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Flashcards in krueger 2 Deck (35)
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1

neuromuscular junction

- which neurotransmitter involved

used to study chemical transmission
- simple, large and easily accessible synapse
- neurotransmitter here is acetylcholine

2

what are end plates?

motor neurons that form large presynaptic terminals

3

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

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

4

what are miniature endplate potentials?

- amplitude

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

5

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

- 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

6

EPPs made up of

- result from..

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

7

electron microscopy of synapses

- 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

8

biochemical evidence that transmitter is stored in synaptic vesicle

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

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

- 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

10

freeze fracture electron microscopy

- 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

11

what is required to elicit a neurotransmitter release

action potential

12

squid giant synapse preparation

- 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

13

voltage gated Ca current

- need voltage gated Ca to cause neurotransmitter release
- need Ca influx


- blockade of voltage-dependent Na and K currents with tetrodotoxin and tetraethylammonium (respectively)

14

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

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

15

activation of voltage-dependent Ca channels (when?)

- activate only slowly in response to membrane depolarization

- Ca channels are slow to open, delay of 1-3ms

16

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

- 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)
-

17

2 types of voltage gated Ca channel involved in neurotransmitter release

fast transmitter release
1) P/Q
2) N type


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

18

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

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

19

Lambert- Eaton myastenic syndrome (LEMS)


- Ca channels

- 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)

20

biogenesis of synaptic vesicles containing small molecule neurotransmitters

- 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

21

neurotransmitter loaded into synaptic vesicle by...?

- process

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

22

neuropeptides synthesized in...

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)

23

do neuropeptides under re-uptake?

NO
- they are degraded by proteolytic enzymes

24

evidence for local recycling for synaptic vesicles

- SV fusion adds new membrane to plasma membrane
- plasma membrane surface area usually held constant by compensatory endocytosis

25

synaptic vesicle cycle

- 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

26

2 different routs for SV exocytosis

1) classical exocytosis

2) kiss-and-run exocytosis

27

what is classical exocytosis

- 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

28

what is kiss and run exocytosis

- 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

29

readily releasable pool of SV

- 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

30

"reserve pool" of SV

- 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