Intracellular fluorescent staining with carboxyfluorescein: a rapid and reliable method for quantifying dye-coupling...

Previous studies investigating electrotonic coupling in mammalian central nervous system have used the fluorescent marker Lucifer Yellow as an indicator of the presence of intercellular junctions between neurons. The fluorescent dye 5,6-carboxyfluorescein is known to have approximately 5 times the fluorescent yield of Lucifer Yellow. We have investigated the use of this dye as a potential alternative fluorescent marker on two types of neurons in the rat hippocampus in vitro. Unfixed hippocampal slices were mounted in a mixture of n-propyl gallate in glycerol and viewed with epifluorescence optics. Injections of small, brief hyperpolarizing currents through carboxyfluorescein-filled glass pipettes reliably produced neuronal fills of good quality. Both dendritic spines and axonal arborizations (including the thin mossy fibers of the dentate gyrus) were frequently observable. In addition to single cell fills, clusters consisting of 2-6 neurons were observed. No correlation was found between the number of cells per cluster and the ejection time. In addition, even cells exhibiting poor electrophysiological characteristics, or cells impaled only briefly, frequently exhibited good quality dye filling. This method will be particularly useful when large sample sizes are necessary to compare regional variations in the extent of electrotonic coupling in the mammalian brain.