The purpose of this study was to investigate the effect of four fluorescent dyes, True Blue (TB), Fluoro-Gold (FG), Fluoro-Ruby (FR), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), in retrograde tracing of rat spinal motor neurons. We transected the muscle branch of the rat femoral nerve and applied each tracer to the proximal stump in single labeling experiments, or combinations of tracers (FG-DiI and TB-DiI) in double labeling experiments.
In the single labeling experiments, significantly fewer labeled motor neurons were observed after FR labeling than after TB, FG, or DiI, 3 days after tracer application. By 1 week, there were no significant differences in the number of labeled neurons between the four groups. In the double-labeling experiment, the number of double-labeled neurons in the FG-DiI group was not significantly different from that in the TB-DiI group 1 week after tracer application. Our findings indicate that TB, FG, and DiI have similar labeling efficacies in the retrograde labeling of spinal motor neurons in the rat femoral nerve when used alone. Furthermore, combinations of DiI and TB or FG are similarly effective. Therefore, of the dyes studied, TB, FG and DiI, and combinations of DiI with TB or FG, are the most suitable for retrograde labeling studies of motor neurons in the rat femoral nerve.