GABA and Excitatory Amino Acids on the Regulation of Glutamic Acid Decarboxylase Expression and Cell Proliferation of in vitro Chick Retina

Abstract

Abstract: : Purpose: GABA added to aggregate retinal cells or to retinal organotypic preparations inhibits the expression of glutamic acid decarboxylase (GAD), the GABA synthetic enzyme (Almeida et al., Brain Res., 925,89:99, 2002). In this work we analyzed the modulation of GAD expression by GABA and by the excitatory amino acids (EAAs), glutamate and kainate, in monolayer cultured retinal cells. In addition, we studied the proliferation of retinal cells under GABA and glutamate (GLU) treatments. Methods: Retinal cells were obtained from chick embryos (E10) and dense monolayer cultures were prepared. For GAD expression analysis, cultures were treated with GABA (5mM) for 96 hours and then GLU (2mM) or kainate (100µM) was added (for 24 hours. After 5 days-in vitro cultures were fixed and processed for GAD immunohistochemistry. For cell proliferation study, we utilized auto-radiographic determination of incorporated [³H]-thymidine previuously added (1µCi/mL) in E10C5 dense monolayer cultures. In some experiments we combined immunohistochemistry for GAD and auto-radiographic [³H]-thymidine. Results: GABA reduced the number of GAD immunoreactive cells in 93%. The treatment of cultures with both EAAs, previously exposed to GABA, promoted the appearance of GAD immunoreactive cells that reached the levels of control. In contrast, GABA, GLU or both together did not interfere with number of cells that incorporate [³H]-thymidine. In double labeled cultures, almost 100% of cells expressing GAD were [³H]-thymidine negative. Conclusions: EAAs promoted GAD re-expression in monolayers retinal preparations even when cells were maintained in the presence of GABA. The absence of [³H]-thymidine in GAD immunoreactive cells indicates that they were post-mitotic at the moment of re-expression of GAD. The increase in the number of GAD immunoreactive cells after treatment with EAA seems to be due to the induction of GABAergic phenotype in differentiated cells.

Keywords: retina: neurochemistry • enzymes/enzyme inhibitors • retinal culture