Lecture 4 - Molecular Postsynaptic Plasticity

Question 1

What does an ionotropic glutamate receptor look like?

Answer 1

• 3 transmembrane domains, 1 sits in the membrane (M2)
• extracellular ligand binding NH3 from M1
• Intracellular C terminus from M4

Question 2

Features of NMDA receptor

Answer 2

• Can be blocked by Mg2+
• Gates sodium and calcium
• Inhibited by AP5 OR mk8o1

Question 3

What can you use to inhibit AMPA receptors?

Answer 3

NBQX

Question 4

What is the Mg block on the NDMA receptor dependent on and what is this for?

Answer 4

Resting membrane potential

So you need glutamate, glycine and depolarisation to open it

Question 5

What is yeast 2 hybrid?

Answer 5

Tests protein protein interactions

• Have a bait protein attached to half a TF
• Make a library expressing different proteins (prey) attached to half a TF
• If the protein binds the TF forms and get a blue readout

Question 6

What did Kim and Sheng (2004) discover and how?

Answer 6

Used Y2H to show there is lots of proteins with PDZ domains that bind to the C terminus of the NDMA receptor showing there is lots of intracellular signalling going on in post synapse

Question 7

What did Seth Grant do?

Answer 7

Characterised all the proteins under the post-synaptic membrane by using an antibody to the AMPA receptor and pulled out 200-300 proteins that make up the post-synaptic density

Question 8

What disability is associated with 6 mutated proteins in the PSD? Examples of proteins

Answer 8

Autism

SynGAP, Shank

Question 9

Outline an LTP experiment

Answer 9

• Record from cell body of CA1 pyramidal cell

- Stimulate axon from CA3 to CA1

Question 10

Why do you record from the cell body and not the synapse?

Answer 10

A synapse is 1 micron across so its too small to record from

Question 11

What is postsynaptic LTP also known as?

Answer 11

Schaffer collateral-CA1 LTP

Question 12

What is MK801

Answer 12

An inhibitor of the NMDA receptor at a particular voltage which wipes out learning

Question 13

What was the 90s model of LTP?

Answer 13

• Depolarisation ejects Mg and opens NMDA receptor
• Ca entry activates second messenger pathways
• Cell becomes more sensitive to glutamate and retrograde signalling causes enhanced neurotransmitter release

Question 14

What else was found about LTP in the 90s which was strange?

Answer 14

LTP causes fewer synaptic failures (when the synapse doesn’t fire) which suggested some synapses didn’t have receptors in them

Question 15

How were synapses shown to develop?

Answer 15

NMDA receptors come first followed by AMPA

Question 16

What experiment did Lledo et al (1998) do?

Answer 16

• Stained AMPA receptors green, NMDA red and this makes yellow when mixed

Question 17

What were Lledo’s results and what did this suggest?

Answer 17

Never had AMPA receptors with out NMDA but had NMDA on its own.
Suggested LTP traffics AMPA receptors to the membrane

Question 18

How did Lledo’s results explain silent synapses?

Answer 18

The NMDA on its own would have Mg blocking it.

Question 19

What treatments did Lledo then use on the synapses to block exocytosis

Answer 19

• NEM inhibits NSF
• N19
• Botulin toxins cut SNAREs

Question 20

What did blocking exocytosis show?

Answer 20

Prevented AMPA receptors going into the membrane and activating synapses

Question 21

What is immunogold electron microscopy?

Answer 21

Way to image proteins
Have an antibody to protein, then a secondary antibody has gold particle.
Can use different sizes of gold to differentiate between proteins

Question 22

How did Shi et al (1999) show AMPA receptor exocytosis?

Answer 22

GFP to AMPA receptors and looked at silent synapses before and 30 minutes after applying tetanus

Question 23

What proteins are involved in endocytosis of AMPA receptors in LTD?

Answer 23

Calcineurin, p38, Rap, Arc

Question 24

What does Richard Huganir do?

Answer 24

Makes phosphorylation mutant mice.

Question 25

What 2 proteins did Richard Huganir show bind to the PDZ domain on the C terminus of AMPA receptor?

Answer 25

PICK1 (protein interacting with PKC)

GRIP1 (glutamate receptor interacting protein)

Question 26

What do PICK1 AND GRIP1 do?

Answer 26

They fight for binding to the PDZ domain.
When the receptor is in the membrane GRIP1 stabilises it
When the receptor is internalised grip falls off and PICK1 pulls it in

Question 27

What happens when you remove the last 7 amino acids from the end of the C terminus?

Answer 27

No LTP and no LTD

Question 28

What is the mechanism for changing from GRIP to PICK?

Answer 28

Phospho-switch.

PKC phosphorylates GRIP causing it to let go, and phosphorylates PICK causing it to pick it up