Mechanotransduction of sensory neurons is of great interest to the scientific community, especially in areas such as pain, neurobiology, cardiovascular homeostasis and mechanobiology. We describe a method to investigate stretch-activated mechanotransduction in sensory nerves through subcellular stimulation. The method imposes localized mechanical stimulation through indentation of an elastomeric substrate and combines this mechanical stimulation with whole-cell patch clamp recording of the electrical response to single-nerve stretching. One significant advantage here is that the neurites are stretched with limited physical contact beyond their attachment to the polymer. When we imposed specific mechanical stimulation through the substrate, the stretched neurite fired and an action potential response was recorded. In addition, complementary protocols to control the molecules at the cell–substrate interface are presented. These techniques provide an opportunity to probe neurosensory mechanotransduction with a defined substrate, whose physical and molecular context can be modified to mimic physiologically relevant conditions. The entire process from fabrication to cellular recording takes 5 to 6 d.
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Chalfie, M. Neurosensory mechanotransduction. Nat. Rev. Mol. Cell. Biol. 10, 44–52 (2009).
Cullen, D.K., Lessing, M.C. & LaPlaca, M.C. Collagen-dependent neurite outgrowth and response to dynamic deformation in three-dimensional neuronal cultures. Ann. Biomed. Eng. 35, 835–846 (2007).
Lechner, S.G., Frenzel, H., Wang, R. & Lewin, G.R. Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development. EMBO J. 28, 1479–1491 (2009).
Welsh, M.J., Price, M.P. & Xie, J. Biochemical basis of touch perception: mechanosensory function of degenerin/epithelial Na+ channels. J. Biol. Chem. 277, 2369–2372 (2002).
Drew, L.J. et al. High-threshold mechanosensitive ion channels blocked by a novel conopeptide mediate pressure-evoked pain. PLoS One 2, e515 (2007).
Drew, L.J. & Wood, J.N. FM1-43 is a permeant blocker of mechanosensitive ion channels in sensory neurons and inhibits behavioural responses to mechanical stimuli. Mol. Pain 3, 1 (2007).
Corey, D.P. & Hudspeth, A.J. Mechanical stimulation and micromanipulation with piezoelectric bimorph elements. J. Neurosci. Methods 3, 183–202 (1980).
Kumar, S. & LeDuc, P. Dissecting the molecular basis of the mechanics of living cells. Exp. Mech. 49, 11–23 (2009).
Bao, G. & Suresh, S. Cell and molecular mechanics of biological materials. Nat. Mater. 2, 715–725 (2003).
Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell. Biol. 7, 265–275 (2006).
Lin, Y.W., Cheng, C.M., Leduc, P.R. & Chen, C.C. Understanding sensory nerve mechanotransduction through localized elastomeric matrix control. PLoS One 4, e4293 (2009).
Cho, H., Shin, J., Shin, C.Y., Lee, S.Y. & Oh, U. Mechanosensitive ion channels in cultured sensory neurons of neonatal rats. J. Neurosci. 22, 1238–1247 (2002).
Leach, J.B., Brown, X.Q., Jacot, J.G., Dimilla, P.A. & Wong, J.Y. Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity. J. Neural. Eng. 4, 26–34 (2007).
Georges, P.C., Miller, W.J., Meaney, D.F., Sawyer, E.S. & Janmey, P.A. Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in mixed cortical cultures. Biophys. J. 90, 3012–3018 (2006).
Balgude, A.P., Yu, X., Szymanski, A. & Bellamkonda, R.V. Agarose gel stiffness determines rate of DRG neurite extension in 3D cultures. Biomaterials 22, 1077–1084 (2001).
Wang, H.B., Dembo, M., Hanks, S.K. & Wang, Y. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc. Natl. Acad. Sci. USA 98, 11295–11300 (2001).
Lo, C.M., Wang, H.B., Dembo, M. & Wang, Y.L. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144–152 (2000).
Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).
Wetzel, C. et al. A stomatin-domain protein essential for touch sensation in the mouse. Nature 445, 206–209 (2007).
Price, M.P. et al. The mammalian sodium channel BNC1 is required for normal touch sensation. Nature 407, 1007–1011 (2000).
Price, M.P. et al. The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice. Neuron 32, 1071–1083 (2001).
Reeh, P.W. Sensory receptors in a mammalian skin-nerve in vitro preparation. Prog. Brain. Res. 74, 271–276 (1988).
Zimmermann, K. et al. Phenotyping sensory nerve endings in vitro in the mouse. Nat. Protoc. 4, 174–196 (2009).
Taguchi, T., Sato, J. & Mizumura, K. Augmented mechanical response of muscle thin-fiber sensory receptors recorded from rat muscle-nerve preparations in vitro after eccentric contraction. J. Neurophysiol. 94, 2822–2831 (2005).
Page, A.J. et al. Different contributions of ASIC channels 1a, 2, and 3 in gastrointestinal mechanosensory function. Gut 54, 1408–1415 (2005).
Khalsa, P.S., Ge, W., Uddin, M.Z. & Hadjiargyrou, M. Integrin alpha2beta1 affects mechano-transduction in slowly and rapidly adapting cutaneous mechanoreceptors in rat hairy skin. Neuroscience 129, 447–459 (2004).
Hoheisel, U., Reinohl, J., Unger, T. & Mense, S. Acidic pH and capsaicin activate mechanosensitive group IV muscle receptors in the rat. Pain 110, 149–157 (2004).
Sanders, R. Torsional elasticity of human skin in vivo. Pflugers. Arch. 342, 255–260 (1973).
von Philipsborn, A.C. et al. Microcontact printing of axon guidance molecules for generation of graded patterns. Nat. Protoc. 1, 1322–1328 (2006).
Weibel, D.B., Garstecki, P. & Whitesides, G.M. Combining microscience and neurobiology. Curr. Opin. Neurobiol. 15, 560–567 (2005).
McDonald, J.C. & Whitesides, G.M. Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc. Chem. Res. 35, 491–499 (2002).
Park, J.W., Vahidi, B., Taylor, A.M., Rhee, S.W. & Jeon, N.L. Microfluidic culture platform for neuroscience research. Nat. Protoc. 1, 2128–2136 (2006).
Stafford, C.M. et al. A buckling-based metrology for measuring the elastic moduli of polymeric thin films. Nat. Mater. 3, 545–550 (2004).
Cheng, C.M. & Leduc, P.R. Creating cellular and molecular patterns via gravitational force with liquid droplets. Appl. Phys. Lett. 93 (2008).
Chou, S.Y., Cheng, C.M. & LeDuc, P.R. Composite polymer systems with control of local substrate elasticity and their effect on cytoskeletal and morphological characteristics of adherent cells. Biomaterials 30, 3136–3142 (2009).
Bhattacharya, M.R. et al. Radial stretch reveals distinct populations of mechanosensitive mammalian somatosensory neurons. Proc. Natl. Acad. Sci. USA 105, 20015–20020 (2008).
Sanchez, D. et al. Localized and non-contact mechanical stimulation of dorsal root ganglion sensory neurons using scanning ion conductance microscopy. J. Neurosci. Methods 159, 26–34 (2007).
Haeberle, H., Bryan, L.A., Vadakkan, T.J., Dickinson, M.E. & Lumpkin, E.A. Swelling-activated Ca2+ channels trigger Ca2+ signals in Merkel cells. PLoS One 3, e1750 (2008).
McCarter, G.C., Reichling, D.B. & Levine, J.D. Mechanical transduction by rat dorsal root ganglion neurons in vitro. Neurosci. Lett. 273, 179–182 (1999).
Drew, L.J. et al. Acid-sensing ion channels ASIC2 and ASIC3 do not contribute to mechanically activated currents in mammalian sensory neurones. J. Physiol. 556, 691–710 (2004).
McCarter, G.C. & Levine, J.D. Ionic basis of a mechanotransduction current in adult rat dorsal root ganglion neurons. Mol. Pain. 2, 28 (2006).
Hu, J. & Lewin, G.R. Mechanosensitive currents in the neurites of cultured mouse sensory neurons. J. Physiol. 577, 815–828 (2006).
Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers. Arch. 391, 85–100 (1981).
Malin, S.A., Davis, B.M. & Molliver, D.C. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat. Protoc. 2, 152–160 (2007).
Dib-Hajj, S.D. et al. Transfection of rat or mouse neurons by biolistics or electroporation. Nat. Protoc. 4, 1118–1126 (2009).
Cummins, T.R., Rush, A.M., Estacion, M., Dib-Hajj, S.D. & Waxman, S.G. Voltage-clamp and current-clamp recordings from mammalian DRG neurons. Nat. Protoc. 4, 1103–1112 (2009).
Kubicek, J.D., Brelsford, S., Ahluwalia, P. & LeDuc, P.R. Integrated lithographic membranes and surface adhesion chemistry for three-dimensional cellular stimulation. Langmuir 20, 11552–11556 (2004).
Lee, D.W. & Choi, Y.S. A novel pressure sensor with a PDMS diaphragm. Microelectronic Eng. 85, 1054–1058 (2008).
Yu, Y.S. & Zhao, Y.P. Deformation of PDMS membrane and microcantilever by a water droplet: Comparison between Mooney-Rivlin and linear elastic constitutive models. J. Colloid Interface Sci. 332, 467–476 (2009).
Bellin, R.M. et al. Defining the role of syndecan-4 in mechanotransduction using surface-modification approaches. Proc. Natl. Acad. Sci. USA 106, 22102–22107 (2009).
Malpass, C.A., Millsap, K.W., Sidhu, H. & Gower, L.B. Immobilization of an oxalate-degrading enzyme on silicone elastomer. J. Biomed. Mater. Res. 63, 822–829 (2002).
Ginn, B.T. & Steinbock, O. Polymer surface modification using microwave-oven-generated plasma. Langmuir 19, 8117–8118 (2003).
This work was supported by the Institute of Biomedical Sciences, Academia Sinica, Taiwan; the National Science Council grant (NSC 98-2321-B-001-043); China Medical University (CMU97-295), as well as the National Science Foundation, NSF-CAREER, Office of Naval Research and the Beckman Young Investigators Program. CMC was supported in part by a PhD Research Scholarship from Taiwan, the Dowd-ICES Scholarship from Carnegie Mellon University, USA and Traveling Fellowship from Journal of Cell Science. We thank Luke Duncan for his work on the protocol for coating PDMS with alternative molecules, and the Medical Art Room of IBMS for help with making the illustrations.
Localized indentation-induced mechanical perturbations tracked using fluorescent beads embedded in PDMS. (a) Bright field image captured before the first pipette indentation. Arrows point the location of two fluorescent beads (A & B). (b) Bright field image captured after the first trial of pipette indentation (100 µm deep indentation). Arrows indicate the displaced positions of these two florescent beads (A’ & B’) after the indentation. (c) Fluorescent microscopy image with blue pseudo coloring before the first pipette indentation. Two representative fluorescent beads (A & B) are tracked when we imposed the indentation. Point “I” represents the indentation point as indicated by a white dot in the panel. (d) Quantitative analysis of the displacement of 2 representative fluorescent beads over a total 10 trials of indentation. The X-axis is the radial distance from the indentation point (I) to the initial position of each fluorescent bead (A, B). The mean and SEM of the bead displacement (A-A’ & B-B’) are on the y-axis. (PDF 274 kb)
(a) ANSYS mesh and (b) indentation map for a 1mm thick PDMS slab. ANSYS finite element modeling software was used to simulate the indentation of a cylindrical indenter on a PDMS substrate. The model consisted of a 12 mm × 12 mm PDMS substrate constrained at the bottom surface indented with a 4.2 micron radius cylindrical indenter. The indenter was constrained in the x and y directions on the periphery. This model was meshed with SOLID187, TARGET170, and CONTACT174 elements. Indentation depth analysis revealed a maximum indentation depth of approximately 125 microns after application of a 57 µN force at the point of indentation. This surface depth deformation decreases as the radial distance away from the indentation area increases. (PDF 447 kb)
ANSYS analysis of PDMS response to slab thickness and radial distance away for the area of indentation. (a) ANSYS finite element modeling software was used to simulate how substrate thickness affects the force required to maintain an indentation depth of 125 microns. (b) The PDMS surface indentation depth when compared to radial distances away from the center area of indentation from the needle (at point 0.1 mm). Negligible surface depths were found at a distance of about 0.1 mm (100 microns) from the area of indentation. (PDF 243 kb)
ANSYS analysis of indentation depth at the area of needle contact compared to the force from the needle. ANSYS finite element modeling software was used to model the dependence of indentation depth at the area of indentation for a 12 mm x 12 mm, 0.75 mm thick PDMS substrate indented with a 4.2 micron radius cylindrical indenter. A linear fit to this data resulted in an approximation of the relationship for this case between the force for the indentation and the indentation depth (R=0.9999). F represents the force applied by the indenter on the PDMS substrate (µN) and h represents the indentation depth at the area of contact (mm). (PDF 164 kb)
Finite Element Model (PDF 66 kb)
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Cheng, CM., Lin, YW., Bellin, R. et al. Probing localized neural mechanotransduction through surface-modified elastomeric matrices and electrophysiology. Nat Protoc 5, 714–724 (2010). https://doi.org/10.1038/nprot.2010.15
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