ATP and ADP induce retinal cell proliferation through activation of PKC and extracellular signal-regulated kinases (ERKs). Here, we characterized the effect of purinergic agonists on the turnover of phosphoinositides and activation of ERKs during development of the chick embryo retina. When intact retinas were incubated with ATP, ADP or UTP, a dose-dependent accumulation of [(3)H]-phosphoinositides was observed (% of control, EC(50): 548+/-20.5%, 0.18 mM; 314+/-53.8%, 0.51 mM; 704+/-139.9%, 0.018 mM, respectively). Only the response promoted by ADP was completely inhibited by the P2 receptor antagonists, PPADS and suramin. All the responses decreased with the progression of retinal development. Western blot assays revealed that ATP, ADP and UTP stimulated the phosphorylation of ERKs in the chick embryo retina very early during development (% of control: 174+/-16; 199+/-16.4 and 206+/-37, respectively). The responses to ADP and UTP were transient and dose-dependent, showing EC(50) values of 0.12 mM and 0.009 mM. The response to ADP was inhibited by the antagonists PPADS and suramin and by U73122 and chelerythrine chloride, which block PLC and PKC, respectively. Conversely, chelerythrine chloride did not block the response induced by UTP. Immunohistochemical analysis revealed that ATP and ADP induced the phosphorylation of ERKs in cells of the neuroblastic layer of retinas from embryos at E8. Our data showed that ATP, ADP and UTP stimulate the turnover of InsPs and promoted the activation of ERKs in the chick embryo retina. ADP, through activation of P2Y(1) receptors, activated ERK pathway through PLC and PKC and UTP, via P2Y(4)-like receptors, induced the phosphorylation of ERKs through a pathway that did not involve PKC.

Previous data suggest that nucleotides are important mitogens in the developing chick retina. Here, we extended the study on the mitogenic effect of ATP to newborn mouse retinal explants. Our results showed that P2Y(1) receptors were widely distributed in C57bl/6 mice retina and that the majority of PCNA positive cells co-localized with P2Y(1) receptor. To evaluate proliferation, retinal explants obtained from newborn mice were incubated with 0.5 microCi [(3)H]-thymidine or 3 microM BrDU 1h before the end of culture. Our data showed that ATP induced a dose-dependent increase in [(3)H]-thymidine incorporation, an effect that was mimicked by ADP but not by UTP and was blocked by the P2 antagonist PPADS in a dose-dependent manner. The increase in [(3)H]-thymidine incorporation induced by ATP was only observed in explants cultured for 3 days or less and was mimicked by the ectoapyrase inhibitor ARL 67156. It corresponded to an increase in the number of BrdU(+) cells in the neuroblastic layer (NL) of the tissue, suggesting that ATP, through activation of P2Y(1) receptors, induced proliferation of late developing progenitors in retinal explants of newborn mice. The increase in the number of BrdU(+) cells was observed across the whole NL when explants were incubated with ATP for 24h and no increase in the number of p-histone H3 labeled cells could be noticed at this time point. In longer incubations of 48h with ATP or 24h with ATP followed by a period of 24h in fresh medium, an increase in the number of BrdU(+) cells promoted by ATP was observed only in the middle and outer, but not in the inner NL. In these conditions, an increase in the number of p-histone H3 labeled cells was detected in the outer NL, suggesting that ATP induced cells to enter S and progress to G2 phase of the cell cycle in the first 24h period of incubation. ATP also induced an increase and a decrease in the expression of cyclin D1 and p27(kip1), respectively, in retinal progenitors of the NL. While the increase in the expression of cyclin D1 was observed when retinal explants were incubated for 3h or longer periods of time, the decrease in the expression of p27(kip1) was noticed only after 6h incubation with ATP. Both effects were blocked by the P2 receptor antagonist PPADS. These data suggest that ATP induces cell proliferation in retinal explants by inducing late developing progenitors to progress from G1 to S phase of cell cycle.

No abstract

The retinotectal projection of rodents presents a precise retinotopic organization that develops, from diffuse connections, from the day of birth to post-natal day 10. Previous data had demonstrated that these projections undergo reorganization after retinal lesions, nerve crush and monocular enucleation. The axonal growth seems to be directly related to growth-associated protein-43 (GAP-43) expression, a protein predominantly located in growth cones, which is regulated throughout development. GAP-43 is presented both under non-phosphorylated and phosphorylated (pGAP-43) forms. The phosphorylated form, has been associated to axon growth via polymerization of F-actin, and synaptic enhancement through neurotransmitter release facilitation. Herein we investigated the spatio-temporal expression of GAP-43 in the rat superior colliculus during normal development and after monocular enucleation in different stages of development. Lister Hooded rats ranging from post-natal day 0 to 70 were used for ontogeny studies. Another group of animals were submitted to monocular enucleation at post-natal day 10 (PND10) or PND21. After different survival-times, the animals were sacrificed and the brains processed for either immunohistochemistry or western blotting analysis. Our data show that GAP-43 is expressed in retinotectal axons in early stages of development but remains present in adulthood. Moreover, monocular enucleation leads to an increase in pGAP-43 expression in the deafferented colliculus. Taken together these results suggest a role for pGAP-43 in retinotectal morphological plasticity observed both during normal development and after monocular enucleation.

Natural cell death is a degenerative phenomenon observed during the normal development of the nervous system. The neuroprotective effects of cytokines produced by neuronal, glial or infiltrating cells on neurons have been extensively studied. In this work we studied the role of interleukin (IL)-2 and IL-4 on the survival of retinal ganglion cells (RGC) after 48 h in culture. Our results demonstrate that the effect of both ILs was dose-dependent and the treatment with either IL-2 (50 U/ml) or IL-4 (5 U/ml) induced a 2-fold increase in RGC survival. The effect of IL-4, but not of IL-2, was totally abolished by either 20 microM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, or by 1 microM telenzepine, an inhibitor of M1 muscarinic receptor. Our results suggest that both cytokines could play an important role during the development of retinal tissue as well as during retina trauma.