Early Expression of Glutamate Transporters in the Developing Human Retina

Abstract

Abstract: : Purpose: Glutamatergic neurotransmission requires the regulation of extracellular glutamate levels by glial or neuronal glutamate transporters. This study investigated when glutamate homeostasis was evident in the human retina as evinced by the expression of the glutamate transporters EAAT1 and EAAT2. Methods: Human retinas aged 10–20 weeks gestational age were prepared either for immunocytochemistry or for Western blotting, then probed using antibodies to the glutamate transporters EAAT1 and EAAT2. Results:Early expression of both EAAT1 and EAAT2 was evident as indicated by Western blotting, suggesting that the retina exhibited significant potential capacity for glutamate homeostasis in the first trimester. Immunocytochemistry for EAAT2 revealed unexpected results. In the adult human retina, EAAT2, which exists as multiple splice variants, is expressed by cone photoreceptors, bipolar cells and populations of amacrine cells. However in foetal retinas, EAAT2 expression was initially observed in populations of cells, the phenotype of which, was suggestive of them being radial glial cells (Müller–cells) or precursors thereof. One key feature of EAAT2 labelling was the presence of a continuous band of immunoreactivity at the apical surface of the retina. This suggested that the apical end feet of the Müller–like cells were tightly apposed and expressed EAAT2. By 17 weeks there was a clear change in the EAAT2 expression pattern, such that whilst the peripheral retina still exhibited Müller– cell–like staining, such staining was diminished in the more mature central regions of the retina and labelling was evident in populations of cells tentatively identified as photoreceptors, bipolar cells and amacrine cells Conclusions: This study demonstrates the unexpectedly early expression of glutamate transporters in the developing human retina. We suggest that the Müller–like cells may form a continuous glutamate–retarding barrier between the retina and the RPE/choroid. This anatomical configuration would potentially prevent the ingress of glutamate from the choroidal circulation into the developing retina. This might facilitate establishment of glutamatergic signalling, which requires low extracellular levels of glutamate. The appearance of EAAT2 in neurons that are likely to express this transporter in adulthood suggests early establishment of a glutamate–transporting phenotype in these cells. We are now investigating which of the splice variants of EAAT2 are expressed (as well as other glutamate transporters such as EAAT5) and their relationship to other markers such as nestin.

Keywords: retinal development • neurotransmitters/neurotransmitter systems • Muller cells