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Recycling Synaptic Vesicles

Part 1: Synaptic Transmission

00:00:08;04 Alright.
00:00:09;11 I'm Erik Jorgensen,
00:00:10;25 I'm from the University of Utah.
00:00:11;24 What I'd like to talk about today is
00:00:14;09 the recycling of synaptic vesicles.
00:00:16;25 So, before we get started,
00:00:19;00 I'd like to tell you a bit about synaptic transmission.
00:00:20;17 So, the way it works is that
00:00:23;04 there's a cell body that can be very far away
00:00:25;27 from the synaptic bouton,
00:00:27;09 an axon that extends
00:00:29;06 all the way to this synaptic bouton,
00:00:32;00 and that can be about a meter long.
00:00:34;13 So, the synaptic bouton
00:00:37;03 needs to act as an independent organelle.
00:00:38;19 So, the way this works is that
00:00:40;24 there are synaptic vesicles
00:00:42;19 that are filled with neurotransmitter molecules,
00:00:44;23 and then these molecules,
00:00:46;14 these synaptic vesicles,
00:00:47;21 are then docked at the membrane.
00:00:50;10 And so the way that synaptic transmission works
00:00:52;17 is that, when there's a calcium influx,
00:00:55;15 the vesicles then are released,
00:00:58;06 the vesicle fuses with the plasma membrane,
00:01:00;07 and releases the neurotransmitter
00:01:02;07 onto the receptor, shown here.
00:01:04;24 So, by doing that,
00:01:07;09 you lose the synaptic vesicle membrane,
00:01:08;26 and you lose the synaptic vesicle protein.
00:01:11;04 And so, because that synapse
00:01:13;05 is so far away from the cell body,
00:01:14;21 the cell body can't replenish synaptic vesicles,
00:01:17;05 so what you need to do is recycle them.
00:01:20;15 And the classic mechanism by which that's done
00:01:22;19 is clathrin-mediated endocytosis,
00:01:25;03 and so I'll tell you a little bit about that today.
00:01:29;17 And then, in the second half,
00:01:31;08 in the second video,
00:01:32;17 I'd like to tell you about
00:01:34;11 some recent work of ours,
00:01:36;11 demonstrating that there is a second mechanism
00:01:39;06 for endocytosis at synaptic boutons,
00:01:41;21 and this is called ultrafast endocytosis.
00:01:44;12 And in this case, what we see is that
00:01:46;25 the synaptic vesicle membrane
00:01:48;15 is recovered very rapidly,
00:01:50;14 about 30-300 milliseconds,
00:01:55;05 generating a synaptic endosome,
00:01:57;07 and then that synaptic endosome
00:01:58;29 is resolved by clathrin-mediated vesicle budding,
00:02:02;03 and this is also a very fast process
00:02:03;18 -- it only takes about 3 seconds.
00:02:05;24 And then, finally,
00:02:07;19 when that budded vesicle is uncoated,
00:02:09;20 we've regenerated a synaptic vesicle
00:02:12;00 after only 5 seconds,
00:02:13;22 and so this makes a synapse
00:02:15;25 be able to run at much faster speeds
00:02:18;02 than with clathrin-mediated endocytosis.
00:02:21;12 So, again, we have two videos
00:02:24;04 that I'll be filming.
00:02:25;08 The first is this demonstration
00:02:27;17 of how synapses work.
00:02:30;22 This is the work of Bernard Katz,
00:02:32;23 showing that vesicles have this exocytic property.
00:02:36;05 Secondly, I'll tell you about the classic work
00:02:40;12 of Heuser and Reese,
00:02:41;27 demonstrating that vesicles are recycled
00:02:43;18 via a clathrin-mediated process.
00:02:45;11 In the second video, I'll tell you about this recent work
00:02:49;28 identifying this ultrafast pathway.
00:02:53;01 So, first of all, this work all relied on
00:02:56;06 the work of Bernard Katz,
00:02:57;20 who was working in the '50s and '60s,
00:02:59;18 and this is work that won him the Nobel Prize,
00:03:02;20 and what he was doing was recording
00:03:04;10 from the frog sartorius muscle.
00:03:05;24 So, he had a sharp electrode,
00:03:08;15 shown here,
00:03:09;21 penetrating the muscle cell,
00:03:11;22 and he could then record
00:03:13;22 changes in the potential of that muscle cell.
00:03:15;25 And so, what he'd see is that,
00:03:17;20 as long as there were no changes,
00:03:19;28 the potential was constant,
00:03:22;22 at about -60 millivolts (mV).
00:03:24;15 However, he found that
00:03:26;25 if he manipulated a pipette
00:03:30;01 with acetylcholine in it (ACh),
00:03:31;21 and spritzed on a little acetylcholine onto the muscle,
00:03:34;29 then he would see a profound depolarization,
00:03:36;12 shown here.
00:03:37;27 And what he didn't know at the time,
00:03:39;21 but we know now,
00:03:41;15 is that on the membrane of the muscle
00:03:43;04 are acetylcholine receptors,
00:03:45;15 so that the acetylcholine molecules,
00:03:47;17 shown in yellow, here,
00:03:49;07 bind receptors,
00:03:51;08 and these receptors are ion channels,
00:03:52;22 so they open and allow cations
00:03:55;17 to enter the cell
00:03:57;07 and cause this depolarization that you see here.
00:04:00;16 What he then found was that,
00:04:02;24 as he reduced the amount of
00:04:05;26 neurotransmitter released onto the muscle,
00:04:07;12 he saw a proportional decrease in the response,
00:04:10;09 and he could continue that until
00:04:13;15 the response was zero.
00:04:14;20 In other words, there is
00:04:17;08 no minimal response for a synapse
00:04:20;25 or for a response to neurotransmitter.
00:04:23;16 So, this was much different than what he saw
00:04:26;19 if he left the motor neuron in place.
00:04:29;12 So, here is a motor axon, shown here...
00:04:31;21 here's the motor neuron
00:04:33;06 and here's the motor axon,
00:04:36;05 and it's now forming a synapse on the muscle cell.
00:04:37;28 And so, now, he simply records.
00:04:40;03 He's not spritzing on neurotransmitter,
00:04:42;05 he's just seeing if there are spontaneous events,
00:04:43;26 and what he saw are
00:04:45;26 these spontaneous depolarizations of the muscle.
00:04:51;05 Moreover, when he looked at them,
00:04:54;04 they were of a fixed size.
00:04:55;17 So, from this, he said that
00:04:58;10 neurotransmission, somewhat like quantum physics,
00:05:01;11 is a quantal response.
00:05:03;06 There is a minimal response
00:05:05;16 that the muscle will respond to.
00:05:07;05 So, Katz was aware of
00:05:11;01 electron micrographs that had just recently been done
00:05:13;19 of the neuromuscular junction
00:05:15;02 -- this one's by David Robertson --
00:05:17;06 and, as you can see here,
00:05:18;20 there are synaptic vesicles
00:05:20;11 that can be seen in this micrograph.
00:05:21;22 And so, the way that Katz thought about this was
00:05:26;01 that maybe these vesicles are filled with neurotransmitter,
00:05:28;18 their size determines that
00:05:30;26 there's a fixed amount of neurotransmitter in them,
00:05:33;05 and that, occasionally,
00:05:34;15 these spontaneously fuse with the plasma membrane.
00:05:37;19 And so that's shown, here.
00:05:39;10 And when that occurs,
00:05:41;00 a few of these ligand-gated ion channels will be opened,
00:05:44;05 and so you'll see a fixed response.
00:05:46;10 So, in this case, it's a depolarization.
00:05:49;08 Here's we're showing a miniature current,
00:05:51;10 and so these events are called minis.
00:05:55;20 And so, the quantal hypothesis
00:05:58;02 has now become the vesicle hypothesis,
00:06:00;15 so the idea now is that
00:06:02;23 the reason that there is a quantal response at synapses
00:06:05;11 is because of these vesicle fusions
00:06:07;20 having a fixed amount of neurotransmitter.
00:06:10;29 And so, these...
00:06:13;08 I just want to step back for a moment
00:06:14;21 and tell you a little bit about how these images were created.
00:06:17;01 They're created by an electron microscope.
00:06:20;13 First, what you have to do is
00:06:22;10 embed your sample in plastic.
00:06:24;00 In this case, I have a worm, a nematode, C. elegans.
00:06:27;18 It's fixed and then embedded in plastic.
00:06:28;29 Those plastic sections are then cut,
00:06:31;22 or the plastic block is then cut into sections.
00:06:35;05 Those sections are then picked up,
00:06:37;03 a ribbon of those is picked up onto this grid,
00:06:39;10 when is mounted on a mount
00:06:42;11 that will go into the electron microscope,
00:06:45;26 and then you get an image of the cross-section of the worm,
00:06:48;28 as shown here.
00:06:50;17 And so the way the electron microscope works
00:06:52;27 is that you have an electron source,
00:06:54;29 the electrons then pass through your sample,
00:06:57;19 which is on a thin film,
00:06:59;07 and then the electrons pass through onto a film,
00:07:03;21 onto photographic film,
00:07:05;10 and then that generates this image
00:07:07;19 that you see.
00:07:09;22 So, let's return, now,
00:07:11;19 to the quantal hypothesis.
00:07:13;10 So, Katz understood that
00:07:16;11 what he needed to demonstrate
00:07:17;28 is that these synaptic vesicles
00:07:19;27 fused with the plasma membrane.
00:07:21;08 And at the time, that was not widely accepted.
00:07:23;24 People imagined that these synaptic vesicles
00:07:26;21 could contain neurotransmitter,
00:07:28;12 but they imagined that the neurotransmitter
00:07:30;24 was being released directly through the plasma membrane,
00:07:33;06 not via these synaptic vesicles.
00:07:36;04 So, to prove the quantal hypothesis,
00:07:38;17 to prove the vesicle hypothesis,
00:07:40;28 Katz needed to demonstrate that
00:07:43;22 these vesicles fused with the membrane,
00:07:45;09 and that had never been seen before.
00:07:47;12 And so he took on a grad student,
00:07:50;07 his name is John Heuser,
00:07:52;04 and John's job was to try to capture these events.
00:07:55;12 And he was never able to do so,
00:07:57;06 and in fact John never got his PhD
00:07:59;14 working with Katz,
00:08:01;09 or ever, for that matter.
00:08:03;06 But he was obsessed with this problem
00:08:04;21 and wanted to pursue it.
00:08:06;16 So, he then moved to the lab of Tom Reese,
00:08:09;24 and together they worked out, I think,
00:08:12;13 one of the most exquisite experiments
00:08:14;02 in the history of science,
00:08:15;16 to prove that these vesicles actually fused.
00:08:18;11 So, let me tell you a little bit about that.
00:08:20;19 So, some of those experiments
00:08:22;08 were done at the Marine Biological Laboratory
00:08:25;00 in Woods Hole,
00:08:26;10 and these experiments were done in the 1970s,
00:08:28;00 and we'll return to the scene of the crime later,
00:08:30;20 in the second video.
00:08:33;09 So, here's Tom and John in the 1970s,
00:08:35;14 when these experiments worked
00:08:36;29 -- beautiful young men,
00:08:39;20 dressed appropriately for the time,
00:08:42;18 so times have changed.
00:08:44;17 So, what I want to show, here,
00:08:46;23 is this device that they built.
00:08:48;11 So, what they did was
00:08:51;03 they took a piece of the frog sartorius...
00:08:53;01 pectoral muscle,
00:08:55;10 which is very thin and could freeze quickly,
00:08:57;19 and that's why these selected it,
00:08:59;25 and they left the nerve attached,
00:09:02;29 so the nerve is still attached, here,
00:09:04;07 and they wrapped that around a stimulating wire, here.
00:09:06;20 And this was all mounted on this cylinder,
00:09:08;29 which was then held in place
00:09:11;28 by a rod on this device, shown here.
00:09:14;03 And so, here is where the sample is,
00:09:17;28 and then if you look up here
00:09:20;21 you can see this rod that is holding this in place,
00:09:22;17 and this was held in place by a solenoid,
00:09:26;02 so just this just a friction connection.
00:09:27;14 Below, here, is a copper block,
00:09:30;17 cooled in liquid helium to 4 Kelvin,
00:09:32;20 so this is the temperature of outer space,
00:09:35;12 right... at this temperature,
00:09:37;08 all molecules stop movement.
00:09:38;26 So, then was they could do is
00:09:41;07 they could release that solenoid,
00:09:43;24 and then, using standard physical equations
00:09:46;13 of acceleration,
00:09:47;26 calculate the time it would take
00:09:49;14 for the sample to hit this copper block.
00:09:51;20 There's a shutter that is removed,
00:09:53;19 and then there's a trip,
00:09:55;02 so that at 5 milliseconds before it hits the surface...
00:09:57;16 so, it's falling, falling, falling, falling...
00:09:59;05 5 milliseconds before it hits the surface,
00:10:00;25 that stimulator then activates that nerve,
00:10:03;13 that nerve then causes neurotransmission to occur,
00:10:07;10 and vesicles are fusing,
00:10:08;24 and then... boom.
00:10:10;22 It hits the copper block and then all movement stops.
00:10:12;04 So, what did they see?
00:10:14;07 These beautiful images.
00:10:16;00 So, here, you can see
00:10:18;01 an unstimulated neuromuscular junction,
00:10:20;23 you can see that there are vesicles
00:10:22;21 that are docked at the membrane,
00:10:24;20 and when they stimulated and then froze,
00:10:26;14 5 milliseconds later,
00:10:28;10 you see that there are these fusions that have occurred.
00:10:31;01 If you look here,
00:10:32;22 here's a vesicle that has just fused,
00:10:34;29 here's another one that's just fused,
00:10:37;22 and then here's one that's beginning to flatten out,
00:10:40;23 so it collapses into the plasma membrane.
00:10:43;01 And so I love this experiment, because it
00:10:44;28 required knowledge of physics,
00:10:46;05 it required engineering
00:10:48;25 to be able to build this device,
00:10:50;05 and they combined this with a deep understanding of biology,
00:10:52;16 to say, how are we going to prove
00:10:54;25 this biological point?
00:10:55;28 We're going to have to invent
00:10:58;10 a new instrument.
00:10:59;19 So, it was really a wonderful experiment.
00:11:01;15 So, by doing so,
00:11:03;07 the quantal hypothesis was proved correct.
00:11:05;27 So, there's a few other
00:11:09;03 techniques that they applied to this problem,
00:11:10;27 and one of these is freeze-fracture electron microscopy.
00:11:15;17 So, in the images I just showed you,
00:11:17;05 those are very thin sections
00:11:19;18 that are mounted in a transmission electron microscope.
00:11:22;14 So, what they also discovered
00:11:24;20 was if they took that frozen block,
00:11:26;14 that they could strike it with a knife
00:11:29;00 and split the ice in half,
00:11:31;23 and what happens is
00:11:33;28 the ice will break along the weak points.
00:11:37;04 So, the weak point
00:11:39;24 is at that lipid bilayer,
00:11:42;20 because there's no covalent bonds between that lipid bilayer, right?
00:11:45;21 They're just interacting with each other
00:11:47;09 because they're greasy.
00:11:48;19 So, what happens when you hit that block of ice
00:11:50;02 is that those split open
00:11:51;20 and you get two faces of that ice block,
00:11:54;14 and so on one face is the extracellular layer,
00:11:57;13 shown here,
00:11:59;08 and the other is this cytoplasmic layer, shown here.
00:12:02;06 And so, if you look at those images
00:12:04;28 independently, if you look at the cytoplasmic layer
00:12:07;01 that's shown, here,
00:12:08;28 and if you look at the extracellular layer...
00:12:12;14 oh, that's the extracellular layer,
00:12:14;09 excuse me,
00:12:15;14 and this is the inside, or the cytoplasmic layer,
00:12:17;05 and you can see these little bumps there,
00:12:18;24 and those are proteins that are
00:12:21;26 embedded in the membrane.
00:12:23;07 So, they can image this because they
00:12:25;11 spatter it with platinum,
00:12:27;15 and what that does is provide contrast
00:12:29;17 that will be visible in an electron microscope,
00:12:31;23 and it also provides a shadow,
00:12:33;04 so things look like they're three-dimensional.
00:12:35;10 And so then they're imaged in a transmission electron microscope,
00:12:39;00 and that's how you get these beautiful images.
00:12:41;10 So, now, they're going to take this technique
00:12:43;13 and they're going to apply it
00:12:45;20 to stimulated synapses
00:12:48;11 and see, what do those events look like
00:12:50;29 in the plasma membrane?
00:12:52;20 And that's shown here.
00:12:54;00 So, here, at 0 milliseconds
00:12:55;20 in an unstimulated synapse,
00:12:56;29 you see these little bumps,
00:12:58;19 and these little bumps
00:13:00;27 are likely to be calcium channels.
00:13:02;14 And, remember that I told you that
00:13:04;11 calcium was required for synaptic vesicle fusion.
00:13:07;09 5 milliseconds after the stimulation,
00:13:10;16 they see that there are these invaginations,
00:13:12;29 and what these invaginations represent
00:13:15;22 is that there's a synaptic vesicle on the other side of this screen,
00:13:19;11 and it's just fused with the membrane,
00:13:21;13 and it's created a neck.
00:13:23;23 And so when you split that,
00:13:26;01 it breaks across that thin part of the neck,
00:13:27;29 because you can't cut all the way
00:13:30;14 into that concave membrane,
00:13:32;25 and you get these little discs,
00:13:34;16 and so that is just simply the ice itself,
00:13:36;29 at that neck.
00:13:38;20 And so, once again, they demonstrated
00:13:40;22 that synaptic vesicles fuse with the plasma membrane.
00:13:48;10 So, at this point, it looked like the fusion took 5 milliseconds,
00:13:50;00 but we now know from electrophysiological experiments
00:13:52;26 that that fusion is actually much faster.
00:13:54;15 After the calcium transient,
00:13:56;01 after the increase of calcium,
00:13:57;28 we know that it only takes
00:13:59;27 one-tenth of a millisecond
00:14:02;28 for that vesicle to fuse with the plasma membrane.
00:14:05;18 Okay, so that is the proof that vesicles
00:14:10;08 contain neurotransmitter
00:14:11;23 and that neurotransmission
00:14:13;10 is mediated by these vesicle fusions.
00:14:16;05 What I'd like to talk about
00:14:18;05 in the second half of this video
00:14:19;22 is what happens after that,
00:14:22;09 what happens to that membrane after it's fused.
00:14:27;15 So, synapses have to be very robust,
00:14:30;00 so they can fire...
00:14:31;17 the neuromuscular function
00:14:33;00 can fire 100 times a second,
00:14:34;26 and so every time you do that
00:14:37;12 you're releasing vesicles.
00:14:38;15 And there's a limited pool,
00:14:40;04 maybe 300 synaptic vesicles at a synapse,
00:14:42;02 so you can see that at these rates
00:14:44;08 you would deplete the synapse of synaptic vesicles.
00:14:47;21 And, again, it's far from the cell body,
00:14:49;27 so the synapse needs to
00:14:53;15 recycle the synaptic vesicles.
00:14:54;17 And so, Heuser and Reese realized that,
00:15:00;11 and so they looked for evidence of that.
00:15:02;16 And so, the first experiments they did
00:15:04;18 is they stimulated very intensely
00:15:06;28 and then they would fix the sample
00:15:09;13 in a fixative,
00:15:11;22 and embed them in plastic,
00:15:13;13 and look at the sections,
00:15:15;00 to see whether there was any evidence of
00:15:17;07 recovery or recycling of these synaptic vesicles.
00:15:19;01 And so that's shown here in this image...
00:15:21;03 you can see that there are
00:15:23;16 these invaginations that are occurring,
00:15:25;01 here's one,
00:15:27;11 here's another one, here...
00:15:29;13 and if you look at those, they have these coats on them,
00:15:31;26 these electron-dense coats,
00:15:34;15 and those had been seen before,
00:15:36;16 and this occurs in any cell that is
00:15:39;18 endocytosing material, like receptors or membrane,
00:15:42;12 and those are clathrin coats,
00:15:44;25 and they'd been seen in electron micrographs before.
00:15:47;11 So, now there was a sort of a unified view
00:15:52;25 of how you recovered proteins in membranes,
00:15:54;17 even at synapses,
00:15:56;01 and it's done via this clathrin-mediated mechanism.
00:15:58;19 So, this was not...
00:16:01;23 it didn't show any timing,
00:16:03;22 this was simply a highly stimulated synapse
00:16:06;10 that was fixed,
00:16:07;18 and so the issue is,
00:16:09;23 how long after stimulation
00:16:12;03 did these vesicles get recovered?
00:16:13;24 And so, once again, they turn to the freeze slammer.
00:16:17;00 So, now, what they do is they have their sample,
00:16:21;27 and they stimulate,
00:16:23;05 and they just hold it,
00:16:24;29 for 1 second, for 3 seconds,
00:16:26;21 for 10 seconds, for 30 seconds,
00:16:28;15 and then they let it go
00:16:30;07 and then they freeze it on this copper block.
00:16:32;18 So, what did they see?
00:16:34;11 So, these again are these freeze-fracture experiments,
00:16:36;22 and at 0 milliseconds --
00:16:38;18 once again, this is just our control,
00:16:40;09 it's unstimulate --
00:16:41;22 you can see that these buds, here,
00:16:45;19 these buttons, are probably calcium channels.
00:16:50;01 At 5 milliseconds, you see these fusions
00:16:51;15 and then nothing happens,
00:16:53;10 so the synapse is basically quiet.
00:16:56;07 Until 30 seconds,
00:16:58;01 you begin to see these invaginations.
00:17:00;03 And so some of these invaginations
00:17:02;16 look like proteins are coalescing.
00:17:05;03 In other images,
00:17:06;23 you can see that they look deeper,
00:17:08;24 as if they're being pinched off,
00:17:10;13 so this suggested that
00:17:13;09 this was the endocytic event,
00:17:14;27 and it's not so dramatic here,
00:17:16;25 but, if they provided a higher stimulation,
00:17:19;20 then they would see many of these invaginations
00:17:22;15 occurring at 30 seconds.
00:17:26;18 So, now we understand...
00:17:28;13 we have a dogma of how synaptic transmission works.
00:17:34;18 Vesicles fuse with the plasma membrane,
00:17:36;20 release neurotransmitter,
00:17:38;06 the proteins that make up that synaptic vesicle
00:17:40;24 and the membrane that makes up that synaptic vesicle,
00:17:43;15 has to be recycled,
00:17:45;02 and so that's via clathrin-mediated mechanism.
00:17:47;25 Clathrin forms this superstructure,
00:17:51;01 a geodesic dome over this piece of membrane,
00:17:53;00 and then removes it from the surface
00:17:56;28 to regenerate a vesicle.
00:17:58;11 This clathrin coat is
00:18:00;03 then removed to regenerate a vesicle.
00:18:01;29 So, the two important conclusions
00:18:04;09 from this video
00:18:06;13 is that endocytosis, synaptic vesicle recycling,
00:18:09;17 occurs via a clathrin-mediated mechanism,
00:18:12;03 and it's rather slow
00:18:13;19 -- it takes about 30 seconds for these vesicles
00:18:16;06 to be regenerated.
00:18:17;18 So, that is the conclusions for this video.
00:18:21;20 I've told you about how
00:18:24;10 synaptic vesicles exocytose,
00:18:26;08 how they fuse with the plasma membrane,
00:18:27;21 and how we understand
00:18:30;01 that that is how a synapse works.
00:18:31;12 And in the second half of this talk,
00:18:35;06 I told you about how clathrin-mediated endocytosis
00:18:38;15 recovers that membrane,
00:18:40;01 recovers those proteins,
00:18:41;20 to regenerate synaptic vesicles
00:18:43;28 that can fuse again.
00:18:45;15 In the second video,
00:18:47;13 I'd like to tell you about some work...
00:18:49;22 that there is a parallel system,
00:18:51;15 that works on a much faster timescale,
00:18:52;27 to recycle synaptic vesicles.
00:18:55;09 Thanks once again for listening,
00:18:57;16 and I encourage you to watch the second video.

Part 2: Recycling Synaptic Vesicles: Ultrafast Endocytosis

00:00:07;22 Hi. I'm Erik Jorgensen from the University of Utah.
00:00:11;12 Welcome to part 2 of recycling synaptic vesicles.
00:00:16;04 And so, what I'd like to tell you about today is about
00:00:18;03 the discovery of the ultrafast endocytosis.
00:00:21;00 So, in the last video,
00:00:23;08 we talked about the discovery of
00:00:25;09 synaptic vesicle exocytosis,
00:00:28;01 which had been proposed by Bernard Katz,
00:00:29;09 and was then really proven
00:00:30;24 by the work of Heuser and Reese,
00:00:32;24 using electron microscopy.
00:00:35;11 And then John Heuser and Tom Reese
00:00:37;07 continued these experiments
00:00:38;17 and discovered that there was
00:00:42;14 synaptic vesicle recycling via endocytosis at the synapse.
00:00:45;14 And the conclusions that they came to was that
00:00:48;11 this is a clathrin-mediated process
00:00:49;24 and took about 30 seconds,
00:00:51;20 so it was a relatively slow process.
00:00:54;07 But the images were beautiful
00:00:55;22 and it's unquestioned that this
00:00:59;01 occurs at synapses.
00:01:00;10 what I'd like to tell you about today,
00:01:01;21 in the second part,
00:01:03;08 is that there is another mechanism
00:01:05;15 that is acting at synapses,
00:01:07;15 and that this is acting on a very fast timescale,
00:01:10;05 between 30-50 milliseconds,
00:01:12;07 so about 1000 times faster
00:01:14;10 than what was seen via clathrin-mediated endocytosis.
00:01:17;11 And so I'll tell you about
00:01:20;05 our experiments in worms and in mice,
00:01:21;22 and then, at the very end,
00:01:23;08 I'll reconcile the work of Heuser and Reese
00:01:25;21 with ultrafast endocytosis.
00:01:28;13 So, what we chose to do was to
00:01:32;00 do our studies in a nematode,
00:01:33;22 a small roundworm, called C. elegans.
00:01:36;04 And the advantage of this is that
00:01:38;00 there are many mutants available,
00:01:39;13 so you can study processes
00:01:41;02 and cause defects in them,
00:01:42;14 and see how the molecular mechanisms
00:01:44;10 work to recycle synaptic vesicles,
00:01:47;22 but what's important for the experiments
00:01:49;13 I'll tell you about today
00:01:50;24 is that the nematode is transparent...
00:01:53;03 so, you... a light...
00:01:55;19 it's perfectly transparent to light,
00:01:57;16 and so that's important because
00:01:59;14 we're going to use optogenetic stimulation
00:02:01;10 of the nervous system.
00:02:02;23 And so, we return to the scene of the crime...
00:02:05;25 we talked about some of these experiments
00:02:07;25 from Heuser and Reese
00:02:09;05 had been done at the Marine Biological Laboratory
00:02:11;08 in Woods Hole,
00:02:13;18 in 1973... starting in 1973,
00:02:15;06 and the experiments I'll tell you about
00:02:17;11 started in 2008
00:02:19;03 from our work, Shigeki Watanabe
00:02:22;08 and my work at MBL.
00:02:26;03 So, what we wanted to do was stimulate,
00:02:27;26 but we didn't want to use electrical stimulation
00:02:29;11 because we wanted to do this in a whole animal,
00:02:31;10 in an animal that was not dissected
00:02:34;05 and we didn't have to remove the nerves
00:02:35;22 and attach them to stimulating wires.
00:02:37;19 So, the way to do this is to use
00:02:39;22 light-activated ion channels.
00:02:41;27 So, there is this single-cell algae
00:02:44;18 called Chlamydomonas
00:02:45;23 and it has phototaxis,
00:02:48;00 meaning it swims to a light source.
00:02:49;19 And the reason it can do this is
00:02:52;10 it has a light-activated ion channel,
00:02:53;20 so you flash it with light
00:02:55;26 and that opens an ion channel
00:02:57;19 and that, because it's DNA
00:03:00;08 and the code is universal,
00:03:01;12 we can cut out the DNA from Chlamydomonas
00:03:05;14 and put it into a nematode.
00:03:07;04 And so that's shown here.
00:03:08;16 So, what we've done is we've
00:03:10;15 put channelrhodopsin into the
00:03:12;18 acetylcholine motor neurons
00:03:14;14 that drive the contraction of muscles,
00:03:16;21 and so here we have... the channelrhodopsin is shown,
00:03:20;05 and here is a cholinergic neuron
00:03:22;02 that is expressing channelrhodopsin.
00:03:24;25 So now, what we can do is
00:03:26;26 we can patch onto a muscle
00:03:28;29 and record the activity of the muscle.
00:03:31;07 That means that we're recording
00:03:33;26 postsynaptic activity
00:03:35;09 that comes from the motor neuron.
00:03:36;24 So, whenever neurotransmitter is released,
00:03:38;17 that will cause a depolarization
00:03:41;23 or a current in these muscles.
00:03:45;00 So, now what we can do is flash a light,
00:03:48;00 and when that light activates that motor neuron,
00:03:49;29 then we see [neurotransmitters]
00:03:52;16 being released here at the synapse,
00:03:52;29 and then we can record that activity
00:03:55;13 using this patch pipette.
00:03:57;02 And those results are shown here.
00:03:59;03 And this is really beautiful,
00:04:01;10 because when we were using electrical stimulation
00:04:03;01 on dissected preps,
00:04:04;07 we found that the neurons often died,
00:04:06;22 but they can respond to
00:04:08;26 very robust trains of light stimuli.
00:04:10;17 So, here, we're using
00:04:12;14 a 30 second train of 10 Hz light stimulus,
00:04:15;11 and you can see that the cell is responding
00:04:18;19 quite well to that
00:04:20;09 -- that means the motor neuron is releasing neurotransmitter
00:04:22;11 every time it sees a light pulse.
00:04:26;06 So, that's great,
00:04:28;00 we can stimulate a whole animal
00:04:30;06 and release neurotransmitter,
00:04:32;06 but how are we going to capture that event?
00:04:35;06 And so we're borrowing a page
00:04:37;23 from Heuser and Reese,
00:04:39;07 and we're going to freeze that sample
00:04:41;03 as fast as we can
00:04:42;26 after a synaptic transmission has been initiated,
00:04:46;07 and for this reason a high-pressure freezer.
00:04:47;19 And these a really wonderful devices.
00:04:50;02 They go from 1 to 2000 atmospheres in 15 milliseconds,
00:04:54;08 and what that does is that... water molecules,
00:04:57;20 if you cool something slowly,
00:05:00;18 will rearrange and then form ice crystals,
00:05:02;24 and those ice crystals will shatter a cell
00:05:05;22 or they will create...
00:05:07;10 they'll exclude solutes and salts,
00:05:11;17 and that will create hypertonic regions,
00:05:13;21 and so water will flow into that hypertonic region
00:05:16;12 and also blow up the cell.
00:05:19;02 So, during slow freezing,
00:05:20;28 there's a lot of tissue damage.
00:05:24;22 But if you could do this under high atmospheric pressure,
00:05:28;00 the water molecules don't have time to rearrange
00:05:30;25 -- they're sluggish --
00:05:30;29 and you drop the temperature,
00:05:36;01 and the water molecules simply stop in place.
00:05:38;04 This is called vitreous ice, because it's not organized into
00:05:40;00 a crystalline form.
00:05:43;00 So, what this allows us to do... so, we go from ambient...
00:05:44;21 so, from room temperature, 22 degrees,
00:05:47;22 to -50 degrees Centigrade
00:05:49;20 in 10 milliseconds,
00:05:50;29 and so that allows us
00:05:52;28 to freeze up to 200 microns deep
00:05:55;03 without ice crystals forming
00:05:56;28 and without any destruction of the tissue.
00:05:59;13 So, what we do after this is then
00:06:02;07 we put that sample that's at -90 degrees,
00:06:04;07 that what we hold it at,
00:06:05;22 and we put in in fixative, in acetone.
00:06:07;26 And so then the water molecules
00:06:09;18 come out slowly
00:06:10;23 and are swapped with acetone,
00:06:12;18 and carrying in with the acetone are
00:06:16;18 fixatives, like osmium.
00:06:18;16 So, how we got this light stimulation to work
00:06:22;17 is shown here.
00:06:23;23 So, normally, it's...
00:06:26;15 shown here in yellow, is the specimen holder,
00:06:28;18 the specimen cup,
00:06:30;04 and so our worms will be held in this little spot here.
00:06:33;01 And the pressure comes in from this plunger,
00:06:36;16 right here,
00:06:38;08 which slams the sample
00:06:40;09 against a black diamond anvil,
00:06:41;26 and that's how we create pressure.
00:06:43;26 But, under these circumstances
00:06:45;26 there's no light path to the sample,
00:06:47;13 so what we did is we replaced that black diamond anvil
00:06:50;12 with a sapphire anvil, shown here,
00:06:52;17 we drilled out that mounting screw, shown here,
00:06:55;10 and we drilled out that bayonet, here,
00:06:57;19 and so we mounted an LED
00:07:00;10 directly behind that sapphire anvil,
00:07:03;00 so now we had a light path to the sample cup.
00:07:07;01 So, we now can stimulate that sample
00:07:09;25 at any point before freezing them.
00:07:13;13 And so, that's shown here...
00:07:15;14 so, my graduate student, Shigeki Watanabe,
00:07:17;00 had mounted the sample,
00:07:18;08 and here's the specimen holder right there,
00:07:20;06 here's the bayonet
00:07:22;12 and you can see the wire that comes out,
00:07:23;23 and that wire goes to a computer, here,
00:07:25;22 which will control when we stimulate the LED,
00:07:30;16 just before the freeze.
00:07:31;25 So, what you'll see is that
00:07:33;13 Shigeki will mount this onto a sled
00:07:35;23 and that sled will run down a rail
00:07:37;17 into the high-pressure chamber, here, at the end.
00:07:39;21 And so in this case
00:07:41;25 we're going to stimulate before we shoot it down the rail,
00:07:44;06 but you can stimulate it any time
00:07:45;20 that it's passing along the rail
00:07:47;12 into the high-pressure chamber.
00:07:48;21 So, let's just watch this video.
00:07:51;24 So, Shigeki has mounted the pod
00:07:54;16 that holds the sample in the specimen cup into the bayonet,
00:07:57;21 and then the bayonet is here on the sled.
00:08:00;19 so, what's going to happen now is
00:08:03;21 he's going to activate the computer,
00:08:05;16 and you'll see flashing, right here -- see that flashing?
00:08:09;00 Now, it's going to shoot down the rail,
00:08:10;26 boom, and then into the high-pressure chamber...
00:08:12;27 sshppoosstt...
00:08:14;13 right, so 2000 atmospheres,
00:08:15;28 the sample is frozen,
00:08:17;19 and it drop into liquid nitrogen, here.
00:08:19;04 So, now we can remove that sample
00:08:20;23 from the liquid nitrogen
00:08:22;11 and we can put it into this freeze-substitution fixation.
00:08:27;12 So, what we'd like to do first
00:08:29;16 is determine where and when
00:08:31;22 exocytosis takes place,
00:08:33;02 so in these experiments
00:08:35;04 what we're doing is I'm showing you
00:08:36;18 a current caused by an electrical stimulation
00:08:38;23 of the motor neuron,
00:08:40;11 and so here is our light stimulus,
00:08:43;22 and so what we're going to do is freeze 20 milliseconds later,
00:08:46;19 while there is still this synaptic transmission going on.
00:08:51;23 So, what do we see?
00:08:54;15 So, first, a little anatomy lesson.
00:08:55;27 So, what you have here is
00:08:58;02 an electron micrograph of a C. elegans neuromuscular junction,
00:09:00;15 and shown in red, here,
00:09:02;29 is the dense projection.
00:09:04;08 That marks the center of the synapse
00:09:06;06 and that is where the calcium channels are.
00:09:08;14 So, when that synapse is excited,
00:09:10;10 calcium will flow through this dense projection
00:09:13;03 and then can activate,
00:09:14;29 or cause the fusion of vesicles that are nearby.
00:09:17;07 So, the other structure that you might notice
00:09:20;16 are the adherens junctions,
00:09:22;00 so, here's an adherens junction here and here,
00:09:24;02 and they mark the edges of the active zone.
00:09:27;22 T he active zone defines where vesicles fuse,
00:09:31;02 so you can see docked vesicles
00:09:32;24 that are now highlighted in blue,
00:09:34;25 and vesicles that are capable of fusion
00:09:37;13 are shown along this entire surface,
00:09:40;17 so, anywhere between the adherens junction
00:09:43;01 and the dense projection,
00:09:44;12 those vesicles can fuse when they're stimulated.
00:09:47;03 And so let me just show you some of those images.
00:09:49;22 So, here you see vesicles that were...
00:09:51;26 are in the process of fusing with the membrane
00:09:54;23 and collapsing into the plasma membrane.
00:09:57;05 So, shown here
00:09:59;09 is one of those vesicles
00:10:00;23 that hasn't fused or flattened very far,
00:10:02;28 here's one that's flattening,
00:10:04;02 and here's one...
00:10:06;14 right there...
00:10:07;26 and just this see this one has just flattened
00:10:10;05 almost completely into the membrane.
00:10:11;15 So, that's 20 milliseconds after stimulation.
00:10:14;13 So, what we wanted to know is
00:10:16;09 where endocytosis takes place,
00:10:17;11 and how soon after stimulation.
00:10:19;25 So, what we found is that
00:10:22;04 there are two places
00:10:24;08 where synaptic vesicle endocytosis takes place.
00:10:25;18 It takes place at the edges of the active zone,
00:10:27;23 so, not within the active zone.
00:10:29;22 So, it occurs, here,
00:10:31;10 at the edge of the dense projection,
00:10:32;29 or it occurs here and here,
00:10:35;18 at the adherens junctions,
00:10:38;19 so, at the edges of the adherens junctions.
00:10:40;09 And those are shown here.
00:10:41;15 So, I'm just showing you endocytosis
00:10:43;26 that's occurring at the dense projection, here,
00:10:46;26 and so it's been just stained lightly with this red color.
00:10:49;14 And you can see here that there's
00:10:52;06 a shallow invagination,
00:10:53;13 and then this one,
00:10:54;25 this endocytic vesicle has already severed
00:10:56;18 from the membrane,
00:10:58;11 and then, eventually,
00:11:00;04 what these vesicles do is that
00:11:02;01 they will migrate into the inside of the synaptic bouton,
00:11:04;23 far from the release site.
00:11:07;29 So, there are our vesicles
00:11:10;06 that have been endocytosed.
00:11:13;17 So, the important thing, here, is that
00:11:16;03 the site of [endocytosis] is not the site of fusion,
00:11:18;29 that endocytosis is taking place at the lateral edges
00:11:20;27 of the active zone.
00:11:23;08 So, the issue, then, is, how fast does this occur?
00:11:25;15 And it occurs extremely quickly
00:11:27;26 after that stimulation.
00:11:29;13 So, here, that shallow invagination
00:11:31;21 was 20 milliseconds after the stimulation,
00:11:34;27 this large vesicle is 50 milliseconds
00:11:37;10 after the stimulation,
00:11:38;18 and then here we see that the vesicles
00:11:40;16 are beginning to translocate
00:11:42;01 into the center of the [synaptic bouton]
00:11:44;03 100 milliseconds after the stimulation.
00:11:46;28 So, here is the quantification of this,
00:11:49;13 and this is important because we have good temporal resolution
00:11:51;25 to demonstrate that this is a product,
00:11:54;25 a precursor-product relationship
00:11:56;24 between each of these steps.
00:11:58;25 So, we se these pits,
00:12:00;18 they peak at 20 milliseconds.
00:12:02;27 We see that there are these large vesicles,
00:12:06;18 they peak at 50 milliseconds.
00:12:08;27 And then we see these vesicles
00:12:10;16 that are beginning to translocate
00:12:12;15 into the center of the [bouton],
00:12:14;00 and those peak at 300 milliseconds.
00:12:15;20 So, we know that this is the process
00:12:17;24 through which the membrane is passing.
00:12:21;05 We see that the first large vesicles
00:12:23;05 appear at 30 milliseconds,
00:12:24;15 so that's when endocytosis,
00:12:26;02 the membrane is actually being cleaved,
00:12:27;18 and the last pits are gone at 50 milliseconds.
00:12:31;16 So, the time period during which endocytosis
00:12:33;24 is actually taking place
00:12:36;04 is 30-50 milliseconds.
00:12:40;25 So, this process is taking about 1000 times...
00:12:43;28 is [going] about 1000 times faster
00:12:46;19 than clathrin-mediated endocytosis.
00:12:49;27 So, instead of 30 seconds for clathrin-mediated endocytosis,
00:12:52;12 we see 30-50 milliseconds
00:12:56;05 for ultrafast endocytosis.
00:12:57;26 So, there appears to be a very fast mechanism
00:12:59;14 that also works at synapses.
00:13:02;27 So, this has demonstrated
00:13:05;29 that this process works in the nematode,
00:13:07;25 and now I'd like to tell you about
00:13:09;15 some work that demonstrates
00:13:11;03 that this process also occurs
00:13:13;15 at mouse synapses.
00:13:14;29 So, we did our work in the nematode, C. elegans,
00:13:18;04 and it's possible that this is an unusual organism,
00:13:22;07 that maybe it has a sped-up form of endocytosis
00:13:26;01 that is not conserved among other organisms.
00:13:29;23 And so, to test this,
00:13:31;08 we needed to look at another organism,
00:13:32;24 one that is more commonly used
00:13:35;23 as a laboratory organism
00:13:38;18 that replicates what might be happening in human synapses,
00:13:41;01 and that's the mouse.
00:13:42;13 And to do this work,
00:13:44;14 we worked with Christian Rosenmund
00:13:45;24 at the Charité in Berlin,
00:13:47;26 and I have to give a big call-out,
00:13:49;24 a shout-out to Christian,
00:13:51;26 this was his idea.
00:13:53;10 Christian said, why don't you come to Berlin
00:13:55;15 and bring your samples and your device
00:13:58;16 and we'll do the experiments
00:14:00;27 on hippocampal neurons
00:14:03;16 that have been cultured onto dishes.
00:14:06;07 And so I brought my graduate student,
00:14:08;11 Shigeki Watanabe,
00:14:09;24 and so what he did was perform a morphometry
00:14:12;20 on over 10,000 synapses,
00:14:14;26 and so the reason we needed to do this
00:14:17;16 was to get statistical differences
00:14:19;25 at each of these timepoints,
00:14:21;17 and so let me show you those data.
00:14:24;20 So, once again, we're stimulating
00:14:26;18 with channelrhodopsin.
00:14:27;21 And so we can take our
00:14:31;19 cultured hippocampal neurons from the mouse brain
00:14:33;29 and then infect them with a virus
00:14:36;13 that expresses the channelrhodopsin gene,
00:14:39;27 and so then we can make the protein,
00:14:42;29 and now we can stimulate with light.
00:14:45;21 And so here is a hippocampal neuron in a dish,
00:14:46;27 it's expressing channelrhodopsin,
00:14:48;09 we stimulate it,
00:14:49;13 and the light stimulation is shown here in blue, right?
00:14:53;29 So, this is the 10 millisecond stimulation,
00:14:56;08 and in this image you can see that
00:14:58;22 there were two action potentials
00:15:00;10 that were caused by this light stimulation.
00:15:03;25 And so this occurs fairly rapidly
00:15:06;10 after the onset of light stimulation,
00:15:07;24 just 5 milliseconds,
00:15:09;24 and it's highly robust.
00:15:11;25 So, only 12% of the time do we see
00:15:14;06 a failure of an action potential.
00:15:15;15 So, we'd like to now demonstrate
00:15:18;00 that that really causes synaptic transmission,
00:15:21;22 and so for this we have a cell that is
00:15:23;26 not expressing channelrhodopsin,
00:15:26;12 so there will be no light response in that cell.
00:15:28;08 The only light response that it will have
00:15:32;04 will come from the presynaptic cell
00:15:34;05 expressing channelrhodopsin.
00:15:35;13 So, now we can flash the light,
00:15:36;24 that activates that presynaptic cell,
00:15:39;04 and then we see these postsynaptic currents
00:15:42;13 in this cell, here.
00:15:44;24 So, you can see that, in this case,
00:15:46;15 here's the light stimulus, shown in blue,
00:15:49;19 and once again there were two action potentials
00:15:52;12 in this sample.
00:15:54;02 And so this is a postsynaptic current, shown here,
00:15:55;23 and we know that this is a synaptic current
00:15:57;08 because we can block it with a drug called TTX,
00:16:00;26 and TTX blocks action potentials.
00:16:03;12 So, if you block the action potential in this presynaptic cell,
00:16:06;10 you have no synaptic transmission.
00:16:11;15 And if you block the receptors
00:16:13;09 for the neurotransmitters
00:16:15;01 in the postsynaptic cell, right here,
00:16:17;05 we also see no response.
00:16:18;26 So, this demonstrates that these are
00:16:21;21 bona fide synaptic events.
00:16:25;01 So, once again, what we'd like to do
00:16:27;14 is determine the speed of exocytosis
00:16:29;19 and the speed of endocytosis.
00:16:32;05 So, we stimulate with light,
00:16:34;18 again, shown in blue,
00:16:35;25 and then we can freeze at 15 millisecond,
00:16:38;08 30 milliseconds,
00:16:39;15 50 milliseconds,
00:16:40;25 100 milliseconds,
00:16:42;09 and so on.
00:16:43;18 And so, for each of these experiments,
00:16:45;06 we analyzed 200 synapses
00:16:47;06 to see whether we could see events.
00:16:49;26 So, let me just give you, again,
00:16:51;11 an anatomy lesson about the
00:16:54;04 mouse hippocampal synapse,
00:16:55;25 and so what you can see here is a pool of vesicles
00:16:59;00 that represents the reserve pool of vesicles.
00:17:03;12 And then there are vesicles that are
00:17:05;18 docked at the membrane, shown here,
00:17:08;06 and they can release along that entire face,
00:17:09;22 so that face, that region,
00:17:11;13 is called the active zone.
00:17:12;29 It's a zone that's active, right? vesicles fuse.
00:17:16;05 On the other side is the postsynaptic density,
00:17:19;15 and what this postsynaptic density is...
00:17:22;20 the positioning of ion channels,
00:17:24;27 ligand-gated ion channels,
00:17:27;10 that will respond to the neurotransmitter being released.
00:17:30;01 In this case, it's glutamate,
00:17:31;22 so, glutamate is the neurotransmitter
00:17:33;18 that will open ligand-gated ion channels,
00:17:36;01 and then there will be a current in that postsynaptic cell.
00:17:38;19 So, these are readily identifiable
00:17:41;09 in these electron micrographs,
00:17:43;20 and these are such exquisite images...
00:17:46;01 look at this.
00:17:47;26 You can see that, at 15 milliseconds after light onset,
00:17:50;07 you can see that one of these vesicles
00:17:52;27 has fused and formed what's called
00:17:54;19 an omega figure, right?
00:17:56;00 And that's a vesicle that's beginning to
00:17:58;09 collapse into the membrane.
00:17:59;17 And then, here, you can see
00:18:01;23 at 30 milliseconds a vesicle that's almost
00:18:04;04 fully collapsed into the membrane.
00:18:05;15 And this is always directly across
00:18:08;00 from the postsynaptic density, right?
00:18:09;18 So, this shows you that's where the receptors are.
00:18:12;02 So, active zone, shown in yellow,
00:18:15;00 postsynaptic density, shown in red.
00:18:16;29 So, where are the vesicles recycling
00:18:20;13 relative to where vesicles fuse?
00:18:22;20 And once again we see that endocytosis,
00:18:26;05 the recycling of vesicles,
00:18:27;25 takes place at the edges of the synapse.
00:18:30;02 So, here we can see...
00:18:32;03 the yellow is the edge of the active zone,
00:18:34;13 you can see vesicles are docked there,
00:18:36;13 and you can already see that there's this indentation,
00:18:38;23 this invagination of the membrane.
00:18:40;25 And then here you see a deeper invagination
00:18:44;15 and here is a vesicle that's being
00:18:46;27 actually cleaved from the membrane,
00:18:48;21 and then in this last image...
00:18:50;16 let me just step back...
00:18:51;29 you can see that there is a vesicle
00:18:53;25 that is freed from the membrane,
00:18:55;15 and this is now translocating
00:18:57;16 into the center of the bouton.
00:18:59;17 So, the process that we saw in C. elegans
00:19:02;04 is also occurring in hippocampal neurons.
00:19:05;20 So, how fast are vesicles recycled?
00:19:08;13 Again, the process is very quick.
00:19:10;14 So, at 50 milliseconds we see these shallow invaginations,
00:19:13;09 100 milliseconds we see these
00:19:15;18 deep invaginations that are being cleaved,
00:19:16;24 and then, finally, at 300 milliseconds
00:19:19;11 we see that these vesicles are beginning
00:19:21;04 to translocate into the [bouton].
00:19:24;02 So, the process is taking about
00:19:26;07 50-100 milliseconds.
00:19:28;25 This is a tomogram, so it's a 3D image
00:19:31;06 of an electron micrograph,
00:19:33;03 and why this is important is because
00:19:35;05 it shows you that while exocytosis is taking place,
00:19:38;04 endocytosis has already begun.
00:19:40;15 So, that's shown here.
00:19:42;00 You can see that these are three vesicles that are fusing,
00:19:44;12 and already you see these invaginations
00:19:46;11 that are taking place.
00:19:47;15 This is at 50 milliseconds,
00:19:49;05 so the vesicles are fusing and collapsing into the membrane,
00:19:52;14 and membrane's already being taken up
00:19:54;04 by ultrafast endocytosis.
00:19:55;20 And so, here you can see that again.
00:19:57;11 You can see that a vesicle...
00:20:00;00 or, an endocytic pit, is here,
00:20:03;19 and then here you can see that there is a vesicle
00:20:06;03 that is completely formed
00:20:07;25 and is being cut from the membrane.
00:20:09;05 So, what this tells us is that
00:20:10;27 the proteins and the membranes
00:20:13;27 that were in the synaptic vesicle
00:20:15;27 are not the same ones that are being endocytosed.
00:20:18;24 So, the process is occurring at the same time,
00:20:21;00 vesicles are fusing,
00:20:22;21 membrane is being taken in.
00:20:26;10 So, we have a temporal...
00:20:30;13 we have data that shows the temporal processing of this,
00:20:33;11 and what we see, first of all,
00:20:35;20 is that there are no coated pits on the plasma membrane.
00:20:39;28 We don't see those beautiful coated pits
00:20:42;17 that Heuser and Reese saw during endocytosis.
00:20:44;22 Rather, we see these pits
00:20:49;07 that form rapidly,
00:20:51;00 and they peak about 100 milliseconds --
00:20:53;01 they do not have coats on them.
00:20:54;16 We see large endocytic vesicles,
00:20:56;01 they also peak at 100 milliseconds.
00:20:57;29 And we can see that the first vesicles
00:21:01;12 are seen at 50 milliseconds
00:21:05;18 and the last ones are seen at 300 milliseconds.
00:21:08;11 And so we now know that endocytosis
00:21:11;00 -- that's actually the process where the membrane is being cut off
00:21:12;21 and removed from the surface --
00:21:14;11 is taking place between 50-300 milliseconds.
00:21:19;17 So, now we have vesicles, 100 milliseconds,
00:21:22;05 we have these vesicles
00:21:23;22 that are in the presynaptic bouton.
00:21:25;17 Well, what happens to them, right?
00:21:27;22 Do they go back to the surface,
00:21:29;29 because this is just a piece of surface membrane,
00:21:33;21 it's a way of recovering, maybe,
00:21:36;19 the tautness of the membrane at the active zone?
00:21:39;29 Or is this is a process by which
00:21:42;10 synaptic vesicles are regenerated?
00:21:44;04 So, to do that, we had to be able to
00:21:46;26 follow this membrane into the cell,
00:21:48;16 and for this we needed a fluid phase marker,
00:21:51;14 something that we could see by electron microscopy,
00:21:53;19 that would be taken up by endocytosis.
00:21:57;17 So, ferritin makes a 12 nanometer particle,
00:22:00;18 it's basically a hollow sphere,
00:22:02;09 and that hollow sphere can hold
00:22:04;29 4000 molecules of iron,
00:22:07;19 and so this stains very darkly in electron micrographs.
00:22:12;20 And that's shown here,
00:22:14;17 so what you see is hippocampal synapses
00:22:18;02 that have been incubated with ferritin in advance,
00:22:22;01 and then they're stimulated.
00:22:23;23 And so you can see these dark ferritin molecules, here,
00:22:26;21 and after stimulation,
00:22:28;11 there's 100 milliseconds after stimulation,
00:22:30;08 you can see a shallow pit forming,
00:22:32;15 there's endocytosis already taking place.
00:22:34;14 And here you can see these ferritin molecules
00:22:36;24 are going into this deep invagination.
00:22:38;28 And this is the most important image, here.
00:22:40;27 In this case, you can see that
00:22:43;25 the ferritin molecules have moved into an endocytic vesicle.
00:22:47;22 This proves that this process is going inward,
00:22:51;12 that these are bits of membrane
00:22:54;18 that are grabbing some of the ferritin particles
00:22:57;28 inside the synaptic cleft
00:22:59;09 and taking them into the [synaptic bouton].
00:23:02;24 So, what I'd like you to do is remember this image.
00:23:06;10 So, this is going to come back
00:23:08;12 at the very end of the talk,
00:23:10;13 and it's an image that will look very similar...
00:23:13;20 so, the presence of ferritin
00:23:15;06 going into these endocytic structures.
00:23:18;22 What happens after that?
00:23:21;25 So, that, again, is what we saw before,
00:23:23;03 this rapid endocytosis,
00:23:24;12 and if we follow that through time
00:23:26;18 we see that after 3 seconds,
00:23:28;16 we see that that ferritin
00:23:31;18 has moved into an endosome,
00:23:32;24 a synaptic endosome,
00:23:33;29 and 10 seconds after that we see that they...
00:23:36;20 ferritin molecules are in synaptic vesicles.
00:23:39;07 It's a little hard to see these,
00:23:41;17 so it if we stimulate more intensely...
00:23:43;09 once again, at 3 seconds
00:23:45;05 you can see ferritin inside these synaptic endosomes,
00:23:48;21 and here, this is after 10 stimuli...
00:23:51;17 at 10 seconds,
00:23:53;17 you can see that there are synaptic vesicles, several of them,
00:23:55;23 that have these ferritin molecules in them,
00:23:57;27 so this really demonstrates that this is a process
00:24:01;08 to regenerate synaptic vesicles.
00:24:05;08 How is that synaptic endosome resolved?
00:24:08;01 And what you can see here is that,
00:24:09;23 now, we see these coats,
00:24:11;15 just like what Heuser and Reese saw,
00:24:14;29 we saw evidence of clathrin coats.
00:24:16;26 And so, here is what, again,
00:24:19;22 a normal clathrin-coated pit looks like,
00:24:24;02 and that occurs on the cell body,
00:24:25;29 and here is one of these coated buds
00:24:28;21 on the endosome,
00:24:30;04 and look how familiar or similar they are.
00:24:31;24 So, here's the process,
00:24:33;23 here's one of these round,
00:24:36;14 spherical synaptic endosomes
00:24:38;27 at 1 second, and then at 1 second, here,
00:24:40;28 we see that buds are forming,
00:24:42;09 and then here's another example, at 3 seconds,
00:24:44;27 of a bud forming.
00:24:47;15 And then this is a... I love this image...
00:24:48;28 here is one of these synaptic endosomes
00:24:50;17 that's being torn apart by wild dogs, right?
00:24:52;18 I mean, there's a bud...
00:24:55;00 four different buds on this process,
00:24:56;16 it's completely tearing the synaptic endosome apart,
00:24:59;11 and it basically becomes invisible to us
00:25:01;26 in electron micrographs after this.
00:25:05;16 So, if we now look at the speed of this process,
00:25:08;29 we see that these deep pits
00:25:11;27 have formed between 50-100 milliseconds,
00:25:14;03 these then form large endocytic vesicles
00:25:17;02 -- these are about 80 nanometers in diameter --
00:25:19;15 and those form at 100 milliseconds,
00:25:22;19 they peak at 100 milliseconds.
00:25:25;00 These synaptic endosomes peak at 1 second,
00:25:27;04 then clathrin-coated vesicles peak at 3 seconds,
00:25:31;27 and then see synaptic endosomes...
00:25:34;12 sorry, synaptic vesicles,
00:25:36;20 and they peak at 10 seconds,
00:25:39;05 and we think that the t1/2,
00:25:41;19 that is, the half-time to create these synaptic vesicles,
00:25:45;09 is only 5 seconds,
00:25:47;00 so the process is very fast.
00:25:49;05 So, what we now have to conclude is that
00:25:52;29 there is other process, this ultrafast endocytosis,
00:25:56;28 which occurs 600 times faster
00:25:59;11 than clathrin-mediated endocytosis, here.
00:26:01;12 So, that takes 30 seconds,
00:26:03;00 and what we've seen is that
00:26:04;29 ultrafast endocytosis is occurring
00:26:07;16 at fast as 50 milliseconds,
00:26:08;27 so, 600 times faster.
00:26:11;25 So, now we have a conflict, right?
00:26:14;23 Heuser and Reese see clathrin-mediated endocytosis,
00:26:18;15 it takes 30 seconds.
00:26:19;20 We see ultrafast endocytosis,
00:26:21;29 that takes about 50 milliseconds.
00:26:24;06 So, the data disagree.
00:26:26;00 So, can we reconcile these conflicting results?
00:26:30;11 And I think we can.
00:26:32;12 So, the issue, then, is, why do our results
00:26:36;27 differ from previous studies on hippocampal neurons?
00:26:38;16 These have been studied for many, many years,
00:26:39;29 and it's always been observed that
00:26:42;23 there are clathrin-mediated endocytic structures.
00:26:47;25 And we think we know the reason why.
00:26:49;16 Because when Shigeki was doing his experiments,
00:26:52;21 he was doing these always at
00:26:54;23 a physiological temperature, either 34 degrees or 37 degrees,
00:26:58;05 that's the temperature that a mouse normally lives at,
00:27:00;14 and in this image at 3 seconds after stimulation,
00:27:04;05 you see the formation of
00:27:05;29 either a large endocytic vesicle
00:27:07;10 or one of these synaptic endosomes,
00:27:09;01 and that, again, was at 34 degrees...
00:27:11;21 we see ultrafast endocytosis.
00:27:13;26 Shigeki then, from reading the literature,
00:27:16;21 realized that everyone else was doing their experiments
00:27:19;05 at room temperature, at 22 degrees,
00:27:21;07 so he then did his experiments at 22 degrees,
00:27:23;25 froze at 3 seconds, and look what you see.
00:27:26;04 Here is a clathrin-coated vesicle right here,
00:27:29;07 a beautiful clathrin-coated vesicle,
00:27:30;14 and here's an enlargement in this inset.
00:27:32;09 Once again, look at that beautiful
00:27:34;27 clathrin-coated pit.
00:27:36;04 So, we now think what's going on is that
00:27:39;03 if you do your experiments at 37 degrees or 34 degrees,
00:27:44;13 you'll see ultrafast endocytosis,
00:27:46;13 but at 22 degrees
00:27:48;12 clathrin-mediated endocytosis predominates.
00:27:51;01 And so that's shown here.
00:27:53;17 If you do your experiments at 34 degrees,
00:27:55;12 we basically see no coated pits
00:27:58;07 on the plasma membrane,
00:27:59;23 that is clathrin-mediated endocytosis,
00:28:02;11 but we see these endosomes,
00:28:04;08 which typify ultrafast endocytosis.
00:28:06;14 At 22 degrees, we now see that
00:28:11;05 there are these coated pits appearing on the membrane,
00:28:12;19 shown here,
00:28:14;07 and that's evidence of clathrin-mediated endocytosis.
00:28:15;24 However, we do not see any of these endosomes,
00:28:18;21 again, the signature of ultrafast endocytosis.
00:28:22;06 So, we think that this explains
00:28:24;11 the difference between our experiments
00:28:26;06 and those from other laboratories.
00:28:29;09 So, how do we put these things together?
00:28:31;10 Well, what we think is going on is that
00:28:34;09 normally what happens is that
00:28:36;18 ultrafast endocytosis is responding
00:28:39;07 for normal stimuli, normal single stimuli.
00:28:43;00 All of our experiments are done
00:28:45;15 with one or ten stimuli, not very many.
00:28:47;24 But, what may happen is that when a synapse is
00:28:51;01 put under extreme, high-frequency stimulation,
00:28:55;18 or higher demands,
00:28:57;29 that you need to kick in another mechanism, and this is clathrin-mediated endocytosis,
00:29:00;24 and it's acting as an emergency backup system
00:29:03;03 when ultrafast endocytosis fails.
00:29:06;16 More experiments need to be done
00:29:08;08 to test this hypothesis,
00:29:10;10 but it's at least a way of thinking about
00:29:12;14 these different results.
00:29:14;19 So, that doesn't get...
00:29:17;04 that gets at why these experiments differed
00:29:18;20 from other people studying hippocampal synapses.
00:29:21;27 It doesn't really clarify
00:29:24;20 why Heuser and Reese got different results.
00:29:28;03 And so I want to go back to the literature
00:29:29;25 and show you that I think
00:29:32;15 even those results can be reconciled.
00:29:35;07 So, these experiments were being done
00:29:37;06 at the frog neuromuscular junction,
00:29:38;20 and we want to know why these results differ.
00:29:42;07 And so the images that I showed you for timing
00:29:45;15 were these freeze-fracture images.
00:29:47;14 And so this is a case where this is stimulated...
00:29:50;15 5 milliseconds we see exocytosis, we see fusion...
00:29:53;19 30 seconds later we see endocytosis,
00:29:56;29 we see that vesicles are being recycled.
00:29:59;20 But the issue is,
00:30:02;06 where are the cross-sections? Right?
00:30:04;15 Normally, you wouldn't be pursuing this
00:30:08;20 via this freeze-fracture.
00:30:10;12 These are beautiful images,
00:30:12;11 but we'd love to know what was going on in the membrane
00:30:15;00 in this case.
00:30:17;00 Are these really clathrin-mediated endocytic vesicles?
00:30:22;15 And so I looked through all of their papers
00:30:24;08 and I was never able to find
00:30:26;15 those cross-sections,
00:30:28;00 but I am sure they would have done them, right?
00:30:29;24 So, finally, I contacted
00:30:32;03 an antiquarian book dealer,
00:30:33;08 because these was one article I couldn't find,
00:30:35;01 I could not get a copy of,
00:30:36;17 and it was this chapter in this book
00:30:39;16 called "Neurocsiences" by MIT Press, in 1979.
00:30:43;14 And so the antiquarian book dealer
00:30:47;25 sold me that book
00:30:49;16 and I looked at that chapter
00:30:51;07 and there were those cross-sections.
00:30:52;18 This is just a beautiful sleuthing story, here.
00:30:56;12 Here is a neuromuscular junction
00:30:59;05 that was stimulated at 10 Hz for 5 minutes,
00:31:01;20 so, high-frequency stimulation.
00:31:04;01 And you can see these beautiful,
00:31:06;28 exquisite clathrin-coated pits.
00:31:08;07 Here's one where the clathrin coat has formed,
00:31:11;02 and here's another one where the vesicle
00:31:13;00 is being squeezed off, about to be cut off.
00:31:15;06 So, that was after an intense stimulation.
00:31:19;17 But in the Miller and Heuser paper,
00:31:21;29 there were also freeze-fracture images
00:31:25;04 that showed something mysterious,
00:31:26;17 that they noted after just 1 second
00:31:29;24 after the stimulation.
00:31:31;06 And those are shown here...
00:31:32;18 so, here, shown in red,
00:31:34;21 I've circled these other indentations
00:31:38;16 that look like they could be endocytic events,
00:31:40;14 and note there's this white structure here,
00:31:42;19 white structure here.
00:31:45;00 That's the ice that's being cut across
00:31:46;16 the neck of that endocytic vesicle,
00:31:51;00 suggesting that, if we could look inside that in a cross-section,
00:31:53;20 we'd be able to see endocytic structures.
00:31:55;05 So, where are those cross-sections?
00:31:57;14 There they are.
00:31:59;16 So, this is one of the images
00:32:01;29 taken from that book chapter,
00:32:03;07 they were using ferritin to track the formation of those vesicles,
00:32:05;26 and that's why I said,
00:32:07;14 "remember this image",
00:32:09;07 because this looks highly similar
00:32:11;09 to the images I showed you.
00:32:12;15 So, here's ferritin being
00:32:15;19 taken up in these invaginations.
00:32:17;16 Here's another one.
00:32:19;09 Some of these invaginations are quite large,
00:32:21;04 shown here,
00:32:22;14 and then here is a vesicle
00:32:24;09 that's been cleaved from the membrane
00:32:25;20 that has ferritin in it,
00:32:27;05 demonstrating that these really are endocytic events.
00:32:30;05 Note that this was a synapse that was stimulated
00:32:33;05 once
00:32:34;19 and frozen 1 second later.
00:32:36;14 So, instead of high stimulation,
00:32:38;02 they're only stimulating once...
00:32:41;03 much more similar to the sort of experiments that I described.
00:32:44;10 So, I think that Heuser and Reese
00:32:48;02 has seen it all, they had the data,
00:32:50;08 they had the images,
00:32:52;08 and the only thing that I think might have been flipped
00:32:55;08 is described in their discussion.
00:32:57;17 So, here, they say,
00:32:59;19 when looking at these fast cisternae,
00:33:03;05 these ones I just showed you with the ferritin in them,
00:33:06;04 they say, "Nearly all the uncoated pits formed
00:33:09;09 during the first few seconds
00:33:11;05 after exocytosis"...
00:33:12;25 "The uncoated pits may not occur at all
00:33:15;09 during the normal functioning of the synapse."
00:33:18;00 Then they talk about these slow coated pits,
00:33:20;08 here, and they say,
00:33:21;20 "Other [membrane] is retrieved
00:33:23;23 over the next 90 seconds
00:33:26;00 via coated pits"...
00:33:28;11 "The path of the coated pits is...
00:33:29;14 physiologically more significant."
00:33:31;29 So, I think that both occur,
00:33:34;03 I think that under low frequency stimulation
00:33:36;12 we see this ultrafast mechanism,
00:33:38;10 and then, under high frequency stimulation,
00:33:41;21 maybe this clathrin-coated mechanism predominates.
00:33:47;01 So, in summary,
00:33:49;17 what I showed you in this second video
00:33:52;03 is that there is this mechanism called ultrafast endocytosis
00:33:55;12 that works at synapses
00:33:57;03 to recover synaptic vesicles.
00:33:59;27 It takes about 30-300 milliseconds
00:34:03;04 to recover the membrane
00:34:04;28 off of the plasma membrane surface.
00:34:07;16 These synaptic endosomes form
00:34:10;17 after about a second...
00:34:12;18 clathrin... these are then resolved by clathrin budding
00:34:14;17 after 3 seconds,
00:34:16;06 and then we see the formation of synaptic vesicles
00:34:18;06 after 5 seconds.
00:34:21;04 So, this is in parallel
00:34:24;04 to this other mechanism,
00:34:25;21 the classic clathrin-mediated endocytosis,
00:34:27;20 which reforms vesicles, complete vesicles,
00:34:31;01 right from the membrane,
00:34:33;09 so that they simply need to be uncoated
00:34:34;15 to regenerate a nascent synaptic vesicle,
00:34:37;10 which can be refilled and then reused.
00:34:41;15 So, I'd like to close by acknowledging
00:34:43;18 the people who did this work.
00:34:44;29 The electron microscopy was done
00:34:47;00 by Shigeki Watanabe,
00:34:48;26 who's now a faculty at Johns Hopkins.
00:34:51;22 All the instrumentation was by Wayne Davis, in my laboratory.
00:34:54;15 And then I really need to thank
00:34:56;17 Christian Rosenmund and the postdocs in his lab
00:35:00;22 for having hosted us and allowed us to do those experiments
00:35:03;16 in their lab,
00:35:04;20 and they conducted all of the physiology,
00:35:06;19 electrophysiology, that was done on these cells.
00:35:09;16 So, thank you for listening.
00:35:11;13 Erik Jorgensen from the University of Utah.

Videos in this Talk
  • Part 1: Synaptic Transmission
    Part 1: Synaptic Transmission
    Audience:
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: Recycling Synaptic Vesicles: Ultrafast Endocytosis
    Part 2: Recycling Synaptic Vesicles: Ultrafast Endocytosis
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
Speaker: Erik Jorgensen
Total Duration: 0:54:47
Recorded: July 2016
All Talks in Neuroscience

Talk Overview

In his first talk, Dr. Jorgensen describes the historic experiments leading to our current understanding of synaptic transmission. In the 1950s, Sir Bernard Katz proposed that stimulation of a neuron caused synaptic vesicles to fuse with the plasma membrane and release neurotransmitter.  Two decades later, John Heuser and Tom Reese produced stunning electron micrographs proving that Katz’ theory was right. In addition, Heuser and Reese made that surprising finding that just 30 sec after nerve stimulation, synaptic vesicles are recycled via clathrin-mediated endocytosis.

Part 2 of Jorgensen’s talk focuses on work from his lab showing that there is a second mechanism for recycling synaptic vesicles which he calls ultrafast endocytosis. Ultrafast endocytosis occurs 1000x faster that the clathrin-mediated endocytosis identified by Heuser and Reese.  Using electron microscopy, Jorgensen and his colleagues found that non-clathrin mediated endocytosis begins so rapidly after stimulation that it overlaps in time with synaptic vesicle exocytosis!  Jorgensen goes on to explain why two mechanisms for recycling synaptic vesicles are necessary in vivo.

Speaker Bio

Erik Jorgensen

Erik Jorgensen

Erik Jorgensen is a Distinguished Professor of Biology and a member of the Program in Neuroscience at the University of Utah, and an Investigator of the Howard Hughes Medical Institute.  His lab studies the molecular mechanisms of synaptic transmission with a focus on synaptic vesicle fusion and recycling.  Jorgensen’s lab uses genetics, biochemistry, light and… Continue Reading

Playlist: Molecular Neuroscience

Related Resources

Watanabe S, Trimbuch T, Camacho-Pérez M, Rost BR, Brokowski B, Söhl-Kielczynski B, Felies A, Davis MW, Rosenmund C, Jorgensen EM.(2014) Clathrin regenerates synaptic vesicles from endosomes.Nature 515:228-33.

Watanabe S, Rost BR, Camacho-Pérez M, Davis MW, Söhl-Kielczynski B, Rosenmund C, Jorgensen EM. (2013) Ultrafast endocytosis at mouse hippocampal synapses. Nature 504:242-7.

Watanabe S, Liu Q, Davis MW, Hollopeter G, Thomas N, Jorgensen NB, Jorgensen EM. (2013) Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2:e00723.

Heuser JE, Reese TS.(1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.J Cell Biol. 57(2): 315-44.

Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, Evans L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol. 81(2):275-300.

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Talk Overview

Erik Jorgensen: Recycling Synaptic Vesicles

I. Synaptic Transmission
II. Recycling Synaptic Vesicles: Ultrafast Endocytosis

Part I: Synaptic Transmission

Download: High Res Low ResSubtitled Videos: English
Resources: Related ArticlesRecorded: 2016Transcript (.txt)(.xls)
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Lecture Overview

In his first talk, Dr. Jorgensen describes the historic experiments leading to our current understanding of synaptic transmission. In the 1950s, Sir Bernard Katz proposed that stimulation of a neuron caused synaptic vesicles to fuse with the plasma membrane and release neurotransmitter.  Two decades later, John Heuser and Tom Reese produced stunning electron micrographs proving that Katz’ theory was right. In addition, Heuser and Reese made that surprising finding that just 30 sec after nerve stimulation, synaptic vesicles are recycled via clathrin-mediated endocytosis.

Part 2 of Jorgensen’s talk focuses on work from his lab showing that there is a second mechanism for recycling synaptic vesicles which he calls ultrafast endocytosis. Ultrafast endocytosis occurs 1000x faster that the clathrin-mediated endocytosis identified by Heuser and Reese.  Using electron microscopy, Jorgensen and his colleagues found that non-clathrin mediated endocytosis begins so rapidly after stimulation that it overlaps in time with synaptic vesicle exocytosis!  Jorgensen goes on to explain why two mechanisms for recycling synaptic vesicles are necessary in vivo.

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Speaker Bio

Erik Jorgensen is a Distinguished Professor of Biology and a member of the Program in Neuroscience at the University of Utah, and an Investigator of the Howard Hughes Medical Institute.  His lab studies the molecular mechanisms of synaptic transmission with a focus on synaptic vesicle fusion and recycling.  Jorgensen’s lab uses genetics, biochemistry, light and electron microscopy to investigate neurotransmission, primarily in C. elegans.

Jorgensen has been honored with the Utah Governor’s Medal for Science and Technology, a Humboldt Research Award from the Humboldt Foundation and he was one of the inaugural recipients of the F.R. Lillie Research Innovation Award from the Marine Biological Laboratory and the University of Chicago. Jorgensen has also received several awards for excellence in teaching from the University of Utah.

Jorgensen received his BS from the University of California, Berkeley and his PhD from the University of Washington.  He was a postdoctoral fellow in the lab of H. Robert Horvitz at the Massachusetts Institute of Technology.

Learn more about Jorgensen’s research here  or here  

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Watanabe S, Trimbuch T, Camacho-Pérez M, Rost BR, Brokowski B, Söhl-Kielczynski B, Felies A, Davis MW, Rosenmund C, Jorgensen EM.(2014) Clathrin regenerates synaptic vesicles from endosomes. Nature 515:228-33.

Watanabe S, Rost BR, Camacho-Pérez M, Davis MW, Söhl-Kielczynski B, Rosenmund C, Jorgensen EM. (2013) Ultrafast endocytosis at mouse hippocampal synapses. Nature 504:242-7.

Watanabe S, Liu Q, Davis MW, Hollopeter G, Thomas N, Jorgensen NB, Jorgensen EM. (2013) Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2:e00723.

Heuser JE, Reese TS.(1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.J Cell Biol. 57(2): 315-44.

Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, Evans L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol. 81(2):275-300.

 

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Speaker Bio

Erik Jorgensen

Erik Jorgensen

Erik Jorgensen is a Distinguished Professor of Biology and a member of the Program in Neuroscience at the University of Utah, and an Investigator of the Howard Hughes Medical Institute.  His lab studies the molecular mechanisms of synaptic transmission with a focus on synaptic vesicle fusion and recycling.  Jorgensen’s lab uses genetics, biochemistry, light and… Continue Reading

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