The N-methyl-D-aspartate (NMDA) 2 type of glutamate receptor plays prominent roles in excitatory neurotransmission (1). In addition to glutamate, the NMDA receptor requires the obligatory binding of a coagonist of the NR1 subunit to mediate ion influx (2). Although glycine was originally suggested as the NMDA receptor coagonist, recent data indicate that endogenous D-serine is a physiologically relevant NMDA receptor ligand at the coagonist site. D-Serine is present at high levels in the brain, with little levels in peripheral tissues (3-5). Destruction of endogenous D-serine by D-amino-acid oxidase in hippocampal cultures promotes a decrease in NMDA receptor responses (6). Likewise, NMDA receptor-mediated responses in the retina and induction of long-term hippocampal potentiation are also diminished by removing D-serine (7,8). Very recently, D-serine was shown to be required for granule cell migration in the developing cerebellum (9). It has been proposed that Bergman glial cells release D-serine and enhance cell migration through activation of NMDA receptors in migrating granule cells (9). In hippocampal organotypic slice cultures, endogenous D-serine was shown to be the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating virtually all cell death elicited by NMDA (10).D-Serine is synthesized from L-serine by serine racemase, a brain enriched enzyme (11-13). Serine racemase activity is stimulated by ATP, and the enzyme also catalyzes deamination of serine into pyruvate and ammonia (14, 15). Both D-serine and serine racemase were shown previously to be enriched in astrocytes (13, 16). Purified astrocytic cultures release D-serine following (Ϯ)-␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor activation (16 -18) and through an amino acid exchange mechanism by the neutral amino acid transporter ASCT (17). It has been proposed that D-serine released from astrocytes that ensheath the synapse will stimulate nearby neuronal NMDA receptors (16).The evidence that D-serine possesses a target receptor as well as a biosynthetic and degradative apparatus implies that D-serine is an important transmitter/neuromodulator in the brain (5, 19). On the other hand, the glial localization of D-serine is not compatible with the classical definition of neurotransmitter, which should be present in neurons. We have explored a possible neuronal localization of D-serine and serine racemase utilizing biochemical methods in cell cultures and new antibodies against D-serine and serine racemase. We demonstrate the unambiguous presence of serine racemase and D-serine in neurons. Neuronal release of D-serine mediates a significant fraction of NMDAelicited excitotoxicity in cortical cultures. Our data indicate that neuronal D-serine may play an important role in NMDA receptor activation, such as occurs in neurotoxicity.
EXPERIMENTAL PROCEDURESMaterials-L-Serine and D-serine were purchased from Bachem. Acetonitrile, AMPA, trans-(Ϯ)-1-amino-1,3-cyclopentanedicarboxylic acid, bafilomycin A 1 , DNase I...