Neurofilament phosphorylation and axon diameter in the squid giant fibre system

Electron energy loss spectroscopic analysis of squid giant axons in a phosphorus energy window yielded bright signals, which were shown to originate from highly phosphorylated neurofilaments. The frequency and distribution of these signals were analysed at defined intervals in cross-sections of the giant axon, starting from its origin in the stellate ganglion and extending distally along the stellar nerve. The analysis revealed a proximodistal gradient of increasing neurofilament phosphorylation. Within the stellate ganglion and for some distance beyond, the increase in frequency of signals correlated with the widening of the neurofilament meshwork and the radial growth of the axon. This agrees with the hypothesis that neurofilament phosphorylation regulates axon calibre by affecting interfilament spacing. In distal axon domains where the axon diameter diminished, contrary to expectations, the spacing of signals increased and the signals were significantly larger. Hyperphosphorylation apparently compensated for a diminishing supply of neurofilament protein. Contrary to predictions, the presynaptic terminal of the giant synapse contained a distinct and highly phosphorylated neurofilament meshwork. We conclude that the growth of the axon diameter is a function of neurofilament phosphorylation, interfilament spacing and neurofilament density. A mature and highly phosphorylated neurofilament cytoskeleton completely filled the presynaptic terminal of the giant synapse.