Resumo

Introduction: In most species, dopamine is the main catecholamine expressed in the retina. It is usually found in amacrine neurons, and modulates visual information through the activation of D1 and D2-like metabotropic receptors, as part of a circuit known as the horizontal pathway. Dopamine formation in this tissue normally happens through the conversion of the amino acid tyrosine by the enzyme tyrosine hydroxylase (TH) into L-DOPA, which is then decarboxylated to dopamine by dopa decarboxylase (DDC). In the chick retina, TH is the rate limiting enzyme in this process and is found only after embryonic day 12 (E12), although dopaminergic activity, as well as transporters and DDC expression can be detected much earlier in the tissue. It has been proposed that an alternative source of L-DOPA, the enzyme tyrosinase, present in the adjacent pigmented epithelium, could supply the neuroretina and allow for early dopamine formation, days before TH expression. This alternative pathway to dopamine synthesis was reported in 2003 (J. Neurochem. 2003, 86: 45-54). In addition to the classical role in visual perception, dopamine has more recently emerged as an important agent in development and cellular differentiation. An early work by our group has shown that in the chick retina, dopaminergic cell development is under the influence of signals that involve the cAMP pathway. Moreover, we demonstrated in vitro that dopamine levels can influence the number and morphogenesis of TH positive cells, the classic marker of the dopaminergic phenotype. Our data established that a mechanism of self-regulation might exist in this system, by which dopamine itself may negatively regulate dopaminergic differentiation (Eur. J. Neurosci. 2001, 13: 1931-1937). The goal of the current study is to analyze the effects of blocking dopamine production in vivo, on the ongoing differentiation of the dopaminergic phenotype in the chick retina. Methods: Injections of the DDC inhibitor hydroxybenzyl hydrazine (HBH) were made into the eggs of E8 chicks (Gallus gallus domesticus; white leghorn), to prevent dopamine production during the period in which the alternative pathway is the only source of retinal dopamine. Controls received PBS injections. The animals were later (E15 or post-hatch) sacrificed and their retinas dissected out to be analyzed through imunohistochemistry, western blotting and a radiometric assay to measure cAMP accumulation. Results and conclusions: Retinas from animals injected with 0,1mM HBH in E8 show a sustained increase of over 60% in cAMP accumulation when later (E15) stimulated with either dopamine or L-DOPA. This effect remains even after the hatching. Using imunohistochemistry we see a significant increase of DDC positive cells, of almost 2-fold, which can be confirmed by western blotting. TH-positive cells also display a clear tendency to increase, in both number and complexity, that is yet to be quantified. These results show an overall potentiation of dopaminergic communication and cellular phenotype in injected animals, strongly suggesting a role as a feedback regulator for the dopamine produced during the earlier stages of development, through the alternative pathway.