Use of Cultured Neurons and Neuronal Cell Lines to Study Morphological, Biochemical, and Molecular Changes Occurring in Cell Death

This chapter discusses the use of cultured neurons and neuronal cell lines to study morphological, biochemical, and molecular changes occurring in cell death. Pheochromocytoma cell line PC12 has been used to study various aspects of neuronal cell death. The chapter describes the ways in which this cell line—termed PC6-3—is maintained and differentiated. Because differentiated PC6-3 cells can be used as a model system for sympathetic neurons, a protocol for growing primary cultures of sympathetic neurons from the superior cervical ganglia is also outlined. PC6-3 cells are subcloned from the PC12 cell line. In their naive state, they grow as single, serum-dependent cells that can be passaged repeatedly. When treated with nerve growth factor (NGF), however, the cells undergo a slow, but relatively steady, differentiation into a neuronal phenotype. Within 6–8 days, the cells have developed neurites, have become nonmitotic, and can be subcultured out of the standard PC12 medium into medium designed to support primary cultures of neurons. After subculturing into DME/Fl2 based medium, the cells assume a more rounded neuronal morphology, grow a very extensive neurite network, and undergo dramatic, transcription-dependent cell death on withdrawal of NGF.