IMMUNOHISTOCHEMICAL LOCALIZATION OF THE NEURON-SPECIFIC GLUTAMATE TRANSPORTER EAAC1 (EAAT3) IN RAT-BRAIN AND SPINAL-CORD REVEALED BY A NOVEL MONOCLONAL-ANTIBODY

Neuronal regulation of glutamate homeostasis is mediated by high-affin ity sodium-dependent and highly hydrophobic plasma membrane glycoprote ins which maintain low levels of glutamate at central synapses. To fur ther elucidate the molecular mechanisms that regulate glutamate metabo lism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain t otal homogenates and isolated synaptosomes from frontal cortex reveale d that the antibody immunoreacted with a protein band of apparent M-r similar to 70 kDa. Deglycosylation of immunoprecipitates obtained usin g the monoclonal antibody yielded a protein with a lower apparent M-r (similar to 65 kDa). These results are consistent with the molecular s ize of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the mon oclonal antibody is specific for the neuron-specific glutamate transpo rter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double -label immunofluorescence using antibody to glial fibrillary acidic pr otein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in d endrites and particularly in cell somata suggests that this transporte r may function in the regulation of other aspects of glutamate metabol ism in addition to terminating the action of synaptically released glu tamate at central synapses. (C) 1997 Elsevier Science B.V.