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Cellular Neuroscience (207)
Ian Parker
Lecture # 18 - Quantal release of
neurotransmitter
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Quantal (vesicular ) release of neurotransmitter:
an historical perspective
Fatt & Katzearly 1950s - chance discovery of miniature endplate potentials
(m.e.p.p.s)
Spontaneous, small depolarizations of muscle fiber resting potential
Occur randomly; ~ 1 per sec
Observed only at the endplate
Blocked by curare (competitive ACh antagonist)
Abolished by denervationEnhanced by blocking ACh esterase
THEREFOREarise from spontaneous release of packets (quanta) of ACh from the nerve
terminal
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Q. Could m.e.p.p.s result from release of single molecules of ACh?
A. No. If apply low dose of ACh in the bathing solution,there is a steadydepolarization, not an increase in frequency of m.e.p.p.-like events
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How many molecules of ACh in a quantum?
About 10,000 : Kuffler & Yoshikami, 1975
Apply ACh from an ionophoretic pipette positioned very close to the
nerve terminal. Evoke artificial m.e.p.p.s that match in size the natural
m.e.p.p.s. Then calibrate how much ACh was released from the pipette.
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ACh release evoked by nerve stimulation is also quantized
In normal extracellular Ca2+ the action potential-evoked endplate potential
(e.p.p.) is ~100x greater than the m.e.p.p. and triggers an action potential.
So, difficult to tell if it is made up of multiple quanta.
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BUT, if extracellular Ca2+ is lowered, the e.p.p. becomes much smaller,
and can be reduced to the size of the m.e.p.p.
(Mean size of the e.p.p. varies as the 4thpower of [Ca2+]because
fusion of a neurotransmitter vesicle requires simultaneous binding of 4Ca2+ions)
At appropriately low [Ca2+], e.p.p.s show a quantized naturesometimes
stimulation evokes release of 1 quantum (e.p.p. the same size as an
m.e.p.p.); sometimes 2 quanta (double the m.e.p.p. size); sometimes a
failure.
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Quantal Analysis of transmitter releaseCalcium increases the statistical PROBABILITY of
release of transmitter quanta
The numbers of e.p.p.s containing kquanta (k= 0, 1, 2, 3 etc) follow a
POISSON distribution (cf. deaths by horse kick in the Napoleonic army)
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Quanta and Vesicles
At the time transmitter quanta were identified, it was not known what they
physically corresponded to. But a few years later (1955), electron microscopy
revealed numerous vesicles in presynaptic nerve terminals.
Vesicular hypothesis: Transmitter (ACh and every other neurotransmitter we
know of) is packaged in vesicles of (roughly) consistent size. Fusion of 1
vesicle releases 1 quantum of transmitter
Evidence:
1. Nerve terminal contains hundreds of vesicles that contain
neurotransmitter
2. Size (volume) of vesicles is big enough to contain the expected number ofmolecules of transmitter
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3. Agents that cause massive release of neurotransmitter (e.g. black widowspider venom) deplete vesicles in terminal
4. E.M. of nerve terminals frozen in the act of releasing transmitter revealsvesicles fusing with surface membrane (omega profile)
5. Measurements of capacitance in cells undergoing exocytosis of transmitter
show stepwise increases, corresponding to addition of one vesicles-worth ofmembrane area.
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Visualizing vesicular fusion with an extracellular
fluorescent dye
Secretion of amylase from
pancreatic acinar cells
(neurotransmitter vesicles are too
small to see in this way, but
mechanism is the same).
Negative staining: dye in
extracellular space enters vesicles
as they fuse.
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The fusion pore
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Vesicular recycling
1. Vesicle fuses with plasma
membrane, releasing quantum of
neurotransmitter
2. Vesicle membrane incorporates
into plasma membrane
3. Vesicle membrane pinched off.
4. Empty vesicle5. Vesicles fuse to form larger
endosome. This is refilled with
transmitter (ACh synthesized by
choline acetyltransferase from
recycled choline)
6. Filled vesicles bud off, and docknear sites of Ca2+channels ready
for release.
