Purpose: To measure the concentrations of polyamines, determine their cellular and subcellular localization, and analyze effects of their depletion in developing rabbit retina.
Methods: Isolated retinas at different developmental stages were analyzed for polyamine content by high-performance liquid chromatography (HPLC). An antibody against polyamines was used to localize endogenous stores in both freshly harvested retinas and neonatal retinal explants. To determine the effects of polyamine depletion on immature retina, neonatal explants were cultured in the presence or absence of alpha-difluoromethylornithine (DFMO), an inhibitor of the polyamine synthetic enzyme ornithine decarboxylase (ODC). Similar studies were also performed on dissociated cell cultures. Tissue was assessed using standard histologic stains as well as cell-specific markers (peanut agglutinin for cone photoreceptors and calbindin for horizontal cells).
Results: Retinal polyamine content was highest at birth, remained relatively high during the first postnatal week, and then steadily decreased to adult levels. At all ages analyzed, spermine concentration was higher than putrescine or spermidine; however, the differential was greatest in the adult. Polyamine immunoreactivity was localized to distal processes of both rods and cones during development. Strong immunoreactivity was maintained in adult cone inner and outer segments; comparatively weak staining was observed in the adult rods. Heavy staining of ganglion cells was present throughout development but was localized in the cytoplasm in immature cells and in the nucleus in the adult. Amacrine cells stained only in the adult. Polyamine depletion caused a disruption of immature cones, evident in the loss of their somata in the outer nuclear layer, in their processes in the outer plexiform layer in retinal explants, and in their decreased association with horizontal cells in dissociated cell culture.
Conclusions: The relatively high concentrations of polyamines in neonatal retina and their discrete localization in developing photoreceptor outer segments and ganglion cells suggests an important role for these compounds in development. The disruption of cone-specific markers in polyamine-depleted retinas indicates a specific reliance on polyamines for expression of normal cone morphology or morphologic development. These developmental effects may involve polyamine-sensitive ion channels, which are known to exist in retina, or direct interactions with specialized cytoskeletal elements within outer segments.