THE KINETICS OF TRACER MOVEMENT THROUGH HOMOLOGOUS GAP-JUNCTIONS IN THE RABBIT RETINA

Observation of the spread of biotinylated or fluorescent tracers follo wing injection into a single cell has become one of the most common me thods of demonstrating the presence of gap junctions. Nevertheless, ma ny of the fundamental features of tracer movement through gap junction s are still poorly understood. These include the relative roles of dif fusion and iontophoretic current, and under what conditions the size o f the stained mosaic will increase, asymptote, or decline. Additionall y, the effect of variations in amount of tracer introduced, as produce d by variation in electrode resistance following cell penetration, is not obvious. To examine these questions, Neurobiotin was microinjected into the two types of horizontal cell of the rabbit retina and visual ized with streptavidin-Cy3. Images were digitally captured using a con focal microscope. The spatial distribution of Neurobiotin across the p atches of coupled cells was measured. Adequate fits to the data were o btained by fitting to a model with terms for diffusion and amount of t racer injected. Results indicated that passive diffusion is the major source of tracer movement through gap junctions, whereas iontophoretic current played no role over the range tested. Fluorescent visualizati on, although slightly less sensitive than peroxidase reactions, produc ed staining intensities with a more useful dynamic range. The rate con stants for movement of Neurobiotin between A-type horizontal cells was about ten times greater than that for B-type horizontal cells. Althou gh direct extrapolation to ion conductances cannot be assumed, tracer movement can be used to give an estimate of relative coupling rates ac ross cell types, retinal location, or modulation conditions in intact tissue.