Piezo-Electrically Driven Mechanical Stimulation of Sensory Neurons
Mechanotransduction, the conversion of a mechanical stimulus into a biological response, constitutes the basis of a variety of physiological functions such as the senses of touch, balance, proprioception, blood pressure, and hearing. In vertebrates, mecha
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Introduction Somatosensory neurons detect a wide variety of mechanical stimuli. Some are specialized to detect external mechanical stimuli (e.g., mechanoreceptors, mechanonociceptors), while others inform the nervous system about self-generated stimuli (e.g., stretch receptors). The ability of these mechanoreceptors to detect mechanical cues relies on the presence on the specialized sensory endings of mechanotransducer channels that rapidly transform mechanical forces into electrical signals and depolarize the sensory ending (1–3). This local depolarization, called receptor potential, eventually leads to the generation of action potentials that propagate toward the central nervous system. Progress has been made in establishing the functional properties, specificity, and perceptual functions of mechanoreceptors.
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_12, © Springer Science+Business Media, LLC 2013
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This progression led to the recognition that mechanoreceptors serve as selective peripheral encoding devices able to extract information about the various parameters of the mechanical stimulus and to supply the central nervous system with a neural “image” of the peripheral situation. However, molecular mechanisms of mechanotransduction remain poorly understood. Compared with other types of ion channels, including voltage-gated and ligand-activated channels, that have been substantially elucidated, we are largely ignorant of the properties of the force transducers that contribute to our perception of mechanical cues. The technique described here (“mechanoclamp”) adds a new dimension to the study of mechanosensation (Fig. 1). It has opened up new pathways for the investigation of molecular mechanisms of mechanosensation. It can be exploited in the effort to bridge the gap between the properties of mechanotransducer channels in vitro and the characteristics of mechanoreceptors in vivo. This technique is applicable to both primary neuronal cultures as well as immortalized cell lines. The aim of this paper is to describe how mechano-clamp can be applied to cultured DRG neurons (4, 5) and to discuss potential applications in mechanotransduction studies with emphasis on the strengths and limitations of the technique.
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Materials
2.1 Reagents for Recording DRG Neurons in Primary Culture
1. Experimental animals: rats (male Wistar, 120–130 g). All animal experiments have to be performed in accordance with the guidelines on the use of animals by the relevant authorities. 2. Halothane (Belamont, Nicholas Piramal). Harmful by inhalation, use adequate ventilation. 3. Collagenase IA (C9891, Sigma). 4. Bovine Serum Albumin (BSA, A9647, Sigma). 5. Cell culture media, DMEM (Invitrogen). 6. Hank’s balanced salt solution, HBSS (Invitrogen). 7. Penicillin/Streptomycin (Invitrogen). 8. Glutamine (Invitrogen). 9. Nerve growth factor, NGF (Millipore). 10. Glial cell derived neurotrophic factor, GDNF (Invitrogen). 11. Lamini
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