Three-dimensional imaging of presynaptic terminals onto amphibian ganglion cells in a live retina

Purpose. We have developed an "in vivo" approach to label and identify synaptic terminals and their distribution onto retinal ganglion cells using optical reconstruction techniques with a confocal microscope. Methods. Activity-dependent dyes, such as sulforhodamine (SR), were dissolved in Ringer and applied in a superfused retina eyecup preparation. Ganglion cells were stained with a yellow fluorescent probe via retrograde labelling or intracellular iontophoresis using a sharp electrode. Serial reconstructions of ganglion cell dendrites and presynaptic terminals were carried out using dual detection with a laser scanning confocal microscope. Three-dimensional surface and volume rendering of the data base was accomplished with commercially available software on an SGI Crimson Workstation. Results. SR was predominantly taken up into presynaptic terminals of bipolar and amacrine cells and photoreceptors. Presumed synapses onto ganglion cell dendrites were estimated using software algorithms which determined the distance between dendrites and clusters of terminals. Conclusions. Results with this approach provide a quantitative view of the numbers of active synapses and their location on the dendritic tree of retinal ganglion cells, in vivo. This approach may lend itself to permit detailed form and function studies of synaptic transmission at retinal synapses.