Activation of N-methyl-D-aspartate receptors (NMDARs) requires glutamate bound to the NR2 subunit as well as a separate co-agonist bound to the NR1 subunit (Johnson and Ascher 1987;Kleckner and Dingledine 1988). Glycine was originally thought to be the endogenous co-agonist, but in recent years D-serine has emerged as the more likely coagonist at several CNS sites, including the retina. D-serine is now known to be abundant in the brain (Hashimoto et al. 1992), especially in regions rich in NMDARs (Schell et al. 1995). The prospects of D-serine as an endogenous coagonist were further strengthened when Wolosker discovered serine racemase, the enzyme that synthesizes D-serine from L-serine (Wolosker et al. 1999). D-serine has since been implicated in numerous mechanisms ascribed to NMDARs, including neuroplasticity ( While the cellular origins of D-serine are presently unclear, it serves as an endogenous co-agonist of retinal ganglion cell (RGC) NMDARs. In the intact retina, applying D-serine deaminase (DsDa), a highly selective D-serine degrading enzyme, has the same effect on light-evoked RGC NMDAR currents as completely blocking NMDARs with conventional antagonist. Adding exogenous co-agonist augments RGC NMDAR currents (Stevens et al. 2003;Gustafson et al. 2007) demonstrating that the co-agonist site is not saturated and additional NMDARs would be recruited by D-serine release.Studies on cultured cortical astrocytes suggest that aamino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor Received October 4, 2010; accepted October 13, 2010.Address correspondence and reprint requests to Steve Sullivan, 6-145 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA. E-mail: sulli419@umn.edu Abbreviations used: AAA, a-aminoadipic acid; AM, acetoxymethyl ester; AMPAR, a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor; ASCT2, alanine-serine-cysteine transporter; CE, capillary electrophoresis; cyclo, cylothiazide; DAO), D-amino acid oxidase mutant; DsDa, D-serine deaminase; EAAT, excitatory amino acid transporter; GlyT1, glycine transporter 1; GYKI 52466, 4-(8-methyl-9H-1,3-dioxolo[4,5-h] AbstractThe N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine is important in a number of different processes in the CNS, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major coagonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated D-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) dependent mechanism. a-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration ...

In the brain, transmembrane AMPAR regulatory proteins (TARPs) critically influence the distribution, gating, and pharmacology of AMPARs, but the contribution of these auxiliary subunits to AMPAR-mediated signaling in the spinal cord remains unclear. We found that the Type I TARP γ-2 (stargazin) is present in lamina II of the superficial dorsal horn, an area involved in nociception. Consistent with the notion that γ-2 is associated with surface AMPARs, CNQX, a partial agonist at AMPARs associated with Type I TARPs, evoked whole-cell currents in lamina II neurons, but such currents were severely attenuated in γ-2-lacking stargazer (stg/stg) mice. Examination of EPSCs revealed the targeting of γ-2 to be synapse-specific; the amplitude of spontaneously occurring miniature EPSCs (mEPSCs) was reduced in neurons from stg/stg mice, but the amplitude of capsaicin-induced mEPSCs from C-fiber synapses was unaltered. This suggests that γ-2 is associated with AMPARs at synapses in lamina II but excluded from those at C-fiber inputs, a view supported by our immunohistochemical colabeling data. Following induction of peripheral inflammation, a model of hyperalgesia, there was a switch in the current-voltage relationships of capsaicin-induced mEPSCs, from linear to inwardly rectifying, indicating an increased prevalence of calcium-permeable (CP) AMPARs. This effect was abolished in stg/stg mice. Our results establish that, although γ-2 is not typically associated with calcium-impermeable AMPARs at C-fiber synapses, it is required for the translocation of CP-AMPARs to these synapses following peripheral inflammation.SIGNIFICANCE STATEMENT In the brain, transmembrane AMPAR regulatory proteins (TARPs) critically determine the functional properties of AMPARs, but the contribution of these auxiliary subunits to AMPAR-mediated signaling in the spinal cord remains unclear. An increase in the excitability of neurons within the superficial dorsal horn (SDH) of the spinal cord is thought to underlie heighted pain sensitivity. One mechanism considered to contribute to such long-lived changes is the remodeling of the ionotropic AMPA-type glutamate receptors that underlie fast excitatory synaptic transmission in the SDH. Here we show that the TARP γ-2 (stargazin) is present in SDH neurons and is necessary in a form of inflammatory pain-induced plasticity, which involves an increase in the prevalence of synaptic calcium-permeable AMPARs.

Non-technical summary Retinal ganglion cells represent a population of neurons that relay information from the retina to the brain. During retinal light responses, the spiking activity of retinal ganglion cells is shaped in part by NMDA receptors, which require a coagonist for activation. There is debate over if glycine or D-serine serves as the endogenous coagonist to retinal ganglion cell NMDA receptors. To address this question, we used a mutant mouse lacking functional serine racemase, the D-serine-synthesizing enzyme. In this study we show that D-serine is required to activate retinal ganglion cell NMDA receptors during light stimulation. Mice lacking serine racemase also appeared to have alterations in the relative contribution of NMDA and AMPA receptors to light responses. Interestingly, behavioural tests showed that mice lacking serine racemase had no apparent visual deficits. Collectively, these findings raise interesting questions about the role of D-serine in shaping excitatory synapses and in visual processing. AbstractGlycine and/or D-serine are obligatory coagonists of the N -methyl-D-aspartate receptor (NMDAR). Serine racemase, the D-serine-synthesizing enzyme, is expressed by astrocytes and Müller cells of the retina, but little is known about its role in retinal signalling. In this study, we utilize a serine racemase knockout (SRKO) mouse to explore the contribution of D-serine to inner-retinal function. Retinal tissue levels of D-serine in SRKO mice are reduced by 85%. Whole-cell recordings from SRKO retinal ganglion cells showed markedly reduced coagonist occupancy of NMDARs and consequently a dramatic reduction in the NMDAR component of light-evoked responses. NMDAR currents in SRKOs could be rescued by applying exogenous coagonist, but SRKO ganglion cells still displayed lower NMDA/AMPA receptor ratios than wild-type (WT) controls when the coagonist site was saturated. Despite having abnormalities in synaptic glutamatergic transmission, SRKO mice displayed no obvious signs of visual impairment in behavioural testing. These findings raise interesting questions about the role of D-serine in inner-retinal function and development.

Experiments were carried out in the retina of the tiger salamander (Ambystoma tigrinum) to evaluate the importance of d-serine synthesis on light-evoked N-methyl d-aspartate (NMDA) receptor-mediated components of ganglion cells and contributions to the proximal negative field potential. We blocked the synthesis of d-serine through brief exposures of the retina to phenazine ethosulfate and validated the changes in the tissue levels of d-serine using capillary electrophoresis methods to separate and measure the amino acid enantiomers. Ten minute exposures to phenazine ethosulfate decreased d-serine levels in the retina by about 50% and significantly reduced the NMDA receptor contribution to light responses of the inner retina. This is the first report of a linkage between d-serine synthesis and NMDA receptor activity in the vertebrate retina.