Expression Pattern of P2Y1 Purinergic Receptor on Intact and Injured Retinas of Zebrafish

MARIA PAULA FAILLACE AND ARIADNA BATTISTA

Fort Lauderdale, Florida

Congreso; ARVO 2012- Translational Research: Seeing the possibilities; 2012

The Association for Research in Vision and Ophtalmology (ARVO)

Expression Pattern of P2Y1 Purinergic Receptor on Intact and Injured Retinas of Zebrafish We previously described that injury-induced cell proliferation and death are significantly regulated by extracellular nucleotides mainly acting via P2Y1 receptors in the zebrafish retina damaged only in the inner layers. We aimed to determine whether a cytotoxic injury that provokes global cell death was able to change the level and pattern of expression of P2Y1 receptor protein in mature layer and ciliary marginal zone (CMZ) cells.   Zebrafish were treated with one intraocular injection of 10 μM ouabain. In situ cell death was detected by TUNEL in injured and apyrase (used as a nucleotide scavenger)- or MRS2179 (P2Y1 receptor antagonist)-treated retinas. Purinergic receptor mRNA expression was determined by RT-PCR. P2Y1 receptor was assessed by immunocytochemistry (ICC) on retinal sections 5 and 7 days following injury. Some zebrafish were lesioned with ouabain and injected with BrdU 24 h before euthanization on day 7. Double labeling ICC assays of the P2Y1 receptor were performed with antibodies against SV2 (presynaptic vesicles), glutamine synthetase (Müller cells), calretinin (ganglion and amacrine cells) or BrdU in intact and injured retinas.   Apyrase and MRS2179 significantly increased injury-induced cell death in all retinal layers. Intact retinas showed mRNA expression of P2X1,2,7 (but not P2X3) and P2Y1,2,12 receptors. P2Y1 receptor protein level exhibited a lesion-induced enhancement during the proliferative phase after injury in plexiform, inner nuclear, and photoreceptor layers and the CMZ. In control retinas, P2Y1 receptor was concentrated in the apical part of ganglion cells, which was not observed at day 5 post injury. In control retinas or at the peak of cell proliferation following injury, P2Y1 receptor was not expressed by Müller glia and was mainly localized in postsynapsis at the plexiform layers. P2Y1 receptor colocalized with BrdU-positive cells in the CMZ of lesioned retinas. Injured retinas showed BrdU-positive cells in the mature nuclear layers that coexpressed P2Y1 receptors.   In sum, a global damage of the adult zebrafish retina causes an increase and modified distribution of P2Y1 plasma membrane receptors, which are also expressed by proliferative cells. These and complementary findings support P2Y1 receptor role as a key plasma membrane signaling molecule for regulating cell proliferation and death during tissue regeneration.