Homeostasis In Hippocampal Neurons Research Articles

DTD1 modulates synaptic efficacy by maintaining D-serine and D-aspartate homeostasis.

D-serine and D-aspartate are involved in N-methyl-D-aspartate receptor (NMDAR)-related physiological and pathological processes. D-aminoacyl-tRNA deacylase 1 (DTD1) may biochemically contribute to D-serine or D-aspartate production. However, it is unclear thus far whether DTD1 regulates D-serine or D-aspartate content in neurobiological processes. In the present research, we found that DTD1 was essential to maintain the D-serine or D-aspartate homeostasis, which was consistent with the phenomenon that DTD1-deficiency resulted in changes in the quantity changes of functional NMDAR subunits in postsynaptic compartments. Moreover, DTD1 played a considerable role in regulating dendritic morphology and synaptic structure. As a consequence, DTD1 affected neurobiological events, including the synaptic strength of the CA3-to-CA1 circuit, dendritic spine density of hippocampal pyramidal neurons, and behavioral performance of mice in the Morris water maze. These findings highlight the important role of DTD1 in synaptic transmission, neuronal morphology, and spatial learning and memory and suggest an undisclosed mechanism of DTD1 that participates the regulation of D-serine or D-aspartate homeostasis in hippocampal neurons.

Early alterations in the MCH system link aberrant neuronal activity and sleep disturbances in a mouse model of Alzheimer’s disease

Early Alzheimer’s disease (AD) is associated with hippocampal hyperactivity and decreased sleep quality. Here we show that homeostatic mechanisms transiently counteract the increased excitatory drive to CA1 neurons in AppNL-G-F mice, but that this mechanism fails in older mice. Spatial transcriptomics analysis identifies Pmch as part of the adaptive response in AppNL-G-F mice. Pmch encodes melanin-concentrating hormone (MCH), which is produced in sleep–active lateral hypothalamic neurons that project to CA1 and modulate memory. We show that MCH downregulates synaptic transmission, modulates firing rate homeostasis in hippocampal neurons and reverses the increased excitatory drive to CA1 neurons in AppNL-G-F mice. AppNL-G-F mice spend less time in rapid eye movement (REM) sleep. AppNL-G-F mice and individuals with AD show progressive changes in morphology of CA1-projecting MCH axons. Our findings identify the MCH system as vulnerable in early AD and suggest that impaired MCH-system function contributes to aberrant excitatory drive and sleep defects, which can compromise hippocampus-dependent functions.

Cigarette smoke triggers calcium overload in mouse hippocampal neurons via the ΔFOSB-CACNA2D1 axis to impair cognitive performance

A growing body of evidence shows that cigarette smoking impairs cognitive performance. The 'Calcium Hypothesis' theory of neuronopathies reveals a critical role of aberrant calcium signaling in compromised cognitive functions. However, the underlying implications of abnormalities in calcium signaling in the neurotoxicity induced by cigarette smoke (CS) have not yet been identified. CACNA2D1, an important auxiliary subunit involved in the composition of voltage-gated calcium channels (VGCCs), was reported to affect the calcium signaling in neurons by facilitating VGCCs-mediated Ca2+ influx. ΔFOSB, an alternatively-spliced product of the Fosb gene, is an activity-dependent transcription factor induced robustly in the brain in response to environmental stimuli such as CS. Interestingly, our preliminary bioinformatics analysis revealed a significant co-expression between ΔFOSB and CACNA2D1 in brain tissues of patients with neurodegenerative diseases characterized by progressive cognitive decline. Therefore, we hypothesized that the activation of the ΔFOSB-CACNA2D1 axis in response to CS exposure might cause dysregulation of calcium homeostasis in hippocampal neurons via VGCCs-mediated Ca2+ influx, thereby contributing to cognitive deficits. To this end, the present study established a CS-induced mouse model of hippocampus-dependent cognitive impairment, in which the activation of the ΔFOSB-CACNA2D1 axis accompanied by severe calcium overload was observed in the mouse hippocampal tissues. More importantly, ΔFOSB knockdown-/overexpression-mediated inactivation/activation of the ΔFOSB-CACNA2D1 axis interdicted/mimicked CS-induced dysregulation of calcium homeostasis followed by severe cellular damage in HT22 mouse hippocampal neurons. Mechanistically speaking, a further ChIP-qPCR assay confirmed the physical interaction between transcription factor ΔFOSB and the Cacna2d1 gene promoter, suggesting a direct transcriptional regulation of the Cacna2d1 gene by ΔFOSB. Overall, our current work aims to deliver a unique insight into the neurotoxic mechanisms induced by CS to explore potential targets for intervention.

Neuroprotective effects of cordycepin inhibit Aβ-induced apoptosis in hippocampal neurons

In Alzheimer’s disease (AD), β-amyloid (Aβ) protein toxicity increases the formation of reactive oxygen species (ROS) and intracellular calcium levels, resulting in neuronal dysfunction, neurodegenerative disorders, and cell death. Cordycepin is a derivative of the nucleoside adenosine; also, it is speculated to exert neuroprotective effects against Aβ-induced oxidative toxicity in hippocampal neurons. In the present study, the fluorescence detection method and whole-cell patch-clamp recordings were used to study the neuroprotective effects against Aβ-induced toxicity in the primary hippocampal cultured neurons. The results revealed that Aβ25–35 toxicity causes increased cellular ROS production and abnormal calcium homeostasis in hippocampal neurons. Moreover, Aβ25–35-induced cytotoxicity led to a series of downstream events, including the activation of acetylcholinesterase, increased p-Tau expression, and increased apoptosis. Cordycepin inhibits ROS production, elevated levels of Ca2+ induced by Aβ25–35, and the activation of acetylcholinesterase; all these are involved in oxidative-induced apoptosis. In addition, it decreases the increased p-Tau expression that plays a key role in the initiation of the AD. Results showed that the anti-apoptotic effects of cordycepin are partially dependent on the activation of adenosine A1 receptor, whereas an antagonist selectively attenuated the neuroprotective effects of cordycepin. Collectively, these results suggest that cordycepin could be a potential future therapeutic agent for neuronal disorders, such as AD.

Role of Cl- -HCO3- exchanger AE3 in intracellular pH homeostasis in cultured murine hippocampal neurons, and in crosstalk to adjacent astrocytes.

A polymorphism of human AE3 is associated with idiopathic generalized epilepsy. Knockout of AE3 in mice lowers the threshold for triggering epileptic seizures. The explanations for these effects are elusive. Comparisons of cells from wild-type vs. AE3-/- mice show that AE3 (present in hippocampal neurons, not astrocytes; mediates HCO3- efflux) enhances intracellular pH (pHi ) recovery (decrease) from alkali loads in neurons and, surprisingly, adjacent astrocytes. During metabolic acidosis (MAc), AE3 speeds initial acidification, but limits the extent of pHi decrease in neurons and astrocytes. AE3 speeds re-alkalization after removal of MAc in neurons and astrocytes, and speeds neuronal pHi recovery from an ammonium prepulse-induced acid load. We propose that neuronal AE3 indirectly increases acid extrusion in (a) neurons via Cl- loading, and (b) astrocytes by somehow enhancing NBCe1 (major acid extruder). The latter would enhance depolarization-induced alkalinization of astrocytes, and extracellular acidification, and thereby reduce susceptibility to epileptic seizures. The anion exchanger AE3, expressed in hippocampal (HC) neurons but not astrocytes, contributes to intracellular pH (pHi ) regulation by facilitating the exchange of extracellular Cl- for intracellular HCO3- . The human AE3 polymorphism A867D is associated with idiopathic generalized epilepsy. Moreover, AE3 knockout (AE3-/- ) mice are more susceptible to epileptic seizure. The mechanism of these effects has been unclear because the starting pHi in AE3-/- and wild-type neurons is indistinguishable. The purpose of the present study was to use AE3-/- mice to investigate the role of AE3 in pHi homeostasis in HC neurons, co-cultured with astrocytes. We find that the presence of AE3 increases the acidification rate constant during pHi recovery from intracellular alkaline loads imposed by reducing [CO2 ]. The presence of AE3 also speeds intracellular acidification during the early phase of metabolic acidosis (MAc), not just in neurons but, surprisingly, in adjacent astrocytes. Additionally, AE3 contributes to braking the decrease in pHi later during MAc in both neurons and astrocytes. Paradoxically, AE3 enhances intracellular re-alkalization after MAc removal in neurons and astrocytes, and pHi recovery from an ammonium prepulse-induced acid load in neurons. The effects of AE3 knockout on astrocytic pHi homeostasis in MAc-related assays require the presence of neurons, and are consistent with the hypothesis that the AE3 knockout reduces functional expression of astrocytic NBCe1. These findings suggest a new type of neuron-astrocyte communication, based on the expression of AE3 in neurons, which could explain how AE3 reduces seizure susceptibility.

Kainate Receptors Coexist in a Functional Complex with KCC2 and Regulate Chloride Homeostasis in Hippocampal Neurons

SUMMARYKCC2 is the neuron-specific K+-Cl− cotransporter required for maintaining low intracellular Cl−, which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.

Disregulation of Calcium Homeostasis Connected with Familial Alzheimer's Disease

Familial Alzheimer's disease (FAD) caused by mutations in presenilin-1 (PS1) gene in approximately 50% of cases and in Amyloid precursor protein (APP) gene in 13 % cases. It was found that FAD genes mutants affect calcium homeostasis in hippocampal neurons by disrupting Ca2+ storage in the lumen of endoplasmic reticulum (ER). In our study we used electrophysiological recordings with a patch-clamp technique in whole-cell mode and calcium imaging with Fura-2AM to study calcium channels activity, ER calcium load and ER calcium leak. We observed different effects of FAD mutated genes expressions on activity of store-operated and L-type voltage-gated calcium channels in mouse hippocampal neurons, mouse neuroblastoma Neuro2a and human neuroblastoma SK-N-SH cell lines. We found that effects of FAD PS1 mutants on calcium channels were driven by misbalance in activity of ER calcium sensors STIM1 and STIM2. The different effects were connected with calcium sensors STIM1 or STIM2 impaired signal transduction from ER to calcium channels in plasmatic membrane (PM) under control of ER calcium levels. The impaired signal transduction was revealed in live confocal imaging experiments. The enzyme activity of PS1 was involved in regulation of store-operated calcium entry in two ways: the activity of channels was depended on PS1 endoproteolysis level and APP cleavage.This work was supported by the program of “Molecular and Cellular Biology” RAS, research grants from the Russian Basic Research Foundation, grant ERA.Net RUS, research grants from the OPTEC LLC and the President of Russia Scholarship.

Presenilin-1 Mutants Connected with Familial Alzheimer's Disease affect Activity of Voltage-Gated Calcium Channels

Familial Alzheimer's disease (FAD) is caused by mutations in presenilin-1 (PS1) gene in approximately 50% of cases. It was found that FAD PS1 mutants disrupt calcium homeostasis in hippocampal neurons disrupting Ca2+ storage in the lumen of endoplasmic reticulum (ER). Recently calcium sensors of ER STIM1 were found to negatively regulate the activity of L-type voltage-gated calcium channels. Therefore it was suggested that FAD PS1 mutants could affect the activity of L-type channels in neurons. To study the activity of voltage-gated calcium channels experiments with a patch-clamp technique in whole-cell mode were performed with human neuroblastoma SK-N-SH cell line transfected with PS1 M146V mutant or PS1 WT. Currents were induced by 10 mV voltage steps per 200 ms from −80 to +40 mV. PS1 M146V mutant expression enchased the amplitude of integral current at positive potentials comparing to cells with expression of PS1 WT and untransfected control cells. Currents were found to be blocked by application 10 uM of nifedipine. Knock-down of STIM1 with shRNA abolished the difference between cells with mutant and PS1 WT expressions but in the same time mock shRNA left the difference unchanged. Knock-down of STIM1 was controlled by western-blot of cell lysates. Expression PS1 M146V enhanced the amplitude of calcium entry in mouse hippocampal neurons induced by depolarization with 140 KCl in calcium imaging experiments with Fura2-AM comparing to PS1 WT expressing cells. It was concluded that PS1 M146V mutant connected with FAD affect activity of L-type calcium channels through the STIM1 calcium sensors.

Disrupting Function of FK506-Binding Protein 1b/12.6 Induces the Ca2+-Dysregulation Aging Phenotype in Hippocampal Neurons

With aging, multiple Ca(2+)-associated electrophysiological processes exhibit increased magnitude in hippocampal pyramidal neurons, including the Ca(2+)-dependent slow afterhyperpolarization (sAHP), L-type voltage-gated Ca(2+) channel (L-VGCC) activity, Ca(2+)-induced Ca(2+) release (CICR) from ryanodine receptors (RyRs), and Ca(2+) transients. This pattern of Ca(2+) dysregulation correlates with reduced neuronal excitability/plasticity and impaired learning/memory and has been proposed to contribute to unhealthy brain aging and Alzheimer's disease. However, little is known about the underlying molecular mechanisms. In cardiomyocytes, FK506-binding protein 1b/12.6 (FKBP1b) binds and stabilizes RyR2 in the closed state, inhibiting RyR-mediated Ca(2+) release. Moreover, we recently found that hippocampal Fkbp1b expression is downregulated, whereas Ryr2 and Frap1/Mtor (mammalian target of rapamycin) expression is upregulated with aging in rats. Here, we tested the hypothesis that disrupting FKBP1b function also destabilizes Ca(2+) homeostasis in hippocampal neurons and is sufficient to induce the aging phenotype of Ca(2+) dysregulation in young animals. Selective knockdown of Fkbp1b with interfering RNA in vitro (96 h) enhanced voltage-gated Ca(2+) current in cultured neurons, whereas in vivo Fkbp1b knockdown by microinjection of viral vector (3-4 weeks) dramatically increased the sAHP in hippocampal slice neurons from young-adult rats. Rapamycin, which displaces FKBP1b from RyRs in myocytes, similarly enhanced VGCC current and the sAHP and also increased CICR. Moreover, FKBP1b knockdown in vivo was associated with upregulation of RyR2 and mTOR protein expression. Thus, disruption of FKBP1b recapitulated much of the Ca(2+)-dysregulation aging phenotype in young rat hippocampus, supporting a novel hypothesis that declining FKBP function plays a major role in unhealthy brain aging.

A bi-directional carboxypeptidase E-driven transport mechanism controls BDNF vesicle homeostasis in hippocampal neurons

Anterograde transport of brain-derived neurotrophic factor (BDNF) vesicles from the soma to neurite terminals is necessary for activity-dependent secretion of BDNF to mediate synaptic plasticity, memory and learning, and retrograde BDNF transport back to the soma for recycling. In our study, overexpression of the cytoplasmic tail of the carboxypeptidase E (CPE) found in BDNF vesicles significantly reduced localization of BDNF in neurites of hippocampal neurons. Live-cell imaging showed that the velocity and distance of movement of fluorescent protein-tagged CPE- or BDNF-containing vesicles were reduced in both directions. In pulldown assays, the CPE tail interacted with dynactin along with kinesin-2 and kinesin-3, and cytoplasmic dynein. Competition assays using a CPE tail peptide verified specific interaction between the CPE tail and dynactin. Thus, the CPE cytoplasmic tail binds dynactin that recruits kinesins or dynein for driving bi-directional transport of BDNF vesicle to maintain vesicle homeostasis and secretion in hippocampal neurons.

Altered Calcium/Calmodulin Kinase II Activity Changes Calcium Homeostasis That Underlies Epileptiform Activity in Hippocampal Neurons in Culture

Epilepsy is characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. A decrease in calcium/calmodulin-dependent protein kinase II (CaMK-II) activity has been shown to occur with the development of SREDs in a hippocampal neuronal culture model of acquired epilepsy, and altered calcium (Ca(2+)) homeostasis has been implicated in the development of SREDs. Using antisense oligonucleotides, this study was conducted to determine whether selective suppression of CaMK-II activity, with subsequent induction of SREDs, was associated with altered Ca(2+) homeostasis in hippocampal neurons in culture. Antisense knockdown resulted in the development of SREDs and a decrease in both immunocytochemical staining and enzyme activity of CaMK-II. Evaluation of [Ca(2+)](i) using Fura indicators revealed that antisense-treated neurons manifested increased basal [Ca(2+)](i), whereas missense-treated neurons showed no change in basal [Ca(2+)](i). Antisense suppression of CaMK-II was also associated with an inability of neurons to restore a Ca(2+) load. Upon removal of oligonucleotide treatment, CaMK-II suppression and Ca(2+) homeostasis recovered to control levels and SREDs were abolished. To our knowledge, the results demonstrate the first evidence that selective suppression of CaMK-II activity results in alterations in Ca(2+) homeostasis and the development of SREDs in hippocampal neurons and suggest that CaMK-II suppression may be causing epileptogenesis by altering Ca(2+) homeostatic mechanisms.

Activity-dependent Expression of Inositol 1,4,5-Trisphosphate Receptor Type 1 in Hippocampal Neurons

There are several lines of evidence showing that synaptic activity regulates the level of expression of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in neurons. In this study, we examined the effect of chronic activity blockade on the localization and level of IP3R1 expression in cultured hippocampal neurons. We found that chronic blockade of NMDA receptors (NMDARs), one of the major Ca(2+)-permeable ion channels, increased the number of neurons that express a high level of IP3R1 without any apparent changes in its intracellular localization. Interestingly, this up-regulation was time-dependent; there was no clear change in IP3R1 expression level up to day 5 of the NMDAR blockade, but expression increased at day 6, and the increased expression level persisted for at least a week. The up-regulation of IP3R1 depended on transcription and protein synthesis and required cAMP-dependent protein kinase activity. Moreover, although most of the control neurons did not respond to the metabotropic glutamate receptor (mGluR) stimulation, the 2-amino-5-phosphonopentanoic acid-treated neurons with high IP3R1 expression became sensitive to mGluR stimulation. Furthermore, we also found that hippocampal neurons transiently overexpressing green fluorescent protein-tagged IP3R1 released Ca2+ in response to mGluR and muscarinic acetylcholine receptor stimulation. These findings suggested that chronic NMDAR blockade increased the IP3R1 expression and enhanced sensitivity to mGluR stimulation. The change in IP3R1 expression level in response to alteration of synaptic activity may be an important determinant of the sensitivity of Ca2+ stores to G-protein-coupled receptor stimulation and would help to maintain intracellular Ca2+ homeostasis in hippocampal neurons.

Peripheral glucose administration stimulates the translocation of GLUT8 glucose transporter to the endoplasmic reticulum in the rat hippocampus.

The expression and localization of glucose transporter isoforms play essential roles in the glucoregulatory activities of the hippocampus and ultimately contribute to cognitive status in physiological and pathophysiological settings. The recently identified glucose transporter GLUT8 is uniquely expressed in neuronal cell bodies in the rat hippocampus and therefore may contribute to hippocampal glucoregulatory activities. We show here that GLUT8 has a novel intracellular distribution in hippocampal neurons and is translocated to intracellular membranes following glucose challenge. Immunoblot analysis revealed that GLUT8 is expressed in high-density microsomes (HDM), suggesting that GLUT8 is associated with intracellular organelles under basal conditions. Immunogold electron microscopic analysis confirmed this observation, in that GLUT8 immunogold particles were associated with the rough endoplasmic reticulum (ER) and cytoplasm. Peripheral glucose administration produced a rapid twofold increase in GLUT8 levels in the HDM fraction while decreasing GLUT8 levels in low-density microsomes. Similarly, peripheral glucose administration significantly increased GLUT8 association with the rough ER in the hippocampus. Conversely, under hyperglycemic/insulinopenic conditions, namely, in streptozotocin (STZ) diabetes, hippocampal GLUT8 protein levels were decreased in the HDM fraction. These results demonstrate that GLUT8 undergoes rapid translocation to the rough ER in the rat hippocampus following peripheral glucose administration, trafficking that is impaired in STZ diabetes, suggesting that insulin serves as a stimulus for GLUT8 translocation in hippocampal neurons. Because glucose is liberated from oligosaccharides during N-linked glycosylation events in the rough ER, we propose that GLUT8 may serve to transport glucose out of the rough ER into the cytosol and in this manner contribute to glucose homeostasis in hippocampal neurons.

Up-regulation of α 1D Ca 2+ channel subunit mRNA expression in the hippocampus of aged F344 rats

There is growing evidence that alterations in calcium (Ca 2+) homeostasis may play a role in processes of brain aging and neurodegeneration. There also is evidence that some of the altered Ca 2+ homeostasis in hippocampal neurons may arise from an increased density of L-type voltage sensitive Ca 2+ channels (L-VSCC). In the present studies, we tested the possibility that previously observed increases in functional L-VSCC with aging might be related to up-regulated gene/mRNA expression for Ca 2+ channel subunits. A significant aging-related increase in mRNA content for the α 1D subunit of the L-type VSCC was observed in hippocampus of aged F344 rats (25 months old) relative to young (4 months old) and middle-aged animals (13 months old), as assessed by both in situ hybridization analyses (densitometry and grain density) and ribonuclease protection assay (RPA). In RPA analyses, the α 1C subunit mRNA also showed a significant increase in 25-month-old rats. No age changes were seen in mRNA for the β 1b subunit of VSCC or for GAPDH, a standard control. The clearest increases in α 1D mRNA expression were observed in subfield CA1, with little or no change seen in dentate gyrus. Although these results alone do not demonstrate that mRNA/gene expression changes contribute directly to changes in functional Ca 2+ channels, they clearly fulfill an important prediction of that hypothesis. Therefore, these studies may have important implications for the role of gene expression in aging-dependent alterations in brain Ca 2+ homeostasis.

Ethanol Effects on Cultured Embryonic Hippocampal Neuronal Calcium Homeostasis Are Altered by Nerve Growth Factor

The neurotoxic effect of acute ethanol treatment (AET) may lead to an alteration in the regulation of calcium (Ca2+) homeostasis in hippocampal neurons. Ca2+ homeostasis could be affected by AET when neurons are at rest or after depolarizing activity during synaptic transmission. It has been shown that nerve growth factor (NGF) can ameloriate some types of neurotoxicity by stabilizing Ca2+ homeostasis. Previously, we observed that ethanol (EtOH) changed unstimulated (basal) and potassium (K+)-stimulated intracellular calcium ([Ca2+]i) in embryonic septohippocampal neurons (Webb et al., Brain Res. 729:176-189, 1996). The purpose of the present study is to determine the effects of NGF and EtOH on neuronal Ca2+ homeostasis in cultured embryonic hippocampal neurons. The hypotheses tested were the following: EtOH alters Ca2+ homeostasis in hippocampal neurons; NGF modulates Ca2+ homeostasis in hippocampal neurons; and NGF treatment alters the effect of EtOH on [Ca2+]i in hippocampal neurons. Our results indicated that hippocampal neuronal cultures treated with EtOH had lower basal [Ca2+]i than untreated neurons. EtOH decreased K+-stimulated (30 mM KCI) changes in [Ca2+]i in a dose-dependent manner. During K+ stimulation, 20 ng/ml of NGF slowed and reduced the increase in [Ca2+]i. Hippocampal neurons treated with NGF increased or did not change basal [Ca2+]i and did not change or increase K+-stimulated [Ca2+]i in response to EtOH. These responses were dose-related and indicated that NGF could alter the response of hippocampal neurons to EtOH. In conclusion, AET results in the alteration of Ca2+ homeostasis in unstimulated and depolarized cultured embryonic hippocampal neurons. NGF and EtOH independently and collectively affected the regulation of Ca2+ homeostasis in this neuronal population. Changes in [Ca2+]i can disrupt normal cellular function and contribute to cell death. Therefore, alteration of Ca2+ homeostasis may be an underlying mechanism involved in EtOH toxicity. NGF may ameliorate the toxic effects of EtOH by regulating Ca2+ homeostasis.

Nitric oxide disrupts Ca2+ homeostasis in hippocampal neurons.

Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca2+ concentration ([Ca2+]i). In [Ca2+]i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S-nitrosocysteine produced a delayed rise in [Ca2+]i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca2+]i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca2+]i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca2+]i fluorimetry experiments, S-nitrosocysteine had no effect on the resting [Ca2+]i or on the recovery kinetics of [Ca2+]i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca2+]i homeostasis in hippocampal neurons by impairing Ca2+ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca2+]i may contribute to the delayed neurotoxicity of nitric oxide.