Expression of GAT‐1, a high‐affinity gamma‐aminobutyric acid plasma membrane transporter in the rat retina

Abstract

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 background 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.

Expression of GABA transporter subtypes (GAT1, GAT3) in the adult rabbit retina

“…GAT3 immunoreactivity was primarily seen in Mü ller cells but also in the somata and processes of amacrine cells. Our observations in rabbit retina are in accordence with results obtained in rat and mouse (Brecha & Weigmann 1994;Ruiz et al 1994;Honda et al 1995;Johnson et al 1996). Double labeling showed that GAT3 immunoreactivity was present in pillarlike cells, which were also labelled with the vimentin antibody, which is a well accepted marker for Mü ller cells.…”

“…In adult rat and mouse retina, GAT1 and GAT3 immunoreactivity is present in amacrine cells, in displaced amacrine cells and in processes in the inner plexiform layer (Brecha & Weigmann 1994;Ruiz et al 1994;Honda et al 1995;Johnson et al 1996). GAT3 is also present in Mü ller cells.…”

Expression of GABA transporter subtypes (GAT1, GAT3) in the developing rabbit retina

“…The immunoreactivity of GAT3 is strong in Mü ller cells, whereas GAT1 immunoreactivity is only weak in them. This result has been confirmed by in situ hybridisation, which demonstrated that the GAT1 mRNA content was lower in Mü ller cells than in neurons (Brecha & Weigmann, 1994).…”

“…GAT1 and GAT3 immunoreactivities are present mainly in amacrine cells, displaced amacrine cells and in the inner plexiform layer (IPL) (Brecha & Weigmann, 1994;Ruiz et al 1994;Honda et al 1995;Johnson et al 1996). GAT2 labeling is present in the optic nerve and the retinal pigment epithelium cells.…”

Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina

“…To date, there is no support for a transporter-mediated mechanism in mammalian horizontal cells. For example, GATs have not been localized to rodent and rabbit horizontal cells (Brecha and Weigmann, 1994;Johnson et al, 1996;Hu et al, 1999), and patchclamp recordings of isolated mammalian (rabbit and mouse) horizontal cells have not revealed the presence of GABA-dependent transporter currents (Blanco et al, 1996;Feigenspan and Weiler, 2004). Finally, no highaffinity transport of [ 3 H] GABA or GABA analogs by horizontal cells has been reported for any adult mammalian retina (Ehinger, 1977;Blanks & Roffler-Tarlov, 1982;Mosinger and Yazulla, 1985;Wä ssle & Chun, 1988Löhrke et al, 1994).…”