In response to an applied tensile load, axons of cultured neurons exhibit a number of morphological responses. We designed and implemented a cell stretching device to study the cellular mechanisms governing these responses. Rat sensory neurons were seeded onto a flexible silicone substrate and imaged during substrate stretch. The positions of stationary mitochondria, docked to the axonal cytoskeleton, were determined before and after 10% stretch, and used to calculate the resulting "instantaneous" strain in regions of the axon. There was dramatic heterogeneity in strain along the length of the stretched axons, particularly in regions shorter than 20 μm. The substrate was then held at 10% strain and the axons imaged for 20 min during "relaxation." Both strain magnitude and variability were larger at small lengths in stretched axons during the initial phase of relaxation, but after 14 min, decreased to levels smaller than those seen in unstretched axons. Mitochondrial pairs in stretched axons showed uncoordinated movement with each other at all lengths, suggesting that cytoskeletal cohesion is reduced after stretch. Collectively, these data present the axonal cytoskeleton as a dynamic structure, which responds to stretch rapidly and locally. Globally, the axon behaves as a viscoelastic continuum. Below a characteristic length, though, it appears to behave as a series of independent linked elements, each with unique mechanical properties which suggests a length scale within which cytoskeletal structural elements may be altered to modulate the biomechanical response of the axon. Finally, testable hypotheses of strain accomodation in the axon are suggested.
Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (−0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.
This work aims to show the utility of EH biomaterials for plasmid delivery for potentially localized skeletal muscle regeneration.
Both Schwann cells (SCs) and neurons dynamically expand and contract their plasma membrane during their extension of projections and movement. However, these cell types have very different motility profiles and physiological function. We developed methods to measure the correlated movement of regions of plasma membrane, based on quantitative analysis of the movement of fluorescently labeled wheat-germ agglutinin (WGA) bound to the extracellular membrane. WGA trajectories were compared between SCs and neurons isolated from neonatal SpragueDawley rats, using both cross-correlation and regression analysis. Schwann cellular membranes exhibited significantly higher correlation (42.37 ± 5.87%, mean ± SEM) compared to neurons (24.51 ± 3.52%). Additionally, Schwann cellular membranes were more mobile (À0.165 ± 0.099 lm/min average velocity) compared to neurons (0.052 ± 0.032 lm/s). Comparison of both cell types upon establishment of contact with another neuron failed to identify any difference with the non-contacting state. Our results are suggestive of a role for forces generated by mobility on the biomechanical continuity of plasma membrane. Such forces are likely to interact with factors, including the cytoskeletal framework and adhesion proteins. This work has implications for interactions of neurons and SCs during development and neuronal regeneration.
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