Today
1. Synapse Formation
– Some early events
– Clustering of postsynaptic receptors at NMJ
Next Lectures
1. Synapse formation
– Clustering of receptors at CNS synapses
– Presynaptic specialization
2. Synaptic maturation and modification
Early events in synapse formation
1. Growth cone filopodia collapse
2. Golgi apparatus moves into postsynaptic
region
•
•
Abundance of transport vesicles
Postsynaptic sight shows first clear
differentiation
3. Synaptic vesicles begin to accumulate
~24h after contact
• As growth cones enter target, they slow
down, retract filopodia, and start extending
branches
Ability to release neurotransmitter is
established early
Record AChr responses
Growth cone
pipette
Membrane with Ach receptors
1
•
Early events in synaptogenesis also use
second messenger systems
1. Calcium
2. cAMP/Protein Kinase A
3. Protein Kinase C
• Calcium is seen to rapidly increase in the
growth cone when appropriate contact is
made
Growth cone filled with Ca-sensitive dye
Experimentally ↑ Calcium or cAMP
•
– Can induce retraction of filopodia
Appropriate contact
inappropriate contact
Synapse Formation
1. Postsynaptic receptor clustering
•
Initially muscles have even distribution of
AchRs
• In the mature synapse:
– 10,000 AchRs / μm2
– Outside synapse 10 AchRs / μm2
1. neurotransmitters cause similar response over
entire muscle
2. Visualized
•
With presynaptic contact receptors cluster
2
Apply Ach
A
A
B
B
• Shift from uniform response → large and
small
muscle
• Suggests receptors cluster with nerve
contact
A
A
B
B
Postsynaptic Potentials
Postsynaptic Potentials
There is a AchR prepattern
prior to innervation
• AchRs labelled with α-bungarotoxin
(component of cobra snake venom
label is radioactive or fluorescent)
Failure to innervate
diaphragm muscles
prepattern
Frog NMJ
3
Is Neurotransmitter Activation
Required for Clustering?
• The ability to induce clustering requires
the correct neuron / target pair
Spinal Cord Motor Neuron
Plus bungarotoxin no difference
Spinal Cord Motor Neuron
Dorsal Root Ganglion Sensory Neuron
No, neurotransmitter action is not required
muscle
Clustering involves both:
•
After innervation clustering involves:
1. Movement of receptors within membrane
2. Insertion of new receptors
Movement of receptors:
1. Apply Ach to small patch and measure
response
2. Apply bungarotoxin to same patch and
measure response (smaller)
3. Wait
4. Apply Ach again (recovers)
4
Movement of receptors
Insertion of new receptors
Control
Red fluorescent label
apply Bungtx
Bungarotoxin
AchR
~5 minutes
green fluorescent label
Y
Y
Y
Y
1. Measure red and green light
Y
1. Bungarotoxin is permanent inhibitor of Ach receptor
2. Since response recovers → suggests receptors can move into patch
Antibody that recognizes AchR
How do AchRs cluster
•
Agrin, a cluster inducing molecule
1. Applied agrin can induce clustering of
receptor
2. Blockade of agrin function blocks clustering
in the presence of neurons
Without neurons
•
With neurons
added
green > red indicates new receptors
– Multiple agrin isoforms from neurons and muscle
– Neural agrin specifically required for clustering
5
How does agrin induce clustering?
Background observations:
• Agrin induces phosphorylation of AchR
β subunit
• Inhibition of tyrosine kinase activity inhibits
clustering
Block agrin function
X
P
P
P
P
How does agrin signal?
Add agrin
Add agrin
P
P
• Aside…
– Receptor tyrosine kinase
• Receptor that has intrinsic signaling abilities
• agrin binds to a lot of candidate molecules
– ie. can directly phosphorylate targets
• Unlike G-protein coupled receptors that activate
kinases indirectly
– Including CAMs, ECM
• Identification of MuSK
Receptor Tyrosine Kinase
G-protein couple receptor
– Muscle Specific Kinase
– Transmembrane receptor tyrosine kinase
target
target-P
target
target-P
6
Evidence MuSK involved in agrindependent clustering
• MuSK knockout mice have no
postsynaptic differentiation
– Neither do agrin KO mice
• Inactive MuSK blocks agrin signalling
• MuSK KO muscles unresponsive to agrin
•
Does agrin bind MuSK directly?
– Agrin can activate MuSK in muscle
– but not when MuSK is expressed in nonmuscle cell
– Suggests an additional muscle derived
factor is required Agrin binds MuSK
– So far unidentified, called Masc
What links MuSK to AchR?
• Rapsyn, a AchR binding protein essential
for clustering
• Rap and AchR will co-cluster in non-neural
cells
– Means rap has ability to cluster indepently
• In rapsyn mouse KO,
– agrin can activate MuSK but AchR don’t
cluster.
– Suggests Rapsyn is required to link agrin
activation of MuSK to AchR receptor clusters
• No AchR clusters in rapsyn KO mice
7
Neural agrin secreted
Masc
clustering
MuSK
P
AchR
rapsyn
8