Lecture 10: Neurotransmitters and Synaptic Vesicle Cycle

Question 1

Life Cycle of Synaptic Vesicle – Steps

Answer 1

1. transmitter is synthesized, and then stored in vesicles
2. AP invades presynaptic terminal
3. depolarization of presynaptic terminal causes opening of voltage-gated Ca2+ channels
4. influx of Ca2+ through channels
5. Ca2+ causes vesicles to fuse with presynaptic membrane
6. transmitter is released into synaptic cleft via exocytosis
7. transmitter binds to receptor molecules in postsynaptic membrane
8. opening or closing of postsynaptic channels
9. postsynaptic current causes excitatory or inhibitory postsynaptic potential that changes the excitability of the postsynaptic cell
10. removal of neurotransmitter by glial uptake or enzymatic degradation
11. retrieval of vesicular membrane from plasma membrane

Question 2

What is the synaptic vesicle cycle?

Answer 2

system for recycling synaptic vesicles at axon terminal

Question 3

Where are synaptic vesicles and their transmembrane proteins originally synthesized and assembled? Why is this a problem?

Answer 3

at cell soma (not at axon terminal) – but this process is not sustainable with ongoing synaptic release

• newly created vesicles are slowly transported down axons to axon terminals using specialized transport proteins that crawl along microtubules – too slow to support the release of multiple quanta for every AP fired by an axon
• every vesicle that fuses with the active zone adds extra surface area to axon terminal membrane

Question 4

How is a constant supply (and reserve) of synaptic vesicles maintained?

Answer 4

axon terminal possesses cellular machinery to recover membrane and vesicle proteins and reform new vesicles quickly

Question 5

What is endocytotic budding?

Answer 5

involves assembly of a special protein coat, which shapes the new vesicle

Question 5

What is endocytotic budding?

Question 6

What is enzymatic cleavage?

Answer 6

pinches off the new vesicle from the axon terminal membrane

Question 7

Heuser & Reese used very strong, prolonged stimulation to obtain their data – subsequent experiments have shown that some aspects of synaptic vesicle cycle shown in these diagrams occur differently in more ‘physiological’ (normal) conditions. What are two aspects?

Answer 7

caveat 1: intermediate endosome stage occurs ONLY if there has been intense, prolonged stimulation

caveat 2: budding process can occur faster (< 1s instead of 10-20s) under physiological conditions of temperature and stimulation

Question 8

What proteins are involved in coating and uncaring of vesicles during reformation?

Answer 8

• clathrin and adaptor protein scaffolds are disassembled by other proteins, and can be recycled for retrieval of new vesicles
• actin fibres of the cytoskeleton provide a track to help speed reformed vesicles away from the terminal membrane, and in some cases may allow for speedy recovery of vesicles without clathrin

Question 9

What do vesicular transporter proteins do?

Answer 9

move neurotransmitters into the vesicle lumen

Question 10

What does proton pump (v-ATPase) do?

Answer 10

found in synaptic vesicle membranes – acidifies lumen of vesicle (pH ~5.4) to help loading of neurotransmitter

Question 11

What is clathrin?

Answer 11

scaffolding protein that shapes the retrieved membrane during vesicle recycling

Question 12

How does clathrin shape the retrieved membrane during vesicle recycling?

Answer 12

• constructs cage-like coats observed around vesicle buds and recently reformed vesicles – individual monomers (triskelia) assemble into repeating geometric structures, which force the membrane to curve with them
• interacts with various adaptor proteins (ie. AP-2), which help it bind to membranes

Question 13

What is dynamin, and what does it do?

Answer 13

ring-shaped protein that creates a ‘neck’ around a budding vesicle, and acts as ‘molecular scissors’ to clip the new vesicle bud from the axon terminal

hydrolyzing GTP to GDP causes dynamin to twist, which tightens ring and severs neck, releasing the vesicle

Question 14

How are synaptic vesicles organized?

Answer 14

into physiologically distinct pools which differ in ‘releasability’ (release pools)

Question 15

What are the 3 types of release pools?

Answer 15

• reserve pool
• recycling pool
• readily releasable pool (RRP)

Question 16

What is the reserve pool?

Answer 16

filled, but often tethered by synapsin proteins, so they are relatively immobile – only released during extreme prolonged stimulation

Question 17

What is the recycling pool?

Answer 17

mobile vesicles that can reach the active zone to maintain steady release levels under moderate stimulation

Question 18

What is the readily releasable pool (RRP)?

Answer 18

docked and primed vesicles that can be immediately released in response to a single elevation of Ca2+

Question 19

In real synapses, how are recycling and reserve pool vesicles arranged?

Answer 19

anatomically mixed – they are not fixed distances away from the active zone

Question 20

How are vesicles experimentally divided into release pools?

Answer 20

1. load synaptic vesicles with fluorescence by bathing axon terminal in fluorescent dye while stimulating
2. after removing the dye from the bath solution, axon terminal is stimulated by a prolonged depolarization
3. percent of total fluorescence LOST from loaded axon terminal is measured over the period of stimulation (∆F)

with steady prolonged stimulation, rate (slope) of fluorescence loss typically changes over time in three distinct stages, indicating three different groups of vesicles with different availability

Question 21

How does prolonged stimulation change the release of vesicles?

Answer 21

1. all terminals are stimulated briefly in presence of dye (enough to label majority of all vesicles in the terminals)
2. rest phase: dye is no longer present
   - control terminals are left alone
   - test terminals are ‘buzzed’ – stimulated enough to empty and refill recycling pool
3. destain phase: all terminals are stimulated for prolonged period (to begin to release the reserve pool)

during destain phase, fluorescence levels in buzzed group of terminals do not decrease in first 10s while control group levels decline steeply, but after ~10s, both groups show similar rates

Question 22

Neurotransmitters are a special case of…

Answer 22

paracrine (between neighbour cells) signalling

Question 23

What are neurotransmitters?

Answer 23

chemicals released at synapses that mediate communication between two neurons, or between a neuron and another excitable cell

• neuron to neuron
• neuron to muscle cell
• receptor cell to neuron

Question 24

What can neurotransmitters be synthesized from?

Answer 24

• phospholipid
• free nucleic acid
• protein
• free amino acids

Question 25

How does the life cycle differ for various neurotransmitters?

Answer 25

each molecule has distinct series of biochemical processes where it is synthesized, packaged, released into synaptic cleft, inactivated, and recycled then repackaged into vesicles

Question 26

Where is glutamine (amino acid) synthesized? Why?

Answer 26

locally, because it does not cross BBB

Question 27

Where is excitatory amino acid transporter (EAAT) present?

Answer 27

present on astrocytes and to a lesser extent on neurons

Question 28

What do astrocytes play an active role in?

Answer 28

• modulating synaptic transmission in glutamatergic synapses –exert a large degree of control over the glutamate concentration in synaptic cleft
• can release glutamate as a ‘gliotransmitter’ via EAAT in response to elevations in [Ca2+]i, enhancing synaptic signalling

Question 29

What is the concept of a tripartite synapse?

Answer 29

three cells are necessary for a synapse to function

• key modulatory roles for astrocytes in synaptic transmission led to this concept

Question 30

Neurons that release biogenic amines have similar…

Answer 30

similar distribution patterns throughout our brain

Question 31

Neurons that use biogenic amines as neurotransmitters are much rarer than…

Answer 31

glutamate, GABA, or glycine

Question 32

Where are cell bodies of neurons that use biogenic amines as neurotransmitters found? What about their axons and axon terminals?

Answer 32

cell bodies are usually only found in small nuclei (groups of neuron somata) deep within brainstem

axons and axon terminals from these small brainstem nuclei are highly divergent throughout the rest of the brain and spinal cord, (contacting many other neurons in many different locations) – usually function as neuromodulators

Question 33

Where is tyrosine hydroxylase (TH) found?

Answer 33

• neurons that release dopamine

- neurons that release norepinephrine

Question 34

How does the dominant mechanism of synaptic vesicle recycling work?

Answer 34

via the membrane shaping protein clathrin, and the GTPase dynamin

Question 35

There are many more vesicles in presynaptic terminals than are ever usually released. Why?

Answer 35

ease of mobilizing different groups of vesicles has consequences for ability of a synapse to maintain signalling during prolonged activity

Question 36

What happens to non-peptidergic neurotransmitters at most synapses?

Answer 36

typically recycled – pathways by which recycling occurs are generally specific to each neurotransmitter and are often targets for medical interventions and psychoactive drugs