MCDB 423 Lecture 34: Lecture 34
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Department
Molecular, Cellular and Developmental Biology
Course
MCDB 423
Professor
John Kuwada
Semester
Winter

Description
Lecture 34 Slide 1 Once synapses are formed, there has to be further maturation Within our nervous system, there’s overgrowth of synapses -More synapses made during development than required -Excess synapses are removed by synapse elimination We’ll start with the NMJ -Early in development, muscle fibers are innervated by more than 1 motor neuron -Later on, it becomes a 1:1 ratio Motor neuron will innervate multiple muscle fibers -Combination of a single motor neuron innervating all of its muscle fibers is called the motor unit So during development, the motor unit decreases in size Slide 2 Once synapses are formed, there has to be further maturation Within our nervous system, there’s overgrowth of synapses -More synapses made during development than required -Excess synapses are removed by synapse elimination We’ll start with the NMJ -Early in development, muscle fibers are innervated by more than 1 motor neuron -Later on, it becomes a 1:1 ratio Motor neuron will innervate multiple muscle fibers -Combination of a single motor neuron innervating all of its muscle fibers is called the motor unit So during development, the motor unit decreases in size Slide 3 In the NMJ, you can make a cut and observe synaptic elimination occuring in real time In this case, these are YFP labeled motor neurons and CFP labeled motor neurons -This particular NMJ is being innervated by two different motor neurons -You can keep looking back at this synapse as time progresses There is no overlap with the two synaptic terminals -They partition the NMJ in a clean fashion A day later, the CFP appears to have retracted a bit and the YFP has taken its place -This process continues and each day the CFP continues to regress and the YFP expands -Until finally, the CFP retracts and degenerates slide 4 How does this happen? There are muscle fibers that are innervated by different kinds of motor neurons early in development -SN and LPN are two types of motor neurons Later in development, muscle fibers become labeled with LPN and the other gets innervated by SN One experiment done to explain this: -Apply TTX (extremely potent binder of Na gated channels which prevents generation of AP) to the LPN thereby depriving activity to these motor neurons -What you see is that both the muscles are innervated by the SN neurons In another experiment, you could eliminate the LPN which will denervate some of the muscle fibers -Under normal situation, there’s a single axon that develops into a NMJ -If you partially denervate it, the reamining pre-synaptic terminals will expand and innervate those now muscle fibers that have been deprived of innervation slide 5 The fact that you can block activity of motor neurons and leads to elimination of LPN innervation and retention of innervation by SN suggests this process is activity dependent of the pre-synaptic terminals Experiments were done were the axons and muscle fibers were completely bathed in ttx during the time that loss of poly-innervation is occuring -It turns out that poly-innervation is retained when doing this -So it is activity dependent It’s not just the fact that a pre-synaptic is active is important, it’s whether you’re active in relationship to the pre-synaptic terminal that you’re competing against When you have ttx, you blocked all activity -So you retain the synapses There has to be a difference in activity for one neuron to take over To look at the possibility if two motor neurons are equally active, this experiment was done -Take a little cuff and have TTX in it -This blocks all AP distal to the block even though the motor neurons are still making APs -Now you can put stimulating electrodes allows you to manipulate the activity of all the axons as you want to manipulate them When you do this where you have stimulated all the motor neurons equally, what you see is that poly-innervation is retained at a higher percentage than previously -It’s the relative activity of two neurons that are fighting over a muscle fiber that is important If you simply stimulated the nerve without the ttx cuff, what you see is you still get a decrease in poly-innervation -This is because when you’re stimulating alone, the motor neurons are also firing on their own so it’s a combination of your stimulation and the asynchronous firing of the motor neurons on their own So synchronous activity (which is equal to no activity) leads to poly-innervation but asynchronous activity suggests it promotes synaptic poly-innervation elimination Slide 6 If you lose activity, the pre-synaptic terminals are lost -This was examined closer by looking at the pre synaptic terminal of a single motor neuron You can put BTX (used to assay for AChR but it is also used to bind to the AChR and block their function -So apply btx to one part of the pre synaptic terminal -What you get is that the pre-synaptic terminals here retract Now we’re seeing that within the terminals of even a single motor neuron, if one part is active and the other part isn’t, the part that isn’t active will lose -This suggests that the loss of activity by the pre-synaptic terminal might affect the maintenance of the NMJ in that region A second experiment -Unlike the first experiment where you limited the btx to a single part on the pre-synaptic terminal, you bathe the whole thing in btx -Under these conditions, there is no affect on the pre-synaptic terminals or AChR This suggests there might be some activity dependent retrograde signaling that goes from the muscle to the pre-synaptic terminals Slide 8 It turns out that only in the development of the NMJ, the motor neurons secret not only Ach but also glutamate and the muscles of glutamate receptors -Later on they disappear Given that the glutamate signaling is placed pretty much during the time when synapse elimination is occurring, this glutamatergic interaction between motor terminals and muscles may be involved in synapse termination -This was tested by applying two drugs: APV and CNQX -APV blocks NMDA receptors (important for synaptic plasticity) -CNQX blocks the AMPA receptor At p11, the percentage of multiple innervation is relatively low but in the presence of the two drugs, the percent dramatically increases -This suggests that the glutametigic is involved in some ways There are two types of glutamertigic receptors: NMDA and AMPA -Glutn1 is specific to the NMDA receptors So you have a mouse where the Glutn1 is knocked out -Look at poly-innervation at the muscle -What you can see is that in the knockout mouse, they have more than doubled levels of poly- innervation This suggests that NMDA receptor mediated transmission is important for synaptic elimination So you should be able to activate this system and by doing so, you can accelerate synapse elimination -You can apply NMDA which is an agonist -You do get fewer in multiple innervation by muscle fibers So glutermatergic transmission at the NMJ mediated by the NMDA receptors is important for synapse elimination -What differentiates the two types of glutermatergic receptors (NMDA and AMPA) is that NMDA receptors pass Ca where as AMPA receptors do not -So an increase in Ca in the muscle is somehow important slide 9 What about in the CNS? -Elimination does occur The purkinje cell is innervated by different pre-synaptic neurons -We’ll focus on the inferior olive -Their axons (called climbing fibers) will innervate a purkinje cell -In a mature cerebellum, a purkinje cell is innervated by a single climbing fiber slide 10 A single purkinje cell is innervated by multiple climbing fibers early on -During synapse elimination, you go from multiple to mono just like in the NMJ You could assay this by elect
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