Spontaneous Calcium Oscillations Research Articles

Sodium oligomannate activates the enteroendocrine-vagal afferent pathways in APP/PS1 mice.

Enteroendocrine cells (EECs) and vagal afferent neurons constitute functional sensory units of the gut, which have been implicated in bottom-up modulation of brain functions. Sodium oligomannate (GV-971) has been shown to improve cognitive functions in murine models of Alzheimer's disease (AD) and recently approved for the treatment of AD patients in China. In this study, we explored whether activation of the EECs-vagal afferent pathways was involved in the therapeutic effects of GV-971. We found that an enteroendocrine cell line RIN-14B displayed spontaneous calcium oscillations due to TRPA1-mediated calcium entry; perfusion of GV-971 (50, 100 mg/L) concentration-dependently enhanced the calcium oscillations in EECs. In ex vivo murine jejunum preparation, intraluminal infusion of GV-971 (500 mg/L) significantly increased the spontaneous and distension-induced discharge rate of the vagal afferent nerves. In wild-type mice, administration of GV-971 (100 mg· kg-1 ·d-1, i.g. for 7 days) significantly elevated serum serotonin and CCK levels and increased jejunal afferent nerve activity. In 7-month-old APP/PS1 mice, administration of GV-971 for 12 weeks significantly increased jejunal afferent nerve activity and improved the cognitive deficits in behavioral tests. Sweet taste receptor inhibitor Lactisole (0.5 mM) and the TRPA1 channel blocker HC-030031 (10 µM) negated the effects of GV-971 on calcium oscillations in RIN-14B cells as well as on jejunal afferent nerve activity. In APP/PS1 mice, co-administration of Lactisole (30 mg ·kg-1 ·d-1, i.g. for 12 weeks) attenuated the effects of GV-971 on serum serotonin and CCK levels, vagal afferent firing, and cognitive behaviors. We conclude that GV-971 activates sweet taste receptors and TRPA1, either directly or indirectly, to enhance calcium entry in enteroendocrine cells, resulting in increased CCK and 5-HT release and consequent increase of vagal afferent activity. GV-971 might activate the EECs-vagal afferent pathways to modulate cognitive functions.

Coupled action potential and calcium dynamics underlie robust spontaneous firing in dopaminergic neurons

Dopaminergic neurons are specialized cells in the substantia nigra, tasked with dopamine secretion. This secretion relies on intracellular calcium signaling coupled to neuronal electrical activity. These neurons are known to display spontaneous calcium oscillations in-vitro and in-vivo, even in synaptic isolation, controlling the basal dopamine levels. Here we outline a kinetic model for the ion exchange across the neuronal plasma membrane. Crucially, we relax the assumption of constant, cytoplasmic sodium and potassium concentration. We show that sodium-potassium dynamics are strongly coupled to calcium dynamics and are essential for the robustness of spontaneous firing frequency. The model predicts several regimes of electrical activity, including tonic and ‘burst’ oscillations, and predicts the switch between those in response to perturbations. ‘Bursting’ correlates with increased calcium amplitudes, while maintaining constant average, allowing for a vast change in the calcium signal responsible for dopamine secretion. All the above traits provide the flexibility to create rich action potential dynamics that are crucial for cellular function.

Bioelectric Reprogramming of the Bone Marrow Niche in Acute Myeloid Leukemia

Background. A large body of evidence indicates that cancer is not comprised solely of malignant cells, but that it represents part of a complex network of transformed and non-transformed cells, including mesenchymal stromal cells (MSCs), immune, nervous, endothelial, stem cells and fibroblasts that, together with soluble mediators, make up the tumor microenvironment. More recently, molecular mechanisms underlying a novel system of developmental control have been described to define the role of bioelectric gradients. Differences in endogenous resting membrane potential (V mem) of non-neural cells have importance in cell-cell interactions and regulation or dysregulation in embryogenesis, regeneration patterns, and cell behavior, especially proliferation and differentiation. Recently, the same issues have been suggested to be related to cancer where abnormal depolarization of resting V mem is associated to neoplastic cells and the role of ion channels conductivity in cancer initiation and progression deserves great attention in tumor microenvironment remodeling and therapy response. Methods and results. We characterized MSCs derived from bone marrow (BM) of pediatric acute myeloid leukemia patients (AML-MSCs) and identified significant changes in ion channel expression profile, including the Voltage-gated L-type calcium channel CaV1.2. We investigated the role of CaV1.2 and documented a reduced expression of CaV1.2 channel in 14 primary AML-MSCs (<53% by flow cytometry, p<0.001) mediating more frequent spontaneous calcium oscillations with higher amplitude than those found in MSCs derived from healthy donors (h-MSCs) (21% and 7%, respectively, p<0.05). Measurement of V mem by patch clamp and a fluorescent potential-sensitive probe DiBAC revealed that AML-MSCs showed a more depolarized V mem (-14.7 mV) with respect to h-MSCs (-30.5 mV, p<0.01). We hypothesized that depolarization may play a role in the AML-MSCs gene expression and functional reprogramming. Hence, we exposed h-MSCs to AML blasts demonstrating that MSCs V mem depolarized (-11.8 mV, n=10), resembling the AML-MSCs V mem, with a concomitant decrease of CaV1.2 channel expression level (<44% by flow cytometry, p<0.01). We treated h-MSCs with depolarizing pharmacological agents (ouabain and potassium gluconate) finding that the pharmacological-induced V mem depolarization promotes a pro-leukemia cell phenotype like the AML-MSCs. Briefly, MSCs exhibited higher proliferation level (ouabain, 2.5-fold; Kgluc, 1.9-fold, p<0.05), higher release of IL-6 (1.7-fold average, p<0.05), lower immunomodulatory potential and reduced the CaV1.2 levels (<35% by flow cytometry, p<0.05) with respect to untreated h-MSCs. On the contrary, we treated AML-MSCs with hyperpolarizing agents (lubiprostone and ivermectin), which reverted their phenotype to that of h-MSCs, including lower proliferation level (ivermectin -1.9-fold, p<0.05), higher immunomodulatory potential, and higher CaV1.2 levels (1.8-fold by flow cytometry, p<0.05) with respect to untreated AML-MSCs, suggesting that depolarization is reversible and that by modeling MSCs V mem we can restore an healthy stroma within the BM niche. We transduced AML-MSCs with a CaV1.2 expressing lentiviral vector (namely re-AML-MSCs, n=7) inducing a stable hyperpolarized V mem and documented that their gene expression profile reverted toward the h-MSCs profile. Finally, we observed that re-AML-MSCs propagated their acquired bioelectrical state to the contiguous AML-MSCs establishing a healthy bio-electric field. Conclusions. We demonstrated an unprecedented AML niche bioelectrical code that governs the state of MSCs, demonstrating that the contact with AML blasts is the driving force inducing the h-MSCs V mem depolarization shift and the acquisition of pro-leukemia functions. In addition, we unveiled CaV1.2 channels as a key player in this process and postulated that rewriting the bioelectric code of the patient' AML-MSCs, through the restoration of voltage-dependent calcium channel CaV1.2 activity, could be a strategy to restore their healthy phenotype. These findings provide the biological rationale to evaluate the potential of novel MSCs gene or pharmacological therapies that could both disadvantage AML progression and restore a permissive BM niche.

Optogenetic stimulation shapes dendritic trees of infragranular cortical pyramidal cells.

Spontaneous or experimentally evoked activity can lead to changes in length and/or branching of neocortical pyramidal cell dendrites. For instance, an early postnatal overexpression of certain AMPA or kainate glutamate receptor subunits leads to larger amplitudes of depolarizing events driven by spontaneous activity, and this increases apical dendritic complexity. Whether stimulation frequency has a role is less clear. In this study, we report that the expression of channelrhodopsin2-eYFP was followed by a 5-day optogenetic stimulation from DIV 5-10 or 11-15 in organotypic cultures of rat visual cortex-evoked dendritic remodeling. Stimulation at 0.05 Hz, at a frequency range of spontaneous calcium oscillations known to occur in the early postnatal neocortex in vivo until eye opening, had no effect. Stimulation with 0.5 Hz, a frequency at which the cortex in vivo adopts after eye opening, unexpectedly caused shorter and somewhat less branched apical dendrites of infragranular pyramidal neurons. The outcome resembles the remodeling of corticothalamic and callosal projection neurons of layers VI and V, which in the adult have apical dendrites no longer terminating in layer I. Exposure to 2.5 Hz, a frequency not occurring naturally during the time windows, evoked dendritic damage. The results suggested that optogenetic stimulation at a biologically meaningful frequency for the selected developmental stage can influence dendrite growth, but contrary to expectation, the optogenetic stimulation decreased dendritic growth.

MAPT genotype-dependent mitochondrial aberration and ROS production trigger dysfunction and death in cortical neurons of patients with hereditary FTLD

Tauopathies are a major type of proteinopathies underlying neurodegenerative diseases. Mutations in the tau-encoding MAPT-gene lead to hereditary cases of frontotemporal lobar degeneration (FTLD)-tau, which span a wide phenotypic and pathological spectrum. Some of these mutations, such as the N279K mutation, result in a shift of the physiological 3R/4R ratio towards the more aggregation prone 4R isoform. Other mutations such as V337M cause a decrease in the in vitro affinity of tau to microtubules and a reduced ability to promote microtubule assembly. Whether both mutations address similar downstream signalling cascades remains unclear but is important for potential rescue strategies. Here, we developed a novel and optimised forward programming protocol for the rapid and highly efficient production of pure cultures of glutamatergic cortical neurons from hiPSCs. We apply this protocol to delineate mechanisms of neurodegeneration in an FTLD-tau hiPSC-model consisting of MAPTN279K- or MAPTV337M-mutants and wild-type or isogenic controls. The resulting cortical neurons express MAPT-genotype-dependent dominant proteome clusters regulating apoptosis, ROS homeostasis and mitochondrial function. Related pathways are significantly upregulated in MAPTN279K neurons but not in MAPTV337M neurons or controls. Live cell imaging demonstrates that both MAPT mutations affect excitability of membranes as reflected in spontaneous and stimulus evoked calcium signals when compared to controls, albeit more pronounced in MAPTN279K neurons. These spontaneous calcium oscillations in MAPTN279K neurons triggered mitochondrial hyperpolarisation and fission leading to mitochondrial ROS production, but also ROS production through NOX2 acting together to induce cell death. Importantly, we found that these mechanisms are MAPT mutation-specific and were observed in MAPTN279K neurons, but not in MAPTV337M neurons, supporting a pathological role of the 4R tau isoform in redox disbalance and highlighting MAPT-mutation specific clinicopathological-genetic correlations, which may inform rescue strategies in different MAPT mutations.

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's disease.

SummaryDysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimer’s disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate local neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex several years before amyloid deposition. We find the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astrocytic calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral disruptions. In conclusion, we show that astrocytes mediate initial features of AD and drive clinically relevant phenotypes.

Mechanisms Underlying Influence of Bioelectricity in Development.

To execute the intricate process of development, cells coordinate across tissues and organs to determine where each cell divides and differentiates. This coordination requires complex communication between cells. Growing evidence suggests that bioelectrical signals controlled via ion channels contribute to cell communication during development. Ion channels collectively regulate the transmembrane potential of cells, and their function plays a conserved role in the development of organisms from flies to humans. Spontaneous calcium oscillations can be found in nearly every cell type and tissue, and disruption of these oscillations leads to defects in development. However, the mechanism by which bioelectricity regulates development is still unclear. Ion channels play essential roles in the processes of cell death, proliferation, migration, and in each of the major canonical developmental signaling pathways. Previous reviews focus on evidence for one potential mechanism by which bioelectricity affects morphogenesis, but there is evidence that supports multiple different mechanisms which are not mutually exclusive. Evidence supports bioelectricity contributing to development through multiple different mechanisms. Here, we review evidence for the importance of bioelectricity in morphogenesis and provide a comprehensive review of the evidence for several potential mechanisms by which ion channels may act in developmental processes.

Acute Neurotoxicity of Antisense Oligonucleotides After Intracerebroventricular Injection Into Mouse Brain Can Be Predicted from Sequence Features.

Antisense oligonucleotides are a relatively new therapeutic modality and safety evaluation is still a developing area of research. We have observed that some oligonucleotides can produce acute, nonhybridization dependent, neurobehavioral side effects after intracerebroventricular (ICV) dosing in mice. In this study, we use a combination of in vitro, in vivo, and bioinformatics approaches to identify a sequence design algorithm, which can reduce the number of acutely toxic molecules synthesized and tested in mice. We find a cellular assay measuring spontaneous calcium oscillations in neuronal cells can predict the behavioral side effects after ICV dosing, and may provide a mechanistic explanation for these observations. We identify sequence features that are overrepresented or underrepresented among oligonucleotides causing these reductions in calcium oscillations. A weighted linear combination of the five most informative sequence features predicts the outcome of ICV dosing with >80% accuracy. From this, we develop a bioinformatics tool that allows oligonucleotide designs with acceptable acute neurotoxic potential to be identified, thereby reducing the number of toxic molecules entering drug discovery pipelines. The informative sequence features we identified also suggest areas in which to focus future medicinal chemistry efforts.

Targeted Discovery of Amantamide B, an Ion Channel Modulating Nonapeptide from a South China Sea Oscillatoria Cyanobacterium.

Amantamide B (1) is a new linear nonapeptide analogue of the cyanobacterial natural product amantamide A (2), and both have methyl ester and butanamide termini. These compounds were discovered in this study from the organic extract of a tropical marine filamentous cyanobacterium, Oscillatoria sp., collected around the Paracel Islands in the South China Sea. The use of LC-MS/MS molecular networking for sample prioritization and as an analytical dereplication tool facilitated the targeted isolation of 1 and 2. These molecules were characterized by spectroscopy and spectrometry, and configurational assignments were determined using chemical degradation and chiral-phase HPLC analysis. Compounds 1 and 2 modulated spontaneous calcium oscillations without notable cytotoxicity at 10 μM in short duration in vitro testing on primary cultured neocortical neurons, a model system that evaluates neuronal excitability and/or the potential activity on Ca2+ signaling. Both molecules were also found to be moderately cytotoxic in longer duration bioassays, with in vitro IC50 values of 1-10 μM against CCRF-CEM human T lymphoblastoid cells and U937 human histiocytic lymphoma cells. These formerly undiscovered bioactivities of known compound 2 expand upon its previously reported function as a selective CXCR7 agonist among 168 GPCR targets.

Homeostatic calcium fluxes, ER calcium release, SOCE, and calcium oscillations in cultured astrocytes are interlinked by a small calcium toolkit

How homeostatic ER calcium fluxes shape cellular calcium signals is still poorly understood. Here we used dual-color calcium imaging (ER-cytosol) and transcriptome analysis to link candidates of the calcium toolkit of astrocytes with homeostatic calcium signals. We found molecular and pharmacological evidence that P/Q-type channel Cacna1a contributes to depolarization-dependent calcium entry in astrocytes. For stimulated ER calcium release, the cells express the phospholipase Cb3, IP3 receptors Itpr1 and Itpr2, but no ryanodine receptors (Ryr1–3). After IP3-induced calcium release, Stim1/2 – Orai1/2/3 most likely mediate SOCE. The Serca2 (Atp2a2) is the candidate for refilling of the ER calcium store. The cells highly express adenosine receptor Adora1a for IP3-induced calcium release. Accordingly, adenosine induces fast ER calcium release and subsequent ER calcium oscillations. After stimulation, calcium refilling of the ER depends on extracellular calcium. In response to SOCE, astrocytes show calcium-induced calcium release, notably even after ER calcium was depleted by extracellular calcium removal in unstimulated cells. In contrast, spontaneous ER-cytosol calcium oscillations were not fully dependent on extracellular calcium, as ER calcium oscillations could persist over minutes in calcium-free solution. Additionally, cell-autonomous calcium oscillations show a second-long spatial and temporal delay in the signal dynamics of ER and cytosolic calcium. Our data reveal a rather strong contribution of homeostatic calcium fluxes in shaping IP3-induced and calcium-induced calcium release as well as spatiotemporal components of intracellular calcium oscillations.

Dynamic Pressure Stimulation Upregulates Collagen II and Aggrecan in Nucleus Pulposus Cells Through Calcium Signaling.

An in vitro study to investigate the effect of pressure stimulation on nucleus pulposus (NP) cells. The aim of this study was to investigate the question whether physical stimulation can be leveraged to enhance extracellular matrix (ECM) synthesis as a preventive measure for intervertebral disc (IVD) degeneration. ECM plays an important role in regulating hydration and pressure balance of the IVD. Cellular stimulation devices with different pressurizing protocols were used to create a pressurized environment to cells cultures. The setup was used to mimic the pressurized conditions within IVD to investigate the effect of pressure stimulation on NP cells. Pressure stimulation at 300 kPa can enhance the synthesis of ECM proteins Collagen II and aggrecan in NP cells and the effect of dynamic pressure stimulation outperformed the static one. The difference between static and dynamic pressure stimulation was due primarily to calcium signaling activated by pressure fluctuation. The superior effect of dynamic pressure holds for a wide range of stimulation durations, relating to the range of spontaneous calcium oscillations in NP cells. The results link mechanotransduction to the downstream ECM protein synthesis and suggest slow exercises that correspond with spontaneous calcium oscillations in NP cells can be effective to stimulate ECM synthesis in IVD.

Diverse inflammatory threats modulate astrocytes Ca2+ signaling via connexin43 hemichannels in organotypic spinal slices

Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells.

Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration.

A multiscale sliding filament model of lymphatic muscle pumping

The lymphatics maintain fluid balance by returning interstitial fluid to veins via contraction/compression of vessel segments with check valves. Disruption of lymphatic pumping can result in a condition called lymphedema with interstitial fluid accumulation. Lymphedema treatments are often ineffective, which is partially attributable to insufficient understanding of specialized lymphatic muscle lining the vessels. This muscle exhibits cardiac-like phasic contractions and smooth muscle-like tonic contractions to generate and regulate flow. To understand the relationship between this sub-cellular contractile machinery and organ-level pumping, we have developed a multiscale computational model of phasic and tonic contractions in lymphatic muscle and coupled it to a lymphangion pumping model. Our model uses the sliding filament model (Huxley in Prog Biophys Biophys Chem 7:255–318, 1957) and its adaptation for smooth muscle (Mijailovich in Biophys J 79(5):2667–2681, 2000). Multiple structural arrangements of contractile components and viscoelastic elements were trialed but only one provided physiologic results. We then coupled this model with our previous lumped parameter model of the lymphangion to relate results to experiments. We show that the model produces similar pressure, diameter, and flow tracings to experiments on rat mesenteric lymphatics. This model provides the first estimates of lymphatic muscle contraction energetics and the ability to assess the potential effects of sub-cellular level phenomena such as calcium oscillations on lymphangion outflow. The maximum efficiency value predicted (40%) is at the upper end of estimates for other muscle types. Spontaneous calcium oscillations during diastole were found to increase outflow up to approximately 50% in the range of frequencies and amplitudes tested.

Carbon Nanotube-Hydrogel Composites Facilitate Neuronal Differentiation While Maintaining Homeostasis of Network Activity.

It is often assumed that carbon nanotubes (CNTs) stimulate neuronal differentiation by transferring electrical signals and enhancing neuronal excitability. Given this, CNT-hydrogel composites are regarded as potential materials able to combine high electrical conductivity with biocompatibility, and therefore promote nerve regeneration. However, whether CNT-hydrogel composites actually influence neuronal differentiation and maturation, and how they do so remain elusive. In this study, CNT-hydrogel composites are prepared by in situ polymerization of poly(ethylene glycol) around a preformed CNT meshwork. It is demonstrated that the composites facilitate long-term survival and differentiation of pheochromocytoma 12 cells. Adult neural stem cells cultured on the composites show an increased neuron-to-astrocyte ratio and higher synaptic connectivity. Moreover, primary hippocampal neurons cultured on composites maintain morphological synaptic features as well as their neuronal network activity evaluated by spontaneous calcium oscillations, which are comparable to neurons cultured under control conditions. These results indicate that the composites are promising materials that could indeed facilitate neuronal differentiation while maintaining neuronal homeostasis.

Dopamine controls neuronal spontaneous calcium oscillations via astrocytic signal

Dopamine is a neuromodulator and neurotransmitter responsible for a number of physiological processes. Dysfunctions of the dopamine metabolism and signalling are associated with neurological and psychiatric diseases. Here we report that in primary co-culture of neurons and astrocytes dopamine-induces calcium signal in astrocytes and suppress spontaneous synchronous calcium oscillations (SSCO) in neurons. Effect of dopamine on SSCO in neurons was dependent on calcium signal in astrocytes and could be modified by inhibition of dopamine-induced calcium signal or by stimulation of astrocytic calcium rise with ATP. Ability of dopamine to suppress SSCO in neurons was independent on D1- or D2- like receptors but dependent on GABA and alpha-adrenoreceptors. Inhibitor of monoaminoxidase bifemelane blocked effect of dopamine on astrocytes but also inhibited the effect dopamine on SSCO in neurons. These findings suggest that dopamine-induced calcium signal may stimulate release of neuromodulators such as GABA and adrenaline and thus suppress spontaneous calcium oscillations in neurons.

Abstract 14219: Oxidation of PKA-RIα Protects Against Ischemia-reperfusion Injury by Inhibiting Lysosomal-triggered Calcium Release

Introduction: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, the regulatory Iα subunit of Protein Kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulfide bonds within the holoenzyme complex. However, the impact of disulfide formation on kinase function, and its influence on PKA signaling in the context of heart disease remains unknown. Methods & Results: Myocardial ischemia-reperfusion (I/R) was found to be a potent inducer of PKARIα disulfide formation in vivo , both in mice and in humans. Using imaging modalities with high spatial and temporal resolution, we found that this conformation did not increase intrinsic PKA catalytic activity, but rather facilitated enhanced AKAP-dependent compartmentation of PKARIα in the adult mouse left ventricular (LV) myocyte, with preferential localization to the lysosome under oxidized conditions (n=38-41 myocytes, N=3 animals, p<0.01). Investigations in isolated LV myocytes revealed disulfide-modified PKARIα to be a significant regulator of lysosomal two pore channel (TPC)-dependent calcium-induced calcium release, with myocytes from ‘redox dead’ PKARIα mice (Cys17Ser) displaying spontaneous sarcoplasmic reticulum calcium release events and pronounced intracellular calcium oscillations. These events were prevented by ryanodine receptor blockade (1 mM tetracaine; n=14, p<0.01), acute depletion of lysosomal calcium stores (100 nM bafilomycin; n=7; p<0.01), or TPC inhibition (5 μM Ned-19; n=9; p<0.05). Absence of I/R-induced disulfide formation in “redox dead” PKARIα mouse hearts resulted in larger infarcts (2-fold increase, p<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, p<0.001), which could be fully prevented by administering the TPC inhibitor, Ned-19, at the time of reperfusion. Conclusions: Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKA oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics.

Glycine Receptor Inhibition Differentially Affect Selected Neuronal Populations of the Developing Embryonic Cortex, as Evidenced by the Analysis of Spontaneous Calcium Oscillations.

The embryonic developing cerebral cortex is characterized by the presence of distinctive cell types such as progenitor pools, immature projection neurons and interneurons. Each of these cell types is diverse on itself, but they all take part of the developmental process responding to intrinsic and extrinsic cues that can affect their calcium oscillations. Importantly, calcium activity is crucial for controlling cellular events linked to cell cycle progression, cell fate determination, specification, cell positioning, morphological development and maturation. Therefore, in this work we measured calcium activity in control conditions and in response to neurotransmitter inhibition. Different data analysis methods were applied over the experimental measurements including statistical methods entropy and fractal calculations, and spectral and principal component analyses. We found that developing projection neurons are differentially affected by classic inhibitory neurotransmission as a cell type and at different places compared to migrating interneurons, which are also heterogeneous in their response to neurotransmitter inhibition. This reveals important insights into the developmental role of neurotransmitters and calcium oscillations in the forming brain cortex. Moreover, we present an improved analysis proposing a Gini coefficient-based inequality distribution and principal component analysis as mathematical tools for understanding the earliest patterns of brain activity.

Calcium dynamics and chromatin remodelling underlie heterogeneity in prolactin transcription.

Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26 h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation, at least in part, drive prolactin transcriptional competence.

HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment.

Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.