In vitro hippocampal gamma oscillation power as an index of in vivo CA3 gamma oscillation strength and spatial reference memory.

Neuronal synchronisation at gamma frequencies (30-100 Hz) has been implicated in cognition and memory. Gamma oscillations can be studied in various in vitro models, but their in vivo validity and their relationship with reference memory remains to be proven. By using the natural variation of wild type C57bl/6J mice, we assessed the relationships between reference memory and gamma oscillations recorded in hippocampal area CA3 in vivo and in vitro. Local field potentials (LFPs) were recorded from area CA3 in behaviourally-characterised freely moving mice, after which hippocampal slices were prepared for recordings in vitro of spontaneous gamma oscillations and kainate-induced gamma oscillations in CA3. The gamma-band power of spontaneous oscillations in vitro correlated with that of CA3 LFP oscillations during inactive behavioural states. The gamma-band power of kainate-induced oscillations correlated with the activity-dependent increase in CA3 LFP gamma-band power in vivo. Kainate-induced gamma-band power correlated with Barnes circular platform performance and object location recognition, but not with object novelty recognition. Kainate-induced gamma-band power was larger in mice that recognised the aversive context, but did not correlate with passive avoidance delay. The correlations between behavioural and electrophysiological measures obtained from the same animals show that the gamma-generating capacity of the CA3 network in vitro is a useful index of in vivo gamma strength and supports an important role of CA3 gamma oscillations in spatial reference memory.

Alterations in Ca2+-buffering in prion-null mice: association with reduced afterhyperpolarizations in CA1 hippocampal neurons.

Prion protein (PrP) is a normal component of neurons, which confers susceptibility to prion diseases. Despite its evolutionary conservation, its normal function remains controversial. PrP-deficient (Prnp(0/0)) mice have weaker afterhyperpolarizations (AHPs) in cerebellar and hippocampal neurons. Here we show that the AHP impairment in hippocampal CA1 pyramidal cells is selective for the slow AHP, and is not caused by an impairment of either voltage-gated Ca(2+) channels or Ca(2+)-activated K(+) channels. Instead, Prnp(0/0) neurons have twofold to threefold stronger Ca(2+) buffering and double the Ca(2+) extrusion rate. In Prnp(0/0) neurons thapsigargin abolished the stronger Ca(2+) buffering and extrusion, and thapsigargin or cyclopiazonic acid abolished the weakening of the slow AHPs. These data implicate sarcoplasmic/endoplasmic reticulum calcium ATPase in the enhanced Ca(2+) buffering, and extrusion into the endoplasmic reticulum, which contains substantial amounts of PrP in wild-type mice. Altered Ca(2+) homeostasis can explain several phenotypes identified in Prnp(0/0) mice.

The importance of being subtle: small changes in calcium homeostasis control cognitive decline in normal aging.

Aging is a complex, multifactorial process. One of the features of normal aging of the brain is a decline in cognitive functions and much experimental attention has been devoted to understanding this process. Evidence accumulated in the last decade indicates that such functional changes are not due to gross morphological alterations, but to subtle functional modification of synaptic connectivity and intracellular signalling and metabolism. Such synaptic modifications are compatible with a normal level of activity and allow the maintenance of a certain degree of functional reserve. This is in contrast to the changes in various neurodegenerative diseases, characterized by significant neuronal loss and dramatic and irreversible functional deficit. This whole special issue has been initiated with the intention of focusing on the processes of normal brain aging. In this review, we present data that shows how subtle changes in Ca(2+) homeostasis or in the state of various Ca(2+)-dependent processes or molecules, which occur in aging can have significant functional consequences.

Sphingosine 1-phosphate receptor expression profile and regulation of migration in human thyroid cancer cells.

S1P (sphingosine 1-phosphate) receptor expression and the effects of S1P on migration were studied in one papillary (NPA), two follicular (ML-1, WRO) and two anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in human thyroid cells in primary culture. Additionally, the effects of S1P on proliferation, adhesion and calcium signalling were addressed in ML-1 and FRO cells. All cell types expressed multiple S1P receptors. S1P evoked intracellular calcium signalling in primary cultures, ML-1 cells and FRO cells. Neither proliferation nor migration was affected in primary cultures, whereas S1P partly inhibited proliferation in ML-1 and FRO cells. Low nanomolar concentrations of S1P inhibited migration in FRO, WRO and ARO cells, but stimulated ML-1 cell migration. Consistently, S1P1 and S1P3, which mediate migratory responses, were strongly expressed in ML-1 cells, and S1P2, which inhibits migration, was the dominating receptor in the other cell lines. The migratory effect in ML-1 cells was mediated by G(i) and phosphatidylinositol 3-kinase. Both S1P and the S1P1-specific agonist SEW-2871 induced Akt phosphorylation at Ser473. However, SEW-2871 failed to stimulate migration, whereas the S1P1/S1P3 antagonist VPC 23019 inhibited S1P-induced migration. The results suggest that aberrant S1P receptor expression may enhance thyroid cancer cell migration and thus contribute to the metastatic behaviour of some thyroid tumours.

Ca2+ regulation and gene expression in normal brain aging.

Understanding the cellular mechanisms that characterize the functional changes of the aged brain is an ongoing and formidable challenge for the neuroscience community. Evidence now links changes in Ca(2+) influx and homeostasis with perturbations induced by the aging process in the function of the main intracellular organelles involved in Ca(2+) regulation: the endoplasmic reticulum and mitochondria. New perspectives are also offered by recent gene microarray studies, illustrating the multifactorial nature of the aging process.

Changes in mitochondrial status associated with altered Ca2+ homeostasis in aged cerebellar granule neurons in brain slices.

In the present work, we investigated the relationship between mitochondrial function and Ca2+ homeostasis in brain slices obtained from mice that aged normally. In acute preparations, the cerebellar neurons had similar values for intracellular free Ca2+ ([Ca2+]i) regardless of their age (range, 6 weeks to 24 months). However, compared with the young slices, the aged neurons (20-24 months) showed an enhanced rate of [Ca2+]i increases as a function of the time the slices were maintained in vitro. When slices were stimulated (KCl depolarization), there were significant differences in the patterns of [Ca2+]i signal displayed by the young and old cerebellar granule neurons. More importantly, the aged neurons showed a significant delay in their capacity to recover the resting [Ca2+]i. The relationship between [Ca2+]i and mitochondrial membrane potential was assessed by recording both parameters simultaneously, using fura-2 and rhodamine-123. In both young and aged neurons, the cytosolic [Ca2+]i signal was associated with a mitochondrial depolarization response. In the aged neurons, the mitochondria had a significantly longer repolarization response, and quantitative analysis showed a direct correlation between the delays in mitochondrial repolarization and [Ca2+]i recovery, indicating the causal relationship between the two parameters. Thus, the present results show that the reported changes in Ca2+ homeostasis associated with aging, which manifest principally in a decreased capacity of maintaining a stable resting [Ca2+]i or recovering the resting [Ca2+]i values after stimulation, are primarily attributable to a metabolic dysfunction in which the mitochondrial impairment plays an important role.

Developmental exposure to ethanol increases the neuronal vulnerability to oxygen-glucose deprivation in cerebellar granule cell cultures.

Prenatal alcohol exposure is associated with microencephaly, cognitive and behavioral deficits, and growth retardation. Some of the mechanisms of ethanol-induced injury, such as high level oxidative stress and overexpression of pro-apoptotic genes, can increase the sensitivity of fetal neurons towards hypoxic/ischemic stress associated with normal labor. Thus, alcohol-induced sequelae may be the cumulative result of direct ethanol toxicity and increased neuronal vulnerability towards metabolic stressors, including hypoxia. We examined the effects of ethanol exposure on the fetal cerebellar granular neurons' susceptibility to hypoxic/hypoglycemic damage. A chronic ethanol exposure covered the entire prenatal period and 5 days postpartum through breastfeeding, a time interval partially extending into the third-trimester equivalent in humans. After a binge-like alcohol exposure at postnatal day 5, glutamatergic cerebellar granule neurons were cultured and grown for 7 days in vitro, then exposed to a 3-h oxygen-glucose deprivation to mimic a hypoxic/ischemic condition. Cellular viability was monitored by dynamic recording of propidium iodide fluorescence over 20 h reoxygenation. We explored differentially expressed genes on microarray data from a mouse embryonic ethanol-exposure model and validated these by real-time PCR on the present model. In the ethanol-treated cerebellar granule neurons we find an increased expression of genes related to apoptosis (Mapk8 and Bax), but also of genes previously described as neuroprotective (Dhcr24 and Bdnf), which might suggest an actively maintained viability. Our data suggest that neurons exposed to ethanol during development are more vulnerable to in vitro hypoxia/hypoglycemia and have higher intrinsic death susceptibility than unexposed neurons.

Hypoxia sensing and pathways of cytosolic Ca2+ increases.

Oxygen-sensing and reactivity to changes in the concentration of oxygen is a fundamental property of cellular physiology. This central role is determined, mainly, by, to the fact that oxygen represents the final acceptor of electrons, derived from the normal cellular metabolism, at the end of the mitochondrial respiratory chain. Despite significant advances in molecular characterization of various oxygen-sensitive processes, the nature of the oxygen-sensor molecules and the mechanisms that link sensors to effects remains unclear. One such controversy is about the role and nature of reactive oxygen species (ROS) changes during hypoxia. Irrespective of the mechanisms of oxygen sensing, one of the constant early responses to hypoxia in almost all cell types is an increase in intracellular Ca2+ ([Ca2+]i). In many instances, this increase is mediated by the activation of various plasma membrane Ca2+ conductances. Some of these channels have specific Ca2+ permeability (e.g. voltage-operated Ca2+ channels), whereas others have non-specific cation conductances and are activated by a variety of ligands (ligand-operated channels). In the last decade, a large superfamily of channels with significant Ca2+ permeability has been progressively identified and characterised: the TRP channels. Through their properties, some groups of the TRP channels provide a link to the other hypoxia-activated mechanism of [Ca2+]i increase: the release of Ca2+ from intracellular Ca2+ stores. Since the [Ca2+]i signals, depending on their localization and intensity, are important regulators of the subsequent cellular responses to hypoxia, a deeper understanding of the mechanisms through which hypoxia regulate the activity of these pathways that increase intracellular Ca2+ could point the way towards the development of new therapeutic approaches to reduce or suppress the pathological effects of cellular hypoxia, such as those seen in stroke or myocardial ischemia.

Neuronal ageing from an intraneuronal perspective: roles of endoplasmic reticulum and mitochondria.

The nature of brain ageing and the age-dependent decline in cognitive functions remains poorly understood. Physiological brain ageing is characterised by mild mental dysfunctions, whereas age-dependent neurodegeneration, as illustrated by Alzheimer disease (AD), results rapidly in severe dementia. These two states of the aged brain, the physiological and the pathological, are fundamentally different as the latter stems from significant neuronal loss, whereas the former develops without significant neuronal demise. In this paper, we review the changes in neuronal Ca(2+) homeostasis that occur during brain ageing, and conclude that normal, physiological ageing is characterised mainly by a decrease of neuronal homeostatic reserve, defined as the capacity to respond effectively to functional and metabolic stressors, but does not reach the trigger required to induce neuronal death. In contrast, during neurodegenerative states, Ca(2+) homeostasis is affected early during the pathological process and result in significant neuronal demise. We also review recent evidence suggesting that the endoplasmic reticulum (ER) might play an important role in controlling the balance between healthy and pathological neuronal ageing.

Mitochondria and Ca(2+) signaling.

Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca(2+) signaling. Mitochondria are capable to sense the levels of cytosolic Ca(2+) and generate mitochondrial Ca(2+) responses. Specific mechanisms for both Ca(2+) uptake and Ca(2+) release exist in the mitochondrial membranes. In turn, the mitochondrial Ca(2+) signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca(2+) transport systems and some of the functional consequences of mitochondrial Ca(2+) uptake.

Mitochondrial polarisation status and [Ca2+]i signalling in rat cerebellar granule neurones aged in vitro.

Mitochondrial membrane potential is a major factor that controls, ultimately, the cellular energy supply. By use of a mitochondrial membrane potential dye (rhodamine 123, R123) and image analysis we show that during long-term (>3 weeks) culture of primary neurones (cerebellar granule neurones) there is a gradual and time-dependent depolarisation of neuronal mitochondria. This process was demonstrated by analysing the changes in the heterogeneity of the cytosolic rhodamine 123 fluorescent signal as a function of the age in culture and by measuring the amplitude of the rhodamine 123 fluorescence evoked by the addition of a mitochondrial protonophore (FCCP). The relationship between cytosolic [Ca(2+)](i) and mitochondrial membrane potential was assessed by recording both parameters simultaneously, in neurones loaded with fura-2 and rhodamine 123. Neuronal stimulation (KCl-evoked depolarisation) induced a mitochondrial depolarisation response resulting from the entry of cytosolic Ca(2+) into mitochondria. In young cultures (10 DIV), the mitochondrial membrane potential recovered fully within 30s from the start of the stimulation, despite the continuous presence of the depolarisation stimulus and the maintained cytosolic [Ca(2+)](i) signal. In contrast, in older neurones (DIV 22), the mitochondrial response was of smaller amplitude and displayed a much longer repolarization period. Also, in these older neurones, the threshold [Ca(2+)](i) level required for the initiation of the mitochondrial depolarisation response was increased by 50%. Thus, the present results indicate that neuronal maturation and ageing in conditions of long-term in vitro culture determine significant changes in the mitochondrial polarisation status that are manifest both in resting conditions and during stimulation and could explain some of the reported changes in neuronal homeostasis in long-term neuronal cultures.

Chronic NGF treatment induces somatic hyperexcitability in cultured dorsal root ganglion neurons of the rat.

Adult rat dorsal root ganglion (DRG) neurons cultured in the presence of 100-ng/mL NGF were reported to show spontaneous action potentials in the cell-attached recording. In this study, underlying mechanisms were examined in the whole-cell and outside-out voltage clamp recording. In 75% neurons with on-cell firing, transient inward current spikes were repetitively recorded in the voltage clamp mode at -50 mV in the whole-cell configuration (named "Isp"). Isp with stable amplitudes occurred in an all-or-none fashion, and was abolished by TTX (< 100 nM), lidocaine (< 1 mM) and a reduction of extracellular Na(+) (154 to 100 mM) in an all-or-none fashion, suggesting that Isp reflects spontaneous dicharges occurring at the loosely voltage-clamped regions. Isp was also observed in the excised outside-out patches and the kinetics and the sensitivity to TTX and lidocaine resembled those in the whole-cell. Spontaneous action potentials were also recorded in the current clamp mode. Small subthreshold spikes often preceded the action potentials. When the localized discharge affected a whole-somatic membrane potential to overcome a threshold, the action potential generated. These results indicate that the triggering sources of the action potential exist in the somatic membrane itself in NGF-treated DRG neurons.

Neuroendocrine signalling: natural variations on a Ca2+ theme.

This special issue on Ca(2+) signalling in neuroendocrine cells is an opportunity to assess, through a range of first-class review articles, the complex world of endocrine signalling, a complexity that is probably best captured by calling it "diversity in unity". The unity comes from the fact that all the endocrine cells are excitable cells, able to generate action potentials and are using Ca(2+) as an essential informational molecule, coupling cell stimulation with the activation of secretion, through the exocytotic process. The 'diversity' element, illustrated by almost all the reviews, stems from the modalities employed to achieve the increase in cytosolic Ca(2+) signal, the balance between the participation of Ca(2+) entry through the plasma membrane voltage-operated Ca(2+) channels and the release of Ca(2+) from intracellular Ca(2+) stores, and the cross-talk between the Ca(2+) and cyclic AMP signalling pathways.

The effect of aging-associated impaired mitochondrial status on kainate-evoked hippocampal gamma oscillations.

Oscillations in hippocampal neuronal networks in the gamma frequency band have been implicated in various cognitive tasks and we showed previously that aging reduces the power of such oscillations. Here, using submerged hippocampal slices allowing simultaneous electrophysiological recordings and imaging, we studied the correlation between the kainate-evoked gamma oscillation and mitochondrial activity, as monitored by rhodamine 123. We show that the initiation of kainate-evoked gamma oscillations induces mitochondrial depolarization, indicating a metabolic response. Aging had an opposite effect on these parameters: while depressing the gamma oscillation strength, it increases mitochondrial depolarization. Also, in the aged neurons, kainate induced significantly larger Ca2+ signals. In younger slices, acute mitochondrial depolarization induced by low concentrations of mitochondrial protonophores strongly, but reversibly, inhibits gamma oscillations. These data indicating that the complex network activity required by the maintenance of gamma activity is susceptible to changes and modulations in mitochondrial status.

Segregation of calcium signalling mechanisms in magnocellular neurones and terminals.

Every cell or neuronal type utilizes its own specific organization of its Ca(2+) homeostasis depending on its specific function and its physiological needs. The magnocellular neurones, with their somata situated in the supraoptic and paraventricular nuclei of the hypothalamus and their nerve terminals populating the posterior hypophysis (neural lobe) are a typical and classical example of a neuroendocrine system, and an important experimental model for attempting to understand the characteristics of the neuronal organization of Ca(2+) homeostasis. The magnocellular neurones synthesize, in a cell specific manner, two neurohormones: arginine-vasopressin (AVP) and oxytocin (OT), which can be released, in a strict Ca(2+)-dependent manner, both at the axonal terminals, in the neural lobe, and at the somatodendritic level. The two types of neurones show also distinct type of bioelectrical activity, associated with specific secretory patterns. In these neurones, the Ca(2+) homeostatic pathways such as the Na(+)/Ca(2+) exchanger (NCX), the endoplasmic reticulum (ER) Ca(2+) pump, the plasmalemmal Ca(2+) pump (PMCA) and the mitochondria are acting in a complementary fashion in clearing Ca(2+) loads that follow neuronal stimulation. The somatodendritic AVP and OT release closely correlates with intracellular Ca(2+) dynamics. More importantly, the ER Ca(2+) stores play a major role in Ca(2+) homeostatic mechanism in identified OT neurones. The balance between the Ca(2+) homeostatic systems that are in the supraoptic neurones differ from those active in the terminals, in which mainly Ca(2+) extrusion through the Ca(2+) pump in the plasma membrane and uptake by mitochondria are active. In both AVP and OT nerve terminals, no functional ER Ca(2+) stores can be evidenced experimentally. We conclude that the physiological significance of the complexity of Ca(2+) homeostatic mechanisms in the somatodendritic region of supraoptic neurones and their terminals can be multifaceted, attributable, in major part, to their specialized electrical activity and Ca(2+)-dependent neurohormone release.

Repeated PTZ treatment at 25-day intervals leads to a highly efficient accumulation of doublecortin in the dorsal hippocampus of rats.

BACKGROUND: Neurogenesis persists throughout life in the adult mammalian brain. Because neurogenesis can only be assessed in postmortem tissue, its functional significance remains undetermined, and identifying an in vivo correlate of neurogenesis has become an important goal. By studying pentylenetetrazole-induced brain stimulation in a rat model of kindling we accidentally discovered that 25±1 days periodic stimulation of Sprague-Dawley rats led to a highly efficient increase in seizure susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: By EEG, RT-PCR, western blotting and immunohistochemistry, we show that repeated convulsive seizures with a periodicity of 25±1 days led to an enrichment of newly generated neurons, that were BrdU-positive in the dentate gyrus at day 25±1 post-seizure. At the same time, there was a massive increase in the number of neurons expressing the migratory marker, doublecortin, at the boundary between the granule cell layer and the polymorphic layer in the dorsal hippocampus. Some of these migrating neurons were also positive for NeuN, a marker for adult neurons. CONCLUSION/SIGNIFICANCE: Our results suggest that the increased susceptibility to seizure at day 25±1 post-treatment is coincident with a critical time required for newborn neurons to differentiate and integrate into the existing hippocampal network, and outlines the importance of the dorsal hippocampus for seizure-related neurogenesis. This model can be used as an in vivo correlate of neurogenesis to study basic questions related to neurogenesis and to the neurogenic mechanisms that contribute to the development of epilepsy.

Vasopressin-induced intracellular Ca²âº concentration responses in non-neuronal cells of the rat dorsal root ganglion.

Arginine-vasopressin (AVP) is a nonapeptide of hypothalamic origin that has been shown to exert many important cognitive and physiological functions in neurons and terminals of both the central and peripheral nervous system (CNS and PNS). Here we report for the first time that AVP induced an increase in intracellular Ca²âº concentration ([Ca²âº](i)) in non-neuronal cells isolated from the rat dorsal root ganglion (DRG) and cultured in vitro. The ratiometric [Ca²âº](i) measurements showed that AVP evoked [Ca²âº](i) responses in the non-neuronal cells and these concentration-dependent (100 pM to 1 µM) responses increased with days in vitro in culture, reaching a maximum amplitude after 4-5 day. Immunostaining by anti-S-100 antibody revealed that more than 70% of S-100 positive cells were AVP-responsive, indicating that glial cells responded to AVP and increased their [Ca²âº](i). The responses were inhibited by depletion of the intracellular Ca²âº stores or in the presence of inhibitors of phospholipase C, indicating a metabotropic response involving inositol trisphosphate, and were mediated by the V1 subclass of AVP receptors, as evidenced by the use of the specific blockers for V1 and OT receptors, (d(CH2)5¹,Tyr(Me)²,Arg8)-Vasopressin and (d(CH2)5¹,Tyr(Me)²,Thr4,Orn8,des-Gly-NH29)-Vasotocin, respectively. V(1a) but not V(1b) receptor mRNA was expressed sustainably through the culture period in cultured DRG cells. These results suggest that AVP modulates the activity of DRG glial cells via activation of V(1a) receptor.

Effect of ageing on CA3 interneuron sAHP and gamma oscillations is activity-dependent.

Normal ageing-associated spatial memory impairment has been linked to subtle changes in the hippocampal network. Here we test whether the age-dependent reduction in gamma oscillations can be explained by the changes in intrinsic properties of hippocampal interneurons. Kainate-induced gamma oscillations, but not spontaneous gamma oscillations, were reduced in slices from aged mice. CA3 interneurons were recorded in slices from young and aged mice using Fura-2-filled pipettes. Passive membrane properties, firing properties, medium- and slow-afterhyperpolarisation amplitudes, basal [Ca(2+)](i) and firing-induced [Ca(2+)](i) transients were not different with ageing. Kainate caused a larger depolarisation and increase in [Ca(2+)](i) signal in aged interneurons than in young ones. In contrast to young interneurons, kainate increased the medium- and slow-afterhyperpolarisation and underlying [Ca(2+)](i) transient in aged interneurons. Modulating the slow-afterhyperpolarisation by modulating L-type calcium channels with BAY K 8644 and nimodipine suppressed and potentiated, respectively, kainate-induced gamma oscillations in young slices. The age-dependent and stimulation-dependent increase in basal [Ca(2+)](i), firing-induced [Ca(2+)](i) transient and associated afterhyperpolarisation may reduce interneuron excitability and contribute to an age-dependent impairment of hippocampal gamma oscillations.