Lecture 8 - Synaptogenesis II

Question 1

What are the three general features of the NMJ which have provided clues about molecular mechanisms of synapse formation?

Answer 1

1. nerve and muscle organize each others differentiation
2. MNs and muscle cells can synthesize and arrange most internal synaptic components without each others help
3. new synaptic components are added in several distinct steps

Question 2

What is established via synaptic dialogue and what is not established?

Answer 2

the size and the location of the presynaptic and postsynaptic terminal relative to each other needs to be determined but the creation of a post and pre synaptic terminal does not depend on the dialogue

Question 3

What are the five distinct stage of synaptic development?

Answer 3

1. growth cone approaches a newly fused myotube
2. an unspecialized but functional contact is established

3.nerve terminal accumulates synaptic vesicles and a basal lamina forms in the synaptic cleft (release of synapses is random)

4. as the muscle matures multiples axons converge on a site
5. all axons but one are eliminate and the surviving terminal matures (the other retracts and saves its material and finds another target)

Question 4

What do you see at the mature NMJ?

Answer 4

-pre and post synaptic membrane are separated by a cleft that contains basal lamina and ecm proteins
-vesicles are clustered at release sites NT receptors are clusters in the postsynaptic membrane and nerve terminals are coated by schwann cells

Question 5

What does the schwann cell do?

Answer 5

caps the nerve terminal and helps to encapsulkate nutrients and helps to recycle the ACh released

Question 6

What is the basal lamina made of?

Answer 6

proteosulfates, proteoglycans, and ECM proteins

Question 7

What does the postsynaptic terminal have to increase sa?

Answer 7

invaginations so ach recpetors can be found in the tips of the folds and you can get more ach receptors

Question 8

What do nerve and muscle cells express on their own and what requires synaptic dialogue?

Answer 8

-the components of the synapse they express intrinsically on their own
-in order to spatially organize these components need synaptic dialogue

Question 9

What is synthesized without neurons with some spontaneous clustering?

Answer 9

acetylcholine receptors

Question 10

Do motor neuron terminal reach pre achR clusters?

Answer 10

no instead innervate the postsynaptic muscle randomly and the achrs cluster under the presynaptic terminal and the clusters that were previously there not under the terminal disperse

Question 11

What clusters at the sites of neurite contact with muscle?

Answer 11

presynaptic vesicles

Question 12

Where do presynaptic vesicles cluster?

Answer 12

at site of neurite contact with muscle

Question 13

What plays a critical role in defining and mediating synaptic dialogue at the NMJ?

Answer 13

the basal lamina

Question 14

If you have an NMJ at the middle of maturity, the MN has bifurcated axon terminal innervating the muscle myotube which is thre result of fusion of tends of different cells which fuse together into a large nad and there are many nuceli which cayses it to converge onto a synaprtic zone - between the MN and the basal lamina are proteins that were expressed

Question 15

If you dennervate the NMj during development and cut the moor axon what happens?

Answer 15

the axons will regenerate and all the new synapses will form at the original synaptic site

Question 16

If you kill the MN and the muscle fibers by poking it and everything leaks out and dies away what happens?

Answer 16

a string preference for innervation at the original syanptic sites persists evn after the muscle fibers have been removed and this leaves behind the basal lamina ghosts

Question 17

Where do regenerated axons develop synapses?

Answer 17

on contact with the orignal sites on the basal lamina- what synaptic proteins or factors are there in the basal lamina to cause this to happen

Question 18

When the muscle remakes itself fiber wise where are its postsynaptic compartments?

Answer 18

on the same region of the basal lamina without the axon there

Question 19

After deneravtion of the skeletal muscle fiber and elmination of mature muscle fibers where are the expression of achrs on the muscle surface concentrated?

Answer 19

the synaptic areas of the basal lamina even when reinnervation is prevented

Question 20

What do factors in the basal lamina do?

Answer 20

synaptogenic and define pre and post synaptic apposition

Question 21

What is localized at synaptic and extrasynaptic sites of the basal lamina?

Answer 21

different laminin isoforms

Question 22

What basal laminin isoform i localized at the synaptic clefts?

Answer 22

laminin beta 2

Question 23

What type of protein is laminin beta 2?

Answer 23

a heterotrimeric protein in which three genes are expressed the alpha beta and gamma gene and they are heterolinked gene wise and a special isoform is found at the synapses of the NMH which is laminin beta 2

Question 24

What is impaired in mice lacking laminin beta 2 meaning a KO?

Answer 24

the maturation of the NMJ is impaired in mice lacking beta2 laminins - they died shrotly after birth because they could not breath - and actually it was seen that the shwann cell was getting in the way of the synaptic clewft and invaded it preventing pre and post synaptic dialogue so the spatiliaization of the synapse was off so laminin beta 2 functions as a physical barrier preventing the schwann cell from invading the synpase and organizes pre and post synaptic taregts

Question 25

How does AChRs secrete clusters at the postsynaptic terminal?

Answer 25

the neuron secretes postsynaptic organizing factors

Question 26

What induces the aggregation of AChRs at synaptic sites?

Answer 26

agrin

Question 27

What is agrin?

Answer 27

agrin is a large ecm proteoglycan secreted by the MN nerve derived whcih binds to Lrp4 which is associated with MuSK which results in the recruition of rapsyn to cause achr clustering

Question 28

What animal model was used to study the clustering of agrin via Lrp4 and MuSK and rapsyn?

Answer 28

the torpedo ray NMJ

Question 29

What does agrin induce?

Answer 29

the aggregation of AChRs at synaptic sites

Question 30

What happens if you take muscle fibers in a pertir sich and throw on diferent protein components of the torpedo ray NMJ milkshake and added agrin to one dish and ommitted it from another dish what would you see?

Answer 30

-in the no agrin added on only a few AchR clusters form in culture under conrol conditions -addition of agrin induces ach receptor clustering

Question 31

What was done in the genetic analysis of agrin and role of activity in AChR clustering?

Answer 31

an agrin KO mutant mouse

Question 32

What was seen in the WT agrin mouse?

Answer 32

ACHRs have formed under each nerve terminal by birth and there are clusters

Question 33

What was seen in the agrin KO mutant mouse?

Answer 33

-in agrin mutants most achrs have dispersed and the presynaptic terminal is very mispread and wide

Question 34

What was seen in a MuSK KO mouse?

Answer 34

there is no AChR clustering in the MuSK KO

Question 35

Is AChR clustering mediated entirely through molecular interactions or is synaptic activity involved?

Answer 35

-seems like you do not need activity for achrs clusters to happen

Question 36

How do you use genetics to block activity at the NMJ?

Answer 36

create a ChAT mutant which is the genetic deletion of choline acetyl transferase and synaptic vesicles do not contain ACh and therefore they do not release neurotransmitter - so the synaptic vesicles fuse and nothing happens though NT release wise

Question 37

What occured in an agrin mutant versus and agrin and ChAT mutant?

Answer 37

-in agrin mutant most AChRs have dispersed but there is still activity since ACh is still released -in agrin and ChAT mutant ACHRs clusters remain which means that agrin works by counteracting receptor dispersion by ACh and stabilizing postsynaptic receptors - there are other non dispersal clustering factors involved in clustering but agrin mediates cliustering in the face of the decluctering effects of ACh

Question 38

What is the role of activity in the organization of synaptic clusters?

Answer 38

does not cause clustering of AChRs but instead what it does is it caues the refinement of the clusters and gets rid of excess clusters and refines the synapse

Question 39

What does a ChAT mutant look like?

Answer 39

will still see clusters but there will be some extra since no activity

Question 40

What type of factor is agrin best understood as?

Answer 40

a factor that aligns the pre and post synaptic specializations

Question 41

Since ACh is the major dispersal factor itself what does agrin do?

Answer 41

agrin protects from the declustering affects of ACh and other factors are involved in clustering in the face of no activity

Question 42

What does the neuron organize?

Answer 42

postsynaptic transcriptional organziation

Question 43

What controls AchR clustering?

Answer 43

local protein trafficking and transcriptional regulation control

Question 44

What is a myotube?

Answer 44

the fusion of a dozen muscles cells whcih fuse into a large sac and have many nuclei and are homogenous and make ACHRs

Question 45

Where on the myotube are ACHRs made the most?

Answer 45

under the presynpatic terminal

Question 46

In the beginning of development prior to receiving presynaptic input where are ACHR clusters on the myotube?

Answer 46

they are distributed diffusely in random cluster on the surface of the embryonic myotube

Question 47

What regulates the transcription of AChR genes?

Answer 47

the motor neuron

Question 48

After a muscle is innervated by a MN, what happens to the receptors whcih are extrasynaptic and the recepotrs at the synapse?

Answer 48

the extrasynaptic receptor density decreases and the synapse receptor density increases - this reflect the aggregation of pre exisiting receptors an enahnced expression of achr genes in nuceli that lie directly beneath the tnerve terminal plus the repression of transcription in extrasynaptic nuclei

Question 49

Explain a mechanism for how something gets specialized in the nuclei near the NMJ and the other nuclei shut down transcription of proteins that are needed for synapses - how do the nuceli at the NMJ know to enhance expression of ACHr gene and how do the extrasynaptic nuclei know how to shut down transcription of the achrs genes?

Answer 49

-when a ap fires there is a release of calcium in the sub synaptic reticulum and this can induce calcium sneisng on the nuceli under the synapose and cause trasncirption of CREB and calcium depndent proteins so the nuclei get enhanced ecxpression of achr genes - while the extrasynaptic nuceli do not sesne this calcium and their transcription is shut down because they experience depolarization without the presence of calcium

Question 50

What does denervation do to transcription of AChrs in the myotube?

Answer 50

following denervation achr gene expression is upregulated in extrasynaptic nuclei and this is not the high level attained by synaptic nuclei - this means the extrasynaptic nuceli express the genes needed for achrs more but not to the same level as the synaptic nuceli cause theyhave been exposed to the upregulation caused by calcium

Question 51

If a loss of muscle actviity mimics denervation how would you induce muscle paralysis?

Answer 51

if you put in an electrode in a specific place electrica; activity in the muscle represses the achr gene expression in nonsynaptic nuclei which causes less density of achrs in this region

Question 52

What are the two things that activity does?

Answer 52

establishes synaptic areas and diminishes extrasynaptic regions

Question 53

What are the three steps in which the postsynaptic membrane matures?

Answer 53

1. during early embryogenesis the achrs exist as loos aggregates - the muscle gets indented a bit and the presynaptic bouton can kinda lie there 2. later these aggregates condense into a plaque like structure -muscle indentations get deepr and the multiple MNs compete 3. after brith the dense cluster opens up as the nerve develops multiple terminals - these axon branches expand as the muscle grows and the p[laque indents to form a gutter and these nvaginations allow for greater sa so more recetors are in the crests of the folds cause more can now fit there and respond to NT causing whole muscle depolarization -winner MN stays

Question 54

Early in development of the NMJ each muscle fibver is innervated by how many motor axons?

Answer 54

several get polyneuronal innervation of muscle

Question 55

After birth how many MNs innervate a single muscle fiber?

Answer 55

after birth all motor axons but ine withdraw from each fiber and the survuing axon becomes more elaborate - axons survives cause gets more trophic supprot from achrs cause it has more achrs beside it

Question 56

Is there an overall loss of axons in synapse elimination?

Answer 56

no because synapse elimination occurs wihout any overall loss of axons and axons that lose at some muscle fibers win at others

Question 57

What is the purpose of this transient stage of polyinnervation?

Answer 57

ensures each muscle is ultimately innervated allow all axons to capture and appropirate set of taregt cells - synapse elimnination shows how actvity can change the strenght of synaptic connections

Question 58

What completes the hardwiring of the nervous system?

Answer 58

synapse formation

Question 59

What must be exquisetly specific to form a functional network?

Answer 59

synaptic connections

Question 60

How is specificity in synaptic connections achieved?

Answer 60

starts with molecular recongiiton and is enhanced by activity

Question 61

How are pre and post synaptic areas high specialized and organized?

Answer 61

through anterograde and retrograde synaptic dialogue - more ahcrs then the MN persists and MN releases agrin and calcium in sub synaptic reticulum to cause achr clustering or up and down regulate its expression respectuvely

Question 62

In what ways must synaptic structure be stable and in what ways must it be plastic?

Answer 62

it must be stable to last a lifetime - molecular recogniton plastic enough to change with experinece - refinements through activity