Expression of GAT-1 a High-Affinity Gamma-Aminobutyric Acid Plasma Membrane Transporter in the Rat Retina

摘要

Gamma-aminobutyric acid (GABA) plasma membrane transporters influence synaptic transmission by high-affinity, Na⁺-dependent transport processes. The cDNA clone, GAT-1, encodes a high-affinity Na⁺- and Cl⁻-dependent GABA plasma membrane transporter, which has kinetic and pharmacological properties similar to those of high-affinity GABA uptake systems associated with neurons. The present study evaluates the distribution and cellular localization of this putative neuronal GABA transporter by RNA blot hybridization and in situ hybridization histochemistry in the rat retina. Northern blot hybridization analysis of total retinal and cerebellar RNA extracts demonstrated a single band of hybridization at 4.2 kilobases. GABA transporter mRNA is expressed by numerous cells that are distributed to the proximal inner nuclear layer and the ganglion cell layer and by a few cells located in the inner plexiform layer. Double label studies combining the retrograde transport of the fluorescent dye Fluorogold from the superior colliculus to identify ganglion cells and in situ hybridization histochemistry demonstrated that most GAT-1 mRNA-containing cells in the ganglion cell layer are displaced amacrine cells, although some ganglion cells containing GAT-1 mRNA were visualized. In freshly dissociated retinal cell preparations, the GAT-1 RNA signal is strong in neurons and weak to moderate in specialized glial cells called Müller cells. Müller cells were identified by both their morphology and the presence of the selective Müller cell marker cellular retinaldehyde-binding protein. Only hackground labeling is seen with the sense GAT-1 RNA probe in both tissue sections and dissociated retinal cell preparations. These findings demonstrate that GAT-1 mRNA is expressed in both the retina and brain. In the retina, this transporter is predominantly localized to amacrine, displaced amacrine and interplexiform cells, and some ganglion cells. This transporter mRNA is also expressed by Müller cells but at a lower level than by neurons. These observations indicate that GABA transport by GAT-1 plasma membrane transporters in the retina is mediated by both neurons and glia cells.