D-serine Levels Research Articles

Pharmacokinetics of Oral d-Serine in d-Amino Acid Oxidase Knockout Mice

D-Amino acid oxidase (DAAO) catalyzes the oxidative deamination of D-amino acids including D-serine, a full agonist at the glycine modulatory site of the N-methyl-d-aspartate (NMDA) receptor. To evaluate the significance of DAAO-mediated metabolism in the pharmacokinetics of oral D-serine, plasma D-serine levels were measured in both wild-type mice and transgenic mice lacking DAAO. Although D-serine levels were rapidly diminished in wild-type mice (t(½) = 1.2 h), sustained drug levels over the course of 4 h (t(½) > 10 h) were observed in mice lacking DAAO. Coadministration of D-serine with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a small-molecule DAAO inhibitor, in wild-type mice resulted in the enhancement of plasma D-serine levels, although CBIO seems to have only temporary effects on the plasma D-serine levels due to glucuronidation of the key hydroxyl group. These findings highlight the predominant role of DAAO in the clearance of D-serine from the systemic circulation. Thus, a potent DAAO inhibitor with a longer half-life should be capable of maintaining high plasma D-serine levels over a sustained period of time and might have therapeutic implications for the treatment of schizophrenia.

D-serine and schizophrenia: an update

Considering the lengthy history of pharmacological treatment of schizophrenia, the development of novel antipsychotic agents targeting the glutamatergic system is relatively new. A glutamatergic deficit has been proposed to underlie many of the symptoms typically observed in schizophrenia, particularly the negative and cognitive symptoms (which are less likely to respond to current treatments). d-serine is an important coagonist of the glutamate NMDA receptor, and accumulating evidence suggests that d-serine levels and/or activity may be dysfunctional in schizophrenia and that facilitation of d-serine transmission could provide a significant therapeutic breakthrough, especially where conventional treatments have fallen short. A summary of the relevant animal data, as well as genetic studies and clinical trials examining d-serine as an adjunct to standard antipsychotic therapy, is provided in this article. Together, the evidence suggests that research on the next generation of antipsychotic agents should include studies on increasing brain levels of d-serine or mimicking its action on the NMDA receptor.

Type 1 diabetes mellitus in mice increases hippocampal d-serine in the acute phase after streptozotocin injection

Diabetes mellitus (DM) is known to be a risk factor in the development of deficits in cognition, learning, and memory. In DM animal models, including the streptozotocin (STZ)-induced diabetic rodent model, abnormalities in the regulation of several neurotransmitters have been reported. However, the role in DM of d-serine, an endogenous co-agonist of glutamatergic N-methyl-d-aspartate receptors, remains unknown. Here, we measured the amounts of d-/l-serine and l-glutamate in the hippocampi of STZ-treated mice using a 2D-HPLC system from acute to chronic phases after the induction of DM. STZ treatment significantly increased the d-serine level by 23.7% in the hippocampus compared with vehicle treatment at 1week after the injection, whereas it did not affect the levels of l-serine. In contrast, l-glutamate levels in the hippocampus were elevated at 3days after STZ injection and rather decreased at 1week after that. Such alterations in the amino acids were not evident in the chronic phases. We further tested whether the STZ-induced d-serine increase was caused by DM pathophysiology. In vivo, subcutaneous insulin implants into STZ-treated mice restored the elevated d-serine levels in the hippocampus. An in vitro study using primary cultured hippocampal neurons revealed that treatments of STZ did not directly affect the level of d-serine secreted in the cultured media. These results indicate that DM pathology caused by insulin deficiency triggers transient d-serine increase and l-glutamate alteration in the hippocampus. Such aberrant regulations of excitatory neurotransmitters may be relevant to the formation of DM-related dysfunction of the central nervous system (CNS).

Depressive-like behavior in adrenocorticotropic hormone-treated rats blocked by memantine

Hyperactivity of the hypothalamic pituitary–adrenal (HPA) axis plays a role in the pathophysiology of major depressive disorder (MDD). Recent studies suggest the role of the glutamatergic system in the pathophysiology of MDD, and N-methyl-d-aspartate (NMDA) receptor antagonists have shown antidepressant effects in both preclinical and clinical studies. However, little is known about the role of adrenocorticotropic hormone (ACTH) specifically in the glutamatergic response to HPA axis activation. Glutamate is an NMDA receptor agonist, and glycine and d-serine act as co-agonists. Here, we measured brain concentrations of these amino acids in rats given repeated administration of ACTH (100μg/rat/day, sc, for 14days). Further, we also evaluated behavioral effects of memantine, a non-competitive NMDA antagonist, on immobility time in the forced swimming test and on locomotor activity in ACTH-treated rats. Compared with control rats, glutamine, glycine, l-serine, and d-serine levels were increased in the hippocampus of ACTH-treated rats; glutamate, glutamine, glycine, l-serine, and d-serine were increased in the cerebellum; and glutamine and glycine were increased in the frontal cortex and striatum, all with statistical significance. Remarkably, these increases in agonists and co-agonists might have led to the augmentation of NMDA receptor activity. ACTH treatment increased immobility time in the forced swimming test and decreased locomotor activity in rats. On the contrary, memantine (10mg/kg, ip) significantly decreased immobility time in the forced swimming test and increased locomotor activity in ACTH-treated rats. Furthermore, imipramine (15mg/kg, ip) did not alter immobility time in the forced swimming test whereas this drug significantly decreased locomotor activity in ACTH-treated rats. These results suggest that depressive-like behaviors by chronic ACTH treatment could be blocked by memantine.

Different serine and glycine metabolism in patients with schizophrenia receiving clozapine

Dysfunction of the N-methyl-d-aspartate receptor, which is modulated by excitatory amino acids (EAA), is involved in the pathophysiology of schizophrenia. The effects of antipsychotics on EAA metabolism are uncertain. Positive clinical effects of treatment with antipsychotics were not always associated with changes in EAA serum levels in patients with schizophrenia in clinical trials. To examine EAA serum levels in relation to the intensity of psychotic symptoms and the type of medication received we compared these variables among patients with schizophrenia (n = 49) treated with first (FGA) or second (SGA) generation antipsychotics or clozapine. Glutamate, aspartate, glycine, total serine and d-serine serum levels were measured by High Performance Liquid Chromatography. The Positive and Negative Syndrome Scale (PANSS) and the Scale for the Assessment of Negative Symptoms (SANS) were used to assess symptoms of schizophrenia. Lower average levels of glycine and total serine were found in the serum of patients receiving clozapine when compared to the groups of patients treated with FGA or SGA. There were no differences in serum glutamate, aspartate or d-serine levels or in the intensity of schizophrenic symptoms assessed by PANSS or SANS among the groups of patients treated with FGA or SGA or clozapine. Lower glycine and total serine serum levels could be caused by the particular characteristics of the population of patients receiving clozapine rather than as an effect of the clozapine. The results suggest selective deficiency of l-serine synthesis in the patients with resistance to non-clozapine treatment. It might be an unique biochemical and pathophysiological characteristic of the treatment-resistance in schizophrenia.

Effects of non-steroidal antiinflammatory drugs on D-serine-induced oxidative stress in vitro

Inflammation is deleterious for organs with reduced capacity of regeneration, such as the brain. Recently, studies have focused on investigating the therapeutic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and multiple sclerosis. Excitotoxicity is the pathological process when receptors for the excitatory neurotransmitter glutamate, such as the N-methyl-D-aspartate (NMDA), receptors are overactivated. This process may be involved in neurodegenerative diseases. D-serine is one of the coagonist of NMDA receptors, and increased levels of D-serine are associated with excitotoxicity. In our study, the potential neuroprotective effects of mefenamic acid, acetaminophen, and naproxen sodium were investigated against D-serine-induced oxidative stress in the rat brain in vitro. To show their potential neuroprotective properties, NSAIDs were incubated with D-serine and reactive oxygen species (ROS), malondialdehyde, and protein carbonyl content of the brain after different treatments were measured. Our results demostrate that NSAIDs used in the present study significantly reduced ROS production, lipid peroxidation, and protein oxidation against D-serine treatment.

Dysfunctional astrocytic regulation of glutamate transmission in a rat model of depression

Depression is usually associated with alterations in the monoaminergic system. However, new evidences suggest the involvement of the glutamatergic system in the aetiology of depression. Here we explored the glutamatergic system in a rat model of depression (i.e., the flinders sensitive line (FSL)) to reveal the mechanism underlying the emotional and cognitive aspects associated with the disease. We showed a dramatically elevated level of baseline glutamatergic synaptic transmission by whole-cell recordings as well as impairment in long-term potentiation induced by high-frequency stimulation in hippocampal slices from FSL rats compared with Sprague-Dawley rats. At behavioural level, FSL rats displayed recognition memory impairment in the novel object recognition test. Enantioselective chromatography analysis revealed lower levels of D-serine in the hippocampus of FSL rats and both synaptic plasticity and memory impairments were restored by administration of D-serine. We also observed dysfunctional astrocytic glutamate regulation including downregulation of the glia glutamate transporter GLAST as shown by western blot. One possibility is that the dysfunctional astrocytic glutamate reuptake triggers a succession of events, including the reduction of D-serine production as a safety mechanism to avoid NMDA receptor overactivation, which in turn causes the synaptic plasticity and memory impairments observed. These findings open up new brain targets for the development of more potent and efficient antidepressant drugs.

Cell Selective Conditional Null Mutations of Serine Racemase Demonstrate a Predominate Localization in Cortical Glutamatergic Neurons

D-serine, which is synthesized by the enzyme serine racemase (SR), is a co-agonist at the N-methyl-D-aspartate receptor (NMDAR). Crucial to an understanding of the signaling functions of D-serine is defining the sites responsible for its synthesis and release. In order to quantify the contributions of astrocytes and neurons to SR and D-serine localization, we used recombinant DNA techniques to effect cell type selective suppression of SR expression in astrocytes (aSRCKO) and in forebrain glutamatergic neurons (nSRCKO). The majority of SR is expressed in neurons: SR expression was reduced by ~65% in nSRCKO cerebral cortex and hippocampus, but only ~15% in aSRCKO as quantified by western blots. In contrast, nSRCKO is associated with only modest decreases in D-serine levels as quantified by HPLC, whereas D-serine levels were unaffected in aSRCKO mice. Liver expression of SR was increased by 35% in the nSRCKO, suggesting a role for peripheral SR in the maintenance of brain D-serine. Electrophysiologic studies of long-term potentiation (LTP) at the Schaffer collateral-CA1 pyramidal neuron synapse revealed no alterations in the aSRCKO mice versus wild-type. LTP induced by a single tetanic stimulus was reduced by nearly 70% in the nSRCKO mice. Furthermore, the mini-excitatory post-synaptic currents mediated by NMDA receptors but not by AMPA receptors were significantly reduced in nSRCKO mice. Our findings indicate that in forebrain, where D-serine appears to be the endogenous co-agonist at NMDA receptors, SR is predominantly expressed in glutamatergic neurons, and co-release of glutamate and D-serine is required for optimal activation of post-synaptic NMDA receptors.

D‐Amino acid oxidase‐mediated increase in spinal hydrogen peroxide is mainly responsible for formalin‐induced tonic pain

Spinal reactive oxygen species (ROS) are critically involved in chronic pain. D-Amino acid oxidase (DAAO) oxidizes D-amino acids such as D-serine to form the byproduct hydrogen peroxide without producing other ROS. DAAO inhibitors are specifically analgesic in tonic pain, neuropathic pain and cancer pain. This study examined the role of spinal hydrogen peroxide in pain and the mechanism of the analgesic effects of DAAO inhibitors. Formalin-induced pain behaviours and spinal hydrogen peroxide levels were measured in rodents. Formalin injected into the paw increased spinal hydrogen peroxide synchronously with enhanced tonic pain; both were effectively prevented by i.t. fluorocitrate, a selective astrocyte metabolic inhibitor. Given systemically, the potent DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) blocked spinal DAAO enzymatic activity and specifically prevented formalin-induced tonic pain in a dose-dependent manner. Although CBIO maximally inhibited tonic pain by 62%, it completely prevented the increase in spinal hydrogen peroxide. I.t. catalase, an enzyme specific for decomposition of hydrogen peroxide, completely depleted spinal hydrogen peroxide and prevented formalin-induced tonic pain by 65%. Given systemically, the ROS scavenger PBN (phenyl-N-tert-butylnitrone) also inhibited formalin-induced tonic pain and increase in spinal hydrogen peroxide. Formalin-induced tonic pain was potentiated by i.t. exogenous hydrogen peroxide. CBIO did not increase spinal D-serine level, and i.t. D-serine did not alter either formalin-induced tonic pain or CBIO's analgesic effect. Spinal hydrogen peroxide is specifically and largely responsible for formalin-induced pain, and DAAO inhibitors produce analgesia by blocking spinal hydrogen peroxide production rather than interacting with spinal D-serine.

Reversal of age‐related oxidative stress prevents hippocampal synaptic plasticity deficits by protecting d‐serine‐dependent NMDA receptor activation

Oxidative stress (OS) resulting from an imbalance between antioxidant defenses and the intracellular accumulation of reactive oxygen species (ROS) contributes to age-related memory deficits. While impaired synaptic plasticity in neuronal networks is thought to underlie cognitive deficits during aging, whether this process is targeted by OS and what the mechanisms involved are still remain open questions. In this study, we investigated the age-related effects of the reducing agent N-acetyl-L-cysteine (L-NAC) on the activation of the N-methyl-D-aspartate receptor (NMDA-R) by its co-agonist D-serine, because alterations in this mechanism contribute greatly to synaptic plasticity deficits in aged animals. Long-term dietary supplementation with L-NAC prevented oxidative damage in the hippocampus of aged rats. Electrophysiological recordings in the CA1 of hippocampal slices indicated that NMDA-R-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) were depressed in aged animals, deficits that could be reversed by exogenous D-serine. Chronic treatment with L-NAC, but not acute application of the reducing agent, restored potent D-serine-dependent NMDA-R activation and LTP induction in aged rats. In addition, it is also revealed that the age-related decrease in D-serine levels and in the expression of the synthesizing enzyme serine racemase, which underlies the decrease in NMDA-R activation by the amino acid, was rescued by long-term dietary treatment with L-NAC. Our results indicate that protecting redox status in aged animals could prevent injury to the cellular mechanisms underlying cognitive aging, in part by maintaining potent NMDA-R activation through the D-serine-dependent pathway.

Paradoxical roles of serine racemase and d‐serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.

Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model

N-methyl-D-aspartic acid receptor (NMDAr) activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase (SR). D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS) and L-erythro-3-hydroxyaspartate (LEHA), among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 μg/10 μL) and LEHA (100 μg/10 μL) in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 μg/10 μL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

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

Glycine 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.

Overexpression of serine racemase in retina and overproduction of D-serine in eyes of streptozotocin-induced diabetic retinopathy

BackgroundRecent data indicate that inflammatory mechanisms contribute to diabetic retinopathy (DR). We have determined that serine racemase (SR) expression is increased by inflammatory stimuli including liposaccharide (LPS), amyloid β-peptide (A-beta), and secreted amyloid precursor protein (sAPP); expression is decreased by the anti-inflammatory drug, dexamethasone. We tested possibility that SR and its product, D-serine, were altered in a rat model of DR.MethodsIntraperitoneal injection of streptozotocin (STZ; 70 mg/kg body weight) to Sprague-Dawley rats produced type-I diabetic mellitus (fasting blood sugar higher than 300 mg/dL). At 3 and 5 months after STZ or saline injection, retinas from some rats were subjected to cryosectioning for immunofluorescent analysis of SR and TUNEL assay of apoptosis. Retinal homogenates were used to detect SR levels and Jun N-terminal kinase (JNK) activation by immunoblotting. Aqueous humor and retina were also collected to assay for neurotransmitters, including glutamate and D-serine, by reverse-phase HPLC.ResultsCompared to saline-injected rats, STZ-injected (diabetic) rats showed elevation of SR protein levels in retinal homogenates, attributed to the inner nuclear layer (INL) by immunofluorescence. Aqueous humor fluid from STZ-injected rats contained significantly higher levels of glutamate and D-serine compared to controls; by contrast, D-serine levels in retinas did not differ. Levels of activated JNK were elevated in diabetic retinas compared to controls.ConclusionsIncreased expression of SR in retina and higher levels of glutamate and D-serine in aqueous humor of STZ-treated rats may result from activation of the JNK pathway in diabetic sequelae. Our data suggest that the inflammatory conditions that prevail during DR result in elevation of D-serine, a neurotransmitter contributing to glutamate toxicity, potentially exacerbating the death of retinal ganglion cells in this condition.

Immunogold Detection of L-glutamate and D-serine in Small Synaptic-Like Microvesicles in Adult Hippocampal Astrocytes

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Turning pyridoxal-5′-phosphate-dependent enzymes into thermostable binding proteins: d-Serine dehydratase from baker’s yeast as a case study

d-serine dehydratase from Saccharomyces cerevisae is a recently discovered dimeric enzyme catalyzing the β-elimination of d-serine to pyruvate and ammonia. The reaction is highly enantioselective and depends on cofactor pyridoxal-5′-phosphate (PLP) and Zn2+. In our work, the aldimine linkage tethering PLP to recombinant, tagged d-serine dehydratase (Dsd) has been reduced by treatment with NaBH4 so as to yield an inactive form of the holoenzyme (DsdR), which was further treated with a protease in order to remove the amino-terminal purification tag. Fourier Transform infrared (FT-IR) spectroscopic analysis revealed that both the reduced form (DsdR) and the reduced/detagged form (DsdRD) maintain the overall secondary structure of Dsd, but featured a significant increased thermal stability. The observed Tm values for DsdR and for DsdRD shifted to 71.5 °C and 73.3 °C, respectively, resulting in nearly 11 °C and 13 °C higher than the one measured for Dsd. Furthermore, the analysis of the FT-IR spectra acquired in the presence of d-serine and l-serine indicates that, though catalytically inert, DsdRD retains the ability to enantioselectively bind its natural substrate. Sequence analysis of d-serine dehydratase and other PLP-dependent enzymes also highlighted critical residues involved in PLP binding. In virtue of its intrinsic properties, DsdRD represents an ideal candidate for the design of novel platforms based on stable, non-consuming binding proteins aimed at measuring d-serine levels in biological fluids.

Levels of d-serine in the brain and peripheral organs of serine racemase ( Srr) knock-out mice

d-Serine, an endogenous co-agonist of the N-methyl- d-aspartate (NMDA) receptor, plays an important role in mammalian brain neurotransmission, via the NMDA receptor. d-Serine is synthesized from l-serine by the pyridoxal-5′ phosphate-dependent enzyme serine racemase (SRR), and d-serine is metabolized by d-amino acid oxidase (DAAO). In this study, we measured levels of the neurotransmission related amino acids, d-serine, l-serine, glycine, glutamine and glutamate in the frontal cortex, hippocampus, striatum and cerebellum as well as in peripheral tissues of blood, heart, pancreas, spleen, liver, kidney, testis, epididymis, heart, lung, muscle and eyeball, in wild-type (WT) and Srr-knockout ( Srr-KO) mice. Levels of d-serine in the frontal cortex, hippocampus, and striatum of Srr-KO mice were significantly lower than in WT mice, while levels in the cerebellum stayed the same. In contrast, levels of l-serine, glycine, glutamine and glutamate remained the same in all tested brain regions. In vivo microdialysis using free-moving mice showed that extracellular levels of d-serine in the hippocampus of Srr-KO mice were significantly lower than in WT mice while the other amino acid levels remained the same between mice. In peripheral organs, levels of d-serine in the kidney, testis, and muscle of Srr-KO mice were significantly lower than in WT mice. Tissue levels of the other tested amino acids in peripheral organs were not altered. These results suggest that SRR is the major enzyme responsible for d-serine production in the mouse forebrain, and that other pathways of d-serine production may exist in the brain and peripheral organs.

Development of a high-throughput method for the determination of pharmacological levels of plasma d-serine

d-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, d-serine must be administered at high doses to observe clinical effects. This is due in large part to d-serine undergoing oxidation by d-amino acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of d-serine with a DAAO inhibitor has been suggested as a way to lower the dose of d-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma d-serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure d-serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor d-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate d-serine from plasma followed by quantitation of d-serine using the DAAO-catalyzed reaction. Plasma d-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.

P.3.c.007 Excitatory amino acids serum levels affected by antipsychotics in patients with schizophrenia

Hyperactivity of the hypothalamic pituitary–adrenal (HPA) axis plays a role in the pathophysiology of major depressive disorder (MDD). Recent studies suggest the role of the glutamatergic system in the pathophysiology of MDD, and N-methyl-d-aspartate (NMDA) receptor antagonists have shown antidepressant effects in both preclinical and clinical studies. However, little is known about the role of adrenocorticotropic hormone (ACTH) specifically in the glutamatergic response to HPA axis activation. Glutamate is an NMDA receptor agonist, and glycine and d-serine act as co-agonists. Here, we measured brain concentrations of these amino acids in rats given repeated administration of ACTH (100 μg/rat/day, sc, for 14 days). Further, we also evaluated behavioral effects of memantine, a non-competitive NMDA antagonist, on immobility time in the forced swimming test and on locomotor activity in ACTH-treated rats. Compared with control rats, glutamine, glycine, l-serine, and d-serine levels were increased in the hippocampus of ACTH-treated rats; glutamate, glutamine, glycine, l-serine, and d-serine were increased in the cerebellum; and glutamine and glycine were increased in the frontal cortex and striatum, all with statistical significance. Remarkably, these increases in agonists and co-agonists might have led to the augmentation of NMDA receptor activity. ACTH treatment increased immobility time in the forced swimming test and decreased locomotor activity in rats. On the contrary, memantine (10 mg/kg, ip) significantly decreased immobility time in the forced swimming test and increased locomotor activity in ACTH-treated rats. Furthermore, imipramine (15 mg/kg, ip) did not alter immobility time in the forced swimming test whereas this drug significantly decreased locomotor activity in ACTH-treated rats. These results suggest that depressive-like behaviors by chronic ACTH treatment could be blocked by memantine.

Glial D-Serine Gates NMDA Receptors at Excitatory Synapses in Prefrontal Cortex

N-methyl-D-aspartate receptors (NMDARs) subserve numerous neurophysiological and neuropathological processes in the cerebral cortex. Their activation requires the binding of glutamate and also of a coagonist. Whereas glycine and D-serine (D-ser) are candidates for such a role at central synapses, the nature of the coagonist in cerebral cortex remains unknown. We first show that the glycine-binding site of NMDARs is not saturated in acute slices preparations of medial prefrontal cortex (mPFC). Using enzymes that selectively degrade either D-ser or glycine, we demonstrate that under the present conditions, D-ser is the principle endogenous coagonist of synaptic NMDARs at mature excitatory synapses in layers V/VI of mPFC where it is essential for long-term potentiation (LTP) induction. Furthermore, blocking the activity of glia with the metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated synaptic transmission and prevents LTP induction by reducing the extracellular levels of D-serine. Such deficits can be restored by exogenous D-ser, indicating that the D-amino acid mainly originates from glia in the mPFC, as further confirmed by double-immunostaining studies for D-ser and anti-glial fibrillary acidic protein. Our findings suggest that D-ser modulates neuronal networks in the cerebral cortex by gating the activity of NMDARs and that altering its levels is relevant to the induction and potentially treatment of psychiatric and neurological disorders.