Glia-derived D-serine controls NMDA receptor activity and synaptic memory

The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the central nervous system. Its activation requires the binding of both glutamate and a co-agonist like D-serine to its glycine site. As D-serine is released exclusively by astrocytes, we studied the physiological impact of the glial environment on NMDA receptor-dependent activity and plasticity. To this end, we took advantage of the changing astrocytic ensheathing of neurons occurring in the supraoptic nucleus during lactation. We provide direct evidence that in this hypothalamic structure the endogenous co-agonist of NMDA receptors is D-serine and not glycine. Consequently, the degree of astrocytic coverage of neurons governs the level of glycine site occupancy on the NMDA receptor, thereby affecting their availability for activation and thus the activity dependence of long-term synaptic changes. Such a contribution of astrocytes to synaptic metaplasticity fuels the emerging concept that astrocytes are dynamic partners of brain signaling.     

A single packet of transmitter does not saturate postsynaptic glutamate receptors

Neurotransmitter is stored in synaptic vesicles and released by exocytosis into the synaptic cleft. One of the fundamental questions in central synaptic transmission is whether a quantal packet of transmitter saturates postsynaptic receptors. To address this question, we loaded the excitatory transmitter L-glutamate via whole-cell recording pipettes into the giant nerve terminal, the calyx of Held, in rat brainstem slices. This caused marked potentiations of both quantal and action potential-evoked EPSCs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. These results directly demonstrate that neither AMPA nor NMDA receptors are saturated by a single packet of transmitter, and indicate that vesicular transmitter content is an important determinant of synaptic efficacy.     

Molecular biology of glycinergic neurotransmission

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem of vertebrates. Glycine is accumulated into synaptic vesicles by a proton-coupled transport system and released to the synaptic cleft after depolarization of the presynaptic terminal. The inhibitory action of glycine is mediated by pentameric glycine receptors (GlyR) that belong to the ligand-gated ion channel superfamily. The synaptic action of glycine is terminated by two sodium- and chloride-coupled transporters, GLYT1 and GLYT2, located in the glial plasma membrane and in the presynaptic terminals, respectively. Dysfunction of inhibitory glycinergic neurotransmission is associated with several forms of inherited mammalian myoclonus. In addition, glycine could participate in excitatory neurotransmission by modulating the activity of the NMDA subtype of glutamate receptor. In this article, we discuss recent progress in our understanding of the molecular mechanisms that underlie the physiology and pathology of glycinergic neurotransmission.     

Interactions between glycine transporter type 1 (GlyT-1) and some inhibitor molecules - glycine transporter type 1 and its inhibitors (review)

Glycine is a mandatory positive allosteric modulator of N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptors in the central nervous system. Elevation of glycine concentrations by inhibition of its reuptake in the vicinity of NMDA receptors may positively influence receptor functions as glycine B binding site on NR1 receptor subunit is not saturated in physiological conditions. Synaptic and extrasynaptic concentrations of glycine are regulated by its type-1 glycine transporter, which is primarily expressed in astroglial and glutamatergic cell membranes. Alteration of synaptic glycine levels may have importance in the treatment of various forms of endogenous psychosis characterized by hypofunctional NMDA receptors. Several lines of evidence indicate that impaired NMDA receptor-mediated glutamatergic neurotransmission is involved in development of the negative (and partly the positive) symptoms and the cognitive deficit in schizophrenia. Inhibitors of glycine transporter type-1 may represent a newly developed therapeutic intervention in treatment of this mental illness. We have synthesized a novel series of N-substituted sarcosines, analogues of the glycine transporter-1 inhibitor NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine). Of the pyridazinone-containing compounds, SzV-1997 was found to be a potent glycine transporter-1 inhibitor in rat brain synaptosomes and it markedly increased extracellular glycine concentrations in conscious rat striatum. SzV-1997 did not exhibit toxic symptoms such as hyperlocomotion, restless movements, respiratory depression, and lethality, characteristic for NFPS. Besides pyridazinone-based, sarcosine-containing glycine transporter-1 inhibitors, a series of substrate-type amino acid inhibitors was investigated in order to obtain better insight into the ligand-binding characteristics of the substrate binding cavity of the transporter.     

NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII

Calcium entry through postsynaptic NMDA-Rs and subsequent activation of CaMKII trigger synaptic plasticity in many brain regions. Active CaMKII can bind to NMDA-Rs, but the physiological role of this interaction is not well understood. Here, we test if association between active CaMKII and synaptic NMDA-Rs is required for synaptic plasticity. Switching synaptic NR2B-containing NMDA-Rs that bind CaMKII with high affinity with those containing NR2A, a subunit with low affinity for CaMKII, dramatically reduces LTP. Expression of NR2A with mutations that increase association to active CaMKII recovers LTP. Finally, driving into synapses NR2B with mutations that reduce association to active CaMKII prevents LTP. Spontaneous activity-driven potentiation shows similar results. We conclude that association between active CaMKII and NR2B is required for different forms of synaptic enhancement. The switch from NR2B to NR2A content in synaptic NMDA-Rs normally observed in many brain regions may contribute to reduced plasticity by controlling the binding of active CaMKII.     

Extracellular glutamate diffusion determines the occupancy of glutamate receptors at CA1 synapses in the hippocampus

Following exocytosis at excitatory synapses in the brain, glutamate binds to several subtypes of postsynaptic receptors. The degree of occupancy of AMPA and NMDA receptors at hippocampal synapses is, however, not known. One approach to estimate receptor occupancy is to examine quantal amplitude fluctuations of postsynaptic signals in hippocampal neurons studied in vitro. The results of such experiments suggest that NMDA receptors at CA1 synapses are activated not only by glutamate released from the immediately apposed presynaptic terminals, but also by glutamate spillover from neighbouring terminals. Numerical simulations point to the extracellular diffusion coefficient as a critical parameter that determines the extent of activation of receptors positioned at different distances from the release site. We have shown that raising the viscosity of the extracellular medium can modulate the diffusion coefficient, providing an experimental tool to investigate the role of diffusion in activation of synaptic and extrasynaptic receptors. Whether intersynaptic cross-talk mediated by NMDA receptors occurs in vivo remains to be determined. The theoretical and experimental approaches described here also promise to shed light on the roles of metabotropic and kainate receptors, which often occur in an extrasynaptic distribution, and are therefore positioned to sense glutamate escaping from the synaptic cleft.     

N-甲基-D-天氡氨酸受体的分子结构与生理功能

NMDA(N-甲基-D-天氡氨酸)受体是离子型谷氨酸受体的一种亚型,在中枢神经系统的突触传递和突触可塑性调节中起着重要的作用。生物体内已经发现了三种NMDA受体亚基,通过基因的选择性剪切可产生多种亚单位。NMDA受体是一个具有多个结合位点的大分子复合物,其生理特性同异聚体通道的装配密切相关。NMDA受体的异常会导致一些认知功能的缺失,这为治疗性药物开发提供了靶点。     

N-Methyl-D-Aspartate Recognition Site Ligands Modulate Activity at the Coupled Glycine Recognition Site

In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.     

Maturation of a PKG-dependent retrograde mechanism for exoendocytic coupling of synaptic vesicles

At presynaptic terminals vesicular membranes are fused into plasma membrane upon exocytosis and retrieved by endocytosis. During a sustained high-frequency transmission, exoendocytic coupling is critical for the maintenance of synaptic transmission. Here, we show that this homeostatic coupling is supported by cGMP-dependent protein kinase (PKG) at the calyx of Held. This mechanism starts to operate after hearing onset during the second postnatal week, when PKG expression becomes upregulated in the brainstem. Pharmacological tests with capacitance measurements revealed that presynaptic PKG?activity is supported by a retrograde signal cascade mediated by NO that is released by activation of postsynaptic NMDA receptors. Activation of PKG also upregulates phosphatidylinositol-4,5-bisphosphate, thereby accelerating endocytosis. Furthermore, presynaptic PKG activity upregulates synaptic fidelity during high-frequency transmission. We conclude that maturation of the PKG-dependent retrograde signal cascade strengthens the homeostatic plasticity for the maintenance of high-frequency synaptic transmission at the fast glutamatergic synapse.     

Fast kinetics of exocytosis revealed by simultaneous measurements of presynaptic capacitance and postsynaptic currents at a central synapse

The rate of release from nerve terminals depends on both the number of release sites and the rate of release at each site. The latter remains largely unknown at central synapses. We addressed this issue by simultaneously measuring the nerve terminal membrane capacitance and the postsynaptic current at single calyceal synapses in rat brainstem. We found that a 10 ms presynaptic step depolarization depleted a releasable pool containing 3300-5200 vesicles. Released vesicles were endocytosed with a time constant of a few seconds to tens of seconds. Release of only one third of this pool saturated both postsynaptic AMPA and NMDA receptors. A release site can release more than three vesicles in 10 ms (>300 vesicles per second). We conclude that both a large number of release sites and a fast release rate at each site enable synapses to release at a high rate.     

gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.     

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.     

Pain Facilitation and Activity-Dependent Plasticity in Pain Modulatory Circuitry: Role of BDNF-TrkB Signaling and NMDA Receptors

Pain modulatory circuitry in the brainstem exhibits considerable synaptic plasticity. The increased peripheral neuronal barrage after injury activates spinal projection neurons that then activate multiple chemical mediators including glutamatergic neurons at the brainstem level, leading to an increased synaptic strength and facilitatory output. It is not surprising that a well-established regulator of synaptic plasticity, brain-derived neurotrophic factor (BDNF), contributes to the mechanisms of descending pain facilitation. After tissue injury, BDNF and TrkB signaling in the brainstem circuitry is rapidly activated. Through the intracellular signaling cascade that involves phospholipase C, inositol trisphosphate, protein kinase C, and nonreceptor protein tyrosine kinases; N-methyl-D-aspartate (NMDA) receptors are phosphorylated, descending facilitatory drive is initiated, and behavioral hyperalgesia follows. The synaptic plasticity observed in the pain pathways shares much similarity with more extensively studied forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD), which typically express NMDA receptor dependency and regulation by trophic factors. However, LTP and LTD are experimental phenomena whose relationship to functional states of learning and memory has been difficult to prove. Although mechanisms of synaptic plasticity in pain pathways have typically not been related to LTP and LTD, pain pathways have an advantage as a model system for synaptic modifications as there are many well-established models of persistent pain with clear measures of the behavioral phenotype. Further studies will elucidate cellular and molecular mechanisms of pain sensitization and further our understanding of principles of central nervous system plasticity and responsiveness to environmental challenge.     

The actions of synaptically released zinc at hippocampal mossy fiber synapses

Zn2+ is present at high concentrations in the synaptic vesicles of hippocampal mossy fibers. We have used Zn2+ chelators and the mocha mutant mouse to address the physiological role of Zn2+ in this pathway. Zn2+ is not involved in the unique presynaptic plasticities observed at mossy fiber synapses but is coreleased with glutamate from these synapses, both spontaneously and with electrical stimulation, where it exerts a strong modulatory effect on the NMDA receptors. Zn2+ tonically occupies the high-affinity binding site of NMDA receptors at mossy fiber synapses, whereas the lower affinity voltage-dependent Zn2+ binding site is occupied during action potential driven-release. We conclude that Zn2+ is a modulatory neurotransmitter released from mossy fiber synapses and plays an important role in shaping the NMDA receptor response at these synapses.     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

Cellular and subcellular localization of the inhibitory glycine receptor in hippocampal neurons

Inhibitory glycine receptors are most abundant in spinal cord and brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Little is known about the function of glycine receptors in cortex and hippocampus, where GABA plays a dominant role in synaptic inhibition. Therefore, we have investigated tissue and cellular expression of glycine receptor alpha-subunits. Western blot and immunohistochemical analyses reveal the presence of glycine receptors in hippocampal tissue. Immunocytochemical experiments in hippocampal cultures show prominent cellular expression of glycine receptors in pyramidal neurons and GAD-positive interneurons similar to the calcium-binding protein VILIP-1 with widespread hippocampal distribution. On the subcellular level we found co-staining of GlyR and the presynaptic marker synapsin I. Furthermore, co-staining with GAD at synaptic terminals indicated partial co-localization of GABA- and glycine receptors.     

Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine

Oxidative stress is a major aspect of Alzheimer disease (AD) pathology. We have investigated the relationship between oxidative stress and neuronal binding of Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in brain tissue of AD patients and are considered centrally related to pathogenesis. Using hippocampal neuronal cultures, we found that ADDLs stimulated excessive formation of reactive oxygen species (ROS) through a mechanism requiring N-methyl-d-aspartate receptor (NMDA-R) activation. ADDL binding to neurons was reduced and ROS formation was completely blocked by an antibody to the extracellular domain of the NR1 subunit of NMDA-Rs. In harmony with a steric inhibition of ADDL binding by NR1 antibodies, ADDLs that were bound to detergent-extracted synaptosomal membranes co-immunoprecipitated with NMDA-R subunits. The NR1 antibody did not affect ROS formation induced by NMDA, showing that NMDA-Rs themselves remained functional. Memantine, an open channel NMDA-R antagonist prescribed as a memory-preserving drug for AD patients, completely protected against ADDL-induced ROS formation, as did other NMDA-R antagonists. Memantine and the anti-NR1 antibody also attenuated a rapid ADDL-induced increase in intraneuronal calcium, which was essential for stimulated ROS formation. These results show that ADDLs bind to or in close proximity to NMDA-Rs, triggering neuronal damage through NMDA-R-dependent calcium flux. This response provides a pathologically specific mechanism for the therapeutic action of memantine, indicates a role for ROS dysregulation in ADDL-induced cognitive impairment, and supports the unifying hypothesis that ADDLs play a central role in AD pathogenesis.     

Regional Variations in [3H]MK801 Binding to Rat Brain N-Methyl-D-Aspartate Receptors

This study examined (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate [( 3H]MK801) binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from six regions of rat brain. Highest levels of binding were found in hippocampus and cortex, whereas much lower densities were found in brainstem and cerebellum. NMDA receptors in cerebellum exhibited a significantly lower affinity for [3H]MK801 than cortical NMDA receptors. To determine whether forebrain and hindbrain NMDA receptors were distinct, the actions of glutamate, NMDA, ibotenate, quinolinate, glycine, and spermine were investigated. These agents increased [3H]MK801 binding in all brain regions examined. However, agonists were uniformly less efficacious in hindbrain compared to forebrain regions. NMDA mimetics and spermine were less potent in cerebellum compared to cortex whereas glycine was equipotent. Antagonists that act at the various modulatory sites on the NMDA receptor were also examined. DL-Amino-phosphonopentanoic acid and 7-chlorokynurenate were approximately equipotent in cortex and cerebellum. However, antagonists that are believed to act inside the NMDA-operated ion channel, including Mg2+ and phencyclidine, were approximately threefold less potent in cerebellum. The diminished regulation of [3H]MK801 binding by glutamate and glycine in the cerebellum was associated with a smaller effect of these agonists on the dissociation of [3H]MK801 from its binding site. The levels of glutamate, aspartate, glycine, serine, and glutamine in the membrane preparations were determined. However, variations in the levels of endogenous amino acids were not sufficient to account for the regional differences in [3H]MK801 binding. These results do not support the hypothesis that a distinct NMDA receptor exists in hindbrian regions of the rat CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

The synaptic and nonsynaptic glycine transporter type-1 inhibitors Org-24461 and NFPS alter single neuron firing rate in the rat dorsal raphe nucleus. Further evidence for a glutamatergic-serotonergic interaction and its role in antipsychotic action

Single neuron firing rate was recorded from dorsal raphe nucleus of anesthetized rats. The firing rate of raphe neurons varied from 4 to 8 discharge per second before drug administration and this neuronal activity was decreased by L-701,324 (2 mg/kg i.v. injection), a competitive antagonist of glycineB binding site of N-methyl-D-aspartate (NMDA) receptors. The glycine transporter type-1 (GlyT1) antagonists Org-24461 (10 mg/kg i.v.) and NFPS (3 mg/kg i.v.) reversed the inhibitory effect of L-701,324 on single neuron activity recorded from dorsal raphe nucleus of the rat. Org-24461 and NFPS both tended to increase the raphe neuronal firing rate also when given alone but their effect was not significant. This finding serves further evidence that glutamate released from axon terminals of the cortico-striatal projection neurons stimulates serotonergic neurons in the raphe nuclei and this effect is mediated at least in part by postsynaptic NMDA receptors. Thus, GlyT1 inhibitors are able to reverse the hypofunctional state of NMDA receptors, suggesting that these drugs may have beneficial therapeutic effects in neurological and psychiatric disorders characterized with impaired NMDA receptor-mediated transmission.