On the use of retrograde tracers for identification of axon collaterals with multiple fluorescent retrograde tracers

Abstract

A common method for identifying collateral projections is to inject different retrograde tracers into two targets and examine labeled cells for the presence of both tracers. Double-labeled cells are considered to have collateral projections to the two injection sites. This method is widely considered to underestimate the extent of collaterals. To test the efficiency of double-labeling, we mixed equal volumes of two tracers, injected them into one site in a guinea-pig brain, and counted the resulting labeled cells. Ideally, the tracers would have precisely overlapping injection sites and all labeled cells would contain both tracers. We tested several combinations of tracers: 1) Fast Blue and fluorescein dextran; 2) fluorescein dextran and FluoroGold; 3) fluorescein dextran and FluoroRuby; 4) FluoroGold and green beads; 5) FluoroGold and red beads; 6) FluoroRuby and green beads; and, 7) green beads and red beads. For each combination, a mixture was injected into the left inferior colliculus. After 1 week to allow for transport, labeled cells were counted in the right inferior colliculus and the left temporal cortex. For each mixture, the results were similar for the two areas. The percentage of cells that were double-labeled varied from 0% to 100%, depending on tracer combination. The highest efficiencies (>96%) were observed with red beads and green beads or with FluoroRuby and fluorescein dextran. The limited efficiency of other mixtures could be accounted for only in part by incomplete overlap of the two tracers at the injection site. The results indicate that the specific combination of tracers used to search for collateral projections can greatly affect the findings.

Anatomical substrates for direct interactions between hippocampus, medial prefrontal cortex, and the thalamic nucleus reuniens

“…Double-labeled cells represented about 1 % of the overall population of retrogradely labeled cells, and the fraction was highest among the mPFC-projecting cells (7.38 % on average also projected to reuniens). Anatomical tracers have occasionally been reported to preferentially bind to certain projections (Schofield et al 2007; Deng and Rogers 1999) and we cannot completely rule out that the differences in the percentage of double-labeled cells in reuniens compared to the subiculum do not result from biases in the binding of CTB-AF conjugates. Nonetheless, CTB is considered a sensitive retrograde tracer (Conte et al 2009) and enters cells by binding to gangliosides in the cell membrane (Stoeckel 1977; Joseph et al 1978).…”

Branched projections in the auditory thalamocortical and corticocortical systems

“…However, to be equally accurate, the tracers used must be matched for membrane affinity, injection site size, visualization methods, the likelihood for producing damage affecting transport, and tracer-specific differences in transport rate (Schofield et al, 2007). CTβ and CTβG are good candidates because of their biochemical similarity and extremely high membrane affinities.…”

“…BAs can create feature-specific maps or synchronize processing within remote brain regions (Schofield et al, 2007). Although there are at least five maps of characteristic frequency (CF) within AC (Morel and Imig, 1987), there are only two within the MGB, one in the rostral pole (RP) and another in the ventral division (MGBv) (Aitkin and Webster, 1972;Imig and Morel, 1985).…”

Enhancing Fluorogold-based neural tract tracing

“…Despite enhanced tracing speed, the FG fluorescence is very low in these long distance targets in the cat compared to that typically reported in studies with rodents (Van Bockstaele et al, 1994;Novikova et al, 1997;Schofield et al, 2007). This is likely a function of the size difference between species.…”

“…While the majority of these papers focused on cell death at the final target location (Garrett et al, 1991;Naumann et al, 2000), tissue damage at the injection site also has been noted (Schmued and Fallon, 1986) with higher FG concentrations in one study in the rat (5-10% vs 2-3%). FG also has been reported as neurotoxic in the guinea pig (Schofield et al, 2007) and even lethal in certain transgenic mice (He, 2009). Further, intravenous or subcutaneous delivery of the chemical that underlies FG, Hydroxystilbamidine, leads to varying degrees of toxicity in the liver, kidney, and central nervous system of rabbits and mice (Festy, 1979).…”