Neurite initiation and elongation is a very important process in brain development and in plasticity of the injured brain. The process of neurite initiation from isolated neurons in culture has been studied with phase contrast time lapse cinematography by Collins and Wessels et al. But the mechanism and the detail of neurite initiation are still unknown.
For clear demonstration of neurite initiation and elongation, isolated dorsal root ganglion cells in culture were observed with phase contrast microscopy, scanning and whole mount transmission electron microscopy. The observed process of neurite initiation and elongation was divided into six stages.
Cell surface of neurons which were fixed with glutaraldehyde before dissociation was almost smooth except 0.05-0.2μ m pits and 0.5-1μm hollows. Stage 1 : Immediately after seeding, neurons had 0.1-0.2μ m microspikes on their surface. In five minutes those microspikes elongated and at the bottom of the cell, reached the substratum. Stage 2 : After 30 minutes microspikes increased and adhered to the sabstratum around the neuron and also to its own cell surface. Pits were also found but smaller than the control. Stage 3 : After 150 minutes from seeding, neurons showed change in microspikes and in cell soma at the bottom. Veils formed between microspikes, enlarged and protruded from the cell body. In microspikes and veils, bundles and networks of microfilament were observed. Stage 4 : Neurons had initial flat growth cones, short neurites and cell body shaped dome. There were many microfilaments, microtubules and vacuoles in growth cones but mitochondria were not found in this stage. Stage 5 : In five to eight hours, many neurons had active large growth cones and microspikes concentrated to growth cones. Cell body flattened to a mound shape and microspikes of cell surface adhered and fused to its own cell membrane. In those active growth cones, many microtubules, mitochondria and other membranous and filamentous materials were found. Stage 6 : After 13-34 hours from seeding, networks of long neurites were remarkable. Terminal small, but thick growth cones had short and a few microspikes. The surface of the flattened cell body was very smooth, but many small pits were still found. In terminal growth cones, many mitochondria and filamentous materials were observed.
From these results it is suggested that microfilaments and microtubules are nessesary for neurite initiation, and also mitochondria and membranous materials are important for neurite elongation. But what determines the site of initiation and what controls the elongation is still unknown. Pit formation may be an important condition. Further studies including thin section electrom microscopy should be performed.
