Retrograde axonal tracing with fluorescent markers

The growth of fluorescence imaging technology and the development of sensitive fluorescent retrograde tracers has provided many new approaches for analyzing neuronal circuits. Fluorescent markers provide unparalleled opportunity for combining axonal tract tracing with techniques such as immunohistochemistry or physiological recording. This unit describes the use of six different fluorescent tracers: Fast Blue, fluorescein dextran, FluoroGold, FluoroRuby, red beads, and green beads. Guidance is provided on how to choose a tracer for a particular experiment, and three methods are described for injecting the tracers, including pressure injection through a microsyringe or a micropipet, and iontophoretic injection through a micropipet. Criteria for selecting the most appropriate method are discussed. The protocols provide the information necessary to take advantage of the numerous fluorescent tracers that are available and to apply them to a wide variety of scientific questions.