Phosphorylation-Dependent epitopes on neurofilament proteins and neurofilament densities differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord

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Abstract
The highest molecular weight neurofilament protein (NF‐H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF‐H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF‐H and poorly with antibodies to dephosphorylated epitopes of NF‐H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF‐H in long‐tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 (μm2) in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 μm2 to 2.0 μm2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 (μm2) size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF‐H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.