Lecture 3 - Molecular Presynaptic Plasticity

Question 1

What are 3 mechanical ways the presynapse could possibly change?

Answer 1

Probability of release
Filling of vesicle
Transmitter conc in cleft

Question 2

How else could the presynapse change and give examples

Answer 2

Retrograde messages

NO, CO, neurotrophins, endocannabinoids

Question 3

How can the postsynapse change mechanically?

Answer 3

Insertion of new receptors

Gating of existing receptors (phosphorylating NMDAR means it gates more)

Question 4

What are the 4 proposed ways a synapse ‘learns’?

Answer 4

Presynaptic mechanisms
Retrograde messages
Postsynaptic mechanisms
Number of synapses

Question 5

What experiment did Zucker and Regehr (2002) perform?

Answer 5

Paired-pulse facilitation

• Gave 2 pulses spaced at a set time
• Plotted interval between them and size of response

Question 6

What was the result of Zucker and Regehr’s (2002) experiment?

Answer 6

Response to second pulse was bigger because residual calcium still in synapse
Short term placticity

Question 7

What is post tetanic potentiation (PTP)?

Answer 7

The stable response carrying on a few minutes after pulses have stopped due to calcium signalling activating CAMKII which phosphorylates proteins

Question 8

Why do you get depression that lasts as long as the stimulus?

Answer 8

Presynapse runs out of synaptic vesicles

Question 9

What protein is involved in mobilisation of synaptic vesicles?

Answer 9

Synapsins

Question 10

What proteins are involved in docking and priming of synaptic vesicles?

Answer 10

Rab3A/RIM1a/14-3-3

Question 11

What proteins are involved in synaptic vesicle fusion?

Answer 11

SNAREs, munc18

Question 12

What animal has a very large synapse and so is good for looking at vesicles?

Answer 12

Lamparay

Question 13

What does RRP stand for and what is it?

Answer 13

Readily releasable pool

Vesicles that are bound to the membrane ready to be released

Question 14

What is the recycling pool?

Answer 14

Vesicles being turned over ready to attach to the membrane

Question 15

What is the reserve pool?

Answer 15

Vesicles tethered to actin cytoskeleton

Question 16

What is synapsin and what is a feature of it?

Answer 16

The bridge between the synaptic vesicle and actin cytoskeleton
Heavily phosphorylated

Question 17

What happens to make the vesicle fall off the cytoskeleton?

Answer 17

Protein kinase A phosphorylates synapsin

Question 18

What did Paul Greengard do and what was the result?

Answer 18

Knocked out synapsin 1 and looked for memory defects.

No effect on the mouse at all.

Question 19

What did George Augustine do and what was the result?

Answer 19

Knocked out all 3 synapsins.

Mouse was fine however it did not recover from synaptic depression.

Question 20

How many are there and what do Rab proteins generally do?

Answer 20

60

Like passports for membrane fusion events

Question 21

What Rab protein is involved in synaptic vesicle exocytosis and what happens?

Answer 21

Rab3

Binds to GTP and binds to an effector bound to the membrane allowing fusion.

Question 22

What are the SNARE proteins?

Answer 22

Syntaxin
SNAP-25-C
VAMP (Synaptobrevin)

Question 23

What protein is involved in calcium sensing on the vesicle?

Answer 23

Synaptotagmin

Question 24

How did Fernandez-Chacon (2001) show that synaptotagmin was the calcium sensor if knocking it out is lethal?

Answer 24

• Mutated residues in the Ca binding pocket but don’t chelate Ca.
• Meant synaptotagmin didn’t bind Ca quite as well
• R233Q was most effective residue
• Measured response, less neurotransmitter release for mutant, reduced EPSC

Question 25

What else did Fernandez-Chacon do and what was strange about the results?

Answer 25

• Did a high frequency pulse test

- Mutant had an increased response to short term placticity

Question 26

Why does the synaptotagmin mutant have an increased response for short term plasticity

Answer 26

As the residual calcium increases it catches up

Question 27

What is the protein process of vesicle fusion?

Answer 27

1 - SNARE-independent vesicle tethering (Rab3)
2 - t-SNARE activation
3 - SNAREpin formation and synaptotagmin binds
4 - SNAREpins zip up mediating fusion
5 - SNAP and NSF use ATP to disassemble SNARE complex

Question 28

How did Sudhof try to find the molecular mechanism for mfLTP?

Answer 28

• Knocked out several presynaptic PKA substrates (synapsins, Rab3A, Rabphilin) but all left normal mfLTP.

Question 29

What is RIM1 alpha and what happened when Sudhof knocked it out?

Answer 29

• Rab interacting molecule Sudhof knocked out
• Required for SV priming
• Knock out mouse had no mfLTP

Question 30

What did Sudhof then discover about RIM1 alpha and how?

Answer 30

• Made a knock in mutant by putting an alanine instead of a serine in
• Mouse had mfLTP showing the knock out not having it was caused by something else

Question 31

What are the features of synaptotagmin 12 (syt12)

Answer 31

• Does not bind Ca
• PKA phosphorylates Ser97
• Binds Syt1 (not PKA-dependent) to regulate its interaction with SNARE complex

Question 32

What are phospho-specific antibodies?

Answer 32

Inject a peptide into a rabbit and hope it will make antibodies against the region that is phosphorylated.
This antibody can then tell you if the protein if phosphorylated or not.

Question 33

How can you stimulate mfLTP?

Answer 33

Add Forskolin which activates adenyl cyclase

Question 34

What is the model for mfLTP?

Answer 34

1 strong stimulation = loads of calcium
2 Ca activates AC = loads of cAMP
3 cAMP activates PKA which phosphorlyates synaptotagmin 12 (Syt12)