Cortical Neuron Outgrowth is Insensitive to Substrate Stiffness
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Changes in substrate compliance affect the cellular behavior of numerous cell types including epithelial, endothelial, fibroblasts, and stem cells. Recently, an emphasis has been placed on understanding the mechanotactic behavior of neurons, in an attempt to treat neurological injury and disease as well as to optimize the development of synthetic biomaterials for neural regeneration. Here, we determine the stiffness of the fetal rat cortex using atomic force microscopy and evaluate the effect of substrate mechanics on cortical neuron behavior using polyacrylamide gels with stiffness around that measured for the cortex. In particular, we evaluate the relationship between substrate compliance and ligand coating to morphology, differentiation, and extension behavior. Remarkably, we see an insensitivity of cortical process length and migration to substrate stiffness. We observe differences in the tortuosity of process extension on laminin vs. poly-d-lysine, as well as differences in cell body migration; however these differences are independent of substrate compliance. Myosin II inhibition revealed effects independent of stiffness, yet dependent on outgrowth behavior. Collectively, this work suggests that cortical neurons are capable of differentiating and extending processes regardless of substrate stiffness, which we attribute to the homogeneity of their native environment and their unwarranted need to distinguish substrate compliance.
KeywordsMechanotaxis Axon differentiation Polyacrylamide gels Atomic force microscopy
This work was supported by NSF Grant CMMI-0643783 to HAE. We thank the Functional Macromolecular Laboratory at the University of Maryland (specifically Dr. Peter Kofinas and Brendan Casey) for training and use of the Dynamic Mechanical Analyzer. We would like to thank Kimberly Stroka for use of the custom-written Matlab programs used to fit AFM force curves, and Emily Shih and Hema Balkaran for assistance with the cell body displacement and outgrowth analysis. We also thank Dr. Herbert Geller for stimulating discussions.
- 3.Basarsky, T. A., V. Parpura, and P. G. Haydon. Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution. J. Neurosci. 14(11 Pt 1):6402–6411, 1994.Google Scholar
- 6.Boal, D. Mechanics of the Cell. Cambridge, UK: Cambridge University Press, p. 406, 2002.Google Scholar
- 7.Bridgman, P. C., et al. Myosin IIB is required for growth cone motility. J. Neurosci. 21(16):6159–6169, 2001.Google Scholar
- 14.Dotti, C. G., C. A. Sullivan, and G. A. Banker. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8(4):1454–1468, 1988.Google Scholar
- 27.Jay, P. Y., et al. A mechanical function of myosin II in cell motility. J. Cell Sci. 108(Pt 1):387–393, 1995.Google Scholar
- 34.Lemmon, V., et al. Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength. J. Neurosci. 12(3):818–826, 1992.Google Scholar
- 39.Majno, G., and I. Joris. Cells, Tissues, Disease: Principles of General Pathology. Worcester: Blackwell Science, 1996.Google Scholar
- 41.Mazuchowski, E., and L. Thibault. Biomechanical properties of the human spinal cord and pia matter. In: 2003 Summer Bioengineering Conference. Key Biscayne, FL: American Society of Mechanical Engineers, 2003.Google Scholar
- 58.Tischler, A. S., et al. Nerve growth factor is a potent inducer of proliferation and neuronal differentiation for adult rat chromaffin cells in vitro. J. Neurosci. 13(4):1533–1542, 1993.Google Scholar