Translational suppression of calpain I reduces NMDA-induced spectrin proteolysis and pathophysiology in cultured hippocampal slices

Transfection of cultured hippocampal slices for five days with antisense oligonucleotides directed against mRNA encoding calpain I resulted in an approximately 60% decrease in the amount of caseinolytic activity stimulated by 10 μM calcium. Increases in a single proteolytic fragment of spectrin produced by 10–20 min of NMDA receptor stimulation were substantially ( 50%) reduced in antisense treated slices; this effect was not obtained in slices exposed to NMDA for 45 min. Attenuation of NMDA receptor-induced spectrin proteolysis by the antisense oligonucleotides was confirmed in immunoassays using antibodies that recognize multiple spectrin breakdown products and in immunocytochemical experiments with an antibody that defects an individual calpain I-mediated fragment. Translational suppression of calpain I did not detectably affect evoked synaptic responses but markedly improved their recovery from a 15 min infusion of NMDA. These results indicate that spectrin breakdown products provide a useful index of in situ calpain I activity and support the hypothesis that the protease plays a significant role in excitoxicity.     

Serotonin Differentially Modulates Responses Mediated by Specific Excitatory Amino Acid Receptors in the Rat Locus Coeruleus

Microiontophoretic application of selective agonists for the three major excitatory amino acid receptors, N -methyl- d -aspartate (NMDA), quisqualate and kainate, increased the discharge rate of noradrenergic locus coeruleus (LC) neurons in vivo. NMDA activation was selectively attenuated by iontophoretic application of 2-amino-5-phosphonopentanoate (AP5), an antagonist at NMDA receptors, whereas kainate- and quisqualate-evoked responses were attenuated by both NMDA and non-NMDA antagonists iontophoresis. NMDA- and quisqualate-evoked responses were significantly decreased by co-iontophoresis of serotonin (5-HT). When the NMDA receptor-mediated component of the response to kainate was blocked with AP5 iontophoresis, 5-HT increased the response of LC neurons to kainate. These results revealed that 5-HT differentially modulates the responsiveness of LC neurons to excitatory amino acids, depending on the receptor subtypes responsible for the neuronal activation.     

Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures.

Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.     

Proteolytic processing of beta-amyloid precursor by calpain I

The beta-amyloid peptide is a core component of the neuritic plaques that accumulate in Alzheimer's disease. Since the beta-peptide resides within a family of precursor proteins (APPs), proteolytic processing of APP is required for beta-amyloid deposition into plaques. Here, we have examined the role played by the calcium-dependent cysteine protease calpain I in APP processing. Immunoblotting with a specific APP antiserum was used to assess the in vitro degradation of rat brain APP, which appears as a triplet of polypeptides of Mr 110-130 kDa. Both soluble and membrane-bound APP were extraordinarily sensitive to activated calpain I. APP contains at least 3 distinct calpain I cleavage sites. The most protease-sensitive site was located within the highly acidic structural motif called the PEST domain, a second site was upstream of the putative N-linked glycosylation sites, and a third generated a 16 kDa carboxy-terminal fragment that contains the beta-peptide. Based on light microscopic immunohistochemistry, APP and calpain I were extensively colocalized within large numbers of neurons distributed throughout the rat brain, with especially high levels of each in neocortical layer 5, subiculum, globus pallidus, entopeduncular nucleus, anterodorsal and reticular thalamic nuclei, motor trigeminal nucleus, deep cerebellar nuclei, and Purkinje cells. Both antigens were most prevalent within neuronal perikarya. Intraventricular kainate infusion, which is known to cause rapid activation of hippocampal calpain I, produced a 32% decline in APP levels after 24 hr, suggestive of in vivo degradation of APP by calpain I. Following kainate-induced neuronal loss, both APP and calpain I immunoreactivities appeared in the surrounding reactive astroglia. These results indicate that calpain I may be involved in the normal and, perhaps, pathological processing of APP, and that this processing could occur in either neurons or reactive astrocytes. Calcium influx and calpain I activation may provide a mechanism by which excitatory neurotransmission regulates APP metabolism.     

Cytotoxic effect of glutamate and its agonists on mouse hippocampal neurons

The neurodegenerative action of the excitatory amino acid neurotransmitter (glutamate) and its exogenous (N-methyl-D-aspartate, kainate) or endogenous (quinolinate) analogues were studied on cultures of dissociated nerve cells from the embryonal mouse hippocampus. The exposure of primary cultures for 3-6 h to these excitotoxins showed that neurons were vulnerable to both glutamate and all tested agonists which induced the swelling and vacuolization of neuronal bodies accompanied by degeneration of their dendrites. This process terminated by complete cell destruction. The neurotoxic effect of glutamate (1 mM) was not suppressed by a competitive NMDA receptor antagonist (D, L-2-amino-5-phosphonovalerate, 0.3 mM) and was only slightly prevented by gamma-D-glutamylglycine (3mM). The protective action of the latter was more evident in the presence of lower glutamate concentration (0.5 mM). The excitotoxic effect of N-methyl-D-aspartate (0.1 mM) or quinolinate (0.5mM) was almost completely blocked by both antagonists. In contrast, D, L-2-amino-5-phosphonovalerate failed to protect hippocampal neurons from damage induced by kainate while partial antagonism of kainate neurotoxicity was observed with gamma-D-glutamylglycine. These finding suggest that glutamate neurotoxicity may be derived, mainly, from the non-NMDA type(s) of glutamate receptor present on hippocampal cell membranes with a low effectiveness to suppress this effect by selective competitive NMDA antagonist. Possible involvement of glutamate receptor(s) in the early dendritic outgrowth of hippocampal neurons and in the process of neuronal "cell death" is discussed.     

Activation of ionotropic glutamate receptors reduces the production of transforming growth factor-beta2 by developing neurons

Neuronal cultures derived from developing rat cerebral cortex were used to investigate the influence of glutamate receptors on the neuronal production of transforming growth factor-beta2 (TGFbeta2), a multifunctional cytokine that modulates neuronal and glial growth. Long-term exposure (48 h) of cortical neurons to selective antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors markedly increased TGFbeta2 levels in the culture medium. Conversely, treatment with NMDA or kainate reduced TGFbeta2 to levels below those in untreated cultures. The effect of kainate did not require NMDA receptor activity. Neuronal depolarization with K+ also reduced TGFbeta2 levels by opening voltage-gated L-type Ca2+ channels. Semi-quantitative RT-PCR measurements of neuronal TGFbeta2 mRNA showed that NMDA or AMPA/kainate receptor stimulation reduced TGFbeta2 mRNA levels. These results demonstrate that tonic activation of glutamate-gated cation channels downregulates neuronal expression of the TGFbeta2 gene and provide evidence for a novel mechanism whereby excitatory amino acids could influence the development of glial and neuronal lineages.     

Excitatory amino acid-evoked release of [3H]GABA from hippocampal neurons in primary culture

We investigated the release of gamma-[2,3-3H(N)]aminobutyric acid ([3H]GABA) from hippocampal neurons in primary cell culture. [3H]GABA release was stimulated by the excitatory amino acid neurotransmitter glutamate as well as by N-methyl-D-aspartate (NMDA) and kainate. Cell depolarization induced by raising [K+]o or by veratridine also stimulated [3H]GABA release. NMDA-induced release was completely blocked by 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP+), Mg2+ and Zn2+ whereas the release induced by glutamate and kainate was much less susceptible to inhibition by these substances. Furthermore, removal of external Ca2+ inhibited NMDA-induced release, but not that induced by glutamate, kainate, veratridine or 50 mM K+. Removal of external Na+ reduced [3H]GABA release evoked by all stimuli, but to different extents. All of the excitatory amino acids tested increased [Ca2+]i within hippocampal neurons as assessed by fura-2 based microspectrofluorimetry. This increase in [Ca2+]i was completely dependent on the presence of external Ca2+. These results suggest that Ca2+-dependent and -independent forms of GABA release from hippocampal interneurons may occur. [3H]GABA release evoked by glutamate, kainate, veratridine or 50 mM K+, appeared to be mediated by the reversal of electrogenic, Na+-coupled GABA uptake. Release was inhibited by nipecotic acid, an inhibitor of the Na+-coupled GABA uptake system. However, release induced by NMDA may also include a Ca2+-dependent component.     

Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons.

The mechanisms regulating the expression and localization of excitatory amino acid (EAA) neurotransmitter receptors in neurons of the developing mammalian brain, and roles for these receptors in the plasticity and degeneration of neural circuits are not well understood. We previously isolated and characterized a 71 kDa glutamate binding protein (GBP) from rat brain, and have recently obtained evidence that this GBP is a component of a functional N-methyl-D-aspartate (NMDA) receptor-ion channel complex. We have now used antibodies to this putative NMDA receptor protein to examine its expression and localization, and consequences of its activation in cultured embryonic (18 day) rat hippocampal neurons. Immunocytochemistry and Western blots using monoclonal antibodies to the GBP demonstrated an increase in GBP-positive neurons and their staining intensity with time in culture. GBP was localized to the somata and dendrites of pyramidal-like neurons and was sparse or absent in the axons. The expression and compartmentalization of GBP occurred in isolated neurons indicating that direct cell interactions were not required for these processes. Cell surface staining for GBP occurred in patches on the soma and dendrites. The developmental expression of GBP immunoreactivity closely paralleled the expression of sensitivity to NMDA neurotoxicity. There was a direct relationship between GBP immunoreactivity and neuronal vulnerability to glutamate-induced degeneration; vulnerable neurons stained heavily whereas resistant neurons showed either low levels of staining or no staining. Finally, a GBP antiserum greatly reduced NMDA neurotoxicity (but not kainate neurotoxicity). Taken together, these findings demonstrate the expression of presumptive NMDA receptors within a subpopulation of embryonic hippocampal neurons, and their segregation to the soma and dentrites of pyramidal neurons. This spatial distribution of glutamate receptors among and within neurons is likely to play important roles in regulating the structure of neural circuitry during development, and may also be an important determinant of selective neuronal vulnerability in pathological conditions.     

Tonic Activation of NMDA Receptors Causes Spontaneous Burst Discharge of Rat Midbrain Dopamine Neurons In Vivo

Midbrain dopamine neurons in vivo discharge in a single-spike firing pattern or in a burst-firing pattern. Such activity in vivo strikingly contrasts with the pacemaker activity of the same dopamine neurons recorded in vitro. We have recently shown that burst activity in vivo of midbrain dopamine neurons is due to the local activation of excitatory amino acid receptors, as microapplication of the broad-spectrum antagonist of excitatory amino acids, kynurenic acid, strongly regularized the spontaneous firing pattern of these dopamine neurons. In the present study, we investigated which subtypes of excitatory amino acid receptors are involved in the burst-firing of midbrain dopamine neurons in chloral hydrate-anaesthetized rats, Iontophoretic or pressure microejections of 6-cyano,7-nitroquinoxaline-2,3-dione (CNQX), a non- N -methyl- d -aspartate (NMDA) receptor antagonist, did not alter the spontaneous burst firing of dopamine neurons ( n = 36). In contrast, similar ejections of (±)2-amino,5-phos-phonopentanoic acid (AP-5), a specific antagonist at NMDA receptors, markedly regularized the firing pattern by reducing the occurrence of bursts ( n = 52). In addition, iontophoretic ejections of NMDA, but not kainate or quisqualate, elicited a discharge of these dopamine neurons in bursts ( n = 20, 12 and 14, respectively). These data suggest that burst-firing of midbrain dopamine neurons in vivo results from the tonic activation of NMDA receptors by endogenous excitatory amino acids. In view of the critical dependency of catecholamine release on the discharge pattern of source neurons, excitatory amino acid inputs to midbrain dopamine neurons may constitute a major physiological substrate in the control of the dopamine level in target areas.     

Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors

Dose-response curves for activation of excitatory amino acid receptors on mouse embryonic hippocampal neurons in culture were recorded for 15 excitatory amino acids, including the L-isomers of glutamate, aspartate, and a family of endogenous sulfur amino acids. In the presence of 3 microM glycine, with no extracellular Mg, micromolar concentrations of 11 of these amino acids produced selective activation of N-methyl-D-aspartate (NMDA) receptors. L-Glutamate was the most potent NMDA agonist (EC50 2.3 microM) and quinolinic acid the least potent (EC50 2.3 mM). Dose-response curves were well fit by the logistic equation, or by a model with 2 independent agonist binding sites. The mean limiting slope of log-log plots of NMDA receptor current versus agonist concentration (1.93) suggests that a 2-site model is appropriate. There was excellent correlation between agonist EC50S determined in voltage clamp experiments and KdS determined for NMDA receptor binding (Olverman et al., 1988). With no added glycine, and 1 mM extracellular Mg, responses to NMDA were completely blocked; responses to kainate and quisqualate were unchanged. Under these conditions, glutamate and the sulfur amino acids activated a rapidly desensitizing response, similar to that evoked by micromolar concentrations of quisqualate and AMPA, but mM concentrations of L-aspartate, homoquinolinic acid, and quinolinic acid failed to elicit a non-NMDA receptor-mediated response. Except for L-glutamate (EC50 480 microM), the low potency of the sulfur amino acids prevented the study of complete dose-response curves for the rapidly desensitizing response at quisqualate receptors. Small-amplitude nondesensitizing quisqualate receptor responses were activated by much lower concentrations of all quisqualate receptor agonists. Full dose-response curves for the nondesensitizing response were obtained for 9 amino acids; L-glutamate was the most potent endogenous agonist (EC50 19 microM). Domoate (EC50 13 microM) and kainate (EC50 143 microM) activated large-amplitude, nondesensitizing responses.     

Protective effects of calpain inhibitors against neuronal damage caused by cytotoxic hypoxia in vitro and ischemia in vivo

The calpains are calcium-dependent intracellular proteases that are activated in a number of pathogenic conditions. We tested the capacities of protease inhibitors, calpain inhibitor I and leupeptin, to protect against the neuronal degeneration caused by cytotoxic hypoxia or transient global cerebral ischemia. Primary neuronal cultures were prepared from embryonic chick telencephalon, and cytotoxic hypoxia was induced by adding 1 mM NaCN to the culture medium for 30 min. Global ischemia was induced in rats by clamping both carotid arteries and lowering the arterial blood pressure to 40 mmHg for 10 min. Both calpain inhibitor I and leupeptin protected neurons against ischemic and hypoxic damage. Neuroprotection was indicated by increased cell viability and protein content in the cultures, and fewer damaged neurons in the hippocampal CA1-subfield. Thus, blockade of proteolysis can protect neurons against cytotoxic and ischemic damage.     

Calpain facilitates the neuron death induced by 3-nitropropionic acid and contributes to the necrotic morphology

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, has been used to model features of neurodegenerative disorders including Huntington disease, as well as acute neuronal insults such as cerebral ischemia. 3NP induces rapid necrosis and delayed apoptosis in primary cultures of rat hippocampal neurons. Low levels of extracellular glutamate shift the cell death mechanism to necrosis, whereas antagonism of NMDA receptors results in predominately apoptotic death. In the present study, the involvement of cysteine proteases in the morphologic and biochemical alterations accompanying 3NP-induced neuron death was investigated. Immunoblots of spectrin breakdown products indicated Ca(2+)-dependent cysteine protease (calpain) activation within the 8 hours of 3NP administration, whereas caspase-3 activation was not evident until 16 to 48 hours after treatment. The NMDA receptor antagonist MK-801 (dizocilpine) decreased 3NP-induced calpain activity, but did not alter caspase-3 activity. Similar to MK-801, calpain inhibitors (Z-Val-Phe.H and Z-Leu-Phe-CONHEt) shifted the cell death morphology towards apoptosis and delayed, but did not prevent, the 3NP-induced cell death. Together, the results indicate that following 3NP administration, increased calpain activity precedes caspase-3 activation, contributes to the necrotic morphology, and facilitates and accelerates the cell death.     

Selective coupling of μ-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons

Excessive μ-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non–central nervous system (CNS) cells, calpain activation is thought to occur following a Ca²⁺-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 μM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-μ-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca²⁺ entry through the NMDA receptor because nonspecific Ca²⁺ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells. J. Neurosci. Res. 54:223–232, 1998. © 1998 Wiley-Liss, Inc.     

Development and regulation of excitatory amino acid receptors involved in the release of arachidonic acid in cultured hippocampal neural cells

Release of [3H]arachidonic acid mediated by excitatory amino acid (EAA) receptors was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. Treatment with N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate resulted in age- and dose-dependent stimulation of [3H]arachidonic acid release. During development, the maximum response for NMDA was observed relatively earlier (at 7 days) than those for QA and kainate (at 14 days) in the hippocampal neuronal cultures. The half maximal effects were obtained at about 15 microM NMDA at all ages studied and about 0.5 microM QA at 14 and 20 days. At optimum concentrations NMDA- and QA-induced releases were additive. Unlike with neurons, treatment with all the 3 EAA receptor agonists, NMDA, QA and kainate, had no significant effect on [3H]arachidonate release in hippocampal astroglial cells. In cultured 14-day-old neurons, the increases in NMDA- and QA-mediated [3H]arachidonic acid release were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and the ionotropic QA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. But the iontropic QA receptor agonist alpha-amino-3-hydroxy-5-methyl-isoxazole-4- propionic acid (AMPA) had no significant effect on [3H]arachidonate release, indicating that interaction between ionotropic QA and metabolotropic QA receptors may be essential for optimal QA-mediated arachidonic acid release. At physiological concentrations of Mg2+ (1.2 mM), AMPA was found to potentiate NMDA-induced release of [3H]arachidonic acid; the effect appeared to be related to a removal of Mg2+ blockade mediated by mild depolarisation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kainate and NMDA toxicity for cultured developing and adult rat spiral ganglion neurons: further evidence for a glutamatergic excitatory neurotransmission at the inner hair cell synapse.

In the inner ear, the excitatory amino acid glutamate is a proposed neurotransmitter acting at the synapse between hair cells and afferent auditory neurons. Using cultures of 5-day-old rat auditory neurons, we show that the afferent auditory neuronal population can be divided, on the basis of its sensitivity to the neuronotoxic effect of glutamate and its analogs, in at least 3 subpopulations, one responding to N-methyl-D-aspartate (NMDA), one responding to kainate and a third minor one unresponsive to NMDA, kainic acid and glutamate. No toxic effect of quisqualate is observed. The use of specific antagonists (kynurenate and 2-amino-5-phosphonovalerate (DAP-5) demonstrates the specificity of the receptors to the excitatory amino acids on the afferent auditory neurons. Afferent auditory neurons from adult rats can also be cultured and in these preparations only the large neurons are sensitive to glutamate, kainate and NMDA while the small neurons are not responsive, suggesting that a glutamatergic neurotransmission occurs only at this synapse between the inner hair cells and the large radial afferent auditory neurons. We also show that, in vitro, the organ of Corti releases, in response to an increased potassium concentration and in the presence of calcium, a toxic activity for the afferent auditory neurons that is antagonized by kynurenate and DAP-5. Pathophysiological implications are discussed.     

Tissue plasminogen activator protects hippocampal neurons from oxygen-glucose deprivation injury

We have previously shown that tissue plasminogen activator (tPA) participates in the neurotoxicity of microglial conditioned medium (MgCM). Killing of hippocampal neurons by MgCM was prevented by both plasminogen activator inhibitor-1 (PAI-1) and anti-tPA antibody. An N-methyl-D-aspartate (NMDA) receptor blocker protected neurons from MgCM, suggesting that this subtype of glutamate receptor is involved. Whereas glutamate receptor-mediated events are important in cerebral ischemia and tPA has previously been shown to enhance excitotoxicity in hippocampus, we hypothesized that tPA would exaggerate oxygen glucose deprivation (OGD) injury in cultures of hippocampal neurons. Dissociated rat hippocampal cells were grown under conditions designed to optimize neuronal growth while minimizing glial replication. At 7--10 days, cultures were subjected to OGD for 2.5 hr. Recombinant human tPA (1,000 IU) was added immediately after OGD. Viability was assessed 24 hr later. Viable, apoptotic, and necrotic cells were classified and quantified based on staining patterns of acridine orange and ethidium bromide under fluorescence microscopy. tPA alone did not alter neuronal integrity. OGD produced significant neuronal death (viability reduced by 45%, P < 0.001). tPA completely protected OGD-exposed cultures. Potential mechanisms of tPA protection were explored. Whereas tPA antibody abolished the protective effect of tPA, its proteolytic inhibitor PAI-1 did not alter the effect. The effect of tPA was tested in separate free radical and excitatory amino acid insults. It did not protect neurons from hydrogen peroxide (1 microM), S-nitro-acetylpenicillamine (10 microM), glutamate (50 microM), or NMDA (10 microM) damage but significantly attenuated injury caused by 250 microM kainate. We conclude that tPA is capable of protecting hippocampal neurons from OGD by a nonproteolytic action. The mechanism of protection was not defined, although attenuation of AMPA/kainate glutamate receptors may play a role.     

Calpain activation and Na+/Ca2+ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na⁺/Ca²⁺ exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate‐induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate‐induced jump in cytosolic Ca²⁺, completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate‐treated hippocampal neurons, the initial increase in cytosolic Ca²⁺ that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD. © 2009 Wiley‐Liss, Inc.     

Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations

There is now convincing evidence that excessive accumulation of the excitatory amino acid glutamate (GLU) in the extracellular space is toxic to central mammalian neurons. However, the role of different GLU receptors in producing this toxicity has not been adequately ascertained. There is also no adequate information about the correlation of free intracellular calcium concentration with eventual excitotoxic death. We have used cultured rat hippocampal neurons to address these issues. Approximately 75% of our neurons died after a 20-min GLU exposure. The potent kainate/quisqualate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione did not significantly ameliorate the GLU toxicity, while the selective noncompetitive N-methyl-D-aspartate (NMDA) antagonist methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801) blocked the GLU toxicity for periods of at least 2 hr. Interestingly, kainate was very toxic to the hippocampal neurons, but this toxicity was markedly attenuated by MK-801. These results suggest that the major toxicity of GLU is mediated by NMDA receptors and that under some conditions kainate toxicity reflects nonspecific opening of NMDA channels. The intracellular calcium concentrations in these neurons at the end of exposure to GLU and kainate (in the presence and absence of different antagonists) correlated poorly with eventual survival. Antagonists that limited the rise in calcium were still ineffective in preventing death. These results confirm earlier observations that stressed the importance of NMDA receptors in mediating GLU toxicity. However, they indicate that the relationship between toxicity and neuronal calcium concentration may be very complicated. An unexpected finding of these experiments was that MK-801, unlike competitive antagonists of GLU, elevated intracellular calcium.     

Excitatory amino acid-induced release of 3H-GABA from cultured mouse cerebral cortex interneurons

A newly developed continuous superfusion model was used for studies of 3H-GABA release from cultured mouse cerebral cortex neurons. It was found that a series of excitatory amino acids (EAAs) representing all receptor subtypes evoked Ca2+- dependent release of 3H-GABA from the neurons. Quisqualate was the most potent agonist tested, with an EC50 value of 75 nM. L-Glutamate, N-methyl-D-aspartate (NMDA), and kainate showed EC50 values of 12, 16 and 29 microM, respectively. The EAA-evoked 3H-GABA release could be blocked by a series of EAA antagonists. The highly selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV) was found to block NMDA responses, whereas the nonselective antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and gamma-D-glutamyl-aminomethyl sulphonic acid (GAMS) blocked responses to all agonists. NMDA responses were found to be sensitive to Mg+ blockade. EAA- as well as potassium-induced 3H-GABA release from the neurons could be detected as early as day 5 in culture. However, during the culture period up to 12 d, the responses to K+, quisqualate, and NMDA were increased. The ontogenetic development of binding sites for quisqualate, kainate, and NMDA in mouse cortex was studied using the radioligands 3H-alpha-amino-3-hydroxy-5-methyl-4-isoxasole propionate (3H-AMPA), 3H-kainate, and 3H-L-glutamate, respectively. The development of binding sites for the different EAA-receptor subtypes showed a good correlation with the development of neuronal 3H-GABA release evoked by the excitatory amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia

Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl- -aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.