Department of Mechanical and Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA.
Nat Protoc. 2010 Apr;5(4):714-24. doi: 10.1038/nprot.2010.15. Epub 2010 Mar 25.
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.
感觉神经元的机械转导引起了科学界的极大兴趣，特别是在疼痛、神经生物学、心血管稳态和机械生物学等领域。我们描述了一种通过亚细胞刺激来研究感觉神经中拉伸激活的机械转导的方法。该方法通过弹性基底的压痕施加局部机械刺激，并将这种机械刺激与单细胞拉伸的全细胞膜片钳记录的电响应相结合。这里的一个显著优势是，在神经突与其附着的聚合物之外的有限物理接触处被拉伸。当我们通过基底施加特定的机械刺激时，拉伸的神经突会发射，并记录到动作电位响应。此外，还提出了用于控制细胞-基底界面分子的补充方案。这些技术为使用定义明确的基底来探测神经感觉机械转导提供了机会，基底的物理和分子环境可以进行修改以模拟生理相关条件。从制造到细胞记录的整个过程需要 5 到 6 天。
