Phenotyping sensory nerve endings in vitro in the mouse

Abstract

This protocol details methods to identify and record from cutaneous primary afferent axons in an isolated mammalian skin–saphenous nerve preparation. The method is based on extracellular recordings of propagated action potentials from single-fiber receptive fields. Cutaneous nerve endings show graded sensitivities to various stimulus modalities that are quantified by adequate and controlled stimulation of the superfused skin with heat, cold, touch, constant punctate pressure or chemicals. Responses recorded from single-fibers are comparable with those obtained in previous in vivo experiments on the same species. We describe the components and the setting-up of the basic equipment of a skin–nerve recording station (few days), the preparation of the skin and the adherent saphenous nerve in the mouse (15–45 min) and the isolation and recording of neurons (approximately 1–3 h per recording). In addition, stimulation techniques, protocols to achieve single-fiber recordings, issues of data acquisition and action potential discrimination are discussed in detail.

Distinctive Changes in Plasma Membrane Phosphoinositides Underlie Differential Regulation of TRPV1 in Nociceptive Neurons

“…Only fibers with nonoverlapping receptive fields were used from the same skin preparation. We used a higher final capsaicin concentration (10 M) than in the patch-clamp experiments (1 M), as customary in these experiments, because the drug needs to penetrate tissue layers to reach the nerve terminals (Zimmermann et al, 2009).…”

Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system

“…The isolated skin saphenous nerve preparation and single-fiber recording technique was used (29,30) on male littermates of WT and TRPC5 −/− mice (n = 32) weighing 20-32 g and on 129S1/SvImJ mice (n = 17; Jackson Labs). TRPM8 −/− mice were from Ajay Dhaka and Ardem Patapoutian (Department of Cell Biology, The Scripps Research Institute, La Jolla, CA).…”

“…We compared the functional properties of C-fiber responses to cold in TRPC5 −/− and TRPM8 −/− mice (11, 13, 28). Skin-nerve preparations allow stimulation of isolated receptive fields of single C-and Aδ-fibers in mouse skin while recording propagated action potentials extracellularly (29,30). Extensive prior work in mammals established three types of coldsensitive afferent fibers: mechanosensitive C-fibers (nociceptors, C-mechano; CM), cold receptors (C-cold; CC), and mechanosensitive Aδ-fibers (Aδ-nociceptors) (31)(32)(33)(34)(35)(36)(37).…”

“…Extensive prior work in mammals established three types of coldsensitive afferent fibers: mechanosensitive C-fibers (nociceptors, C-mechano; CM), cold receptors (C-cold; CC), and mechanosensitive Aδ-fibers (Aδ-nociceptors) (31)(32)(33)(34)(35)(36)(37). In the mouse, CM-fibers can be subdivided further into two cold-sensitive subclasses, the cold nociceptors (C-mechano cold, CMC) and the multimodal cold nociceptors (C-mechano-cold-heat, CMCH) (29). In primates, CMC-and CMCH-fibers are believed to relay information to the brain that is perceived as painful cold, whereas CC-fibers are likely associated with innocuous cold (33,35).…”

Methylglyoxal Activates Nociceptors through Transient Receptor Potential Channel A1 (TRPA1)

“…Single Fiber Electrophysiology of the Skin Nerve PreparationSingle fiber recordings of identified primary afferents from skin of C57Bl/6 and TRPA1 knock-out mice were carried out using the skin nerve preparation in vitro as has been described previously (28,35). Briefly, after sacrificing mice by exposure to a pure CO 2 atmosphere, the saphenous nerve was dissected downward from the upper thigh and excised together with the hairy skin of the dorsal hind paw and lower leg containing its receptive fields.…”