Does Motor Learning Affect the Neuromuscular Junction?
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The activity-dependent regulation and maintenance of the neuromuscular junction, where the chemical synapse between motor neurons and muscle fibers initiating contraction occurs, play a critical role in the formation of memory-like effect with or without the presence of the spinal cord. Prior work has shown that neurons within the spinal cord can learn about environmental relations without input from the brain (Grau et al., 2014, Neurobiol. Learn Mem, 108, 155-171). For example, rats that have undergone a spinal transection can learn to maintain a hindlimb in a flexed position to minimize exposure to a noxious shock. Interestingly, after this learning has occurred, cutting communication with the muscle (i.e. sciatic denervation) does not eliminate the flexion response. This implies that a peripheral modification within the muscle helps maintain the flexion response over time. My hypothesis was that this involved an alteration at the NMJ by acetylcholine receptor (AChR)-mediated neural communication. The degree to which the AChR is engaged depends upon both its subunit composition and whether it is positioned in the neural membrane. Past work has shown that inactivity leads to the movement (trafficking) of AChR out of the membrane, which would reduce the strength of the response elicited by the release of acetylcholine (ACh). My premise was that learning leads to a lasting increase in flexion duration because it has the opposite effect, and traffics AChR into the neural membrane. To explore this possibility, I sought to compare the ratio of AChR protein within the cellular membrane portion to that contained within its interior (cytosol). This required a fractionation procedure to separate the membrane and cytosolic components. Once the tissue was separated, protein antibodies and Western blotting were used to assess AChR protein levels in muscle tissue from rats that have, or have not, undergone training. While fractionation is routinely used to assess receptor trafficking in neural tissue (e.g., Huang et al., 2016, Exp Neurol, 285, 82-95), this procedure has not been widely applied to muscle tissue. Thus, the majority of my thesis has focused upon developing an effective fractionation procedure to explore the protein changes within the neuromuscular junction in the skeletal muscle. We specifically developed a protocol for subcellular fractionation of rat skeletal muscle tissue. The purity of the membrane and cytosolic fractions was validated by Western blot analysis and probing with “house-keeper” marker proteins specific for each cellular compartment. The results showed relatively high purities of each fraction and significant difference in the levels of marker proteins among two fractions. This protocol allows the membrane and cytosolic cellular compartments from muscle samples to be rapidly isolated without the use of an ultracentrifuge and thus is time-efficient.
Lu, Chunchen J (2017). Does Motor Learning Affect the Neuromuscular Junction?. Undergraduate Research Scholars Program. Available electronically from