Work on how PC12 cells can be neuronally differentiated has taken two directions. (A) The use of doubly treated cells is a model system for synaptic vesicle properties. When PC12 cells are treated with both nerve growth factor (NGF) and ras-oncogene, the small 40-60 nm-wide vesicles in neurite varicosities more closely resemble such vesicles in vivo. The number of vesicles increases and, for the first time in a cell line, form clusters as they do in vivo. Aggregation enabled unequivocal immunocytochemical localization of synapsin I and synaptophysin to the vesicles and establish them as synaptic. By gel electrophoresis, the amounts of synapsin I levels rose in the treated PC12 cells while synaptophysin fell in NGF- treated cells. Other properties of synaptic vesicles can be probed with this system. (B) Identification of transcription patterns created by NGF and ras. Most, but not all, of the effects of NGF on PC12 cells can be mimicked by transduction with ras. It is still unknown as to which elements are critical for NGF differentiation and which genes are induced in the process. The technique of differential display of reverse transcribed mRNA from PC12 cells is being used to identify changes in mRNA expression. Eight transcripts were increased in NGF and ras cells and a ninth was abundantly expressed in ras but not in NGF treated cells. The pathway of neuronal differentiation by the two stimulations are closely parallel but not equivalent.