Glutamate neurotoxicity in cortical cell culture

The central neurotoxicity of the excitatory amino acid neurotransmitter glutamate has been postulated to participate in the pathogenesis of the neuronal cell loss associated with several neurological disease states, but the complexity of the intact nervous system has impeded detailed analysis of the phenomenon. In the present study, glutamate neurotoxicity was studied with novel precision in dissociated cell cultures prepared from the fetal mouse neocortex. Brief exposure to glutamate was found to produce morphological changes in mature cortical neurons beginning as quickly as 90 sec after exposure, followed by widespread neuronal degeneration over the next hours. Quantitative dose-toxicity study suggested an ED50 of 50-100 microM for a 5 min exposure to glutamate. Immature cortical neurons and glia were not injured by such exposures to glutamate. Uptake processes probably do not limit GNT in culture, as the uptake inhibitor dihydrokainate did not potentiate GNT. Possibly reflecting the lack of uptake limitation, glutamate was found to be actually more potent than kainate as a neurotoxin in these cultures, a dramatic reversal of the in vivo potency rank order. Some neurons regularly survived brief glutamate exposure; these possibly glutamate-resistant neurons had electrophysiologic properties, including chemosensitivity to glutamate, that were grossly similar to those of the original population.     

Glutamate neurotoxicity in mesencephalic dopaminergic neurons in culture

The neurotoxic effect of glutamate in cultured mouse mesencephalic dopaminergic neurons was investigated. Neuron-rich cell cultures were prepared from 13–14-day-old fetal mouse ventral mesencephalic tissue. Cultures were exposed to glutamate for 10 min and evaluated for glutamate neurotoxicity (GNT) 18–24 hr later by tyrosine hydroxylase (TH) immunostaining, microtubule associated protein-2 (MAP2)immunostaining, and radiolabeled dopamine uptake assay. In glutamate-exposed cultures, the number of TH-positive neurons and the level of dopamine uptake were reduced to 40% (35–45%) and 50% (47–52%), respectively, of control cultures. The number of MAP2-positive neurons was also reduced to 47%, indicating that the GNT was not restricted or selective to dopaminergic neurons. It is concluded that GNT was mediated by the N-methyl-D-aspartic acid (NMDA) receptor from the following observations: (1) GNT was completely blocked by MK-801, an NMDA receptor antagonist; (2) NMDA itself was as toxic as glutamate; (3) 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), an antagonist of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor, did not block GNT; (4) kainate did not show neurotoxicity at a low concentration; and 5) two modulators of the NMDA receptor, 7-chlorokynurenic acid and magnesium, were effective in blocking GNT. Protective effects of phorbol myristate acetate, a tumor promoter, and gangliosides (G_M1 and G_T1b on GNT were also demonstrated. Possible interactions between GNT and several protein kinase cascades were also investigated. Forskolin, an activator of adenyl cyclase and protein kinase A, showed some protective effect on GNT. But okadaic acid, an inhibitor of phosphatases, and genistein, a tyrosine kinase inhibitor, did not show any protective effect. These results suggest that (1) glutamate is capable of causing neuronal death in the substantia nigra; (2) GNT on dopaminergic neurons is mainly mediated by the NMDA receptor under the conditions of our study; (3) protein kinase C translocation is a key mechanism of GNT; and (4) there is an interplay of a signal transduction system in the pathomechanism of GNT. © 1993 Wiley-Liss, Inc.     

Comparison of plate reader-based methods with fluorescence microscopy for measurements of intracellular calcium levels for the assessment of in vitro neurotoxicity

The intracellular calcium concentration ([Ca²⁺]_i) is an important readout for in vitro neurotoxicity since calcium is critically involved in many essential neurobiological processes, including neurotransmission, neurodegeneration and neurodevelopment. [Ca²⁺]_i is often measured with considerable throughput at the level of cell populations with plate reader-based assays or with lower throughput at the level of individual cells with fluorescence microscopy. However, these methodologies yield different quantitative and qualitative results. In recent years, we demonstrated that the resolution and sensitivity of fluorescence microscopy is superior compared to plate reader-based assays. However, it is currently unclear if the use of plate reader-based assays results in more ‘false negatives’ or ‘false positives’ in neurotoxicity screening studies. In the present study, we therefore compared a plate reader-based assay with fluorescence microscopy using a small test set of environmental pollutants consisting of dieldrin, lindane, polychlorinated biphenyl 53 (PCB53) and tetrabromobisphenol-A (TBBPA). Using single-cell fluorescence microscopy, we demonstrate that all test chemicals reduce the depolarization-evoked increase in [Ca²⁺]_i, whereas lindane, PCB53 and TBBPA also increase basal [Ca²⁺]_i, though via different mechanisms. Importantly, none of these effects were confirmed with the plate reader-based assay. We therefore conclude that standard plate reader-based methods are not sufficiently sensitive and reliable to measure the highly dynamic and transient changes in [Ca²⁺]_i that occur during chemical exposure.     

Glutamate neurotoxicity in the developing rat cochlea: physiological and morphological approaches

The neurotoxic effects of exogenous glutamate were studied in the rat cochlea. Glutamate-treated rats (4 g/kg/day i.p., postnatal days 2-9) exhibited electrophysiologically-measured elevations in high frequency thresholds usually associated with hair cell loss in the basal region of the cochlea. While surface preparations of the organ of Corti revealed no loss of hair cells, there was a dramatic and selective reduction of neurons in the basal, high frequency-related portion of the spiral ganglion. This sensitivity of developing spiral ganglion cells to the neurotoxicity of glutamate is consistent with the hypothesis that glutamate or a structurally related substance is a neurotransmitter at afferent synapses of cochlear hair cells.     

Glutamate transporter‐associated anion channels adjust intracellular chloride concentrations during glial maturation

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride‐sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl^?]_int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na⁺‐K⁺‐2Cl^? cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl^?]_int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion‐selective channels during glutamate transport, and thus represent a class of ligand‐gated anion channels. Age‐dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT‐1 increased [Cl^?]_int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl^?]_int. Other tested chloride channels or chloride transporters do not contribute to [Cl^?]_int under our experimental conditions. Neither genetic removal of ClC‐2 nor pharmacological block of K⁺‐Cl^? cotransporter change resting Bergmann glial [Cl^?]_int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388–400     

Polychlorinated biphenyls and methylmercury alter intracellular calcium concentrations in rat cerebellar granule cells

Assessments of the effects of exposure of human populations to complex environmental contaminants, such as those found in contaminated fish, necessitate the investigation of contaminant interactions. We have recently demonstrated that polychlorinated biphenyls (PCBs) and methylmercury (MeHg) synergistically reduce rat brain striatal slice dopamine (DA) and increase media DA concentrations in vitro. To better understand the mechanism(s) by which these effects occur we examined the effects of these two contaminants, either alone or in combination, on intracellular calcium concentrations ([Ca2+]) in rat cerebellar granule cells using flow cytometry. Exposure of granule cells to either 2,2'-dichlorobiphenyl (2,2'-DCB) or MeHg dose-dependently increased [Ca2+]i. Granule cells exposed to 1.5 microM MeHg and 2.5 or 5.0 microM 2,2'-DCB showed synergistic increases in [Ca2+]i which were greatest at exposure times of 5 and 10 min. Higher dose combinations, including 2.0 microM MeHg and 10 or 20 microM 2,2'-DCB, or longer duration of exposure to lower concentrations of contaminant mixtures, reduced [Ca2+]i in the granule cells compared to elevations seen following exposure to MeHg only, suggesting a dose-dependent antagonism between PCBs and MeHg. These data provide evidence for the synergistic and antagonistic interactions of PCBs and MeHg at the level of [Ca2+]i regulation that may ultimately lead to alterations in cellular function, including changes in dopamine regulation.     

Cerebrospinal fluid from human immunodeficiency virus-infected individuals facilitates neurotoxicity by suppressing intracellular calcium recovery

Neurologic decline associated with penetration of human immunodeficiency virus type 1(HIV-1) into the central nervous system is thought to be due, in large part, to inflammation and local secretion of neurotoxic substances. To examine the cellular processes that mediate neurotoxicity in vivo, the authors valuated the ability of neurons to maintain intracellular calcium homeostasis in the presence of toxic cerebrospinal fluid (CSF) (CSF(tox)) collected from a subset of HIV-infected individuals. Exposure of rat neural cultures to CSF(tox) resulted in a gradual increase in intracellular calcium in neurons (+63%), microglia (+251%), and astrocytes (+52%). Pretreatment of neural cultures with CSF(tox) resulted in an exaggerated calcium response to a brief pulse of glutamate and a > 90% suppression of the rate of recovery of intracellular calcium. Attempts to model the deficit using inhibitors of calcium transport across endoplasmic reticulum, mitochondrial, or plasma membrane indicated that blockade of the plasma membrane sodium/calcium exchanger was best able to reproduce the deficits seen during exposure to CSF(tox). Because the inability of cells to maintain calcium homeostasis would lead to exaggerated responses from a wide variety of stimuli, therapeutics designed to facilitate calcium transport from the cell may provide more comprehensive and effective intervention than strategies targeted to specific receptor pathways.     

Alterations of intracellular calcium homeostasis and mitochondrial function are involved in ruthenium red neurotoxicity in primary cortical cultures

Ruthenium red (RR) is a polycationic dye that induces neuronal death in vivo and in primary cultures. To characterize this neurotoxic action and to determine the mechanisms involved, we have analyzed the ultrastructural alterations induced by RR in rat cortical neuronal cultures and measured its effect on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and on mitochondrial function. RR produced a dose-dependent, progressive disruption of neurites and plasma membrane of neuronal somata after 8-24 hr of incubation. RR caused also an elevation of both the basal [Ca(2+)](i) and its maximal levels after K(+) depolarization. Mitochondrial oxidative function, assessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and by changes in dihydrorhodamine-123 fluorescence, was significantly diminished after treatment with RR, both in cultured neurons and in isolated brain mitochondria. La(3+) did not prevent but rather potentiated RR-induced cell death. Glutamate receptor antagonists also failed to prevent RR neurotoxicity. Apoptotic electron microscope images were not observed, and protein synthesis inhibitors did not show any protective effect. It is concluded that RR penetrates neurons and that its neurotoxic damage probably is due to intracellular Ca(2+) dishomeostasis and disruption of mitochondrial oxidative function. These results enhance our understanding of the intracellular mechanisms underlying neuronal death.     

人参皂甙Rb1对模拟缺血环境中的大鼠神经元胞内游离钙的影响

目的探讨人参皂甙Rb1(GSRb1)对模拟缺血环境中的大鼠神经元胞内游离钙的影响。方法对大鼠胚胎脑海马神经元进行体外培养,将其置于正常细胞外液(正常对照组)、模拟缺血的细胞外液(缺血组)、无钙的模拟缺血液(缺血+无钙组)、以及模拟缺血液+不同浓度的GSRb1溶液[缺血+GSRb1(20、40、60、80μmol/L组)],采用激光共聚焦技术测定各组细胞内钙荧光强度,并计算与正常对照组相比荧光强度变化百分比。结果与正常对照组相比,荧光增强度缺血组为(73.5±10.31)%,缺血+无钙组为(4.5±2.58)%,缺血+不同浓度GSRb1组(20、40、60、80μmol/L组)分别为(20.2±3.41)%、(13.6±2.98)%、(10.5±3.62)%和(12.7±4.51)%;缺血+各浓度GSRb1组的荧光增强度明显小于缺血组(均P<0.01)。结论缺血缺氧时神经细胞内游离钙的上升主要是来自细胞外钙离子内流;GSRb1可通过抑制细胞外钙离子内流,减轻细胞内的钙超载,保护缺血神经元;保护作用呈浓度依赖性,在60μmol/L时达到最大。     

Nitroprusside in vitro inhibits platelet aggregation and intracellular calcium translocation. Effect of haemoglobin

The biologically final active compound of nitrovasodilators is now supposed to be nitric oxide (NO), a labile substance identical to EDRF. The effects of nitroprusside on platelet functions were studied in vitro. Platelet aggregation induced by several stimuli (ADP, collagen, arachidonic acid and PAF) was inhibited by increasing concentrations of the drug (1-50 uM); interestingly, the potency of nitroprusside is higher when PAF is employed as stimulating agent in comparison with the other agonists (ED50 = 2 uM for ADP, 2.5 uM for A.A., 4.5 uM for collagen and 0.3 uM for PAF-induced aggregations). The concomitant addition of haemoglobin is able to reverse the inhibitory effect of nitroprusside, according to the view that haemoglobin possesses a high affinity for NO, thus antagonizing the effect of this compound. Nitroprusside was also able to inhibit intracellular calcium translocation, as studied with the Quin 2 technique, induced by PAF and arachidonic acid. Fron these observations the hypothesis may be suggested that nitroprusside inhibits platelet functions by mimicking the endogenous NO, and that the intracellular calcium metabolism is involved in the inhibitory activity of the drug.     

Glutamate neurotoxicity: Perturbations of ionic homeostasis, mitochondrial dysfunction, and changes in cell functioning

The hyperstimulation of neuronal glutamate receptors during different pathologies results in CNS injuries. This review summarizes the data concerning changes in the concentrations of sodium, potassium, calcium, and protons in the cytosol and mitochondrial matrix of cultured neurons subjected to prolonged glutamate treatment. Development of delayed calcium deregulation (DCD) plays a leading role in the initiation of neuronal death during toxic glutamate treatment. We review the mechanisms of DCD development, the role of mitochondria in the regulation of neuronal calcium homeostasis during stimulation of glutamate receptors, and intracellular events that accompany DCD and may result in neuronal death.     

Receptor mechanisms and circuitry underlying NMDA antagonist neurotoxicity.

NMDA glutamate receptor antagonists are used in clinical anesthesia, and are being developed as therapeutic agents for preventing neurodegeneration in stroke, epilepsy, and brain trauma. However, the ability of these agents to produce neurotoxicity in adult rats and psychosis in adult humans compromises their clinical usefulness. In addition, an NMDA receptor hypofunction (NRHypo) state might play a role in neurodegenerative and psychotic disorders, like Alzheimer's disease and schizophrenia. Thus, understanding the mechanism underlying NRHypo-induced neurotoxicity and psychosis could have significant clinically relevant benefits. NRHypo neurotoxicity can be prevented by several classes of agents (e.g. antimuscarinics, non-NMDA glutamate antagonists, and alpha(2) adrenergic agonists) suggesting that the mechanism of neurotoxicity is complex. In the present study a series of experiments was undertaken to more definitively define the receptors and complex neural circuitry underlying NRHypo neurotoxicity. Injection of either the muscarinic antagonist scopolamine or the non-NMDA antagonist NBQX directly into the cortex prevented NRHypo neurotoxicity. Clonidine, an alpha(2) adrenergic agonist, protected against the neurotoxicity when injected into the basal forebrain. The combined injection of muscarinic and non-NMDA Glu agonists reproduced the neurotoxic reaction. Based on these and other results, we conclude that the mechanism is indirect, and involves a complex network disturbance, whereby blockade of NMDA receptors on inhibitory neurons in multiple subcortical brain regions, disinhibits glutamatergic and cholinergic projections to the cerebral cortex. Simultaneous excitotoxic stimulation of muscarinic (m(3)) and glutamate (AMPA/kainate) receptors on cerebrocortical neurons appears to be the proximal mechanism by which the neurotoxic and psychotomimetic effects of NRHypo are mediated.     

GABAB receptors increase intracellular calcium concentrations in chromaffin cells through two different pathways: Their role in catecholamine secretion

The activation of GABA_B receptors of adrenal chomaffin cells produces an increase of [Ca²⁺]_i measured by fura-2 AM techniques. GABA_B agonists 3-aminopropylphosphinic acid or (-)baclofen, at concentrations of 0.5 mM, increased basal Ca²⁺, values 332 ± 60.9 and 306 ± 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca²⁺ buffer. The GABA_B-induced increase of [Ca²⁺]_i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L-type voltage-dependent Ca²⁺ channels as the dihydropiridine nifedipine 50 μM was able to decrease it more than 60%, while ω-conotoxin, which blocks N-type channels, did not produce any change in the GABA_B-evoked Ca²⁺ increment. The second component was due to a release of Ca²⁺ from intracellular pools and was about one-third of the total GABAB-induced increase of [Ca²⁺]_i. GABA_B receptors stimulated inositol 1,4,5-trisphosphate-sensitive and not the caffeine-sensitive Ca²⁺ store. In a low Ca²⁺ buffer after treatment with 2 μM angiotensin II, neither 0.5 mM 3-APPA nor baclofen were able to produce an additional increase of [Ca²⁺]_i, whereas 4 mM caffeine had no effect on GABA_B response. This intracellular Ca²⁺ mobilization could be due to inositol 1,4,5-trisphosphate accumulation produced by the activation of GABA_B receptors. In fact, the specific agonists after 10 minutes incubation produced a dosedependent increase of inositol 1,4,5-trisphosphate. The maximal effect was obtained at 100 μM baclofen and 3-APPA, and it was 3.63 ± 0.75 and 3.2 ± 1.5 times the basal levels (7.3 ± 0.3 pmol/10⁶ cells), respectively. In the absence of extracellular Ca²⁺, GABA_B-evoked catecholamine secretion and cyclic AMP formation were reduced more than 70%, suggesting an important role of extracellular Ca²⁺ in GABA_B mechanisms in adrenal chromaffin cells. © 1995 Wiley-Liss, Inc.     

Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion

The role of mitochondrial energy metabolism in glutamate mediated neurotoxicity was studied in rat neurones in primary culture. A brief (15 min) exposure of the neurones to glutamate caused a dose-dependent (0.01–1 mM) increase in cyclic GMP levels together with delayed (24 h) neurotoxicity and ATP depletion. These effects were prevented by either the nitric oxide (^·NO) synthase (NOS) inhibitor Nω-nitro- -arginine methyl ester (NAME; 1 mM) or by the N-methyl- -aspartate (NMDA) glutamate-subtype receptor antagonist -(−)-2-amino-5-phosphonopentanoate (APV; 0.1 mM). Glutamate exposure (0.1 mM and 1 mM) followed by 24 h of incubation caused the inhibition of succinate–cytochrome c reductase (20–25%) and cytochrome c oxidase (31%) activities in the surviving neurones, without affecting NADH–coenzyme-Q₁ reductase activity. The rate of oxygen consumption was impaired in neurones exposed to 1 mM glutamate, either with glucose (by 26%) or succinate (by 39%) as substrates. These effects on the mitochondrial respiratory chain and neuronal respiration, together with the observed glutathione depletion (20%) by glutamate exposure were completely prevented by NAME or APV. Our results suggest that mitochondrial dysfunction and impairment of antioxidant status may account for glutamate-mediated neurotoxicity via a mechanism involving ^·NO biosynthesis.     

In vitro status epilepticus causes sustained elevation of intracellular calcium levels in hippocampal neurons

Calcium ions and calcium-dependent systems have been implicated in the pathophysiology of status epilepticus (SE). However, the dynamics of intracellular calcium ([Ca2+]i) levels during SE has not yet been studied. We have employed the hippocampal neuronal culture (HNC) model of in vitro SE that produces continuous epileptiform discharges to study spatial and dynamic changes in [Ca2+]i levels utilizing confocal laser scanning microscopy and the calcium binding dye, indo-1. During SE, the average [Ca2+]i levels increased from control levels of 150-200 nM to levels of 450-600 nM. This increased [Ca2+]i was maintained for the duration of SE. Following SE, [Ca2+]i levels gradually returned to basal values. The duration of SE was shown to affect the ability of the neuron to restore resting [Ca2+]i levels. Both N-methyl-D-aspartate (NMDA) receptor-gated and voltage-gated Ca2+ channels (VGCCs) contributed to the increased calcium entry during SE. Moreover, this elevation in [Ca2+]i occurred in both the nucleus and cytosol. These results provide the first dynamic measurement of [Ca2+]i during prolonged electrographic seizure discharges in an in vitro SE model and suggest that prolonged epileptiform discharges give rise to abnormal sustained increases in [Ca2+]i levels that may play a role in the neuronal cell damage and long-term plasticity changes associated with SE.     

Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons.

Glutamate-induced changes in intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single rat hippocampal neurons grown in primary culture by employing the Ca2+ indicator indo-1 and a dual-emission microfluorimeter. The [Ca2+]i was monitored in neurons exposed to 100 microM glutamate for 5 min and for an ensuing 3 hr period. Ninety-two percent (n = 64) of these neurons buffered the glutamate-induced Ca2+ load back to basal levels after removal of the agonist; thus, the majority of cells had not lost the ability to regulate [Ca2+]i at this time. However, following a variable delay, in 44% (n = 26) of the neurons that buffered glutamate-induced Ca2+ loads to basal levels, [Ca2+]i rose again to a sustained plateau and failed to recover. The changes in [Ca2+]i that occur during glutamate-induced delayed neuronal death can be divided into three phases: (1) a triggering phase during which the neuron is exposed to glutamate and the [Ca2+]i increases to micromolar levels, followed by (2) a latent phase during which the [Ca2+]i recovers to a basal level, and (3) a final phase that begins with a gradual rise in the [Ca2+]i that reaches a sustained plateau from which the neuron does not recover. This delayed Ca2+ overload phase correlated significantly with cell death. The same sequence of events was also observed in recordings from neuronal processes. The delayed Ca2+ increase and subsequent death were dependent upon the presence of extracellular Ca2+ during glutamate exposure. Calcium influx during the triggering phase resulted from the activation of both NMDA and non-NMDA receptors as indicated by studies using receptor antagonists and ion substitution. Treatment with TTX (1 microM) or removal of extracellular Ca2+ for a 30 min window following agonist exposure failed to prevent the delayed Ca2+ overload. The delayed [Ca2+]i increase could be reversed by removing extracellular Ca2+, indicating that it resulted from Ca2+ influx. The three phases defined by changes in the [Ca2+]i during glutamate-induced neuronal toxicity suggest three distinct targets to which neuroprotective agents may be directed.