Sodium-dependent high-affinity uptake of glutamate is thought to play a major role in the rapid termination of synaptic activity and the maintenance of very low extracellular concentrations of excitatory amino acids (EAA) in the central nervous system. However, few studies have directly or systematically addressed the role of high-affinity glutamate uptake in excitatory synaptic transmission. This may be due, in part, to the lack of selective and potent inhibitors of the sodium-dependent high- affinity glutamate transport system. The recently developed selective, highly potent uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L- trans-PDC) will be used in the proposed experiments to test the hypothesis that the high-affinity glutamate transport system plays a major role in modulating excitatory synaptic transmission in cultured hippocampal neurons. The involvement of EAA neurotransmission in rapid information transfer, synaptic plasticity and neurotoxicity make the study of the modulation of normal excitatory synaptic transmission particularly relevant for understanding mechanisms underlying changes associated with neuropathologies such as Parkinson's and Alzheimer's diseases and brain aging. Electrophysiologic approaches will be used to elucidate the effects of pharmacological inhibition of the high-affinity glutamate transporter by L-trans-PDC on both spontaneous and evoked synaptic events in cultured hippocampal neurons. Miniature excitatory postsynaptic currents (mEPSCs) reflect the spontaneous quantal release of neurotransmitter at single presynaptic terminals. Whole-cell patch clamp recording methods allow these currents to be resolved with sufficiently high resolution to allow analysis of their frequencies, distribution of amplitudes and time courses. Examining these parameters of the mEPSCs will provide useful information of any changes in the sensitivity of the postsynaptic EAA receptors and/or changes in presynaptic release resulting from inhibition of high-affinity glutamate uptake. Evoked excitatory postsynaptic currents (EPSCs) may also be examined by recording from isolated pairs of monosynaptically coupled hippocampal neurons maintained in very low density cultures. Various presynaptic stimulus paradigms including single stimulation, paired-pulse stimulation and high-frequency tetanic stimulation will be used to determine the effects of the glutamate uptake inhibitor L-trans-PDC on NMDA and non-NMDA components of the EPSC. The hypothesis is that inhibition of high-affinity glutamate transport will result in an increase in the ambient concentration of glutamate in the extracellular space of the cultured hippocampal neurons; the experiments are designed to determine the effects of the elevated levels or prolonged exposure to glutamate on non-NMDA receptor desensitization, the time course and amplitude of the slower NMDA component of the EPSC , and the involvement of presynaptic metabotropic receptors.
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