Lecture 24- Synaptogenesis I

Question 1

What are synapses?

Answer 1

•Specialized junctions between neurons (or between neurons and other cells e.g. muscle cells)

Question 2

What are synapses for?

Answer 2

-Information is transferred across synapse in one direction -from pre-synaptic neuron to post-synaptic cell ALSO used -from afferent neuron (input) to efferent neuron (output)

Question 3

What are the vast majority of synapses like?

Answer 3

-Vas tmajority are chemical

Question 4

What happens at the chemical synapse?

Answer 4

-neurotransmitter is released at the chemical synapse

Question 5

What are the features of chemical synaptic transmission?

Answer 5

1. Action potential reaches terminal
2. Voltage gated Ca2+ channels opens
3. Ca2+ enters the axon terminal
4. Ca2+ makes the Neurotransmitter release and diffusion
5. Neurotransmitter binds to postsynaptic receptors
6. Neurotransmitter is removed from synaptic cleft

Question 6

What is a neuromuscular junction?

Answer 6

• synapse onto skeletal muscle cell from motor neuron
• specialised synapse
• also called motor end plate
• part of motor unit (single motor neuron and all of the skeletal muscle cells it controls, motor unit is a single motor neuron and all the muscle fibres it controls, each fibre conrolled by just one neuron)
• lower motor neuron cell bodies are located in spinal cord (ventral horn) or brainstem (motor nuclei)
• lower motor neurons directly stimulate their target muscle

Question 7

What is the structure of the neuromuscular junction?

Answer 7

• neuromuscular junction consists of multiple neurotransmitter release sites
• each is a swelling on a branch of a motor axon terminal
• postsynaptic= the muscle tissue form junctional fold, highly folded= mechanism to increase area, get more Ach

Question 8

What does neuromuscular junction at high resolution?

Answer 8

• left= striated muscle
• bouton= the motor end plate= where the neuron terminates onto the muscle

Question 9

PIC4What are three characteristic elements of synapses?

Answer 9

• synaptic vesicles releasing neurotransmitter
• synaptic cleft
• postsynaptic thickening or density with neurotransmitter receptor at the NMJ on tips of junctional folds (excitatory synapses have denser density than inhibitory)

Question 10

What is the neuromuscular junction synaptic function?

Answer 10

• neurotransmitter is acetylcholine (ACh)
• postsynaptic receptor is nicotinic acetylcholine receptor (AChR)
• ACh opens nicotinic ACh receptor channels
• Na+ and K+ ions flow inward causing the muscle depolarization (the end plate potential) (less negative) which then triggers an action potential in the muscle fibre and muscle contraction
• nicotinic receptors at 10 000/per micrometer squared at motor end plate (much lower density on extra junctional membrane)= very dense! the post synaptic density

Question 11

What is used as a model for synapse development?

Answer 11

• Development of the NMJ as a model for synapse development
• NMJ widely used to investigate synapse development due to its large size, accessibility and relative simplicity
• great techniques to label it
• over time becomes more complex, more branched etc. in the beginning called plaque stage in the end called pretzel stage= looks like a pretzel (the branches of the axon= the terminals)

Question 12

What is the development of skeletal muscle cells?

Answer 12

• many small myoblasts fuse to form myotube multinucleated
• myotube develops myofibrils (composed of many thousands of repeated sarcomeres)
• Muscle cell grows to many cm long, 100μm diameter

Question 13

How are Ach receptors produced by muscles?

Answer 13

• Fusion of myoblasts into myotubes activates gene for AChR
• Nicotinic AChRs made and inserted into muscle cell membrane
• At the myotube stage, receptor density is uniform (~1000/μm2 over entire surface)
• At the mature NMJ, receptor density is 10,000/μm2 at the synapse and only 10/μm2 in non-synaptic membrane
• AChRs become highly aggregated or clustered
• at the earlier stage the receptors are distributed quite equally, then the reeceptors get clustered into the nerve terminal

Question 14

How does the ACh receptor aggregation in embryonic muscle happen?

Answer 14

• • Early: some receptor aggregation into “hot spots” or “aneural AChR clusters” before innervation; termed pre-patterning, since they don’t occur too far from where they should be as mature
• all the pre-patterned hot spots consolidate into larger hot spots, it is the presence of the axons that make sthis happen
• • Prepatterned “hot spots” help direct the site of NMJ formation (direct in-growing axon to the central region of the muscle fibre)
• • Presence of axons leads to reduction of extra-synaptic “hot spots” and increases receptor aggregation in the sub-synaptic region by stabilizing clusters

Question 15

Can ACh receptors can aggregate in regenerating muscle even after functional nerve terminals removed?

Answer 15

-yes

• Basal lamina between muscle and terminal at synapse
• If nerves are cut and muscle destroyed, basal lamina remains
• Muscle regrows and clusters AChRs at original sites of synapse (even though muscle denervated - no nerve activity)
• What aggregates AChRs?
• muscle already developed Ach receptor clustering, only lamina remains, the clustering occurs due to the basal lamina (sth on it)
• agrin does it!

Question 16

What is agrin?

Answer 16

-a large proteoglycan (glycosylated protein)

• Involved in aggregation of AChRs

• it is in the basal lamina and makes the receptors aggregate
• involved in aggregation of Ach receptors
• is it agrin from muscle or from nerve?

Question 17

What does agrin do?

Answer 17

-Agrin stabilizes AChR clusters -• Involved in aggregation of AChRs • Antibodies that bind to and block agrin inhibit AChR aggregation

Question 18

Which source of agrin is needed to stabilize AChR clusters?

Answer 18

• Use antibody to agrin that only binds(&blocks)chicken agrin
• Neuronal agrin is required to maintain ACh Rclusters Clustering of muscle cell AChRs not maintained 
• muscle from chicken, rat motor neurons and vice versa
• became apparent that if antiobody to bloc agrin from chicken then when the neuron is from chicken the clustering is not maintained

Question 19

What was the investigation into the role of neuronal agrin investigated further using agrin knockout mice?

Answer 19

• Still observed “hot spots” prior to arrival of axon therefore agrin not required for AChR pre-patterning
• In agrin KO mouse, treatment of pre-patterned muscle with ACh to stimulate AChRs led to receptor dispersal EXCEPT when neuronal-type agrin was produced (expressed) in muscle cell
• AChR dispersal was prevented Conclusion: agrin from neurons important for maintaining AChR clusters
• now only muscle, small clusters= like the prepatterned hot spots
• agrin not needed for prepatterning step,but need nerve agrin to get the aggregation
• agrin from neurons is important
• if take the system and have pre patterned cluster and then put Ach on there, then no clusters form!

Question 20

What is agrin signalling like?

Answer 20

• Agrin released from motor neuron terminals into extracellular space
• Interacts with laminin in basal lamina
• Agrin co-receptors are Lrp4 (low-density lipoprotein receptor-related protein 4) and MuSK (muscle specific kinase)
• Agrin binding activates MuSK sending an“anti- dispersal” signal so that ACh receptor clustering can be stabilized under active (and agrin-secreting) axon terminals)
• Agrin activates a receptor complex which signals to phosphorylate AChRs
• Clustering also requires the “scaffolding protein”, rapsyn
• agrin released from the nerve, integrates into the basal lamina (basal lamina is mainly laminin and agrin can bind to that), then activates a combined receptor lr4 and MuSK
• then agrin sends signal via the receptors to the Ach receptors to cluster, ultimately caus ephosphorylation of the receptors and rapsyn holds them together

Question 21

What happens in MuSK knockout mice?

Answer 21

• AChR pre-patterning and clustering is lost and motor nerve terminal branches continue to grow (rapsyn KO similar)
• Conclusion: AChR pre-patterning requires MuSK and rapsyn, but not agrin, whereas the formation of nerve-induced AChR clusters and NMJs requires all three proteins
• if knockout MuSK or rapsyn don’t even get pre-patterning, and the axon terminals grow further then they should and don’t stop where they should
• pre patterning need MuSK and rapsyn
• for clustering and maturation need agrin, MuSK and rapsyn

Question 22

What is myasthenia gravis?

Answer 22

• an auto-immune disease
• Characterised by impaired NMJ synaptic transmission and muscle weakness
• In some patients, auto-antibodies to agrin are detected while in others, the main target is MuSK
• Agrin and MuSK are auto-antigens in myasthenia gravis
• some patients attack agrin

Question 23

What competing signals regulate AChR levels at developing NMJ?

Answer 23

1. Agrin-MuSK/Lrp4: • aggregation of AChRs • increased transcription of mRNAs in sub-synaptic nuclei → much higher density of AChRs at NMJ
2. Acetylcholine-AChR: • opposing effect: inhibiting transcription and causing endocytosis (removal “aneural” AChRs from muscle cell surface)
   - When cluster-stabilizing agrin not present (e.g. in aneural regions), AChRs become dispersed (global effect – low receptor levels generally)
   - light blue:agrin and ach released, agrin helps signal to cluster ach, also activating MuSK and Lrp4, also helps the transcription of the Lrp and MuSK genes so get mor eof them and better signalling, can strengthem the postsynaptic signalling
   - the beige= if agrin not present but have acetylcholine it does the opposite thing, it prevents the transcriptionof Lrp4 and MuSK genes and has opposing effects,

Question 24

What is the local neural AChR clustering vs global aneural dispersal?

Answer 24

• Local motor neuron derived agrin – AChR clustering at NMJs
• Global electrical activation (depolarization) along whole muscle fibre – removal of AChRs from surface and dispersal

-electric activity in the absence of agrin is blocking the positive things

Question 25

What happens during the maturation of synaptic zone?

Answer 25

- Synaptic zone develops from a simple plaque into an elaborate “pretzel” shaped structure as pre-synaptic nerve fibre branches - plaque to pretzel transformation - pretzel= has mature distribution of AchR

Question 26

How can you see AChr distribution?

Answer 26

-A labelled toxin from Taiwanese cobra venom reveals AChR distribution -

Question 27

Does AChR distribution align with the terminal branches of the motor neuron axon?

Answer 27

- AChR distribution exactly aligns with the terminal branches of the motor neuron axon - must develop together - each axon has more than one terminal branch and will innervate several muscle fibres - in development each muscle fibre will be innervated by several axonal terminals, this will be pruned down to each muscle fibre being innervated by only one nerve= refinement of the system, better control over when the fibre is recruited

Question 28

How many muscle fibres innervate a mature NMJ?

Answer 28

-in mature NMJ, each muscle fibre only innervated by one axon

Question 29

How many nerve fibres are are at a developing NMJ?

Answer 29

- Multiple innervation at developing NMJs - • Initially each muscle gets inputs from two or more motor nerve terminals * Terminals from different neurons “fight” for each NMJ * Competition results in one neuron winning and other neuron withdrawing -each of the terminals fight to get hold over the acetylcholine terminals, the winner= depends on how much neurotransmitter and activity

Question 30

What is the competition at NMJs part 1?

Answer 30

* Initially,both sets of terminals are intermingled * Each axon establishes terminals of roughly similar area * AChRs under terminals of each axon similar density * Synaptic strengths similar -two axons, branched, and active, both able to depolarize the muscle fibre = the synpatic strengths is similar

Question 31

What is competition at NMJs part 2?

Answer 31

* Shortly after birth, terminals begin to segregate into different junctional regions * Still have equal synaptic strengths -become less intermingled,roughly half half on the territory

Question 32

What is the competition at NMJs part 3?

Answer 32

* One terminal starts to invade territory of neighbouring terminal * May go back and forth for some time * One terminal starts to lose area and synaptic strength -not clear if the loser is pushed out or stochastic process

Question 33

What is the competition at NMJs part 4?

Answer 33

- Normally, AChR expression remains constant under both terminals and the “winner” takes over the AChRs of the loser - assymetry of synaptic strengths gets greater - the loser retracts from the NMJ, has retraction bulb= doesn't emean the motor neurons is dead, it will have otehr successful branche son other fibres

Question 34

What is the competition at NMJs part 5?

Answer 34

• Finally the“loser”withdraws • Usually the loser does not die but has won the competition on some other muscle cell

Question 35

What are the observations of competition and hypothetical mechanism at the NMJs?

Answer 35

* At level of individual NMJ (not whole motor units) * Axon with largest coverage at start doesn’t always win * Axon activity matters, but it is timing not total amount Proposed mechanism: * Different terminals at NMJ fire asynchronously * Each firing of a terminal produces a punishment signal that weakens AChRs that are not activated and a protective signal that supports AChRs that are active - lot to do with the strength and firing of the axons, the competition is at level of each NMJ, each NMJ is initially innervated by more than one - sth to do qith activity and timing of the activity, the axons are firing but won't be firing at the same time, by firing first it will have a negative effect on the other one= reward and punishment

Question 36

How are more active inputs made inthe NMJ?

Answer 36

* Relative timing of activity critical * Protection lasts for up to 50 msec * Identity of signals? – Maintenance of AChR clusters requires neurotrophin receptor TrkB (and NT4 or BDNF ligands) so TrkB signalling under active terminals could be “reward” - reward signals strengthen more active inputs - likely to be neurotrophins acting via neurotrophic receptos that act here, the active terminal by firing first= causes the temrinals to strengten via neurotrophin signalling

Question 37

Summary?

Answer 37

* From myotube stage, AChR present in muscle membrane * Pre-patterning (small AChR clusters) requires MuSK and rapsyn but not neuronal agrin * Neuron-derived agrin (through co-receptors MuSK and Lrp4) stabilizes the accumulation of AChR at synaptic sites * In the early post-natal period, poly-innervation of muscle cells is lost through an activity-dependent competitive process * The mature innervation pattern (only one motor neuron innervating each myofibre) is important for efficient and precisely controlled force generation