NEUROFILAMENT SPACING, PHOSPHORYLATION, AND AXON DIAMETER IN REGENERATING AND UNINJURED LAMPREY AXONS

It has been postulated that phosphorylation of the carboxy terminus si dearms of neurofilaments (NFs) increases axon diameter through repulsi ve electrostatic forces that increase sidearm extension and interfilam ent spacing. To evaluate this hypothesis, the relationships among NF p hosphorylation, NF spacing, and axon diameter were examined in uninjur ed and spinal cord-transected larval sea lampreys (Petromyzon marinus) . In untransected animals, axon diameters in the spinal cord varied fr om 0.5 to 50 mu m. Antibodies specific for highly phosphorylated NFs l abeled only large axons (>10 mu m), whereas antibodies for Lightly pho sphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inve rsely related to NF packing density, but the interfilament distances o f the largest axons were only 1.5 times those of the smallest axons. I n addition, the lightly phosphorylated NFs of the small axons in the d orsal columns were widely spaced, suggesting that phosphorylation of N Fs does not rigidly determine their spacing and that NF spacing does n ot rigidly determine axon diameter. Regenerating neurites of giant ret iculospinal axons (GRAs) have diameters only 5-10% of those of their p arent axons. If axon caliber is controlled by NF phosphorylation via m utual electrostatic repulsion, then NFs in the slender regenerating ne urites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although Linear density of NFs (the number of NFs per micrometer) in these sle nder regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons c lose to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphor ylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF si dearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, su ch as the slowing of NF transport and the formation of a stationary cy toskeletal lattice, as has been proposed by others. Such a mechanism c ould be overridden during regeneration, when a more compact, phosphory lated NF backbone might add mechanical stiffness that promotes the adv ance of the neurite tip within a restricted central nervous system env ironment. (C) 1996 Wiley-Liss, Inc.