Probing localized neural mechanotransduction through surface-modified elastomeric matrices and electrophysiology

Abstract

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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Figure 1: Schematic of the mechanical stretching imposed on a neurite in the recording chamber.
Figure 2: Digital images of the recording chamber.
Figure 3: Preparation of the DRG culture on fibronectin-coated PDMS-covered coverslips.
Figure 4: Modification of the PDMS substrate with antibodies against cell surface proteins, an alternative procedure to fibronectin coating.
Figure 5: Imposing mechanical stretching on a single neurite of a patched neuron.
Figure 6: Effects of distal indentation on neurite-free and neurite-bearing neurons.
Figure 7: Measuring the mechanical threshold in neurite stretching, which will evoke an action potential.
Figure 8: Effects of fibronectin on neurite outgrowth for PDMS substrates.

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Acknowledgements

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.

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Authors

Contributions

C.-M.C., P.R.L. and C.-C.C. conceived and designed the experiments. C.-M.C., Y.-W.L., R.M.B., Y.-R.C. and R.L.S. performed the experiments. C.-M.C., Y.-W.L., R.M.B., Y.-R.C. and R.L.S. analyzed the data. C.-M.C., Y.-W.L., R.M.B., P.R.L. and C.-C.C. contributed reagents/material/analysis tools. R.M.B., P.R.L. and C.-C.C. wrote the paper.

Note: Supplementary information is available via the HTML version of this article.

Corresponding authors

Correspondence to Philip R LeDuc or Chih-Cheng Chen.

Supplementary information

Supplementary Figure 1

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)

Supplementary Figure 2

(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)

Supplementary Figure 3

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)

Supplementary Figure 4

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)

Supplementary Material

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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