Concentrations Of N-methyl-D-aspartate Research Articles

Ex vivo comparative investigation of suprachiasmatic nucleus excitotoxic resiliency

Background: Glutamate signaling in the brain is regulated by release, reuptake, and receptor responsiveness. In diseased conditions, glutamate signaling can exceed normal regulatory processes, giving rise to a condition called excitotoxicity. Although regional differences in the excitotoxic effects of glutamate in the brain have been reported, the extent and characteristics of these potential differences are not clear. Here we compared the excitotoxic resiliency of the suprachiasmatic nucleus (SCN), anterior hypothalamus (AH) and cortex. Methods: We treated acute brain slices containing either the SCN and AH or the cortex from adult male mice at different times across the diurnal cycle with varying concentrations of N-methyl-D-aspartate (NMDA), NMDA+ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or control medium. The extent of cell damage was assessed using propidium iodide (PI), a cell death marker. Results: The results indicate that all three brain regions exhibited increasing cell damage/death when treated with increasing concentrations of NMDA. However, higher concentrations of NMDA were needed to significantly increase cell damage in the SCN compared to the cortex and AH. All three brain regions also exhibited greater cell death/damage when treated in the nighttime compared to the daytime, although the SCN exhibited increased cell death during a more restricted time interval compared to the AH and cortex. Conclusions: Together, these data confirm previous studies showing excitotoxic resiliency in the SCN, while extending them in two ways. First, we demonstrate a dose-dependency in excitotoxic susceptibility that differentiates the SCN from the surrounding AH and the cortex using a brain slice preparation. Second, we demonstrate a diurnal rhythm in excitotoxic susceptibility with a broadly similar phase across all three brain regions. These data increase our understanding of the extent and nature of the SCN excitotoxic resiliency, which will inform future studies on the cellular mechanisms underlying this phenomenon.

Ex vivo comparative investigation of suprachiasmatic nucleus excitotoxic resiliency

Background: Glutamate signaling in the brain is regulated by release, reuptake, and receptor responsiveness. In diseased conditions, glutamate signaling can exceed normal regulatory processes, giving rise to a condition called excitotoxicity. Although regional differences in the excitotoxic effects of glutamate in the brain have been reported, the extent and characteristics of these potential differences are not clear. Here we compared the excitotoxic resiliency of the suprachiasmatic nucleus (SCN), anterior hypothalamus (AH) and cortex. Methods: We treated acute brain slices containing either the SCN and AH or the cortex from adult male mice at different times across the diurnal cycle with varying concentrations of N-methyl-D-aspartate (NMDA), NMDA+ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or control medium. The extent of cell damage was assessed using propidium iodide (PI), a cell death marker. Results: The results indicate that all three brain regions exhibited increasing cell damage/death when treated with increasing concentrations of NMDA. However, higher concentrations of NMDA were needed to significantly increase cell damage in the SCN compared to the cortex and AH. All three brain regions also exhibited greater cell death/damage when treated in the nighttime compared to the daytime, although the SCN exhibited increased cell death during a more restricted time interval compared to the AH and cortex. Conclusions: Together, these data confirm previous studies showing excitotoxic resiliency in the SCN, while extending them in two ways. First, we demonstrate a dose-dependency in excitotoxic susceptibility that differentiates the SCN from the surrounding AH and the cortex using a brain slice preparation. Second, we demonstrate a diurnal rhythm in excitotoxic susceptibility with a broadly similar phase across all three brain regions. These data increase our understanding of the extent and nature of the SCN excitotoxic resiliency, which will inform future studies on the cellular mechanisms underlying this phenomenon.

Ex vivo comparative investigation of suprachiasmatic nucleus excitotoxic resiliency

Background: Glutamate signaling in the brain is regulated by release, reuptake, and receptor responsiveness. In diseased conditions, glutamate signaling can exceed normal regulatory processes, giving rise to a condition called excitotoxicity. Although regional differences in the excitotoxic effects of glutamate in the brain have been reported, the extent and characteristics of these potential differences are not clear. Here we compared the excitotoxic resiliency of the suprachiasmatic nucleus (SCN), anterior hypothalamus (AH) and cortex. Methods: We treated acute brain slices containing either the SCN and AH or the cortex from adult male mice at different times across the diurnal cycle with varying concentrations of N-methyl-D-aspartate (NMDA), NMDA+ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or control medium. The extent of cell damage was assessed using propidium iodide (PI), a cell death marker. Results: The results indicate that all three brain regions exhibited increasing cell damage/death when treated with increasing concentrations of NMDA. However, higher concentrations of NMDA were needed to significantly increase cell damage in the SCN compared to the cortex and AH. All three brain regions also exhibited greater cell death/damage when treated in the nighttime compared to the daytime, although the SCN exhibited increased cell death during a more restricted time interval compared to the AH and cortex. Conclusions: Together, these data confirm previous studies showing excitotoxic resiliency in the SCN, while extending them in two ways. First, we demonstrate a dose-dependency in excitotoxic susceptibility that differentiates the SCN from the surrounding AH and the cortex using a brain slice preparation. Second, we demonstrate a diurnal rhythm in excitotoxic susceptibility with a broadly similar phase across all three brain regions. These data increase our understanding of the extent and nature of the SCN excitotoxic resiliency, which will inform future studies on the cellular mechanisms underlying this phenomenon.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Effects of Intraperitoneal N-methyl-D-aspartate (NMDA) Administration on Nociceptive/Repetitive Behaviors in Juvenile Mice.

Dysregulation of excitatory neurotransmission has been implicated in the pathogenesis of neuropsychiatric disorders. Pharmacological inhibition of N-methyl-D-aspartate (NMDA) receptors is widely used to model neurobehavioral pathologies and underlying mechanisms. There is ample evidence that overstimulation of NMDA-dependent neurotransmission may induce neurobehavioral abnormalities, such as repetitive behaviors and hypersensitization to nociception and cognitive disruption, pharmacological modeling using NMDA has been limited due to the induction of neurotoxicity and blood brain barrier breakdown, especially in young animals. In this study, we examined the effects of intraperitoneal NMDA-administration on nociceptive and repetitive behaviors in ICR mice. Intraperitoneal injection of NMDA induced repetitive grooming and tail biting/licking behaviors in a dose- and age-dependent manner. Nociceptive and repetitive behaviors were more prominent in juvenile mice than adult mice. We did not observe extensive blood brain barrier breakdown or neuronal cell death after peritoneal injection of NMDA, indicating limited neurotoxic effects despite a significant increase in NMDA concentration in the cerebrospinal fluid. These findings suggest that the observed behavioral changes were not mediated by general NMDA toxicity. In the hot plate test, we found that the latency of paw licking and jumping decreased in the NMDA-exposed mice especially in the 75 mg/kg group, suggesting increased nociceptive sensitivity in NMDA-treated animals. Repetitive behaviors and increased pain sensitivity are often comorbid in psychiatric disorders (e.g., autism spectrum disorder). Therefore, the behavioral characteristics of intraperitoneal NMDA-administered mice described herein may be valuable for studying the mechanisms underlying relevant disorders and screening candidate therapeutic molecules.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

Concentration-Dependent Inner Retina Layer Damage and Optic Nerve Degeneration in a NMDA Model.

The intravitreal injection of N-methyl-D-aspartate (NMDA), a glutamate analogue, is an established model for fast retinal ganglion cell (RGC) degeneration. Yet, NMDA does not cause specific RGC damage. Now, the effects on the whole retina were analyzed. Additionally, the related effects for the structure and apoptotic levels of the optic nerve were investigated. Therefore, different NMDA concentrations were intravitreally injected in rats (20, 40, or 80nmol NMDA or PBS). At days 3 and 14, Brn-3a+ RGCs were degenerated. A damage of calretinin+ amacrine cells was also recognized at day 14. Only a slight damage was observed in regard to PKCα+ bipolar cells, while rhodopsin+ photoreceptors remained intact. A long-lasting retinal microglia response was observed from day 3 up to day 14. Furthermore, a partial degeneration of the optic nerve was noted. At day 3, the SMI-32+ neurofilaments were just slightly affected, whereas the neurofilament structure was further degenerated at day 14. However, the luxol fast blue (LFB)-stained myelin structure remained intact from day 3 up to day 14. Interestingly, apoptotic mechanisms, like FasL and Fas co-localization as well as caspase 3 activation, were restricted to the optic nerve of the highest NMDA group at this late stage of degeneration. The degeneration of the optic nerve is probably only a side effect of neuronal degeneration of the inner retinal layers. The intact myelin structure might form a barrier against the direct influence of NMDA. In conclusion, this model is very suitable to test therapeutic agents, but it is important to analyze all inner retina layers and the optic nerve to determine their efficacy in this model more precisely.

A method for objectively quantifying propidium iodide exclusion in organotypic hippocampal slice cultures

BackgroundOrganotypic hippocampal slice cultures (OHSCs) are an attractive in vitro model to examine mechanisms of neuronal injury, because the normal hippocampal architecture, function and cellular diversity are mostly preserved. The effects of exposure to excitotoxins such as N-methyl-d-aspartate (NMDA) on cell viability can be determined by propidium iodide (PI) staining. New methodWe describe a simple method to objectively quantify cell death in NMDA exposed slice cultures using PI that provides a standardized means of quantifying cell death in hippocampal subfields without the need to induce maximal cell death in each slice. The method employs separation of subfields using simple landmarks and densitometric quantification of PI intensity in 10 template-oriented counting fields. ResultsWe show that exposure to increasing concentrations of NMDA results in a dose-dependent increase in PI uptake. Additionally, our method facilitates the comparison of cell death in different hippocampal subfields, such as dentate gyrus, CA1 and CA3. Our results show marked differences of PI uptake in the hippocampal regions with the CA1 area being most sensitive to NMDA-induced injury. Comparison with existing method(s)The method provides a standardized format for quantifying PI exclusion in OHSCs that can be applied to cultures of differing shapes and sizes, permits comparisons between hippocampal subfields and does not require induction of maximal cell death. ConclusionThe method of quantifying PI uptake described herein allows for an objective, quantitative and reproducible analysis and comparison of cell death in distinct regions of OHSCs.

Single-Cell Resolution Imaging of Retinal Ganglion Cell Apoptosis In Vivo Using a Cell-Penetrating Caspase-Activatable Peptide Probe

Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging standard for functional evaluation of future probe analogues and provides a basis for extending this strategy into glaucoma-specific animal models.

4-hydroxy-2(E)-Nonenal facilitates NMDA-Induced Neurotoxicity via Triggering Mitochondrial Permeability Transition Pore Opening and Mitochondrial Calcium Overload

N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is one of the major causes for neuronal cell death during cerebral ischemic insult. Previously, we reported that the final product of lipid membrane peroxidation 4-hydroxy-2E-nonenal (HNE) synergistically increased NMDA receptor-mediated excitotoxicity (J Neurochem., 2006). In this study, we investigated the mechanism involved in the synergistic neuronal cell death induced by co-treatment with HNE and NMDA. Although neither HNE (1 µM) nor NMDA (2 µM) alone induced the death of cortical neurons, simultaneous treatment of neuronal cells with HNE and NMDA synergistically evoked the death of the cells. However, the synergistic effect on neuronal death was observed only in the presence of calcium. HNE neither increased the cytosolic calcium level ([Ca2+]i) nor altered the NMDA-induced intracellular calcium influx. However, HNE together with NMDA elevated the mitochondrial calcium level and depolarized the mitochondrial transmembrane potential. Furthermore, HNE evoked damage of isolated mitochondria at the cytosolic calcium level (200 nM), which is maximally induced by 2 µM NMDA. Consistently, ATP was depleted in neurons when treated with both HNE and NMDA together. Ciclopirox, a potent inhibitor of mitochondrial permeability transition pore opening (Br. J. Pharmacol., 2005), largely prevented the synergistic damage of mitochondria and death of cortical neurons. Therefore, although low concentrations of HNE and NMDA cannot individually induce neuronal cell death, they can evoke the neuronal cell death by synergistically accelerating mitochondrial dysfunction.

Endogenous acetylcholine rescues NMDA-induced long-lasting hippocampal cell damage via stimulation of muscarinic M1 receptors: Elucidation using organic hippocampal slice cultures

This study aimed to investigate a recuing role of cholinergic systems in the excitotoxicity-induced hippocampal cell damage. Organotypic hippocampal slice cultures (OHSCs) were prepared from 7-day-old mice and exposed to N-methyl-d-aspartate (NMDA) for 24h. After washing out the NMDA, OHSCs were incubated in medium containing test drugs for 0–6 days. Hippocampal cell damage was evaluated by propidium iodide staining, immunofluorescence, and Western blotting. NMDA (1–10μM) dose-dependently damaged hippocampal cells. The toxic effect of 3μM NMDA was also observed at 3–6 days, even after washing out NMDA, and was blocked by MK-801 from day 3 to day 6. Post-treatments with tacrine, donepezil, and galantamine reduced the NMDA-induced long-lasting hippocampal cell damage. The effect of tacrine was induced in a manner dependent on the incubation period after NMDA treatment and was confirmed by Nissl staining and immunostaining with NeuN, a marker of mature neurons. The effect of tacrine was attenuated by scopolamine and a muscarinic M1 receptor antagonist, pirenzepine, but not by a muscarinic M3 receptor antagonist, darifenacin, or a nicotinic receptor antagonist, mecamylamine. The protein kinase C inhibitor Ro-31-8220 abolished the effect of tacrine. The pretreatment with 3μM NMDA had no effect on the expression level of presynaptic cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter, in OHSCs. These results suggest that a low concentration of NMDA causes long-lasting hippocampal cell damage and that endogenous acetylcholine plays, via muscarinic M1 receptor, a rescuing role in the excitotoxicity-induced long-lasting hippocampal cell damage.

Bi‐directional regulation of CaMKIIα phosphorylation at Thr286 by NMDA receptors in cultured cortical neurons

The N-methyl-D-aspartate (NMDA) receptor (NMDAR)-stimulated autophosphorylation of calmodulin-dependent kinase IIα at Thr286 may regulate many aspects of neuroplasticity. Here, we show that low NMDA concentration (20 μM) up-regulated Thr286 phosphorylation, and high concentration (100 μM) caused dephosphorylation. We next modulated the strength of NMDAR activation by manipulating NMDAR 2A subunit (NR2A) and NMDAR 2B subunit (NR2B), which represent the major NMDAR subtypes in forebrain regions. Pharmacological inhibition and molecular knockdown of NR2A or NR2B blocked 20 μM NMDA-induced phosphorylation. Conversely, over-expression of NR2A or NR2B enhanced phosphorylation by 20 μM NMDA. The 100 μM NMDA-induced dephosphorylation was suppressed by inhibition or knockdown of NR2A or NR2B, and enhanced by over-expression of NR2A or NR2B. Compared to NR2A, NR2B showed a higher impact on the NMDA-stimulated bi-directional regulation of Thr286 phosphorylation. We further found that activation of NR2A and NR2B by 100 μM NMDA-induced dephosphorylation through protein phosphatases (PP) that are inhibited by high concentration okadaic acid (1 μM), but not by PP2A and PP2B inhibitors. This novel function of NMDAR in dynamic regulation of calmodulin-dependent kinase IIα activity provides new evidence to support the current understanding that, depending on the degree of activation, NMDAR may lead to different and even opposing effects on intracellular signaling.

Episodic swimming in the larval zebrafish is generated by a spatially distributed spinal network with modular functional organization

Despite the diverse methods vertebrates use for locomotion, there is evidence that components of the locomotor central pattern generator (CPG) are conserved across species. When zebrafish begin swimming early in development, they perform short episodes of activity separated by periods of inactivity. Within these episodes, the trunk flexes with side-to-side alternation and the traveling body wave progresses rostrocaudally. To characterize the distribution of the swimming CPG along the rostrocaudal axis, we performed transections of the larval zebrafish spinal cord and induced fictive swimming using N-methyl-d-aspartate (NMDA). In both intact and spinalized larvae, bursting is found throughout the rostrocaudal extent of the spinal cord, and the properties of fictive swimming observed were dependent on the concentration of NMDA. We isolated series of contiguous spinal segments by performing multiple spinal transections on the same larvae. Although series from all regions of the spinal cord have the capacity to produce bursts, the capacity to produce organized episodes of fictive swimming has a rostral bias: in the rostral spinal cord, only 12 contiguous body segments are necessary, whereas 23 contiguous body segments are necessary in the caudal spinal cord. Shorter series of segments were often active but produced either continuous rhythmic bursting or sporadic, nonrhythmic bursting. Both episodic and continuous bursting alternated between the left and right sides of the body and showed rostrocaudal progression, demonstrating the functional dissociation of the circuits responsible for episodic structure and fine burst timing. These findings parallel results in mammalian locomotion, and we propose a hierarchical model of the larval zebrafish swimming CPG.

The Subtype of GluN2 C-terminal Domain Determines the Response to Excitotoxic Insults

SummaryIt is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca2+ influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD2B) and GluN2A (CTD2A). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD2B enhances NMDAR toxicity, compared to CTD2A. Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca2+ influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD2B exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity.

Corrigendum

CorrigendaCorrigendumPublished Online:01 Mar 2012https://doi.org/10.1152/ajpcell.zh0-6904-corr.2012Original articleMoreSectionsPDF (197 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 300, January 2011Volume 69, January 2011Anderson M, Suh JM, Kim EY, Dryer SE. Functional NMDA receptors with atypical properties are expressed in podocytes. Am J Physiol Cell Physiol 300: , 2011. First published August 25, 2010; doi:10.1152/ajpcell.00268.2010 (http://ajpcell.physiology.org/content/300/1/C22).—In results, the same confocal image was inadvertently presented twice in Fig. 1A. The image on the right has now been replaced with the appropriate one, and the correct image of Fig. 1 is shown below. The authors apologize for this error. The changes made do not affect the conclusions presented in this study.Fig. 1.Responses to N-methyl-d-aspartate (NMDA) in differentiated cells of an immortalized mouse podocyte cell line. A: confocal immunofluorescence images showing expression of NR1 subunit of NMDA receptors in podocyte cell lines. B: reciprocal coimmunoprecipitation of NR1 subunits and the podocyte marker synaptopodin (Synpo). Input lane shows diluted sample of lysate used for preparation of immunoprecipitates. No signals of appropriate molecular weight were seen in immunoprecipitates prepared with IgG. C: whole cell recordings from 2 different podocytes. A micropipette filled with 100 μM NMDA was placed adjacent to the recorded cells and used to deliver NMDA in response to brief pressure pulses (horizontal bars above traces). Once the pipette was placed, responses to NMDA in a given cell were very consistent, as shown by response to 3 separate NMDA applications in each of the 2 cells. Vm, membrane (holding) potential. D: responses to NMDA do not desensitize or fade during prolonged application. Record shows a response to a continuous 60-s application of 100 μM NMDA to a podocyte at a holding potential of −60 mV. E: concentration-response curve for NMDA in podocytes. Data points show mean ± SE of currents evoked by a given NMDA concentration applied to groups of 4 cells. Superimposed curve is a nonlinear least-squares fit to the logistic equation with an EC50 of 36 μM NMDA and a Hill coefficient of 1.98 (R2 = 0.96413).Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationRelated ArticlesFunctional NMDA receptors with atypical properties are expressed in podocytes 01 Jan 2011American Journal of Physiology-Cell Physiology More from this issue > Volume 302Issue 5March 2012Pages C834-C834 Copyright & PermissionsCopyright © 2012 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.zh0-6904-corr.2012History Published online 1 March 2012 Published in print 1 March 2012 Metrics

Role of Retinal Glial Cell Glutamate Transporters in Retinal Ganglion Cell Survival Following Stimulation of NMDA Receptor

Purpose: To investigate the role of glutamate transporters (GLTs)in retinal glial cells that were treated with N-methyl-D-aspartate (NMDA), in retinal ganglion cell (RGC) survival.Methods: Primary cultures of retinal glial cells or RGCs from 3-day-old Sprague-Dawley rats were employed in the present study. Retinal glial cells were treated with NMDA and changes in GLT mRNA and protein expression were analyzed. The effects of pretreating retinal glial cells with the GLAST-specific inhibitor, rottlerin (ROT), and the GLT-1-specific inhibitor, dihydrokainic acid (DHK), on RGC survival were investigated under exposure to NMDA. The amount of glutamate in the culture medium of retinal glial cells was measured by high-performance liquid chromatography.Results: NMDA treatment increased GLAST and GLT-1 expression. GLAST and GLT-1 mRNA expression increased by 2.94-fold and 3.36-fold at 12 h after treatment with the highest concentration of NMDA (33 mM), and by 1.41-fold and 1.39-fold at 24 h, respectively. GLT-1 and GLAST protein expression also increased. MK801, an NMDA-receptor antagonist, inhibited the NMDA-induced upregulation of GLT mRNA expression. Co-culture with retinal glial cells increased the survival rate of RGCs. ROT decreased the survival rate of RGCs, whereas DHK significantly increased the survival rate of RGCs treated with 33 mM NMDA. NMDA treatment reduced the total amount of glutamate in the culture medium, particularly when 33 mM NMDA was added to the medium. ROT pretreatment increased the amount of glutamate in the culture medium, whereas DHK pretreatment decreased it.Conclusion: GLAST and GLT-1 may have different roles in the survival of RGCs mediated by retinal glial cells. These results suggest that the NMDA-associated induction of GLTs plays an important role in RGC survival.

Functional expression of ionotropic glutamate receptors in the rabbit retinal ganglion cells

It has been known that retinal ganglion cells (RGCs) with distinct morphologies have different physiological properties. It was hypothesized that different functions of RGCs may in part result from various expressions of N-methyl- d-aspartate (NMDA), α-amino-3-hydroxyl-5-methyl-isoxazole-4-propinoic acid (AMPA), and kainic acid (KA) receptors on their dendrites. In the present study, we aimed to characterize the functional expression of AMPA and NMDA receptors of morphologically identified RGCs in the wholemount rabbit retina. The agmatine (AGB) activation assay was used to reveal functional expression of ionotropic glutamate receptors after the RGCs were targeted by injecting Neurobiotin. To examine the excitability of these glutamate receptors in an agonist specific manner, the lower concentrations of AMPA (2 μM) and NMDA (100 μM) were chosen to examine G7 (ON–OFF direction selective ganglion cells) and G11 (alpha ganglion cells) types of RGCs. We found that less than 40% of G7 type RGCs had salient AGB activation when incubated with 2 μM AMPA or 100 μM NMDA. The G11 type RGCs also showed similar activation frequencies, except that all of the OFF subtype examined had no AGB permeation under the same AMPA concentration. These results suggest that RGCs with large somata (G7 and G11 types) may express various heterogeneous functional ionotropic glutamate receptors, thus in part rendering their functional diversity.

Neurotensin regulates cortical glutamate transmission by modulating N‐methyl‐D‐aspartate receptor functional activity: An in vivo microdialysis study

The aim of the present in vivo microdialysis study was to investigate whether the tridecapeptide neurotensin (NT) influences the N-methyl-D-aspartate (NMDA) receptor-mediated increase of cortical glutamate transmission in freely moving rats. Intracortical perfusion with NT influenced local extracellular glutamate levels in a bell-shaped, concentration-dependent manner. One hundred and three hundred nanomolar NT concentrations increased glutamate levels (151% ± 7% and 124% ± 3% of basal values, respectively). Higher (1,000 nM) and lower (10 nM) NT concentrations did not alter extracellular glutamate levels. The NT receptor antagonist SR48692 (100 nM) prevented the NT (100 nM)-induced increase in glutamate levels. NMDA (100 and 500 μM) perfusion induced a concentration-dependent increase in extracellular glutamate levels, the lower 10 μM NMDA concentration being ineffective. When NT (10 nM, a concentration by itself ineffective) was added in combination with NMDA (100 μM) to the perfusion medium, a significant greater increase in extracellular glutamate levels (169% ± 7%) was observed with respect to the increase induced by NMDA (100 μM) alone (139% ± 4%). SR48692 (100 nM) counteracted the increase in glutamate levels induced by the treatment with NT (10 nM) plus NMDA (100 μM). The enhancement of cortical glutamate levels induced by NMDA (100 and 500 μM) was partially antagonized by the presence of SR48692, at a concentration (100 nM) that by itself was ineffective in modulating glutamate release. These findings indicate that NT plays a relevant role in the regulation of cortical glutamatergic transmission, especially by modulating the functional activity of cortical NMDA receptors. A possible role in glutamate-mediated neurotoxicity is suggested.

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.