Blockade of calcium entry accelerates arsenite-mediated apoptosis in rat cerebellar granule cells

Arsenical exposure can cause defects in the central nervous system, yet the underlying cellular and molecular mechanisms are largely unknown. We have recently demonstrated that sodium arsenite induces apoptosis of cultured cortical and cerebellar neurons, suggesting that arsenite-induced neuronal apoptosis may contribute to at least some of its neurotoxic effects. Here we investigated the effect of Ca2+ on arsenite-mediated cerebellar granule neuron death. Sodium arsenite induced apoptosis in cerebellar neurons which were maintained in the presence of serum and depolarizing concentrations of potassium chloride (25 mM KCI). Under these conditions, inhibition of calcium entry by N-methyl-D-aspartate (NMDA) receptor blocker DL-aminophosphonovalerate (APV) or calcium channel antagonist nifedipine increased arsenite-induced apoptosis, while APV or nifedipine alone had little effect on cell viability. In cortical neurons or cerebellar neurons maintained at low potassium (5 mM), arsenite also induced apoptosis. However, the addition of APV or nifedipine did not alter levels of arsenite-induced apoptosis. These data suggest that arsenite-mediated apoptosis is regulated by intracellular calcium levels.     

Production of Reactive Oxygen Species and Release of l-Glutamate During Superoxide Anion-Induced Cell Death of Cerebellar Granule Neurons

Abstract: Enhanced production of superoxide anion (O₂⁻) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O₂⁻ generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA) for O₂⁻ and other ROS and hydroethidine (HEt) specifically for O₂⁻ by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO-induced cell death. Catalase effectively reduced C-DCDHF-DA but not HEt fluorescence, whereas SOD reduced HEt but not C-DCDHF-DA fluorescence, indicating that HEt and C-DCDHF-DA fluorescence correlated with O₂⁻ and hydrogen peroxide, respectively. The NMDA antagonist MK-801 prevented the death. XA + XO induced an increase in l -glutamate release from cerebellar granule neurons. These results indicate that elevation of O₂⁻ induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l -glutamate.     

Pharmacologically distinct glutamate receptors on cerebellar granule cells

Cultured cerebellar granule cells were found to exhibit calcium-dependent release of 3H-D-aspartate when stimulated with excitatory amino acids. L-glutamate and L-aspartate were found to be potent stimulators of 3H-D-aspartate release, D-aspartate was weaker and only minor effects were seen with D-glutamate, quisqualate, kainate, N-methyl-D-aspartate (NMDA) and L-alpha-aminoadipate (L-alpha AA). It was also found that only L-glutamate and L-aspartate showed high affinity for the 3H-L-glutamate binding sites on granule cell membranes. Stimulation by L-glutamate of 3H-D-aspartate release could be blocked by various excitatory amino acid antagonists. From the relative potencies of agonists and antagonists on D-aspartate release it is suggested that cerebellar granule cells express functionally active glutamate receptors with pharmacological characteristics different from all known excitatory amino acid receptors.     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Low‐level laser therapy (810 nm) protects primary cortical neurons against excitotoxicity in vitro

Excitotoxicity describes a pathogenic process whereby death of neurons releases large amounts of the excitatory neurotransmitter glutamate, which then proceeds to activate a set of glutamatergic receptors on neighboring neurons (glutamate, N‐methyl‐D‐aspartate (NMDA), and kainate), opening ion channels leading to an influx of calcium ions producing mitochondrial dysfunction and cell death. Excitotoxicity contributes to brain damage after stroke, traumatic brain injury, and neurodegenerative diseases, and is also involved in spinal cord injury. We tested whether low level laser (light) therapy (LLLT) at 810 nm could protect primary murine cultured cortical neurons against excitotoxicity in vitro produced by addition of glutamate, NMDA or kainate. Although the prevention of cell death was modest but significant, LLLT (3 J/cm² delivered at 25 mW/cm² over 2 min) gave highly significant benefits in increasing ATP, raising mitochondrial membrane potential, reducing intracellular calcium concentrations, reducing oxidative stress and reducing nitric oxide. The action of LLLT in abrogating excitotoxicity may play a role in explaining its beneficial effects in diverse central nervous system pathologies. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons

Using neurotoxicity and inositol phosphate release as criteria for receptor expression, we report the differential expression of excitatory amino acid receptor subtypes in cerebellar granule cells grown in serum-free media containing either high (25 mM) or low (5 mM) KCl. NMDA receptors are expressed in neurons grown in high, but not low, KCl. In contrast, ionotropic quisqualate receptors are expressed in neurons grown in low KCl, but not in those grown in high KCl. Addition of NMDA to cultures containing low KCl appears to mimic high KCl conditions: NMDA receptors are expressed, but ionotropic quisqualate receptors are not. Glutamate and kainate are toxic to cells grown in either condition.     

Sources of reactive oxygen species production in excitotoxin- stimulated cerebellar granule cells

Reactive oxygen species (ROS) production in rat cerebellar granule cells in the presence of the excitotoxins N-methyl-d-aspartate (NMDA) and kainic acid (KA) and by the protein kinase C activator phorbol myristate acetate (PMA) was Ca2+-dependent and resulted in decreased cell viability. Exposure of stimulated cells to rotenone (a respiratory chain inhibitor) did not decrease ROS levels and did not affect short-term cell viability. In cells stimulated by NMDA and KA, exposure to indomethacin (a cyclooxygenase inhibitor) and nialamide (a monoamine oxidase inhibitor) caused a decrease in ROS levels and increased cell viability occurred in NMDA-treated cells. In contrast, PMA-stimulated neurons did not show decreased ROS levels when exposed to indomethacin and nialamide. These studies suggest that there is a multiplicity of routes for Ca2+-dependent ROS production in neurons but that ROS generation by cyclooxygenase and monoamine oxidase is not controlled by protein kinase C.     

Magnesium-Dependent Inhibition of Agonist-Stimulated Phosphoinositide Breakdown in Rat Cortical Slices by Excitatory Amino Acids

The excitatory amino acid agonists kainate, N-methyl-D-aspartate (NMDA), and quisqualate inhibited ligand-stimulated phosphoinositide hydrolysis in rat cortical slices. The NMDA channel blocker MK-801 antagonized the inhibition by NMDA but had no effect on the inhibition due to kainate or quisqualate. The antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the effects of quisqualate and kainate but not the effect of NMDA. These data indicate that activation of the NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate types of ionotropic receptors has the same effect. In membranes prepared from cortical slices, there was no inhibition of carbachol-stimulated phosphoinositidase C activity by excitatory amino acids, suggesting that excitatory amino acids indirectly affect carbachol-stimulated phosphoinositide hydrolysis. The inhibition by excitatory amino acids of carbachol-stimulated phosphoinositide breakdown was dependent on extracellular Mg2+ and was abolished by procedures that increase intracellular Ca2+. Veratridine inhibition of carbachol-stimulated phosphoinositide hydrolysis was reversed by ouabain but not by other procedures that increase intracellular Ca2+. In contrast to excitatory amino acids, veratridine potentiated carbachol-stimulated phosphoinositide breakdown in the presence of 10 mM extracellular Mg2+. These data suggest that excitatory amino acids inhibit carbachol-stimulated phosphoinositide breakdown in rat cortex by lowering intracellular Ca2+ through a mechanism dependent on extracellular Mg2+.     

Reactive oxygen species induce different cell death mechanisms in cultured neurons

Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In this study we evaluated the role of different ROS in neuronal death. Superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with the glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners on neuronal death.     

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N -methyl- d -aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.     

Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture

The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca(2+) level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca(2+) rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca(2+) rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pre-treatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBX-induced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that both in astrocytes and neurons gap junctions provide protection against NMDA-induced cytotoxicity.     

Loss of nitric oxide-mediated down-regulation of NMDA receptors in neurofilament aggregate-bearing motor neurons in vitro: implications for motor neuron disease

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder in which excitotoxicity has been implicated as a cause for cell death. To examine neurofilament (NF) aggregate-mediated sensitization of motor neurons to NMDA excitotoxicity, we examined NMDA receptor expression and the impact of NO donors (NOC12 or NOC5) or sodium cyanide (NaCN) on calcium influx and viability in dissociated motor neurons derived from wt and hNFL+/+ (NF aggregate-forming) mice. Alterations in intracellular calcium were assayed using Oregon Green calcium dye and the extent of apoptosis using active caspase-3 immunoreactivity. Although NF aggregate-bearing neurons demonstrated increased intracellular calcium levels and enhanced cell death in response to NMDA receptor activation, this was not associated with increased NMDA receptor expression. The down-regulation of the NMDA receptor using NO donors decreased calcium influx and caspase-3 activation in aggregate-bearing neurons, but had no effect on wt cultures. The converse was observed with NaCN in which intracellular calcium levels increased significantly in wt cultures in association with increased cell death. No effect was observed in aggregate-bearing neurons. These findings suggest that the presence of NF aggregates renders motor neurons more susceptible to NMDA-mediated excitotoxicity, and that this can be reversed by NO.     

Excitotoxic cell death.

Excitotoxicity refers to the ability of glutamate or related excitatory amino acids to mediate the death of central neurons under certain conditions, for example, after intense exposure. Such excitotoxic neuronal death may contribute to the pathogenesis of brain or spinal cord injury associated with several human disease states. Excitotoxicity has substantial cellular specificity and, in most cases, is mediated by glutamate receptors. On average, NMDA receptors activation may be able to trigger lethal injury more rapidly than AMPA or kainate receptor activation, perhaps reflecting a greater ability to induce calcium influx and subsequent cellular calcium overload. It is possible that excitotoxic death may share some mechanisms with other forms of neuronal death.     

Evidence for Activation of Caspase-3-Like Protease in Excitotoxin- and Hypoxia/Hypoglycemia-Injured Neurons

Abstract: Caspase activation has been shown to be a critical step in several models of neuronal apoptosis such as staurosporine treatment of human neuroblastoma SH-SY5Y cells and potassium deprivation of rat cerebellar granule neurons. One common event is the appearance of caspase-mediated 120-kDa nonerythroid α-spectrin breakdown product (SBDP120). Second, inhibitors of the caspase family are effective blockers of such neuronal death. In this study, we report the appearance of caspase-mediated SBDP120 in excitotoxin-challenged fetal rat cerebrocortical neurons [ N -methyl- d -aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate] and rat cerebellar granule neurons (NMDA and kainate). A general caspase inhibitor, carbobenzoxy-Asp-CH₂OC(O)-2,6-dichlorobenzene (Z-D-DCB), blocked the formation of SBDP120 under these conditions and attenuated the observed NMDA-induced lactate dehydrogenase (LDH) release in both cell types. Furthermore, hydrolytic activity toward a caspase-3-preferred synthetic peptide substrate, acetyl-DEVD-7-amido-4-methylcoumarin, was significantly elevated in NMDA-treated granule neurons. Lastly, oxygen-glucose deprivation (OGD)-challenged cerebrocortical cultures also showed the appearance of SBDP120. Again, Z-D-DCB blocked the SBDP120 formation as well as attenuated the LDH release from the OGD-challenged neurons. Taken together, the presence of caspase-specific SBDP120 and the neuroprotective effects of Z-D-DCB strongly suggest that caspase activation contributes at least in part to excitotoxin- and OGD-induced neuronal death.     

NMDA-, but not kainate- or quisqualate-dependent increases in cerebellar cGMP are dependent upon monoaminergic innervation.

As previously reported, intracerebellar injections of D-serine, quisqualate and kainate elevated mouse cerebellar cGMP levels. Similarly, activation of endogenous excitatory amino acid utilizing neurons with harmaline or pentylenetetrazole also increased cerebellar cGMP. We previously have demonstrated that the harmaline- and pentylenetetrazol-dependent cGMP increases are NMDA receptor mediated. In this report we further demonstrate that NMDA-dependent increases in cGMP are dependent upon monoaminergic innervation of the cerebellum, while kainate- and quisqualate-dependent cGMP increases are independent of such innervation.     

Effect of hypoxia upon intracellular calcium concentration of human endothelial cells.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.     

Excitotoxic cell death

Excitotoxicity refers to the ability of glutamate or related excitatory amino acids to mediate the death of central neurons under certain conditions, for example, after intense exposure. Such excitotoxic neuronal death may contribute to the pathogenesis of brain or spinal cord injury associated with several human disease states. Excitotoxicity has substantial cellular specificity and, in most cases, is mediated by glutamate receptors. On average, NMDA receptors activation may be able to trigger lethal injury more rapidly than AMPA or kainate receptor activation, perhaps reflecting a greater ability to induce calcium influx and subsequent cellular calcium overload. It is possible that excitotoxic death may share some mechanisms with other forms of neuronal death. © 1992 John Wiley & Sons, Inc.     

Antagonism of Ionotropic Glutamate Receptors Attenuates Chemical Ischemia-Induced Injury in Rat Primary Cultured Myenteric Ganglia

Alterations of the enteric glutamatergic transmission may underlay changes in the function of myenteric neurons following intestinal ischemia and reperfusion (I/R) contributing to impairment of gastrointestinal motility occurring in these pathological conditions. The aim of the present study was to evaluate whether glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R. Primary cultured rat myenteric ganglia were exposed to sodium azide and glucose deprivation (in vitro chemical ischemia). After 6 days of culture, immunoreactivity for NMDA, AMPA and kainate receptors subunits, GluN₁ and GluA_1–4, GluK_1–3 respectively, was found in myenteric neurons. In myenteric cultured ganglia, in normal metabolic conditions, -AP5, an NMDA antagonist, decreased myenteric neuron number and viability, determined by calcein AM/ethidium homodimer-1 assay, and increased reactive oxygen species (ROS) levels, measured with hydroxyphenyl fluorescein. CNQX, an AMPA/kainate antagonist exerted an opposite action on the same parameters. The total number and viability of myenteric neurons significantly decreased after I/R. In these conditions, the number of neurons staining for GluN₁ and GluA_1–4 subunits remained unchanged, while, the number of GluK_1–3-immunopositive neurons increased. After I/R, -AP5 and CNQX, concentration-dependently increased myenteric neuron number and significantly increased the number of living neurons. Both -AP5 and CNQX (100–500 µM) decreased I/R-induced increase of ROS levels in myenteric ganglia. On the whole, the present data provide evidence that, under normal metabolic conditions, the enteric glutamatergic system exerts a dualistic effect on cultured myenteric ganglia, either by improving or reducing neuron survival via NMDA or AMPA/kainate receptor activation, respectively. However, blockade of both receptor pathways may exert a protective role on myenteric neurons following and I/R damage. The neuroprotective effect may depend, at least in part, on the ability of both receptors to increase intraneuronal ROS production.     

Intermittent Hypothermia Is Neuroprotective in an in vitro Model of Ischemic Stroke

Objective: To investigate whether the intermittent hypothermia (IH) protects neurons against ischemic insult and the potential molecular targets using an in vitro ischemic model of oxygen glucose deprivation (OGD).Methods: Fetal rat cortical neurons isolated from Day E18 rat embryos were subjected to 90-min OGD and hypothermia treatments during reoxygenation before examining the changes in microscopic morphology, cell viability, microtubule- associated protein 2 (MAP-2) release, intracellular pH value and calcium, reactive oxygen species (ROS) generation, mitochondrial membrane potential (△Ψm) and neuronal death using cell counting kit (CCK-8), enzyme-linked immunosorbent assay (ELISA), BCECF AM, Fluo-3 AM, DCFH-DA and dihydroethidium (DHE), JC-1 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively.Results: 90-min OGD induced morphologic abnormalities, cell viability decline, MAP-2 release, intracellular acidosis, calcium overload, increased ROS generation, △Ψm decrease and cell death in primary neurons, which was partially inhibited by continuous hypothermia (CH) and intermittent hypothermia (IH). Interestingly, 6-h CH was insufficient to reduce intracellular calcium overload and stabilize mitochondrial membrane potential (△Ψm), while 12-h CH was effective in reversing the above changes. All IH treatments (6×1 h, 4×1.5 h or 3×2 h) effectively attenuated intracellular free calcium overload, inhibited ROS production, stabilized mitochondrial membrane potential (△Ψm) and reduced delayed cell death in OGD-treated cells. However, only IH intervals longer than 1.5 h appeared to be effective in preventing cell viability loss and intracellular pH decline.Conclusion: Both CH and IH were neuroprotective in an in vitro model of ischemic stroke, and in spite of shorter hypothermia duration, IH could provide a comparable neuroprotection to CH.     

Prolonged inhibition of glutamate reuptake down-regulates NMDA receptor functions in cultured cerebellar granule cells

In the present study, we have examined the effects of prolonged (up to 72 h) inhibition of high-affinity glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 100 microM) on glutamate receptor functions in primary cultures of rat cerebellar granule neurons. This was done by comparing the effects of various glutamate receptor agonists on neuronal 45Ca2+ uptake, free cytoplasmic Ca2+ concentration ([Ca2+]i), and cell viability. We also determined the parameters of[3H]MK-801 binding as well as the expression of the NMDAR1 subunit protein in control and PDC-exposed cultures. The blockade of glutamate reuptake by PDC led to a gradual increase of ambient glutamate to concentrations that are neurotoxic when applied acutely to control cells. In PDC-exposed cells, however, the acute glutamate-induced NMDA receptor-mediated calcium fluxes were strongly diminished and no toxicity was observed. The down-regulation of the functional effects of glutamate was dependent on the duration of PDC exposure and was accompanied by a reduced NMDAR1 subunit expression and decreased [3H]MK-801 binding, indicative of a pronounced structural rearrangement of NMDA receptors. The possibility that the decrease of NMDA glutamate receptor sensitivity can be explained on the basis of a reduced density or altered subunit composition of NMDA receptors is discussed.