Intravital Two-Photon Imaging of Touch Sensory Axon Morphology in Mouse Skin

We are investigating the molecular and cellular mechanisms of the somatosensory circuit formation by in vivo imaging of the somatosensory axons in the mouse skin. Recent developments in mouse genetics for somato sensation research allowed us to study somatosensory axon development in vivo with subtype specificity. To begin, turn on the laser and allow sufficient time for it to warm up.

Then, turn on the two photon microscope and open the control and acquisition software on the connected computer. Turn on the heating pad and set the temperature to 37 degrees Celsius to prevent hypothermia during anesthesia. Now, transfer the anesthetized mouse to the heating pad on the imaging platform.

Align the mouse body on a platform adjacent to the imaging stage so that it does not touch the area where the paw is mounted, minimizing movement artifacts during imaging. Now, apply ophthalmic ointment to both eyes of the mouse to prevent dryness during imaging. Next, using a cotton swab, gently apply depilatory cream to the mouse paw to remove hair.

After one minute, remove the cream and clean the paw using 70%ethanol followed by water. Then with one hand, gently stretch the skin to stabilize the paw surface. Using a sterile 30 gauge needle and black ink, pierce the skin and tattoo two adjacent dots on either side of the target imaging region, spaced approximately two millimeters apart.

After tattooing, dispose of the used needle in a sharps container. Take a glass microscope slide and place four dots of vacuum grease at the corners. Then, position a one millimeter thick piece of modeling clay at the center of the slide.

Place the slide under the objective lens and center the mouse's paw directly on top of the clay. Gently roll the mouse's paw on the slide to flatten it and press it into the clay. Then, carefully place a 22 by 60 millimeter cover slip over the paw to secure it.

Adjust the angle of the mouse skin by applying light pressure on each corner of the cover slip until the skin surface is parallel to the imaging plane. To image the glabra skin, use tweezers to rotate the paw, so the glabra skin faces upward, and then use the flat end of the tweezers to gently press the paw into the clay. Once flattened, place the cover slip on top and press down carefully.

Confirm that the paw is centered. Place a drop of distilled water on the cover slip directly above the mouse paw. Lower the objective lens until a water column forms between the lens and the cover slip.

Once the objective lens is centered over the mouse paw, close the microscope box and use the LED screen to focus on the sample by adjusting the X, Y, and Z controllers. The LED screen will show a live image illuminated by the microscopes built in LED. After locating the sample, turn off the LED screen.

To begin two-photon imaging, flip the lever on the upper right side of the microscope to switch from the mirror setting to the dichroic setting. Then, close the blackout curtains, and switch on the detector to visualize yellow fluorescent protein, or YFP. In prairie view, open the shutter and switch the software to imaging mode.

For imaging thy1-YFP, turn on the tunable laser and set the wavelength to 960 nanometers, with a gain between 700 and 800. For green fluorescent protein, or GFP imaging, adjust the laser to 920 nanometers. For mice aged postnatal day 10 or younger, set the initial laser power to 50, and gradually increase the power until the desired signal intensity is achieved.

This minimizes light penetration and reduces the risk of photo damage. Once the laser wavelength, gain and power are set, begin scanning the tissue. Now, use the X, Y and Z controllers to locate the imaging field and adjust the zoom as necessary.

Start image acquisition at a position approximately 10 to 20 micrometers beneath the skin surface. Finally, remove the mouse paw from the glass slide after imaging, and return the mouse to a recovery cage for postoperative care. In the hairy skin of adult mice, the overall axonal terminal organization in the four paw was revealed through a maximum intensity projection, showing fully developed lanceolate endings surrounding hair follicles.

The average density of lanceolate endings surrounding hair follicles in these mice was 15.8 endings per square millimeter. A sparse, crescent shaped A delta LTMR lanceolate ending was observed innervating a hair follicle at postnatal day four. A longitudinal lanceolate complex in hairy skin was imaged in an adult mouse at postnatal day 39, and high resolution Z plane images showed individual A beta rapidly adapting LTMR lanceolate endings, each separated by three micrometers.

Immature lanceolate complexes were observed in the hairy skin at postnatal day 10 with fewer A beta LTMR axons, and individual Z planes revealed sparse nascent lanceolate endings. In the adult mouse fingertip, A beta RA LTMR axons were visualized innervating meissner corpuscles as ellipsoid structures beneath the epidermis and individual optical sections highlight discrete axon terminals at different depths within the skin. Across three consecutive days, the same nerve bundles in adult mouse hairy skin were identified with slight shape variations, likely due to paw positioning.

The same axonal morphologies around hair follicles in the hairy skin were consistently imaged over three days. Our protocol addresses a challenge in the field to visualize somatosensory axon structure and dynamics in living animals during development. Our technique enables high resolution chronic imaging of somatosensory axon development and also in adult stage in vivo.

This study enables the investigation of the molecular and the cellular mechanisms of somatosensory axon development and regeneration in vivo.