An in vivo electrophysiological preparation for mechanical, electrical and optical stimulation of sensory neurons that innervate murine bone

In this study, we aimed to develop an in vivo electrophysiological bone-nerve preparation to record the activity of peripheral sensory neurons that innervate the murine tibia. A small nerve that innervates the tibial marrow cavity was identified in isoflurane-anesthetized C57BL/6 mice, and placed over a platinum hook electrode for extracellular recording. Whole-nerve activity was amplified, filtered and sampled at 20 kHz using PowerLab (ADInstruments). A cannula was placed into the marrow cavity to deliver mechanical stimuli (by pressurizing with injection of saline) and/or capsaicin. Optical stimulation was achieved by application of 473 nm blue light (1 Hz, 0.25-0.5 ms, 0-12.5 mW/mm) to the tibial marrow cavity in Wnt1-Cre; loxP-ChR2 mice. Murine bone afferent neurons responded to high threshold noxious mechanical stimulation, coded for the intensity of mechanical stimulation, could be sensitized by capsaicin, and did not suffer stimulus-evoked fatigue at 10-minute interstimulus intervals. Electrical and optical stimulation within the marrow cavity evoked action potentials with conduction velocities in the Aδ and/or C fiber range. These new approaches to recording the activity of bone afferent neurons will allow us to take advantage of transgenic and optogenetic tools to further our understanding of mechanisms that generate and maintain bone pain in the future.