In vitro imaging of retinal whole mounts.

Neuronal circuits of the vertebrate retina are organized into stereotyped laminae. This orderly arrangement makes the retina an ideal model system for imaging studies aimed at understanding how circuits assemble during development. In particular, live-cell imaging techniques are readily applied to the developing retina to monitor dynamic changes over time in cell structure and connectivity. Such imaging studies have collectively revealed novel strategies by which retinal neurons contact their presynaptic and postsynaptic partners to establish synaptic connections. We describe here the procedures developed in our laboratory for confocal and multiphoton live-cell imaging of the developing retina using in vitro retinal explants. Retinas can be removed from the eye and kept in culture conditions for several days with limited disruption to the retinal circuit. The explanted retina is amenable to a variety of labeling techniques and provides a large, flat, unobstructed surface that is ideal for optical imaging experiments. This protocol describes procedures for mounting and imaging the isolated mouse retina. The same general procedure, with only minor modification (composition of culture medium), has been used to image retinas from a variety of vertebrates (e.g., chick, ferret, and rabbit).