Optical Recording from Individual Neurons in Culture

Methods for optically recording dynamic processes in single living neurons must be considered in light of two fundamental questions: what to record and how to record it. Specifically, deciding what to record involves determining a parameter of interest (e.g., membrane potential or ion concentration), the nature of the information required (e.g., qualitative or quantitative) and the optical indicator best suited to making these measurements. Likewise, deciding how to record these signals involves consideration of recording methodologies (i.e., photometry or imaging), experimental procedures (e.g., loading and staining protocols) and data processing techniques (i.e., signal processing and analysis). Irrespective of which combination of methods are chosen it is important to understand the essential factors that contribute to obtaining high quality optical signals. By fully understanding the fundamental limits of this recording methodology, the novice investigator should be able to maximize signal quality and effectively solve any technical problems that might arise. This chapter considers both instrumentation and experimental factors and their implications for making both qualitative and quantitative optical recordings from individual neurons in culture. In particular, appropriate methods for making fast recordings of various physiological parameters, with subcellular resolution, are documented. Two classes of indicators, voltage-sensitive dyes and calcium indicators, are used to illustrate the principles underlying successful optical recording. These principles can easily be extrapolated to other indicator types. The scope of this chapter is limited to the consideration of methods for making physiological recordings from individual neurons or small group of cells in culture. Therefore, we do not consider methods used to examine fine structural details or localize cellular markers. Nor do we consider the cell culture methods necessary to produce neurons suitable for optical recording (instead, see Chapter 10). However, several cell culture properties that should be optimized to facilitate this kind of recording are documented. Optical recording methods for use in more complex tissues such as brain slices or in vivo preparations are considered in other chapters in this volume. In particular, the reader is referred to Sinha and Saggau (Chapter 16) and Grinvald et al. (Chapter 34). There are a number of preliminary steps that must be undertaken prior to conducting an experiment employing optical recording methods. The remainder of this section examines several such issues. These include: – Desirable properties of cell cultures for optical recording – Methods for visualizing single neurons