During the past five decades the practice of cell and tissue culture has become a cornerstone of research in cell biology. Growing cells for use in microscopic or biochemical studies is an entirely routine laboratory procedure. Yet many cell biologists who are comfortable handling immortalized cell lines remain intimidated by the aura surrounding the use of neurons, especially as primary cultures. This is unfortunate because the continual refinement of culture techniques and reagents has produced reliable procedures for obtaining, growing, and studying dozens of neuronal cell types with diverse and useful properties. It is the intention of this volume to stimulate the interest of cell biologists in utilizing neurons in their studies by demystifying the methods necessary to handle them. It is hoped that the collection here of detailed, current cell culture methods for neurons of all sorts will make working with neurons more appealing to the uninitiated.

But why should a cell biologist bother to study neurons in culture? What purpose can the neuron serve that the HeLa cell cannot? In fact, many important cell biological questions have been pursued differently—and sometimes more effectively—in neurons than in other cell types. For example, it is hard to imagine Drosophila our current knowledge of cellular morphogenesis, signal transduction, intracellular trafficking, exocytosis, or the cytoskeleton without the critical studies that have been performed in neurons. Even many areas once thought to be special features of neurons are in fact amply worthy of study by cell biologists. Some of these have, upon close analysis, proven to be more general features of cells that simply reach a pinnacle of development—and therefore accessibility—in neurons. It has long been clear, for instance, that fast axonal transport involves the same mechanisms as organelle traffic in other cells. And what is synapse formation except a specialized kind of cell—cell interaction? It behooves us to study neurons anytime that a phenomenon that interests us appears particularly well exposed there. To this end, this volume contains not only methods for growing a variety of neurons, but also procedures for employing them in specific experimental applications. In addition, we have included two in vivo systems— one of which is amenable to genetic manipulations—for those who need to study their cell biological question in a developmental or organismal context.

So what is it that makes the use of neurons seem daunting? They are typically primary cultures, and the dissection usually necessary to acquire them can seem laborious compared to thawing a tube of 3T3 cells. But this alone need not be an obstacle—primary culturing of nonneuronal cells, such as fibroblasts, is routine in laboratories around the world. Unlike fibroblasts, however, neurons are postmitotic cells, often with a very limited lifetime in culture, and thus require regular schedules of dissection coordinated with the timing of experiments. But freezing procedures for primary tissue and the availability of neuronlike transformed cells ameliorate some of these problems, and these are treated in several chapters of this volume. Another part of the lore of neuronal cell culture is that they are difficult cells to transfect. However, in recent years a variety of vectors and procedures have been developed that will allow neurons to be used by most workers whose studies require the introduction and expression of exogenous DNA and proteins, and this volume also contains several chapters on these methods. Finally, there is the perception that the culture media and growth conditions for neurons are more finicky than for other cells, or even that they are incompletely defined. Although we neuronal cell cultists have not always helped to dispel this ''eye of newt'' reputation, the methods presented in this volume have been chosen for their robustness and reliability. If you can grow BHK cells, you can grow sensory neurons, and the others described here as well. Because there have been other volumes in this series dedicated to cell culture methods, we have chosen not to repeat the general information on biological safety cabinets, media, incubators, etc., an excellent review of which may be found in Volume 57 of this series.

The editors heartily thank the contributors of this volume for sharing their hard-won expertise at using neurons for cell biological studies. We also thank the Methods in Cell Biology series editors for their help and advice in assembling this volume.

Peter Hollenbeck Jim Bamburg

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