Lecture 12: Neurotransmitter (Release and Retrieval)

List the steps for neurotransmitter release.

1. docking at the active zone

2. SNARE proteins alter conformation upon a rise in intracellular calcium

3. vesicle exocytosis and neurotransmitter release (5000 per vesicle)

4. vesicle membrane recovered by endocytosis

True or False: Neurotransmitter release leads to synaptic delay. There is a delay from the AP arriving at the axon terminal to creating a postsynaptic response.

true

True or False: Neurons maintain a readily releasable pool of vesicles so release can occur extremely rapidly.

true

What state are vesicles in before stimulation?

a docked state

True or False: Vesicle fusion occurs within milliseconds after stimulation.

true

Which proteins are present at the active zones?

• synaptotagmin

• VAMP (synaptobrevin)

• synapsin

• Rab3

What is synaptotagmin? What is its function?

• calcium sensor

• detects incoming calcium

What is VAMP? What is its function?

• vesicle SNARE

• participates directly in fusion

What is synapsin’s function?

tethers reserve vesicles to the actin cytoskeleton

What is Rab3?

controls vesicle availability and docking

Is vesicle interior basic or acidic?

acidic

True or False: The proton gradient helps load neurotransmitter into vesicles.

true

What is required for triggering the exocytosis of synaptic vesicles?

microdomains with high Ca2+ concentrations in the active zone near opened Ca2+ channels

List the steps from when the AP arrives to when fusion ceases.

1. voltage-gated Ca2+ channels open

2. tiny regions Ca2+ microdomains form

3. vesicles sit within -50 nm of multiple channels

4. local Ca2+ becomes high enough to trigger fusion

5. channels close

6. microdomains disappears

7. fusion stops

True or False: Neurotransmitter release depends on highly localized calcium microdomains near active zones.

true

How does fusion of synaptic vesicles with the presynaptic membrane occur?

through SNARE complexes

What do SNARE proteins do?

physically pull the vesicle membrane and presynaptic plasma membrane toward each other until fusion occurs

Which protein is a v-SNARE protein?

synaptobrevin (VAMP)

Where are v-SNAREs located?

vesicle membranes

Which proteins are t-SNAREs?

• syntaxin

• SNAP-25

Where are t-SNAREs located?

target membranes

What anchors t-SNAREs?

a single TM domain

What anchors v-SNAREs?

a single TM domain

v-SNAREs and t-SNAREs form what?

a four-helix bundle

How did researchers study the role of SNAREs at the synapse?

• yeast mutations

• drosophila and C. elegans

• 8 neurotoxins

Trans-SNARE complexes exert which kind of force?

exert inward force (F)

Cis-SNARE complexes exert which kind of force?

no force

What do trans-SNARE complexes (SNAREpins) generate?

generate an inward force vector that pulls the bilayers together—forcing them to fuse

When fusion has occurs, what happens to the complex?

the force vanishes and the SNAREs are in the low-energy cisSNARE complex

True or False: SNARE zippering provides the mechnical force required for membrane fusion.

true

What produces tetanus and botulinum toxins?

Clostridium bacteria

What do tetanus and botulinum toxins do?

act as Zn2+-dependent proteases that cleave SNARE proteins

True or False: A single toxin molecule of either tetanus or botulinum can block the activity of an entire nerve terminal.

true

What does tetanus and botulinum toxicity cause?

• vesicles cannot fuse

• neurotransmitter release stops

What are the major targets of neurotoxins?

• synaptobrevin/VAMP

• syntaxin

• SNAP-25

List the molecular steps of exocytosis.

1. v-SNARE and t-SNARE interact

2. nucleation begins

3. zippering proceeds

4. Ca2+ signal arrives

5. fusion pore opens

6. neurotransmitter exits

Briefly describe vSNARE.

• on surface of vesicle

• binds tSNARE

• mediates fusion

Briefly describe tSNARE.

• on surface of target membrane

• binds vSNARE

• mediates fusion

What are some important proteins for synaptic vesicle exocytosis?

• complexin

• synaptotagmin

• munc 18

What does complexin do?

• binds SNARE complex

• clamps it prior to Ca2+ signal

• arrests the fusion and serves as a brake

What does synaptotagmin do?

• senses calcium

• triggers rearrangement of SNARE complex

• displaces complexin from SNARE complex

What does munc 18 do?

• binds syntaxin

• regulates SNARE complex formation

• essential for neurotransmitter release

What is NSF?

an ATPase that disrupts the SNARE complex

True or False: NSF/SNAP complex binding to SNARE complex will dissociate and recycle SNARE proteins.

true

What are the 2 modes of synaptic vesicle recycling?

• kiss and run

• clathrin-mediated reuptake

What is the kiss and run mode?

vesicle partially fuses with the membrane, taken up, and refilled without sorting through endosomes

Describe kiss-and-run.

• vesicle: opens briefly, releases transmitter, and quickly reseals

• advantages: fast and efficient

Describe clathrin-mediated endocytosis.

• vesicle: fully collapses into membrane and reformed through endocytic machinery

• advantage: more complete sorting and recycling

How can we measure synaptic vesicle endocytosis and endocytosis?

• capacitance measurements

• FM1-43 dye imaging

What do changes in capacitance reflect?

changes in surface area

What is shown when capacitance increases in a stepwise fashion?

complete exocytosis

What is shown when capacitance decreases in a stepwise fashion?

endocytosis

True or False: During kiss-and-run exocytosis, capacitance flickers.

true

Where does FM1-43 insert into?

the membrane

What happens with FM1-43 dye during endocytosis?

• membrane containing dye is internalized

• vesicle becomes fluorescent

What happens with FM1-43 dye during exocytosis?

• dye leaves the membrane

• fluorescence decreases

What is the process for FM1-43 dye?

1. unlabeled synaptic terminal

2. add FM1-43

3. stimulation

4. after endocytosis

5. wash out FM1-43

6. stimulation

Describe the FM1-43 experiment in the frog neuromuscular junctions.

• control: minimal uptake

• endocytosis: dye enters vesicles

• exocytosis: labeled vesicle fuse and fluorescence pattern changes

True or False: At the NMJ, a motor neuron normally releases hundreds of synaptic vesicles per impulse.

true

True or False: ACh binding to postsynaptic nicotinic ACh receptors on the muscle generate end plate potentials (EPPs).

true

What is an EPP provoked by? What does this lead to?

evoked by nerve stimulation, and depolarizes the muscle cell plasma membrane—leading to muscle contraction

True or False: To evoke an EPP, presynaptic depolarization and calcium-influx is required for ACh release.

true

What is an MEPP?

a spontaneous postsynaptic depolarization caused by spontaneous release of small quantifies of ACh; miniature EPP

True or False: No presynaptic depolarization and no calcium-influx is required for MEPP.

true

Briefly describe BAPTA.

• fast calcium chelator

• EPP decreases

Briefly describe EGTA.

• slow calcium chelator

• little effect on the EPP

Describe the classic Katz experiment.

• experimentally reduced the amplitude of EPPs by placing muscle preparation to a low calcium solution

• now evoked response EPP coincided with integral multiples of the mini amplitude

• the frequency with which different EPP amplitudes are observed follows a Poisson distribution

True or False: Each synaptic vesicle releases one quantum.

true

What is the amount of neurotransmitter in a single vesicle?

one quantum

EPP correlates to what?

a higher rate of fusion, many vesicles, evoked

mEPP correlates to what?

a low rate of fusion, a single vesicle, spontaneous

How can someone mimic the MEPP?

with direction application of ACh

A single quantum contains how many molecules of ACh?

about 7,000 molecules

For what reasons does the standard Katz model not always apply to the CNS synapses?

• nonuniformity of quanta:

  • vesicle content and diverse receptor population

• nonuniformity of release site:

  • different subunit or posttranslational modification of Ca2+ channels

• postsynaptic membranes that distort responses:

  • dendritic spine (local electronic properties) and spine neck

  • silent synapses (which contains NMDARs but not AMPARs, no depolarization)

• saturation of receptors:

  • glutamatergic synapses contain only about 100 receptors