Apoptotic Cell Death and Caspase-3 Activation Induced by N-Methyl-d-Aspartate Receptor Antagonists and Their Prevention by Insulin-Like Growth Factor I

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.     

Blockade of ionotropic glutamate receptors produces neuronal apoptosis through the Bax-cytochrome C-caspase pathway: the causative role of Ca2+ deficiency

Blockade of ionotropic glutamate receptors induces neuronal cell apoptosis. We investigated if mitochondria-mediated death signals would contribute to neuronal apoptosis following administration of glutamate antagonists. The administration of MK-801 and CNQX (MK-801/CNQX), the selective antagonists of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, produced widespread neuronal death in neonatal rat brain and cortical cell cultures. MK-801/CNQX-induced neuronal apoptosis was prevented by zVAD-fmk, a broad inhibitor of caspases, but insensitive to inhibitors of calpain or cathepsin D. Activation of caspase-3 was observed within 6-12 h and sustained over 36 h after exposure to MK-801/CNQX, which cleaved PHF-1 tau, the substrate for caspase-3. Activation of caspase-3 was blocked by high K+ and mimicked by BAPTA-AM, a selective Ca2+ chelator. Reducing extracellular Ca2+, but not Na+, activated caspase-3, suggesting an essential role of Ca2+ deficiency in MK-801/CNQX-induced activation of caspases. Cortical neurons treated with MK-801/CNQX triggered activation of caspase-9, release of cytochrome c from mitochondria, and translocation of Bax into mitochondria. The present study suggests that blockade of ionotropic glutamate receptors causes caspase-3-mediated neuronal apoptosis due to Ca2+ deficiency that is coupled to the sequential mitochondrial death pathway.     

NMDA-receptor blockade enhances cell apoptosis in the developing retina of the postnatal rat

During visual system development, programmed cell death occurs in order to facilitate the establishment of correct connections and synapses. During this period, glutamate plays a very important role as an excitatory neurotransmitter. With a view to evaluating if NMDA glutamate receptors participate in the regulation of apoptosis which occurs during the development of the rat retina, we subcutaneously injected the NMDA receptor antagonist MK-801 into rats at different stages of early postnatal development (P2 to P9). Ensuing cell death in the retina and superior colliculus was analyzed by using the Feulgen method. MK-801 administration had no effect on the survival of photoreceptor cells. In contrast, the presence of this antagonist induced a significant increase in the number of apoptotic cells in the neuroblastic layer (P7 and P8) and ganglion cell layer (P6-P8), as well as in the superior colliculus which receives afferent contacts from retinal ganglion cells during P7-P9. We conclude that during development, specific types of cells in the mammalian retina are critically dependent for their survival on glutamate stimulation through NMDA receptors. These findings thus throw fresh light on the mechanisms of development of the rat visual system by identifying NMDA glutamate receptors as participants in the regulation of apoptotic processes which occur during the initial stages of development.     

Activation of NMDA receptors induces protein kinase A-mediated phosphorylation and degradation of matrin 3. Blocking these effects prevents NMDA-induced neuronal death

Activation of NMDA receptors leads to activation of cAMP-dependent protein kinase (PKA). The main substrates phosphorylated by PKA following NMDA receptor activation remain unidentified. The aim of this work was to identify a major substrate phosphorylated by PKA following NMDA receptor activation in cerebellar neurones in culture, and to assess whether this phosphorylation may be involved in neuronal death induced by excessive NMDA receptor activation. The main PKA substrate following NMDA receptor activation was identified by MALDI-TOFF fingerprinting as the nuclear protein, matrin 3. PKA-mediated phosphorylation of matrin 3 is followed by its degradation. NMDA receptor activation in rat brain in vivo by ammonia injection also induced PKA-mediated matrin 3 phosphorylation and degradation in brain cell nuclei. Blocking NMDA receptors in brain in vivo with MK-801 reduced basal phosphorylation of matrin 3, suggesting that it is modulated by NMDA receptors. Inhibition of PKA with H-89 prevents NMDA-induced phosphorylation and degradation of matrin 3 as well as neuronal death. These results suggest that PKA-mediated phosphorylation of matrin 3 may serve as a rapid way of transferring information from synapses containing NMDA receptors to neuronal nuclei under physiological conditions, and may contribute to neuronal death under pathological conditions.     

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.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Neurotrophic and neurotoxic effects of zinc on neonatal cortical neurons

Although zinc exerts direct neurotoxic action, this metal is also essential for the activity of numerous biological systems and zinc deficiency has been associated with various pathologies. We investigated the cellular responses and neuronal viability following exposure to different concentrations of zinc in primary cultures of neonatal rat cortical neurons. Higher concentrations of zinc (0.15 and 0.2 mM) triggered excessive zinc influx, glutathione depletion and ATP loss leading to necrotic neuronal death. In contrast, lower concentrations of zinc (0.05 and 0.1 mM) attenuated serum-deprivation induced apoptotic neuronal death. The antiapoptotic action of low amounts of zinc was found both in mixed cultures and neuron-enriched cultures indicating the independence of glial mediator. Neurotrophic action was not accompanied by significant alteration in those cellular responses but required chelatable zinc. The N-methyl-D-aspartate (NMDA) antagonist, MK-801, mimicked the beneficial effect of zinc in protecting neuronal death. Moreover, both MK-801 and zinc eliminated NMDA-induced neuronal injury. The results suggest that zinc is an intrinsic factor for neuron survival and exogenous zinc, in low amounts, is an active neuroprotectant against serum deprivation in part through the antagonism of NMDA receptor activation.     

Effects of NMDA Receptor Blockade During the Early Development Period on the Retest Performance of Adult Wistar Rats in the Elevated Plus Maze

The elevated plus maze (EPM) is an animal model of anxiety used to test the effects of anxioselective drugs. The loss of the anxiolytic effect of drugs during the second exposure to the EPM is called the “one trial tolerance” (OTT) phenomenon. The present study was designed to investigate the relationship between the OTT phenomenon and N-methyl-d-aspartate (NMDA) receptor blockade in the early developmental period of rats. NMDA receptor blockade was accomplished using MK-801 treatment given between postnatal days 20–30. Beginning on postnatal day 20, the rats were subcutaneously injected with MK-801 twice a day at the nape of the neck for a period of 10 days (0.25 mg/kg). Increased open arm exploration was observed in MK-801-treated rats during trial 1 (p = 0.001) and trial 2 (p = 0.003). The rats spent less time in the closed arms as compared to the saline animals in trial 1 (p = 0.006), and this time decreased further in trial 2 (p = 0.02). The fecal boli of the MK-801 group was decreased in trial 1 as compared to the saline group (p = 0.01), but was not significantly different in trial 2 (p = 0.08). In conclusion, NMDA receptor blockade using MK-801 produced an anxiolytic-like effect in trials 1 and 2. Furthermore, OTT was not affected by NMDA receptor blockade.     

Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation

N-Methyl-d-aspartate (NMDA) receptor overactivation has been proposed to induce excitotoxic neuronal death by enhancing membrane phospholipid degradation. In previous studies, we have shown that NMDA releases choline and reduces membrane phosphatidylcholine in vivo. We now observed that glutamate and NMDA induce choline release in primary neuronal cortical cell cultures. This effect is Ca(2+)-dependent and is blocked by MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). In cortical neurons, the NMDA receptor-mediated choline release precedes excitotoxic cell death but not neuronal death induced by either osmotic lysis or serum deprivation. Glutamate, at concentrations that release arachidonic acid, does not release choline in cerebellar granule cells, unless these cells are rendered susceptible to excitotoxic death by energy deprivation. The NMDA-evoked release of choline is not mediated by phospholipases A(2) or C. Moreover, NMDA does not activate phospholipase D in cortical cells. However, NMDA inhibits incorporation of [methyl-(3)H]choline into both membrane phosphatidylcholine and sphingomyelin. These results show that the increase in extracellular choline induced by NMDA receptor activation is directly related with excitotoxic cell death and indicate that choline release is an early event of the excitotoxic process produced by inhibition of phosphatidylcholine synthesis and not by activation of membrane phospholipid degradation.     

Pharmacological analysis of ionotropic glutamate receptor function in neuronal circuits of the zebrafish olfactory bulb

Although synaptic functions of ionotropic glutamate receptors in the olfactory bulb have been studied in vitro, their roles in pattern processing in the intact system remain controversial. We therefore examined the functions of ionotropic glutamate receptors during odor processing in the intact olfactory bulb of zebrafish using pharmacological manipulations. Odor responses of mitral cells and interneurons were recorded by electrophysiology and 2-photon Ca(2+) imaging. The combined blockade of AMPA/kainate and NMDA receptors abolished odor-evoked excitation of mitral cells. The blockade of AMPA/kainate receptors alone, in contrast, increased the mean response of mitral cells and decreased the mean response of interneurons. The blockade of NMDA receptors caused little or no change in the mean responses of mitral cells and interneurons. However, antagonists of both receptor types had diverse effects on the magnitude and time course of individual mitral cell and interneuron responses and, thus, changed spatio-temporal activity patterns across neuronal populations. Oscillatory synchronization was abolished or reduced by AMPA/kainate and NMDA receptor antagonists, respectively. These results indicate that (1) interneuron responses depend mainly on AMPA/kainate receptor input during an odor response, (2) interactions among mitral cells and interneurons regulate the total olfactory bulb output activity, (3) AMPA/kainate receptors participate in the synchronization of odor-dependent neuronal ensembles, and (4) ionotropic glutamate receptor-containing synaptic circuits shape odor-specific patterns of olfactory bulb output activity. These mechanisms are likely to be important for the processing of odor-encoding activity patterns in the olfactory bulb.     

Enhanced expression of RNase L as a novel intracellular signal generated by NMDA receptors in mouse cortical neurons

Recently we showed that the level of mitochondrial mRNA was decreased prior to neuronal death induced by glutamate. As the level of mRNA is regulated by ribonuclease (RNase), we examined RNase activity and its expression in the primary cultures of cortical neurons after glutamate treatment in order to evaluate the involvement of RNase in glutamate-induced neuronal death. A 15-min exposure of the cultures to glutamate at the concentration of 100muM produced marked neuronal damage (more than 70% of total cells) at 24-h post-exposure. Under the experimental conditions used, RNA degradation was definitely observed at a period of 4-12-h post-exposure, a time when no damage was seen in the neurons. Glutamate-induced RNA degradation was completely prevented by the N-methyl-d-aspartic acid (NMDA) receptor channel blocker MK-801 or the NR2B-containing NMDA receptor antagonist ifenprodil. Glutamate exposure produced enhanced expression of RNase L at least 2-12h later, which was absolutely abolished by MK-801. However, no significant change was seen in the level of RNase H1 mRNA at any time point post-glutamate treatment. Immunocytochemical studies revealed that RNase L expressed in response to glutamate was localized within the nucleus, mitochondria, and cytoplasm in the neurons. Taken together, our data suggest that expression of RNase L is a signal generated by NMDA receptor in cortical neurons. RNase L expression and RNA degradation may be events that cause neuronal damage induced by NMDA receptor activation.     

The Effects of N-Methyl-d-Aspartate Receptor Blockade During The Early Neurodevelopmental Period on Emotional Behaviors and Cognitive Functions of Adolescent Wistar Rats

The N-Methyl-d-Aspartate (NMDA) receptor is expressed abundantly in the brain and plays an important role in neuronal development, learning and memory, neurodegenerative diseases, and neurogenesis. In this study, we evaluated the effects of NMDA receptor blockade during the early neurodevelopmental period on exploratory locomotion, anxiety-like behaviors and cognitive functions of adolescent Wistar rats. NMDA receptor hypofunction was induced 7–10 days after birth using MK-801 in rats (0.25 mg/kg twice a day for 4 days via intraperitoneal injection). The open-field (OF), elevated plus maze (EPM) and passive avoidance (PA) tests were used to evaluate exploratory locomotion, anxiety-like behaviors and cognitive functions. In the OF test, MK-801 caused an increase in locomotion behavior (p < 0.01) and in the frequency of rearing (p < 0.05). In the EPM test, MK-801 treatment increased the time spent in the open arms, the number of open arm entries and the amount of head dipping (p < 0.01). MK-801 treatment caused no statistical difference compared to the control group in the PA test (p > 0.05). Chronic NMDA receptor blockade during the critical period of maturation for the glutamatergic brain system (postnatal days 7–10) produces locomotor hyperactivity and decreased anxiety levels, but has no significant main effect on cognitive function during adolescence.     

Low extracellular zinc increases neuronal oxidant production through nadph oxidase and nitric oxide synthase activation

A decrease in zinc (Zn) levels increases the production of cell oxidants, affects the oxidant defense system and triggers oxidant sensitive signals in neuronal cells. However, the underlying mechanisms are still unclear. This work tested the hypothesis that the increase in neuronal oxidants that occurs when cellular Zn decreases is mediated by the activation of the NMDA receptor. Differentiated PC12 cells were cultured in control, Zn-deficient or Zn-repleted media. The incubation in Zn deficient media led to a rapid increase in cellular calcium levels, which was prevented by a NMDA receptor antagonist (MK-801). Cellular calcium accumulation was associated with NADPH oxidase and nitric oxide synthase (NOS) activation, an increase in cell oxidant levels, and an associated activation of a redox-sensitive signal (AP-1). In cells incubated in the Zn deficient medium, NADPH oxidase activation was prevented by MK-801 and by a protein kinase C inhibitor. The rise in cell oxidants was prevented by inhibitors of NADPH oxidase, of the NOS and by MK-801. A similar pattern of inhibitor action was observed for zinc deficiency-induced AP-1 activation. Results demonstrate that a decrease in extracellular Zn leads to an increase in neuronal oxidants through the activation of the NMDAR that leads to calcium influx and to a calcium-mediated activation of protein kinase C/NADPH oxidase and NOS. Changes in extracellular Zn concentrations can be sensed by neurons, which using reactive oxygen and nitrogen species as second messengers, can regulate signaling involved in neuronal development and function.     

NMDA receptor activation modulates programmed cell death during early post-natal retinal development: a BDNF-dependent mechanism

Glutamate is a classical excitotoxin of the central nervous system (CNS), but extensive work demonstrates neuroprotective roles of this neurotransmitter in developing CNS. Mechanisms of glutamate-mediated neuroprotection are still under scrutiny. In this study, we investigated mediators of glutamate-induced neuroprotection, and tested whether this neurotransmitter controls programmed cell death in the developing retina. The protective effect of N-methyl-d-aspartate (NMDA) upon differentiating cells of retinal explants was completely blocked by a neutralizing antibody to brain-derived neurotrophic factor (BDNF), but not by an antibody to neurotrophin-4 (NT-4). Consistently, chronic activation of NMDA receptor increased the expression of BDNF and trkB mRNA, as well as BDNF protein content, but did not change the content of NT-4 mRNA in retinal tissue. Furthermore, we showed that in vivo inactivation of NMDA receptor by intraperitoneal injections of MK-801 increased natural cell death of specific cell populations of the post-natal retina. Our results show that chronic activation of NMDA receptors in vitro induces a BDNF-dependent neuroprotective state in differentiating retinal cells, and that NMDA receptor activation controls programmed cell death of developing retinal neurons in vivo.     

NMDA receptor blockade attenuates CCK-induced reduction of real feeding but not sham feeding

Systemic injection of MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor ion channels, increases meal size and delays satiation. We examined whether MK-801 increases food intake by directly interfering with actions of cholecystokinin (CCK). Prior administration of MK-801 (100 microg/kg ip) reversed the inhibitory effects of CCK-8 (2 and 4 microg/kg ip) on real feeding of both liquid and solid foods. MK-801 alone did not alter 30-min sham intake of 15% sucrose compared with intake after saline. Furthermore, while CCK-8 (2 or 4 microg/kg ip) reduced sham intake, this reduction was not attenuated by MK-801 pretreatment. To ascertain whether MK-801 attenuation of CCK-induced reduction of real feeding was associated with attenuated inhibition of gastric emptying, we tested the effect of MK-801 pretreatment on CCK-induced inhibition of gastric emptying of 5-ml saline loads. Ten-minute gastric emptying was accelerated after MK-801 (3.9 +/- 0.2 ml) compared with saline vehicle (2.72 +/- 0.2 ml). CCK-8 (0.5 microg/kg ip) reduced 10-min emptying to 1.36 +/- 0.3 ml. Pretreatment with MK-801 did not significantly attenuate CCK-8-induced reduction of gastric emptying (0.9 +/- 0.4 ml). This series of experiments demonstrates that blockade of NMDA ion channels reverses inhibition of real feeding by CCK. However, neither inhibition of sham feeding nor inhibition of gastric emptying by CCK is attenuated by MK-801. Therefore, increased food intake after NMDA receptor blockade is not caused by a direct interference with CCK-induced satiation. Rather, increased real feeding, either in the presence or absence of CCK, depends on blockade of NMDA receptor participation in other post-oral feedback signals such as gastric sensation or gastric tone.     

NMDA receptor antagonism produces antinociception which is partially mediated by brain opioids and dopamine.

Inhibition of nitric oxide synthase (NOS) activity results in opioid-mediated supraspinal analgesia in the rat, as indicated by increased reaction time in the hot plate test. It is documented that a relationship exists between NMDA receptor activation and the activity of NOS. The present investigation sought to determine if inactivation of the NMDA receptor produced antinociception of supraspinal origin, as was observed in response to inhibition of NOS, and if this response was mediated by brain opioids, by activation of receptors for the neurotransmitter, dopamine, or both. Administration of MK-801, a non-competitive antagonist of the NMDA receptor, produced significant antinociception as measured by reaction time in the hot plate test of analgesia. Antinociception resulting from treatment with MK-801 appeared to be mediated by brain opioids, as indicated by the ability of the opioid antagonist, naloxone, to partially reverse the effect of MK-801 administration. This analgesic response was also partially diminished by administration of the dopamine D1 receptor antagonist, SCH 23390 and the dopamine D2 receptor antagonist, sulpiride. The analgesia resulting from NMDA receptor antagonism was found to be only partially attributable to dopamine and brain opioids, since co-administration of naloxone and SCH 23390 or naloxone and sulpiride, were unable to completely reverse the antinociceptive response to MK-801. The present findings suggest that inhibition of NMDA receptor activity produces supraspinal analgesia. Furthermore, it appears that antinociception induced by blockade of the NMDA receptor results, at least in part, from activation of endogenous brain opioids and stimulation of D1 and D2 subtypes of the dopamine receptor.     

NMDA channels control meal size via central vagal afferent terminals

The N-methyl-D-aspartate (NMDA) ion channel blocker MK-801 administered systemically or as a nanoliter injection into the nucleus of the solitary tract (NTS), increases meal size. Furthermore, we have observed that ablation of the NTS abolishes increased meal size following systemic injection of dizocilpine (MK-801) and that MK-801-induced increases in intake are attenuated in rats pretreated with capsaicin to destroy small, unmyelinated, primary afferent neurons. These findings led us to hypothesize that NMDA receptors on central vagal afferent terminals or on higher-order NTS neurons innervated by these vagal afferents might mediate increased food intake. To evaluate this hypothesis, we examined 15% sucrose intake after 50-nl MK-801 injections ipsilateral or contralateral to unilateral nodose ganglion removal (ganglionectomy). On the side contralateral to ganglionectomy, vagal afferent terminals would be intact and functional, whereas ipsilateral to ganglionectomy vagal afferent terminals would be absent. Three additional control preparations also were included: 1) sham ganglionectomy and 2) subnodose vagotomy either contralateral or ipsilateral to NTS cannula placement. We found that rats with subnodose vagotomies increased their sucrose intake after injections of MK-801 compared with saline, regardless of whether injections were made contralateral (12.6 +/- 0.2 vs. 9.6 +/- 0.3 ml) or ipsilateral (14.2 +/- 0.6 vs. 9.7 +/- 0.4 ml) to vagotomy. Rats with NTS cannula placements contralateral to nodose ganglionectomy also increased their intake after MK-801 (12.2 +/- 0.9 and 9.2 +/- 1.1 ml for MK-801 and saline, respectively). However, rats with placements ipsilateral to ganglionectomy did not respond to MK-801 (8.0 +/- 0.5 ml) compared with saline (8.3 +/- 0.4 ml). We conclude that central vagal afferent terminals are necessary for increased food intake in response to NMDA ion channel blockade. The function of central vagal afferent processes or the activity of higher-order NTS neurons driven by vagal afferents may be modulated by NMDA receptors to control meal size.     

Influence of cytosolic and mitochondrial Ca2+, ATP,mitochondrial membrane potential,and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 microM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 microM glutamate, 3NP administration resulted in a massive elevation in [Ca(2+)](c) and [Ca(2+)](m), decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca(2+)](c) within the 24h time period examined, nor increase [Ca(2+)](m) within 1h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16h. Calpain and caspase activity were first evident at 24h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8h), and elevated [Ca(2+)](m) as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca(2+)](c), nor with early changes in [Ca(2+)](m), mitochondrial membrane potential, ATP levels, or calpain activity.     

NR2A induction and NMDA receptor-dependent neuronal death by neurotrophin-4/5 in cortical cell culture

We have previously shown that prolonged exposure to neurotrophins induces oxidative neuronal death. In the present study, we further examined the cascades involved in neurotrophin-4/5 (NT-4/5)-induced neuronal death. Exposure of mature cortical cultures for 48 h to NT-4/5 induced neuronal death through TrkB activation. The NT-4/5-induced neuronal death was largely attenuated by addition of MK-801, indicating a critical role for NMDA receptors. Western blots revealed the induction of NR2A by NT-4/5. In addition, levels of phospho-NR2A and 2B increased, suggesting the upregulation of the NMDA receptor function. Whereas glutamate levels in the media changed little, levels of D-serine and L-glycine, co-agonists at NMDA receptors, increased significantly following NT-4/5 treatment. Exposure to NT-4/5 resulted in the activation of Src and extracellular signal-regulated kinase-1/2 (Erk-1/2). Their inhibitors blocked NR2A induction and phosphorylation as well as neuronal death induced by NT-4/5. In addition, Egr-1 was induced in an Src- and Erk-1/2-dependent manner. Anti-sense oligodeoxynucleotides to egr-1 attenuated NR2A induction as well as neuronal death. Although induction of NADPH oxidase and neuronal nitric oxide synthase (nNOS) contributes to NT-4/5-induced neuronal death, inhibition of their activity did not reduce NR2A induction. Conversely, blockade of NMDA receptors did not attenuate induction of NADPH oxidase or nNOS. These results indicate that two events are largely independent of each other. Our results demonstrate that the signaling cascade of TrkB leads to increase in NMDA receptor activity. Whereas this cascade may play an important role in the modulation of NMDA receptors in physiologic conditions, in the context of TrkB overactivation, it may contribute to neuronal death.