Cell culture techniques have been used to investigate some aspects of neural development. These include the expression of neuronal phenotypes, factors controlling ontogenic nerve cell death, the development of electrical and chemosensitivity functions, synaptogenesis, and to identify neural specific proteins which may mediate some developmental functions.
Cells of a transformed neuronal line, neuroblastoma, were used as model neuroblasts to study the expression of genes for neuronal phenotypes. Different agents which induced differentiation in these cells produced a differential emergence of neuronal properties. Thus the development of active or passive electrical functions was not always parallel to a similar degree by development of acetylcholinesterase activity or neurite extension.
These findings suggest that genes for neuronal properties may not be under coordinate control.
Ontogenic cell death in the sympathetic nervous system was found to occur in two phases: an initial period when most neurones degenerated and a second period of longer duration where the loss was less marked. This pattern of neuronal death was maintained when the neurones were transferred to the culture environment. In vitro, the degeneration of neurones during the initial phase was not halted by treatment with nerve growth factor (NGF), but could be controlled by factors which accelerated morphological differentiation, such as prolonged depolarization, or soluble factors derived from heart and glioma cells. Synapse formation did not appear to be obligatory for neuronal survival. Cell death during the second phase was circumvented in vitro by treatment with NGF.
In keeping with data from the neuroblastoma studies, the development of neuronal characters in embryonic sympathetic neurones was uncorrdinated. In dissociated culture, these cells showed an arrested development of electrical functions but a marked increase in cholinoceptive activity. The membrane density of acetylcholine receptors was measured by quantitative iontophoresis and the changes observed correlated with those values obtained by (125I)-α-bungarotoxin binding. Quantitative iontophoresis was used to determine a value for the Hill coefficient of the acetylcholine response in these cells and this, together information from the literature has led to a model for the acetylcholine receptor complex in these cells.
Synaptogenesis was also studied using cell culture techniques. Evidence was obtained that sympathetic preganglionic neurones established inappropriate but stable synapses on skeletal myotubes and that motor neurones form stable inappropriate synapses on sympathetic neurones. The results are discussed in relation to models which attempt to account for the origin of specificity during synaptogenesis.
In an attempt to locate molecular species which might mediate some neuronal developmental changes, embryonic sympathetic neurones were used as an immunogenic complex to prepare monoclonal antibodies against neural-specific determinants. Screening of the products from hybridoma cell lines identified one clone, H3, that produced antibodies which bound to the plasma membrane and to cytoplasmic sites in neurones, but only to cytoplasmic sites in glial cells. When added to newly dissociated sympathetic neurones in cell culture, H3 antibodies blocked neurite extension and diminished long-term neuronal viability. When H3 antibodies were added to established cultures of sympathetic neurones to detrimental effects resulted. The nature and function of the H3 antigen remains to be determined but preliminary studies suggest the antigen is a protein of molecular weight approximately 4 0,000.