Manuel Esguerra

d-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors

d-serine has been proposed as an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors in many brain regions, but its presence and function in the vertebrate retina have not been characterized. We have detected d-serine and its synthesizing enzyme, serine racemase, in the retinas of several vertebrate species, including salamanders, rats, and mice and have localized both constituents to Müller cells and astrocytes, the two major glial cell types in the retina. Physiological studies in rats and salamanders demonstrated that, in retinal ganglion cells, d-serine can enhance excitatory currents elicited by the application of NMDA, as well as the NMDA receptor component of light-evoked synaptic responses. Application of d-amino acid oxidase, which degrades d-serine, reduced the magnitude of NMDA receptor-mediated currents, raising the possibility that endogenous d-serine serves as a ligand for setting the sensitivity of NMDA receptors under physiological conditions. These observations raise exciting new questions about the role of glial cells in regulating the excitability of neurons through release of d-serine.

Retinogeniculate EPSPs recorded intracellularly in the ferret lateral geniculate nucleus in vitro: role of NMDA receptors.

We used an in vitro preparation of the ferret lateral geniculate nucleus (LGN) to examine the role of the NMDA class of excitatory amino acid (EAA) receptors in retinogeniculate transmission. Intracellular recordings revealed that blockade of NMDA receptors both shortened the time course and reduced the amplitude of fast and slow components of excitatory postsynaptic potentials (EPSPs) evoked by optic tract stimulation. The amplitude and width of the EPSPs mediated by NMDA receptors increased as membrane potential was depolarized towards spike threshold. Individual LGN cells were influenced to varying extents by blockade of NMDA receptors; NMDA and non-NMDA receptor blockade together attenuated severely the entire retinogeniculate EPSP. The dependence of all components of retinogeniculate EPSPs (and action potentials) on NMDA receptor activation supports the hypothesis that the NMDA receptor participates in fast (less than 10 ms) synaptic events underlying conventional retinogeniculate transmission. The voltage dependence of the NMDA receptor-gated conductance suggests strongly that the transmission of retinal information through the LGN is subject to modulation by extraretinal inputs that affect the membrane potential of LGN neurons.

CD38 expression and NAD+-induced intracellular Ca2+ mobilization in isolated retinal Müller cells

Müller cells of the vertebrate retina are prominent radial glia that provide essential support to sustain homeostasis of the tissue, including redistribution of external potassium, uptake and metabolism of neurotransmitters, and secretion of factors that stabilize the retina. Meeting this diversity of functional supports requires that Müller cells express numerous receptors, transporters, enzymes, and tissue factors. In this study, we provide evidence that adds to the dimensions of Müller cell function by demonstrating a unique relationship between external NAD+ and the mobilization of internal calcium, expressed in the form of calcium waves. The cellular mechanism that supports internal mobilization of calcium appears to depend on a complex multifunctional ectoenzyme, CD38, which converts NAD+ into the intracellular Ca2+‐mobilizing second‐messenger cyclic ADP‐ribose (cADPR) and could function as a detector for extracellular NAD+, thus providing a novel signal detection system for evaluating the extracellular environment. Our results are consistent with a model of intracellular Ca2+ mobilization in which membrane‐bound CD38 binds extracellular NAD+ and triggers intracellular Ca2+ waves either by direct conversion of NAD+ to cADPR or by activating intracellular cADPR synthesis. Our preliminary results indicate that the Ca2+ waves induced by external NAD+ propagate through an internal pathway that depends on the activation of ryanodine receptors, which appear to be distributed throughout the Müller cell cytosol. Because NAD+ is likely to be enhanced when cells are stress or damaged, CD38 could enable Müller cells to detect NAD+ under these circumstances and respond appropriately. Alternatively, NAD+ could also represent a novel extracellular, paracrine function that mediates signaling between glial cells and/or other cellular elements of the retina. GLIA 39:314–319, 2002.

The glycine transporter GlyT1 controls N-methyl-D-aspartic acid receptor coagonist occupancy in the mouse retina

We examined the role of GlyT1, the high‐affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N‐methyl‐D‐aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1−/+) and compared those results with wild‐type (WT) littermate controls (GlyT1+/+). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; pharmacologically blocking GlyT1 with N‐[3‐([1,1‐biphenyl]‐4‐yloxy)‐3‐(4‐fluorophenyl)propyl]‐N‐methylglycine in the WT retina generated a significantly larger accumulation of glycine into the bathing environment when compared with the GlyT1−/+ retinas. The relative occupancy state of the NMDA receptor coagonist sites was tested using whole‐cell recordings from ganglion cells while bath applying D‐serine or D‐serine + NMDA. The interpretation of these studies was simplified by blocking post‐synaptic inhibition with picrotoxinin and strychnine. NMDA receptor coagonist sites were more saturated and less enhanced by D‐serine in the GlyT1−/+ mice compared with the WT controls. Immunoblots of NMDA receptor subunits (NR1, NR2A and NR2B) in WT and GlyT1−/+ animals showed that the NR1 subunits were identical. These observations are discussed in view of contemporary issues about NMDA receptor coagonist function in the vertebrate retina and the role of glycine vs. D‐serine as the endogenous coagonist.

Brivaracetam augments short-term depression and slows vesicle recycling.

Brivaracetam (BRV) decreases seizure activity in a number of epilepsy models and binds to the synaptic vesicle glycoprotein 2A (SV2A) with a higher affinity than the antiepileptic drug levetiracetam (LEV). Experiments were performed to determine if BRV acted similarly to LEV to induce or augment short‐term depression (STD) under high‐frequency neuronal stimulation and slow synaptic vesicle recycling.

Light-evoked NMDA receptor-mediated currents are reduced by blocking D-serine synthesis in the salamander retina

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.

Serine racemase deletion abolishes light-evoked NMDA receptor currents in retinal ganglion cells.

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.

Retinal NMDA receptor function and expression are altered in a mouse lacking d-amino acid oxidase

D-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-D-aspartate (NMDA) receptors of ganglion cells. Although the enzyme D-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating D-serine levels using a mutant mouse line deficient in DAO (ddY/DAO(-)) and compared these results with their wild-type counterparts (ddY/DAO(+)). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of D-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of D-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce D-serine levels in the retina, and 2) D-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio.

Spike trains and signaling modes of neurons in the ferret lateral geniculate nucleus

Intracellular recordings were used to examine the action potential firing modes of cells in the ferret lateral geniculate nucleus (LGN) in vitro. We compared the effects of altered membrane voltage on patterns of action potential trains evoked by direct current injection and by retinal afferent (synaptic) stimulation. The results confirm that LGN cells in the ferret can fire action potentials in the “burst” and “tonic” modes that have been described previously for other species. At depolarized membrane potentials, LGN neurons respond to sustained depolarization with short-latency trains of action potentials whose frequency is directly proportional to the amount of current injected. At hyperpolarized membrane potentials, LGN cells enter burst mode, in which depolarizing inputs are differentiated into brief high-frequency discharges whose latency varies with membrane potential. We also observed a “mixed” mode, in which LGN cell responses to synaptic or injected currents within a narrow range of membrane potentials reflect aspects of both burst and tonic firing simultaneously. Thus a striking consequence of the interplay among voltage-dependent membrane conductances in thalamic cells is wide variability in length, duration, and latency of spike discharges elicited by identical stimuli. These results also suggest that the concept that LGN cells display only two active response modes must be expanded to include varying amounts of delay and the possibility of mixed discharges.

Role of Postsynaptic Activity in Retinogeniculate Pattern Formation

There is considerable evidence now that activity in retinal afferents plays an important role in the formation of connections between retinal ganglion cell axons and their target cells in the lateral geniculate nucleus (LGN). Thus, postnatal eyelid suture in kittens prevents retinogeniculate X and Y axon arbors from developing normally (Sur et al., 1982), and causes postsynaptic X and Y LGN cells to have abnormal structural and functional features (Friedlander et al., 1982; Sherman and Spear, 1982). Blocking action potentials in retinal afferents by infusing tetrodotoxin (TTX) intraocularly in postnatal kittens also alters the morphology of retinogeniculate axon arbors (Sur et al., 1985), and causes LGN cells to have abnormal physiological properties (Dubin et al., 1986). Infusion of TTX into the vicinity of the optic chiasm in prenatal kittens prevents the formation of eye-specific laminae (Shatz and Stryker, 1988), and causes retinogeniculate axon arbors to be abnormally large (Sretavan et al., 1988).