In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca2+ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012–2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types  that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca2+, in addition to Ca2+ signals evoked by an InsP3-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca2+ store emptying and store-operated Ca2+ entry following application of the Ca2+ATPase inhibitor thapsigargin. Multiple parameters  were analysed to explore potential impact of radiofrequency field exposure on Ca2+ signals. Our data indicate that 900 MHz GSM fields do not affect either basal Ca2+ homeostasis or provoked Ca2+ signals. Even at the highest field strengths applied, which exceed typical phone exposure levels, we did not observe any changes in cellular Ca2+ signals. We conclude that under the conditions employed in our experiments, and using a highly-sensitive assay, we could not detect any consequence of RF exposure.In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an “electromagnetic smog”, with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca Some of this concern was fostered by the results of experiments performed 30–40 years ago, which examined the biological effects of radio frequency (RF) waves on the brain, and in particular on the influence of modulated RF fields on Ca2+ fluxes. Adey and colleagues reported that RF electromagnetic fields influenced Ca2+ within tissues following experiments measuring 45Ca2+ fluxes in excised chick brain 45Ca2+ efflux in samples exposed to specific RF signals.In recent years, the widespread use of mobile phones has been accompanied by concern over their possible effects on health  and modulation frequencies (e.g. 16 Hz), was found to have a significant effect on Ca2+ ion efflux In addition to studies using excised brain tissues, putative effects of RF fields have been examined using cultured neurons where experimental conditions can be rigorously controlled. For example, exposure of 2+ ion concentration The development of fluorescent indicators capable of reporting intracellular Ca2+ changes in cardiac myocytes in the presence of GSM pulsed RF fields, and reported a small effect when the cells were exposed to 900 MHz fields that were modulated at 50Hz 2+ currents 2+ in cultured neurons 2+ was also monitored in cultured rat cerebellar granule cells and cardiac myocytes. The biological effects of both CW and TETRA  pulsed RF fields were studied at a carrier frequency of 380 MHz and a wide range of power densities (5–400 mW/kg) 2+ were observed in cells exposed to either the CW or TETRA pulsed RF conditions.Meyer and colleagues examined Ca2+ homeostasis in mammalian cells. Given that Ca2+ is a critical cellular messenger, it is vitally important to establish whether such an interaction is plausible. We therefore applied a novel microscope-based high throughput screening system that allowed the automatic analysis of mammalian cells under different exposure regimes. In addition, we used fluorescent indicators that provide high sensitivity and good signal to noise reporting of cellular calcium concentration in within living cells.The studies described above illustrate a discrepant and controversial literature concerning the impact of RF fields on CaAll experiments conformed to guidelines laid out by the UK Home Office; animals were killed using authorised Schedule 1 procedures according to Home Office guidelines. Furthermore, all procedures were approved by Babraham Institute Animal Welfare and Ethics Committee.2 atmosphere of the imaging chamber to be monitored, controlled and matched to the buffers appropriate for the cell types under study.A customized version of the Discovery-1  fluorescence imaging screening system was used in all experiments. A schematic drawing of all the major components is presented in philippe.leveque@unilim.fr). An experimental estimate of the SAR was also obtained by measuring the temperature change in the sample with a fluoroptic temperature probe  when exposed to a continuous power of 20 W. The SAR was derived from the change in temperature (ΔT°) produced over time (Δt), given the specific heat capacity (C) of the sample and following the relation, SAR = C ΔT°/Δt [W/kg], as shown in The RF exposure system was based around an open transverse electromagnetic (TEM) cell . SimilarThe TEM cells were mounted side by side on the motorized stage of the Discovery-1 microscope. The arrangement of the TEM cells was chosen to facilitate placement of the mounted coverslips into the microscope light path. Acquisition software alternated the image capture between the two TEM cells during the course of an experiment. In this way, exposed and sham-exposed samples were subjected to identical physical conditions. An Agilent E4432B Signal Generator produced an industry standard GSM 900 MHz (1/8 time) signal with a 217 Hz recurrence frequency, as well as the 900 MHz continuous wave (CW) signal when GSM modulation was toggled off. The state of the RF switch was set and logged by software, which directed the signal into either the left-hand or right-hand TEM line. A vector network analyzer was used to measure the isolation between the two TEM cells and evaluate whether the RF field in the energized condition could influence the other TEM cell. The transmission coefficient between the two TEM cells was −30 dB (data not shown), indicating a negligible interaction.The majority of experiments used 900 MHz GSM moduated RF fields. The SARs of handsets differ; however, mobile phones sold in the UK produce an average SAR of less than 2 W/kg to meet guidelines set by ICNIRP 5 cells per ml in 25 cm2 culture flasks (Nunc). Cells were maintained under standard culture conditions  in a conventional culture incubator. Fresh media was introduced every 3–4 days and the cell line maintained by reseeding 1–2×105 cells per ml into a fresh 25 cm2 flask during the exponential growth phase. At the same time, ∼2–4000 cells were seeded onto 22 mm glass coverslips  and placed into individual 35 mm culture dishes (Nunc) containing 2 ml of fresh culture media. Cells were allowed to grow to 80% confluence.EA.hy926 human endothelial cells  were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% foetal bovine serum (Invitrogen) and 2% L-Glutamine-Penicillin-Streptomycin solution (Sigma). In an attempt to reduce variability, a single batch of foetal bovine serum was used to supplement the culture media used during all experiments. Cells were seeded at an initial density of 1–2×10PC12 cells were obtained from the American Type Culture Collection, and have been described previously 2+ oscillations.Primary rat hippocampal neurons (isolated from rats postpartum day 2–3) were prepared using established procedures and seeded onto 22 mm coverslips 2+ indicator Fura-2 AM (Molecular Probes) as described previously 2 0.8 mM; CaCl2 1.8 mM; NaHCO3 6 mM; D-glucose 5.5 mM; HEPES 25 mM, pH 7.3) containing 1 µM Fura-2 ester for 45 min followed by a 30 min de-esterification period in HBSS solution at room temperature. Imaging was performed using a 10× objective with the Discovery-1 by alternately exciting the Fura-2 dye at 340 nm and 380 nm. Emitted light was filtered at 540 nm and collected with a cooled CCD camera . The intracellular Ca2+ concentration was calculated from background corrected fluorescence images (F) according to the equation, Ca2+ concentration = Kd×[(R−Rmin)/(Rmax−R)] 2+ (Rmin) was determined by incubating cells with 1 µM ionomycin solution in Ca2+ -free buffer containing 1 mM EGTA. The fluorescence at maximum Ca2+ (Rmax) was determined by incubating cells with 1 µM ionomycin in Ca2+-containing buffer.Coverslips containing a monolayer of adherent EA.hy926 or PC12 cells were loaded with the ratiometric Ca2+ indicator. Cells were incubated in standard HBSS solution containing 2 µM Fura-PE3 AM for 45 min followed by a 30 min de-esterification period in HBSS solution. Imaging was performed at 37°C using the Discovery-1 system by alternately cells loaded with Fura-PE3 at 340 nm and 380 nm. Emitted light >540 nm and collected with a cooled CCD camera and analyzed in the same manner as mentioned for other cell types.In a similar manner, coverslips containing primary hippocampal neurons were loaded with the ratiometric dye Fura-PE3 AM . Fura-PE3 is a leakage-resistant CaImaging experiments were typically carried out in two stages. The experimental design consisted of a screening phase and provocation phase, as illustrated in 2+ concentration. During the next 30 minutes of image acquisition, cells were exposed to a 900 MHz GSM-modulated RF field, or received no field in a sham exposure condition. A final 30-minutes of imaging was acquired after the exposure period to determine the persistence and reversibility of any putative effects observed in the preceding period. In the case of cultured PC12 and primary hippocampal cells, the vitality and responsiveness of neurons was assessed at the end of the imaging protocol by application of 54 mM KCl. KCl-mediated depolarization causes large, rapid and reversible Ca2+ transients in vital neurons. Only cells displaying a robust, reversible response to KCl were included in the analysis presented herein.In this set of experiments, imaging protocols consisted of 3 periods lasting 30 minutes each . A 340 nm and 380 nm image was acquired every 30 seconds during the course of the experiment. Images acquired in the first 30 minutes were used to establish a baseline Ca2+ signals were actively induced with a number of pharmacological manipulations to study the effect of RF exposure on various components of cellular Ca2+ handling. EA.hy926 cells were chosen for these experiments and imaged every 30 seconds in Ca2+-free HBSS solution for 30 minutes to determine baseline Ca2+ concentration. The cells were then exposed to a GSM-modulated RF field, or to no field in the sham exposure condition, for 30 minutes.Following the screening phase, a second series of ‘provocation’ experiments was performed. In this case, Ca2+ release from intracellular stores was evoked by application of histamine. Whilst passive Ca2+ store depletion and Ca2+ entry were stimulated by incubation with thapsigargin.Ca2+ concentration was the dependent variable. When cells were compared across different time periods (ie. baseline vs. exposure vs. washout), a mixed model ANOVA with repeated measures was used to analyze significant differences across the time periods and compare this between the exposed and control samples. Dependent variables were distribution tested for normality, homogeneity of variance and sphericity; the latter in the case of repeated measures. In cases where dependent measures violated these assumptions, the data was transformed or non-parametric statistical tools were used. Statistical significance was accepted where p<0.05. All statistical analyses were performed with SPSS 16 for Windows. >150 cells were analysed on each of the coverslips tested.Statistical analyses were performed using analysis of variance tests (ANOVA) with the SAR of RF exposure and modulation conditions (GSM or CW) as the independent variables. Cytosolic Ca2+ was measured in human endothelial cells exposed to RF fields at different levels of SAR. Coverslides of cells were exposed to 1 of 7 SARs  for durations of 30 minutes with either CW or GSM-modulated RF power   = 0.75, p = 0.63).Cytosolic CaRF power . A contr (±6.34)   = 1.15, p = 0.34) = 2.19, p = .05)  = 1.33, p = 0.24)(There was no significant difference in the baseline Cap = 0.34). Furtherp = .05) . In addip = 0.24).2+ concentration in the human endothelial cells (2+ level in the 10 minutes before exposure (basemn), the first 10 minutes of exposure (exposmn) and in the final ten minutes after exposure had ceased (washmn). The repeated measures of Ca2+ concentration did not meet the assumption of sphericity, having failed Mauchly's test, therefore, Greenhouse-Geiser corrected statistics were used. The mixed model repeated measures analysis of Ca2+ concentration showed no significant differences between the unexposed coverslides and any of the SARs, even considering the three time periods, for either the GSM  = 1.77, p = 0.10), or CW power condition  = 0.21, p = .99)  and analyzed with the mixed model repeated measures approach. The slopes of the Ca2+ concentration in the 30-minute periods were normally distributed . Analysis of the slopes showed no significant differences between the unexposed coverslides and those exposed to any of the SARs tested, considering the three repeated measures of slope for each sample  = 1.64, p = 0.10; results not shown). These results indicate that cytosolic Ca2+ concentration in EA.hy926 human endothelial cells neither increases nor decreases in response to a 30-minute exposure to 900 MHz GSM or CW RF power at either 2 W/kg or the six lower SARs tested.A further statistical analysis was performed to take advantage of the repeated measurements of Caal cells . A repeap = .99) . To take2+ signals, a threshold concentration (1 µM) of the agonist histamine was applied to cause Ca2+ release from intracellular stores during the exposure period. The experimental protocol is illustrated in In order to test the effect of RF exposure on provoked Ca2+ concentration of exposed and sham-exposed coverslides  = 1.53, p = .16). Histamine treatment caused an expected rapid increase in cytosolic Ca2+, which peaked at 177 nM (±26 nM) and was normally distributed , as shown in 2+ response to histamine in coverslides exposed to any of the 7 SARs tested when compared to controls  = 1.23, p = .29); shown in 2+ signal in exposed and control coverslides at any of the SARs tested  = 0.87, p = 0.53) . Finally, no significant differences were observed in the time constant for recovery of the Ca2+ signals at any of the SARs in either CW or GSM exposed samples  (No significant differences were observed between the baseline Ca = 0.53) . There w p>.10.) .2+ efflux from intracellular stores and Ca2+ entry was studied using thapsigargin, which disrupts the sequestration of Ca2+ by the ER Ca2+ATPases. Inhibition of the Ca2+ATPases by thapsigargin causes Ca2+ to slowly leak from ER stores, eventually leading to their complete emptying. The protocol that we 2+ signals is depicted in 2+, thapsigargin causes a transient Ca2+ signal. This is due to loss of the finite Ca2+ content within the ER and the progressive removal of the ion by plasma membrane ATPases that are insensitive to thapsigargin. The depletion of the ER Ca2+ stores activates a Ca2+ influx pathway known as ‘store operated Ca2+ entry’. As a consequence, the re-addition of Ca2+ to the extracellular buffer is associated with a rapid Ca2+ entry signal  = 0.297, p = 0.95) and amount of total Ca2+ sequestered and slowly released from ER stores  = 0.258, p = 0.97)  was quantified by fitting mono-exponential decay curves. There were no significant differences in the time constants for the recovery of thapsigargin-evoked Ca2+ signals when the RF exposed coverslides were compared to the control samples at any of the SARs tested  = 0.471, p = .911; Cells exposed to GSM RF fields at 1 of 7 SARs  for 30 minutes had similar peak thapsigargin-evoked Ca = 0.97) . The rec2+ to thapsigargin-treated coverslides induced a large store-operated Ca2+ entry signal, which was not significantly different across the various RF exposure SARs  = 0.568, p = .778; 2+ entry signal in exposed coverslides was also not significantly different than control samples . A control coverslide was run in parallel with each experiment in the unenergized TEM cell (SAR = 0). A final 30-minute period was recorded at the end of the exposure to study the persistence of any observed effects. 2+ in a coverslide of PC12 cells exposed to the GSM RF condition with an SAR of 2 W/kg for 30 min, and its matched control sample.The influence of GSM RF exposure on neuronal Ca2 cells, . Using a2+ concentration of PC12 cells was normally distributed around a mean of 35.9 nM with a SD of 5.43  = 0.05, p = 0.99), nor were there any differences across groups with respect to their resting Ca2+ concentration at the start of the experiment  = 0.125, p = 0.972).The resting Ca of 5.43 . There w2+ concentration during the exposure period failed to show any influence of RF exposure at the SARs tested when compared to controls  = 0.243, p = 0.912) (2+ concentration over the first 5 min of the experiment (basemn), last 5 min of the exposure period (expomn) and final 5 min of the experiment (washmn). No significant effect for RF exposure was observed  = 0.271, p = 0.894), showing that there were no differences in Ca2+ concentration across the SARs tested. Interactions between SAR and the repeated measure factor were not observed  = 0.578, p = 0.7, Greenhouse-Geiser corrected), indicating that neither SAR had an effect on Ca2+ concentration at either of the 3 times chosen  = 0.817, p = .621, Greenhouse-Geiser corrected) with the additional repeated temporal measures of Ca2+ concentration added to the model, suggesting that exposure failed to induce any significant changes in Ca2+ in PC12 cells at the SARs tested  . In ordes chosen . A more s tested .2+ indicator Fura2-PE3. Cultures of hippocampal neurons were placed in the dual TEM cell imaging system and the Ca2+ concentration was measured over the course of a 30-minute baseline, 30-minute exposure and 30-minute washout period. At the onset of the exposure period, one coverslide of hippocampal neurons was exposed to a 900 MHz GSM field at an SAR of 2 W/kg, while the other coverslide of neurons served as a matched control sample. A typical experimental trace of the cytosolic Ca2+ in a coverslide of hippocampal neurons exposed to the GSM RF condition with an SAR of 2W/kg for 30 min, and its matched control sample is depicted in To screen a class of primary cells from the central nervous system, acutely isolated hippocampal neurons were exposed to GSM RF fields. Dissociated hippocampal neurons form extensive networks in culture, as shown in 2+ concentration for the hippocampal neurons exposed to the control or GSM exposed conditions was normally distributed around a mean of 84 nM with a SD of 7.8  was compared between hippocampal neurons in the control condition and those receiving the GSM pulse at an SAR of 2 W/kg. No significant difference in Ca2+ concentration was observed between the two groups during this period  = 0.06, p = 0.81), as shown in 2+ concentration between the baseline, exposure and washout periods that might be attributable to the GSM exposure, a mixed model repeated measures analysis of variance was performed. A significant linear increase in Ca2+ concentration occurred between the three temporal epochs periods tested  = 74.93, p<0.01)  = 0.65, p = 0.47). Further analyses with repeated measures using smaller temporal increments (5 min) failed to show any transient differences in Ca2+ concentration between hippocampal neurons exposed to the GSM 2 W/kg or control condition  = 0.87, p = 0.44).The mean Ca p<0.01) . However2+ binding and homeostasis 2+ signals by changing the phase, or packing, of membrane lipids, or by somehow directly modulating Ca2+ influx channels 2+ signalling and radicals have been demonstrated in various biological systems, with positive feedback evident in many situations 2+ fluxes.Over the past decades, a substantial number of reports have claimed that electromagnetic field exposure can modify cellular Ca2+ homeostasis, there are a substantial number of published studies that found no interaction of such radiation with cellular Ca2+ signalling systems 2+ imaging to establish whether the type of pulsed GSM emissions produced by mobile phone handsets can influence Ca2+ signalling under highly-controlled in vitro conditions.In contrast to the many reports of demonstrable electromagnetic field effects on Ca2+-binding indicators, such as Fura-2 used in this study, have been extensively employed to monitor cellular Ca2+ signals. These indicators can detect rapid changes in Ca2+ concentration without significantly altering cellular buffering. In the wider studies of Ca2+ signaling in our laboratory, we are routinely able to use such indicators to detect modest Ca2+ rises of ∼10 nM amplitude, which represents a 0.1-fold change in Ca2+ concentration over baseline and is typically >2 times the standard deviation of the basal signal. In the present study, the use of a sensitive fluorescence imaging system to monitor thousands of cells afforded us the ability to discriminate subtle Ca2+ changes.Fluorescent Ca2+ signalling that might be influenced by the GSM exposure. This cell line was chosen due to several recent reports of their sensitivity to non-thermal GSM RF exposure and speculation that these effects might be mediated by Ca2+ signalling events 2+-associated cytoskeletal and scaffolding proteins. In the present study, we used an acute GSM RF exposure (30 minutes) to investigate modulation of Ca2+ homeostasis that might be up-stream of phenotypic changes.EA.hy926 human endothelial cells were used to explore specific aspects of Ca2+. This was the case when the RF field was presented in the form of pulsed 900 MHz GSM signals similar to those emitted from mobile handsets or when presented as un-modulated 900 MHz RF power. In addition, RF exposure had no influence on the kinetics, amplitude, clearance or re-sequestration of Ca2+ released from intracellular stores caused by the application of pharmacologic agonists. Changes in Ca2+ homeostasis can therefore be ruled out in any potential biological effects observed in EA.hy926, at least in experiments using a similar duration (30 minutes) and SARs of up to 2 W/kg.In our experiments, exposure of EA.hy926 cells to GSM RF fields across a wide range of SARs did not lead to any significant changes in the homeostasis of cytosolic Ca2+ proposed as a potential mechanism 2+ were observed for either of these cell types in any of the conditions tested.To explore the possibility that different mammalian cell types have varying sensitivity to RF exposure, experiments were repeated using cultured PC12 cells and neurons acutely isolated from the hippocampii of rats. PC12 cells were chosen as they had previously been shown to exhibit transient changes in gene expression in response to GSM RF fields, with modulation of Ca2+ signalling? The majority of these results can be traced to one or two groups who used a very specific biological preparation; whole or excised chick brain. There has been much criticism of this work. In particular, in the physiological state of the cells located deep within tissues, which would have poor oxygen perfusion, energy metabolism and homeostasis. It is plausible that the compromised physiology of such tissue samples is critical for observation of RF-induced effects on Ca2+. If this were the case, it would explain why improvements in the methodology and tissue conditions led to failed replications of this work We conclude that under the conditions employed in our experiments, and using a highly-sensitive assay, we could not detect any consequence of RF exposure. How then can one explain the published results showing that RF fields influence cellular Ca2+ were analogue, amplitude-modulated fields. In fact, a key finding of Adey's work was that the analogue modulation within specific frequency windows  may stimulate MAPK (mitogen-activated protein kinase) cascades 2+ concentration, is the most sensitive measure of GSM RF-EMF effects.Although acute changes in intracellular Ca


Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I.The function of synaptotagmins (syt) in CaTo test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect.These data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished. To accomplish this fusion of vesicle and plasma membranes, the SNARE (soluble NSF receptor) complex of proteins is required as well as a Ca2+ sensor that can bind Ca2+ i by release of Ca2+ from intracellular stores or influx of extracellular Ca2+, we used fura-2 to image Ca2+ and measured relative levels of resting and stimulated [Ca2+]i. Resting Ca2+ levels in untreated and forskolin-treated control, syt I knockdown and CT cells were unaffected by forskolin-treatment (data not shown). With high K+ stimulation, each group of cells responded with an expected ~4-fold increase in [Ca2+]i. Treatment with forskolin did not enhance or prevent influx of extracellular Ca2+ in response to depolarization in any of the cell groups (data not shown). Thus, forskolin does not alter extracellular Ca2+ entering the cell, nor does it induce or alter intracellular release of Ca2+ from stores. This is in agreement with previous results that show that some clonal lines of PC12 cells do not have major intracellular Ca2+ stores i) were measured using ratiometric fluorescence imaging with fura-2 . Cells were replated 24 h before experimentation, loaded with fura-2, and prepared for recording as previously described [2+]i by comparing data to fura-2 calibration curves made in vitro by adding fura-2 (50 μM free acid) to solutions that contained known [Ca2+] . Average resting and stimulated [Ca2+]i were determined from numerous independent experiments. Resting [Ca2+]i was measured during the first 180 sec of recording prior to stimulation with a high K+ solution. Stimulated [Ca2+]i was calculated from the five points during the peak change in Ca2+ levels that occurred in response to stimulation.Intracellular Caescribed ,62. CellCells that were untreated or forskolin-treated for 4 hrs were pelleted by centrifugation and fixed with ice-cold 3.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.25) containing 5% sucrose and 2 mmol/L calcium chloride for 16 hours at 4°C. Cell pellets were washed in 0.1 mol/L sodium cacodylate buffer containing 5% sucrose  and postfixed in 1% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer containing 5% sucrose for 3 hours. Cell pellets were then washed twice in distilled water, incubated for 1 hour in 2% aqueous uranyl acetate, dehydrated through graded ethanols to 100%, rinsed twice in propylene oxide, and infiltrated with a 1:1 mixture of Polybed resin  and propylene oxide for 3 hours. The cell pellets were then incubated in fresh Polybed resin for 3 hours, transferred to BEEM capsules filled with fresh resin, and polymerized overnight at 70°C. Ultrathin sections (0.05 μm) were cut with a diamond knife using a Reichert Ultracut E ultramicrotome , collected on 200 mesh copper grids, post stained with uranyl acetate and lead citrate, and viewed and photographed with a JEOL  100CX transmission electron microscope.All data are displayed as mean ± SEM. Statistical significance was determined by a t-test with unequal variance.(SYT): synaptotagmin, (CA): catecholamine, (NPY): neuropeptide Y, (LDCV): large dense core vesicle, (FSK): forskolin, (HBSS): Hank's buffered saline solution, (HPLC-EC): high performance liquid chromatography-electrochemical detection.2+ imaging experiments and assisted with the cell culture, DNA preparation and drafted the manuscript. JBP prepared most of the cells for experimentation, transfections, all of the HPLC, some of the immunocytochemistry and NPY experiments, and most of the plasmid DNA preparation. ABH helped to conceive the experiment, performed some of the cell culture, transfection, amperometry, and Ca2+ imaging experiments, and prepared the final figures and manuscript. All authors have read and approved the final manuscript.JMM helped conceive the experiment, performed all of the immunoblot experiments, most of the amperometry, NPY, immunocytochemistry, and Ca


It is a fantastic time for the field of robotic exoskeletons. Recent advances in actuators, sensors, materials, batteries, and computer processors have given new hope to creating the exoskeletons of yesteryear's science fiction. While the most common goal of an exoskeleton is to provide superhuman strength or endurance, scientists and engineers around the world are building exoskeletons with a wide range of diverse purposes. Exoskeletons can help patients with neurological disabilities improve their motor performance by providing task specific practice. Exoskeletons can help physiologists better understand how the human body works by providing a novel experimental perturbation. Exoskeletons can even help power mobile phones, music players, and other portable electronic devices by siphoning mechanical work performed during human locomotion. This special thematic series on robotic lower limb exoskeletons and orthoses includes eight papers presenting novel contributions to the field. The collective message of the papers is that robotic exoskeletons will contribute in many ways to the future benefit of humankind, and that future is not that distant. In 2004, Rodney Brooks, then director of the Massachusetts Institute of Technology Computer Science and Artificial Intelligence Laboratory and general robot guru, proclaimed that "the robots are here" . He madeAdding a corollary onto Brooks' prediction, I firmly believe that robotic exoskeletons are today where computers were in 1978. Currently, popular media outlets routinely herald new robotic exoskeletons such as HAL, BLEEX, and XOS . Even moExoskeleton development has an advantage over robot development in general because exoskeletons can rely on the intelligence of the human user. Exoskeletons can take advantage of all the sensors, computational power, control system, and mechanics that humans possess. As a result, the types of controllers that need to be created for exoskeletons are quite different from the types of controllers that need to be created for autonomous independent robots.Exoskeleton development also has a disadvantage over general robot development in that exoskeletons have to work in cooperation with the physiology and biomechanics of the human body. This is a major disadvantage because there is a great deal not understood about the physiology and biomechanics of human movement . If it ist century. In a second review in the thematic series, Crespo and Reinkensmeyer focus on control strategies that have been used for robotic movement training after neurological injury [In this special thematic series, eight papers contribute new advances on robotic exoskeleton technology and our understanding of how humans respond to mechanical assistance from robotic exoskeletons. Herr starts off with a review on exoskeletons and orthoses, highlighting major accomplishments and discussing future directions in the field . Herr isl injury . The conl injury , and Wesl injury . In the l injury , and Noel injury . The thel injury , and thel injury .To advance exoskeleton technology at the fastest rate possible, it is critical that scientists and engineers document and share their successes and failures with the research community. This special thematic series is intended to highlight that need. A major factor limiting the development of powered prostheses in the past has been the lack of carefully controlled scientific studies and open publication of technological advancements. While it is understandable that for-profit companies do not readily publish their research and development work, researchers at universities and institutes need to focus more on how they can move the field forward in cooperation with for-profit companies. University and institute researchers can identify basic principles governing human movement with robotic technologies  with carefully designed experimental studies. They can also provide unbiased assessment of new technologies with controlled tests using adequate sample sizes. Lastly, they can reach out to for-profit companies working on research and development to offer their services and expertise in mutually beneficial collaborations. While the collaboration work may still not yield peer-reviewed publications, it does provide opportunities for scientists and engineers to learn from each other in a way that would greatly benefit the field.


We find that taurine effectively reduces glutamate-induced Ca2+ influx via ionotropic glutamate receptors and voltage-dependent Ca2+ channels in the neurons, and the effect of taurine was selectively inhibited by strychnine and picrotoxin, but not GABA receptor antagonists, although GABA receptors are present in the neurons. A CaMKII inhibitor partially reversed the effect of taurine, suggesting that a Ca2+/calmodulin-dependent pathway is involved in taurine regulation. On the other hand, a rapid influx of Ca2+ through ionotropic glutamate receptors could inhibit the amplitude and kinetics of taurine-elicited currents in the third-order neurons, which could be controlled with intracellular application of BAPTA a fast Ca2+ chelator. This study indicates that taurine is a potential neuromodulator in glutamate transmission. The reciprocal inhibition between taurine and glutamate in the postsynaptic neurons contributes to computation of visual signals in the retinal neurons.Although taurine and glutamate are the most abundant amino acids conducting neural signals in the central nervous system, the communication between these two neurotransmitters is largely unknown. This study explores the interaction of taurine and glutamate in the retinal third-order neurons. Using specific antibodies, both taurine and taurine transporters were localized in photoreceptors and Off-bipolar cells, glutamatergic neurons in retinas. It is possible that Off-bipolar cells release juxtaposed glutamate and taurine to activate the third-order neurons in retina. The interaction of taurine and glutamate was studied in acutely dissociated third-order neurons in whole-cell patch-clamp recording and Ca Taurine is a sulfur containing amino acid structurally similar to the neurotransmitters glycine and GABA (Gamma aminobutyric acid). It is the most abundant free amino acid in retina and the second most abundant free amino acid in the central brain after glutamate . Althoug2+]i levels, leading to a large amplification of glutamate signals; whereas activation of ionotropic receptors directly changes the cell membrane potential by cation influx. Some ionotropic glutamate receptors are Ca2+ permeable. Ca2+ entrance triggers intracellular second-messenger pathways that lead to alterations in cellular and molecular levels in neurons. Therefore regulation of Ca2+ permeable glutamate receptors in neurons can exert a large influence in neuronal signals. This study is to examine the effect of taurine on regulation of Ca2+ permeable ionotropic glutamate receptors in the retinal neurons.Glutamate is the major excitatory neurotransmitter conducting visual signals within retina. In general glutamate releases from presynaptic neurons and transmits signals upon activation of metabotropic and ionotropic receptors in the postsynaptic neurons. Activation of metabotropic receptors usually triggers intracellular transduction pathways associated with changes of i increase via AMPA and kainate receptors in the third-order neurons. The effects of taurine and glutamate reciprocally inhibit each other, linked by [Ca2+]i. Our study indicates that taurine is important for control of glutamate-induced Ca2+ overload in the retinal third-order neurons.We used amphibian retinal amacrine and ganglion cells, the third-order neurons, as a model system to study the interactions of taurine and glutamate signals. To avoid retinal network effects, isolated neurons were used in this study. We characterized taurinergic neurons using specific antibodies against taurine and the taurine transporter. The results indicate that photoreceptors and Off-type bipolar cells are taurinergic neurons in the retina. We find that the effect of taurine on the third-order neurons suppresses CaAmbystoma tigrinum), were purchased from Kons Scientific  and Charles Sullivan . The animals were kept in aquaria at a constant 12oC under a 12-hour dark-light cycle with continuous filtration. The retinas were collected from animals that had been kept at least six hours in the dark. Briefly, the animals were decapitated, double-pithed and the eyes were enucleated. All procedures were performed in accordance with the guidelines of National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University's Animal Care Committee.Larval tiger salamanders , 400μl of Ringer’s solution containing (in mM) 5 L-Cysteine and 1 EDTA (adjusted to pH 7.4) for 20-35 minutes at room temperature. The enzymatically treated retina was washed before being mechanically dissociated, by gentle shaking, in Ringer’s solution. The dissociated cells were seeded on 18mm glass cover slips freshly coated with lectin and allowed to set for 20 minutes before use. The cover slip was then placed in the recording chamber containing Ringer solution on an Olympus BX51WI microscope equipped with a CCD camera linked to a monitor. All experimentation was done at room temperature and within a few hours of preparation.The retinal tissue was removed from the eyecup in Ringer’s solution consisting of (in mM):111 NaCl, 2.5 KCl, 1.8 CaCl2, 5 EGTA and 10 HEPES, with an ATP regenerating cocktails consisting of (in mM): 20 ATP, 40 Phosphocreatine and 2 Creatine Phosphokinase (adjusted to pH 7.4). Cell responses were recorded at a holding potential of -60mV.Whole-cell recordings were performed on amacrine and ganglion cells using an EPC-10 amplifier and HEKA Patchmaster software. These third-order neurons were identified based on their distinctive large transient sodium currents for whole cell recording. Low resistance (5-10MΩ) electrodes were pulled from borosilicate glass (Drummond Scientific Co) with the P-97 micropipette puller (Sutter Instrument Co). The electrodes were filled with a high-potassium solution containing (in mM): 100 K-gluconate, 1 MgClThe isolated cell preparation was constantly perfused with oxygenated Ringer’s solution. A DAD-VM automated superfusion system (ALA Scientific Instruments) was used for local drug application. Glutamate, NMDA, AMPA and kainic acid with or without Taurine pre-perfusion were all applied for 3-second intervals. Any antagonist that was also applied during experimentation was first pre-perfused at a low pressure. All drugs were prepared in stock solutions before being diluted to their final concentrations (listed in results section) in the perfusion solution on the day of use.Freshly enucleated eyeballs were fixed in either 2.25% glutaraldehyde with 2% paraformaldehyde (for anti-taurine labeling) or 4% paraformaldehyde (for anti-taurine transporter labeling) in Ringer’s solution for 10 minutes, before 10 minutes fixation of the eyecups after corneas were removed. The eyecups were then thoroughly washed in the Ringer’s solution. The fixed eyecups were gradually dehydrated in 15%, 20% and then 30% sucrose solutions (in the Ringer’s solution) before being kept overnight in 30% sucrose. The eyecups were embed in the optimal cutting temperature (OCT) compound , frozen and dissected at 12-20µm thickness. The frozen sections were collected on silane coated slides, dried and stored at -80°C.Retinal frozen sections were rinsed with PBS and treated with an antibody blocking reagent “cocktail” consisting of 10% goat serum, 1% BSA in 0.3% Triton X-100 with 0.1% Tween in PBS (PBST) for 20 minutes. The retinal sections were then incubated with an affinity purified rabbit anti-taurine polycolonal antibody , with a 1:200 concentration, or rabbit anti-taurine transporter polyclonal antibody  with 10µg/mL for 2 hours. After being washed for 35 minutes, the sections were incubated with a secondary antibody, the goat-anti-rabbit Cy-3-conjugated (1:600) antibody for 40 minutes in a dark room. The retinal sections were rinsed with PBS and were mounted with Vectorshield . A Zeiss LSM 700 confocal microscope system was used to observe fluorescence labeling in the retinal sections. Single scanning and multiple scanning along Z-direction were applied on the sections with the Zen software. The images were collected and processed with the software.Isolated retinal cells seeded on lectin-coated cover slips were incubated for 20-25 minutes in the cell-permeable calcium sensitive Fluo-4 AM dye (3μM in ringer solution). The cover slip was washed in Ringer’s solution before being placed in the recording chamber. Cells were excited at 480nm with the emission collected at 520nm. A Rolera-MGi Plus camera (Q-imaging) was used to view and collect fluorescence signals. Frame images were taken every 3 seconds with cells exposed for 50 milliseconds at each interval. A Lambda 10-2 (Sutter Instrument Co) controlled by IP Lab 4.0 software was used to open and close the filter shutter at each interval. All cell preparation and experimentation was done at room temperature in complete darkness.The third-order neurons were identified by their morphology. Usually, dissociated ganglion cells appeared to have a long axon process extending from the cell somas; the dissociated amacrine cells have larger somas than that of bipolar cells. However, with the morphological profiles, we could not rule out the possibility that some bipolar and horizontal cells might be mistakenly counted in our experiments since most of dissociated cells lost their clear morphology.2+ imaging data was measured using IP Lab 4.0 software. In these experiments individual frames from a single experiment were combined into sequences before specific regions of interest were selected. For each cell in each sequence that showed a response the intensity value was normalized by first subtracting the baseline value before dividing by the maximum intensity for that cell. The normalized values for each experiment set were then combined to produce a mean value trace. Mean, Standard Error and paired t-test values were all calculated using Microsoft Office 2007.Electrophysiological data was obtained using HEKA Patchmaster software and analyzed using Igor Pro Software. CaTriticum vulgaris, Picrotoxin, Potassium D-gluconate, K-gluconate, BAPTA, EGTA, Adenosine 5’-Triphosphate (ATP), Phosphocreatine, Creatine Phosphokinase Type I, CoCl2, NaCl and Dextrose were purchased from Sigma-Aldrich Inc . L-Cysteine, EDTA, HEPES, MgCl2 and KCl were purchased from J.T. Baker . CaCl2 was purchased from Fisher Scientific . Papain and Deoxyribonuclease I were purchased from Worthington Biochemical . Kainic Acid, (S)-AMPA, NMDA, SR 95531, Bicuculline, TPMPA, CGP 55845, GF 10920X, H-7, H-89, KN-62, PKI 14-22 amide, Thapsigargin, Ruthenium Red, CNQX and L-AP7 were all purchased from Tocris Bioscience . Fluo-4AM was purchased from Invitrogen Corporation .Taurine, Glycine, L-Glutamic acid, Lectin from The taurine-positive neurons in tiger salamander retina were labeled by the specific antibody of taurine in frozen vertical sections. Figure To further characterize taurinergic neurons, a specific taurine transporter antibody was used. Figure + currents in multiple depolarizing voltage steps. Photoreceptors, bipolar cells and horizontal cells were lacking Na+ currents, enabling the third-order neurons in isolated cell preparations to be distinguished. Third-order neurons can be further characterized as Na+ currents are much smaller in amacrine cells compared to those in ganglion cells. The cells were voltage-clamped at -70mV, near the resting potential of the cells. Inward currents were recorded from the cells elicited with taurine concentrations of 10µM, 20µM, 50µM, 100µM, 500µM, 1mM. 2mM and 4mM puffed locally on the neurons. Figure 50) determined from the dose response relationship curve . Application of 200µM glutamate evoked a large [Ca2+]i increase from the third-order neurons. When taurine was co-applied with glutamate in 10µM, 350µM and 2mM, the minimum, EC50 and maximum concentrations respectively, the [Ca2+]i was suppressed in a dose-dependent manner. Figure 2+]i changes in the third-order neurons activated by glutamate and glutamate with varying concentrations of taurine. On average, 10µM taurine reduced 23% of glutamate-elicited free [Ca2+]i levels, with the EC50 and maximum concentration of taurine blocking 40% and 67% of the glutamate-elicited free [Ca2+]i respectively in the third-order neurons (n=40). This indicates that the effect of taurine effectively suppressed glutamate-elicited [Ca2+]i in the neurons.The effect of taurine on glutamate response was studied in the third-order neurons using Ca2+ imaging studies indicate that low concentrations of kainate and AMPA specifically activate their own receptors that are highly permeable to Ca2+ in the third-order neurons [2+]i in the neurons , but had no significant effect on kainate-induced [Ca2+]i with no reduction in the level of [Ca2+]i (n=23). This indicates that the low concentration of taurine selectively suppressed AMPA-sensitive receptor response in the neurons. Higher taurine concentrations gradually suppressed AMPA-induced [Ca2+]i, but acutely reduced kainate-induced [Ca2+]i in the neurons . NMDA-induced [Ca2+]i was completely blocked by 2mM taurine , which was significantly reversed by 2µM strychnine to 75% of the control . The remaining strychnine-insensitive effect indicates that taurine might activate different types of receptors. The effect of taurine on AMPA- and kainate-induced [Ca2+]i in the third-order neurons was also tested. In the control, 2mM taurine significantly suppressed both 15µM AMPA- by 65%  and 15µM kainate-induced [Ca2+]i by 85%  in the third-order neurons, as shown in figure 2+]i compared to kainate-induced [Ca2+]i, the reversal was significant for both AMPA  and kainate . These results suggest that the action of taurine on glutamate receptors could therefore be mediated by strychnine-sensitive receptors.As the third-order neurons possess both glycine and GABA receptors, the effect of taurine on suppression of glutamate-induced i from 55% to 30% , showing in figure 2+]i. Figure 2+]i is active through a Cl- permeable receptor blocked by picrotoxin.The effect of strychnine could be duplicated by 100µM picrotoxin, a non-selective blocker for Cl2+]i . Figure A receptor antagonists, 10µM bicuculline  and 20µM SR95531  had no significant effect on reversing taurine-produced suppression on glutamate-induced [Ca2+]i . In addition the GABAC receptor antagonist 50µM TPMPA  and the GABAB receptor antagonist 10µM CGP55845  were also unable to reverse taurine-produced suppression  and AP-7  were used to block all ionotropic glutamate receptors. The glutamate-induced [Ca2+]i was completely blocked by the presence of these ionotropic glutamate receptor antagonists, and in addition when 2mM taurine was co-applied it had no effect on [Ca2+]i in the neurons . In the control, taurine suppressed glutamate-induced [Ca2+]i. by 62%+3% (n=40). Application of KN-62 had no effect on glutamate-induced [Ca2+]i., however with KN-62, taurine suppression was reversed significantly to 47%+3% . In the case of PKI (14-22) amide, taurine suppressed glutamate-induced [Ca2+]i by 63%+5% (n=38) in the control, which was reduced to 52%+4%  with blockade of PKA. This indicates that both CaMKII and PKA are the intracellular proteins involved in the taurine signaling transduction.If intracellular signal transduction pathways are involved in taurine regulation, then their inhibitors should reverse the suppressive potential of taurine. Several intracellular protein inhibitors were used to block taurine produced effect and among these inhibitors the selective cell-permeable CaMKII inhibitor KN-62 and protein kinase A inhibitor PKI (14-22) amide (both 1µM) could partially reverse the taurine-produced suppressive effect on glutamate responses. Figure 2+]i was via both the strychnine-sensitive and -insensitive receptors, CaMKII and PKA and could involve the downstream pathways of these taurine-activated receptors. To specify which type of taurine-activated receptors triggers the CaMKII-sensitive regulation in glutamate-induced [Ca2+]I, we performed the experiments with strychnine to block strychnine-sensitive taurine receptors, isolating strychnine-insensitive taurine receptor response. Figure 2+]i. With strychnine, KN-62 further blocked about 11% of the taurine suppression on glutamate-induced [Ca2+]i , indicating that CaMKII mediates strychnine-insensitive taurine regulation in the third-order neurons.As shown in figure 2+ influx via glutamate receptors also regulates taurine response. This was studied in a whole-cell voltage-clamp recording mode. Taurine-elicited currents were measured in the presence and absence of glutamate receptor agonists. As the results in figure 2+] levels, both 15µM AMPA and kainate were used to elevate [Ca2+]i levels in the neurons. A taurine dose of 350µM was applied before the application of AMPA or kainate. Taurine elicited a large inward current with fast desensitization at a holding voltage -70mV, and adding AMPA or kainate had no significant change on the substandard taurine currents  was applied into the cytosol through recording electrodes during whole-cell recording. It was determined that BAPTA reduced the effects of AMPA and kainate on taurine-elicited currents  Ca2+ is the second messenger for modulation of taurine-sensitive receptors in the neurons. Glutamate-induced accumulation of [Ca2+]i could activate both Ca2+/calmodulin and PKA through a Ca2+/calmodulin-dependent mechanism. Also Ca2+-dependent PKC might be the mechanism for regulation of taurine-sensitive receptors. The detail mechanism for Ca2+ -related regulation of taurine-sensitive receptors needs to be further investigated.Several findings are present in this study: 1) taurine regulates Careceptor . 2) Likeons Fig. . The strway Fig.  indicateeviously . As consate Fig. . Both NM studies ,44. We d2+ directly binds to Ca2+/calmodulin to activate CaMKII, then Ca2+/calmodulin dependent activation of adenylyl cyclase stimulates cAMP synthesis that in turn activates PKA [2+]i. Previous studies have indicated that CaMKII modulates glycine and GABAA receptor activations by regulation of phosphorylation states i to protect cerebellar granule cell damages in excitotoxicity [2+ overload via voltage-gated Ca2+ channels and NMDA glutamate receptors via intracellular calpain-dependent mechanisms [2+]i overloading might be the key function for neuroprotection of taurine against cell damage by excessive glutamate levels in pathological conditions. The results from our study suggest that taurine might have similar effects as in the central brain and spinal cord to protect retinal neurons form glutamate toxicity damage in disease.Taurine has multiple functions in neuroprotection against glutamate toxicity in the central brain ,51,53. Ttoxicity . Wu et achanisms . This lechanisms . This miThe authors declare that they have no competing interests.


In CA1 pyramidal neurons, CaM regulates two opposing Ca2+-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca2+-CaM-dependent enzymes: Ca2+/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca2+ and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca2+ ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca2+ and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca2+ signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca2+-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca2+ channels, and to the microscopic injection rate of Ca2+ ions. We also demonstrate that Ca2+ saturation takes place via two different pathways depending on the Ca2+ injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca2+ sensors that can differentially transduce Ca2+ influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca2+-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.Calmodulin (CaM) is a ubiquitous Ca 2+ signal mediator and a buffer in a wide variety of body organs including the heart and brain. In the brain, calmodulin regulates intracellular molecular processes that change the strength of connectivity between neurons, thus contributing to various brain functions including memory formation. The exact molecular mechanism as to how calmodulin regulates these processes is not yet known. Interestingly, in other excitable tissues, including the heart, each of two lobes of calmodulin responds differentially toward Ca2+ influx and toward its target molecules . This way, calmodulin precisely controls the Ca2+ dynamics of the cell. We wish to test if a similar mechanism may be operational in neurons so that two lobes of calmodulin interact differentially with Ca2+ ions to activate different downstream molecules that control the strength of connections between neurons. We constructed a detailed simulation of calmodulin that allows us to keep track of its interactions with Ca2+ ions and target proteins at the single molecule level. The simulation predicts that two lobes of calmodulin respond differentially to Ca2+ influx both in space and in time. This work opens a door to future experimental testing of the lobe-specific control of neural function by calmodulin.Calmodulin is a versatile Ca In the excitatory synapse of hippocampal CA1 pyramidal neurons, the activation of CaM dependent enzymes results in the induction of synaptic plasticity  and long-term depression (LTD)) 2+ and subsequent activation of CaM-dependent downstream enzymes: CaM-dependent protein kinase II (CaMKII) and calcineurin. Injection of CA1 pyramidal cells with peptides that block CaMKII activity inhibited the induction 2+ and it appears that the CaM-dependent phosphatase, protein phosphatase 2B  is involved in LTD induction 2+ that activate CaM and subsequently calcineurin but not CaMKII. Conversely, higher levels of Ca2+ initiate CaM-dependent CaMKII activation and autophosphorylation, leading to LTP induction. However, it is still unknown how Ca2+ and CaM regulate two opposing processes as distinct as LTP or LTD in such a precise and controlled manner.Calmodulin (CaM) is a ubiquitous Ca2+ buffer in CA1 pyramidal neurons 2+ binding proteins  , the concentration of 1 µM of any chemical species corresponds to ∼40 molecules. The basal (resting) level of spine Ca2+ is 50∼100 nM which corresponds to 2∼4 molecules of Ca2+ ions. Under such a circumstance, the behavior of single molecules within synaptic spines is not well described by the concentration-based mathematical approach such as reaction diffusion equation.Many elegant experimental measurements have been made of dendritic spine Ca2+-CaM interaction within a dendritic spine using a novel particle-based event-driven Monte Carlo algorithm, which we call Cellular Dynamics Simulator  state. In fact, a mathematical formula is already available to calculate this mean first passage time  form, State “1” as one Ca2+ ion bound form, and State “2” as a two Ca2+ ion bound form of a given lobe. The symbols kXij in i and State j  of lobe X ( = N or C). Thus, each lobe has three states and the whole CaM molecule has nine states  first passage time: the (average) length of transition time required for each lobe of CaM molecule to reach the Cae states .2+ . Here we assume the system is well-stirred and the concentration of Ca2+ is constant (time-invariant). Then, the mean first passage time (<t>) can easily be found through differentiation . As predicted, the mean first passage time for the C-terminal lobe (magenta) is much longer than the N-lobe (blue). For comparison, we show the first passage time for full Ca2+ saturation of CaM; the mean first passage time to reach the state N2C2 in 2+ association and dissociation pathways for both lobes and is influenced by the lifetime of the Ca2+ saturated states of each lobe. The corresponding mathematical formula will be much more complicated than Eq. 1 and 2 and therefore, we calculated this quantity numerically using an extended version of the Gillespie type stochastic algorithm  may not result in significant CaM saturation in the spine. In fact, at a ∼1 µM Ca2+ concentration, the mean first passage time for the C-terminal lobe (or full Ca2+ saturation of CaM) is much longer than the duration of the Ca2+ transient . The second term in the right-hand side of Eq. 3  of the first passage time. In addition, we ignored the fact that the number of Ca2+ saturation, respectively, taken from a single stochastic simulation . 2+ saturation of the C-terminal lobe is possible even if the mean first passage time is shorter than that of Ca2+ transient. However, the inset of 2+ saturation predominates and precedes that of the C-terminal lobe during the short Ca2+ transient. Knowing that two lobes of CaM compete for the limited amount of available Ca2+ ions in the dendritic spines, we predict that the N-terminal dominance for the short Ca2+ transient is more prominent in neurons.2+ ions enter the spine head through a Ca2+ channel, a steep spatial gradient of Ca2+may be formed around the channel mouth (depending on the Ca2+ injection rate). At a single molecule level, it is the transient local (microscopic) “concentration” of Ca2+  felt by a CaM molecule that determines the probability of Ca2+ saturation of a given lobe of each CaM molecule. A CaM molecule can experience much higher  Ca2+ “concentration” than indicated by the bulk Ca2+ transient depending on its location with respect to the Ca2+ source.This type of analysis, however, is further complicated when taking into account the non-homogeneous spatial distribution of molecules. When Ca2+ profile in the spines. Without such a systematic dissection, the interpretation of simulation results when trying to determine the spatial/temporal profile of CaM activation would not be possible. We next explored how fast Ca2+ ions escape from the spine.The present work aims to describe a detailed analysis of this spatial stochastic phenomenon. However, before going into the detailed simulations, it is necessary to dissect each of the biophysical factors that we discussed at the beginning of this section. The last two of these factors determine the space- and time- dependent Ca2+ from the spine. Ca2+ ions that enter the spine through ion channels will eventually diffuse into the dendrites or be extruded by the Ca2+ pumps 2+ pumps on the escape rate of Ca2+ from the spines. We carry out this analysis in a stepwise manner. We first analyze the escape of Ca2+ via pure diffusion without Ca2+ pumps (or buffers) and establish the impact of spine morphology on the Ca2+ escape rate  in the later sections when we combine all known biophysical factors in the detailed simulations.The second factor that will determine the spatial profile of CaM activation is the escape rate of Caape rate . Then wer impact . This wa2+ decay for three different spine neck geometries. Here, we randomly placed a fixed number of Ca2+ ions ( = 400 that corresponds to ∼10 µM) in the head of a spherical spine and let them diffuse out of the spine to the dendrite. The diffusion coefficient (2+ was set to 200∼225 µm2/s (nm2/µs) 2+ decay process. This is a so-called narrow escape problem and has been extensively investigated 2+ decay transient is well approximated by a single exponential decay term. These decay time constants fit well (the relative error <5%) with one of the pre-existing mathematical formula (the left-hand side of Eq. 4 below):http://synapses.clm.utexas.edu/) (data not shown).2+ decay from spines is Ca2+ pumps 2+ extrusion mechanisms in CA1 spines are Na+/Ca2+ exchangers  and plasma membrane Ca2+ ATPase (PMCA) 2+ clearance process with standard spine morphology  with (dashed black line) and without pumps (solid black line). The fast decay time constant of Ca2+ in the presence of pump is ∼45% of the narrow escape time without pumps (∼5–6 ms). In this analysis, we have included NCX/NCKX and PMCA at the concentration close to the highest level known in the literature to examine the maximal impact that Ca2+ pumps would have on Ca2+ clearance. The Ca2+ transients with reduced number of pumps lie between the dashed and solid lines (data not shown).Another biophysical factor that regulates the Ca2+ without buffers in a standard spine in the presence of pumps is ∼5 ms or shorter. In the subsequent section, we will show that a major Ca2+ buffer in CA1 pyramidal neurons  slows down the Ca2+ decay to ∼10∼20 ms  rate and we examine the relation between the Ca2+ injection rate and the spatio-temporal profile of Ca2+ transients in the spine. To realize the impact of Ca2+ injection rate in isolation on the spatiotemporal Ca2+ profile, there are no pumps or Ca2+ binding buffers in this model spine. Once injected, Ca2+ ions travel via simple diffusion until they are absorbed from the compartment at the spine-dendrite boundary  at designated time points after the start of Ca2+ injection. The mean Ca2+ injection rates in Panel A and B are 1.4 and 0.07 Ca2+ ions per microsecond, respectively. At the higher injection rate (1.4 ions/µs), there is a build-up of Ca2+ ions near the channel (Panel A) while such a build up is not evident in Panel B. Note that the time points chosen for Panel A and B are 20-fold different so that the total number of Ca2+ injected by the indicated time points in Panel A  and B  are identical. Ca2+ ions can travel ∼140 nm from the channel via diffusion before the next Ca2+ ion exits the channel at injection rate of 0.07 ions/µs. At a higher Ca2+ injection rate, Ca2+ ions will accumulate near the channel pore before they diffuse away (red in 2+ injection rate (black) leads to a much lower peak Ca2+ number (concentration) than the higher Ca2+ injection rates. Ca2+ ion can travel more than 1 µm away from the channel during 1 ms. During a 10 ms Ca2+ injection period, a significant fraction of Ca2+ ions has already left the spine. Thus, we have lower Ca2+ peak than at the higher Ca2+ injection rate. After the peak, the Ca2+ level decreases with a time constant of ∼7–8 ms for all Ca2+ injection rates. This decay process is controlled by the diffusion and is consistent with the narrow escape rate we calculated in y red in . As anti2+ injection rates on the spatial and temporal dynamics of Ca2+ transients in dendritic spines. The relative lack of a Ca2+ gradient in 2+-saturated C-terminal lobe may not form. However, as mentioned at the beginning of this section, we need to include CaM and examine the combined effect of all of these biophysical factors on the spatial profile of Ca2+-CaM interactions. The second half of Results provides this analysis.2+ injection rate on the Ca2+ micro-domain  uniformly within the spine volume . At this basal condition, majority of CaM molecules are Ca2+ free or in a single Ca2+ bound form and none of their lobes are Ca2+ saturated. The diffusion coefficient of CaM varies between 2∼20 µm2/s (nm2/µs) 2/s (nm2/µs) (but see our comments below).In the previous sections, we studied the impact of three biophysical factors: the first passage time , the naro-domain . In this2+/CaM with a channel of high Ca2+ injection rate  and fully Ca2+-saturated CaM. The number of free Ca2+ ions in the spine is shown in 2+ much faster than the C-terminal lobe , the N-terminal lobes quickly release Ca2+ and the C-terminal lobes slowly bind the available Ca2+ (2+ remains associated with the C-lobe for a relatively long time (the decay time constant is ∼120 ms) and the C-lobes therefore trap Ca2+ in the spine . Another important point to note is that even at this high Ca2+ injection rate, the total number of fully Ca2+-saturated CaM molecule is less than ∼7. This number varies from simulation to simulation, but with a single Ca2+ channel, the number remains below 10 (over 100 simulation runs), a remarkably low number.The results in nal lobe . As a coble Ca2+ . Once bohe spine . The fre2+ saturation, each lobe of CaM exhibits a nano-domain near the channel pore. For example, in first Ca2+ saturated. We plot these accumulated locations of “first Ca2+ saturation event” up to the different designated time point in the figure  and for the fully Ca2+-saturated CaM.Panels C, E, and G of 2+ saturation events . The N-terminal lobe responds to and senses the Ca2+ gradient much faster than the C-lobe . Note we recorded and analyzed only the first Ca2+ saturation events for each lobe of each CaM molecules. The relatively widespread C-terminal lobe Ca2+ saturation in Panel C, therefore, is not because the high affinity C-terminal lobe carries Ca2+ ions while diffusing away from the channel.In summary, the high Caobe blue . The C-l2+ injection rate? 2+ is less prominent with a reduced Ca2+ injection rate. One possible scenario is that, under such a condition, only N-terminal lobe with higher Ca2+ binding kinetics , and free Ca2+ ions (B). The difference in the rising phase of Ca2+ saturated N- and C- terminal lobes observed in 2+ influx. The Ca2+ saturated N- and C-terminal lobes increase at a similar rate but the N-terminal lobe exhibits a larger fluctuation due to its fast Ca2+ dissociation rate. Again, the number of fully Ca2+ saturated molecules is small (less than 5∼10) over the course of a 25 ms simulation experiment.What if we reduce the Cakinetics  can dete2+ saturation for each lobe becomes less localized around the channel  and repeated simulations in 2+ influx. It appears that the Ca2+ binding kinetics of CaM (first passage time) is the major determinant of the lobe specific spatial pattern formation during Ca2+ influx.In conclusion, the N-terminal lobe exhibits a transient Canal lobe  and 2 isnal lobe  and obtanal lobe . We also2+ saturated CaM was also influenced by the Ca2+ injection rate  during which CaM (or any Brownian particle of the same diffusion coefficient) can travel a distance equal to or larger than the entire spine head volume. CaM can reach its fully Ca2+ saturated state when additional Ca2+ binds to the N-terminal lobe . Alternatively, if Ca2+ injection rate is high and the transient Ca2+ concentration is adequate, CaM can reach the fully Ca2+ saturated state via N-terminal lobe Ca2+ saturation before Ca2+ saturates the C-terminal lobe because the first passage time for the N-terminal lobe is shorter than the C-terminal lobe , yellow for State N1C1, N0C2 and N2C0 (two Ca2+ bound state), green for State N0C1 and N1C0 (one Ca2+ bound state), and blue for State N0C0 (apo CaM) (see CaM) see . The cho2+ saturated state , and then fully Ca2+ saturated  near the channel , N1C2 (magenta), and N0C2 (yellow) states as it explores the space close to the channel (The CaM molecule we selected for  channel .2+ ions/µs). We randomly selected a CaM molecule from the simulation presented in 2+ saturated state of the C-terminal lobe  via states N1C2 (magenta) to reach the fully Ca2+ saturated state  , N0C1 (green), and N1C1 (yellow) states  . After Cates see .2+ saturation pathways: N-terminal first pathway and C-terminal first pathway (see 2+ saturated state (for the first time) up to each time point (cumulative sum). We plot the number of CaM molecules who have reached saturation via N-terminal lobe saturation first (blue) and via C-terminal lobe first (magenta). At the lower Ca2+ injection rate, the C-terminal lobe first is the dominant pathway  of NMDA receptors dictates the Ca2+ transient as predicted by previous work 2+ (or Ca2+ binding to CaM) transients. To show the spatial patterns of Ca2+ saturation, we compiled the results of 20 simulation runs  and plot the locations of the Ca2+ saturated N- and C-lobe and fully Ca2+ saturated CaM as before  undergoes a series of conformational changes upon Ca2+ and/or target binding. In fact, the Ca2+ binding and target association are thermodynamically coupled (see 2+ of CaM while Ca2+ binding in turn changes the binding kinetics of CaM towards its targets (see below for more discussion). The changes in the Ca2+ binding kinetics upon target binding  is a critical factor that may affect the spatial profile of Ca2+-CaM-target activation.What are the implications of the lobe specific functionalities of CaM, especially for the CaM-and NMDA receptor-dependent synaptic plasticity that involves CaMKII and calcineurin? In order to understand this issue, one must pay close attention to the details of Capled see . Target 2+ concentrations. Interestingly, recent experimental and modeling work suggested that the N-terminal lobe of CaM preferentially interacts with CaMKII before the C-terminal lobe 2+ concentrations via the N-terminal lobe. This mode of CaM-CaMKII interaction is different from the so-called CaM-trapping by auto-phosphorylated CaMKII  and shorter first passage time for Ca2+ saturation (Panel B). The present work  of calcineurin 2+ binding kinetics of CnB is similar to that of the C-terminal lobe of CaM, one would expect a spatial and temporal pattern of calcineurin activation to be similar to the C-lobe specific Ca2+-CaM activation domain. Detailed experimental characterization of the Ca2+ binding kinetics of the “CaM-like” subunit of calcineurin (CnB) is necessary.What if the C-terminal lobe preferentially interacts with calcineurin which underlies LTD (long-term depression) induction? Then, each of the two lobes of CaM differentially regulates these two opposing processes of synaptic plasticity. This may seem like an attractive hypothesis and in fact, our preliminary modeling study indicated that the C-terminal lobe of CaM has a higher affinity toward calcineurin than the N-terminal lobe. However, the affinity of calcineurin for CaM is extremely high 2+-CaM. RC3 binds CaM (even in the absence of Ca2+) and accelerates the Ca2+ dissociation from the C-terminal lobe thereby decreasing its affinity toward Ca2+2+ binding and target interaction that we mentioned earlier may play an important role in determining the spatial dynamics of Ca2+-CaM-RC3 interactions. The released Ca2+ ion can bind the N-terminal lobe of the same or another CaM molecule. We predict that RC3 has a positive impact on the N-terminal specific Ca2+-CaM nano-domain and on the nano-domain of CaMKII bound CaM. In addition, RC3 is known to interact with membrane phosphatidic acid 2+-CaM activation. Overall, genetic studies clearly suggest a critical role of RC3 in the regulation of Ca2+ dynamics in spines In CA1 pyramidal neurons, another critical factor, RC3 , regulates the induction of NMDA-receptor and CaM-dependent synaptic plasticity. RC3 is highly enriched in CA1 spines and is known to regulate the transition between the induction of LTP vs. LTD 2+-CaM nano-domain  or being extruded. Only under certain conditions, for example, when the Ca2+ binding rate is faster than the diffusion of Ca2+, are “Ca2+ domain” and “Ca2+-buffer” domain closely related in space. Clearly, the C- and N- terminal lobe specific Ca2+-CaM domains respond differently for the same Ca2+ input , in the presence of excess unsaturable mobile buffers, is given by the following equation 2+ current, 2+ (defined earlier), 0 is the bulk Ca2+ concentration, and 2+ ion travels before being captured by buffer, B is the buffer concentration 2+ binding rate, and F is the Faraday constant. This and many other mathematical formulas have been developed  as we did in this study.Furthermore, in a small sub-cellular compartment, like CA1 spines, the number but not the concentration of molecules is important. As an illustration, when the equation for the steady-state Ca2+-CaM nano-domains, reside in the same molecule and are competing for a limited amount of Ca2+ as we discussed in the 2+ signals in a different manner, and potentially serve distinct cellular functions. The current work is the first step to understand this unique functionality of CaM at the single molecule level.In addition, it is important to note that the two lobes of CaM, with almost opposite impacts on Ca2+ transient in dendritic spines is regulated by highly nonlinear interactions between voltage-gated Ca2+ channels, K+ channels, and glutamate receptors. This important issue was recently reviewed in 2+-activated K+ channels (SK channels) in hippocampal neurons shape the Ca2+ transients in spines and a direct coupling between voltage-gated Ca2+ channels and SK channels via “Ca2+ nano-domains” is a critical regulatory factor of spine Ca2+ metabolism. In addition, CaM itself regulates the activities of Ca2+ channels and Ca2+ pumps (PMCA) 2+ dynamics. It is also difficult to make correct interpretations of pre-existing Ca2+ imaging experimental data . For these reasons, in this study we focused on the initial rising phase of Ca2+ transients and therefore only studied the impacts of Ca2+ injection rate that are relevant for any Ca2+ channels.The Ca2+ pumps and discovered that the resultant spatial profile of lobe specific Ca2+-CaM domains were similar to the results with Ca2+ pumps (data not shown). As long as Ca2+ pumps are uniformly distributed, the Ca2+ binding kinetics of CaM dictates the spatial and temporal pattern of the Ca2+-CaM interaction. We have not, however, tested spatially non-uniform distribution of Ca2+ pumps such as clusters of PMCA/NCX/NCXK tightly coupled to Ca2+ channels. This is an open area of future research.With these limitations in mind, we repeated all simulations in 2+ that potentially influences Ca2+ transients in the spine. Although our simulator is fully capable of implementing SER structures and Ca2+ release from this source, only a small subset of dendritic spines on CA1 pyramidal neurons contain SER Finally, the smooth endoplasmic reticulum (SER) is another source of Ca2+-binding sites within each lobe. 2+ ions: (apo)-CaM, (Ca2+)-CaM and (Ca2+)2-CaM  and inactive closed (tense) states of each EF-hand of CaM as proposed by Stefan et al. 2+/CaM/target interactions.CaM is a bi-lobed molecule that has two Cag states . We assu2+ transient in the spine (head) is regulated by a highly complicated set of nested feedback loops 2.3 voltage-sensitive Ca2+ channels, small conductance Ca2+-activated K+ channel (SK channels), and voltage-gated Na+ channels. The role of voltage-gated CaV2.3 channels and Na channels have been largely unknown until recently 2+ influx into the spine compartment, has been extensively studied in the past and we used a recently published model for our simulation  distribution, and detailed kinetics properties of these Ca2+ pumps. The membrane densities of the plasma membrane Ca2+-ATPase (PMCA) and the Na+-Ca2+ exchanger (NCX) are 150∼300/µm2 of membrane and 32∼60/µm2 membrane, respectively 2+ dynamics . Finally, we randomly incorporated Ca2+ leak channels so that the net flux of Ca2+ is 0 at rest (50 nM Ca2+).The reaction scheme for the Ca2+/CaM activation in the spine. The stochastic fluctuation of Ca2+ influx is then due to the stochastic kinetics of NMDA receptors.The NMDA receptor kinetics was taken from previous modeling work All other numerical analyses including spatial point pattern analysis and first passage time calculation were carried out under the Matlab environment .http://nba.uth.tmc.edu/cds). The CDS algorithm uses the discretized Brownian motion and relies on the first passage theory and event-driven simulation scheme. The overview of pre-existing particle-based Monte Carlo simulations  is described in  Smoldyn  and diff2+ ion during this time period is smaller than the size of CaM molecule).Under the CDS algorithm scheme, we calculate the first passage time (and probability) of molecular collisions and chemical reactions for each molecule in the simulation and create a list of all possible future events and their timing. We execute all of these molecular collisions and chemical reactions exactly as they happen one-by-one while moving all molecules simultaneously in the space. Every time we execute an event, we update the event list based on the new location or chemical status of the molecules. The time interval between two consecutive events varies from one simulation step to the other. Therefore, unlike time-driven Monte Carlo algorithms , there is no fixed time step in CDS. This event-driven scheme is the only accurate way to handle molecular collisions in a crowded cellular environment. In some cases, the interval between two successive events  becomes long and may result in the non-Brownian motion of molecules. To avoid this situation, we add “change of direction of move” to the event list so that the direction of molecular motion is constantly randomized at least once every10 ns (the jump length of Ca2+ ion was set to 0.2∼0.25 nm (larger than its atomic radius) taking into account its hydration shell 2+ as a solvated ion while simulating its diffusion and interactions with proteins. The diffusion coefficient (2+ in non-buffered cytoplasm is 200∼225 µm2/s (nm2/µs) In the CDS simulations, the radius of gyration of CaM (2.2 nm) was used to set the size of CaM molecules. The radius of Ca2+ channel (instead of 2+ saturation) can happen in the middle of the spine head when multiple channels exist or when multiple cycles of Ca2+ binding and unbinding to the same CaM molecule take place (2+-saturation point) distance distribution at all distance scales and over the entire spine compartment. The important point to note is that in a confined and complicated geometry such as dendritic spines, a simple mathematical formula of Besag's L-function for complete spatial randomness is unavailable. To overcome this constraint, we created 1000 sets of randomly distributed points in the spine of the same number of data points and then calculated the L-function for the data and for the simulated random point patterns. If the resultant L-function of the data deviates from the simulated point pattern, we can conclude that the data points are not randomly distributed.The idea behind the Ripley's K-function, or its derivative Besag's L-function, is that if the distribution of the points is random, the number of points within a distance ke place  and 9. OFigure S12+-CaM in a spine with a long neck. We randomly placed the same number of CaM molecules  as in 2+-CaM interactions. Here we show the summary of these analyses. Panel A shows the locations of Ca2+ saturation of C- terminal or N-terminal lobes up to the designated time point with a higher . No spatial gradient is formed for the C-terminal lobe with a lower Ca2+ injection rate. At a higher Ca2+ injection rate, Ca2+-saturation of the N-terminal lobe exhibits a gradient around the channel. The results did not depend on the diffusion coefficient of CaM as long as the CaM molecules were randomly placed in the spine.Domain of Caecules 1,00 as in ecules 1,00 as in er as in  or lowerer as in  Ca2+ injecules 1,00 as in (2.57 MB EPS)Click here for additional data file.Figure S22+-CaM in a spine with short neck. The same analyses Click here for additional data file.Figure S32+ binding to CaMKII-bound CaM. (A) A kinetic diagrams showing the interactions between Ca2+ and each lobe of CaMKII-bound CaM. The kinetic rates of Ca2+ binding to each lobe of CaMKII-bound CaM are shown in 2+ association rate and the leftward arrows are the Ca2+ dissociation rates. These values were obtained via parameter optimization as in 2+ association rate for the C-terminal lobe is no longer slow . Target free lobes are indicated by solid lines and CaMKII bound lobes are shown by dashed lines of the same color. The unit of first passage time (y-axis) is seconds. The range of Ca2+ concentrations considered is from 0.05 µM (resting level)∼12 µM (close to the peak Ca2+ concentration during the synaptic stimulation). The inset shows the blow up of mFPT near ∼1 µM Ca2+ concentrations.Mean first passage time of Ca(1.32 MB EPS)Click here for additional data file.Figure S42+ and lobe-specific Ca2+-CaM domain. These panels are snapshots from the simulations showing the spatial correlation between free Ca2+ ions with respect to Ca2+ saturated CaM. For illustration purpose, the sizes of molecules in the figure were artificially enlarged and are not proportional to their physical dimensions. In fact, the large light brown dots are free Ca2+ ions at the designated time points. Each of these panels are taken from a single simulation run used in 2+ injection rate) and 2+ injection rate). The small blue dots are the locations of (first) N-terminal lobe saturation (for each CaM molecule) taken from the same simulation. The red points are the locations of (first) full Ca2+ saturation of CaM up to the designated time point. Note Ca2+ ions are still being injected in Panel D at the designated time point but there is no significant spatial gradient of Ca2+ or the N-terminal lobe Ca2+ saturation. In Panel B (high Ca2+ injection rate), the (first) full Ca2+ saturations of CaM took place away from the channel.The spatial domain of Ca(1.56 MB EPS)Click here for additional data file.


Although it has been demonstrated that CaR  activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.Communication between the SR  and mitochondria is important for cell survival and apoptosis. The SR supplies Ca Mitochondrial Ca2+ is taken up from the cytosol via a low-affinity Ca2+ uniporter at mitochondrial membranes [2+ concentration ([Ca2+]i) is not high enough to initiate the uniporter under physiological conditions. Therefore, it has been postulated that activation of the inositol 1,4,5-trisphosphate receptors (IP3Rs) signaling pathway could release Ca2+ from the sarcoplasmic reticulum (SR) to increase the microdomain Ca2+ concentration ([Ca2+]) at focal contacts, known as mitochondria-associated ER membranes (MAM), between the SR and mitochondria, and then activate the uniporter. Recent studies have suggested that IP3Rs are highly compartmentalized at MAMs, providing direct mitochondrial Ca2+ signaling. Cardiomyocytes contain an abundance of mitochondria, many of which are in close apposition to SR Ca2+ release sites SR would change with CaR activation in response to hypoxia/reoxygenation in cardiomyocytes. We specifically focused on the relationship between SR Ca2+ depletion, mitochondrial Ca2+ uptake and cardiomyocyte apoptosis during hypoxia/reoxygenation (H/Re).The calcium-sensing receptor (CaR) is a member of the family of G protein-coupled receptors (GPCRs). One of the effects of CaR signal transduction is the activation of phospholipase C, which leads to the generation of the secondary messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate  supplemented with 10% fetal bovine serum (FBS) and then cultured in a 5% CO2 incubator at 37°C. Hypoxic conditions were produced using D-Hanks solution  saturated with 95% N2 and 5% CO2. The pH was adjusted to 6.8 with lactate to mimic ischemic conditions. The dishes were put into a hypoxic incubator that was equilibrated with 1% O2/5%CO2/94%N2. After hypoxic treatment, the culture medium was rapidly replaced with fresh DMEM with 10% FBS (10% FBS/DMEM) to initiate reoxygenation i was determined as previously described i and an emission wavelength of 530 nm for [Ca2+]i, using the equation [Ca2+]i = Kd[(F -Fmin)/(Fmax - F)], where Kd is the dissociation constant (345 nM for fluo-4), F is the fluorescence at intermediate Ca2+ levels (corrected from background fluorescence), Fmin is the fluorescence intensity of the indicator in the absence of Ca2+and is obtained by adding a solution of 10 mM EGTA for 15 min, and Fmax is the fluorescence of the Ca2+-saturated indicator and is obtained by adding a solution of 25 μM digitonin in 2.2 nM CaCl2 for 15 min. Final values for [Ca2+]i are expressed in nanomoles.[Caescribed . Briefly2+]SR, cardiomyocytes were treated with Fluo-5N acetoxymethylester (10 μM) for 2 h and deesterified for 1.5 h. For intact myocytes, the superfusate contained (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose . For permeabilization, myocytes were exposed to solution  and then permeabilized using saponin (50 μg/ml) for 20 seconds. Excitation was set at 488 nm and emission was measured at 530 nm at room temperature [2+]i and [Ca2+]SR were recorded using a laser confocal scanning microscope . There were more than 10 cells to be analyzed in each view and quantified using the analysis software for the microscope.To determine [Caperature . Images 2+ concentration ([Ca2+]m) consistently increases during reoxygenation [2+]m was measured at 60 min post-reoxygenation. [Ca2+]m was determined according to the manufacturer's instructions (Molecular Probes). In brief, the cultured cardiomyocytes (1 × 106 cells/sample) were initially washed with HEPES buffer containing (in mM) 130 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 10 HEPES, 11 glucose, and 0.2 CaCl2 at pH 7.4 and then stained with 5 μmol/L X-rhod-1 AM for 30 min at room temperature. To avoid deesterification of intracellular X-rhod-1 AM in the cytosolic compartment, which would interfere with the detection of [Ca2+]m, the cardiomyocytes were rinsed and incubated with 100 μM MnCl2-HEPES for an additional 20 min to quench the cytosolic Ca2+ signal [2+]m. To validate the measurement of [Ca2+]m, the cultured cardiomyocytes were transferred into a slide chamber after X-rhod-1 AM staining and were placed on the stage of a fluorescence microscope . The images from the slides were captured using a digital camera connected to Image-Pro Plus software . There were more than 10 cells to be analyzed in each view.Recent study showed that the mitochondrial Cagenation . Therefo+ signal . Fluoresm) was measured with a unique cationic dye of 5,5',6,6'-tetrachloro 1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), as previously described [2+]m might continuously increase during the process of reoxygenation and result in mitochondrial △ψm collapse i data were obtained from 2-3 experiments, and 10-12 images were analyzed in each group.Significance was evaluated using student's 3Rs were expressed in cardiomyocytes, while type 1 IP3R expression was undetectable , 78.3 ± 6.8% (Ca + Ni + Cd-H/Re) and 77.6 ± 5.1% (Gd + Ni + Cd-H/Re), respectively, compared with that of control cells using the MTT assay. Cell viability in NPS-2390 + Ca + Ni + Cd-H/Re (91.7 ± 4.6%), NPS-2390 is an allosteric antagonist of group 1 metabotropic glutamate receptors. 2-APB + Ca + Ni + Cd-H/Re  and Ru + Ca + Ni + Cd-H/Re  groups was more than that of the H/Re, Ca + Ni + Cd-H/Re and Gd + Ni + Cd-H/Re groups . The calcium concentration of the H/Re (346 ± 35 nM) and Ca + Ni + Cd-H/Re (321 ± 29 nM) groups was significantly increased compared to the control (81 ± 9 nM), NPS-2390 + Ca + Ni + Cd-H/Re (163 ± 15 nM) and 2-APB + Ca + Ni + Cd-H/Re (142 ± 11 nM) groups  that is only bright where [Ca2+] is very high, such as in the SR [3Rs and prevented PE-induced enhancement of Ca2+ sparks in neonatal cardiomyocytes [2+]i through the inhibition of Ca2+ release from the SR via IP3R. Thus, 2-APB treatment could maintain the fluorescence intensity in the SR of cardiomyocytes during reperfusion. These results suggested that the activation of CaR by CaCl2 or H/Re induced SR release of Ca2+.We have demonstrated that CaCln the SR . Rat neomyocytes . Our stu2-activated CaR significantly reduced [Ca2+]SR, the role of type 3 IP3Rs at the MAM in mediating Ca2+ uptake to mitochondria is less clear. To address this question, [Ca2+]m was measured at 60 minutes post-reoxygenation by X-rhod-1 AM staining. The [Ca2+]m was markedly low in the control group  and Ca + Ni + Cd-H/Re (589 ± 52 nM) groups than in the NPS-2390 + Ca + Ni + Cd-H/Re (331 ± 27 nM), 2-APB + Ca + Ni + Cd-H/Re (277 ± 29 nM), or Ru + Ca + Ni + Cd-H/Re (233 ± 26 nM)groups.Although CaClThe mitochondrial membrane potential was detected with JC-1 staining Fig. . The rat2 treatment during H/Re  and Ca + Ni + Cd-H/Re (0.25 ± 0.04) groups was significantly decreased compared with the control (1.0 ± 0.1), NPS-2390- + Ca + Ni + Cd-H/Re (0.75 ± 0.09) and Ru + Ca + Ni + Cd-H/Re (0.69 ± 0.08) groups  Activation of CaR induced the release of Ca2+ from the SR and, simultaneously, the increase of Ca2+ uptake into the mitochondria through MAM during H/Re. (ii) The CaR activation increased the expression of the p20-BAP31 fragment, the translocation of bax/bak from the cytoplasm to the mitochondria and the release of cytochrome c from the mitochondria during H/Re.This study was designed to address the potential involvement of the sarcoplasmic reticulum and mitochondria in regulating cardiomyocyte Ca3 from phosphatidylinositol 4,5-bisphosphate (PIP2). The major function of IP3 is to induce endogenous Ca2+ release through IP3Rs SR, which could be reversed by either the CaR inhibitor NPS-2390 or the IP3Rs inhibitor 2-APB. Furthermore, the type 3 isoform of the IP3R localized to the SR membranes. Taken together, these results suggest that activation of CaR is involved in the release of Ca2+ from the SR through the IP3R during H/Re.IPe to IP3 . During 3Rs play an important role in establishing macromolecular complexes on the surface of the SR membranes and in modulating the linkage between the SR and mitochondrial membranes. Mitochondria respond rapidly to physiological increases in [Ca2+]e, and stimulation with Gq-coupled receptor agonists, which induce IP3 production and the subsequent release of Ca2+ from ER, causes a rapid rise in [Ca2+]m [2+ release from ER and then in induced the [Ca2+]m rise. Our results suggest that [Ca2+]m was elevated and mitochondrial membrane potential collapsed in the Ca + Ni + Cd-H/Re group, whereas [Ca2+]m and mitochondrial membrane potentials were maintained in the 2-APB + Ca + Ni + Cd-H/Re group. The rapid mitochondrial Ca2+ uptake is related to the low affinity of the Ca2+ transport system. Therefore, Ruthenium red, an inhibitor of the mitochondrial calcium transporter, was used in our experiment. The results reveal that [Ca2+]m and mitochondrial potentials were maintained in the Ru + Ca + Ni + Cd-H/Re group. These results suggest that both the SR and the mitochondria orchestrate the regulation of Ca2+ signaling between these two organelles.Rizzuto et al. have provided a structural basis for this hypothesis by showing that mitochondria and ER form an interconnected network in living cells with a restricted number of close contacts . It has  [Ca2+]m . This ef [Ca2+]m ,28. CaR,2+ has been implicated in many models of apoptosis, a primary role for IP3 generation and the activation of IP3Rs in this process has been examined in only a few instances. Caspase-8 cleavage of BAP31 at the SR leads to the generation of a p20 fragment, which directs pro-apoptotic signals between the SR and mitochondria, resulting in early discharge of Ca2+ from the SR and its concomitant uptake into the mitochondria. Early and critical events in apoptosis occur in mitochondria and in the ER, and the release of elements acting as caspase cofactors, such as cytochrome c (from mitochondria) and Ca2+ (from the ER), into the cytosol are requisites for cell death in many cases i was increased, inhibiting the expression of Bcl-2 and elevating the expression of the pro-apoptotic protein caspase-3 in cytoplasm [2+-dependent model of apoptosis was subsequently supported by a series of observations with the pro-apoptotic Bcl-2 family members Bax and Bak. Cells deriving from knockout mice lacking Bax and Bak that are very resistant to apoptotic death have a dramatic reduction in the [Ca2+] within the ER and a drastic reduction in the transfer of Ca2+ from the ER to mitochondria [c release into the cytosol. Green et al. demonstrated that [Ca2+]SR depletion caused bax- and bak-mediated permeability of the outer mitochondrial membrane, thereby releasing pro-apoptotic factors and particularly cytochrome c [c release from the mitochondria during H/Re.Although a role for the SR in the mitochondrial redistribution of Cany cases . The mitc signal ,31. Jianytoplasm . Howeverchondria .This chachrome c . Our pre2+ signaling through the IP3Rs, which are also involved in apoptosis during H/Re.In conclusion, our results constitute the first report that CaR plays an important role in the SR-mitochondrial inter-organelle Ca3Rs: inositol 1,4,5-trisphosphate receptors; MAM: mitochondrion-associated ER membrane; H/Re: hypoxia/reoxygenation; CaR: calcium sensing receptor; GPCR: G protein-coupled receptors; PIP2: phosphatidylinositol 4,5-bisphosphate; MTT: 3--2,5-diphenyltetrazolium bromide; JC-1: 5,5',6,6'-tetrachloro 1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodideIPThe authors declare that they have no competing interests.WZ and CX drafted the manuscript, FL and ZT participated in the design of the study and did most of the experiments, YZ conceived of the study, HL, HR, HZ, CL and GH participated in its design and coordination, YT, BY and RW revised the paper and gave some suggestions. All authors read and approved the final manuscript.CaR inducing apoptosis via the sarcoplasmic reticulum-mitochondrion crosstalk in hypoxia/reoxygenation.Click here for file