Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicles

Begin with a dissected mouse pinna, or outer ear, with its connected nerves exposed.

Insert the nerve intended for recording into the recording electrode.

Remove the fat layer near the pinna margin to expose the hair follicle base, then fold the skin to expose the hairs for stimulation.

Position a mechanical stimulation probe to touch and deflect the hairs.

This stimulation activates sensory nerve endings, opens mechanically-gated ion channels, allows positive ion influx, and generates action potentials.

The action potentials propagate through these endings to the nerve being recorded. Visualize the signal.

Next, add a fluorescent dye and continue hair stimulation.

The dye binds to the stimulated neuronal membranes and is internalized via endocytosis without affecting neuronal activity.

Post-stimulation, wash to remove unbound dye.

Add a sequestering agent to remove the non-internalized membrane-bound dye.

Using fluorescence microscopy, visualize the staining in the hair follicles to identify mechanically stimulated nerve endings.