Lecture 9 - Neurotransmitter Release and the Active Zone

Question 1

What is the NMJ a well studied exmaple of?

Answer 1

directly gated synaptic transmission

Question 2

How does the MN excite the muscle?

Answer 2

opens ligand gated ion channels at the end plate via acetylcholine

Question 3

What are the three maoin reasons the NMj is such a powerful system for studying synaptic transmission?

Answer 3

1. the muscle cell is large enough to accomodate 2 or more electrodes
2. the muscle receives signals from 1 presynaptic neuron (two in flies)
3. simple transmission of a single type of transmitter and single type of receptor

Question 4

What is direct gating and what does it lead to?

Answer 4

that a ligand opens up ion channels causing postsyaptic depolarization
-achrs are packed at the top of folds and the achrs bind to ach and this allows sodium to rush in and depolarize the cell

Question 5

Where is acetylcholine made and how is released?

Answer 5

made in the presynaptic terminal and is released in synaptic vesicles

Question 6

How is acetylcholine removed from the synaptic cleft?

Answer 6

it is degraded by the enzyme acteylcholinesterase which breaks it down to acetate and choline

Question 7

What inhibits acetylcholinesterase causing more ACh to remain at synapse and excite muscle?

Answer 7

sarin and VX nerve gas

Question 8

Where are Ach receptors concentrated at?

Answer 8

the top one third of the junctional folds

Question 9

What is alpha bungarotoxin?

Answer 9

is secreted by snakes and mongoose are immune to it and it binds to achrs and prevents synaptic transmission cause now ach cannot bind and if you radioactively label alpha bungarotoxin you can see the junctional folds of an EM section

Question 10

What underlies synaptic plastciity?

Answer 10

modulation of transmitter release

Question 11

What three things help modulate NT release?

Answer 11

1. factors on the synaptic vesicle - synaptotagmin - the calicum sensor
2. the active zone - has proteins and nanostructure
3. synaptic vesicle endocytosis - 4 mechanisms

Question 12

Where is calcium flowing into the presynaptic terminal concentrated?

Answer 12

at the active zone

Question 13

If you radioactively label presynaptic calcium channels with snail toxin and postsynaptic ACHRs with alpha bungarotoxin what will you see?

Answer 13

that they are precisely aligned

Question 14

Why is the fly NMJ a good model to study?

Answer 14

because you can see these stereotyped repeats in it and these repeats are the alignment of the presynaptic calcium channel with the postsyanptic receptors - this establishes spatual precisioon and it you move a receptor or calcium channels 10nkm off then can cause a disease

Question 15

If you load the presynaptic terminal with calcium indicator dye what do you see?

Answer 15

calcium influx happens at discrete specific sites and it is localized at active zones - there is close spatial correspondence between areas of presynaptic calcium influx and postsynaptic receptors

Question 16

What is the synaptic vesicle cycle?

Answer 16

1. synapotic vesicles are filled with NT where they dock at the active zone
2. undergo an atp dependent priming recation
3. fuse followng calcium influx
4. recycled via clathrin mediated endocytosis

Question 17

What is the most energy taxing process in the brain and what was it believed to be?

Answer 17

-it was believed to be the AP and synaptic transmission and fusion
-it is actually the atp dependent process to pump and keep NT in vesicles since it keeps leaking out

Question 18

Which vesicles are most likely to be released?

Answer 18

docked vesicles which are then primed

Question 19

What happens during the recycling of vesicles?

Answer 19

50% of the vesicles are refilled with NT and the other 50% become an endosome because when vesicles fuse there is a lot of damage to the protein and they assemble and unwind which is why they go into an endosome where they are fixed and broken parts are degraded

Question 20

What controls the SV fusion?

Answer 20

a collaboration between synaptic vesicles and the active zone

Question 21

What is the purpose of minis or MEPPs?

Answer 21

tells the postsyanptic compartment you are there and you might need more synapses and the frequncy of minis is how many postsyanptic rceptor you have and synapses you have

Question 22

What happens if you block minis?

Answer 22

get major problems in issues with postsyanptic structure

Question 23

What regulates the synaptic vesicle exocytosis and membrane cycling?

Answer 23

a protein network
-calcium channels and active zone scaffolds
-SNARE complex - sv fusion

Question 24

What are the three snare complex proteins and what do they do?

Answer 24

synaptobrevin - interacts with two plasma membrane proteins is found on the synaptic vesicle
-syntaxin - is a transmembrane protein
-SNAP-25 is a peripheral membrane protein

Question 25

How does the snare complex work?

Answer 25

1. synaptibrevin on the sv binds to syntaxin a transmmebrane proteins and snap-25 a peripheral membrane protein
2. the three proteins form a tight complex which brings the vesicle and presynaptic membrane in close apposition

Question 26

What does munc18 do?

Answer 26

binds to the snare complex

Question 27

How many alpha helices in the snare complex?

Answer 27

4 alpha helices
-one from synaptobrevin one from syntaxin and two from snap-25

Question 28

What drives fusion of the SV and plasma membrane?

Answer 28

formation and dissociation of the SNARE complex - a fused SV is cleared from the active zone and enables a new round of SV docking and priming

Question 29

What takes the most energy in SV fusion and dissociation to the plasma membrane?

Answer 29

-fusion does not take much ATP cause of calcium but dissociation and unwinding of the snare complex takes alot of atp

Question 30

What is the calcium sensor and what does it work with to drive the snare complex?

Answer 30

synaptotagmin and it drives membrane fusion

Question 31

Post an influx of calciium and the rapid fusion of the SV and plasma membrane where does the snare complex reside post fusion?

Answer 31

in the plasma membrane

Question 32

What binds to the snare cmplex and unwinds it?

Answer 32

NSF and SNAP and this requires alot of ATP

Question 33

What is the SNARE complex targeted by?

Answer 33

tetanus and botulinum toxins

Question 34

What do the tetanus and botulinum toxins do?

Answer 34

the targets the snare complex and proteolytically cleave it

Question 35

What does synaptotagmin do?

Answer 35

binds calcium and faciliitates synaptic vesicle fusion

Question 36

How many calcium binding or C2 domains does synaptotagmin have?

Answer 36

has two C2 domains which are contained in the cytoplasmic tail - known as C2A and C2B

Question 37

How many calcium ions does C2A bind?

Answer 37

3 calcium ions

Question 38

How many calcium ions does C2B bind?

Answer 38

2 calcium ion

Question 39

Where is synaptotagmin located?

Answer 39

it is a synaptic vesicle protein

Question 40

What do the C2 domains of synaptotagmin also bind?

Answer 40

phospholipids

Question 41

What was observed in synaptotagmin mutants?

Answer 41

fast caclium truggers NT release is abolished but asynchronous release of neurotransmitter is increased in absence of synaptotagmin

Question 42

In EM tomography what are the pegs, ribs, and beams?

Answer 42

pegs are the calcium channels and ribs and beams are AZ components

Question 43

What are synaptic vesicles closely intertwined with?

Answer 43

the active zone

Question 44

What are the four mechanisms of SV endocytosis?

Answer 44

1. reversible pore fusion also known as kiss and run - the sv membrane foes not does not fuse with the plasma membrane and this may predominate at low release rate -most rapid
2. clathrin mediated - slower occurs outside the actuve zone and is important at normal to high frequency stimulation

3.bulk retrieval - excess membrane reenters the terminal from buddling uncoated pits or endosomes - only at excessively high activity

4. ultra fast endocytosis - shock waves in membrane