Trophic factors short synopsis: idea was that there are factors which are produced by post cells which do something to promote the health of a pre-cell. We talked about this before when we talked about the possibility of signals going from post to pre cells in the case of LTP. There has to be some sort of cue that can control whether the pre cell forms a synapse, whether it has an axon branch that goes to a synapse. Many nerve cells, after they make synapses, become sensitive to the post-cell. If you take away target tissues, both the sensory and motor neurons that would normally innervate that limb go through massive cell death. The only way to go around this is to make them temporarily insensitive to the trophic factor or to find something to take the place of that limb. Nerve growth factor - target derived growth factors. Tissues produce these things to allow survival of pre-cell neurons. Most target derived trophic factors are things where you've grown to the target tissue and you are trying to make synapses - then make it so that the pre-cells are sensitive to how much trophic factor they receive from the post cell. Creates competition between the neurons trying to synapse onto the post-cell. How does pre-cell know how and where to make a synapse? Synaptic specializations are not present all over the surface of the cell. They are usually only concentrated in the region where you get synapses from the pre-cell. So how do you get synaptogenesis: the formation or preservation of synapses. People study a synapse of the nerve cells onto muscles (vertebrates). This is called the neuromuscular junction (NMJ). The nice thing about this is that they are big. They are really easy to study, happen at the surface of the muscle, easy to visualize. You can also take an isolated population of motor neurons and put them next to cells that are becoming muscle cells and see how they interact with each other. Neurotransmitter at the NMJ is Acetocholine (Ach). Muscle fibers are wrapped by a single cell membrane, but have multiple nuclei in it. This are called syncytial cell because it is formed by several cells merging together. Individual myoblasts fuse to make the muscle fiber. Junctional folds try to increase the surface area because the post cell will pack a whole bunch of Ach receptors into the space that is right across from the pre-cell nerve terminal. In between the pre- and post- cells, there is a sheet of extracellular matrix (ECM) that runs all the way around the muscle cell. This is called basal lamina in the NMJ - particular type of ECM. So basically there is a lot of special stuff here at the NMJ. So how did it all get there in the first place? You can start this off in cell culture and grow some myotubes (immature nerve fibers) in petri dish with some medium to keep it happy. The easiest thing to look at is the Ach receptors. It was very easy to label these on the myoblast cell surface. To start with, they were all over the place. To label the Ach receptors, you use a toxin called alpha bungarotoxin labelled with horse radish peroxidase. You can also do this with a fluorescent tag. This is how you see that the Ach receptors are scattered all over. But as the cell starts to mature, you start to see little clusters of Ach receptors instead of scattered all over the place. What happens is that when you finally have a nerve cell come in and make contact, you lose all the other clusters of Ach and you only get Ach receptors underneath the synapse site. Why do you get them there? It turns out there are two different reasons: Ach receptors are migrating through the cell surface to the synaptic site. You can see this in a time-lapse movie, but they actually figured this out by knowing the bungarotoxin binds irreversibly to the Ach receptors. So what you can do is bathe the muscle in bungarotoxin and then wash that with solution that has no label in it. So bungarotoxin is going to be bound to the receptors that were already there before, but it won't be bound to any new ones that might develop later on. It turns out the ones you labelled way back when are the same ones that are concentrating underneath the synapse. Insertion of new Ach receptors at the synapse. What happens if you don't have a nerve coming along? Some of the clusters will maintain themselves, but you don't get this unified decision to all congregate in the same place. You usually have too many clusters. The idea is that the nerves must be producing some kind of signal that when it comes in tells the Ach receptors where to go. So what is this signal? Lab that started looking at this by looking at a situation of regeneration of the NMJ in a living organism. Here you have a NMJ, and the experimenters wanted to find the cue, so they cut the axon. If you cut the axon, the synaptic end degenerates because it isn't getting the things it needs anymore. If you don't crush too hard, though, the cell body and the axon that's connected to the cell body stay just fine. So if you wait long enough, these will regenerate. Axon will grow out back to the same site that the synaptic terminal used to be. This means that there is something at the synaptic site that is relatively long-lasting that's either being supplied by the muscle cell or it is being preserved there. What happens when you kill off the muscle? Irradiate the muscle to kill it, and in some of your muscles there are muscle stem cells that wait around to see if any muscles are missing, and when they find that one is missing, they form a new muscle fiber and you can look to see where it starts to make junctional folds and where it concentrates its Ach receptors. You find that it regenerates those things right underneath the synapse. So this makes it look like the signal is coming from the axon. So what happens when you cut the axon and you irradiate the muscle at the same time? The basal lamina is still left. You have preserved a basal lamina "ghost". Then what you can do is keep killing off the axon, even after it starts regenerating, and let the muscle fiber regenerate inside the basal lamina ghost. See if it forms a junctional specialization and where it is. You can see where the old junctional specialization was because you can look for molecules in the basal lamina that are specialized for synaptic sites. One of these molecules is an enzyme called Ach esterase, which breaks down Ach. It is there to make sure there is no long-lasting Ach after one action potential. And when the muscle cell regrows, the junctional specialization appeared at the same site AGAIN! So this means that there must be a cue left in the basal lamina "ghost" that says, "Receptors cluster here." It turns out this is not Ach Esterase, even though this seems logical. Must be a molecule present in the basal lamina that is capable of clustering Ach receptors. What molecule clusters Ach receptors? Researcher first said that this was too difficult to do in vivo. So try to develop a bio assay and dump a bunch of molecules on it and see if there is a molecule in there that will cause the clustering of Ach receptors. Take myotube and took various types of extracts from living organisms and dumped it on the myotube. Have to have the extract cause some kind of biological effect. Wanted it to cause clusters earlier than normal on this myotube. He also needed to extract from something that looked like the NMJ. So you go to a place that has the densest concentration of cholinergic synapses - Torpedo, which has an electric organ that produces a large voltage to shock you. Torpedo has an organ that is a huge collection of cholinergic synapses that are all stacked up in series. If you stack up a bunch of synapses and you put them in the right orientation, you can get a pretty whopping voltage across the electric organ as long as you fire all the cholinergic synapses at the same time. So the researchers guessed that these cholinergic synapses much share enough similarities that they might share some of the same stuff for the concentrating of the Ach receptors. So you take this electric organ and grind it up and dump it on your bio assay, and sure enough, you get clumping of Ach receptors. So then you start breaking the molecules from this ground up organ into different pools of things and you dump them back on the bio assay one by one and figure out which pure molecule is the one that is actually causing the clumping of the Ach receptors. Finally got a single molecule out that was an ECM component: this is called Agrin. This is found in the basal lamina and if you look back at the NMJ, it is concentrated in the basal lamina right at the synaptic site. This stuff is sufficient in vitro and that it is in the right place. So now we have to test necessity. If you go into the normal situation and you take agrin away, what happens to the system? So go back to your in vitro situation and have the myotube sitting there with a nerve next to it. Developed antibodies that bound to agrin (basically making the organism allergic to agrin). Counting on the fact that these antibodies will bind to agrin and prevent it from binding to its receptors. So if you take these antibodies and add them to the culture with the myotube and the nerve sitting next to it, it blocks Ach receptor clustering. It is blocking the type of clustering that is due to movement of old Ach receptors to the synaptic site. Where is agrin actually being produced? It is made not only by the axon but also by the muscle. Problem in the experiments: which agrin is it that is the important one - the stuff coming from the axon or the stuff coming from the muscle? They developed antibodies that were specific to agrin from specific species. Put a chicken motor neuron next to a rat myotube. Now you could throw in an antibody to the rat agrin (being made by the muscle) and see if that screws up the formation of these clusters. Turns out that doesn't make any difference. If you put in the antibody to the chicken agrin (which is coming from the motor neuron), then you do not get the clustering. So the agrin coming from the axons is the necessary stuff. What happens if you do this in vivo? Developed various ways of getting rid of molecules in vivo. Knockout mouse - very specific kind of genetic trick. You can make DNA that looks like DNA from a normal organism, but it has a defect in it. Then you put this into a precursor cell, and it binds to and hybridizes with normal DNA and every once in a while the DNA repair machinery gets really confused and does homologous recombination - this lets you mutate the endogenous gene that is in the normal cell. Since the cell is a stem cell, you can stick it in a normal embryo and the cell will make a mouse that has one of its genes mutated or "knocked out". On purpose, just using DNA sequence, you can go in and mutate any gene you want in a mouse. Mice are really the only place you can do this. You can make a mouse that is a knockout for agrin. Does its NMJ go away? Sort of. It gets worse, but it doesn't completely go away. You still get a concentration of Ach receptors and they are still in approximately the right place, but it doesn't have anything to do with where the nerve terminal is. What is lacking is the coordination between where the pre-cell is and where the post-cell puts it Ach receptors. This can also happen in some places in the CNS. Agrin turns out to be one of many cues. This one is responsible for concentrating Ach receptors, but it is not the one that is responsible for stimulating the synthesis of new Ach receptors that are getting put into the new synaptic site. The one that does this is called Ach receptor Inducing Activity (ARIA) - also called Neureguilin. They called it ARIA because then it allows you to write articles that are entitled "ARIA sings a sweet song at the synapse". Oh ha ha. ARIA is released by the axon and there are receptors for it in the NMJ that leads to a signaling chain that increases the synthesis of Ach receptors. Does this locally in the NMJ, so most of those Ach receptors get inserted into the membrane right next to where the synapse is. What about signals coming back the other way? You can do the basal lamina growth experiment the other way, by making the axon grow back inside the basal lamina and stop the muscle cell from growing.