Visualizing Synaptic Vesicles Signaling

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00:00:16.04 I'm going to tell you about 4 delightful decades I've had
00:00:21.09 examining the structure of the synapse.
00:00:23.24 Actually, I'm standing in front of a giant picture of a synapse
00:00:27.28 that I took about 40 years ago.
00:00:30.22 Inside the synapse, you can see these little circles. Those are synaptic vesicles.
00:00:36.26 And then you see those dense areas down at the bottom.
00:00:39.20 That's a place where the one nerve cell, the pre-synaptic nerve cell
00:00:43.28 contacts the other nerve cell, the post-synaptic nerve cell,
00:00:47.08 and it's right there that the pre-synaptic nerve cell
00:00:50.04 releases transmitter to send a signal to the post-synaptic nerve cell.
00:00:56.15 Now, once we had these pictures,
00:01:00.27 and the group at University College, London had shown
00:01:07.04 that this release of transmitter comes in little squirts.
00:01:09.29 They called them quanta.
00:01:12.04 It seemed obvious from the pictures that the vesicles might contain the quanta.
00:01:17.10 So, that was known as the vesicle hypothesis,
00:01:20.08 and this is where I came into the picture.
00:01:22.24 We had no idea how vesicles might release a transmitter
00:01:27.00 or how new vesicles might be reformed after they released their transmitter.
00:01:32.25 At that point, John Heuser joined my lab
00:01:37.17 from University College, London, where they were working
00:01:39.21 on this beautiful preparation of frog neuromuscular junction
00:01:42.29 which is a sausage-shaped structure
00:01:45.22 I've got my hand on it right here,
00:01:47.11 and you can see it has synaptic vesicles, just like I said.
00:01:50.16 It's a synapse between a nerve and a muscle,
00:01:53.00 rather than between a nerve and a nerve.
00:01:55.12 And, the structure is beautifully clear,
00:01:58.09 and so we decided that we would stimulate it and see what changes
00:02:03.22 in structure might occur upon stimulation that might tell us something
00:02:07.17 about how the quanta are released
00:02:09.19 or at least remade.
00:02:11.13 So, when we stimulated,
00:02:13.05 we got an amazing result.
00:02:15.15 Many of the synaptic vesicles disappeared,
00:02:18.21 and in their place, these big vacuole things appeared.
00:02:22.09 And the vacuoles filled inside the synapse,
00:02:27.28 and also inside the synapse there were little tiny vesicles with fuzzy stuff on them.
00:02:32.28 These are called coated vesicles.
00:02:34.21 And they were known to be the way that cells recover things from the outside.
00:02:38.18 This made us think that maybe the vesicles are being released somehow
00:02:43.12 or used up somehow, and then they're being recovered
00:02:45.14 by bringing in more surface membrane.
00:02:48.04 And, if that's the case, we should be able to test it by using
00:02:51.12 a dense material that you can see in the electron microscope.
00:02:54.04 Now, the synapse that's been stimulated here
00:02:57.08 recovers within minutes.
00:02:58.25 And so, if we put the dense material around the outside and let it recover,
00:03:02.09 if the outside is the source of new synaptic vesicles,
00:03:06.19 then the synaptic vesicles should have the dense material in it.
00:03:10.06 And, indeed they did.
00:03:11.29 So, here are synaptic vesicles with dense material.
00:03:15.08 This was a synapse that was stimulated. It's recovered,
00:03:17.23 and it's taken up the dense material in the synaptic vesicles.
00:03:20.23 This led us to propose a theory
00:03:23.12 of synaptic vesicle recycling.
00:03:26.17 The idea is that the synaptic vesicles are released
00:03:29.27 and become part of the surface, and we thought this might be the case
00:03:33.10 because when you stimulate the synapse, it gets bigger.
00:03:35.18 But, actually, vesicles on the surface... it's recovered
00:03:39.05 by these little coated vesicles pinching off
00:03:41.29 and also perhaps by large vesicles pinching off directly
00:03:45.06 when you have very high levels of release.
00:03:47.07 And once inside, the membranes get sorted out
00:03:50.24 and made back into synaptic vesicles.
00:03:53.17 So that's the theory of synaptic vesicle recycling.
00:03:56.18 That paper was very well received. John went back to London.
00:04:00.12 And we were left scratching our heads, because this theory
00:04:04.03 was very unsatisfying and incomplete from both of our points of view.
00:04:08.04 The problem being that we could see vesicles lined up on the surface,
00:04:12.18 but we couldn't see how they were released.
00:04:14.02 The reason we couldn't see it was because we were using chemical fixatives.
00:04:18.09 They're very, very slow, and so by the time the fixative got there,
00:04:22.09 everything was over.
00:04:23.05 We couldn't really catch that fleeting event that would involve
00:04:27.27 the release of a squirt of transmitter.
00:04:31.06 We thought that maybe we could do this with freezing.
00:04:33.13 And there was an old freezing machine in my lab
00:04:35.13 that never worked, and so while John was in London, I got it down
00:04:38.20 and started working with it,
00:04:40.09 and I introduced a new technique, I'll come back to in a minute later.
00:04:43.11 I'm not going to say a lot about it. It's called freeze fracture.
00:04:46.03 It allows you to see the surface of the synapse,
00:04:48.27 rather than looking inside the synapse.
00:04:50.23 And looking at the surface of the synapse,
00:04:52.22 I could see this freezing machine was sort of working.
00:04:54.20 We could occasionally see what looked like vesicles coming out.
00:04:58.01 So, I call up John in London, and I say,
00:05:00.06 "John, get over here, and let's get to work on this."
00:05:03.02 And he came over for about 2 months, and we worked with this machine
00:05:06.04 with very, very spotty results.
00:05:08.19 And, we were scratching our heads. We didn't know what was wrong.
00:05:11.19 But, John gets a call from University California, San Francisco,
00:05:19.02 "Would you like to have a position out here?"
00:05:21.01 So, John goes out to San Francisco,
00:05:23.17 and that begins 5 years of he and I going back and forth
00:05:27.03 across the country to try to work out this problem.
00:05:30.11 Now, this is John
00:05:35.28 who came back from London, and we spent a lot of time together.
00:05:39.29 Very intense time at University of California, San Francisco.
00:05:42.20 Our time was so intense that people... it sort of cleared the oxygen out of the lab.
00:05:48.07 A lot of people left. We argued like an old married couple every day.
00:05:51.29 And, people said it was just terrible.
00:05:54.12 In fact, some people thought we might be an old married couple,
00:05:57.11 but we weren't, but we were just intensely linked
00:06:00.00 in this quest to try and figure this thing out,
00:06:03.11 and we kept doing freezing rounds and freezing rounds
00:06:06.00 and getting every once in a while, we'd get a little result.
00:06:09.00 It suddenly occurred to both of us that when you're in this close of a relationship,
00:06:13.13 you never know whose idea it is.
00:06:15.27 The ideas just come together... and
00:06:21.08 that if you drop something on something, that it's going to bounce,
00:06:25.27 and so these machines that were trying to drop things on the cold metal plate
00:06:29.06 were going to bounce, and so, we built a machine that was designed
00:06:35.04 not to bounce, made by a remarkable machinist
00:06:40.02 at University of California called Jim Wall
00:06:43.02 who made electrode pulling machines, and so this is an adaptation
00:06:47.01 of the electrode puller. It's essentially an arm that holds the tissue
00:06:51.15 and drops it down onto a
00:06:52.28 block cooled with liquid helium.
00:06:55.00 Now, the key here was that we used a big heavy metal ring and a big strong magnet.
00:07:01.06 And we also used spring loading, and by adjusting the springs and the magnets,
00:07:06.26 and we used electrical contact ridges
00:07:09.15 to see what was actually happening when it hit.
00:07:11.16 We figured out that with certain combinations of forces,
00:07:15.02 we could get rid of the bounce.
00:07:16.01 It was very hard.
00:07:17.00 But once we got rid of the bounce,
00:07:18.21 we started getting beautiful freezing every time.
00:07:21.13 And we could then go and look at the neuromuscular junction.
00:07:26.09 Now this machine that Jim built us had a... the frog neuromuscular junction
00:07:31.29 was mounted on that little plunger thing on the end,
00:07:36.06 and on that plunger was a little wire hook ... 2 little wire hooks,
00:07:39.27 and we could hook the nerve to the muscle.
00:07:41.12 So, on the way down, there's a trigger
00:07:43.10 and we could trigger a stimulus and stimulate the nerve
00:07:46.08 right before the muscle got frozen,
00:07:49.01 and we could explore different time periods.
00:07:51.26 We did some electrical measurements of the freezing rate,
00:07:55.21 and we found that we were freezing in less than 1/10 of a millisecond.
00:07:58.08 So, we were able to do structure for the first time in sub-millisecond resolution.
00:08:03.07 Now, when we looked at the neuromuscular junction at rest,
00:08:08.13 with no stimulation, we saw what we expected:
00:08:10.27 vesicles... full of vesicles, some lined up on the surface.
00:08:13.29 But, when we stimulated 4 milliseconds before it hit...
00:08:18.14 At 2 milliseconds, we saw nothing.
00:08:21.06 4 milliseconds, all of a sudden, the whole surface of the nerve begins to bubble,
00:08:26.01 and you see these little things that look like vesicles opening.
00:08:29.07 They're already down there and over there.
00:08:31.22 They're little flasks, and this vesicle is fused and open.
00:08:36.02 Now, I want to stress, this happens within milliseconds,
00:08:41.15 and if you wait a few more milliseconds,
00:08:43.10 these vesicles proceed to flatten out and dissolve
00:08:46.26 into the surface. Now, using the freeze fracture technique
00:08:49.22 which shows us the surface, we could actually see that.
00:08:52.21 You remember now, the vesicle is a little flask,
00:08:55.19 and if you look at it from the surface, it'll look like a ring,
00:08:57.26 and as it flattened out, the ring would expand.
00:09:00.26 So, what you see here at different times from left to right
00:09:06.00 is the ring expanding, and finally it flattens out, completely flattens out
00:09:09.29 and you just see a little indentation.
00:09:12.10 So, this is a sequence of events that shows that the vesicles
00:09:16.22 are actually fusing and becoming part of the surface.
00:09:21.08 Now, this is exactly what we wanted to see
00:09:25.17 to fill in the gap in our recycling theory, because
00:09:29.19 the recycling theory said that the new vesicles were coming from the surface.
00:09:33.16 So, how'd the vesicles get to the surface?
00:09:35.27 Well, now we could actually see vesicles join the surface
00:09:40.17 and flattening out on the surface. Furthermore,
00:09:43.11 we could count the number of these events.
00:09:45.22 And, we could measure physiologically how many squirts
00:09:49.13 there were, and we related the number of squirts
00:09:52.14 to the number of structural events
00:09:55.15 at different levels of release, and they matched perfectly.
00:09:59.21 So, it seemed that we had very direct evidence of the
00:10:05.24 vesicle recycling hypothesis.
00:10:08.21 Now, any theory you come up with is going to have its detractors.
00:10:13.02 And, people are going to start in on you, which is good,
00:10:17.16 because that's what science is supposed to be about.
00:10:19.00 And so, people started to think, well,
00:10:24.02 maybe some vesicles recycle.
00:10:25.12 Maybe other vesicles just come up to the surface and make a little opening
00:10:28.26 and release a little squirt of transmitter.
00:10:32.13 And that was called kiss-and-run theory.
00:10:34.24 And John, remarked, "Why would anybody kiss-and-run when you could stay?"
00:10:40.04 But, there is more and more evidence coming up for kiss-and-run,
00:10:44.13 and it seems to m that it had a small part in some synapses
00:10:48.21 for some reasons. We don't really know why yet.
00:10:50.18 And investigating it structurally, such a small, rapid event,
00:10:56.13 would be difficult even with these freezing techniques.
00:10:59.09 So, that was a delightful decade of examining synaptic structure.
00:11:06.06 And one night, late one night,
00:11:07.21 John and I were sitting there, exhausted,
00:11:09.23 and I turned to John and said,
00:11:11.01 "Why the hell are we doing this? Our lives are falling apart outside the lab.
00:11:14.10 "Our wives hate us. And, why are we doing this?"
00:11:18.17 And John turned to me,
00:11:19.16 and he said, "Well, you know, Tom. The most important thing in science
00:11:23.03 "is to be together with a colleague at that moment when you discover something.
00:11:30.02 "When some little piece of nature unfolds, and you see something new."
00:11:36.22

This Talk

Speaker: Thomas Reese

Audience:

Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: September 2012

Talk Overview

Thomas Reese describes a decade of experiments with John Heuser which led to their theory of synaptic vesicle recycling and allowed them to directly visualize synaptic vesicles fusing with the plasma membrane. The result was a triumph of technical wizardry and scientific insight.

Speaker Bio

Thomas Reese

Tom Reese is a Senior Investigator at the National Institute of Neurological Diseases and Stroke at the National Institutes of Health and a summer researcher at the Marine Biological Laboratory. He continues to study synaptic structure and function using advanced light and electron microscopy techniques. Reese was elected to the National Academy of Sciences in… Continue Reading