Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer

This report describes a method of marking nerve cells which is approximately 100 times more sensitive than those previously available. The method depends upon intracellular injection of a new, highly fluorescent dye, Lucifer Yellow CH, which can be viewed both in living tissue and after fixation and embedding. The intense fluorescence of the dye makes injected neurons visible in cleared wholemounts, where the complex three-dimensional structure of neurons is readily apparent.Three new observations have been made with Lucifer Yellow. First, many of the invertebrate neurons studied possess an extensive and complex array of fine processes not visible with other techniques. Second, dye spreads rapidly within an injected cell. Third, dye frequently spreads from the injected cell directly to certain other cells. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily, but not exclusively, between cells known to be electrically coupled.Dye-coupling in the turtle retina revealed striking and distinctive patterns of connections. Type I horizontal cells appear to be multiply connected to each other in an extensive net. Type II horizontal cells are often connected to each other in a hexagonal array. Individual type I and type II cells, widely separated, are frequently dye-coupled; in one case, they were connected by a dyefilled axon.Dye-coupling, readily observed because of the low molecular weight and the intense fluorescence of the new dye, may serve as a general method of tracing certain functional connections by morphological means, and of studying the transfer of small molecules between cells. Preliminary results suggest that systems of dye-coupled cells are substantially more common than was previously believed.