Neuronal Intracellular Calcium Research Articles

Leaky RyR2 channels unleash a brainstem spreading depolarization mechanism of sudden cardiac death

Cardiorespiratory failure is the most common cause of sudden unexplained death in epilepsy (SUDEP). Genetic autopsies have detected "leaky" gain-of-function mutations in the ryanodine receptor-2 (RyR2) gene in both SUDEP and sudden cardiac death cases linked to catecholaminergic polymorphic ventricular tachycardia that feature lethal cardiac arrhythmias without structural abnormality. Here we find that a human leaky RyR2 mutation, R176Q (RQ), alters neurotransmitter release probability in mice and significantly lowers the threshold for spreading depolarization (SD) in dorsal medulla, leading to cardiorespiratory collapse. Rare episodes of sinus bradycardia, spontaneous seizure, and sudden death were detected in RQ/+ mutant mice in vivo; however, when provoked, cortical seizures frequently led to apneas, brainstem SD, cardiorespiratory failure, and death. In vitro studies revealed that the RQ mutation selectively strengthened excitatory, but not inhibitory, synapses and facilitated SD in both the neocortex as well as brainstem dorsal medulla autonomic microcircuits. These data link defects in neuronal intracellular calcium homeostasis to the vulnerability of central autonomic brainstem pathways to hypoxic stress and implicate brainstem SD as a previously unrecognized site and mechanism contributing to premature death in individuals with leaky RYR2 mutations.

5α-reduced progestogens ameliorate mood-related behavioral pathology, neurotoxicity, and microgliosis associated with exposure to HIV-1 Tat

Human immunodeficiency virus (HIV) is associated with motor and mood disorders, likely influenced by reactive microgliosis and subsequent neural damage. We have recapitulated aspects of this pathology in mice that conditionally express the neurotoxic HIV-1 regulatory protein, trans-activator of transcription (Tat). Progestogens may attenuate Tat-related behavioral impairments and reduce neurotoxicity in vitro, perhaps via progesterone’s 5α-reductase-dependent metabolism to the neuroprotective steroid, allopregnanolone. To test this, ovariectomized female mice that conditionally expressed (or did not express) central HIV-1 Tat were administered vehicle or progesterone (4mg/kg), with or without pretreatment of a 5α-reductase inhibitor (finasteride, 50mg/kg). Tat induction significantly increased anxiety-like behavior in an open field, elevated plus maze and a marble burying task concomitant with elevated protein oxidation in striatum. Progesterone administration attenuated anxiety-like effects in the open field and elevated plus maze, but not in conjunction with finasteride pretreatment. Progesterone also attenuated Tat-promoted protein oxidation in striatum, independent of finasteride pretreatment. Concurrent experiments in vitro revealed Tat (50nM)-mediated reductions in neuronal cell survival over 60h, as well as increased neuronal and microglial intracellular calcium, as assessed via fura-2 AM fluorescence. Co-treatment with allopregnanolone (100nM) attenuated neuronal death in time-lapse imaging and blocked the Tat-induced exacerbation of intracellular calcium in neurons and microglia. Lastly, neuronal-glial co-cultures were labeled for Iba-1 to reveal that Tat increased microglial numbers in vitro and co-treatment with allopregnanolone attenuated this effect. Together, these data support the notion that 5α-reduced pregnane steroids exert protection over the neurotoxic effects of HIV-1 Tat.

Effects of Leptin on Na+/Ca2+ Exchanger in PC12 Cells

Background/Aims: Alzheimer's disease (AD) is known to be related to alterations in neuronal intracellular calcium activity ([Ca²⁺]_i). The present study revealed the distinct role of leptin in Na⁺/Ca²⁺-exchanger activity. Methods: [Ca²⁺]_i was determined utilizing Fura-2 fluorescence. The activity of NCX was measured by removal of extracellular Na⁺ in the presence of external Ca²⁺. Na⁺/Ca²⁺-exchanger activity was further quantified from whole cell currents following removal of extracellular Na⁺. Na⁺/Ca²⁺-exchanger isoform NCX1 transcript levels and protein abundance were quantified by RT-PCR and Western blotting, respectively. Results: Exposure of PC12 cells to 30 µM amyloid (Aβ₄₂) increased [Ca²⁺]_i, an effect significantly blunted by 6 hours incubation with leptin before Aβ₄₂ treatment. Moreover, leptin treatment significantly increased Na⁺/Ca²⁺-exchanger mediated Ca+ transport and current, NCX1 transcript level as well as NCX1 membrane protein abundance. Conclusion: We show that leptin blunts Aβ₄₂-evoked [Ca²⁺]_i increase by increasing expression and activity of Na⁺/Ca²⁺-exchanger NCX1.

Peripheral neuron plasticity is enhanced by brief electrical stimulation and overrides attenuated regrowth in experimental diabetes

Peripheral nerve regrowth is less robust than commonly assumed, particularly when it accompanies common clinical scenarios such as diabetes mellitus. Brief extracellular electrical stimulation (ES) facilitates the regeneration of peripheral nerves in part through early activation of the conditioning injury response and BDNF. Here, we explored intrinsic neuronal responses to ES to identify whether ES might impact experimental diabetes, where regeneration is attenuated. ES altered several regeneration related molecules including rises in tubulin, Shh (Sonic hedgehog) and GAP43 mRNAs. ES was associated with rises in neuronal intracellular calcium but its strict linkage to regrowth was not confirmed. In contrast, we identified PI3K–PTEN involvement, an association previously linked to diabetic regenerative impairment. Following ES there were declines in PTEN protein and mRNA both in vitro and in vivo and a PI3K inhibitor blocked its action. In vitro, isolated diabetic neurons were capable of mounting robust responsiveness to ES. In vivo, ES improved electrophysiological and behavioral indices of nerve regrowth in a chronic diabetic model of mice with pre-existing neuropathy. Regrowth of myelinated axons and reinnervation of the epidermis were greater following ES than sham stimulation. Taken together, these findings identify a role for ES in supporting regeneration during the challenges of diabetes mellitus.

PBT2 inhibits glutamate-induced excitotoxicity in neurons through metal-mediated preconditioning

Excitotoxicity is the pathological process by which neuronal death occurs as a result of excessive stimulation of receptors at the excitatory synapse such as the NMDA receptor (NMDAR). Excitotoxicity has been implicated in the acute neurological damage from ischemia and traumatic brain injury and in the chronic neurodegeneration in Alzheimer's disease (AD) and Huntington's disease (HD). As a result NMDAR antagonists have become an attractive therapeutic strategy for the potential treatment of multiple neurodegenerative diseases. However NMDAR signaling is dichotomous in nature, with excessive increases in neuronal intracellular calcium through excessive NMDAR activity being lethal but moderate increases to intracellular calcium levels during normal synaptic function providing neuroprotection. Subsequently indiscriminant inhibition of this receptor is best avoided as was concluded from previous clinical trials of NMDAR antagonists. We show that the metal chaperone, PBT2, currently in clinical trials for HD, is able to protect against glutamate-induced excitotoxicity mediated through NMDARs. This was achieved by PBT2 inducing Zn2+-dependent increases in intracellular Ca2+ levels resulting in preconditioning of neurons and inhibition of Ca2+-induced neurotoxic signaling cascade involving calpain-activated cleavage of calcineurin. Our study demonstrates that modulating intracellular Ca2+ levels by a zinc ionophore is a valid therapeutic strategy to protect against the effects of excitotoxicity thought to underlie both acute and chronic neurodegenerative diseases.

Role of hippocampal neuronal intracellular calcium overload in modulating cognitive dysfunction and the neuronprotective effect of mematine in a mouse model of chronic intermittent hypoxia

To investigate the role of hippocampal intracellular calcium overload in modulating cognitive dysfunction and the neuronprotective effect of mematine in a mouse model of chronic intermittent hypoxia. 45 ICR male mice were randomly divided into 3 groups: the unhandled control group (UC group, n = 15), the chronic intermittent hypoxia (CIH group, n = 15) and the pretreatment memantine group (MEM group, n = 15). CIH and MEM mice were subjected to intermittent hypoxia while UC mice to room air for 8 h per day during 4 weeks. Mice in the MEM group were pretreated with memantine (5 mg/kg) by intraperitoneal injection before the cycle started, and those in the UC group and the CIH group were treated with same volume of physiological saline. Neurobehavioral assessments were performed by Open filed and Morris water maze, [Ca²⁺]i in hippocampal neurons was evaluate by flow cytometry, and the expression of cleaved caspase-3, phospho-ERK1/2 in hippocampus were detected by Western blotting. Compared with the UC group, CIH mice displayed markedly more locomotor activity (P < 0.05) in Open filed test, longer mean escape latency (P < 0.05), less number of times of crossing the platform (P < 0.01) and less percentage of time in target quadrant (P < 0.01). Furthermore, exposure to CIH enhanced [Ca²⁺]i (vs. UC mice, 155 ± 12 vs. 92 ± 8, P < 0.01), and up-regulated the expression of cleaved caspase-3 (P < 0.01), but down-regulated the level of phospho-ERK1/2 (P < 0.05) in the hippocampus. Pre-treatment with memantine significantly decreased hyperlocomotion (P < 0.05), attenuated memory deficit (P < 0.05), mitigated [Ca²⁺]i (vs. CIH mice, 90 ± 8 vs. 155 ± 12, P < 0.01), decrease the expression of cleaved caspase-3 (P < 0.01), but increased the level of phospho-ERK1/2(P < 0.05) comparing to the CIH group. The neurobehavioral impairments induced by CIH are mediated, at least in part, by intracellular calcium concentration overload, neuron apoptosis, dephosphorylation of ERK1/2, which can be attenuated by memantine. Memantine may have a therapeutic effect in the neurocognitive impairment associated with OSAHS.

Establishment of SD neonatal rat hippocampal neuron models with human cytomegalovirus infection in vitro and preliminary study on calcium metabolism

Objective To investigate the influence of primary cultured neonatal rat hippocampal neurons caused by human cytomegalovirus (HCMV AD169)infection on intracellular calcium and its mechanism. Methods Twenty SPF SD rats born within 24 hours(10 cases of male and 10 cases of female) were assigned to establish the primary rat hippocampal neuronal monolayer cells; After cultured 8 days in vitro, the eligible cells were randomly divided into HCMV infection group, HCMV+ MK-801 group, MK-801 group and control group, with 10 wells in each group.The fluorescence intensity values of the intracellular free calcium were detected after 24 hours of treatment with Fluo-3AM fluorescence staining. Results Inoculation of HCMV neurons after 24 h turned to round and swollen gradually, and 4 days later, most of the cells disappeared; by immunohistochemistry in cultures of hippocampal neurons in HCMV, visible early proteins, brownish yellow granules, hematoxylin were found after being stained with brown pigment.The fluorescence intensity values of neuronal intracellular calcium (215.5±14.9) in HCMV group was higher than that of control group(116.4±5.9) (t=15.2, P<0.01), whilerise, that in MK-801 group (88.1±4.5) was significantly lower than that of control group, with decreased rate of (24.0±6.7)%(t=-9.3, P<0.01). The fluorescence intensity values of neuronal intracellular calcium in HCMV+ MK-801 group (135.5±8.6) was significantly decreased compared with that of HCMV group(215.5±14.9), with decreased rate of (37.0±3.4)%(t=11.3, P<0.01). Conclusions Intracellular calcium overload of cultured rat hippocampal neurons in vitro with HCMV AD16 strains infection can be detected.One of its main mechanisms is the N-methyl-D-aspartic acid receptor channel-mediated calcium influx. Key words: Laser scanning confocal microscope; Human cytomegalovirus; Rat neuron; Cell model; Calcium overload

Changes in Neuronal Excitability and Intracellular Calcium Concentration by Reactive Oxygen Species on Spinal Substantia Gelatinosa Neurons

Changes in Neuronal Excitability and Intracellular Calcium Concentration by Reactive Oxygen Species on Spinal Substantia Gelatinosa Neurons

Activation of N-Methyl-d-aspartate (NMDA) Receptors in the Dorsal Vagal Complex Lowers Glucose Production

Diabetes is characterized by hyperglycemia due partly to increased hepatic glucose production. The hypothalamus regulates hepatic glucose production in rodents. However, it is currently unknown whether other regions of the brain are sufficient in glucose production regulation. The N-methyl-D-aspartate (NMDA) receptor is composed of NR1 and NR2 subunits, which are activated by co-agonist glycine and glutamate or aspartate, respectively. Here we report that direct administration of either co-agonist glycine or NMDA into the dorsal vagal complex (DVC), targeting the nucleus of the solitary tract, lowered glucose production in vivo. Direct infusion of the NMDA receptor blocker MK-801 into the DVC negated the metabolic effect of glycine. To evaluate whether NR1 subunit of the NMDA receptor mediates the effect of glycine, NR1 in the DVC was inhibited by DVC NR1 antagonist 7-chlorokynurenic acid or DVC shRNA-NR1. Pharmacological and molecular inhibition of DVC NR1 negated the metabolic effect of glycine. To evaluate whether the NMDA receptors mediate the effects of NR2 agonist NMDA, DVC NMDA receptors were inhibited by antagonist D-2-amino-5-phosphonovaleric acid (D-APV). DVC D-APV fully negated the ability of DVC NMDA to lower glucose production. Finally, hepatic vagotomy negated the DVC glycine ability to lower glucose production. These findings demonstrate that activation of NR1 and NR2 subunits of the NMDA receptors in the DVC is sufficient to trigger a brain-liver axis to lower glucose production, and suggest that DVC NMDA receptors serve as a therapeutic target for diabetes and obesity.

Are Patients with Darier and Haily-Haily Diseases Susceptible to Alzheimer's Disease? A Theory Based on Abnormal Intraneuronal Ca2+ Homeostasis

Calcium is one of the most important intracellular messengers in human brain. Studies show that there is a relationship between altered Ca2+-homeostasis, especially elevation of intracellular calcium, and formation of the hallmark pathological lesions of Alzheimer's disease. Additionally, Ca2+ is crucial for normal function of the skin, and an abnormal rise in intracellular Ca2+ can consequently lead to the development of two skin disorders: Darier and Hailey-Hailey diseases. As these mutated genes are also highly expressed in the brain and these patients are reported to experience some neuropsychiatric problems, we have hypothesized that patients with these dermatologic diseases may be more prone to the development of Alzheimer's disease. Further investigation may give us clues to find novel therapeutic targets and agents for modulation of intracellular calcium in neurons.

High Concentrations of Morphine Sensitize and Activate Mouse Dorsal Root Ganglia via TRPV1 and TRPA1 Receptors

BackgroundMorphine and its derivatives are key drugs in pain control. Despite its well-known analgesic properties morphine at high concentrations may be proalgesic. Particularly, short-lasting painful sensations have been reported upon dermal application of morphine. To study a possible involvement of TRP receptors in the pro-nociceptive effects of morphine (0.3 – 10 mM), two models of nociception were employed using C57BL/6 mice and genetically related TRPV1 and TRPA1 knockout animals, which were crossed and generated double knockouts. Hindpaw skin flaps were used to investigate the release of calcitonin gene-related peptide indicative of nociceptive activation.ResultsMorphine induced release of calcitonin gene-related peptide and sensitized the release evoked by heat or the TRPA1 agonist acrolein. Morphine activated HEK293t cells transfected with TRPV1 or TRPA1. Activation of C57BL/6 mouse dorsal root ganglion neurons in culture was investigated with calcium imaging. Morphine induced a dose-dependent rise in intracellular calcium in neurons from wild-type animals. In neurons from TRPV1 and TRPA1 knockout animals activation by morphine was markedly reduced, in the TRPV1/A1 double knockout animals this morphine effect was abrogated. Naloxone induced an increase in calcium levels similar to morphine. The responses to both morphine and naloxone were sensitized by bradykinin.ConclusionNociceptor activation and sensitization by morphine is conveyed by TRPV1 and TRPA1.

Initiation and propagation of a neuronal intracellular calcium wave

The ability to image calcium movement within individual neurons inspires questions of functionality including whether calcium entry into the nucleus is related to genetic regulation for phenomena such as long term potentiation. Calcium waves have been initiated in hippocampal pyramidal cells with glutmatergic signals both in the presence and absence of back propagating action potentials (BPAPs). The dendritic sites of initiation of these calcium waves within about 100 microm of the soma are thought to be localized near oblique junctions. Stimulation of synapses on oblique dendrites leads to production of inositol 1,4,5-trisphosphate (IP(3)) which diffuses to the apical dendrite igniting awaiting IP(3) receptors (IP(3)Rs) and initiating and propagating catalytic calcium release from the endoplasmic reticulum. We construct a reduced mathematical system which accounts for calcium wave initiation and propagation due to elevated IP(3). Inhomogeneity in IP(3) distribution is responsible for calcium wave initiation versus subthreshold or spatially uniform suprathreshold activation. However, the likelihood that a calcium wave is initiated does not necessarily increase with more calcium entering from BPAPs. For low transient synaptic stimuli, timing between IP(3) generation and BPAPs is critical for calcium wave initiation. We also show that inhomogeneity in IP(3)R density can account for calcium wave directionality. Simulating somatic muscarinic receptor production of IP(3), we can account for the critical difference between calcium wave entry into the soma and failure to do so.

Proteomic Analysis of Mouse Brain Microsomes: Identification and Bioinformatic Characterization of Endoplasmic Reticulum Proteins in the Mammalian Central Nervous System

The endoplasmic reticulum (ER) is the main source for the storage and release of intracellular calcium in neurons and, thus, contributes to the functionality of a diverse set of pathways that control critical aspects of central nervous system function including but not limited to gene expression, neurotransmission, learning, and memory. ER-derived proteins obtained after subcellular fractionation of mouse brain homogenate were digested with trypsin and the corresponding peptides fractionated by strong cation exchange chromatography followed by LC-MS/MS analysis on a hybrid linear ion trap--Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. A comprehensive catalogue representing 1914 proteins was generated from this particular proteomic analysis using identification criteria that corresponded to a false positive identification rate of 0.4%. Various molecular functions and biological processes relevant to the ER were identified upon gene ontology (GO)-based analysis including pathways associated with molecular transport, protein trafficking and localization, and cell signaling. Comparison of the 2D-LC-MS/MS results with those obtained from shotgun LC-MS/MS analyses demonstrated that most molecular functions and biological processes were represented via GO analysis using either methodology. Results from this comparison as well as a focused investigation into components of calcium-mediated signaling in the mouse brain ER are also presented.

NCX3 knockout mice exhibit increased hippocampal CA1 and CA2 neuronal damage compared to wild-type mice following global cerebral ischemia

There is uncertainty as to whether the plasma membrane Na +/Ca 2+exchanger (NCX) has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue we compared hippocampal neuronal injury in NCX3 knockout mice ( Ncx3 -/- ) and wild-type mice ( Ncx3 +/+ ) following global cerebral ischemia. Using a bilateral common carotid artery occlusion (BCCAO) model of global ischemia we subjected NCX3 knockout and wild-type mice to 17 and 15 minutes of ischemia. Following the 17 minute period of ischemia, wild-type mice exhibited ≈ 80% CA1 neuronal loss and ≈ 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed > 95% CA1 neuronal loss and ≈ 95% CA2 neuronal loss. Following the 15 minute period of ischemia, wild-type mice did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed ≈ 45% CA1 neuronal loss and ≈ 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient in the NCX3 protein are more susceptible to global cerebral ischemia than wild-type mice. Our findings suggest NCX3 has a positive role in maintaining neuronal intracellular calcium homeostasis following ischemia, and that when exchanger function is compromised neurons are more susceptible to calcium deregulation and cell death.

Estrogen evokes a rapid effect on intracellular calcium in neurons characterized by calcium oscillations in the arcuate nucleus

Rapid estrogen effects became an interesting topic to explain estrogen effects not associated with the classical nuclear pathway. The rapid estrogen effect on intracellular calcium oscillations was characterized in neurons of the arcuate nucleus. Ratiometric calcium imaging (fura-2AM) was used to measure intracellular calcium in brain slices of female Swiss Webster mice (median of age 27 days p.n.). Calcium oscillations were dependent on intracellular calcium and also on calcium influx from the extracellular space. The perfusion of slices with calcium-free solution inhibited spontaneous calcium oscillations. The metabotropic glutamate receptor agonist t-ACPD (5 microM) and low concentrated ryanodine (100 nM) induced intracellular calcium release when slices were perfused with calcium-free solution. 17beta-estradiol (10 nM) also induced intracellular calcium release in calcium-free ACSF. This effect was inhibited by the preceding administration of thapsigargin (2 microM) indicating the association of the rapid estrogen effect with intracellular calcium stores. The administration of the non-selective phospholipase C-inhibitor ET-18 (30 microM), but not U73122 (10 microM), and the inhibition of protein kinase A by H-89 (0.25 microM) suppressed the rapid estrogen effect. Analyses indicated a qualitative, but not quantitatively significant effect of 17beta-estradiol on calcium oscillations.

Characterization of transcriptional regulation of neurogranin by nitric oxide and the role of neurogranin in SNP-induced cell death: implication of neurogranin in an increased neuronal susceptibility to oxidative stress

Neurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca(2+)/CaM complex and modulates the homeostasis of intracellular calcium in neurons. Previous work showed Ng oxidation by NO donor induces increase in [Ca(2+)](i). The current study demonstrated that the gene transcription of Ng could be up-regulated by various nitric oxide (NO) donors via a NO-soluble guanylyl cyclase (sGC)-mediated pathway. Furthermore, ectopic expression of neuronal nitric oxide synthase (nNOS) in human embryonic kidney 293 cells (HEK 293) exhibited a nNOS-concentration-dependent biphasic regulatory effect on Ng gene transcription. One of the NO donors, sodium nitroprusside (SNP), however, induced cell death of neuroblastoma Neuro-2a cells. The potency of SNP-induced cell death was shown to be higher in Neuro-2a cells expressing recombinant Ng, as compared with Neuro-2a control cells without Ng expression in cell viability and apoptosis assays. Single-cell fluorescence imaging and site-directed mutagenesis studies suggest that Ng promotes SNP-induced cell death through an amplification of calcium-mediated signaling, which requires the interaction between CaM and IQ motif of Ng. Increased neuronal susceptibility rendered by Ng in response to pathophysiological NO production is suggested to be involved in the selective vulnerability of neurons to oxidative insults in the CNS.

Reading vascular changes in brain imaging: Is dendritic calcium the key?

A key goal in functional neuroimaging is to track the neuronal correlates of mental activity based on signals related to local changes in metabolism and blood flow. Recent findings indicate that the dendritic processing of excitatory synaptic inputs correlates more closely than the generation of spikes with the brain imaging signals. The correlation is often non-linear and context-sensitive, and cannot be generalized for every condition or brain region. The vascular signals are mainly produced by rises in intracellular calcium in neurons and possibly astrocytes, which activate key enzymes that produce vasodilators to generate increments in flow and, in turn, the positive BOLD (blood-oxygen-level-dependent) signal. Our new knowledge of the cellular mechanisms of functional brain imaging signals place constraints on the interpretation of the data.

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

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

Reading vascular changes in brain imaging: is dendritic calcium the key?

A key goal in functional neuroimaging is to use signals that are related to local changes in metabolism and blood flow to track the neuronal correlates of mental activity. Recent findings indicate that the dendritic processing of excitatory synaptic inputs correlates more closely than the generation of spikes with brain imaging signals. The correlation is often nonlinear and context-sensitive, and cannot be generalized for every condition or brain region. The vascular signals are mainly produced by increases in intracellular calcium in neurons and possibly astrocytes, which activate important enzymes that produce vasodilators to generate increments in flow and the positive blood oxygen level dependent signal. Our understanding of the cellular mechanisms of functional imaging signals places constraints on the interpretation of the data.

Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons

Although large increases in neuronal intracellular calcium concentrations ([Ca 2+] i) are lethal, moderate increases in [Ca 2+] i of 50–200 nM may induce immediate or long-term tolerance of ischemia or other stresses. In neurons in rat hippocampal slice cultures, we determined the relationship between [Ca 2+] i, cell death, and Ca 2+-dependent neuroprotective signals before and after a 45 min period of oxygen and glucose deprivation (OGD). Thirty minutes before OGD, [Ca 2+] i was increased in CA1 neurons by 40–200 nM with 1 nM–1 μM of a Ca 2+-selective ionophore (calcimycin or ionomycin-“Ca 2+ preconditioning”). Ca 2+ preconditioning greatly reduced cell death in CA1, CA3 and dentate during the following 7 days, even though [Ca 2+] i was similar (approximately 2 μM) in preconditioned and control neurons 1 h after the OGD. When pre-OGD [Ca 2+] i was lowered to 25 nM (10 nM ionophore in Ca 2+-free medium) or increased to 8 μM (10 μM ionophore), more than 90% of neurons died. Increased levels of the anti-apoptotic protein protein kinase B (Akt) and the MAP kinase ERK (p42/44) were present in preconditioned slices after OGD. Reducing Ca 2+ influx, inhibiting calmodulin, and preventing Akt or MAP kinase p42/44 upregulation prevented Ca 2+ preconditioning, supporting a specific role for Ca 2+ in the neuroprotective process. Further, in continuously oxygenated cultured hippocampal/cortical neurons, preconditioning for 30 min with 10 nM ionomycin reduced cell death following a 4 μM increase in [Ca 2+] i elicited by 1 μM ionomycin. Thus, a zone of moderately increased [Ca 2+] i before a potentially lethal insult promotes cell survival, uncoupling subsequent large increases in [Ca 2+] i from initiating cell death processes.