Ultrastructure of an identified molluscan neuron in organ culture and cell culture following axotomy.

We examined the ultrastructure of neuron 5 from the buccal ganglion of the mollusc Helisoma trivolvis after axotomy and organ culture, and after isolation of the same neuron in culture. Buccal ganglia containing axotomized neurons 5 were cultured either in host snails or in Leibovitz medium conditioned with ganglia. In addition, some neurons 5 were isolated from buccal ganglia by micro-dissection and plated into culture. Neuron 5 and its processes were identified in both whole mounts and plastic sections of buccal ganglia after intracellular injection with Lucifer Yellow or horseradish peroxidase. Five days after axotomy of neuron 5, thick sections of buccal ganglia stained with toluidine blue revealed that densely staining basophilic bodies (Nissl bodies) within the cytoplasm had dispersed, i.e., they had undergone chromatolysis. Coincident with chromatolysis was an overall increase in diffuse basophilic staining within the cytoplasm of neuron 5 when maintained in organ culture. The dispersion of Nissl bodies viewed by light microscopy correlated with a more freely arranged rough endoplasmic reticulum and associated polysomes within neuron 5 as seen by electron microscopy. Isolated neurons 5 did not possess densely staining Nissl bodies when examined after 2 days in vitro, thus indicating that chromatolysis occurred earlier in isolated neurons. Furthermore, no increase in diffuse cytoplasmic basophilia was observed within isolated neurons 5 cultured in vitro. However, isolated neurons 5 exhibited a marked increase in the number of lipid-like bodies (0.5-1.5 micron in diameter) that were particularly evident in scanning electron micrographs. Scanning and transmission electron micrographs revealed that the isolated neurons were free of associated glia, but non-neuronal cells (hemocytes) would attach themselves to the somata and neurites. Glia surrounding neuron 5 within buccal ganglia exhibited a marked hypertrophy following axotomy and organ culture. Hypertrophy of glia was absent, however, if ganglia were axotomized and left within the animal or axotomized ganglia were implanted into host animals and examined 5 days later by electron microscopy. These observations indicate that, following axotomy, a molluscan neuron may exhibit different morphological features depending on its microenvironment. In addition, the hypertrophy of glia surrounding neurons in Helisoma was not associated with axotomy per se, but with organ culture.