just keep On scanning across us.
So this Is a calyx here that we're going for. What I've made is two marks on the screen, which is where the pipette is gonna go. And you can see that there's the piece of calyx that I'm gonna go after pipette iss gonna come down.
I'm gonna apply positive pressure so that it pushes up against the calyx, and then I'm gonna relieve the positive pressure. The calyx is then going to push up against the pipette, and at that point I apply very gentle suction and try to suck and push the membrane or get the membrane to seal onto the pipette tip. All right, relieved the positive pressure.
And now I'm gonna apply suction. So here we're pro, we're applying a five millivolt pulse, or actually a four millivolt pulse. Okay, That's it.Okay.
Now we lower the membrane potential minus 80, cancel the fast transient boom. Now we're gonna go whole cell. I'm gonna apply gentle suction and try to go wholesale.
Those charging transients indicate that he's, that the pipette and the cell are now continuous that he is got a, a wholesale recording. That's another beautiful shot. So the lighter red there was the capacitance trace, and you can see there's a sudden jump And then it declined.
Okay, I've just shown you the basic techniques for getting recordings from a calyx upheld presynaptic terminal. I've shown you how to produce the slices that contain the medial nucleus of the trapezoid body. That's where you'll find the calyxes have held.
And I've shown you the the techniques for looking through the slices. You're gonna go through each of the slices, looking for the slice that has the largest number of caly that are on the surface. You're then gonna look for the largest caly membrane that you can find and patch clamp record onto it.
And I've also showed you the techniques for patch clamp recording, and those Are standard patch clamp recording techniques.
