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Cultures of mouse Neuro-2a neuroblastoma cells treated with 3-6 mM extracellular Ca2+ exhibited enhanced neurite extension characterized by increased neurite numbers and lengths. The ganglioside GM1 potentiated the effect of extracellular Ca2+ by increasing further the number and length of the neurites formed in response to exogenous Ca2+. Maximal neuritic numbers were achieved with 4 mM Ca2+ while the longest neurites were observed in medium containing 4-6 mM Ca2+. Stimulation of the Ca2+ influx with the ionophore A23187 or the amino acid taurine also enhanced neurite formation and GM1 potentiated these actions. Transmission electron microscopy revealed numerous microtubules and neurofilaments in neurites and microfilaments with the spine-like processes along fine neuritic branches and in the filopodia of growth cones. Neuritic varicosities and growth cones contained a variety of vesicles. All of these structures were increased in the presence of GM1 and were increased further by extracellular Ca2+ or A23187. The ability of GM1 to enhance neuritogenesis was diminished by EGTA or Ruthenium red. Similarly, the effect of GM1 was diminished or abolished by Ca2+ channel blockers such as CdCl2 or LaCl3. X-ray microprobe analysis revealed that GM1 alone enhanced intracellular levels of total ionic and membrane bound Ca2+, perhaps accounting for the increased neuritogenesis observed under conditions in which Ca2+ was manipulated. The present study suggest that the neuritogenic action of GM1 is Ca2+ dependent.

These studies demonstrate that while microtubules are essential for BBG-mediated neurite initiation and elongation, they are not involved in microfilament-dependent ganglioside-mediated surface activity. Microfilaments may be more directly altered by exogenous gangliosides than microtubules since they are the major structural elements of microvilli and are required for neurite branching. Our studies suggest that normal neuritogenesis requires a delicately balanced interaction between various cytoskeletal elements. Since there is a close relationship between membrane-associated lipid molecules and submembranous cytoskeletal elements, the incorporation of gangliosides into membranes may alter this balance and result in neurite formation. The use of gangliosides to enhance neurite production provides a unique model for the study of nerve development. We have shown that bovine brain gangliosides stimulate an immediate sequence of surface-related changes as well as microtubule and microfilament dependent neurite formation in Neuro-2a cells. However, the precise molecular events by which gangliosides enhance neuritogenesis await further study.

A new structure associated with the surfaces of neuronal microtubules is described which connects microtubules to neurofilaments in the axonal processes of cultured chick sensory ganglia. These couplers consist of a spherical core particle (15 nm in diameter) from which radiate several thin filaments (4 nm in diameter). Connection of adjacent microtubules and neurofilaments is achieved by thin filaments radiating from core particles positioned between these cytoskeletal elements. Couplers are most conspicuous in regions of axonal processes containing widely separated microtubules and neurofilaments. The structure and distribution of these couplers suggests that they are directly involved in intra-axonal organelle movements, possibly by modulating the spatial separation of adjacent microtubules and neurofilaments, thereby allowing the passage of transported organelles.

Elevated concentrations of adenosine 3',5'-monophosphate induce a variety of cell movements. The role of adenosine 3',5'-monophosphate in promoting those movements associated with growth prompted our study of in vitro microtubule-dependent axonal elongation. Ganglia treated with adenosine 5'-monophosphate show no enhancement over controls; treatment with adenosine 3', 5'monophosphate or its dibutyryl derivative significantly enhances elongation, as measured by increases in both axonal numbers and length. Our study suggests that adenosine 3',5'-monophosphate promotes elongation by stimulation of microtubule assembly.

Adenosine 3',5'-monophosphate (cyclic AMP) and its dibutyryl derivative induce a variety of morphological changes, including those associated with in vitro axonal maturation. Established sensory ganglia treated with dibutyryl cyclic AMP show significant increases in average axonal length and number in comparison with controls; those treated with maintenance doses of Colcemid show no increases in either parameter; simultaneous treatment with both agents results in growth statistically similar to that produced by dibutyryl cyclic AMP alone. The data are consistent with our hypothesis that cyclic AMP promotes axonal elongation by stimulating microtubule assembly from a preexisting subunit pool.