Study of the regenerating newt retina by electrophysiology and immunohistochemistry (bipolar- and cone-specific antigen localization).

Immunohistochemical and electrophysiological examinations were carried out to investigate the sequence of appearance of the retinal neurons during regeneration after a complete surgical removal of the original retina of the newt. We produced a monoclonal antibody, RB-1, specific for cone photoreceptors and a subtype of bipolar cells in adult newt retina. This antibody was used as a major tool for this analysis. Appearance of spiking activity as a possible marker of ganglion cell differentiation was examined with whole-cell patch-clamp techniques. Spiking cells, which possessed voltage-dependent Na+, K+, and Ca2+ channels similar to those of mature ganglion cells, appeared in the regenerating retina by 24 days before cone photoreceptors had been labeled by the RB-1 antibody. Cones and ganglion cells differentiated before the retina had been segregated into distinct synaptic layers. The RB-1-labeled bipolar cells as well as PKC-immunoreactive bipolar cells appeared in the regenerating retina after the segregation of the synaptic layers. Their appearance seemed to coincide with the appearance of immunoreactive amacrine cells described previously (Negishi et al. [1992] Dev. Brain Res. 68:255-264). During embryonic development of the newt retina, cone photoreceptors appeared prior to bipolar cells. Thus the process of reformation of a functional retina seems to follow the same steps as differentiation of retina during development.