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

• Published in: Neuroscience Vol. 146; no. 2; pp. 773 - 783
• Main Authors: Schofield, B.R, Schofield, R.M, Sorensen, K.A, Motts, S.D
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 11.05.2007; Elsevier
• Subjects: Amidines; Animals; axon branching; Axons - metabolism; Axons - physiology; Biological and medical sciences; Brain - anatomy & histology; Brain Mapping; Coloring Agents - administration & dosage; Coloring Agents - metabolism; Dextrans - administration & dosage; Dextrans - metabolism; Fast Blue; Female; Fluoresceins - administration & dosage; Fluoresceins - metabolism; fluorescent dextran; fluorescent microspheres; fluorescent tracers; FluoroGold; Fundamental and applied biological sciences. Psychology; Guinea Pigs; Indicators and Reagents - administration & dosage; Male; Neural Pathways - anatomy & histology; Neural Pathways - metabolism; Neurology; Vertebrates: nervous system and sense organs; FG; fluorescent microspheres; fluorescent tracers; axon branching; FluoroGold; MW; molecular weight; FR; RB; red beads; FluoroRuby (tetramethylrhodamine dextran); fluorescent dextran; Fast Blue; inferior colliculus; IC; GB; green beads; FB; FD; fluorescein dextran; Dextran; Axonic collateral; Axon
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2007.02.026

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.