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Neuroscience with Pharmacology 2

Neuromuscular Junction 1: Structure and Function

Galvani, 1850 Ranvier, 1880

NMJ’s in the “Brainbow” transgenic mouse

J Livet, JW Lichtman et al (2007) Nature

30 µm

Importance of NMJ’s:

1. Every breath you take, every move youmake…..

2. NMJ’s are accessible models of synapticstructure and function elsewhere (brain,spinal cord, autonomic ganglia)

3. Many general principles of synaptic functionwere established by studies of the NMJ.(They still are!)

The “Final Common Path”….

...leading to the “ultimate” synapses

Motor neurones

Electrical stimulation of nerves causes muscles to contract

2

5 mN

5 s

Muscle contractions in response to single (twitch) and repetitive (tetanic) stimuli

1. Imaging the “Ultimate Synapse”

2. Ultrastructure and electrophysiology of the NMJ

3. Synthesis, storage, release, action and inactivation of ACh

4. Quantal secretion and exocytosis

5. The Safety Factor for neuromuscular transmission

Neuromuscular Junction - Structure and Function(or… How We Eat Pizza….)

RobertHartley/Adrianna Teriakidis

The (mouse) motor unit

500 µm

50µm

27,412 µm3

Vr=1

5,88

2 µ

m3

Vr=30

2,377 µm3

Vr=4

100µm

3 cm

30µm

4DL muscle

50 µm

Neuromuscular junctions viewed with confocal microscopyNeuromuscular junctions comprise four types of cells

20 µm

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1. Imaging the “Ultimate Synapse”

2. Ultrastructure and electrophysiology of the NMJ

3. Synthesis, storage, release, action and inactivation of ACh

4. Quantal secretion and exocytosis

5. The Safety Factor for neuromuscular transmission

Neuromuscular Junction - Structure and Function(or… How We Eat Pizza….)

Junctional Folds

Basal lamina

Synaptic vesicles

Pre

Post

Muscle Fibre

Terminal Schwann cell

Nerve terminal(presynaptic)

Motor end-plate(postsynaptic)

AChRNaV

Action Potential

End-Plate Potential (EPP)

Threshold

Muscle fibre action potentials are normally triggered by EPP’s,leading to muscle contraction

Action potentials are necessary because subthreshold voltages decay with distance

1. Imaging the “Ultimate Synapse”

2. Ultrastructure and electrophysiology of the NMJ

3. Synthesis, storage, release, action and inactivation of ACh

4. Quantal secretion and exocytosis

5. The Safety Factor for neuromuscular transmission

Neuromuscular Junction - Structure and Function(or… How We Eat Pizza….)

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Synthesis

Storage

Release

Action

Inactivation

Synthesis

~5000 molecules

Storage

Synaptic vesicles

Release

Ca2+

EM tomography reveals association of vesicles with “active zones”

http://en.wikipedia.org/wiki/SNARE_(28protein)

Several SNARE proteins have been identified

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Action

~50 pA

Inactivation

Basal lamina

1. Imaging the “Ultimate Synapse”

2. Ultrastructure and electrophysiology of the NMJ

3. Synthesis, storage, release, action and inactivation of ACh

4. Quantal secretion and exocytosis

5. The Safety Factor for neuromuscular transmission

Neuromuscular Junction - Structure and Function(or… How We Eat Pizza….)

5 mV

10.00 ms

Ca2+

Mg2+

EPP’s (muscle action potential blocked)

EPP’s in low Ca/high Mg

Action potentials are “all-or-nothing” signals...… but EPPs are variable responses

5 mV

10.00 ms

Quantal secretion...

… by exocytosis

MEPPs

EPPs

1 mV 50 nm

1 vesicle MEPP = 1 “quantum”

m vesicles EPP = m quanta =

=

The Quantal (Vesicle) Hypothesis

5 mV

10.00 ms

m =EPP

q

Quantal Content:

q = MEPP

Quantal Size:

Quantification of EPPs: Quantal Analysis

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m = n.p

n=3p<0.001

If x=1P1=p1.(1-p)2

If x=2P2=p2.(1-p)1

If x=3P2=p3.(1-p)0

If x=0P0=p0.(1-p)3

Testing the Quantal/Vesicle Hypothesis

!

Px

=e"mm

x

x!

PoissonEquation

MEPPEPP

Stim.

MEPPs

EPPs

Quantal analysis: Fitting the Poisson Equation

!

Px

=e"mm

x

x!

1. Imaging the “Ultimate Synapse”

2. Ultrastructure and electrophysiology of the NMJ

3. Synthesis, storage, release, action and inactivation of ACh

4. Quantal secretion and exocytosis

5. The Safety Factor for neuromuscular transmission

Neuromuscular Junction - Structure and Function(or… How We Eat Pizza….) Actual m

Threshold m

Quantal analysis of EPPs shows a “safety factor” of 2-5

Gillingwater D. Thomson

5 ms

EPPs - Facilitation

300 ms

10 mV

EPPs - Short-term Depression

Frog

Rat

Man

0 250 500 750 1000 1250 15000

50

100

150

200

Synaptic area

Synaptic strength depends on synaptic size

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Summary

1. Neuromuscular junctions comprise a motor nerve terminal (axon ending), motor end-plate (muscle fibre contact region) and are capped by terminal Schwann cells andkranocytes.

2. Acetylcholine (ACh) is synthesised in motor nerve terminals by choline acetyltransferase (ChAT)

3. ACh is pumped into and stored in synaptic vesicles.

4. ACh is released at the NMJ by Ca-dependent exocytosis from synaptic vesicles.These vesicles are recycled by endocytosis.

5. ACh action occurs by binding to receptors (AChR, ligand-gated ion channels) in thepost-synaptic membranes producing depolarising MEPP’s and EPP’s

6. ACh is broken down (inactivated) by acetylcholinesterase (AChE) enzyme in thesynaptic basal lamina. Choline is recycled into the nerve terminal and used in thesynthesis of ACh.

7. A high safety factor (3-5 fold excess of transmitter release) ensures action potentialsare triggered in muscle fibres, even during synaptic depression caused by repetitiveexcitation.