Study Guide- Synaptogenesis

 What are the specializations that occur at synapses in presynaptic and postsynaptic cells, especially at
the Neuro-Muscular Junction (NMJ)?
In the presynaptic cell: You have nerve terminals containing active zones; these are regions on
the presynaptic membrane with high concentrations of vesicle release.

In the neuro-muscular junction in between the presynaptic and post synaptic cell (synaptic
cleft): You have a basal lamina (this is a sheet of extracellular matrix containing large proteins that
are woven together). The basal lamina allows secreted proteins to accumulate in the synaptic cleft. On
example is Acetyl-choline esterase (AChE) which degrades Acetylcholine (ACh) that’s been released by
the presynaptic cell and contributes to neurotransmitter recycling.

In the post-synaptic cell: You have junctional folds. These are folds in the post-synaptic membrane
that allows ACh receptor to be enriched (more folds in the membrane means more surface area for ACh
receptor). This is kind of like the folds in the human brain, which allows the outer layer of neurons to
have a high surface area relative to brain volume.
Refresher on ACh-AChR signaling in the synaptic cleft (this example below is between two neurons;
but we are going to talk about neuron-muscle synapses)

 What is the experimental evidence that the cues that induce the clustering of AChRs at the NMJ
are present in the basal lamina? In other words, how does the basal lamina ghost experiment
work? How was the ghost created, what happened next, and how did scientists identify the site of
the old synapse in the basal lamina ghost?
o The experiment that determined the cues responsible for the clustering of AChRs at the NMJ are
present in the basal lamina is called the basal lamina ghost experiment. But first we have to
talk about a series of experiments that set the principles to allow the basal lamina ghost
experiment to work:
o If you allow a NMJ to form between a presynaptic neuron and a postsynaptic muscle cell, you
get deposition of basal lamina followed by clustering of active zones in the presynaptic
neuron and AChRs and folds in the postsynaptic muscle.
o So the first principle is the fact that if you allow a NMJ to form, and then cut the presynaptic
neuronal axon, this axon can regenerate and will “know” where the original NMJ was located
and form another NMJ where the original one was.
o The second principle is the fact that if you allow a NMJ to form, and then kill the postsynaptic
muscle fiber, a new regenerating muscle fiber will also “know” where the original NMJ was
located and concentrate AChRs to the site of the original NMJ to form a new one at the same
location.
o The above two experiments means that the cues responsible for forming a NMJ by clustering of
AChRs are not coming from the presynaptic neuron or the postsynaptic muscle, but likely from
 the basal lamina in between the two. This is where the third experiment (the basal lamina ghost
experiment) comes into play:
 You allow a NMJ to form, and then cut the presynaptic neuronal axon AND kill the
postsynaptic muscle fiber, so all that is left is the basal lamina in between. You then
keep cutting the presynaptic neuronal axon so that it can never grow back, but allow the
muscle fiber to regenerate. The muscle fiber is able to concentrate AChRs where the
original NMJ was, independent of the presynaptic neuron. This means that the basal
lamina itself contains some cue that allows the cells to “know” where the original NMJ
was.

o Some key points to also know: In these experiments, the scientists knew where the original
NMJs were located by labeling Acetyl-choline Esterase (AChE) on the basal lamina. NMJs
should have an enrichment of AChE in the basal lamina. However, AChE is NOT the cue that
drives NMJ formation, it is simply used to identify NMJs. The professor also talked about clever
ways to identify old versus newly formed NMJs by labeling AChRs. Alpha-bungarotoxin (a-
BTX) from snakes will irreversibly bind to AChRs (this allows the snake to paralyze its prey
by preventing ACh binding to its receptor for motor function). You can attach a fluorophore
onto a-BTX and use this to label all the AChRs on a muscle fiber, then wash away the excess.
You can then wait a period of time to allow a NMJ to form, and label both old and new AChRs
with a fluorescent antibody. In this scenario, all the original AChRs will be labeled with both the
a-BTX and antibody, while only the newly formed AChRs will be labeled with only the
antibody.

What in vitro bioassay was used to isolate Agrin? Why was the Torpedo ray electric organ used as a
source for Agrin?
Similar to the bioassay done to identify neurotrophic factors, you first need a ton of starting
material (in this case a ton of material that contains your cue that induces clustering of AChRs at the
muscle fiber). For this, the scientists used the electric organ from Torpedo rays: these electric organs
have a super high number of cholinergic synapses (and are thus enriched for NMJs which should have a
lot of the mystery cue that induces AChR clustering). Torpedo ray electric organ extracts when added to
a culture of myotubes was sufficient to induce AChR clustering. You can then run your bioassay on this
Torpedo ray electric organ extract (isolate specific proteins and through process of elimination identify
Agrin: a signaling protein molecule made by the nerve and muscle fiber that once secreted can
accumulate in the basal lamina).

 What experiment shows that is it neuronal Agrin, rather than muscle Agrin, that is necessary for
clustering AChRs at the NMJ?
First it’s important to note that Agrin is sufficient to induce clustering of AChRs at the NMJ: if
you treat a myotube culture with BTX and add Agrin, you get NMJ formation and AChR clustering
through the formation of new AChR.
Both the neuron and muscle will produce different forms of Agrin, so the problem is how do we
determine that the Agrin coming specifically from the neuron is necessary for AChR clustering?
It turns out that a neuron from a chicken or rat can form a NMJ with a muscle fiber from the other
species. So what you can do is treat the cells with an antibody that blocks Agrin specifically from the
chicken cell to determine if Agrin coming from the presynaptic nerve, or the postsynaptic muscle, or
both is important for AChR clustering through several different combinations:
1) Have a presynaptic neuron from a chicken paired with a postsynaptic muscle fiber from a rat. With
no chicken Agrin antibodies, you get AChR clustering at the NMJ. But if you do add antibodies
against the Agrin from the chicken cell, you block AChR clustering. This means that Agrin coming
specifically from the presynaptic neuron is important for AChR clustering at the NMJ.
2) Have a presynaptic neuron from a rat paired with a postsynaptic muscle fiber from a chicken. With
no chicken Agrin antibodies, you get AChR clustering at the NMJ. With the presence of antibodies
 against the Agrin from the chicken cell, you still get AChR clustering at the NMJ. This means that
Agrin coming from the motor neuron is not needed for AChR clustering at the NMJ.

 What is the receptor for Agrin?

The receptor for Agrin is Muscle-specific receptor kinase (Musk I) which forms a complex with
LRP4. This signals to a molecule called Rapsyn to allow AChR clustering.
 What does ARIA (Neuregulin) do? Is it necessary for this? Sufficient?
*Important to know: Agrin is important for the clustering of pre-existing AChRs. But there is
also the formation of new AChRs that get shuttled to the NMJ. This molecule that is important for the
formation of new AChRs is known as ACh R Inducing Activity (ARIA), which is just the name of a
molecule that was already discovered: Neuregulin. ARIA or Neuregulin is sufficient but not necessary
to induce AChR synthesis. (Sufficient: it is capable of inducing AChR synthesis, but not necessary: you
still get AChR synthesis after removal of Neuregulin). This is because you may have redundancy
(multiple molecules doing the same thing).

 What happens to AChRs when you block motoneuron activity to a muscle fiber? What causes this?
If you block motorneuron activity to a muscle fiber, you get the synthesis of a lot of new AChRs
that are termed “extrasynaptic AChRs”. Extrasynaptic just meaning that these new AChRs are formed
not only in the synapse but also outside the synapse or extrasynaptic. It turns out that ACh itself is
needed for controlling the extrasynaptic production of AChRs: ACh signals to AChR which allows Ca+
+ to enter the cell which activates CamKII which activates myogenin to reduce extrasynaptic AChR.
So, inhibiting the presynaptic motorneuron with TTX reduces ACh release and causes a bunch of
extrasynaptic AChR formation at the muscle fiber. Interestingly, you can reduce the extrasynaptic
formation of AChR after inhibiting the presynaptic motorneuron by stimulating the muscle fiber: this
increases Ca++ influx and through the pathway described above reduces extrasynaptic AChR formation.

 What happens to AChRs if you remove both Agrin and motoneuron activity? What does this mean?
If you remove Agrin alone, you lose the ability of AChR clustering. If you remove motorneuron
activity alone, you get production of extrasynaptic AChR. If you remove both, get clustering of AChR
(less than when you do have Agrin, but you still get less). This means that there must be some other cue
in addition to Agrin that can contribute to AChR clustering.

**There was also additional information at the end of this lecture that isn’t covered in this study guide: Stuff
about different signals at other types of synapses like Cell Adhesion Molecules (CAMs) including Neurexin and
Neuroligan. Wouldn’t hurt to know these as well.