Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings in vivo. The main controversies regard the types of calcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the in situ acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidence that even a large amplitude calcium signal was not sufficient and that metabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future.In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous  Moreover, when cells in a tissue are interconnected through gap junctions, Ca2+ signals can spread between them to coordinate the activity of a large number of cells 2+ is the common denominator for metabolic, neuronal, hormonal, as well as pharmacological influences, regulating exocytosis of islet hormones 2+ in endocrine cells from islets of Langerhans, predominantly in the most abundant cell type, insulin-secreting beta cells. In these cells, according to the current consensus model, glucose and other nutrients increase production of ATP, thereby decreasing the open probability of the ATP-sensitive potassium (KATP) channels in the plasma membrane. This in turn depolarizes the plasma membrane and leads to an influx of Ca2+ through voltage-dependent calcium channels. Ca2+ influx has been described to occur rhythmically due to a rhythmic nature of the underlying electrical activity. The exact pattern of changes of intracellular concentration of Ca2+ ([Ca2+]i) upon stimulation depends on the experimental model employed.Calcium Ca2+]i oscillations of comparable frequency, regularity, and durations 2+]i have been demonstrated, indicating a very good temporal correlation between electrical activity, Ca2+ signaling, and insulin secretion in isolated islets 2+]i as well as membrane potential oscillations recorded in vivo were very similar to the ones described in isolated islets studied shortly after isolation In isolated mouse islets, which are currently the most often used experimental model, a considerable overlap has been demonstrated between the glucose-stimulated electrical activity occurring in the form of membrane potential bursts and the i oscillations (frequency = 2–7 min−1) that correspond to bursts of membrane potential are the predominant type of response, in islets exposed to stimulatory glucose concentrations during longer culture, slow [Ca2+]i oscillations (frequency = 0.2–1 min−1) prevail, either as the sole pattern of activity, or with superimposed fast oscillations 2+]i oscillations closely resemble slow [Ca2+]i oscillations described in dispersed beta cells 2+]i pattern after culture 2+]i oscillations, and cultured isolated islets remain the most important experimental model, despite the possibility that the observed [Ca2+]i patterns might partially be an experimental artifact.In experiments where frequent solution changes and a high perifusion rate are necessary, islets need to be cultured longer to adhere to the cover slips in vivo, a considerable degree of synchronicity in electrical activity in different cells in the same islet has been shown 2+]i oscillations occurring synchronously throughout the islet, with only short phase lags of up to 1–2 seconds detected between oscillations in different islet regions 2+]i oscillatory behavior was shown to emerge from waves originating in most sensitive cells and spreading across or alongside periphery of islets 2+]i oscillations in different parts of an islet or pairs of cells, the speed of progression of [Ca2+]i waves has been estimated at 20–200 µms−12+]i waves are absent and that they appear after several days of culture, although in many studies in shortly cultured islets, time resolution of [Ca2+]i imaging systems did not permit detection of possible rapid [Ca2+]i waves 2+]i waves also exist in fresh islets.In experiments in fresh isolated islets as well as in islets 2+]i imaging experiments. The uptake of fluorescent indicators into the core of isolated islets is limited by diffusion and dye trapping in outermost cells In addition to the confusion regarding types of oscillations present in normal islets and the mechanistic substrate of synchronicity, there is an important technical drawback associated with the use of isolated islets in [Ca2+]i imaging employing confocal laser scanning microscopy (CLSM). In the tissue slice, by virtue of using thin transversal sections, one inherently gains access to cells distributed across the whole cross-sectional area of an islet To shed new light on the controversy regarding types of oscillations present in beta cells, to find out whether calcium waves normally spread across islets and synchronize the activity of beta cells, and to be able to characterize responses in many cells from all layers of islets, we decided to combine the acute pancreas tissue slice technique 2+]i oscillations superimposed on a sustained plateau are the predominant type of response and that these oscillations are synchronized via [Ca2+]i waves originating in a group of cells and repeatedly spreading across islets. The absence of clear spatial patterns and long time lags between individual cells during activation, despite large amplitudes of [Ca2+]i changes, confirm heterogeneity of beta cells and provide evidence that local [Ca2+]i increases cannot be efficiently communicated throughout an islet until the majority of cells are metabolically activated. Once the islet as a whole is active, [Ca2+]i waves as well as large and rapid [Ca2+]i decreases efficiently spread to all cells, producing synchronized oscillatory activity and a synchronous deactivation.Applying this novel approach, we gained access to all layers of cells, characterized their responses, and showed that in islets of Langerhans from acute pancreas tissue slices, fast i imaging. Enzymes from the exocrine part did not appear to have any significant effect upon viability and did not significantly interfere with dye loading. Therefore no enzyme inhibitors were needed. At least partially this can be ascribed to the subphysiological temperatures used throughout isolation, cutting, and dye-loading, to lack of stimuli to activate the enzymes, and to dilution due to large and constant perifusion volumes during experimentation at physiological temperatures. Notably, due to its porous structure, agarose did not importantly impede the exchange of extracellular solution and the access of dye molecules. We checked cell viability and morphology using the LIVE/DEAD® Viability/Cytotoxicity Kit  As described before We took advantage of discriminatory functional differences reported in previous studies 2+]i level and returned to the baseline [Ca2+]i upon removal of stimulatory glucose . Among the latter, approximately a half responded with a transient increase in [Ca2+]i followed by a sustained plateau at a level higher than the baseline and lower than the peak of the transient, with [Ca2+]i oscillations superimposed on the plateau . In 2+]i oscillations is shown for a single representative islet, demonstrating the absence of any clear trend towards changing frequencies even over a longer time period. To quantify the constancy of frequency, for 12 different islets median interval durations were calculated for five 100-seconds-long time intervals and compared with each other 2+]i oscillations did not significantly change over the five time intervals . After taking into account 6 consecutive spikes in the same set of cells, the same average speed was obtained  is in the upper range of frequencies determined in isolated islets and in islets in vivo. It has been suggested that normal paracrine relationships are retained and the level of important intracellular signaling molecules is less disrupted in fresh preparations and that this could account for the higher frequencies 2+]i oscillations without a clear plateau and with only some or none superimposed [Ca2+]i oscillations. The presence of fast oscillations has been shown to depend on cyclic adenosine monophosphate (cAMP) and it was proposed that their gradual disappearance in culture is a consequence of loss of alpha cells and endogenous glucagon that normally conveys a paracrine cAMP-sustaining tonus to beta cells 2+]i oscillations, we hypothesize that an important loss of paracrine tone does not occur with our preparation and that our work further supports the possibility that slow oscillations could be an experimental epiphenomenon.As expected, our protocol allowed for detection of beta cells  is in excellent agreement with previous reports [Ca2+]i oscillations during the plateau. So why does a cell invariably respond to a relatively small increase or decrease in [Ca2+]i in its neighbor by promptly producing or terminating a [Ca2+]i oscillation during the plateau phase, while tolerating huge increases in [Ca2+]i in its neighbor during activation and deactivation and responding with a delay, one or two orders of magnitude larger? In addition to Ca2+ that might well suffice to align the heterogeneity of cells during plateau, something else seems necessary during activation and deactivation. The very magnitude of time delays and the hint of cooperativity that comes from the cumulative distributions point to metabolic activation and possibly to a gradually increasing efficiency of tonically spreading de- and hyperpolarizing electrical signals. The latter might be due to a decrease in membrane conductance or an increase in gap junctional conductance. After a critical level of metabolically driven decrease in membrane permeability (due to closure of the KATP channels) is reached or gap junctional coupling is increased, the beta cell network can gradually start functioning as a network. Once the whole syncitium is well coupled, hyperpolarizing stimuli due to removal of glucose are expected to be communicated throughout the network much more efficiently than depolarizing ones during activation. This is exactly what we observe by comparing the cumulative distributions of time delays during activation and deactivation. The evidence obtained in favor of these ideas is largely circumstantial and further research will be needed to properly address them, as well as to provide clues to some as yet unresolved issues, such as the asymmetry in delays before first responses during activation and deactivation, the relative invariability of frequency and duration of [Ca2+]i oscillations during the plateau, and the striking intercellular homogeneity in duration of oscillations. Namely, while the interval between consecutive oscillations is expected to be constant in excitable systems with [Ca2+]i waves spreading in roughly defined directions, the duration of individual oscillation can still be largely determined by the repertoire of membrane channels in individual cells. One notable example of such an excitable system is the myocardium. In beta cell network on the other hand, it seems that the group of cells in which the waves originate determine not only the frequency of oscillations but possibly to at least some extent also their duration.Additionally, our findings shed new light on function of beta cells. Coupling of cells has long been believed to play a crucial role in aligning the responsiveness of a heterogeneous population of beta cells. We confirmed that this is the case. The heterogeneity of cells was clearly visible from differences in time required for activation, but once a beta cell or a group of cells had been activated, all other cells followed and synchronized eventually. Scrutinizing activation and deactivation before and after the sustained plateau where beta cells are well synchronized, we found that the activation process requires almost two orders of magnitude longer time interval (≈100 seconds) to activate beta cells than the synchronization during the plateau phase (≈1 second). Deactivation, while being much more synchronous than activation, still requires at least an order of magnitude longer time interval (≈10 seconds) than the synchronization of [Ca2+]i  that is based on two probes measuring intracellular esterase activity and integrity of the plasma membrane, which are recognized parameters of cell viability. Live cells (green) are labeled with component A of the kit , since intracellular esterase activity is needed for the presence of the dye in the cytosol. The component B (ethidium homodimer-1) binds to nuclei of dead cells (red) when plasma membrane integrity is lost and enables entry of component B into the cell. Slicing of the agarose-embedded pancreatic islet resulted in damage to the superficial layer of cells. In deeper layers, starting at 2–3 cells below the cutting surface (i.e. 15–20 microns below the surface) mostly calcein labeled cells were detected, with only a few ethidium labeled cells, indicating that a great majority of cells were viable at the optical section from which the recordings of the [Ca2+]i were made. Yellow lines indicate the position of the image within the Z-stack. Depth of the Z-stack was 45 µm. Scale bar indicates 50 µm.(TIF)Click here for additional data file.Figure S2Morphology of cells in islets of Langerhans in tissue slices. To assess cell morphology in our preparation, we labeled the membranes of cells within an islet of Langerhans with the lipophilic dye di-4-ANNEPS. Shown are imaging layers at 10 µm (A), 20 µm (B) and 30 µm (C) below surface of the tissue slice. Note that characteristic polyhedral shapes of cells are well preserved. Scale bar indicates 25 µm.(TIF)Click here for additional data file.Figure S3Concentration changes in the chamber and in the intercellular space. A Geometry of the bath chamber used in our experiments. The tissue slice was superfused constantly at a rate of 13 µl/s. The volume of extracellular solution (ECS) in the chamber was 1.5 ml. ECS entered the chamber at one border (Inflow) and exited it at the other (Outflow). Points at which concentration changes were assessed are indicated in black, red, and green. B Employing the polar extracellular fluorescent tracer sulforhodamine B , the temporal evolution of fluorescence at points indicated in A was tracked in the bulk ECS just above the islet. At the recording rate of 1 Hz, no clear delays could be detected between onsets of increases in the fluorescence, indicated in colors corresponding to colors of the three points indicated in A. C Fluorescence signals recorded from intercellular spaces at the three points indicated in A, at a depth within the islet at which recordings of [Ca2+]i were typically performed. The concentration of sulforhodamine B increased slower than in the solution above the slice. However, the temporal evolution at different locations followed practically the same time course. D [Ca2+]i-dependent fluorescence was recorded simultaneously with the fluorescence of the extracellular indicator sulforhodamine B. Cells situated at the points indicated in A activated with clearly pronounced time delays despite virtually identical increases in extracellular fluorescence at these points, practically excluding the possibility that unequal access of glucose to individual cells could be responsible for the time delays between activations of individual cells observed in our study. An effective equilibration with the concentration in the solution above the slice was typically reached by the time when high frequency [Ca2+]i oscillations on the sustained elevation of [Ca2+]i were established. Scale bar indicates 60 seconds.(TIF)Click here for additional data file.Video S1Spatiotemporal characterization of activation of beta cells. Ca2+-dependent fluorescence signal in the islet of Langerhans shown in −1. Individual frames are presented as F/F0 and pseudocolored with blue and white representing low and high intensity signals, respectively.(MP4)Click here for additional data file.Video S22+]i oscillations superimposed on the sustained plateau.Spatiotemporal characterization of i waves repeatedly spread from top left to bottom right. Towards the end of the recording, the [Ca2+]i wave did not always spread to the region at the right end of the islet, indicating a variable efficiency of the synchronizing mechanism.(MP4)Click here for additional data file.Video S3Viability of cells in islets of Langerhans in tissue slices. A Z-stack of the islet of Langerhans labeled with the LIVE/DEAD® Viability/Cytotoxicity Kit  and shown in (MP4)Click here for additional data file.


Whereas the NCX activity was measured by observing oocyte activation following culture with NCX inhibitor or activator, the NCX contents were determined by immunohistochemical quantification. Although NCX was active in freshly-ovulated rat oocytes recovered 13 h post hCG injection and in aged oocytes recovered 19 h post hCG in both species, it was not active in freshly-ovulated mouse oocytes. However, NCX became active when the freshly-ovulated mouse oocytes were activated with ethanol before culture. Measurement of cytoplasmic Ca2+ revealed Ca2+ increases always before NCX activation. Whereas levels of the reactive oxygen species (ROS) and the activation susceptibility increased, the density of NCX member 1 (NCX1) decreased significantly with oocyte aging in both species. While culture with H2O2 decreased the density of NCX1 significantly, culture with NaCl supplementation sustained the NCX1 density in mouse oocytes. It was concluded that (a) the NCX activity was involved in the modulation of oocyte aging and spontaneous activation; (b) ROS and Na+ regulated the NCX activity in aging oocytes by altering its density as well as functioning; and (c) cytoplasmic Ca2+ elevation was essential for NCX activation in the oocyte.We studied the role of the Na Mammalian oocytes are arrested at the meiotic metaphase II (MII) stage following ovulation. If not fertilized in time, the ovulated oocytes undergo a time-dependent process of aging In spite of intensive efforts, many studies have failed to obtain rat offspring by nuclear transfer of somatic cells 2+ oscillations both at fertilization 2+ increases in rat oocytes during spontaneous activation 2+ extrusion across the plasma membrane: the Ca2+–ATPase and Na+/Ca2+ exchange +/Ca2+ exchange has been observed in immature and mature mouse +/Ca2+ exchange in the spontaneous activation of rat oocytes +/Ca2+ exchanger (NCX) activity in controlling postovulatory oocyte aging of different species. Furthermore, in previous studies the NCX activity was usually determined by functioning assays , with little attention paid to the dynamic changes in NCX contents.Mammalian oocytes are activated by intracellular Ca2+ oscillations in fertilized oocytes 2+ homeostasis mainly through direct effects on the machinery involved in intracellular Ca2+ storage Post-ovulatory aging of oocytes is associated with significant decreases in cytoplasmic reduced glutathione The objective of the current study was to determine the role of NCX in modulating oocyte postovulatory aging by observing changes in NCX contents and activities in aging mouse and rat oocytes and to study the effect of oxidative stress on the NCX activity during oocyte aging.To test the NCX activity in freshly-ovulated oocytes, mouse and rat oocytes collected 13 h post hCG injection were cultured for 6 h in CZB and mR1ECM medium, respectively, with different supplements to modulate the NCX activity. At the end of culture, while rat oocytes were observed for spontaneous activation immediately, mouse oocytes were treated with 10% ethanol for 10 min, cultured in CZB without supplements and observed for activation 6 h later. Whereas percentages of the spontaneously activated rat oocytes increased significantly, percentages of the ethanol activated mouse oocytes did not change after treatment with different concentrations of bepridil . AlthougTo test the NCX activity in aged oocytes, mouse and rat oocytes collected 19 h post hCG injection were cultured for 6 h with supplementation of 50 μM bepridil or different concentrations of NaCl. While rat oocytes were examined for spontaneous activation immediately after culture, mouse oocytes were treated with 5% ethanol for 10 min after culture and observed for activation 6 h later. Activation rates increased significantly in both mouse and rat oocytes after culture with 50 μM bepridil . SupplemThe objective of this experiment was to find out (a) why NCX was not active in freshly-ovulated mouse oocytes while it was in freshly-ovulated rat oocytes and (b) whether the NCX could be activated by activating mouse oocytes. Mouse oocytes collected 13 h post hCG were first treated for activation with 10% ethanol for 10 min and then cultured for 6 h in CZB medium supplemented with different concentrations of ouabain or NaCl. Ouabain was used in place of bepridil because many mouse oocytes lysed when treated with bepridil following ethanol activation treatment. At the end of culture, oocytes were examined for activation. Activation rates increased with increasing concentrations of ouabain but decreased with increasing NaCl concentrations significantly . Results2+ rises were reported at oocyte activation, we proposed that Ca2+ increases might be essential for NCX activation. To test this hypothesis, cytoplasmic concentrations of Ca2+ were measured in mouse and rat oocytes recovered at different times after hCG injection. Whereas rat oocytes recovered either 13 or 19 h after hCG injection showed marked Ca2+ rises, freshly-ovulated mouse oocytes recovered 13 h post hCG did not show any Ca2+ increase . As the extracellular concentrations of Na+ and Ca2+ remain close to constant, NCX activity is largely under the influence of the intracellular Ca2+ and Na+ concentrations + is mainly controlled by NKA 2+ from cells In the present study, whereas NaCl was used to increase the NCX activity, bepridil or ouabain was used to inhibit it. Bepridil was used for rat and mouse oocytes when no ethanol activation was involved, while ouabain was used for mouse oocytes after ethanol activation treatment because many mouse oocytes lysed when treated with bepridil following ethanol treatment. It is known that the NCX uses the electrochemical gradient of Na2+ increases in oocytes in which the NCX was proven active. Thus, whereas the freshly-ovulated mouse oocytes showed neither NCX activities nor Ca2+ increases when observed immediately after recovery, they displayed marked cytoplasmic Ca2+ rises as well as NCX activities when observed after pre-culture ethanol activation. We therefore concluded that a cytoplasmic Ca2+ elevation was essential for NCX activation in the oocyte. There are two major mechanisms to extrude Ca2+ across the plasma membrane: the Ca2+–ATPase and the Na+/Ca2+ exchanger 2+ for the activation of the two transporters is different; whereas the Ca2+–ATPase needs a low threshold level, the Na+/Ca2+ exchanger requires a high threshold level of cytoplasmic Ca2+ to activate 2+ concentration is approximately 100 nM 2+–ATPase plays an important role in maintaining resting cytoplasmic Ca2+, NCX is activated to transport Ca2+ when the cytoplasmic concentration of Ca2+ increased to the micromolar range The present results showed that although NCX was active in aged oocytes recovered 19 h post hCG injection in both mice and rats, and in freshly-ovulated rat oocytes as well, it was not active in freshly-ovulated mouse oocytes. However, NCX was activated when the freshly-ovulated mouse oocytes were activated with ethanol. Our further observations on different oocytes indicated that there were always cytoplasmic Ca2+ rises, which activated NCX under a low ROS level . Similarly, whereas most of the newly-ovulated rat oocytes showed cytoplasmic Ca2+ oscillations 2+ oscillations.Many studies have shown that oocyte activation is associated with intracellular Ca+ inhibited oocyte increase of activation susceptibility by maintaining the NCX density, suggesting that extra-cellular molecules regulated the NCX activity of aging oocytes by modifying its density as well as functioning; and (c) a cytoplasmic Ca2+ elevation was essential for NCX activation in the oocyte. The data are important for our understanding of not only the mechanisms for aging and spontaneous activation of oocytes but also the cellular mechanisms regulating the NCX activity.In summary, we have studied the role of the NCX activity in modulating postovulatory aging of mouse and rat oocytes. The results showed that (a) the NCX activity was involved in the modulation of oocyte aging and spontaneous activation; (b) whereas ROS increased oocyte activation susceptibility by decreasing the NCX density, NaMouse car and use were conducted exactly in accordance with the guidelines and approved by the Animal Research Committee of the Shandong Agricultural University, P. R. China (Permit number: 20010510). According to the guidelines of the committee, the animal handling staff  must be trained before using animals. Mice must be housed in a temperature-controlled room with proper darkness-light cycles, fed with a regular diet, and maintained under the care of the Experimental Animal Center, Shandong Agricultural University College of Animal Science and Vet Medicine. In the present study, mice were sacrificed by cervical dislocation. The only procedure performed on the dead animals was the collection of oocytes from the ovaries.Chemicals and reagents used in this study were purchased from Sigma Chemical Co.  unless otherwise specified.Mice of the Kunming breed and rats of the Sprague-Dawley breed were kept in an air-conditioned room with 14 h/10 h light-dark cycles, the darkness starting from 8 pm. The animals were handled by the rules stipulated by the Animal Care and Use Committee of Shandong Agricultural University. Female mice (8–10 wk after birth) and rats (23–26 days after birth) were induced to superovulate by i.p. injection of equine chorionic gonadotropin (eCG) , followed 48 h later by injection of human chorionic gonadotropin (hCG) . Both eCG and hCG used in this study were from Ningbo Hormone Product Co., Ltd. The superovulated mice or rats were sacrificed at different times after hCG injection and the oviductal ampullae were broken to release oocytes. After dispersed and washed three times in M2 medium, the oocytes were denuded of cumulus cells by pipetting with a thin pipette in a drop of M2 containing 0.1% hyaluronidase.2O2 were diluted to 100 μM with CZB immediately before use. The aging culture was conducted in wells (20–35 oocytes per well) of a 96-well culture plate containing 200 μl of aging medium covered with mineral oil at 37°C under 5% CO2 in humidified air.For in vitro aging, mouse or rat oocytes recovered at different times after hCG were cultured for 6 h in the aging medium supplemented with different substances. The aging medium used for mouse oocytes was a modified Chatot-Ziomek-Bavister (CZB) medium Whereas rat oocytes were assessed for spontaneous activation immediately after aging culture, mouse oocytes were treated with ethanol to induce activation. For ethanol activation, mouse oocytes were first treated with ethanol in M2 medium for 10 min at room temperature, then washed three times and cultured in the CZB medium for 6 h. Whereas oocytes recovered 13 h post hCG were treated with 10% ethanol, oocytes collected 19 h post hCG were activated with 5% ethanol. This was done because whereas the freshly-ovulated mouse oocytes were not activated well with 5% ethanol, almost all the aged oocytes were activated with 10% ethanol regardless of treatments. At the end of culture, oocytes were observed under a microscope for activation. Only those oocytes that had one or two pronuclei or two cells each having a nucleus were considered activated. To observe spontaneous activation, rat oocytes were fixed with 3.7% paraformaldehyde in M2 for 30 min at room temperature before being stained with 10 μg/ml Hoechst 33342 and mounted on glass slides. The state of chromosomes was observed under an epifluorescence microscope (Leica DMLB) and was classified into two types. Oocytes with chromosomes compacted at the metaphase plate were considered to be at the metaphase II (MII) stage, whereas oocytes with chromosomes dispersed in the cytoplasm were classified as activated. Dispersed chromosomes in the cytoplasm, rather than the formation of pronuclei, were used as an indication of oocyte activation because the spontaneously activated rat oocytes are often arrested in metaphase III instead of forming pronuclei 2+ was measured using the Ca2+-sensitive dye fluo-3. For loading, oocytes were incubated for 20 min at 37°C with 30 μM of the acetoxymethyl (AM) form of the dye made up in CZB (mouse) or mR1ECM (rat) with 0.02% pluronic F-127. After loading, mouse and rat oocytes were washed with and placed in a 22.5 μl drops of M2 and Hepes-buffered mR1ECM (HR1), respectively, under paraffin oil in a Fluoro dish  with its base coated with phytoagglutinin. The dish was transferred to a heated stage (37°C) of a Leica laser-scanning confocal microscope . In order to detect the mouse oocytes reaction to ethanol, 2.5 μl of absolute ethanol was injected into the M2 drop to initiate ethanol stimulation. The 10 min ethanol stimulation was terminated by diluting the drop with 1000 μl M2. Oocytes were observed using a 10× objective. An argon laser was used for excitation at 488 nm and signals emitted at 505–540 nm were collected for 40 min by the laser scanning confocal imaging system. Traces of Ca2+ oscillations were plotted using SigmaPlot 2000 software.Intracellular Ca4, pH 7.0) for at least 30 min, followed by treatment with 0.25% protease in M2 for 1 to 2 seconds to remove zona pellucida; (ii) permeabilized with 0.1% Triton X-100 in PHEM for 5 min; (iii) blocked in PHEM containing 3% BSA for 1 h; (iv) incubated overnight with mouse monoclonal anti-NCX1  in 3% BSA in M2 at 4°C; (v) incubated for 1 h with Cy3-conjugated goat-anti-mouse IgM  in 3% BSA in M2.All the procedures were conducted at room temperature unless otherwise specified. Oocytes were washed 3 times in M2 between treatments. Oocytes were (i) fixed with 3.7% paraformaldehyde in PHEM buffer . Helium/neon  lasers were used to excite Cy3, fluorescence was detected with bandpass emission filter (560–605 nm), and the captured signals were recorded as red. The relative content of NCX1 was quantified by measuring fluorescence intensities. For each experimental series, all high-resolution z-stack images were acquired with identical settings. The relative intensities were measured on the raw images using Image-Pro Plus software  under fixed thresholds across all slides.2O2 levels were measured using 20′,70′-dichlorodihydrofluorescein diacetate (DCHFDA) as described by Nasr-Esfahani et al In order to quantify ROS in individual oocytes, intraoocyte HThere were at least three replicates for each treatment. Percentage data were arc-sine transformed and analyzed with ANOVA; a test of Duncan multiple comparisons was used to locate differences. The software used was Statistics Package for Social Science . Data were expressed as mean ± S.E.M. and P<0.05 was considered significant.


Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi-component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi-component γ-haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca2+ concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura-2 Ca2+ indicator dye. Using pharmacological antagonists of receptors and Ca2+ channels, the variations in intracellular Ca2+ concentration were found independent of the activation of voltage-operatedCa2+ channels or glutamate receptors. Drugs targeting Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA) or H+-ATPase and antagonists of the store-operated Ca2+ entry complex blunted, or significantly reduced, the leukotoxin-induced elevation in intracellular Ca2+. Moreover, activation of the ADP-ribosyl cyclase CD38 was also required to initiate the release of Ca2+ from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca2+ from lysosomes, modifies the steady-state level of reticular Ca2+ stores and finally activates the Store-Operated Calcium Entry complex.Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate  Staphylococcus aureus is a common host in human flora that asymptomatically colonizes one in three healthy individuals; children having higher persistent carriage rates than adults  to systemic propagation (bacteraemia) and metastatic infections . The emergence and spread of community-acquired, drug-resistant (CA-MRSA) and extremely virulent strains reinforces the morbidity of S. aureus infections  , a bi-component toxin encoded by S. aureus and from other origins  were initiated by a discharge of Ca2+ from acidic stores and followed by a Ca2+-induced Ca2+ release from the reticulum. These effects were further amplified by the activation of the store-operated Ca2+ entry complex. The initial signal linking leukotoxin binding to the acidic stores was due to the activation of the ADP ribosyl cyclase CD38. This cellular response to the presence of the leukotoxin in neuronal cells preceded the formation of toxin pores into the plasma membrane.In the present study, the question of the sensitivity of neurones to bi-component leukotoxins of et al., S. aureus leukotoxins, glutamate release by cerebellar granular neurones was quantified after a 10 min exposure to the toxins. Dose–response curves for α-toxin, leukotoxins HlgC/HlgB and HlgA/HlgB and the PVL are shown in 2+]i, since pre-incubation of neurones with the intracellular calcium chelator BAPTA-AM (10 μM) (30 min loading followed by 20 min hydrolysis) strongly reduced the amount of glutamate released into the medium  enzymatic activity released into the medium was quantified. An example of G6PDH activity measured after incubation at increasing concentrations of HlgC/HlgB leukotoxin is illustrated in 2+]i variations in cerebellar granular neurones and in sensory neurones from dorsal root ganglia (DRG), using the Fura-2 Ca2+ indicator dye, to analyse the activity of leukotoxins HlgC/HlgB and HlgA/HlgB and PVL . PVL (2 nM) was active in 24% of recorded granular neurones  and the time needed by the cell to reach the [Ca2+]i peak after the addition of the leukotoxin (latency). These values are shown in box plots with the mean, the median and percentiles.The typical reaction of a neurone to the presence of leukotoxin HlgC/HlgB was a progressive elevation in free [Cang value A and B. 2+]i peak was dependent on leukotoxin concentration, as illustrated in examples in 2+]i peak amplitude values increased, while the latency of the response decreased with increasing concentrations of leukotoxin, as shown in 2+]i homeostasis and that its activation may depend on a particular cell's status.In granular neurones, the amplitude of the [Ca2+ channels (VOCC) or glutamate receptors may lead to rise in [Ca2+]i in granular neurones , 0.1 μM ω-GVI-A (N-Type channels) or 0.1 μM ω-AgaTK (P/Q-Type channels) whereas ionotropic glutamate receptors were antagonized with 10 μM CNQX (AMPA receptors) or 10 μM D-AP5 (NMDA receptors) while recording [Ca2+]i variations induced by leukotoxin HlgC/HlgB. Amplitude and latency of [Ca2+]i variations are presented in 2+]i peak (P < 0.05), effect observed also when Na+ channels and glutamate receptors were additionally blocked . The selective blockade of voltage-gated Na+ channels (0.3 μM TTX) doubled the latency of the response . Altogether, these results rule out both VOCC and glutamate receptors as direct passageways for extracellular Ca2+ influx induced by leukotoxin. However, given the impact of the blockade of these channels on the kinetics of the leukotoxin effect, a role for resting [Ca2+]i or membrane potential cannot be excluded.Activation of any of the various types of voltage-operated Ca+]i peak A. Howeve blocked . When th2+, neurones were equilibrated in buffers of various extracellular Ca2+ concentration ([Ca2+]e) prior to incubation with the leukotoxin HlgC/HlgB. As shown in 2+]i peak was reduced by 80% in 30 μM [Ca2+]e relative to controls , with no significant change observed for the latency of the effect. Leukotoxin-induced [Ca2+]i changes could still be observed in neurones incubated in 3 μM [Ca2+]e but not in cells bathing in Ca2+-free buffer , where resting [Ca2+]i remained constant.To ascertain the contribution of extracellular Ca2+ stored in internal compartments was assessed by incubating the neurones in the presence of 1 μM thapsigargin (endoplasmic reticular stores) or 0.2 μM bafilomycin A (acidic stores)  whereas the latency of the response was prolonged . Inhibition of vesicular H-ATPase by 0.2 μM bafilomycin A abolished nearly all leukotoxin-induced [Ca2+]i movements; approximately half of the recorded cells showed delayed low-amplitude transient increases in [Ca2+]i that barely reached twice the amplitude of resting [Ca2+]i. To confirm that Ca2+ release from acidic organelles indeed contributed to the leukotoxin effect, osmotic lysosomal degradation was induced with Glycyl-1-phenylalanine 2-naphthylamide (GPN)  mechanism. Moreover, the contribution of Ca2+ from both the external medium and intracellular stores points towards the Store-Operated Ca2+ Entry (SOCE) complex as a final outcome of Ca2+ mobilization initiated by the leukotoxin.The contribution of Ca stores) B prior t2+]i variations were monitored in the presence of drugs or ions known to interfere with SOCE  reduced the [Ca2+]i amplitude by 44%  and slightly affected the time of the response, but totally abolished the second Ca2+ rise present in 96% of control cells challenged with 4 nM leukotoxin (not shown). The SOCE antagonist econazole (5 μM), totally prevented the toxin-induced rise in [Ca2+]i  and increased the latency of the response by more than 10-fold   and a sixfold delay in latency  (2+ release-activated Ca2+ (CRAC) channels , while the latency of the effect remained unaltered. CRAC channels are formed in the plasma membrane by the association of Orai1 monomers, mediated by the reticular protein STIM1 that senses the ER Ca2+ filling state  responded to the leukotoxin through a low amplitude rise in [Ca2+]I whereas in controls, 90% of the 155 recorded cells mobilized [Ca2+]i  B. Cells < 0.001) B. The py [Ca2+]i C.2+ depletion, the translocation of STIM1 molecules to endoplasmic reticulum–plasma membrane junctions activates the Orai1-formed CRAC channels; simultaneously the formation of STIM1 puncta can be observed using fluorescence labelling methods , an intracellular messenger produced by the ADP-ribosyl cyclase CD38  and a delayed latency   A. Simila< 0.001) A. NAADP  et al.,  that mayet al., et al., n = 94, P < 0.001) along with a reduction in the peak of [Ca2+]i   B.Granular neurones incubated in Alexa 488-tagged HlgC/HlgB leukotoxin (4 nM) were observed by confocal microscopy after fixation and paired labelling the anti-CD38 antibody or Alexa 594-conjugated Cholera toxin-B subunit C and D. 2+ signalling in a variety of cells  from Helicobacter pylori induces the oscillation of free cytosolic Ca2+ through the mobilization of internal stores . The final step (4) involves the activation of CRAC channels induced by the Ca2+ reticular stores depletion.The contribution of intracellular acidic stores as an early step of Ca2+ initiate a related mechanism, regardless of other effects they may induce. Similarly to S. aureus bi-component leukotoxins that evoke intracellular Ca2+ rise in neurones, even though they are known to discriminate between target cells  animal facilities (French government agreement No. B-67-482-25). All procedures regarding housing, feeding and sacrifice of the animals were in accordance with approved European guidelines (No. 86/609/CEE) on animal care and experimentation.S. aureus α-toxin, γ-haemolysins HlgC/HlgB and HlgA/HlgB and the Panton – Valentine leukocidin LukS-PV/LukF-PV where purified as described previously -1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (YM-59483) was from Tocris Bioscience . The voltage-gated channel blockers Tetrodotoxin, ω-Conotoxin GVI-A, ω-Agatoxin TK as well as ryanodine and thapsigargin were from Alomone Labs (Israel). Fura2-AM was from TEFLabs , while Pluronic-127 and BAPTA-AM were from Molecular Probes/Life Technologies . Stock solutions of the drugs were prepared in their appropriate solvent  at 1000× the final working concentration. The Ca2+-free bath solution used in selected experiments was prepared by omitting CaCl2 from the standard solution and adding 10 mM EGTA.All standard chemicals, the glutamate receptor antagonists CNQX and APV, the calcium blockers and modulators nifedipine, dantrolene, 2-Aminoethoxydiphenyl borate D-AP5, Bafilomycin A1, Glycyl-1-phenylalanine 2-naphthylamide and the PI3Kinase antagonist LY-294002 were purchased from Sigma-Aldrich . −1 and DNase (250 U ml−1). Enzymes were inactivated by adding fetal calf serum (10%) and removed by three successive washes in culture media. The tissue was mechanically dissociated through polished Pasteur pipettes of decreasing tip diameter. After removal of tissue debris, cells were centrifuged and the pellet resuspended in culture medium. Cells were seeded on either poly-l-lysine-coated (10 μg ml−1) 24-well plates (Corning) at a density of 3.5 × 105 cells per well for glutamate release studies, in 35 mm glass bottom Petri dishes for microfluorimetry, or on 11-mm-diameter glass coverslips for immunocytochemical studies. Cells were then incubated at 37°C in a 5% CO2-air humidified atmosphere. The culture medium consisted of B27-supplemented Neurobasal medium (Gibco/Life Technologies) and 25 mM KCl, 2 mM l-glutamine and penicillin/streptavidin antibiotics. Growth medium was further complemented with 1 μM insulin, 20 nM progesterone, 0.1 mM putrescine, 80 μg ml−1 transferrin and 0.5 μM sodium selenite. Cultures were also supplemented with 5 μM cytosine β-d-arabinofuranoside 24 h after plating in order to prevent glial proliferation; culture media were renewed every 3 days. Experiments were performed after 8-day differentiation in vitro to allow neuronal stabilization of intracellular Ca2+ stores . Insulin, progesterone, putrescine and human apo-transferrin were from Sigma . Cerebellar cortices from postnatal day 5 (P5) rats were incubated for 5 min at 37°C in trypsin 0.05%–EDTA 0.02% solution, followed by 20 min at 37°C in papain 10 U mlet al., l-lysine-coated bottom-glass Petri dishes at a density of 1 × 105 cells per dish. Culture media consisted of complete Neurobasal medium supplemented with 100 ng ml−1 Nerve Growth Factor . The culture medium was renewed 4 days after plating. Experiments were performed using neurones cultured for 4–7 days.Dorsal root ganglia from as many spinal levels as possible were collected from euthanized P5 rats. The cells were dissociated from the ganglia as described previously  pre-warmed at 37°C. The appropriate toxin concentration was added for an incubation of 10 min. For KCl stimulation, cells were maintained in the depolarizing buffer (same as incubation buffer but with 60 mM KCl and 73 mM NaCl) for less than 10 s, before collecting the media for glutamate determination.−1 HRP, 0.08 U ml−1 glutamate oxydase, 0.5 U ml−1 glutamate pyruvate synthetase, 0.2 mM l-alanine and 100 μM 10-acetyl-3,7-dihydroxyphenoxazine  in 0.1 mM Tris-HCl buffer pH 7.4. The cycling reaction was allowed to proceed at 37°C for 30 min and the increase in resorufin fluorescence was measured using a Mithras LB 940 fluorescence plate reader  at 530 nm excitation and 590 nm emission.Glutamate cycling assay was based on the Amplex Red Glutamic Acid method . Cells were incubated at room temperature for 1–10 min in the presence of 1 nM native or 5 nM tagged HlgC/HlgB. Neurones were then fixed in 4% (v/v) paraformaldehyde and 4% (w/v) sucrose in phosphate-buffered saline (PBS) for 15 min at room temperature and subsequently rinsed with PBS. Neurones were permeabilized and non-specific epitopes were blocked in PBS buffer containing 0.1% (v/v) Triton X-100, 1% (w/v) bovine serum albumin and 5% (v/v) normal goat serum  for 30 min prior to 2 h incubation at room temperature with antibodies diluted in the same buffer. The following primary antibodies were used: the anti-STIM1 (N-19) goat polyclonal antibody and A8 mouse monoclonal antibody; the anti-mouse CD38 (2Q1628) rat monoclonal antibody and M-19 goat polyclonal antibody (Santa Cruz Biotechnology). The plasma membrane was labelled by the cholera toxin B subunit conjugated to Alexa Fluor 594 (Life Technologies). After three washes in PBS, secondary antibodies tagged with Alexa 488 or Alexa 546  were incubated in PBS for 2 h at room temperature. After the immunostaining process, cell nuclei were stained using Hoechst 32258 (Sigma) at a final concentration of 40 nM. After washing in PBS, coverslips were stored at 4°C until observation. A Leica SP5-II confocal microscope was used.The subcellular localization of leukotoxin in granular neurones was detecting using the HlgB subunit tagged with Alexa Fluor 488 C2+] recordings were performed on cells grown on glass coverslips and loaded with 5 μM Fura-2 AM – 0.04% Pluronic 127 for 30 min in the dark at 37°C, washed twice in HBSS and transferred to an inverted epifluorescence microscope  equipped with a UPlanFL 40/0.75 objective. The cells were alternatively illuminated at 350 nm and 380 nm and image pairs of the 520 nm light emission were recorded every 2 s for 30 min. Cells were bathed in HBSS buffer containing 10 mM Hepes buffer (pH 7.2) or, if needed, continuously superfused with the same buffer at a rate of 1 ml min−1. The toxins were applied directly into the bath. For DRG neurones, a solution of ATP  preceded of 1 min the administration of the toxin; this ATP application never induced [Ca2+]i changes. The ratio of fluorescence intensities (Ex350 nm/Ex380 nm) was calculated on a pixel basis for each image pair. The calcium concentration was then estimated by the formula , β = (I380 max)/(I380min) . Values of Rmin and Rmax were determined by periodical calibrations . Differences were considered significant at


In recent years research suggests that astrocyte networks, in addition to nutrient and waste processing functions, regulate both structural and synaptic plasticity. To understand the biological mechanisms that underpin such plasticity requires the development of cell level models that capture the mutual interaction between astrocytes and neurons. This paper presents a detailed model of bidirectional signaling between astrocytes and neurons (the astrocyte-neuron model or AN model) which yields new insights into the computational role of astrocyte-neuronal coupling. From a set of modeling studies we demonstrate two significant findings. Firstly, that spatial signaling via astrocytes can relay a “learning signal” to remote synaptic sites. Results show that slow inward currents cause synchronized postsynaptic activity in remote neurons and subsequently allow Spike-Timing-Dependent Plasticity based learning to occur at the associated synapses. Secondly, that bidirectional communication between neurons and astrocytes underpins dynamic coordination between neuron clusters. Although our composite AN model is presently applied to simplified neural structures and limited to coordination between localized neurons, the principle , and the modeling strategy may be extended to coordination among remote neuron clusters. In particular, the interaction of glutamate with astrocytic receptors leads to transient elevation in astrocytic intracellular Ca2+ levels 2+ transients, astrocytes release gliotransmitters which in turn modulate synaptic transmission by acting both on pre- and post-synaptic receptors. As well as intracellular communication, astrocytes communicate with each other through the propagation of Ca2+ waves, a process which is thought to be mediated via extracellular ATP diffusion and the transmission of inosotil 1, 4, 5-trisphosphate (IP3) through gap junctions. However, the exact nature of this process is still unclear For many years, astrocytes, a subgroup of glial cells found in the brain, have been thought to support neurons by providing them with vital elements needed for their survival tripartite synapse3. This in turn regulates the release of Ca2+ from internal stores, creating a transient increase in Ca2+  on the postsynaptic neuron, mainly located at dendritic spines 2+ waves throughout a glial network Astrocytes also communicate in a feedback mode with neurons and have been found to play key roles in Long Term Potentiation/Depression (LTP/LTD) In this paper, we model neuron-astrocyte interaction and provide evidence which shows that astrocytes have a role to play in LTP/LTD where neuron-astrocyte interactions at a synaptic site can cause plasticity at other remote sites via SICs. Also we show how an astrocytic induced signal can cause dynamic synchronization between neurons. Much evidence indicates that cognitive and behavioral functions rely on flexible coordination among distributed neural activities within and between cortical areas  model uses astrocyte-driven SICs  to provide a teaching signal for learning and to synchronize neural activity between neurons.A feature of the present modeling approach is its constructive nature: it combines and constructs multiple detailed models in order to reveal the regulatory dynamics of astrocytes at a network level. In this it contrasts to larger-scale network approaches in which statistical descriptions of neural populations allow one to derive generic differential equations that describe the evolution of the averaged activity of pools of equivalent neurons (see 3 into the astrocytic cytoplasm. IP3 subsequently binds to IP3 receptors (IP3Rs) on the Endoplasmic Reticulum (ER), a long network of tubes and vesicles used to store Ca2+ within the cell 3 with IP3Rs opens channels that allow the release of Ca2+ from the ER in to the cytoplasm . Whereas individual Ca2+ puffs are incapable of propagating intracellularly, several puffs can raise Ca2+ levels in the cytoplasm beyond a threshold ). From equation (1) it is clear that IP3 levels will be maintained as long as there is an input stimulus to the synapse. Furthermore, IP3 levels will reach a steady state based on the maintained input stimulus frequency, i.e. the higher the input stimulus frequency, the higher the level of IP3 , via the adjustment of different model parameters To describe the Ca2+ dynamics within the Li-Rinzel model are described by three channels. pumpJ which models how Ca2+ is stored within the ER by pumping Ca2+ out of the cytoplasm into the ER via Sarco-Endoplasmic-Reticulum Ca2+-ATPase (SERCA) pumps, leakJ which describes the amount of Ca2+ released by leakage through the ER membrane and chanJ which models the opening of Ca2+ channels by the mutual gating of Ca2+ and IP3 concentrations. Since the model only considers the case of a single cell which exists in a Ca2+-free extracellular environment, no account is taken of any Ca2+ flux across the cell membrane chanJ is the 3IP and 2+Ca dependent Ca2+ release, pumpJ is the amount of Ca2+ pumped from the cytoplasm into the ER via the SERCA pumps, leakJ is the Ca2+ which leaks out of the ER and h is considered to be the fraction of activated IP3Rs. The parameters chanJ channel is given by:Cr is the maximal CICR rate, 0C is the total free Ca2+ cytosolic concentration, 1C is the ER/cytoplasm volume ratio and ∞m and ∞n are the IP3 Induced Calcium Release (IICR) and CICR channels respectively and are given by:Lr is the Ca2+ leakage rate, ERv is the maximum SERCA pump uptake rate and ERk is the SERCA pump activation constant. 0C and ERk as described by Cax, y, and z are the fractions of resources in the recovered, active, and inactive states of the synapse respectively, recτ and inτ are the recovery and inactive state time constants respectively, spt is the time series of presynaptic action potentials, δ is the Dirac delta function and seU is the utilization of synaptic efficacy. In the case of an excitatory synapse, which releases glutamate, these variables can be equated to the dynamics of glutamate release; y can be considered to be the amount of released glutamate while x can be equated to the amount of glutamate stored in the presynaptic vesicle pool ready for transmission. The EPSC that is received by a neuron from synapse i is proportional to the fraction of resources remaining in the active state and is given by:i, seA is the absolute synaptic efficacy and i(t)y is the amount of neurotransmitter released by the synapse at time t.Synaptic information transfer is considered to be probabilistic: on arrival of an AP a vesicle is either released or it is not 2+. Rather than describe this mechanism in a biophysical manner, the gatekeeper model f. When Ca2+ levels within the cytoplasm exceed a set threshold  2+ channels which in turn reduces the flux of Ca2+ during incoming spike trains, therefore reducing the amplitude of EPSCs. The gatekeeper model f, subsequently controlling the amount of neurotransmitter released into the cleft during activity. To model the effects of the glutamate released by the astrocyte in the tripartite synapse the following modifications to equations (12) and (13) are made:Astrocytes influence synaptic transmission/modulation based on levels of intracellular Ca2+ levels within the astrocyte exceed SIC is the NMDA slow inward current, Am controls the magnitude of SIC, S is a stimulus current used to form SIC and decτ adjusts the decay time of SIC. S is modeled by:sm is the magnitude of the stimulus, τS is the decay rate of S and Cat is the time that 2+Ca crosses the activation threshold from below. Research has shown that astrocytic glutamate release is correlated with Ca2+ oscillation peaks. Furthermore, conditions which result in a single Ca2+ peak followed by a sustained Ca2+ plateau lead to a single glutamate release 2+ levels exceed a threshold and no further release is possible until the threshold is crossed again from below. By adjusting the magnitude and decay time of S along with the decay time of SIC it is possible to produce a SIC with similar kinetics as those observed in CA1 neurons Evidence suggests that astrocytic released glutamate acts upon extrasynaptic NMDARs on the postsynaptic neuron to produce large SICs Research has also shown the existence of extrasynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) Δt is the time difference between the crossing of the Ca2+ threshold and the previous independent presynaptic stimulation. F(Δt) is given by:2+ crossing. Note that 100 ms is typical of a plasticity window.To reflect these SICs in the total current supplied to the postsynaptic neuron, we modify equation (15) to:m is the membrane time constant, v is the membrane potential and mR is the membrane resistance. If v is greater than the firing threshold (thv) then v is clamped at 0 V for 2 ms; this implements the refractory period of the neuron.Although many neuron models exist δω(Δt) is the weight update of the synapse, Δt is the time difference between pre- and postsynaptic events, +A and −A are the maximum value of weight potentiation and depression respectively and +τ and −τ control the decay rate of δω(Δt). Equation 21 suggests that seA can be interpreted as the weight of the synapse: by varying this value it is possible to change synaptic strength. Therefore, we relate seA to δω and formulate an update rule as:SOA is the previous synaptic weight, δω is the amount the weight has changed according to STDP and K ( = 10−12) is a scaling factor.Since Donald Hebb first suggested that a neuron must fire shortly before or at the same time as a neuron to which it is connected in order to strengthen the connection between them 3 levels become too large there is a cessation of Ca2+ oscillation 3 levels within the astrocyte can be degraded depending on the level of Ca2+ within the cytosol 3 may still reach levels which cause cessation of Ca2+ oscillations. In contrast, it is believed that Ca2+ oscillations can be initiated within discrete microdomains and may be localized or propagate intracellularly by activating neighboring microdomains 2+ oscillation based on the stimulus level present at the synapse. Although the Ca2+ level within the cell will differ depending on the spatial location of the stimulated microdomains and the propagation properties of the oscillations, capturing delay in the absence of supporting experimental data seems premature. Therefore, propagation delays are ignored in our model and the aggregate Ca2+ level remains the same everywhere throughout the cell given by:2+ within the cytosol at time t and 2+ within the ith microdomain at time t.Recent research suggests that when employing the Li-Rinzel if IP3 in equation (1) is set at 0.16 µM n):Given that the baseline level of IPIn all the results presented here, Matlab 2009a platform (Windows version) by Mathworks was used to realize the AN model and the Euler method of integration was used for simulation. A fixed time step of 2+ oscillation in each of the AM, FM and AM-FM modes. This is followed by an investigation into the role of extrasynaptic NMDARs in providing a remote supervisory learning signal. Finally, we explore how the combination of pre- and post-astrocytic feedback affects coordination of neural activity. All astrocyte and synapse parameters used in these experiments can be found in Initial simulations provide insight into the valid range of synaptic input frequencies that cause Ca2+ model i.e. AM, AM-FM and FM. The purpose of the simulation (the full AN model including the gating function f and NMDA SICs) is to determine the frequency ranges that result in sustained Ca2+ oscillation for each of the three modes. 3 to enable a burst of Ca2+ release from the ER (see example shown in 3 (0.16 µM) is very close to the level at which Ca2+ release can occur 3 production to such a degree that the negative feedback signal f can not prevent IP3 levels reaching a steady state. Consequently, oscillations fail to cross 2+ threshold. Examples of this can be seen in In this simulation, presynaptic neuron A stimulates a tripartite synapse with a sustained Poisson spike train for 100 s see . The freHere we show how a learning signal, distributed spatially via an astrocyte, can promote STDP-based plasticity at remote synaptic sites.2+ oscillation using the parameters provided in One major requirement of STDP is that the pre- and post-synaptic neurons must fire within the plasticity window. If we consider the case where the synaptic efficacy of the synapse is insufficient to produce a postsynaptic AP then the STDP rule cannot activate learning. We propose that spatial neuron to neuron signaling using large astrocytic NMDA SICs promotes postsynaptic neuron activation. While some doubts have been expressed 2+ oscillation within the astrocyte and the gating function f. When the Ca2+ oscillation passes 2+ oscillation (y) released by the axon  and N2 (B) while in AM mode. Given that there is a microdomain associated with each synapse, the response of which will be very similar to the same Poisson stimulus, f function. 2+ level within the astrocyte while (E) and (F) describe the output firing activity of N1 and N2 as a result of the AN model. Observe that while the background firing activity of N1 and N2 is not coordinated, both N1 and N2 burst at the same time provided presynaptic inputs are present at N2  and the gating activity of f (red line). It is interesting to note that when the Caled by f . It shouThis type of simulation was repeated for all frequencies within the valid frequency range see  of both 2+ threshold within our model can be treated as an open parameter we investigated its effect on coordination. The same simulations as previously presented were again repeated with the threshold varying from 1.44 µM to 3.94 µM in steps of 0.5 µM per trial. The Ca2+ threshold was found to have a significant impact on the coordination of the neurons. Firstly, if the threshold was beyond the level reached by the total Ca2+ oscillation, no coordination in the form of synchronized bursts from N1 and N2 occurred. 2+ oscillation and neural coordination when the threshold is set at 3.94 µM and 1.44 µM with all synapses stimulated with a 7 Hz Poisson spike train.Since the Ca2+ threshold was raised it was found that the timing of the onset of the first burst in both the AM and AM-FM modes was changed: the higher threshold delayed the onset. In both cases the initial burst was followed by bursting that was synchronized to the total Ca2+ oscillation. Furthermore, in both cases the activity of N1 and N2 changed between the periods of bursting activity: as the threshold was increased the f function was activated for much shorter periods. Therefore, the duration of the silent periods of neural activity was reduced as a result of shorter periods of depression by f. Secondly, as the Ca3 levels and the individual Ca2+ oscillations of these synapses ceased. Reduction in synaptic depression was also found to change the valid frequency range. As the threshold was increased it was found that when frequencies at the upper limit of the valid input frequency range were used to stimulate the synapse, the reduction in synaptic depression could not prevent the synapses associated with N1 from producing saturated IP2+ oscillation and IP3 transients of each synaptic microdomain while 2+ oscillation, (B) the f function and (C–D) the output firing activity of N1 and N2. From these results we note that the phases of the individual Ca2+ oscillations are changed. This is especially evident from 2+ is much more sinusoidal than in previous experiments yet coordination is still maintained.Next we investigated dynamic coordination when Poisson spike trains with different average frequencies were applied to each synapse of N1 and N2 in both modes. 2+ oscillation and IP3 transients of each synaptic microdomain while 2+ oscillation, (B) the f function and (C–D) the output firing activity of N1 and N2. From these results we note that there is no noticeable phase locking of the individual Ca2+ oscillations: the coordination of N1 and N2 is not maintained at regular periods as in the AM mode due to the irregular total Ca2+ level. The results of these simulations suggest that the negative feedback and IP3 regulation provided by the f function help maintain the coordination of N1 and N2. Phase locking of the individual Ca2+ oscillations appears to occur and the total Ca2+ oscillation resembles a sinusoid. Further simulations in which the f function was removed revealed in all cases that coordination was lost and the total Ca2+ oscillation became much less sinusoidal.2+ oscillations, the previous two simulations were repeated with all synaptic stimuli in succession with a delay of 2 s between each stimulus. Despite the fact that the phase of each individual Ca2+ oscillation is now different, in AM mode it was found that the individual oscillations are successfully phase locked  to one synapse. We then investigated each combination of input stimulus frequency (in steps of 1 Hz) and different phases by repeating each experiment with a different starting time for the synapse of N2  for the valid input frequency range of each mode. In the AM mode it was found that phase locking of the individual waves was achieved for the entire valid input frequency range as long as the phases were not 2+ oscillations were between 2+ oscillation still crossed the threshold from above and below. However, coordination of the N1 and N2 was often infrequent and not maintained.Finally we investigated the extent to which phase locking can occur between different CaThe AN model presented in this paper captures the bidirectional coupling between astrocytes and neurons and in so doing demonstrates that positive and negative feedback to extrasynaptic NMDARs and presynaptic mGluRs significantly contributes to the regulatory capability of astrocytes.2+ levels rise above a threshold due to stimulation from a neuron or neurons, glutamate is released which in turn activates large SICs which can induce postsynaptic firing in other neurons, thereby causing STDP related weight potentiation/depression. This learning relies on a presynaptic stimulus and astrocytic induced SICs. However, after the synaptic weight is strengthened significantly, presynaptic stimulation alone is sufficient to cause firing of the presynaptic neuron and the synaptic weight grows uncontrollably. Such instability is in agreement with other research Experimental evidence indicates that astrocytes have a role to play in LTP/LTD. Deficiency of GFAP , which is predominantly expressed by astrocytes in the CNS, has been found to enhance LTP and impair LTD 2+ into the dendrite causing endocannabinoids to be synthesized and subsequently released from the dendrite. However, the exact release machinery related to this process is not fully understood When the postsynaptic neuron is sufficiently depolarized voltage gated-channels allow the influx of Ca2+ oscillations and the Ca2+ threshold responsible for the release of gliotransmitter directly impact on the pattern of synchronization. Extrasynaptic NMDAR activation was needed to provide coordinated periods of bursting activity. We also found that negative feedback of presynaptic mGluR activation was important for maintaining neural coordination via phase locking of the Ca2+ oscillations. In exploring the effects of out of phase oscillations we expected the total Ca2+ oscillation to be a complex wave comprising the sum of the individual waves. These should be out of phase due to the different initiation times of the input spike trains. However, our results suggest that over a period of time and under certain conditions Ca2+ oscillations may become phase locked due to the negative feedback provided by f. The latter arises from modulation of neurotransmitter in the cleft of all synapses associated with the astrocyte and consequently the IP3 level in the astrocyte. This process leads to gradual alignment of Ca2+ oscillations in each microdomain that, when successful, provides a simple sinusoidal like total Ca2+ oscillation. Moreover, it has recently been suggested that oscillations within microdomains of astrocytic distal processes differ from oscillations at the soma 2+ oscillation which may be considered to occur at the soma. Despite this simplistic assumption our results show that when phase locking cannot be achieved, oscillations within the distal processes and the soma are indeed different.The AN model also shows that astrocytes have a key role in the dynamic synchronization of neurons. The SICs and neurotransmitter modulation induced by astrocytic release of glutamate causes coordinated neural activity in remote neurons. Our results suggest that the frequency of input stimuli, the phase relation of the astrocytic Ca2+ oscillation becoming much flatter and less sinusoidal. However, when both input stimulus frequencies were below 12 Hz and again in antiphase, coordination was still possible. The reason is that lower frequencies produce lower amplitudes: as a result, it was still possible for the fluctuation of Ca2+ to cross the threshold and release glutamate, thus producing global SICs responsible for coordination. On the other hand, when both frequencies were above 12 Hz and in antiphase, coordination was no longer possible as the fluctuations of the total Ca2+ were not sufficient to continually cross the Ca2+ threshold. Consequently, no further global release of glutamate from the astrocyte was possible and the SICs died off. In AM-FM mode phase locking occurred much less frequently and was only achieved when the individual Ca2+ oscillations were between 2+ oscillation crossed the threshold from above and below.Our results also suggest that even though phase locking can occur it is not vital for coordination. In AM mode, when both waves were approximately in antiphase, phase locking could not be achieved, the total Ca2+ signal pathways are dynamically changing causing ongoing phase shifting between bursting activity across the clusters. If we assume that the “envelope” of a rhythm correlates with the times of maximum bursting then the frequency of the brain oscillation depends on cluster locations and consequently on delays. Moreover, the phase locking characteristics of our model may also provide a mechanism for dynamically changing the coordination between neuron ensembles. Since phase locking was found to occur between microdomain oscillations with approximately the same frequency of oscillation, it is not inconceivable that stimuli of similar frequency may cause a unique pattern of oscillation which changes as the stimuli frequencies change.Although propagation delays were not included in the present version of the AN model it would be interesting to investigate intercellular delays and the propagation time for waves across astrocyte networks. For example, it is likely that dynamic coordination across neuron clusters, mediated via astrocyte networks, is important for the brain rhythms underlying cognitive function. Dynamically altering the cluster size and with that their spatial location may mean that the Ca2+ threshold responsible for releasing glutamate from the astrocyte has an effect on the valid input frequency range that causes Ca2+ oscillations in each microdomain. Furthermore, this threshold may also have an impact on the pattern of coordination between neurons. When phase locking occurs the total Ca2+ oscillation is sinusoidal like and therefore the bursting activity is regular and periodic. In both modes many of the higher input frequencies caused the total Ca2+ oscillation to cease crossing the threshold. This condition may be avoided, however, if the threshold is raised. More biological experimentation is required to establish the key parameters governing the threshold level.Our model shows that the Ca2+ oscillations are related to the frequency of the input stimuli and dependent on IP3 and its rate of change (ip3r) which, in our model, is not constrained and therefore can be treated as an open parameter. Therefore, changing the value of ip3r would make the Ca2+ oscillations sensitive to a band of different input frequencies thereby providing a possible biophysical mechanism for receptive fields.CaIt is intriguing that the frequency of phase synchronization in AM mode is of the order ∼0.1 Hz, in line with the low frequency Blood Oxygen Level-Dependent (BOLD) oscillations used to identify large scale brain networks and their properties from functional Magnetic Resonance Imaging (fMRI) e.g. . This fi2+ oscillations to other non stimulated astrocytes in situ experimentation which showed that glutamate released as a result of hippocampal neuronal activity results in both intracellular Ca2+ oscillations and intercellular waves (a wave which propagates through an astrocytic network) in vivo studies showed that astrocytes exhibit Ca2+ transients that travel much faster within the astrocyte syncytium than previously expected. Furthermore, Ca2+ transients have also been reported to be spontaneous and independent of neural activity 2+ waves are a result of external synaptic stimulation, coordinated spontaneous activity or a mixture of both 2+ waves, their ability to traverse relatively long distances suggests that coordination may not be solely local in nature, but may also be a means to realize flexible global communication across remote networks of neurons.Astrocytes, like neurons, also form interconnected networks where the communication between astrocytes is via gap junctions. Early research using cultures of hippocampal astrocytes showed that they were not only excitable by external stimulation but were also able to transmit or propagate intracellular CaIt is worth noting that the various model systems encompassed by the AN model were chosen to reduce computational overhead while still remaining biophysically meaningful. Thus, the AN model can form an important building block to explore global communication across large remote clusters of neurons via astrocyte ensembles. Extension of the AN model to include intercellular signaling similar to those described in 2+ concentration in astrocytes 2+ fluctuations and the changes that these fluctuations trigger.The present modeling results strengthen the hypothesis that astrocyte networks provide much more than structural support to neural networks. Indeed astrocytes are viewed as regulators of neural circuitry through coordination of transmission at synaptic junctions. Moreover, it is also believed that retrograde messengers induced in the postsynaptic neuron can be fed back either directly or via an astrocyte to receptors on the presynaptic neuron Figure S13 generation within the astrocyte cytoplasm.IP (A) The evolution of IP3 within the cytoplasm of the astrocyte as a result of a range of Poisson generated spike trains stimulating the tripartite synapse. Note that IP3 builds much faster than it decays which can be seen after 10 s when the input ceases. (B) Same experiment as (A) except the Poisson distributed spike train is maintained. Note how the level of generated IP3 is limited to a steady state value and is dependent on the stimulus frequency.(TIF)Click here for additional data file.Figure S22+ oscillation.Examples of no Ca (A) AM mode with input stimulus frequency set at 3 Hz. (B) FM mode with input stimulus set at 7 Hz. Note that in both cases the levels of IP3 are insufficient to cause a Ca2+ oscillation.(TIF)Click here for additional data file.Figure S32+ oscillations outside of the valid frequency range.Examples Ca (A) AM mode with the input stimulus frequency set at 18 Hz. (B) FM mode with the input stimulus frequency set at 36 Hz. (C) AM-FM mode with the input stimulus frequency set at 11 Hz. In (A) AM mode this frequency causes Ca2+ to oscillate at a point at which it no longer crosses the threshold from above, therefore the gating function ( f ) remains active and in a state of depressing neurotransmitter release from the synapse. However, this negative feedback is insufficient to reduce the transmitter to which IP3 is degraded sufficiently to allow Ca2+ to drop below the threshold level. As a result IP3 reaches a steady state at which no further crossing of the threshold is possible. This is also the case with (B) and (C); however, in these cases the steady state level of IP3 now causes a cessation of the Ca2+ oscillation also.(TIF)Click here for additional data file.


RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+] <1 µM, ß-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The aK and maximum levels of cytoplasmic Ca2+ activation site were not affected by ß-adrenergic stimulation.Here we investigate how ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß During this response, increased catecholamine concentrations stimulate cardiac β-adrenergic receptors, resulting in adenylyl cyclase activation, increased cyclic AMP and increased activity of cyclic AMP-dependent protein kinase A (PKA). Increased intracellular  causes contraction. Diastolic relaxation occurs with cessation of Ca2+ release and Ca2+ sequestration by the SR Ca2+ uptake transporter (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) Systolic contraction follows activation of sarcolemmal voltage-gated L-type Ca32P incorporation assays and known to be phosphorylated in vivo; namely S2808/S2809, S2814/S2815 (depending on the species) and S2030 in vitro phosphorylation sites on RyR2 -adrenergic stimulation is limited by a lack of knowledge of how RyR2 is regulated by phosphorylation within the cell. Most investigations show increased RyR2 activity in lipid bilayers with in vitro phosphorylation at these sites by exogenous PKA and CaMKII 2+2+ inhibition -adrenergic stimulation. Therefore, just how ß-adrenergic signaling alters regulation of RyR2 by luminal and cytoplasmic Ca2+ and Mg2+ is unknown. In this study, we perfused isolated rat hearts with the ß-adrenergic agonist, isoproterenol, then incorporated RyR2 from these hearts into artificial lipid bilayers whilst preserving their phosphorylation state. The response of RyRs to luminal and cytoplasmic Ca2+ and Mg2+ indicated significant and novel changes in RyR2 function associated with increased RyR2 phosphorylation at S2808 and S2814 induced by ß-adrenergic stimulation. Since it is not feasible to measure RyR2 regulation under diastolic [Ca2+] and [Mg2+] (their activity is too low), we used a RyR2 gating model to predict the effects of ß-adrenergic stimulation on RyR2 activity within cells under diastolic conditions.Three phosphorylation sites have been confirmed by Healthy male adult rats (Sprague-Dawley) were heparinized , and anesthetized with Isoflurane. Hearts were rapidly removed and immediately perfused via the Langendorff method . Homogenization was carried in 3×15 s bursts with a rotor-stator homogenizer (HD Scientific) followed by 10 manual strokes of a loose glass/glass Dounce homogenizer. The homogenate was then centrifuged at 7000 rpm for 20 min in Beckman Optima L-100XP Ultracentrifuge. The resulting supernatant was centrifuged at 50000 rpm for 30 min. The pellet from this step was re-homogenized with a tight manual glass/glass Dounce homogenizer in homogenization buffer also containing 0.65 M KCl , incubated for 30 min and then centrifuged at 7000 rpm for 15 min. The supernatant was centrifuged at 50000 rpm for 1 h, and the resulting pellet was resuspended in storage buffer, snap frozen in liquid nitrogen and stored at −80°C. The NaF present in our buffers was to prevent ongoing activity of phosphatases (PP1 and PP2A) during SR vesicle preparation. The whole procedure was carried out at 4°C.cis (cytoplasmic) and trans . For vesicle fusions the cis solution contained (mM): 250 Cs+ , 1–5 CaCl2 , and 500 Mannitol ; while trans solution contained (mM): 50 Cs+ , 0.1 CaCl2 and 10 TES. For channel recordings, [Cs+] in trans solution was raised to 250 mM by adding an appropriate volume of 4 M CsCH3O3S. During experiments the composition of the cis solution was altered by local perfusion, which provided solution exchange within ∼1 s trans solutions was altered by addition of aliquots of stock solutions. All experiments were performed at room temperature (21–26°C).Lipid bilayers were formed form a mixture of phosphatidylethanolamine and phosphatidylcholine (8∶2 wt/wt) in 50 mg/ml n-decane. Lipid bilayers were formed across a delrin hole (approximately 100 µm diameter), separating two experimental compartments, N-tris [hydroxymethyl] methyl-2-aminoethanesulfonic acid; ICN Biomedicals) and adjusted to pH 7.4 by CsOH , using a TPS digital pH meter. Free [Ca2+] ≤3 µM was buffered using 4.5 mM BAPTA  ethane- N, N, N’, N’- tetraacetic acid; Invitrogen) and additional 1 mM dibromo BAPTA was used for 10 µM free Ca2+. Sodium citrate was used to adjust free [Ca2+] between 10–50 µM. A Ca2+ electrode (Radiometer) was used to determine free [Ca2+] in the experimental solutions and the purity of Ca2+ buffers and Ca2+ stock solutions using titrations against a 100 mM CaCl2 standard (Fluka).All solutions were made with MilliQ water and were pH buffered with 10 mM TES . The ratio of fluorescence intensities at 340 and 380 nm were calibrated in the experimental solutions  also containing 5 µM Mag-fura-2, 4.5 mM BAPTA, (free [Ca2+] <1 µM) and MgCl2 from aliquots of a calibrated stock.All recording solutions contained ATP , which buffers MgStandard techniques were used for SDS PAGE and Western Blotting . We also used an antibody against dephosphorylated S2808 (DepS2808) as a cross check for pS2808. The average degree of S2808 phosphorylation was quantified as both pS2808 immunostaining relative to maximum PKA phosphorylation and also DepS2808 staining (1- DepS2808 staining) relative to minimal phosphorylation in PP1 incubated samples. Similarly, S2814 phosphorylation was expressed as pS2814 immunostaining relative to maximal phosphorylation levels achieved using calcium calmodulin (CaCaM) to activate endogenous CaMKII. Maximal and minimal phosphorylation levels were determined from the time courses of phosphorylation during incubations with PP1, PKA and CaCaM . Significance was calculated by Student’s All experimental procedures were approved by the University of Newcastle Animal Care and Ethics Committee (A-2009-153).Mean heart rate was 220±18 bpm . S2814 phosphorylation in control hearts was not different to RyR2 dephosphorylated by PP1  with 2 mM ATP and the luminal bath contained 0.1 mM Ca2+. We investigated the concentration dependencies of cytoplasmic and luminal Ca2+]. RyR2 was dephosphorylated in-bilayer by adding PP1 (7–10 units) to the cis bath for 5 min either by local perfusion or by aliquot addition of PP1 cis chamber was perfused with solutions lacking PP1 and with cytoplasmic [Ca2+] of 100 nM or 10 µM and the same channel was recorded again (recordings shown in 2+ (2+ (n = 7), PP1 did not change either opening rate or mean open duration . oP) due to an increase in RyR opening rate whereas PP1 incubation led to a 15-fold decrease in oP due to decreases in both opening rate and mean open duration.RyR2 was dephosphorylated to assess whether changes in RyR2 activity after ß-adrenergic stimulation were due to increased RyR2 phosphorylation. The activity of RyR2 from control and isoproterenol stimulated hearts was measured with 100 nM and 10 µM cytoplasmic  (minP) with no significant effect on aK for activation or maximal Ca2+ activation (maxP), suggesting that the Ca2+ sensitivity of the cytoplasmic Ca2+ activation site of RyR2 was not affected by isoproterenol. The Ca2+ dependencies of oP were also reflected in RyR2 opening rate .RyR2 from control and isoproterenol stimulated hearts were strongly activated by micromolar cytoplasmic Ca µM Ca2+  and inhiing rate . At sub 2+ from 10 to 100 µM. 2+]-dependencies of the oP, opening rate (ok) and mean open time (oT) of control and isoproterenol treated RyR2 with diastolic (100 nM) cytoplasmic [Ca2+]. In the absence of luminal Ca2+, RyR2 from control and stimulated groups had a mean o = P0.003 and exhibited a bell-shaped luminal [Ca2+]-dependence with peak activity occurring in the presence of ∼100 µM. Peak oP of RyRs from the isoproterenol-stimulated hearts was 4-fold larger (oP = 0.045) than control  was previously shown to be due to Mg2+ competing with Ca2+ at cytoplasmic and luminal Ca2+ activation sites;  in RyRs from control and stimulated hearts (2+ inhibition of oP (iK (p = 0.003) by 4.5-fold compared with control  than control, indicating decreased Mg2+ inhibition via the low affinity divalent binding site.We also investigated effects of adrenergic stimulation on Mgmic Ca2+ . We foun2+ activation properties of rat RyR2s in the presence of physiological concentrations of cytoplasmic Mg2+ (1 mM) and in the presence of 1 mM luminal Ca2+  and cytoplasmic domains (A-site) of RyR2 have Ca2+ affinities of 8 µM and 2 µM, respectively. These sites trigger a common gating mechanism to produce synergistic activation by luminal and cytoplasmic Ca2+. The cytoplasmic domain also possesses two inhibitory sites with Ca2+ affinities of 0.4 µM (I1-site) and 1.5 mM (I2-site). Magnesium, which competes with Ca2+ at the L-, A- and I2-sites, inhibits RyRs and shapes the Ca2+-dependent activation of RyRs 2+ and Mg2+ can pass through open channels and act at cytoplasmic facing sites.The RyR2 gating model considers Ca2+  The opening rate in response to Ca2+ binding to the luminal Ca2+ activation site (L-site) increased 20-fold, accounting for the increased opening rate at low cytoplasmic [Ca2+]  was slowed 2-6-fold, accounting for the increase in mean open duration (2+/Mg2+ inhibition site (I1-site) was reduced by 40%, accounting for the reduced Ca2+ inhibition (2+ inhibition in the presence of 10 µM cytoplasmic Ca2+ (2+ activation site (A-site). Model fits to the data indicated a 25% change in the Mg2+ affinity of the A-site which accounted for the systematic, though non-significant increase in RyR activity in RyR opening rates and mean open durations , and 6 w2+ . None thc [Ca2+]  and incring rate . 2) Therhibition , 3) RyR2duration . 4) The hibition  and Mg2+mic Ca2+ . Interes2+ in cytoplasm and lumen is 1 mM, 2) Ca2+ and Mg2+ fluxes through RyR2 are slightly larger in the presence of intracellular [K+] than with 250 mM Cs+ and that 3) intracellular Ca2+ buffering is substantially weaker and slower in the cell than in our bilayer solutions. Assumptions 2 and 3 affect the feed-through parameters, AX and IX in the model , the model predicts a RyR2 open probability in the range 10−7 to 10−6 which is far too small to be measured by single channel recording. Under these conditions, ß-adrenergic stimulation increases RyR2 oP by 20-fold, which was due to an increase in the ability of luminal Ca2+ to activate the RyR (The model predicts that under systolic conditions where cytoplasmic [Ca RyR2 Po . This isand Mg2+ . Under d the RyR .1- and I2-sites during ß-adrenergic stimulation contribute to increasing RyR2 oP under both diastolic (2+ binding to the L-site contributes a 3-fold increase in RyR2 activity during ß-adrenergic stimulation (2+)), decreased Mg2+ inhibition at the L-site contributed another 50% increase (2++Mg2+)) and reduced cytoplasmic Mg2+ inhibition contributed another 3-fold increase in activity (1-site and A-site contributed ∼1.7-fold each). The I2- and A-sites made no significant contribution to ß-adrenergic stimulation. In systole, the only significant contribution to ß-adrenergic stimulation came from a decrease in cytoplasmic Mg2+ inhibition  and supra-physiological luminal Ca2+ (50 mM). However, the degree of RyR activation seems to depend on the presence of ATP and Mg2+. In the absence of ATP and Mg2+, isoproterenol stimulation caused a 15-fold increase in RyR2 activity and less than a 2-fold increase in the presence of ATP 2+. Here, we present the first investigation of how ß-adrenergic stimulation alters the various Ca2+ and Mg2+ regulation mechanisms in RyR2 and their relative roles in physiological Ca2+ release from the SR. We identify four actions of ß-adrenergic stimulation on RyR2 gating which we associate with three mechanisms: #1) a 3- to 5-fold increase in RyR2 activation by luminal Ca2+ (2+ (2+ binding site (L-site) 2+ inhibition at mM concentrations  increased RyR2 mean open durations (2+ inactivation (I2-site). The cytoplasmic Ca2+ activation, associated with the cytoplasmic Ca2+ activation site ; consistent with the finding that exogenous CaMKII removes Mg2+ inhibition with 5–10 µM Ca2+et al.Regulation of RyR2 by phosphorylation and luminal Cain vitro Langendorff perfusion method so that hearts could be snap frozen within one minute of administering isoproterenol.  perfusion of isolated rat hearts, 2) data acquisition and analysis, 3) SDS PAGE and Western Blotting, 4) in-vitro Exogenous PP1, PKA and endogenous CaMKII activity assays and 5) a summary of heart rates and phosphorylation levels determined by Western Blots for each heart used in this study (Table S1). Additional details of the model are included in Tables S2 and S3.(DOCX)Click here for additional data file.File S2Figures of supporting information on the analysis of phosphorylation of RyR2 in Western Blots. These figures demonstrate 1) the feasibility of reprobing Western Blots, 2) the time course of PP1 incubation of SR vesicles, 3) the time course of binding by antibodies to phospho-S2808 and phospho-S2814 in response to PKA and CamKII incubation, 4) that endogenous phosphatases do not alter S2808 phosphorylation in bilayer experiments and 5) that ATP does not alter S2814 phosphorylation in bilayer experiments.(PDF)Click here for additional data file.