This protocol allows direct imaging of the neurofilament population in myelinated axons from juvenile or adult mice using fluorescence microscopy, making it possible to measure the directionality and velocity of neurofilament transport in isolated nerve segments. This method depends on the availability of mice expressing a photoactivatable fluorescent fusion of the protein of interest, but can be adapted for use in any readily dissected nerve. To begin, pour oxygenated saline into a 60 milliliter syringe and ensure that there is minimal air remaining in the syringe.
Connect the syringe to the tubing, then place the outflow tube into a waste flask. Place the outer gasket into the perfusion chamber housing, ensuring that the flow inlet and outlet posts are aligned with the holes in the gasket, then lay the inner gasket on a number 1.5 circular coverslip and carefully smooth out any wrinkles in the gasket to ensure a tight seal. Place the coverslip and gasket on a paper towel or a task wipe with the gasket facing up.
Use a pair of large dissection scissors to make a dorsal incision in the skin near the middle of the spine and continue to cut around the ventral aspect of the animal. Slowly pull the skin away from the muscle and cut the fascia. Next, use microdissection scissors to make an incision in the thigh muscles midway between the tail and knee to expose the sciatic nerve.
Make sure that the nerve, which is visible through the muscle, is not cut. Extend the incision dorsally and ventrally to remove the muscle, then remove the muscles of the calf, keeping the cuts shallow and short to avoid damaging nerves. Continue removing the muscles until the tibial nerve is fully exposed from the point where it branches from the sciatic nerve to the heel.
Grasp the tibial nerve at the spine proximal end with a pair of forceps and cut it with a pair of microdissection scissors, making sure to not apply too much tension to the nerve. Carefully lift it away from the muscle and cut any attachments, taking care not to put tension on the nerve. Cut the spine distal end of the tibial nerve and transfer it to a small Petri dish of room temperature oxygenated saline.
Starting from the proximal end of the nerve, gently grasp the exposed axon ends with a pair of very fine tip forceps. With a second pair of forceps, grasp the nerve sheath proximally and slowly pull towards the distal end of the nerve making sure that no undue tension is applied during this process. Grasp the proximal end of the nerve and slowly lay it down onto a coverslip, then straighten it under gentle tension.
Place the microaqueduct slide over the nerve with the grooved slide down and position the direction of flow parallel to the nerve. Flip the coverslip and microaqueduct assembly over and place it in the perfusion chamber housing with the slide opposed to the outer gasket. To secure the profusion chamber, place it in the metal housing and rotate the locking ring.
Slowly depress the saline syringe plunger and fill the perfusion chamber. Keep the inlet and outlet tubing, outlet flask, and syringe elevated above the chamber itself at all times during the setup and imaging. Transfer the profusion assembly to an inverted microscope stage and mount the saline syringe into the syringe pump, then start the motor and adjust the speed for a flow rate of 0.25 milliliters per minute.
Connect the inline solution heater and set it to 37 degrees Celsius. Connect the objective heater and set it to 37 degrees Celsius. Apply oil to the objective and insert the profusion chamber into the stage mount.
Apply oil to the chamber heater pad and attach it to the perfusion chamber, then connect it and set it to 37 degrees Celsius. Lock the profusion chamber into the stage adapter and bring the objective oil into contact with the coverslip on the underside of the chamber. Use Brightfield illumination to focus on the layer of axons on the bottom surface of the nerve closest to the coverslip surface.
Myelinated axons can be identified by the presence of a myelin sheath which is visible under Brightfield transmitted light illumination without contrast enhancement. Acquire a Brightfield reference image, recording the directionality of the nerve, then acquire a confocal image using a 488 nanometer laser and an emission filter appropriate for photoactivatable GFP. Set the laser power to approximately five times normal imaging power and acquire an image with an exposure time of three to four minutes, then acquire another confocal image to record the pre-activation autofluorescence after the bleaching step.
Draw a line parallel to the axons with a length equal to the desired activation window size of the Brightfield image. Using this line as a guide, draw a rectangular region of interest across the field of view perpendicular to the axons. Activate the GFP fluorescence in this region by pattern excitation with the 405 nanometer light, making sure that an image is acquired just prior to and immediately following activation.
As the activation finishes, start a one-minute timer. When it goes off, start acquisition of a timelapse series. Add 250 milligrams of fluorescein powder to 0.5 milliliters of double distilled water and mix it until there are no visible particles, then spin the solution for 30 seconds in a tabletop centrifuge to sediment any undissolved material.
Add eight microliters of fluorescein solution to a slide and apply a number 1.5 coverslip. Blot the excess liquid, seal the coverslip with nail polish, and allow the slide to dry. Place the slide coverslip side down on the inverted microscope stage and adjust the focus on the thin plane of fluorescence at the surface of the coverslip.
Move the slide around to find a field of view that does not contain air bubbles or large fluorescein particles and acquire a Z-stack spanning six micrometers at 0.2 micrometer intervals such that the middle image is the original focus plane. Close all light path shutters including the camera shutter and set the laser power and the exposure time to zero seconds. Acquire a stack of 100 images with these settings, which will be averaged to generate the dark field image.
Representative images from pulse-escape and pulse-spread experiments are shown here. For the pulse-escape method, the fluorescence decay kinetics in the activated region can yield information on long-term and short-term pausing behavior. The activated region can be short for these experiments.
A longer activated region is used for the pulse-spread method in order to provide a larger pool of fluorescent neurofilaments. This lengthens the time over which the fluorescence increase in the flanking regions remains linear. 15 micrometer flanking windows are used here.
The total fluorescence from the measuring window was quantified. The decay is biphasic for the pulse-escape method with an initial exponential decay representing the departure of on-track neurofilaments and a second slower exponential decay at 10 to 20 minutes that represents the departure of off-track filaments. For the pulse-spread strategy, the calculations of the velocity and directionality of the movement are dependent only on the slopes in the flanking windows.
For the window lengths used here, the linear phase extends for about five minutes. A timelapse from a glycolytically inhibited nerve is shown here, demonstrating an apparent reduction in transport out of the activated region. Indeed, the distal and proximal slopes are significantly lower in nerves treated with inhibitor.
There is also a significant decrease in the population velocity between these two conditions. When attempting this protocol, remember that it is crucial to be hasty in conducting the dissection and chamber assembly portions as nerve health declines following sacrifice of the animal. Neuronal cell culture can allow measurement and analysis of the movement of individual neurofilaments, which would supplement the population scale data gathered using this protocol.
