MCDB 423 Lecture 31: Lecture 31

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Molecular, Cellular and Developmental Biology
MCDB 423
John Kuwada

Lecture 31 Slide 1 So is the pre synaptic neuron needed for the development of the post synaptic specialization? If we remove the motor neuron, will post-synaptic specialization still develop in the skeletal muscle? Alpha-BGT blocks all NMJ function so you’ll stop breathing at very low concentrations of the toxin and die (irreversible, highly specific reaction to AChR and is used as a dye to label the AChR) -You see that the clusters of the AChR are right under the pre-synaptic neuron which makes sense In a mouse which the motor neurons have never developed due to a mutation called HB9 (critical transcription factor needed for the differentiation of motor neurons) -These causes no pre-synaptic terminals -But there are AChR -So their development doesn’t require the pre-synaptic patter -However the pattern of the AChR is no longer restricted to a tight little zone as in the WT So a signal from the pre-synaptic neuron regulates some aspect of muscle development Slide 2 Muscles are multi nucleated -Fibers form by a number of myoblast (which are mono-nucleated) will fuse together to form a larger muscle cell In the synaptic folds, you have the addition of AChE which are made and secreted by the muscle fiber -Put specifically at the NMJ -So this can be a marker for the NMJ -They hydrolyze ACh Within the basal lamina, not only do you find a muscle fiber but you find another myoblast Slide 3 Regeneration can happen in some places of the CNS Jack studied the frog muscle right along the chest -These are highly accessible -It’s a monolayer of muscle fiber Innervated by motor neurons that come from the spinal cord First thing he did: took a pair of forceps and crushed the motor neuron on one side -Then what he found was that crushing the nerve would severe the motor axons -The distal motor axon would project growth cone to grow back to the muscle fiber -Grew back to the same sites So regeneration occurs and it occurs in a highly specific fashion Slide 4 Since regeneration did occur and it occurs at the site of the original NMJ, he wanted to know why the motor cones grow back to the same sites So he did a second experiment -When the motor growth cone in a regeneration system grow back to the synapse, he found that not only did the motor neurons develop presynaptic specialization but the muscle had post synaptic specialization at the same time -He wondered if the motor nerve was necessary to establish the posy-synaptic specialization This is the experiment he did -He took slabs of muscle which killed the muscle fiber -Then he cut out a piece of the nerve and sutured it to some other place to prevent motor growth cones from growing back -So he eliminated the motor neurons in this case What happens when the muscle fiber dies, the basal lamina remains and has the folds in it -If you assay for ACh esterase, you would find esterase located in those folds However, the key to this experiment is that the myoblast are still there -They aren’t damaged -The death of the muscle fiber initiates the myoblast to undergo proliferation -The progeny will fuse to form new muscle fibers in addition to keeping one of the myoblast around By looking at the distribution of ACh, the receptors are located where they are normally located -So the muscle put the Ach receptors at the original sites So why? -He reasoned that it must be the basal lamina -So the synaptic basal lamina appears to be sufficient to dictate where the new Ach receptors will be slide 5 Then he asked if the muscle fibers are required for the development of the pre-synaptic specialization of motor neurons Did a different experiment -Exercised slabs of muscle fibers to kill the muscle fiber -Also radiated the muscle fiber with X-rays which would kill the myoblast -Then he crushed the nerve (he knew if he did this, the growth cone would be produced to grow the nerve back to its original location) In this situation, you still have the original basal lamina with its normal fold and Ach esterase What he found -Regenerated motor axons would grow to that site, develop clusters of synaptic vesicles So the muscle isn’t necessary to direct the growth cones to their original site and to induce them to develop pre-synaptic specialization In fact, the synaptic basal lamina seems to be sufficient This demonstrated that there must be something in the synaptic basal lamina that can confer all this information -So what was this stuff? Slide 6 He thought: The surface area of the basal lamina was way less than 1% of the total surface area of a typical basal lamina Electric rays are fish that generate a voltage shock to their prey/predators by the electronic organ -This organ is a modified muscle Unlike a modified muscle where the synaptic region is way less than 1%, the electric organ’s synaptic area is almost 50% of the surface area -This is because the cells look like a dime and the electric organ is a stack of dime -The motor axons grow in between and cover the entire top surface of the electric organ He reasoned -These are modified muscle -So maybe the basal lamina has the molecule I’m looking for slide 7 He needed to decide what to assay (what specialization?) -Assay for localization of Ach receptors cause he knew that was dependent on the synaptic basal lamina Under normal conditions, the Ach receptors are highly enriched at the synapse -However you can take muscle fibers and grow them in culture -When you do this, early on, there are Ach receptors on the surface of that muscle fiber but they aren’t aggregated So he used this as an assay system -What he hypothesized is if he added the magic stuff onto the muscle fiber, the Ach receptors would aggr
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