This summary contains information about: the synaptic vesicle cycle, transmission electron microscopy, BoTX, literature research, and types of fixation (chemical vs. cyro-fixation).
Experimental Cell Biology: Synaptic vesicle cycling
Synaptic transmission is initiated when an Transmission Electron Microscopy (TEM)
action potential triggers neurotransmitter Single-molecule imaging can be used to study one
release from a presynaptic nerve terminal. An vesicle, a microscope that can be used to study
action potential induces the opening of Ca2+ only one vesicle is the TEM. ~Dx,y stands for
channels, which results in the exocytosis of uncertainty.
calcium. After exocytosis, synaptic vesicles
undergo endocytosis, recycle, and refill with
neurotransmitters for a new round of
exocytosis. Action potentials, initiated in the
neuronal cell body, travel to all of the cell body’s
nerve terminals to be transformed into synaptic
secretory signals.
TEM is an imaging technique that uses a reference
space. In this case, that’s the side where the ER
and mitochondria are. The difference between
TEM and fluorescence microscopy is that the
image of the TEM needs to be labelled. The
All presynaptic functions involve synaptic advantages of using TEM as an imaging technique:
vesicles. Synaptic vesicles undergo a trafficking • Superior resolution in x, y and z
cycle in the nerve terminal. • Huge magnification range
1) Neurotransmitters are actively transported • Reference space
into synaptic vesicles Considerations:
2) Synaptic vesicles cluster in front of the active • 2D projected image of a 3D object
zone • High vacuum so only fixed specimens, no
3) Then synaptic vesicles dock at the active zone dynamics (frozen)
4) Vesicles are primed
5) Vesicles are converted into a state of How can you study a dynamic process, such as the
competence for calcium-triggered fusion-pore synaptic vesicle cycle with EM?
opening. • Take a lot of pictures and look at faces
• Add dye to synaptic cleft + electron blocker, HRP
for instance
Contrasting synaptic vesicles: DAB oxidation by
HRP produces an electron-dense precipitate. Frogs
were used in this experiment, cause frogs don’t
have myelin. (Heuser & Reese, 1973, J Cell Biol)
Synaptic recycling in the frog’s NMJ
With the dye, you can see that all the vesicles that
come out of the synapse, take up the membrane.
After stimulation (action potential) of the
synapse, the dark dye should disappear more.
Vesicles are transported through neurites. Black= HRP. 1 hour later
Proteins and lipids are synthesized in the soma
of the neuron. During transport through the
neurites, they also need to get rid of false
proteins in the cell. The vesicle cycle can
transport vesicles to synapses further away
from the cell body (max 40 cm/day).
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