Comparative analysis of calbindin D-28K and calretinin in the retina of anuran and urodele amphibians: Colocalization with choline acetyltransferase and tyrosine hydroxylase

Abstract

Previous studies in amphibians yielded contradictory results about the distribution of calbindin-D28k (CB) and calretinin (CR) in retinal neurons, most likely due to the different antibodies used. The present comparative study aimed to characterize the distribution of CB and CR in relation to retinal neurons in six species of anuran and urodele amphibians by using the same immunohistochemical protocol with specific poly- and monoclonal antibodies. CB was specifically found in cones, in subpopulations of bipolar and amacrine cells and in sparse neurons in the ganglion cell layer. All photoreceptors were negative for CR, whereas subpopulations of horizontal, bipolar and amacrine cells as well as cells in the ganglion cell layer contained CR. CB/CR colocalization occurred in some amacrine cells and in cells of the ganglion cell layer, with variable proportions among species. Most of the ganglion cells identified by retrograde labeling from the optic nerve contained CB and/or CR. Cholinergic cells, visualized by choline acetyltransferase (ChAT) immunoreactivity, constituted a subpopulation of the CR-positive amacrine cells in anurans and a high percentage (40-90%) of cholinergic cells were CR immunoreactive in urodeles. CB/ChAT colocalization was between 10 and 30% in anurans and lower in urodeles (7-10%). Finally, CB colocalized in 6-8% of the tyrosine hydroxylase (TH) amacrine cells only in anurans, whereas CR and TH colocalized in 5% of TH cells in the urodele retina. Our data suggest a specific pattern for CB and CR distribution in the retinal neurons of amphibians comparable to amniotes in some cell types showing, however, peculiar features not observed previously in other vertebrates.