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

The lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label neuronal pathways in fixed goldfish brains. The normal procedure involving 4% paraformaldehyde as a fixative, applying DiI and storing the brain in the fixative at 40 degrees C resulted in many cases in a rather diffuse labeling of fiber pathways and the occurrence of transneuronally labeled cells and fibers. We found that calcium and heat both facilitate the diffusion of DiI out of membranes in vibratome sections. We modified the protocol by adding the calcium binding substance ethylenediamine tetraacetate to all solutions and incubated at room temperature. This improved the sharpness of labeled structures and eliminated the transneuronal labeling in our material. Although transneuronal transport of the tracer may still occur under certain circumstances, the present modification of the DiI staining procedure substantially increased the staining quality and reproducibility and decreased the occurrence of transneuronal labeling.