GAT-3, a High-Affinity GABA Plasma Membrane Transporter, Is Localized to Astrocytic Processes, and It Is Not Confined to the Vicinity of GABAergic Synapses in the Cerebral Cortex

Abstract

The termination of GABA synaptic action by high-affinity, Na(+)-dependent, neuronal, and glial plasma membrane transporters plays an important role in regulating neuronal activity in physiological and pathological conditions. We have investigated the cellular localization and distribution in the cerebral cortex of adult rats of one GABA transporter (GAT), GAT-3, by immunocytochemistry with affinity-purified polyclonal antibodies directed to its predicted C terminus that react monospecifically with a protein of approximately 70 kDa. Light microscopic studies revealed specific GAT-3 immunoreactivity (ir) in small punctate structures, and it was never observed in fibers or cell bodies. No changes in immunostaining were observed in sections incubated with GAT-3 antibodies preadsorbed with the related rat GAT-1 or mouse GAT-2/ BGT-1 C-terminal peptides, whereas in sections incubated with GAT-3 antibodies preadsorbed with rat GAT-3 C-terminal peptide, ir was not present. The highest number of GAT-3-positive puncta was in layer IV and in a narrow band corresponding to layer Vb, followed by layers II and III. Many GAT-3-positive puncta were in close association with pyramidal and nonpyramidal neuron cell bodies. Ultrastructural studies showed that GAT-3 ir was localized exclusively to astrocytic processes, which were found in the neuropil and adjacent to axon terminals having either symmetric or asymmetric specializations. In sections processed by both preembedding labeling for GAT-3 and postembedding immunogold labeling for GABA, only some of the GAT-3-positive astrocytic processes were found close to GABAergic profiles. These findings on the localization of GAT-3 in the cerebral cortex indicate that this transporter mediates GABA uptake into glial cells, and suggest that glial GABA uptake may function to limit the spread of GABA from the synapse, as well as to regulate overall GABA levels in the neuropil.

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine

“…Homogenate processing, protein concentration determination, and protein electroblotting onto nitrocellulose filters (0.22 m) probed with antiGLT-1 antibodies (0.025 g ml −1 ) were performed as described previously. 26 Optical densities of the bands were determined using the 1.61 NIH Image analysis software. Results were normalized to GLT-1 levels in controls prepared in parallel and loaded onto the same gel.…”

“…10 Among the five GluTs that have been characterized in the mammalian CNS, GLT-1, a GluT localized to distal astrocytic processes, exhibits the highest level of expression, it is responsible for the greatest proportion of total glutamate transport and its functional inactivation raises extracellular glutamate levels. 11,12 We therefore examined whether chronic oral administration 13 of the widely used and highly effective antipsychotic clozapine 14,15 (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) mg kg −1 day −1 in the drinking water) reduces the expression of GLT-1 in rats.…”

Basic Mechanisms of Gabitril (Tiagabine) and Future Potential Developments

“…In some regions, however, it is found additionally or exclusively in glia. For example, in the thalamus GAT-1 and GAT-3 are found in astrocytes but not in neurons (9). GAT-3 is found in many brain regions, especially the cerebellum.…”

“…Immunocytochemical studies using light and electron microscopy have provided insight into the localization of the GABA transporter GAT-1 (8)(9)(10)(11)(12)(13)(14)(15). GAT-1 is the predominant GABA transporter in the rat forebrain (cortex, striatum, hippocampus), where it is predominantly localized to GABAergic terminals (8).…”

“…GAT-3 is found in many brain regions, especially the cerebellum. Immunocytochemical studies in rats and mice have localized it to astrocytes in cortex (1 0), thalamus (9), and striatum (Table 2).…”