GAT-3, A HIGH-AFFINITY GABA PLASMA-MEMBRANE TRANSPORTER, IS LOCALIZEDTO ASTROCYTIC PROCESSES, AND IT IS NOT CONFINED TO THE VICINITY OF GABAERGIC SYNAPSES IN THE CEREBRAL-CORTEX

The termination of GABA synaptic action by high-affinity, Na+-dependen t, neuronal, and glial plasma membrane transporters plays an important role in regulating neuronal activity in physiological and pathologica l conditions. We have investigated the cellular localization and distr ibution 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 monospecifi cally with a protein of similar to 70 kDa. Light microscopic studies r evealed specific GAT-3 immunoreactivity (ir) in small punctate structu res, and it was never observed in fibers or cell bodies. No changes in immunostaining were observed in sections incubated with GAT-3 antibod ies preadsorbed with the related rat GAT-1 or mouse GAT-2/BGT-1 C-term inal peptides, whereas in sections incubated with GAT-3 antibodies pre adsorbed with rat GAT3 C-terminal peptide, ir was not present. The hig hest number of GAT-3-positive puncta was in layer IV and in a narrow b and corresponding to layer Vb, followed by layers II and III. Many GAT -3-positive puncta were in close association with pyramidal and nonpyr amidal neuron cell bodies. Ultrastructural studies showed that GAT-3 i r 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 preembed ding labeling for GAT-3 and postembedding immunogold labeling for GABA , only some of the GAT-3-positive astrocytic processes were found clos e to GABAergic profiles. These findings on the localization of GAT-3 i n the cerebral cortex indicate that this transporter mediates GABA upt ake 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 o verall GABA levels in the neuropil.