krueger 2

Question 1

neuromuscular junction

• which neurotransmitter involved

Answer 1

used to study chemical transmission

• simple, large and easily accessible synapse
• neurotransmitter here is acetylcholine

Question 2

what are end plates?

Answer 2

motor neurons that form large presynaptic terminals

Question 3

what is the EPP (when is it recorded, elicits what?)

Answer 3

EPP= end plate potential

• intracellular recoding in muscle fibre near endplate
• when presynaptic axon is stimulated, an excitatory postsynaptic potential is recorded
• EPP usually elicits action potential in muscle

Question 4

what are miniature endplate potentials?

• amplitude

Answer 4

mEEPS (Katz and coworkers)

• spontaneous changes in muscle membrane potential occur even in absence of motor nerve stimulation
• smaller then EEP
• amplitude is homogeneous, averaging 0.5 mV
• too big to represent potential change in response to opening of single acetycholine receptor

Question 5

measuring EPP to prevent muscle contraction from dislodging the microelectrode (what do you need to do-2 possible things to do)

Answer 5

• lower [Ca] in extracellular medium
• partially block postsynaptic Ach receptors with drug curare
• lowering [Ca] reduces neurotransmitter secretion, reducing magnitude of EPP below threshold for postsynaptic action potential

Question 6

EPPs made up of

• result from..

Answer 6

made up of individual units elicited by exocytosis of a “quantum” of neurotransmitter

• result from spontaneous, action potential-independent release of one quantum of neurotransmitter

Question 7

electron microscopy of synapses

Answer 7

• EM studies reveal accumulations of small vesicles in presynaptic terminals
• Katz hypothesized, neurotransmitter is stored in these vesicles, one quantum of neurotransmitter corresponds to the amount of transmitter release upon exocytosis on synaptic vesicle

Question 8

biochemical evidence that transmitter is stored in synaptic vesicle

Answer 8

• SV biochemically isolated from brain tissue by density gradient centrifugation; acetylcholine enriched in synaptic vesicle fractions

Question 9

ultrastructural evidence that exocytosis of a single SV is responsible for release of one quantum of neurotransmitter

• how to find out that vesicles have to fuse to membrane to release neurotransmitters

Answer 9

• used drug 4-AP(K channel inhibitor) to increase # vesicle in fusion events produced by single AP
• neuromuscular junction was stimulated, frozen and analyzed using FREEZE FRACTURE ELECTRON MICROSCOPY
• exocytosis of single synaptic vesicle leads to release of one quantum of neurotransmitter

Question 10

freeze fracture electron microscopy

Answer 10

• breaking of frozen tissue under high vacuum
• plasma membrane break between lipid layers
• large expanses of presynaptic membrane exposed, facilitating detection of fusion synaptic vesicles
• fusing vesicles appear as pockets in membrane leaflets

Question 11

what is required to elicit a neurotransmitter release

Answer 11

action potential

Question 12

squid giant synapse preparation

Answer 12

• allow simultaneous voltage-clamp of the presynaptic terminal and intracellular recording of the membrane potential of the postsynaptic cell
• voltage-gated Na channels blocked with tetrodotoxin, neurotransmitter release elicited by injecting current into presynaptic terminal
• depolarizations of 50mV or less – no EPSP
• depolarizations of 70mV or more– maximal EPSP

Question 13

voltage gated Ca current

Answer 13

• need voltage gated Ca to cause neurotransmitter release
• need Ca influx
• blockade of voltage-dependent Na and K currents with tetrodotoxin and tetraethylammonium (respectively)

Question 14

evidence that voltage-gated Ca currents are required for neurotransmitter release (4)

Answer 14

1) buffering intracellular Ca with a fast Ca chelator abolishes synaptic transmission
2) injection of Ca into presynaptic terminal leads to postsynaptic potential in absence of presynaptic membrane depolarization
3) increasing extracellular Ca concentration increase the amplitude of postsynaptic currents, whereas decreasing [Ca] abolishes synaptic transmission
4) blockade of voltage-gated Ca channels abolishes synaptic transmission

Question 15

activation of voltage-dependent Ca channels (when?)

Answer 15

• activate only slowly in response to membrane depolarization
• Ca channels are slow to open, delay of 1-3ms

Question 16

why presynaptic voltage-gated Ca channels need to be close in proximity to synaptic vesicles

Answer 16

• extracellular [Ca] high, intracellular low
• neurons keep intracellular Ca low, by expressing Ca-binding proteins
• ## Ca sensory responsible for triggering synaptic vesicles fusion has fairly low affinity to Ca – has to be close to an open Ca channel (to elicit release)

Question 17

2 types of voltage gated Ca channel involved in neurotransmitter release

Answer 17

fast transmitter release

1) P/Q
2) N type

• other involved in slow release of dense-core vesicles= L-type (peptide transmitter)

Question 18

mutations in P/Q type Ca channels leading to congenital diseases (3)

Answer 18

Familial Hemiplegic Migraine

Ataxia- inability to coordinate voluntary movements, cerebellar/spinal cord defects

absence epilepsy- seizures characterized by unconsciousness, without voluntary muscle contractions

• no disease- causing mutations for N-type Ca channel known

Question 19

Lambert- Eaton myastenic syndrome (LEMS)

• Ca channels

Answer 19

• complications in patients with certain cancer (especially small-cell lung carcinoma)
• weakness and fatigability of skeletal muscles
• lower density of Ca channels
• autoimmune disease: blood of LEMS contains high concentrations of antibodies to presynaptic Ca channels (bind to channels)

Question 20

biogenesis of synaptic vesicles containing small molecule neurotransmitters

Answer 20

• synthesis/uptake occur locally within presynaptic terminals
• some neurotransmitters (ex: glutamate) taken up by extracellular space by plasma membrane transporters
• some neurotransmitters, precursors are taken up from extracellular space. enzymes produced in soma, transported to terminal via slow axonal transport, locally synthesize the neurotransmitter from precursor

Question 21

neurotransmitter loaded into synaptic vesicle by…?

• process

Answer 21

vesicular neurotransmitter transporter
- against concentration gradient

• energy for transport comes from electrochemical gradient across the SV membrane that is created by vesicular proton pump
• pump hydrolyzes ATP to transport H into SV lumen, creating pH and membrane potential

Question 22

neuropeptides synthesized in…

Answer 22

Soma

• made in the ER (synthesized by ribosomes)
• converted into neuropeptides in Golgi
• transported to synapse in peptide-filled vesicle (fast axonal transport along microtubules)

Question 23

do neuropeptides under re-uptake?

Answer 23

NO

- they are degraded by proteolytic enzymes

Question 24

evidence for local recycling for synaptic vesicles

Answer 24

• SV fusion adds new membrane to plasma membrane

- plasma membrane surface area usually held constant by compensatory endocytosis

Question 25

synaptic vesicle cycle

Answer 25

• vesicular membrane is retrieved by clathrin mediated endocytosis (endocytosis is completed 10-20 seconds following exocytosis)
• SV stored in reserve pool within cytoplasm, until need to participate in neurotransmitter release
• SV mobilized from pool, have to dock to the active zone, undergo priming step before becoming fusion-competant
• SV can complete whole endocytosis-exocytosis cycle in approx 1 min

Question 26

2 different routs for SV exocytosis

Answer 26

1) classical exocytosis

2) kiss-and-run exocytosis

Question 27

what is classical exocytosis

Answer 27

• full collapse of vesicle membrane into plasma membrane
• clathrin-mediated endocytosis to retrieve SV membranes required 20s
• high frequency stimulation quickly leads to depletion of vesicles at synapses with relatively small SV pools

Question 28

what is kiss and run exocytosis

Answer 28

• transient fusion pore, vesicle membrane never fully collapses into plasma membrane
• possibly repeated fusions of individual SV with plasma membrane in short time frame
• may allow for sustained release in response to repetitive stimulation

Question 29

readily releasable pool of SV

Answer 29

• pool of SV immediately available for release
• at CNS synapses, only 2-4% of SV
• readily releasable vesicles may correspond to SV docked to active zone

Question 30

“reserve pool” of SV

Answer 30

• SV available for exocytosis, not for immediate release

- readily releasable and reserve pool constitute the recycling pool of SV, representing on avg. 20% of all SV

Question 31

“resting pool” of SV

Answer 31

non recycling SV

- largest pool

Question 32

Describe how small-molecule neurotransmitters and neuropeptides are synthesized and
packaged into synaptic vesicles.

What happens to these neurotransmitters once they are released?

Answer 32

Small-molecule transmitters (e.g. glutamate, GABA, acetylcholine, monoamines):

• Uptake or synthesis at the synapse, packaging into synaptic vesicles by transporter that utilizes H+ gradient to accumulate neurotransmitter in synaptic vesicle lumen.
• NT undergo re-uptake after release.

Peptide neurotransmitters:

• Synthesis in the soma; processing in ER and Golgi. Fast axonal transport.
• Neuropeptides are degraded following exocytosis

Question 33

What experimental approach can be utilized to demonstrate that action potential-evoked neurotransmitter release requires calcium influx through voltage-gated calcium channels? (4)

Answer 33

To show that rises in the cytoplasmic calcium concentration required for release, one can record postsynaptic potentials (or currents) from the postsynaptic neuron while

(a) injecting calcium chelators (substances that bind calcium) into the presynaptic terminal to block NT
release

(b) injecting calcium into presynaptic terminal which elicits neurotransmitter release

(c) changing the concentration of calcium in the extracellular solution, leading to concomitant changes in
the amount of neurotransmitter released

(d) To demonstrate the involvement of voltage-gated calcium channels one can block these channels (with
cadmium or with w-agatoxin and w-conotoxin) which abolishes neurotransmitter release.

Question 34

Why does 4-aminopyridine, a potassium channel blocker, increase the amount of neurotransmitter released at the neuromuscular junction?

Answer 34

• Voltage-gated potassium channels are required for the repolarization of the plasma membrane.
• A partial inhibition of voltage-gated potassium channels will slow down the depolarization phase and lead to
  prolonged opening of presynaptic voltage-gated calcium channels.
• This leads to larger increases in the
  presynaptic calcium concentration, leading to an increase in neurotransmitter release.

Question 35

How many acetylcholine molecules elicit a “miniature endplate potential”?

Answer 35

• A miniature endplate potential (mEPP) results from the spontaneous (i.e. action potential independent)
  fusion of a synaptic vesicle with the membrane, releasing the entire acetylcholine content of the synaptic vesicle into the synaptic cleft.
• The postsynaptic plasma membrane contains a large number of nicotinic acetylcholine receptors, and a large fraction of these receptors will bind acetylcholine and open
  in response to the release of one quantum (one synaptic vesicle full of) acetylcholine.

Exactly how many?
— According to estimates of Katz and Miledi, the current passing through a single nicotinic acetylcholine
receptor in response to acetylcholine binding is approximately 0.3 µV. The average mEPP has an amplitude
around 0.5 mV, corresponding to the opening of approximately 2000 receptors.