Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: Applications and pitfalls

We have examined five conjugated 10,000 mol.wt. dextrans as potential anterograde tract tracers: Lucifer Yellow, Texas Red, fluorescein, Cascade Blue and tetramethylrhodamine. Pressure injections were made into the brain, dorsal root ganglia or footpads of adult rats. The retrograde tracer Fluoro-Gold was injected alone or mixed with the dextrans before injection. Three-14 days after injection, animals were perfused and sections cut with a freezing microtome. Texas Red-,fluorescein- and tetramethylrhodamine-conjugated dextrans produced intense labeling of neuronal cell bodies, axons and dendritic processes at the injection site and were transported by neurons predominantly in an anterograde direction to yield terminal and preterminal labeling. Relative to the fluorescein and tetramethylrhodamine conjugates, the quality and intensity of the anterograde labeling produced by Texas Red was variable. Results with Lucifer Yellow and Cascade Blue conjugates were negative. Optimal results were produced by slow-pressure injections via glass micropipettes. In comparison with Fluoro-Gold, retrograde transport by the dextran conjugates was present, but limited in its extent. Injections of the tetramethylrhodamine conjugate into dorsal root ganglia produced anterograde labeling of afferent fibers in visceral organs and injections into the nucleus ambiguus labeled motor fibers in the esophagus. Double/triple labeling was observed in the brain and spinal cord following multiple injections of fluorescein, tetramethylrhodamine and Fluoro-Gold. Also, Fluoro-Gold could be mixed with one of the dextrans in order to produce specific retrograde and anterograde labeling from the same injection site. The conjugates were compatible with fluorescent immunocytochemical procedures, but proved unsuitable for peripheral injections. All central injections produced intensely dye-labeled macrophage or pericytes associated with blood vessels, but these cells were easily distinguished from labeled neuronal structures. The potential for labeling fibers of passage represents a major pitfall with these dextran conjugates and must be considered when using these sensitive tracers.