Visualization of active synaptic terminals onto ganglion dendrites using confocal microscopy and three-dimensional optical reconstruction techniques

Purpose. Apply optical reconstruction methods, combined with activity-dependent dyes to visualize and estimate the numbers and positions of synaptic terminals onto ganglion cell dendrites. Methods. Activity-dependent dyes were applied to the amphibian retina to label presynaptic terminals in the IPL. Ganglion cell labeling was achieved through back labeling with dextran conjugated fluorescein, rhodamine green or lucifer yellow (LY). Serial optical sections with a confocal microscope were combined with dual detection methods for separate imaging data from each of two dyes. The two datasets generated by this method were fused using commercially available software packages running on an SOI computer. A special software package Dfield was used to localize and estimate the number of terminals, their proximity and localization along the dendritic tree of a single, reconstructed ganglion cell. Results. Activity-dependent dyes are taken up into terminals of amacrine and bipolar cells in the inner retina, with negligible labeling of ganglion cells dendrites. Optical reconstruction methods reveal that ganglion cells appear to have a relatively small number of presynaptic terminals associated with their dendritic trees, averaging about 12 contacts/1000 /tm2. Computer simulations with realistic ganglion cell morphologies indicate that relatively small conductance changes (-130 pS) injected into these identified sites are sufficient to strongly activate the cell in a manner consistent with physiological observations. Conclusions. Activity-dependent dyes, combined with optical, three dimensional reconstructions techniques provides a powerful, visual insight into the physiological basis of ganglion cell activation.