QUANTIFICATION OF EXCITATORY AMINO-ACID-UPTAKE AT INTACT GLUTAMATERGIC SYNAPSES BY IMMUNOCYTOCHEMISTRY OF EXOGENOUS D-ASPARTATE

To study the localization and efficiency of glutamate/aspartate membra ne transport in the vicinity of intact glutamatergic synapses, the ava scular lamprey spinal cord was incubated with D-aspartate, a metabolic ally inert transporter substrate, The exogenous D-aspartate was locali zed by immunocytochemistry after aldehyde fixation. Incubation at 50 o r 500 mu M D-aspartate for 1 hr caused a prominent D-aspartate labelin g of glial processes at glutamatergic synapses, while presynaptic axon s and postsynaptic dendrites remained unlabeled, The glial processes s urrounding glutamatergic sensory axons with a predominantly tonical fi ring pattern contained significantly higher levels of D-aspartate than did processes surrounding glutamatergic reticulospinal axons, which f ire rarely and in brief bursts, Preparations incubated for 10 hr with 500 mu M D-aspartate showed D-aspartate immunolabeling in glia as well as in the two types of glutamatergic axon, but no evidence was obtain ed for uptake into synaptic vesicles, Nor was such evidence obtained a fter high-frequency electrical stimulation. The observations suggest t hat excitatory amino acids delivered diffusely to the extracellular sp ace in the intact CNS are transported almost exclusively into glia, Th e avid uptake in glial processes, combined with their spatial arrangem ent around glutamatergic synapses, appears to limit the access of exog enous D-aspartate to the nerve terminal glutamate/aspartate transporte r, In physiological conditions, the glial processes are likely to impe de the exchange of glutamate between the synaptic cleft and the rest o f the extracellular space, The transport was more efficient in glial p rocesses located near tonically active synapses than in ones located n ear synapses releasing transmitter sporadically, D-Aspartate is not a substrate of vesicular glutamate transport sites at these intact synap ses.