Abstract
Neurofilaments are abundant space-filling cytoskeletal polymers that are transported into and along axons. During postnatal development, these polymers accumulate in myelinated axons causing an expansion of axon caliber, which is necessary for rapid electrical transmission. Studies on cultured nerve cells have shown that axonal neurofilaments move rapidly and intermittently along microtubule tracks in both anterograde and retrograde directions. However, it is unclear whether neurofilament transport is also bidirectional in vivo Here, we describe a pulse-spread fluorescence photoactivation method to address this in peripheral nerves dissected from hThy1-paGFP-NFM transgenic mice, which express a photoactivatable fluorescent neurofilament protein. Neurofilaments were photoactivated in short segments of myelinated axons in tibial nerves at 2, 4, 8, and 16 weeks of age. The proximal and distal spread of the fluorescence due to the movement of the fluorescent neurofilaments was measured over time. We show that the directional bias and velocity of neurofilament transport can be calculated from these measurements. The directional bias was ∼60% anterograde and 40% retrograde and did not change significantly with age or distance along the nerve. The net velocity decreased with age and distance along the nerve, which is consistent with previous studies using radioisotopic pulse labeling. This decrease in velocity was caused by a decrease in both anterograde and retrograde movement. Thus, neurofilament transport is bidirectional in vivo, with a significant fraction of the filaments moving retrogradely in both juvenile and adult mice.