Evidence for calcium-reducing and excito-protective roles for the calcium-binding protein calbindin-D28k in cultured hippocampal neurons.

Neuronal systems for calcium homeostasis are crucial for neuronal development and function and may also contribute to selective neuronal vulnerability in adverse conditions such as exposure to excitatory amino acids or anoxia, and in neurodegenerative diseases. Previous work demonstrated the presence and differential distribution of calcium-binding proteins in the CNS. We now report that a subpopulation of neurons in dissociated cell cultures of embryonic rat hippocampus expresses calbindin-D28k (Mr 28,000 calcium-binding protein) immunoreactivity and that these neurons are relatively resistant to neurotoxicity induced by either glutamate or calcium ionophore. Direct comparisons of dynamic aspects of intracellular calcium levels and calbindin-D28k immunoreactivity in the same neurons revealed that calbindin-D28k-positive neurons were better able to reduce free intracellular calcium levels than calbindin-D28k-negative neurons. These findings indicate that the differential expression of calbindin-D28k in hippocampal neurons occurs early in development and may be one determinant of selective neuronal vulnerability to excitotoxic insults.     

Cellular defenses against excitotoxic insults

The cellular events mediating necrotic neuron death are now reasonably well understood, and involve excessive extracellular accumulation of glutamate and free cytosolic calcium. When such necrotic neurological insults occur, neurons are not passively buffeted, but instead mobilize a variety of defenses in an attempt to decrease the likelihood of neuron death, or to decrease the harm to neighboring neurons (by decreasing the likelihood of inflammation). This review considers some of these defenses, organizing them along the lines of those which decrease neuronal excitability, decrease extracellular glutamate accumulation, decrease cytosolic calcium mobilization, decrease calcium-dependent degenerative events, enhance neuronal energetics, and bias a neuron towards apoptotic, rather than necrotic, death. Although these are currently perceived as a disparate array of cellular adaptations, some experimental approaches are suggested that may help form a more unified subdiscipline of cellular defenses against neurological insults. Such an advance would help pave the way for the rational design of therapeutic interventions against necrotic insults.     

Tyrosine phosphatase SHP-2 is a mediator of activity-dependent neuronal excitotoxicity

Calcium influx can promote neuronal differentiation and survival, at least in part by activating Ras and its downstream targets, including the Erk pathway. However, excessive calcium influx can initiate molecular signals leading to neuronal death during excitotoxicity or in neurodegenerative diseases. Here we describe a new signaling pathway associated with calcium influx that contributes to neuronal cell death in cerebellar neurons. Influx of calcium, mediated either by L-type voltage-sensitive calcium channels or glutamate receptors, is associated with the suppression of brain-derived neurotrophic factor (BDNF) activation of Ras and its effectors Erk and Akt. This is the result of enhanced association of the tyrosine phosphatase Shp-2 with TrkB receptors, which inhibits BDNF-induced TrkB autophosphorylation and activation. Deletion of the Shp2 gene in neuronal cultures reverses inhibition of TrkB function and increases neuronal survival after extended depolarization or glutamate treatment. These findings implicate Shp-2 in a feedback system initiated by calcium that negatively regulates neurotrophin signaling and sensitizes neurons to excitotoxicity.     

Metabotropic Glutamate Receptors in Glial Cells

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and exerts its actions via a number of ionotropic glutamate receptors/channels and metabotropic glutamate (mGlu) receptors. In addition to being expressed in neurons, glutamate receptors are expressed in different types of glial cells including astrocytes, oligodendrocytes, and microglia. Astrocytes are now recognized as dynamic signaling elements actively integrating neuronal inputs. Synaptic activity can evoke calcium signals in astrocytes, resulting in the release of gliotransmitters, such as glutamate, ATP, and d-serine, which in turn modulate neuronal excitability and synaptic transmission. In addition, astrocytes, and microglia may play an important role in pathology such as brain trauma and neurodegeneration, limiting or amplifying the pathologic process leading to neuronal death. The present review will focus on recent advances on the role of mGlu receptors expressed in glial cells under physiologic and pathologic conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition induces neurotoxicity via dysregulation of glutamate/calcium signaling and hyperexcitability

Aberrant glutamate and calcium signalings are neurotoxic to specific neuronal populations. Calcium/calmodulin-dependent kinase II (CaMKII), a multifunctional serine/threonine protein kinase in neurons, is believed to regulate neurotransmission and synaptic plasticity in response to calcium signaling produced by neuronal activity. Importantly, several CaMKII substrates control neuronal structure, excitability, and plasticity. Here, we demonstrate that CaMKII inhibition for >4 h using small molecule and peptide inhibitors induces apoptosis in cultured cortical neurons. The neuronal death produced by prolonged CaMKII inhibition is associated with an increase in TUNEL staining and caspase-3 cleavage and is blocked with the translation inhibitor cycloheximide. Thus, this neurotoxicity is consistent with apoptotic mechanisms, a conclusion that is further supported by dysregulated calcium signaling with CaMKII inhibition. CaMKII inhibitory peptides also enhance the number of action potentials generated by a ramp depolarization, suggesting increased neuronal excitability with a loss of CaMKII activity. Extracellular glutamate concentrations are augmented with prolonged inhibition of CaMKII. Enzymatic buffering of extracellular glutamate and antagonism of the NMDA subtype of glutamate receptors prevent the calcium dysregulation and neurotoxicity associated with prolonged CaMKII inhibition. However, in the absence of CaMKII inhibition, elevated glutamate levels do not induce neurotoxicity, suggesting that a combination of CaMKII inhibition and elevated extracellular glutamate levels results in neuronal death. In sum, the loss of CaMKII observed with multiple pathological states in the central nervous system, including epilepsy, brain trauma, and ischemia, likely exacerbates programmed cell death by sensitizing vulnerable neuronal populations to excitotoxic glutamate signaling and inducing an excitotoxic insult itself.     

Transient glucose and amino acid deprivation induces delayed preconditioning in cultured rat cortical neurons

Several studies have demonstrated that glucose deprivation, combined either with anoxia or with the inhibition of oxidative phosphorylation, leads to the development of ischemic tolerance in neurons. The aim of our experiments was to investigate whether similar effects could be achieved by transient energy deprivation without either anoxia or the inhibition of the electron transfer chain. Preconditioning was carried out by incubating primary rat cortical neuronal cultures for 3, 6 or 9 h in a glucose- and amino acid-free balanced salt solution supplemented with B27 in normoxic conditions. After 24 h, neuronal cultures were exposed to oxygen-glucose deprivation, glutamate or hydrogen peroxide. Cell viability was measured 24 h after the lethal insults. Potential mechanisms that can influence free radical production were also examined. Energy deprivation protected neuronal cells against lethal stimuli (e.g. cell survival after oxygen-glucose deprivation was 33.1 +/- 0.52% in the untreated group and 80.1 +/- 1.27% in the 9-h energy deprivation group), reduced mitochondrial membrane potential, decreased free radical formation, attenuated the intracellular free calcium surge upon glutamate receptor stimulation, and resulted in an elevated level of GSH. Our findings show that transient energy deprivation induces delayed preconditioning and prevents oxidative injuries and neuronal cell death.     

The influence of the astrocyte field on neuronal dynamics and synchronization

Astrocytes can sense local synaptic release of glutamate by metabotropic glutamate receptors. Receptor activation in turn can mediate transient increases of astrocytic intracellular calcium concentration through inositol 1,4,5-trisphosphate production. Notably, the perturbation of calcium concentration can propagate to other adjacent astrocytes. Astrocytic calcium signaling can therefore be linked to synaptic information transfer between neurons. On the other hand, astrocytes can also modulate neuronal activity by feeding back onto synaptic terminals in a fashion that depends on their intracellular calcium concentration. Thus, astrocytes can also be active partners in neuronal network activity. The aim of our study is to provide a computationally simple network model of mutual neuron–astrocyte interactions, in order to investigate the possible roles of astrocytes in neuronal network dynamics. In particular, we focus on the information entropy of neuronal firing of the whole network, considering how it could be affected by neuron–glial interactions.     

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.     

Neuroprotective immunity: T cell-derived glutamate endows astrocytes with a neuroprotective phenotype

A well-controlled T cell response to CNS injury may result in increased neuronal survival. However, the precise mechanism of T cell-induced neuroprotection is unknown. In this study, we report the unexpected finding that during culture of T cells, high levels of glutamate accumulate, which are efficiently cleared if T cells are cocultured with astrocytes. The T cell-derived glutamate elicits in turn, the release of neuroprotective thiols (cysteine, glutathione, and cysteinyl-glycine) and lactate from astrocytes. Media obtained from astrocytes conditioned in the presence of T cells reduce neuronal apoptosis induced by oxidative stress in primary neuronal cultures from 48 +/- 14 to 9 +/- 4% (p < 0.001). Inhibition of glutamate-dependent signaling during astrocyte-T cell cocultivation by a glutamate uptake inhibitor, l-aspartic acid beta-hydroxamate, abolishes this neuroprotective effect. The ability of astrocytes to clear extracellular glutamate is impaired under conditions of oxidative stress. We demonstrate that T cells, via secreted cytokines, restore glutamate clearance capacity of astrocytes under oxidative conditions. Furthermore, under normoxic conditions, glutamate-buffering capacity of astrocytes is increased upon cocultivation with T cells. It is known that, following CNS injury, astrocytes can respond with beneficial or destructive effects on neurons. However, the context and signaling mechanisms for this dual astrocytic response are unknown. Our results implicate T cells as potential determinants of the context that elicits a protective role for astrocytes in the damaged CNS.     

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.     

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.     

Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Homer proteins, a new member of the postsynaptic scaffolding proteins, regulate glutamatergic signaling and intracellular calcium mobilization in the central nervous system. Here we investigated the effects of down-regulating Homer1b/c, a constitutively expressed long form of Homer proteins, on glutamate excitotoxicity-induced neuronal injury. In our in vitro excitotoxic models, we demonstrated that glutamate insults led to a dose-dependent neuronal injury, which was mediated by the intracellular calcium-dependent reactive oxygen species (ROS) production. We found that down-regulation of Homer1b/c with specific small interfering RNA (siRNA) improved neuronal survival, inhibited intracellular ROS production, and reduced apoptotic cell death after neurotoxicity. Homer1b/c knockdown decreased the intracellular calcium overload through inhibition of the group I metabotropic glutamate receptor (mGluR)/inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) in injured neurons. In addition, Homer1b/c siRNA transfection attenuated the activation of eukaryotic initiation factor 2α (eIF2α), RNA-dependent protein kinase-like ER kinase (PERK) and caspase-12, and inhibited the up-regulation of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) after glutamate treatment. Homer1b/c knockdown also preserved the mitochondrial membrane potential (MMP), reduced cytochrome c (Cyt. c) release, and partly blocked the increase of capase-9 activity and Bax/Bcl-2 ratio. Taken together, these results suggest that down-regulation of Homer1b/c protects cortical neurons against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the inhibition of calcium-dependent ROS production and the preservation of the ER and mitochondrial function.     

Integrating Cytosolic Phospholipase A2 with Oxidative/Nitrosative Signaling Pathways in Neurons: A Novel Therapeutic Strategy for AD

The pathophysiology of Alzheimer's disease (AD) is comprised of complex metabolic abnormalities in different cell types in the brain. To date, there are not yet effective drugs that can completely inhibit the pathophysiological event, and efforts have been devoted to prevent or minimize the progression of this disease. Much attention has focused on studies to understand aberrant functions of the ionotropic glutamate receptors, perturbation of calcium homeostasis, and toxic effects of oligomeric amyloid beta peptides (Aβ) which results in production of reactive oxygen and nitrogen species and signaling pathways, leading to mitochondrial dysfunction and synaptic impairments. Aberrant phospholipase A(2) (PLA(2)) activity has been implicated to play a role in the pathogenesis of many neurodegenerative diseases, including AD. However, mechanisms for their modes of action and their roles in the oxidative and nitrosative signaling pathways have not been firmly established. In this article, we review recent studies providing a metabolic link between cytosolic PLA(2) (cPLA(2)) and neuronal excitation due to stimulation of ionotropic glutamate receptors and toxic Aβ peptides. The requirements for Ca(2+) binding together with its posttranslational modifications by protein kinases and possible by the redox-based S-nitrosylation, provide strong support for a dynamic role of cPLA(2) in serving multiple functions to neurons and glial cells under abnormal physiological and pathological conditions. Therefore, understanding mechanisms for cPLA(2) in the oxidative and nitrosative pathways in neurons will allow the development of novel therapeutic targets to mitigate the detrimental effects of AD.     

Protection by pyruvate against glutamate neurotoxicity is mediated by astrocytes through a glutathione-dependent mechanism

Pyruvate, an endogenous metabolite of glycolysis, is an anti-toxicity agent. Recent studies have suggested possible roles for pyruvate in protecting CNS neurons from excitotoxic and metabolic insults. Utilizing cultures derived from embryonic rat cortex, the studies presented in this paper indicate that an astroglia-mediated mechanism is involved in the neuroprotective effects of pyruvate against glutamate toxicity. Glutamate-induced toxicity could be reversed by pyruvate in a mixed culture of cortex cells. Importantly, in pure neuronal cultures from the same tissue, pyruvate failed to protect against glutamate toxicity. Addition of astroglia to the pure neuronal cultures restores the ability of pyruvate to protect neurons from glutamate-induced toxicity. Our results further suggest that pyruvate can induce glia to up-regulate the synthesis of glutathione (GSH), an antioxidant that protects cells from toxins such as free radicals. Taken together, our data suggest that astroglia in mixed cultures are essential for mediating the effects of pyruvate, revealing a novel mechanism by which pyruvate, an important intermediate of tricarboxylic acid cycle in the body, may act to protect neurons from damage during insults such as brain ischemia.     

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels

Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-β peptide (Aβ) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3?h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aβ-treated microglia (CM-Aβ). The effects of CM-Aβ were mimicked by TNF-α and IL-1β and were abrogated by neutralizing TNF-α with soluble receptor and IL-1β with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aβ made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aβ induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia.     

Effect of Acute Stress on Hippocampal Glutamate Levels and Spectrin Proteolysis in Young and Aged Rats

Abstract: Aging in rats is associated with a loss of hippocampal neurons, which may contribute to age-related cognitive deficits. Several lines of evidence suggest that stress and glucocorticoids may contribute to age-related declines in hippocampal neuronal number. Excitatory amino acids (EAAs) have been implicated in the glucocorticoid endangerment and stress-induced morphological changes of hippocampal neurons of young rats. Previously, we have reported that acute immobilization stress can increase extracellular concentrations of the endogenous excitatory amino acid, glutamate, in the hippocampus. The present study examined the effect of an acute bout of immobilization stress on glutamate levels in the hippocampus and medial prefrontal cortex of young (3–4-month) and aged (22–24-month) Fischer 344 rats. In addition, the effect of stress on spectrin proteolysis in these two brain regions was also examined. Spectrin is a cytoskeleton protein that contributes to neuronal integrity and proteolysis of this protein has been proposed as an important component of EAA-induced neuronal death. There was no difference in basal glutamate levels between young and old rats in the hippocampus or medial prefrontal cortex. During the period of restraint stress a modest increase in glutamate levels in the hippocampus of young and aged rats was observed. After the termination of the stress procedure, hippocampal glutamate concentrations continued to rise in the aged rats, reaching a level approximately five times higher than the young rats, and remained elevated for at least 2 h after the termination of the stress. A similar pattern was also observed in the medial prefrontal cortex with an augmented post-stress-induced glutamate response observed in the aged rats. There was no increase in spectrin proteolysis in the hippocampus or medial prefrontal cortex of young or aged rats after stress or under basal nonstress conditions. The enhanced poststress glutamate response in the aged rats may contribute to the increased sensitivity of aged rats to neurotoxic insults.     

NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglycemic damage by stabilizing calcium homeostasis.

NGF and bFGF have recently been shown to have biological activity in central neurons, but their normal functions and mechanisms of action are unknown. Since central neurons are particularly vulnerable to hypoglycemia that occurs with ischemia or insulin overdose, we tested the hypothesis that growth factors can protect neurons against hypoglycemic damage. NGF and bFGF each prevented glucose deprivation-induced neuronal damage in human cerebral cortical and rat hippocampal cell cultures (EGF was ineffective). Protection was afforded when the growth factors were administered before (NGF and bFGF) or up to 12 hr following (NGF) the onset of hypoglycemia. Direct measurements of intracellular calcium levels and manipulations of calcium influx demonstrated that sustained elevations in intracellular calcium levels mediated the hypoglycemic damage. NGF and bFGF each prevented the hypoglycemia-induced elevations of intracellular calcium. These findings indicate that growth factors can stabilize neuronal calcium homeostasis in central neurons and thereby protect them against environmental insults.     

Oligodendroglial Signal Transduction Systems Are Regulated by Neuronal Contact

Abstract: Previous reports indicate that oligodendrocytes express signaling systems activated by classical neurotransmitters. Several signaling systems linked to mobilization of intracellular calcium have been demonstrated, and some of these are developmentally lost in vitro and in vivo. The experiments described here use oligodendrocyte-neuron cocultures to examine the effects of neuronal contact on the expression of these signaling pathways. Neonatal rat cerebral oligodendrocytes in contact with dorsal root ganglia (DRG) neurites responded to bath application of histamine, ATP, carbachol, glutamate, or bradykinin with increases in intracellular Ca²⁺ concentration. Similar results were obtained in coculture with superior cervical ganglia neurons. Preventing neuronal contact by transection of DRG neurites significantly reduced the percentage of oligodendrocytes responsive to each ligand, with the exception of bradykinin responsiveness, which was unaffected. Oligodendroglia isolated from adult rat spinal cord were also examined for responsiveness to these neuroligands. Few isolated adult oligodendroglia were responsive to these ligands, and coculture with DRG neurons failed to restore responsiveness. Neuroligand responsiveness was not induced in oligodendrocytes maintained 8 days in purified culture before establishment of cocultures. A significant reduction in the number of neuroligand-responsive oligodendroglia was noted for histamine, carbachol, glutamate, and ATP after including tetrodotoxin for the final 6 days of coculture. These results suggest that both neuronal contact and neuronal activity contribute to the maintenance of functional neurotransmitter-activated signaling pathways coupled to mobilization of intracellular calcium in oligodendrocytes.     

Amyloid-β Decreases Nitric Oxide Production in Cultured Retinal Neurons: A Possible Mechanism for Synaptic Dysfunction in Alzheimer’s Disease?

The neurotoxicity of the amyloid-β peptide (Aβ) appears to be, at least in part, related to pathological activation of glutamate receptors by Aβ aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimer’s disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by Aβ₄₂ and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that l-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either Aβ₄₂ (20 μM) or glutamate (1 mM). Surprisingly, at short incubation times both Aβ and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by Aβ and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by Aβ or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by Aβ and glutamate suggests a possible mechanism leading to synaptic failure in AD.