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.

To make controlled recordings of stimulus-evoked action potentials, use a mechanical probe of fire-polished 10-centimeter borosilicate microelectrode glass attached to a ceramic piezoelectric actuator. Position the probe so it moves parallel to the skin fold and touches the hairs, not the skin. Put it about one-half to one millimeter above the skin surface. Then, manually verify the stimulation by listening for audio output when the probe moves.

Now, set up the software to drive mechanical stimulation of a small area of hairs. For example, program three-second stimulations at five Hertz sinusoids every 10s. This requires a probe displacement between 200 and 2000 microns. Then, proceed using repeated automated stimulation to check consistency of recorded responses in the nerves. Repeatable results should be achievable.

When the results are consistent, drop the repeat rate to every 30s. Further instructions are provided in the text protocol. To investigate the properties of the mechanosensory channels and label the mechanosensory terminals around the hair follicles, add 10 micromolar FM143 to the solution and leave for 60 minutes, while continuing to stimulate the hairs with the oscillating probe.

This dye blocks candidate transducer channels in sensory neurons in culture and in cochlea hair cells. However, it does not block the firing in mature hair follicle afferents in situ. The labeling of the endings shows the dye acts as the terminals, which continue to respond to mechanical stimulation throughout dye exposure.