Transmitter Release

Question 1

Where are nicotinic receptors expressed

Answer 1

brain and spinal cord

Question 2

Speed of action potentials

Answer 2

up to 120 metres/sec

Question 3

Role of neurotransmitters

Answer 3

• carry signal across the synaptic cleft
• cause excitation or inhibition

Question 4

Steps of synaptic transmission

Answer 4

1) presynaptic AP
2) calcium influx via voltage-gated calcium channels
3) vesicular transmitter release
4) action of transmitter-gated ion channels
5) postsynaptic depolarisation of cation conducting transmitter-gated ion channels activate postsynaptic voltage-gated ion channels & initiate AP

Question 5

How is fast synaptic transmission mediated

Answer 5

transmitter-gated ion channels

Question 6

What are transmitter-gated ion channels (cys-loop family)

Answer 6

• e.g. nAChR
• integral ion channel
• agonist binding to receptor induces rapid conformational change to open channel
• selective for certain ions
• extremely rapid signalling

Question 7

How are nAChRs identified on alpha-bungarotoxin at the NMJ

Answer 7

red fluorescent, binds irreversibly

Question 8

Origins of a mepp

Answer 8

• results from a spontaneous release of just a single neurotransmitter packet
• at the end plate region vesicles
• open simultaneously -> sodium flux (depolarisation, AP)

Question 9

How can we use electron microscopy to study synapses

Answer 9

reveal vesicles at the synapse in exocytosis

Question 10

Action of Vesamicol

Answer 10

• inhibits vesicular uptake of ACh and decreases amplitude of mepps
• response to iontophoretically applied ACh remains unchanged by vesamicol, suggesting that the decrease in the amount of ACh in the vesicle is the reason for the decrease of mepp amplitude

Question 11

How alpha-LTX influences transmitter release

Answer 11

• depletion of vesicles
• inhibition of endocytosis
• distended terminal paralysis

Question 12

Mechanism of alpha-LTX

Answer 12

• involving both calcium dependent and independent pathways
• binds to specific presynaptic receptors (Neurexin and Latrophilin)
• toxin forms a cation (Na/Ca) conducting pore/channel
• 2 specific binding sites on presynaptic nerve

Question 13

Steps of recycling synaptic vesicles

Answer 13

1. Budding of endosome
2. Docking of neurotransmitter
3. Priming vesicle for release
4. Fusion of vesicle to membrane
5. Endocytosis via “Kiss and Run” / “Clathrin pathway”)

Question 14

Calcium role in neurally-evoked neurotransmitter release

Answer 14

• localised calcium entry via coltage-gated calcium channels
• calcium triggers vesicle fusion

Question 15

Technical issue for studying nerve stimulation at NMJ

Answer 15

nerve stimulation causes muscle contraction which results in breakage of glass microelectrode (use high Mg / low Ca buffered solution)

Question 16

Why is it beneficial to use high Mg / low Ca extracellular solution to measure nerve stimulation

Answer 16

it brings the epp to below threshold for AP firing

Question 17

Describe what is meant by neurotransmitter release at the NMJ is quantal

Answer 17

• amplitude of smallest event = amplitude of mepp
• stimulating multiple times provides varying responses depending on conditions -> shows repetitive peaks that are multiples of each other

Question 18

Define the quantal content

Answer 18

QC = mean epp amplitude / mean mepp amplitude

Question 19

Factors influencing the amplitude of the mepp

Answer 19

• number of nicotinic receptors
• amount of ACh stored in each vesicle

Question 20

How is a muscle contraction stimulated?

Answer 20

mepps summate to give epp, which, if large enough, exceeds threshold and initiates an AP, causing muscle contraction

Question 21

How is neuronal excitation stimulated in the CNS

Answer 21

• miniature excitatory postsynaptic potentials (mEPSPs) summate to produce excitatory post synaptic potential (EPSP), causing neuronal excitation

Question 22

Mechanism of action of dendrotoxin

Answer 22

• works presynaptically
• large increase in EPP amplitude
• large increase in QC
• no change MEPP
• blocks voltage-gated potassium channels
• limits repolarisation by potassium channels (control duration of AP)
• longer AP, increased Ca influx, increase vesicle release

Question 23

Mechanism of action of Tubocurarine

Answer 23

• works postsynaptically
• competitive antagonist for nAChRs
• reduces EPP below threshold
• reduces MEPP
• no change QC
• stops skeletal muscle contracting (skeletal neuromuscular blocker)

Question 24

Mechanism of action of botulinum toxin

Answer 24

• works presynaptically
• decreases QC
• decreases EPP
• no change MEPP
• decrease neurally-evoked ACh release via 4 steps

Question 25

4 steps of Botulinum toxin vesicular inhibiton

Answer 25

1. Attachment onto cholinergic presynaptic nerve 2. Toxin enters by endocytosis 3. Light chain in cytoplasm 4. Proteolytic cleavage of synaptobrevin

Question 26

Role of light chain

Answer 26

acts like protease and cleaves important components of proteins expressed on vesicles that are essential for vesicular fusion

Question 27

What is synaptotagmin (VAMP)

Answer 27

- calcium sensor - binds calcium cooperatively and undergoes conformational change - "knock-out" mice calcium-dependent transmitter release is impaired - Drosophila synaptotagmin mutants exhibit impaired release and impaired sensitivity to calcium

Question 28

Describe a simple model of vesiclar release

Answer 28

1) docking: complex of synaptobrevin, syntaxin, and SNAP 25 form to bring vesicle into the active zone adjacent to calcium channel 2) priming: synaptotagmin is recruited to the complex 3) fusion: calcium influx triggers fusion and transmitter release

Question 29

Role of magnesium in neurally-evoked transmitter release

Answer 29

- magnesium blocks the voltage-gated calcium ion channel and allows us to study neurally-evoked transmitter release - fusing solution of high mg and low ca reduces epps