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Shape-shifting, a phenomenon wide-spread in folklore, refers to the ability to physically change from one identity to another, typically from an innocuous entity to a destructive one. The amino acid D-serine over the last 25 years has "shape-shifted" into several identities: a purported glial transmitter activating N-methyl-D-aspartate receptors (NMDARs), a co-transmitter concentrated in excitatory glutamatergic neurons, an autocrine that is released at dendritic spines to prime their post-synaptic NMDARs for an instantaneous response to glutamate and an excitotoxic moiety released from inflammatory (A1) astrocytes. This article will review evidence in support of these scenarios and the artifacts that misled investigators of the true identity of D-serine.

d-serine is the primary NMDAR coagonist at mature forebrain synapses and is synthesized by the enzyme serine racemase (SR). However, our understanding of the mechanisms regulating the availability of synapticd-serine remains limited. Though early studies suggestedd-serine is synthesized and released from astrocytes, more recent studies have demonstrated a predominantly neuronal localization of SR. More specifically, recent work intriguingly suggests that SR may be found at the postsynaptic density, yet the functional implications of postsynaptic SR on synaptic transmission are not yet known. Here, we show an age-dependent dendritic and postsynaptic localization of SR andd-serine by immunohistochemistry and electron microscopy in mouse CA1 pyramidal neurons. In addition, using a single-neuron genetic approach in SR conditional KO mice from both sexes, we demonstrate a cell-autonomous role for SR in regulating synaptic NMDAR function at Schaffer collateral (CA3)-CA1 synapses. Importantly, single-neuron genetic deletion of SR resulted in the elimination of LTP at 1 month of age, which could be rescued by exogenousd-serine. Interestingly, there was a restoration of LTP by 2 months of age that was associated with an upregulation of synaptic GluN2B. Our findings support a cell-autonomous role for postsynaptic neuronal SR in regulating synaptic NMDAR function and suggests a possible autocrine mode ofd-serine action.SIGNIFICANCE STATEMENTNMDARs are key regulators of neurodevelopment and synaptic plasticity and are unique in their requirement for binding of a coagonist, which isd-serine at most forebrain synapses. However, our understanding of the mechanisms regulating synapticd-serine availability remains limited.d-serine is synthesized in the brain by the neuronal enzyme serine racemase (SR). Here, we show dendritic and postsynaptic localization of SR andd-serine in CA1 pyramidal neurons. In addition, using single-neuron genetic deletion of SR, we establish a role of postsynaptic SR in regulating NMDAR function. These results support an autocrine mode ofd-serine action at synapses.

The neurotoxic action of glutamic acid was first described by Lucas and Newhouse, who demonstrated neural degeneration in the inner layers of the neonatal mouse retina after systemic treatment with L-glutamate. Olney extended these findings by showing that neuronal degeneration affected other brain structures including neurons within the arcuate nucleus of the hypothalamus and the area postrema, that the lesion spared axons passing through these areas, and that the neurotoxic potency of glutamate analogs correlated with their excitatory potency, resulting in the designation "excitotoxins." As this method affected only a small number of brain regions, it was not suitable for targeted brain lesions. The Coyle laboratory showed that direct injection of the potent glutamate receptor agonist, kainic acid, into the rat striatum caused a rapid degeneration of intrinsic neurons while sparing axons of passage or termination including the unmyelinated dopaminergic terminals. Kainic acid also exhibited this perikaryal-specific and axon-sparing profile when injected into the cerebellum, hippocampus and eye. However, neuronal vulnerability was highly variable, with hippocampal CA3, pyriform cortex and amygdala neurons exhibiting great sensitivity due to kainate's high convulsive activity. In a comparison study, ibotenic acid, a potent glutamatergic agonist isolated from the amanita muscaria mushroom, was found to have excitotoxic potency comparable to kainate but was far less epileptogenic. Ibotenate produced spherical, perikaryal-specific lesions regardless of the site of injection, and experiments with specific glutamate receptor antagonists showed that its effects were mediated by the N-methyl-D-aspartate receptor. Because of this uniform neurotoxicity and near ubiquitous efficacy, ibotenic acid became the excitotoxic lesioning agent of choice. The discovery of the excitotoxic properties of the tryptophan metabolite quinolinic acid and of the anti-excitotoxic, neuroprotective effects of the related metabolite kynurenic acid in the Schwarcz laboratory then gave rise to the concept that these endogenous compounds may play causative roles in the neuropathology of a wide range of neurological and psychiatric disorders.

D-serine is the primary NMDA receptor (NMDAR) co-agonist at mature forebrain synapses and is synthesized by the enzyme serine racemase (SR). However, our understanding of the mechanisms regulating the availability of synaptic D-serine remains limited. Though early studies suggested D-serine is synthesized and released from astrocytes, more recent studies have demonstrated a predominantly neuronal localization of SR. More specifically, recent work intriguingly suggests that SR may be found at the postsynaptic density, yet the functional implications of postsynaptic SR on synaptic transmission are not yet known. Here, we show an age-dependent dendritic and postsynaptic localization of SR and D-serine by immunohistochemistry and electron microscopy in mouse CA1 pyramidal neurons, as well as the presence of SR in human hippocampal synaptosomes. In addition, using a single-neuron genetic approach in SR conditional knockout mice, we demonstrate a cell-autonomous role for SR in regulating synaptic NMDAR function at Schaffer collateral (CA3)-CA1 synapses. Importantly, single-neuron genetic deletion of SR resulted in the elimination of LTP at one month of age. Interestingly, there was a restoration of LTP by two months of age that was associated with an upregulation of synaptic GluN2B. Our findings support a cell-autonomous role for postsynaptic neuronal SR in regulating synaptic NMDAR function and suggests a possible autocrine mode of D-serine action.

d -Serine plays an important role in modulating N-methyl- d -aspartate receptor (NMDAR) neurotransmission in the mammalian brain by binding to the receptor’s glycine modulatory site (GMS). The cytosolic enzyme serine racemase (SR) converts L-serine to d -serine, while the peroxisomal enzyme d -amino acid oxidase (DAAO) catalyzes the breakdown of d -serine. Although it is important to understand how the activities of SR and DAAO regulate d -serine levels, very little is known about the mechanisms that regulate the expression of SR and DAAO. In this study, we investigated whether the different centrally active drugs affect the expression of SR and DAAO in adult mouse brain. We found that the NMDAR antagonist, MK801, and cocaine, psychotropic drugs that both augment glutamate release, reduce the expression of SR and DAAO. This regulation is brain region selective, and in the case of cocaine, is reversed in part byα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). However, d -serine and antipsychotics do not regulate SR and DAAO protein levels. In a genetic model of SR disruption, we found that DAAO expression was unaltered in SR conditional knockout mice, in which tissue d -serine content remains fairly stable despite marked reduction in SR expression. This study reveals a new mechanism by which AMPAR activity could regulate NMDAR function via d -serine availability.

d-serine is synthesized by serine racemase (SR) and is a co-agonist at forebrain N-methyl-d-aspartate receptors (NMDARs). d-serine and SR are expressed primarily in neurons, but not in quiescent astrocytes. In this study, we examined the localization of d-serine and SR in the mouse striatum and the effects of genetically silencing SR expression in GABAergic interneurons (iSR−/−). iSR−/− mice had substantially reduced SR expression almost exclusively in striatum, but only exhibited marginal d-serine reduction. SR positive cells in the striatum showed strong co-localization with dopamine- and cyclic AMP-regulated neuronal phosphoprotein (DARPP32) in wild type mice. Transgenic fluorescent reporter mice for either the D1 or D2 dopamine receptors exhibited a 65:35 ratio for co-localization with D1and D2 receptor positive cells, respectively. These results indicate that GABAergic medium spiny neurons receiving dopaminergic inputs in striatum robustly and uniformly express SR. In behavioral tests, iSR−/− mice showed a blunted response to the hedonic and stimulant effects of cocaine, without affecting anxiety-related behaviors. Because the cocaine effects have been shown in the constitutive SR−/− mice, the restriction of the blunted response to cocaine to iSR−/− mice reinforces the conclusion that d-serine in striatal GABAergic neurons plays an important role in mediating dopaminergic stimulant effects. Results in this study suggest that SR in striatal GABAergic neurons is synthesizing d-serine, not as a glutamatergic co-transmitter, but rather as an autocrine whereby the GABAergic neurons control the excitability of their NMDARs by determining the availability of the co-agonist, d-serine.

Both schizophrenia (SZ) and substance abuse (SA) exhibit significant heritability. Moreover, N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathophysiology of both SZ and SA. We hypothesize that the high prevalence of comorbid SA in SZ is due to dysfunction of NMDARs caused by shared risk genes. We used transgenic mice with a null mutation of the gene encoding serine racemase (SR), the enzyme that synthesizes the NMDAR co-agonist d-serine and an established risk gene for SZ, to recreate the pathology of SZ. We determined the effect of NMDAR hypofunction resulting from the absence of d-serine on motivated behavior by using intracranial self-stimulation and neurotransmitter release in the nucleus accumbens by using in vivo microdialysis. Compared with wild-type mice, SR-/- mice exhibited similar baseline intracranial self-stimulation thresholds but were less sensitive to the threshold-lowering (rewarding) and the performance-elevating (stimulant) effects of cocaine. While basal dopamine (DA) and glutamate release were elevated in the nucleus accumbens of SR-/- mice, cocaine-induced increases in DA and glutamate release were blunted. γ-Amino-butyric acid efflux was unaffected in the SR-/- mice. Together, these findings suggest that the impaired NMDAR function and a consequent decrease in sensitivity to cocaine effects on behavior are mediated by blunted DA and glutamate responses normally triggered by the drug. Projected to humans, NMDAR hypofunction due to mutations in SR or other genes impacting glutamatergic function in SZ may render abused substances less potent and effective, thus requiring higher doses to achieve a hedonic response, resulting in elevated drug exposure and increased dependence/addiction.

The N-methyl-D-aspartate receptor (NMDAR) is unique in requiring two agonists to bind simultaneously to open its cation channel: the neurotransmitter, glutamate, and the coagonists, glycine, or D-serine. The Snyder laboratory was the first to clone serine racemase (SR), the enzyme that synthesizes D-serine, and to localize it immunocytochemically. Our laboratory has focused on the role of D-serine in brain disorders. Silencing the expression of SR, a risk gene for schizophrenia (SCZ), in mice (SR−/−), results in a phenotype that closely resembles SCZ including: cortical atrophy, reduced dendritic spine density and complexity, downregulation of parvalbumin-positive cortical GABAergic neurons, and cognitive impairments. This pathology can be reversed by treatment of SR−/− mice with D-serine in adulthood. SR−/− mice also exhibit abnormal response toward abusable substances, such as stimulants. They show reduced behavioral sensitization to D-amphetamine, but fail to extinguish it. Place preference to cocaine is altered, and the hedonic response to it is profoundly impaired as assessed by intracranial self-stimulation. D-cycloserine, a partial agonist at the NMDAR glycine modulatory site, shows therapeutic benefit for treating pathologic anxiety in combination with behavioral therapies. Studies in vitro with cortical culture and in vivo with middle cerebral artery occlusion show that silencing SR provides substantial protection against ischemic neuronal death. Finally, the switch of SR expression from neurons to reactive astrocytes after closed head trauma accounts for the reduced in vivo neuro-plasticity, electroencephalogram abnormalities, and cognitive impairments.

In the hippocampus of mice lacking the gene for serine racemase (SR), a D-serine synthesizing enzyme, in the CaMKIIα-expressing neurons, we observed a significant decrease in the extracellular concentration of D-serine, a coagonist for the N-methyl-D-aspartate type glutamate receptor (NMDAR), and NMDAR hypofunction as revealed by diminished extracellular taurine concentrations after an intra-hippocampal NMDA infusion when compared to the wild type controls. Therefore, the neuronal SR could regulate the extracellular D-serine signaling responsible for NMDAR activation in the hippocampus.

d-Serine is a co-agonist at forebrain N-methyl-d-aspartate receptors (NMDAR) and is synthesized by serine racemase (SR). Although d-serine and SR were originally reported to be localized to glia, recent studies have provided compelling evidence that under healthy physiologic conditions both are localized primarily in neurons. However, in pathologic conditions, reactive astrocytes can also express SR and synthesize d-serine. Since cultured astrocytes exhibit features of reactive astrocytes, we have characterized d-serine synthesis and the expression of enzymes involved in its disposition in primary glial cultures. The levels of SR were quite low early in culture and increased markedly in all astrocytes with the duration in vitro. The concentration of d-serine in the culture medium increased in parallel with SR expression in the astrocytes. Microglia, identified by robust expression of Iba1, did not express SR. While the levels of glial fibrillary acidic protein (GFAP), glycine decarboxylase (GLDC) and phosphoglycerate dehydrogenase (PHGDH), the initial enzyme in the pathway converting glycine to l-serine, remained constant in culture, the expression of lipocalin-2, a marker for pan-reactive astrocytes, increased several-fold. The cultured astrocytes also expressed Complement-3a, a marker for a subpopulation of reactive astrocytes (A1). Astrocytes grown from mice with a copy number variant associated with psychosis, which have four copies of the GLDC gene, showed a more rapid production of d-serine and a reduction in glycine in the culture medium. These results substantiate the conclusion that A1 reactive astrocytes express SR and release d-serine under pathologic conditions, which may contribute to their neurotoxic effects by activating extra-synaptic NMDARs.

After traumatic brain injury (TBI), glial cells have both beneficial and deleterious roles in injury progression and recovery. However, few studies have examined the influence of reactive astrocytes in the tripartite synapse following TBI. Here, we have demonstrated that hippocampal synaptic damage caused by controlled cortical impact (CCI) injury in mice results in a switch from neuronal to astrocytic d-serine release. Under nonpathological conditions, d-serine functions as a neurotransmitter and coagonist for NMDA receptors and is involved in mediating synaptic plasticity. The phasic release of neuronal d-serine is important in maintaining synaptic function, and deficiencies lead to reductions in synaptic function and plasticity. Following CCI injury, hippocampal neurons downregulated d-serine levels, while astrocytes enhanced production and release of d-serine. We further determined that this switch in the cellular source of d-serine, together with the release of basal levels of glutamate, contributes to synaptic damage and dysfunction. Astrocyte-specific elimination of the astrocytic d-serine-synthesizing enzyme serine racemase after CCI injury improved synaptic plasticity, brain oscillations, and learning behavior. We conclude that the enhanced tonic release of d-serine from astrocytes after TBI underlies much of the synaptic damage associated with brain injury.

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 Muller 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.

All current drugs approved to treat schizophrenia appear to exert their antipsychotic effects through blocking the dopamine D2 receptor. Recent meta-analyses and comparative efficacy studies indicate marginal differences in efficacy of newer atypical antipsychotics and the older drugs, and little effects on negative and cognitive symptoms. This review integrates findings from postmortem, imaging, and drug-challenge studies to elucidate a corticolimbic "pathologic circuit" in schizophrenia that may be particularly relevant to the negative symptoms and cognitive impairments of schizophrenia. Potential sites for pharmacologic intervention targeting glutatatergic, GABAergic, and cholinergic neurotransmission to treat these symptoms of schizophrenia are discussed.

Enhancement of N-methyl-D: -aspartate receptor (NMDAR) activity through its glycine modulatory site (GMS) is a novel therapeutic approach in schizophrenia. Brain concentrations of endogenous GMS agonist D: -serine and antagonist N-acetyl-aspartylglutamate are regulated by serine racemase (SR) and glutamic acid decarboxylase 2 (GCP2), respectively. Using mice genetically, under-expressing these enzymes may clarify the role of NMDAR-mediated neurotransmission in schizophrenia.We investigated the behavioral effects of two psychotomimetic drugs, the noncompetitive NMDAR antagonist, phencyclidine (PCP; 0, 1.0, 3.0, or 6.0 mg/kg), and the indirect dopamine receptor agonist, amphetamine (AMPH; 0, 1.0, 2.0, or 4.0 mg/kg), in SR -/- and GCP2 -/+ mice. Outcome measures were locomotor activity and prepulse inhibition (PPI) of the acoustic startle reflex. Acute effects of an exogenous GMS antagonist, gavestinel (0, 3.0, or 10.0 mg/kg), on PCP-induced behaviors were examined in wild-type mice for comparison to the mutants with reduced GMS activity.PCP-induced hyperactivity was increased in GCP2 -/+ mice, and PCP-enhanced startle reactivity was increased in SR -/- mice. PCP disruption of PPI was unaffected in either mutant. In contrast, gavestinel attenuated PCP-induced PPI disruption without effect on baseline PPI or locomotor activity. AMPH effects were similar to controls in both mutant strains.The results of the PCP experiments demonstrate that convergence of pharmacological and genetic manipulations at NMDARs may confound the predictive validity of these preclinical assays for the effects of GMS activation in schizophrenia. The AMPH data provide additional evidence that hyperdopaminergia in schizophrenia may be distinct from NMDAR hypofunction.

d-Serine, formed froml-serine by serine racemase (SR), is a physiologic coagonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role ford-serine in NMDA receptor-mediated neurotoxicity and stroke. Brain cultures of SR-deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knock-out mice, NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity.

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.

Many risk genes interact synergistically to produce schizophrenia and many neurotransmitter interactions have been implicated. We have developed a circuit-based framework for understanding gene and neurotransmitter interactions. NMDAR hypofunction has been implicated in schizophrenia because NMDAR antagonists reproduce symptoms of the disease. One action of antagonists is to reduce the excitation of fast-spiking interneurons, resulting in disinhibition of pyramidal cells. Overactive pyramidal cells, notably those in the hippocampus, can drive a hyperdopaminergic state that produces psychosis. Additional aspects of interneuron function can be understood in this framework, as follows. (i) In animal models, NMDAR antagonists reduce parvalbumin and GAD67, as found in schizophrenia. These changes produce further disinhibition and can be viewed as the aberrant response of a homeostatic system having a faulty activity sensor (the NMDAR). (ii) Disinhibition decreases the power of gamma oscillation and might thereby produce negative and cognitive symptoms. (iii) Nicotine enhances the output of interneurons, and might thereby contribute to its therapeutic effect in schizophrenia.

This review examines the large body of toxicological and epidemiological information on human exposures to chlorpyrifos, with an emphasis on the controversial potential for chlorpyrifos to induce neurodevelopmental effects at low doses. The results of this review demonstrate that the use of urinary 3,5,6-trichlorpyridinol (TCPy), a metabolite of chlorpyrifos as a biomarker of nonoccupational exposure is problematic and may overestimate nonoccupational exposures to chlorpyrifos by 10-to 20-fold because of the widespread presence of both TCPy and chlorpyrifos-methyl in the food supply. Current "background" (nonoccupational) levels of exposure to chlorpyrifos are several orders of magnitude lower than those required to inhibit plasma cholinesterase activity, which is a more sensitive target than nervous system cholinesterase. However, several in vitro studies have identified putative neurodevelopmental mechanisms that are altered at concentrations of chlorpyrifos below those that inhibit cholinesterases. Although one human cohort study reported an association between maternal and cord blood chlorpyrifos levels and several measures of neurodevelopment, two other cohort studies that utilized urinary TCPy as a surrogate for chlorpyrifos exposure did not demonstrate an association. Although the weight of the scientific evidence demonstrates that current levels of chlorpyrifos exposure will not have any adverse effects on neurodevelopment that might result from inhibition of nervous system cholinesterases, several recent studies propose alternative mechanisms. Thus, further in vivo investigation on neurodevelopment in an appropriate animal model is needed; additional epidemiological studies may be warranted if a suitable, chlorpyrifos-exposed cohort can be identified and more rigorous measures of exposure are utilized.

Galantamine is an approved treatment for mild to moderate Alzheimer's disease, with demonstrated benefits for cognition and functional ability in human studies. The mechanism of action that is most generally recognized as underlying the clinical benefits of galantamine is inhibition of brain acetylcholinesterase (AChE). However, an increasing body of evidence suggests that an additional mechanism, most likely allosteric modulation of nicotinic acetylcholine receptors (nAChRs), may contribute to the therapeutic effects of galantamine. This review summarizes the research on this additional mechanism, with emphasis on data derived from in vivo animal studies and open-label hypothesis-generating studies in humans. In general, these studies provide evidence of effects beyond those of AChE inhibition alone, most notably in comparisons with other AChE inhibitors, in which galantamine produced similar or greater effects at doses that provided lower levels of brain AChE inhibition. The use of nAChR agonists and antagonists in some of these studies lends support to the proposed allosteric potentiating ligand activity of galantamine at nAChRs. This dual action of galantamine may account for its therapeutic profile.

Schizophrenia is associated with high prevalence of substance abuse. Recent research suggests that dysregulation of N-methyl-d-aspartate receptor (NMDAR) function may play a role in the pathophysiology of both schizophrenia and drug addiction, and thus, may account for this high comorbidity. Our laboratory has developed two transgenic mouse lines that exhibit contrasting NMDAR activity based on the availability of the glycine modulatory site (GMS) agonists d-serine and glycine. Glycine transporter 1 knockdowns (GlyT1(+/-)) exhibit NMDAR hyperfunction, whereas serine racemase knockouts (SR(-/-)) exhibit NMDAR hypofunction. We characterized the behavior of these lines in a cocaine-induced (20 mg/kg) conditioned place preference (CPP) and locomotor sensitization paradigm. Compared with wild-type mice, GlyT1(+/-) mice displayed hastened extinction of CPP and robust cocaine-induced reinstatement. SR(-/-) mice appeared to immediately "forget" the learned preference, because they did not exhibit cocaine-induced reinstatement and also displayed attenuated locomotor sensitization. Treatment of GlyT1(+/-) mice with gavestinel (10 mg/kg on day 1; 5 mg/kg on days 2-17), a GMS antagonist, attenuated cocaine-induced CPP and caused them to immediately "forget" the learned preference. Treatment of SR(-/-) mice with d-serine (300 mg/kg on day 1; 150 mg/kg on days 2-17) to normalize brain levels caused them to avoid the cocaine-paired side of the chamber during extinction. These results highlight NMDAR dysfunction as a possible neural mechanism underlying comorbid schizophrenia and substance abuse. Also, these findings suggest drugs that directly or indirectly activate the NMDAR GMS could be an effective treatment of cocaine abuse.

To investigate the effects of persistent elevation of synaptic glycine at Schaffer collateral–CA1 synapses of the hippocampus, we studied the glutamatergic synaptic transmission in acute brain slices from mice with reduced expression of glycine transporter type 1 (GlyT1+/−) as compared to wild type (WT) littermates using whole-cell patch-clamp recordings of CA1 pyramidal cells. We observed faster decay kinetics, reduced ifenprodil sensitivity and increased zinc-induced antagonism in N-methyl-d-aspartate receptor (NMDAR) currents of GlyT1+/− mice. Moreover, the ratio α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)/NMDAR was decreased in mutants compared to WT. Surprisingly, this change was associated with a reduction in the number of AMPARs expressed at the CA1 synapses in the mutants compared to WT. Overall, these findings highlight the importance of GlyT1 in regulating glutamatergic neurotransmission.

N-acetylaspartylglutamate (NAAG) is an abundant neuropeptide in the nervous system, yet its functions are not well understood. Pyramidal neurons of the CA1 sector of acutely prepared hippocampal slices were recorded using the whole-cell patch-clamp technique. At low concentrations (20 microM), NAAG reduced isolated N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic currents or NMDA-induced currents. The NAAG-induced change in the NMDA concentration/response curve suggested that the antagonism was not competitive. However, the NAAG-induced change in the concentration/response curve for the NMDAR co-agonist, glycine, indicated that glycine can overcome the NAAG antagonism. The antagonism of the NMDAR induced by NAAG was still observed in the presence of LY-341495, a potent and selective mGluR3 antagonist. Moreover, in dissociated pyramidal neurons of the CA1 region, NAAG also reduced the NMDA current and this effect was reversed by glycine. These results suggest that NAAG reduces the NMDA currents in hippocampal CA1 pyramidal neurons.

Mice that are transgenic for and overexpress human copper/zinc superoxide dismutase were used to investigate the role of this enzyme in the pathophysiology of Down's syndrome (DS; trisomy 21). Previous studies have indicated that overexpression of copper/zinc superoxide dismutase leads to deficits in peripheral markers of neurochemical function, which are consistent with the hypothesis that this enzyme plays a role in the pathophysiology of DS. We have measured concentrations of amino acids and biogenic amines (catecholamines, serotonin, and their metabolites), uptake of biogenic amines into crude synaptosomes, and activities of synthetic enzymes in both control mice and mice transgenic for human copper/zinc superoxide dismutase that overexpress it by two- to fivefold above control values. We find that these transgenic mice exhibit higher concentrations of the biogenic amines in specific brain regions, with little or no change in amino acid concentration. Furthermore, tyrosine hydroxylase activity is increased in the striatum of the transgenics, whereas glutamic acid decarboxylase and choline acetyltransferase activities are unchanged in all but one brain region. These findings indicate that over-expression of copper/zinc superoxide dismutase, by itself, is not sufficient to cause the synaptic neurochemical deficits reported in DS.

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

Cerebellar granule cells (CGC) deprived of serum or trophic factors develop sensitivity to kainate neurotoxicity that is mediated by the α-amino-3-hydroxy-5-methyl-isoxazole proprionic acid (AMPA) subtypes of glutamate receptors (GluR). The L-type voltage-gated calcium channel (L-type VGCC) blocker nifedipine increases the potency of kainate 50-fold. Thus, one goal of this laboratory is to determine the underlying protective mechanism triggered by calcium influx through this channel. The cell-permeable heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine effected complete protection against kainate treatment in the presence of nifedipine, as did the iron chelator deferoxamine. The chelatable heavy metal pool decreased ∼70% immediately following treatment with kainate, but did not change following kainate/nifedipine treatment. Tetramethylrhhodamine ethyl ester (TMRE) fluorescence, an indicator of mitochondrial membrane potential, decreased ∼70% following kainate treatment but displayed a more modest decrease (∼15%) when CGC were treated with kainate/nifedipine. Reactive oxygen species (ROS) formation decreased in CGC immediately following kainate treatment but was slightly elevated following kainate/nifedipine treatment. Electron microscopic examinations of the CGC indicated severe swelling and distortion of mitochondria immediately following kainate/nifedipine treatment and the appearance of mitochondrial herniations, whorls, and bridges 2 h later, features that were rarely observed following kainate treatment. These results support the hypothesis that calcium entry through L-type VGCCs protects CGC during kainate treatment by lowering the chelatable heavy metal pool and the mitochondrial membrane potential, thereby mitigating the formation of ROS. Synapse 43:30–41, 2002. © 2001 Wiley-Liss, Inc.

Despite the proven efficacy of acetylcholinesterase inhibitors in Alzheimer's disease, there is a need for new and more effective treatments. Galantamine is a novel treatment for Alzheimer's disease that inhibits acetylcholinesterase and modulates nicotinic receptors. In randomized, double-blind, placebo-controlled studies of up to 6 months duration, galantamine significantly improved cognitive function. Galantamine also had beneficial effects on instrumental and basic activities of daily living, and postponed the progression of behavioral symptoms. Patients who completed one of the 6-month, placebo-controlled studies were eligible to enter a 6-month, open-extension study of the 24-mg/day dose of galantamine. At the end of 12 months, cognitive function and activities of daily living were preserved in those patients who had been treated throughout the study with galantamine 24 mg/day. At 12 months, this group of patients had significantly better cognitive functions than patients who had been treated with a placebo for 6 months before receiving galantamine. These studies indicate that galantamine postpones the progression of symptoms in Alzheimer's disease. Since galantamine shows the greatest benefits when treatment is started early, its long-term benefits may result from an effect on the underlying disease process; such an effect might be mediated by galantamine's concomitant action on nicotinic receptors.

Receptor supersensitivity is an important concept for understanding neurotransmitter and receptor dynamics. Traditionally, detection of receptor supersensitivity has been performed using autoradiography or positron emission tomography (PET). We show that use of magnetic resonance imaging (MRI) not only enables one to detect dopaminergic supersensitivity, but that the hemodynamic time course reflective of this fact is different in different brain regions. In rats unilaterally lesioned with intranigral 6-hydroxydopamine, apomorphine injections lead to a large increase in hemodynamic response (cerebral blood volume, CBV) in the striato-thalamo-cortico circuit on the lesioned side but had little effect on the intact side. Amphetamine injections lead to increases in hemodynamic responses on the intact side and little on the lesioned side in the same animals. The time course for the increase in CBV after either amphetamine or apomorphine administration was longer in striatum and thalamus than in frontal cortex. 11C-PET studies of ligands which bind to the dopamine transporter (2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane 1,5-naphthalnendisulfonate, WIN 35, 428 or CFT) and D2 receptors (raclopride) confirm that there is a loss of presynaptic dopamine terminals as well as upregulation of D2 receptors in striatum in these same animals. Pharmacologic MRI should become a sensitive tool to measure functional supersensitivity in humans, providing a complementary picture to that generated using PET studies of direct receptor binding. Synapse 36:57–65, 2000. © 2000 Wiley-Liss, Inc.

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels.

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N -methyl- d -aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro , demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo , thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

The cytoplasmic copper-zinc superoxide dismutase (Cu, Zn SOD; SOD-1) is an abundant and well-conserved intracellular antioxidant enzyme which has been implicated in a number of oxidative stress mediated phenomena, especially Down Syndrome, in which SOD-1 activity is increased due to triplication of chromosome 21 containing the gene and, in hereditary amyotrophic lateral sclerosis, in which the gene is mutated. Overexpression of SOD-1 could theoretically, therefore, lead to increased vulnerability to oxidative stress in two distinct manners: increasing steady-state hydrogen peroxide levels or increasing toxic side reactions. We used two mouse neuronal culture systems--one in which the murine chromosome containing SOD-1 is triplicated and one in which human SOD-1 is a transgene--to test the effect of overexpression of this enzyme on antioxidant status in general and specifically on glutamate mediated oxidative stress. We found that SOD-1 overexpression increases antioxidant status at the same time it decreases vulnerability to glutamate.

Glutamate carboxypeptidase II may modulate excitatory neurotransmission through the catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) and possibly other endogenous peptide substrates. To investigate the molecular properties of cloned human GCP II (hGCP II), we analyzed the NAAG-hydrolytic activity conveyed by transfection of a full-length hGCP II cDNA into PC3 cells, which do not express GCP II endogenously. Membrane fractions from these cells demonstrated activity with an apparent Km of 73 nM and Vmax of 35 pmol/(mg protein*min). Activity was inhibited by EDTA and stimulated by the addition of CoCl2. Addition of GCP II inhibitors β-NAAG, quisqualic acid and 2-(phosphonomethyl)pentanedioic acid (PMPA) inhibited hydrolysis of 2.5 nM NAAG with IC50s of 201 nM, 155 nM and 98 pM, respectively. In competition experiments designed to infer aspects of hGCP II substrate selectivity, NAAG was the most potent alpha peptide tested, with an IC50 of 26 nM. Folate derivatives and some other γ-glutamyl peptides showed comparable affinity to that of NAAG, also displaying IC50s in the low nM range. Taken together with previous evidence demonstrating their presence in GCP II-expressing tissues, these data suggest that both NAAG and folates are good candidate substrates for GCP II in vivo.

Recent evidence suggests that ethanol abuse produces its diverse effects on the brain to a substantial degree by disrupting the function of the major excitatory neurotransmitter, glutamate. Ethanol, at concentrations associated with behavioral effects in humans, inhibits the N-methyl-d-aspartate (NMDA) receptor, which mediates the post-synaptic excitatory effects of glutamate. Tolerance to ethanol results in up-regulation of the NMDA receptor so that abrupt withdrawal produces a hyperexcitable state that leads to seizures, delerium tremens, and excitotoxic neuronal death. Ethanol's inhibition of the NMDA receptor in the fetal brain likely contributes to the CNS manifestations of fetal alcohol syndrome. Therapeutic strategies aimed at correcting glutamatergic dysregulation in alcoholism need to be explored.

We examined the effects of N -acetylaspartylglutamate (NAAG), an endogenous peptide thought to be involved in neurotransmission and neuromodulation, on striatal quinolinate lesions, a rodent model of Huntington's disease. We found that NAAG (500 and 1000 nmol) co-injected with quinolinic acid significantly reduced lesion volumes (by 50% and 65%, respectively). A 1000 nmol dose of the non-hydrolyzable analogue, β -NAAG, also reduced quinolinic acid lesion volumes by 78.4%, indicating that the protection observed was not secondary to cleavage of NAAG into N -acetyl-aspartate (NAA) and glutamate. Likewise, co-injection of both NAA and glutamate (1000 nmol each) with quinolinic acid did not significantly alter the size of lesions. NAAG's protective effect may be mediated through actions on N -methyl- d -aspartate receptors or metabotropic glutamate receptors.

Publisher Summary This chapter examines the structural chemistry and the biological aspects of glutamate carboxypeptidase II. This enzyme was first characterized in the rat nervous system by its hydrolysis of the neuropeptide N-acetylaspartylglutamate and was termed N-acetylated-alpha-linked acidic dipeptidase. The prostate cancer marker known as the prostate-specific membrane antigen also possesses NAALADase activity. Subcellular fractionation of brain reveals enrichment of the enzyme in neurosynaptosomal complexes. Electron microscopy localizes glutamate carboxypeptidase II immunoreactivity to the outer surface of the plasma membrane. The results indicate that the rat enzyme is expressed exclusively in astrocytic glial cells. Prostate-Specific Membrane (PSM) was characterized originally as the ligand of the monoclonal antibody 7E11.C5, which exhibited immunohistochemical staining restricted among human tissues to the prostate epithelium. The analyses have reported 7E11.C5 immunoreactivity and/or positive RNAase protection assays with PSM-derived probes in human salivary gland, brain, small intestine, seminal plasma and blood serum. Two gene loci closely related to PSM have been identified on human chromosome 11. The PSM form of glutamate carboxypeptidase II is expressed in normal and neoplastic prostate epithelial cells, and PSM has been examined for utility in the diagnosis, assessment and treatment of prostatic carcinoma.

Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainic acid neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainic acid-induced neuronal degeneration. We found DNA laddering by agarose gel electrophoresis, cellular DNA fragmentation by in situ end labeling of DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainic acid (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainic acid-induced death. While the morphological and biochemical features of neuronal death induced by kainic acid resembled low K(+)-induced apoptosis in cerebellar granule cells, the time interval from the institution of the death promoting condition to neuronal death was shorter with kainic acid and did not require new protein or RNA synthesis. These results demonstrate that kainic acid receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainic acid receptor activation with apoptosis.

Growing evidence suggests that non- N -methyl- d -aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainate neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainate-induced neuronal degeneration. We found DNA laddering by agarose gel electrophoresis, cellular DNA fragmentation by in situ end labeling of DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainate (100 μM). Aurintricarboxylic acid protected cerebellar granule cells from kainate-induced death. While the morphological and biochemical features of neuronal death induced by kainate resembled low-K + -induced apoptosis in cerebellar granule cells, the time interval from the institution of the death-promoting condition to neuronal death was briefer with kainate and did not require new protein or RNA synthesis. These results demonstrate that kainate receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainate receptor activation with apoptosis.

There is an increasing amount of experimental evidence that oxidative stress is a causal, or at least an ancillary, factor in the neuropathology of several adult neurodegenerative disorders, as well as in stroke, trauma, and seizures. At the same time, excessive or persistent activation of glutamate-gated ion channels may cause neuronal degeneration in these same conditions. Glutamate and related acidic amino acids are thought to be the major excitatory neurotransmitters in brain and may be utilized by 40 percent of the synapses. Thus, two broad mechanisms--oxidative stress and excessive activation of glutamate receptors--are converging and represent sequential as well as interacting processes that provide a final common pathway for cell vulnerability in the brain. The broad distribution in brain of the processes regulating oxidative stress and mediating glutamatergic neurotransmission may explain the wide range of disorders in which both have been implicated. Yet differential expression of components of the processes in particular neuronal systems may account for selective neurodegeneration in certain disorders.

We conducted structural and pharmacological studies of galanthamine, a cortical acetylcholinesterase (AChE) inhibitor, and 19 structural analogs. Systematic derivatization of galanthamine at the cyclohexene ring, tertiary amino, hydroxyl and methoxyl functions indicated that these structural features are essential for biological activity. Molecular modeling studies suggested that the low energy conformations of the analogs are similar to that of the parent. One derivative, galanthamine n-butyl carbamate, had an LD 50 of over 100 mg/kg (ip) in mice. In a passive avoidance paradigm, this analog improved performance in a dose-dependent fashion with a peak effect at 0.1 mg/kg in control and 0.5 mg/kg in basal forebrain lesioned mice. In the same paradigm, the peak effect of the parent compound is a 6-fold higher dose. With this surprisingly high therapeutic ratio, this compound may be of interest in treating cholinergic deficits of the central nervous system such as Alzheimer's disease.

In the presence of physiological concentrations of Mg 2+ and in glycine-free buffer, the relationship between KA-mediated generation of NO and neurotoxicity in cultures of cerebellar granule cells of the rat was examined. The neuronal damage elicited by KA was not dependent on the presence of l -arginine, a precursor of NO, since neither the potency nor magnitude of KA-mediated cell death was altered in either the absence or presence of exogenously applied l -arginine. Similarly, with the exception of 4-hydroxy-azobenzene-4'-sulfonic acid, disodium salt dihydrate (HBS), the salt associated with N G -monomethyl- l -arginine (di-( p -hydroxyazobenzene- p ′-sulfonate) (MA(HBS)), treatment with several different competitive NO synthetase inhibitors did not provide protection against the toxicity of KA. However, the ability of KA to induce neuronal damage was significantly decreased in cerebellar granule cells treated with either HBS or α-tocopherol (VE). On the basis of these results, it is concluded that the generation of free radicals may be involved in the process of KA-elicited neuronal death in cultures of cerebellar granule cells but that this is unrelated to the synthesis of NO. This conclusion agrees with both in vivo and in vitro studies, implicating the involvement of free radicals in non-NMDA mediated neuronal damage.

In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.

Inter-neuronal communication is mediated primarily by chemical neurotransmitters, which are released from the nerve terminal, diffuse across the synaptic cleft and interact with specific receptors on adjacent neurons. The development of the biochemical machinery for neurotransmission is closely linked to the functional maturation of the brain's neuronal circuitry. Components essential for neurotransmission (e.g., synthetic enzymes, endogenous neurotransmitters, re-uptake processes and receptors) serve as specific biochemical markers for neuronal systems. The appearance of and developmental increases in these markers during fetal and postnatal life occur with the cessation of neuronal replication and initiation of neuropil elaboration. Discrete groups of neurotransmitter-specific neurons develop according to different timetables, resulting in a shifting pattern of their relative influence in the maturing brain. Human and animal studies demonstrate an early innervation of the neocortex by catecholaminergic axons while neurons using gamma-aminobutyric acid (GABA) mature somewhat later; and the ontogeny of the acetylcholine neurons lags behind both of these. Within each neuronal group the individual biochemical components for neurotransmission also follow differing time courses of maturation. Animal studies, in which cortical neurons were ablated by administering a toxin to the fetus, illustrate the interplay between intrinsic programmes and environmental influences in the assembly of neuronal circuits. The brain's preparation for independent life is characterized by a continual reorganization of neurotransmitter pathways.

N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.

The involvement of phospholipase A2 (PLA2) and fatty acid release in the regulation of sodium-dependent high-affinity choline uptake in rat brain was assessed in vitro through the use of the specific binding of 3H-hemicholinium-3 (3H-HCh-3). Addition of arachidonic acid and other unsaturated fatty acids to rat striatal membranes in vitro resulted in a dose-dependent, temperature-independent activation of 3H-HCh-3 binding. Scatchard analysis revealed that these changes in binding result from a 2-fold increase in the affinity and capacity of 3H-HCh-3 binding. Saturated fatty acids, lysophospholipids, and phospholipids did not affect specific 3H-HCh-3 binding. Addition of defatted BSA to membranes, which had been treated previously with arachidonic acid, completely reversed the increase in specific 3H-HCh-3 binding. However, several inhibitors of fatty acid metabolism, including nordihydroguaiaretic acid, indomethacin, catalase, and superoxide dismutase, did not alter arachidonic acid-induced changes in 3H-HCh-3 binding, suggesting that unsaturated fatty acids, and not their metabolites, are directly responsible for the observed activation of specific 3H-HCh-3 binding. Additionally, unsaturated fatty acids dose- dependently inhibited high-affinity 3H-choline uptake in rat striatal synaptosomes, apparently due to the disruption of synaptosomal integrity. The phospholipase A2 inhibitors quinacrine hydrochloride, trifluoperazine, and 4-bromophenacylbromide dose-dependently inhibited potassium depolarization-induced activation of specific 3H-HCh-3 binding in slices of rat brain in vitro. Similarly, both quinacrine and trifluoperazine inhibited the metabolism of phospholipids and the release of fatty acids evoked by either elevated KCl or calcium ionophore A23187. These results support the involvement of PLA2 and subsequent fatty acid release in the increase of 3H-HCh-3 binding in cholinergic neurons and suggest that activation of PLA2 may be the penultimate step in regulating the velocity of sodium-dependent choline transport.

Kainic acid, an acidic pyrolidine isolated from the seaweed Digenea simplex, is the most potent of the commonly used exogenous excitotoxins. The neurotoxic threshold of kainic acid is nearly two magnitudes lower than that of the other receptor-specific agonists, N-methyl-D-aspartic acid and quisqualic acid. Neurophysiological and ligand-binding studies indicate that the neurotoxic action of kainic acid is mediated by a specific receptor which exhibits a remarkably broad phylogenetic distribution in the nervous system of vertebrates and invertebrates. The mechanism of neurotoxicity of kainic acid appears to be indirect and requires the functional integrity of excitatory afferents to vulnerable neurons. Consistent with the excitotoxin hypothesis, kainic acid depletes high-energy phosphates and glucose at sites of neurotoxic action; nevertheless, the proximate cause of neurotoxicity may involve increases in intraneuronal calcium levels and the activation of calcium-dependent proteases. Kainic acid neurotoxicity provides a useful animal model for selective neuronal vulnerability that may shed light on the pathophysiology of a number of neurodegenerative disorders, including Huntington's disease and temporal lobe epilepsy.

Background Glutamatergic dysregulation has been documented in schizophrenia but has received less systematic study in affective illness. Methods Cerebrospinal fluid (CSF) levels of the excitatory amino acids glutamate (Glu) and aspartate (Asp) and the N-methyl-D-aspartate (NMDA) receptor modulator, glycine (GLY) were measured by high performance liquid chromatography in 32 patients with refractory affective disorder (16 female/16 male, 12 bipolar I, 12 bipolar II, and 8 unipolar) and in 14 age-matched controls. Results There was a significant reduction in CSF glutamate and glycine in patients versus controls. A diagnosis by sex interaction was present for CSF glycine with lower levels in female patients compared to female controls. Levels of the excitatory amino acids were highly inter-correlated in patients, but not in controls. In patients studied after 6 weeks of lamotrigine, there was a trend for CSF glutamate levels to increase. Conclusions These data suggest that in patients with refractory affective disorder, excitatory amino acids are dysregulated, as exemplified both by the decreased CSF glutamate and glycine and their high intercorrelation compared to controls. Further controlled study of glutamatergic dysregulation and its relationship to the pathophysiology of affective disorders and potential mechanism of action of mood stabilizers appears indicated.