Lecture 8: Synaptic vesicle cycling Flashcards
Explain shortly what happens in a chemical synapse upon stimulation of an action potential on the presynaptic neuron.
- Upon action potential arrival in the presynaptic terminal, voltage-gated calcium channels open and calcium flows into the presynaptic terminal.
- This stimulates the fusion of synaptic vesicles that contain neurotransmitters.
- Neurotransmitters are released in the synaptic cleft and bind to and activate their postsynaptic receptors.
Describe what you can see in this picture.
See picture.
- Also seen in the picture (that is not described in the picture) is the thick brushy dark band → these are all the neurotransmitter regulating ion channels.
By what are vesicles transported?
Through neurites, as can be seen in the picture.
What can be concluded based on this picture and if taken into regard that transport of vesicles through neurites has a maximum speed of 40 cm/day?
The longest axon in our body is the one in your leg (from spine to foot). The maximum speed of 40 cm/day, would impy that it will take a vesicle from the cell body 3 days to reach to synapse in the foot. This would not be convenient for fast neurotransmission, so you need local recycling.
Describe the cycle of a vesicle (including recycling of the vesicle).
- Ready-made vesicles with neurotransmitters are located in the presynaps.
- Upon stimulation of the presynaps, docking and priming occurs.
- Here, through a complex of synaptic proteins, the vesicles get attached to the membrane (docking).
- The vesicles that dock have a higher energy status and are thus more likely to be secreted (priming).
- After fusion, empty synaptic vesicles are recycled through endocytosis. After this, neurotransmitters are pumped back into the vesicle (not depicted in the picture).
- The filled-up and recycled vesicles are then transported back to the ready-made pool of vesicles.
Fill in:
The amount of synaptic vesicles fusing and releasing neurotransmitters closely correlates with the amount of …
The amount of synaptic vesicles fusing and releasing neurotransmitters closely correlates with the amount of vesicles that are being recycled.
What problem arises when you try to visualize a synapse to look at synaptic vesicles with the help of GFPs?
You get a very blurry picture of the synapse
How does this problem of poor visualization through the use of GFP occur?
The wavelenght of GFPs is about 500 nm, which is about ten fold larger then the synaptic vesicles (40 nm). With only one vesicle in the synaps, you can still deduct where the GFP light came from. But since there are much more vesicles in the synaps, they all will emit green light. This wil disturb the picture.
What can be used as an alternative to visualize vesicles in the synapse? And what is tricky about this method?
Electron microscopy.
- Synaptic vesicle cycling is a dynamic process, while electron microscopy is very static.
Explain this picture.
The picture depicts the uncertainty of a signal (what chances are there that the light has spread out more then wanted/needed).
- CLSM (confocal imaging) → has high uncertainty, which makes it very hard to conclude where the ligth is coming from.
- STED, SIM or PALM/STROM (super-resolution techniques) → have a much lower uncertainty compared to confocal imaging.
- TEM (electron microscopy) → very high resolution/certainty.
What is a disadvantage of PALM/STORM that is not a disadvantage in electron microscopy (TEM)?
PALM/STORM has high spatial resolution, but the disadvantage is that you cannot see anything else, but the object that is visualized (picture bottom left).
TEM doesn’t have this, it has a reference space where other structures also become visible.
Name advantages and disadvantages of TEM/electron microscopy.
- Advantages → superior resolution in x, y and z plane, huge magnification range, reference space.
- Disadvantages → 2D projected image of a (sectioned) 3D object, high vacuum (only fixed specimens → static).
How can you study a dynamic process, such as the synaptic vesicle cycle, with EM?
Researchers bathed a neuron in a bath with horseradisch peroxidase (HRP). DAB oxidizes HRP so that it can be visualized under an electron microscope. They then stimulated the presynaptic terminal to release their vesicles. If there’s local recycling of synaptic vesicles, there also should be a little HRP inside the recycled vesicles.
When this neuron was looked at under a electron microscope, they indeed found that there was local recycling, as why they found HRP inside vesicles.
Just study.
This is another picture from the experiment with the electron microscope where they bathed neurons in HRP to visualize the vesicles.
Ok
So the researchers proved with the HRP experiment that there’s internalization of vesicles after release of neurotransmitters.
What didn’t they prove with this and what can be done to complete the conclusion of vesicle recycling that they saw in the experiment?
You cannot conclude based on the first experiment if there’s really recycling of the same vesicle. So next, the researchers removed the outside bath of HRP. If there’s recycling of the same vesicles, the vesicles that took up HRP in the first experiment, should be able to release HRP to the extracellular evironment again. This way, the experiments prove that there’s internalization of the same vesicles, but also that these vesicles are able to undergo a second round of neurotransmitter release.
What are the steps of the synaptic vesicle cycle after neurotransmitters have been released into the synaptic cleft?
- Endocytosis of empty vesicles
- Sorting
- Loading (refilling it with neurotransmitters)
- Vesicles wait for the next stimulation and activation of the presynaptic terminal
- Stimulation of presynaptic terminal
- Docking (transport to secretion site)
- Priming (vesicles are made fusion-competent and form readily releasable pools (RRPs))
- Calcium sensing → calcium influx triggers the release of neurotransmitters
- Release of neurotransmitters
Several proteins are needed for synaptic vesicle exocytosis. Name these proteins and also whether they’re linked to the vesicle or to the plasma membrane.
SNARE proteins:
- On the synaptic vesicle → synaptobrevin-2 and synaptotagmin (calcium sensor)
- On the plasma membrane → syntaxin-1 and SNAP-25
Explain the steps of synaptic vesicle exocytosis in regard to the SNARE proteins.
- Vesicle docking → the vesicle comes closer to the membrane
- Synaptobrevin, syntaxin-1 and SNAP-25 undergo loose association with each other and pull membranes together.
- When an action potential arrives, calcium influx is activated that are able to bind to synaptotagmin.
- Calcium-bound synaptotagmin catalyzes membrane fusion by binding to SNAREs and the plasma membrane.
How does the influx of calcium and the binding of calcium to synaptotagmin catalyze the fusion of two membranes?
Membranes are negatively charged due to the phosphate groups on their phospholipids. Bringing together negatively charged membranes costs energy. Since calcium is a positive ion, binding of calcium to synaptotagmin counteracts the negative charge of both the vesicle and plasma membrane. Therefore, the membranes can come closer to each other.
What toxines can cleave SNARE proteins?
Botox/BoTX → secreted by Clostridium botulinum.
BoTX temporarily blocks SV secretion and thereby face-muscle contraction.
Researchers wanted to define the minimal machinery needed for secretory vesicle docking. They used adrenal chromaffin cells that contain neurotransmitters for this experiment.
What was the method of this experiment?
They isolated adrenal gland cells from mouse pups and used light/fluorescent microscopy. Here, every dot is a chromaffin cell. They also measured the total surface of the membrane, because every time a vesicle fuses with the membrane, the surface of the membrane expands.
With this, it can be studied how quickly these vesicles can actually secrete neurotransmitters.
What proteins were expected to be important in vesicle docking?
SNAREs, syntaxin-binding protein Munc18-1 and synaptotagmin.
How was the function of these proteins suspected to be important in vesicle docking researched?
- By making null mutant mice, lacking the gene of interest and then replacing the knockout gene with another overexpressing gene.
- By using BoTX
Then: measuring the vesicle docking and secretion of neurotransmitters in living cells in aldehyde fixed cells by EM.
What did they see for Munc18-1 in regard to neurotransmitter secretion?
That Munc18-1 is essential for neurotransmitter secretion. This can be seen when looking at the null mutant for Munc18-1, where all neurotransmission stops.
