NMDA Receptors Enhance Spontaneous Activity and Promote Neuronal Survival in the Developing Cochlea.

Spontaneous bursts of activity in developing sensory pathways promote maturation of neurons, refinement of neuronal connections, and assembly of appropriate functional networks. In the developing auditory system, inner hair cells (IHCs) spontaneously fire Ca(2+) spikes, each of which is transformed into a mini-burst of action potentials in spiral ganglion neurons (SGNs). Here we show that NMDARs are expressed in SGN dendritic terminals and play a critical role during transmission of activity from IHCs to SGNs before hearing onset. NMDAR activation enhances glutamate-mediated Ca(2+) influx at dendritic terminals, promotes repetitive firing of individual SGNs in response to each synaptic event, and enhances coincident activity of neighboring SGNs that will eventually encode similar frequencies of sound. Loss of NMDAR signaling from SGNs reduced their survival both in vivo and in vitro, revealing that spontaneous activity in the prehearing cochlea promotes maturation of auditory circuitry through periodic activation of NMDARs in SGNs.

NMDA receptor signaling in oligodendrocyte progenitors is not required for oligodendrogenesis and myelination.

Oligodendrocyte precursor cells (OPCs) express NMDA receptors (NMDARs) and form synapses with glutamatergic neurons throughout the CNS. Although glutamate influences the proliferation and maturation of these progenitors in vitro, the role of NMDAR signaling in oligodendrogenesis and myelination in vivo is not known. Here, we investigated the consequences of genetically deleting the obligatory NMDAR subunit NR1 from OPCs and their oligodendrocyte progeny in the CNS of developing and mature mice. NMDAR-deficient OPCs proliferated normally, achieved appropriate densities in gray and white matter, and differentiated to form major white matter tracts without delay. OPCs also retained their characteristic physiological and morphological properties in the absence of NMDAR signaling and were able to form synapses with glutamatergic axons. However, expression of calcium-permeable AMPA receptors (AMPARs) was enhanced in NMDAR-deficient OPCs. These results suggest that NMDAR signaling is not used to control OPC development but to regulate AMPAR-dependent signaling with surrounding axons, pointing to additional functions for these ubiquitous glial cells.

Ncm-D-aspartate: a novel caged D-aspartate suitable for activation of glutamate transporters and N-methyl-D-aspartate (NMDA) receptors in brain tissue

The D-isomer of aspartate is both a substrate for glutamate transporters and an agonist of N-methyl-D-aspartate (NMDA) receptors. To monitor the behavior of these receptors and transporters in intact tissue we synthesized a new photo-labile analogue of D-aspartate, N-[(6-nitrocoumarin-7-yl)methyl]-D-aspartic acid (Ncm-D-aspartate). This compound was photolyzed rapidly (t(1/2)=0.11 micros) by UV light with a quantum efficiency of 0.041 at pH 7.4. In acute hippocampal slices, photolysis of Ncm-D-aspartate by brief (1 ms) exposure to UV light elicited rapidly activating inward currents in astrocytes that were sensitive to inhibition by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA). Neither Ncm-D-aspartate nor the photo-released caging group exhibited agonist or antagonist activity at glutamate transporters, and Ncm-D-aspartate did not induce transporter currents prior to photolysis. Glutamate transporter currents were also elicited in cerebellar Purkinje cells in response to photolysis of Ncm-D-aspartate. Photo-release of D-aspartate from Ncm-D-aspartate did not induce alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor or metabotropic glutamate receptor (mGluR) currents, but triggered robust NMDA receptor currents in neurons; Ncm-D-aspartate and the photolzyed caging group were similarly inert at NMDA receptors. These results indicate that Ncm-D-aspartate can be used to study NMDA receptors at excitatory synapses and interactions between transporters and receptors in brain tissue.

Synthesis and characterization of 4-methoxy-7-nitroindolinyl-D-aspartate, a caged compound for selective activation of glutamate transporters and N-methyl-D-aspartate receptors in brain tissue.

The D-isomer of aspartate is efficiently transported by high-affinity Na(+)/K(+)-dependent glutamate transporters and is an effective ligand of N-methyl-d-aspartate (NMDA) receptors. To facilitate analysis of the regulation of these proteins in their native membranes, we synthesized a photolabile analogue of D-aspartate, 4-methoxy-7-nitroindolinyl-D-aspartate (MNI-D-aspartate). This compound was photolyzed with a quantum efficiency of 0.09 at pH 7.4. Photorelease of d-aspartate in acute hippocampal slices through brief (1 ms) UV laser illumination of MNI-d-aspartate triggered rapidly activating currents in astrocytes that were inhibited by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA), indicating that they resulted from electrogenic uptake of D-aspartate. These transporter currents exhibited a distinct tail component that was approximately 2% of the peak current, which may result from the release of K(+) into the extracellular space during counter transport. MNI-D-aspartate was neither an agonist nor an antagonist of glutamate transporters at concentrations up to 500 muM and was stable in aqueous solution for several days. Glutamate transporter currents were also elicited in Bergmann glial cells and Purkinje neurons of the cerebellum in response to photolysis of MNI-D-aspartate, indicating that this compound can be used for monitoring the occupancy and regulation of glutamate transporters in different brain regions. Photorelease of D-aspartate did not activate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or metabotropic glutamate receptors (mGluRs) in neurons, but resulted in the selective, but transient, activation of NMDA receptors in hippocampal pyramidal neurons; MNI-D-aspartate was not an antagonist of NMDA receptors. These results indicate that MNI-D-aspartate also may be useful for studying the regulation of NMDA receptors at excitatory synapses.