Neural development in dissociated cell cultures of fetal rat brain can be expected to depend on synaptic interactions between cultured neurons. Therefore, an attempt was made to obtain a quantitative measure of the time course of synaptogenesis in such a culture system by assessing the level of the secretory vesicleassociated protein synaptophysin (p38). The developmental schedule of p38 was compared to that of neuron-specific enolase (NSE), an established marker of neuronal differentiation.
Cultures were raised from dissociated 14 day-old fetal rat diencephalon. In cultures grown for 1-2 days in vitro (DIV), p384mmunoreactivity was preferentially located in neuronal perikarya. After 10-16 DIV, neurons in culture had formed a dense neuritic network, and almost all of the p384mmunoreactivity occurred in the form of fine punctate deposits associated with neuronal processes that often outlined neuronal cell bodies in a basket-like fashion. Electron-microscopic immunocytochemistry proved the punctate deposits to be presynaptic elements, mostly in the form of axonal varicosities. Quantitative immunoblotting showed that levels of p38 increased from the start of cultivation to DIV 4, stayed fairly constant from DIV 4 to DIV 8, and rose again steeply to peak at DIV 12. In contrast, levels of NSE rose continuously up to DIV 12. After DIV 12, levels of both p38 and NSE fell again.
Treatment of cultures with dimethyl sulfoxide (DMSO), an agent known to induce differentiation in various normal and malignant cell types, resulted in a significant increase of p38 levels and in a decrease of NSE levels. The amount of p38 continued to increase beyond DIV 12, whereas NSE diminished after having reached a maximum at DIV 12.
Assessment of p38 levels as a measure to quantify synaptogenesis in culture has been validated through combined morphological observations and quantitative immunoblotting. The results show that expres-sion of NSE and p38 during neuronal development follows different time schedules and that the two neuron-specific proteins can be manipulated independently from each other. DMSO treatment appears to selectively promote synaptogenesis and/or maturation of specific synaptic properties.