Improved detection of fluorogold-labeled neurons in long-term studies

Fluorogold (FG) is a widely used neuroanatomical tracer. However, because FG-labeled neurons become undetectable over time, its use has been limited in long-term studies. We investigated whether the detection of FG in retrogradely labeled neurons in long-term studies can be improved by immunohistochemistry (IHC) using an antibody to FG. We performed intraperitoneal injections of a FG solution to retrogradely label all parasympathetic preganglionic neurons (PPNs) and motoneurons (MNs) in the S1 spinal cord segment in adult rats. At 1, 6, and 12 weeks after the tracer injection, sections were immunohistochemically processed for FG and choline acetyltransferase (ChAT), an endogenous marker for all PPNs and MNs. Stereological counts demonstrated no cell loss of FG-labeled PPNs and MNs at 6 and 12 weeks. Cell size measurements showed that FG-immunolabeled neurons were smaller at 12 weeks, but not at 6 weeks. However, it is likely that there was no neuronal atrophy, but loss/degradation of the dye at a timepoint between 6 and 12 weeks, as ChAT-immunolabeled neurons showed no cell size reduction at 12 weeks. Our results suggest that the use of an antibody against FG improves the detection of FG for reliable neuronal counts and that the dye is not toxic to the retrogradely labeled neurons. We conclude that FG-labeling is a useful tool to determine neuronal counts in long-term studies, but should be used cautiously for neuronal size measurements.