Effect of temperature and calcium on transneuronal diffusion of DiI in fixed brain preparations

The lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanin e perchlorate (DiI) was used to label neuronal pathways in fixed goldfish b rains. The normal procedure involving 4% paraformaldehyde as a fixative, ap plying DiI and storing the brain in the fixative at 40 degrees C resulted i n many cases in a rather diffuse labeling of fiber pathways and the occurre nce of transneuronally labeled cells and fibers. We found that calcium and heat both facilitate the diffusion of DiI out of membranes in vibratome sec tions. We modified the protocol by adding the calcium binding substance eth ylenediamine tetraacetate to all solutions and incubated at room temperatur e. This improved the sharpness of labeled structures and eliminated the tra nsneuronal labeling in our material. Although transneuronal transport of th e tracer may still occur under certain circumstances, the present modificat ion of the DiI staining procedure substantially increased the staining qual ity and reproducibility and decreased the occurrence of transneuronal label ing. (C) 1999 Elsevier Science B.V. All rights reserved.