Accordingly, using patch-clamp whole cell recording during silver ion application, inward current/s (IAg) at −90 mV were detected and cells were permeable to Ca2+ and monovalent ions such as Na+. IAg activation and the intracellular Ca2+ increase promoted by silver ions (Ag+) were prevented by co-application of 20 µM cysteine and 300 µM DIDS , suggesting a critical role of thiol groups in the biological effects of silver ions. IAg was partially inhibited by 1 mM Gd3+, an unspecific inhibitor of cationic currents. Cysteine, Gd3+ and extracellular free Ca2+ solution completely abolished blebbing formation promoted by Ag+. Furthermore, extracellular Na+ ion replacement with TEA or an increase of extracellular tonicity by sucrose (100 mM) reduced both size and growth of membrane blebbing. Our data suggest that Ag+ promotes the formation necrotic blebs as consequence of the increase of intracellular Ca2+ and intracellular hydrostatic pressure associated to the activation of cationic currents. Since silver-induced blebs were less evident in benign glial human Müller MIO-M1 cells, silver compounds could represent new adjuvant to anticancer agents to improve GBM therapies.Glioblastoma (GBM) is the most common and aggressive human brain cancer with low prognosis and therefore the discovery of new anticancer agents is needful. Sulfydryl reagents, such as silver, have been shown to induce membrane vesiculation in several cellular models through a mechanism that has not been yet completely clarified. Using U251 glioblastoma cells, we observed that silver induced irreversible bleb formation of the plasma membrane. This morphological event was anticipated by an increase of intracellular Ca Glioblastoma cells can migrate through different ways based on various interactions between cells and extracellular matrix. Three-dimensional or amoeboid glioblastoma migration was observed when the cells are forced to pass through tight spaces. In particular, cells change their shape and volume following a highly regulated process such as membrane blebbing3. The formation of blebs protrusions promotes amoeboid migration that typically occurs around the vessels4. Notably, blebbing based motility is essential for metastatic cells to escape anti-tumor treatments5. Several ion channels and transporters have been reported to display a role in the malignancy of glioblastoma cells by modulating morphological changes. Transporters and ion channels can regulate these mechanisms through two different ways: by changing the concentration of internal Ca2+ and by modifying the volume of the cell, a process closely related to a two-dimensional migration6. Recently, it has been reported that intracellular Ca2+ agonist bradykinin promotes blebs formation and invasion in glioblastoma cells7.Glioblastoma is the most common and aggressive type of human brain tumor. Its poor prognosis largely derives from its diffused invasiveness into normal brain tissues which precludes successful surgical resection8. The temporary behaviour of the bleb is associated to a transient increase of hydrostatic pressure in the cytoplasm close to plasma membrane as a consequence of acto-myosin cortex contraction9. The dissipation of hydrostatic pressure in the elastic network is delayed by the biophysical properties of the cytoplasm (poroelastic model). In the presence of defects of the plasma membrane, the transitory pressure increase causes a cytosol flow and bleb protrusion, caused by the interaction with the underlying cytoskeleton. During this phase, actin undetectably on bleb surface only becomes evident when cytosol inflation is slow. The membrane blebbing retraction is a consequence of the new formation of the myosin contraction machinery on the bleb inner surface9. Depending on the nature of the inducing agent, its concentration or cell line model, blebbing can become not transient, but can continuously grow until it produces extracellular vesicles10. For example, low concentrations of hydrogen peroxide promote transitory blebs whereas high concentrations promote a continuous blebs growth11. Bleb induction has been associated to membrane protrusion during apoptosis whereas continuous blebs growth can lead to cell damage and necrosis11. The result of a continuous bleb growth is the release of membrane vesicles, in the culture medium of cells from different tissues12. Same indications support the idea that both the blebs (transient and continuous) start from a common mechanism, but differ in the hydrostatic intracellular pressure profile11.Membrane blebbing is an outward budding process where a plasma membrane protrusion is transitory formed in timescale of tens of seconds13. Depending on the nature of the compound and the cell type, various cellular blebbing morphological responses have been described . The involvement of thiolic groups in the sulfhydryl reagents inducing vesicle release was demonstrated by studying thiols protective agents such as cysteine and glutathione. It has been suggested that the formation of blebs is related to defects in the functionality of specific thiolic (SH) groups having a fundamental role in controlling the plasma membrane permeability and the actin-myosin interaction13. Silver ion (Ag+) is among the sulfhydryl reagents able to induce blebbing. The present work addresses blebs formation in glioblastoma U251 cells and the alteration of plasma membrane permeability following Ag+ exposure. Herein, we studied the properties of ionic currents associated to the treatment with silver ions and the involvement of the intracellular Ca2+ homeostasis.Sulfhydryl reagents represent a class of compounds able to induce blebbing and vesicles release2-humidified atmosphere. For all experiments U251 cells were plated at 15,000/ml in a 35 mm petri dish and used on day 3 of culture.The human glioblastoma multiform cell line U251 , human Müller cell line MIO-M1 and epithelioid cervix carcinoma HeLa cell line were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin/streptomicyn, and 200 mM of L-Glutammine. The flasks were incubated at 37 °C in a 5% CO14, cells were incubated with FURA-2-AM  for 45 min and extensively washed with external Ringer’s solution of the following composition (in mM): NaCl 140, KCl 5, CaCl2 2, MgCl2 2, MOPS 5, glucose 10, at pH 7.4. Cells were continuously perfused using a gravity-driven perfusion system, focally oriented onto the field of interest. The estimation of intracellular free Ca2+ concentration, was reported as change of the ratio between fluorescence emission at 510 nm obtained with 340 and 380-nm excitation wavelengths . Ratiometric data were acquired every 3 sec and fluorescence determinations were performed using fluorescence microscopy system Zeiss (Axiozoom V16 and Axiocam 502 mono). The acquisition and analysis were driven by ZEN 2 software (Zeiss). Intracellular calibration of FURA-2 to obtain exact free intracellular Ca2+ concentration was carried out at the end of the experiments determining maximum and minimum 340/380 ratio of FURA-2 in the field of cells being studied. The maximum and minimum ratio was obtained with ionomicyn co-applicated with Ca2+ extracellular solution and with Ca2+ free plus EGTA respectively as described in O’Connor and Silver15. The Ca2+ concentration was calculated by using the relationship described for FURA-2 by Grynkiewicz et al.16. In some experiments the FURA-2 signal was expressed as % change of the 340/380 ratio, normalized to maximal signal obtained following 3 µM ionomycin application.Accordingly with our previous work17, whole-cell perforated patch clamp configuration was used for electrophysiological recordings from U251 cells. Currents and voltages were amplified with a HEKA EPC-10 amplifier, and analyzed with the PatchMaster and Origin 4.1 softwares. For online data collection, currents were filtered at 3 kHz, and sampled at 100 µsec/point. Membrane capacitance measurements were made by using the transient compensation protocol of PatchMaster. The external solution contained (in mM): NaCl 106.5, KCl 5, CaCl2 2, MgCl2 2, MOPS 5, glucose 20, Na-gluconate 30, (pH 7.25). Octanol (1 mM) + TRAM-34 (3 µM) and TEA (3 mM) were added to the external bathing solution to block gap-junctions and intermediate and Big conductance Ca2+-activated potassium channels18. The absence of Ca2+-activated K channel activation following Ag+ application was assessed by monitoring outward currents at 0 mV (Vh = 0) according to our previous reports19. The pipette solution contained (in mM) 57.5 K2SO4, 55 KCl, 5 MgCl2, 10 MOPS (pH 7.20). Amphotericin B (200 µM) was added to the pipette solution to achieve electrical access to the cytoplasm18 ranging between 10 and 20 MOhm within 10 min after seal formation. Electrophysiological recording was performed before visible bleb formation was observed. This allowed to carry out experiments without the heavy leakeage currents associated to increased membrane permeabilization.Accordingly with our previous work3 in Ringer’s solution. Pictures of cells were taken every 30 seconds at 20× magnification using an Axio Examiner (Zeiss) with CCD Axiocam 502 mono digital camera (Zeiss) for at least 30 minutes. Blebs number and area were measured using Zen 2 image processing software.U251 cells were washed twice with PBS and treated with 3 μM of AgNO3+) and Tetraethylammonium (TEA) were from Sigma Chemical Co . DIDS and DCPIB were purchased from Tocris Bioscience. TRAM-34 was a kind gift of Dr. Heike Wulff. TRAM-34 and Amphotericin B were prepared as stock solutions in DMSO at 20 mM and 50 mM respectively whereas DIDS was prepared in carbonate buffer at 100 mM. The maximal DMSO concentration used was 0.1% v/v for recording solutions. Solutions of AgNO3 (Sigma-Aldrich) were prepared immediately before application. Solutions were applied by a gravity-fed superfusion system at a flow rate of 2 ml/min. Experiments were carried out at room temperature (18–22 °C). Data are presented as mean ± SE.All analytical grade chemicals were used. Amphotericin B, Dimethyl sulfoxide (DMSO), L-Cysteine, Gadolinium  . The intracellular Ca2+ increase was dose dependent with EC50 of 2.1 µM  Fig. . All theels Fig. . Bath ap µM Fig.  and alsoely Fig. . The inc2+ mobilization promoted by Ag+  suggests the occurrence of an extracellular Ca2+ influx. In accordance with this observation, the removal of extracellular Ca2+ ions prevented the silver-induced intracellular Ca2+ increase , L-type Ca2+ currents, but involved a thiol-sensitive store-independent Ca2+ influx20. On the base of these observations, we tested the effects exerted on silver-induced Ca2+ increase by the incubation of the cells with: (i) the aspecific SOCE inhibitor Gd3+21, (ii) the L type voltage dependent Ca2+ currents antagonist nitrendipine22, (iii) the thiol reducing reagent cysteine23. Co-application of Ag+ with Gd3+ (30 and 300 µM) or nitrendipine (30 µM) did not abolish silver-induced Ca2+ increase (data not shown), whereas 1 mM Gd3+ preserved silver-induced Ca2+ increase  and Big (KCa1.1) calcium activated potassium currents, we performed electrophysiological recording in presence of the inhibitors TRAM-34 and TEA18 at the membrane voltage near the equilibrium potential of K+. In a physiological solution, in whole cell perforated patch-clamp configuration, 10 µM of Ag+ application activates an inward current at −90 mV  of 3.3 µM, 1.6 and 34.6 pA/pF, respectively  before . I-V relationship was built by estimation of the currents obtained from digital subtraction between currents in Ag+ and CTRL conditions at the end of the voltage steps  ions shifted the reversal potential of about 20 mV in negative direction , we verified whether this could also occurred for our currents. 300 µM DIDS inhibits about 80% the silver-activated cationic currents  in control conditions, which dropped down to −1.7 ± 9.2 (n = 7) following application with Ag+ . Since the increase of the intracellular Na+ accumulation and membrane depolarization has been demonstrated to be involved in intracellular Ca2+ increase by promoting reverse mode of Na/Ca exchanger28, we verified its involvement of Na/Ca exchanger in silver-induced Ca2+ increase. Pre-incubation of the cells with the inhibitor of Na/Ca exchanger YM24476928, did not abolish the ability of Ag+ to increase intracellular Ca2+ (data not shown), thus suggesting that it was not dependent on reverse mode of Na/Ca exchanger.Since IAg currents promoted Na+ application, we detected the appearance of membrane blebs. As shown in Fig. 2 within 45 minutes  with a similar diameter of the neck linking the membrane protrusion to the cytoplasm membrane11. In our experiments, cell exposure to micromolar silver concentration promotes blebs formation with a constant growth rate, a process similar to that associated with necrosis. According to this interpretation, application of trypan blue dye on blebbing cells marks cells nuclei due to the change of membrane permeability associated to the necrotic event11.In this paper, we studied the electrophysiological and morphological effects of Ag+ influx driven by nonselective cation channels and cell swelling31 defined as Necrotic Volume Increase (NVI). Similarly, we found that silver ions cell treatment activates, in a concentration dependent manner, a nonselective cationic currents (IAg) permeable to Na+, K+ and Ca2+. The role of Na+ influx associated to IAg in bleb formation was demonstrated by using pharmacological and biophysical approaches. The IAg inhibitors, such as DIDS, Gd3+ and cysteine prevented blebs appearance, whereas the Na+ extracellular substitution with TEA or the increase of extracellular osmoalarity, reduced the blebs growth. These data demonstrate that the involvement of IAg currents in blebs formation may be associated to Na+ and water influx that increase the intracellular pressure. Surprisingly, we observed that the increase of intracellular hydrostatic pressure, obtained by applying hypotonic conditions, resulted in cell swelling without blebs formation. This suggests that, beside hydrostatic pressure, a further mechanism induced by silver ions is necessary to promote necrotic blebbing event. It is likely that it involves the Ca2+ signalling, since IAg Ca2+-permeable current and extracellular Ca2+ removal abolished the silver induced blebbing. In some cases, the intracellular Ca2+ increase was demonstrated to cause the local destabilization of the link of the plasma membrane with the actin in the submembrane cortex and to promote bleb formation33. Intracellular hydrostatic pressure is balanced by membrane tension dependent upon the cytoskeletal network under normal conditions. During bleb formation the reduction of local membrane tension caused by cytoskeletal destabilization, represents the point where cytosol flows into the bleb. The cytosol invasion according to Bernoulli’s principle is driven by the hydrostatic pressure gradient between the interior of the cell and the bleb11. Our proposed mechanism for silver-induced blebbing formation is based on IAg-driven Na+ and Ca2+ influxes responsible of hydrostatic pressure and Ca2+ concentration increase respectively. In particular, the intracellular Ca2+ increase is necessary for bleb induction, whereas Na+ influx is necessary to bleb growth.Apoptotic blebbing coincides with cell shrinkage whereas necrotic blebs are accompanied to Na+ have been reported in different cellular models. In Helix pomatia neurons, Ag+ application was described to increase intracellular Ca2+ concentration and to activate aspecific cationic currents34 but their molecular nature was not identified. Similarly, in Xenopus oocytes Ag+ application increases an aspecific cationic Na+ permeable current conductance that was prevented by chloride current blockers DIDS and SITS but not by NPPB and 9AC. More interesting, reagents that reduce disulfide bridge like dithiothreitol (DTT) and cysteine abolished inward cation currents activated by Ag27. The IAg currents reported here share similar pharmacological and biophysical proprieties, such as DIDS, Gd3+ and cysteine sensibility, as compared to those described in Xenopus oocytes. It is however possible that the IAg currents recorded in our experiments flow through TRP-like channels sensible to redox status. In fact, it has been demonstrated that a class of TRP Ca-permeable cation channels is subjected to modifications of protein sulfhydryl groups and that these are sensitive to changes in the redox status35. Since silver-induced Ca2+ increase and IAg activation are both quickly reversible on wash-out, the receptor/s for Ag+ may be localized on the external side of plasma membrane. However, we cannot exclude the presence of cytosolic receptors for Ag+ based on the observation that an active sodium dependent transport of Ag+ was reported36.Several electrophysiological effects of Ag+ has been shown to be able to promote cell death and could represent a potential anticancer agent. We observed that U251 glioblastoma cells display a greater susceptibility to blebbing as compared to normal glial Müller MIO-M1 cell line. Since blebs formation is associated to necrotic induction, these data suggest the possible use of sulfhydryl reagents such as silver ions as new anticancer agent to induce selective necrotic program in cancer cells versus normal cells. The ability of Ag+ to generate hydroxyl radicals has been suggested to represent the underlying mechanism in inducing cytotoxicity and apoptosis by silver nanoparticles37. In fact, their action may be related to the capability to enter cells and release Ag+38. Understanding the molecular nature of the IAg activation, as the triggering factor of the necrotic bleb formation in glioblastoma cell lines, could be useful in developing a silver-based new agent, as a promising strategy for anti-cancer therapy and to improve the knowledge about the emerging application of silver in nanomedicine.AgSupplementary FiguresSupplementary Video S1


The membrane potential (Vm) and intracellular calcium (Ca2+) transient (CaT) are usually investigated separately, because incorporating different techniques to acquire both aspects concurrently is challenging. In this study, we recorded Vm and CaT simultaneously to understand the interrelation between these parameters in hiPSC-CMs. For this, we used a conventional patch clamp technique to record Vm, and synchronized this with a Ca2+ imaging system to acquire CaT from same hiPSC-CMs. Our results revealed that the CaT at 90% decay (CaT90) was longer than action potential (AP) duration at 90% repolarization (APD90). In addition, there was also a strong positive correlation between the different parameters of CaT and AP. The majority of delayed after depolarizations (DADs) observed in the Vm recording were also characterized by elevations in the intracellular Ca2+ level, but in some cases no abnormalities were observed in CaT. However, simultaneous fluctuations in CaT were always observed during early after depolarizations (EADs) in Vm. In summary, simultaneous recording of Vm and CaT broadens the understanding of the interrelation between Vm and CaT and could be used to elucidate the mechanisms underlying arrhythmia in cardiac disease condition.Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer a unique  Summary: We performed simultaneous recordings of action potentials and calcium transients from the same cardiomyocytes to improve understanding of the mechanisms underlying arrhythmias and the correlation between these two parameters. At the molecular level, hiPSC-CMs express major cardiac ion channels (2+ cycling components (2+. Inward current through sodium channels (INa) starts the rapid depolarization of the CM cell membrane, which triggers inward current through L-type calcium channels . This leads to Ca2+ induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptors. Ca2+ binds to sarcomeres, the contractile units of myofibrils, and allows the movement of myofilaments and contractile motion. The cell membrane is then repolarized by several repolarizing potassium currents and Ca2+ is reloaded into SR via SR Ca2+ ATPase. (The discovery of human induced pluripotent stem cells (hiPSCs)  from somchannels , Ca2+ cymponents  and adremponents . Thus, tmponents , LQT2  and intracellular Ca2+ concentration [Ca2+]i are the most crucial elements in the normal physiology and also in arrhythmias in cardiology. Ca2+ ion plays a major role in activation and modulation of contraction in CMs. The measurement of [Ca2+]i using a Ca2+ indicator during contraction and relaxation of CMs reflects the SR Ca2+ release and uptake. Furthermore, [Ca2+]i influences the shape and duration of AP via Ca2+-sensitive ionic channels such as ICa, Na+-Ca2+ exchanger (NCX) and Ca2+-activated nonselective cation channels, thereby modifying the electrophysiological properties, for example, the refractoriness and membrane depolarization rate  from the same hiPSC-CMs  (green), CaV1.2 (red) and cardiac ryanodine receptor (RyR2) (red) . These iNa is responsible for the rapid upstroke phase in AP, and peak INa current density was −23.1±5.2 pA/pF . Following rapid depolarization, transient outward potassium current (Ito) starts the repolarization of AP. We found that the peak Ito current density at 70 mV was 7.3±1.4 pA/pF . The calcium current (ICa) is responsible for the plateau phase of AP, and peak ICa current density at 10 mV was 2.6±0.7 pA/pF . As the plateau phase moves toward more negative membrane potential, mainly two types of potassium currents, rapid rectifier potassium current (IKr) and slow rectifier potassium current (IKs), are activated. IKr peak current was calculated at the end of 3 s depolarization test potential and IKr tail current is calculated as peak current in response to step depolarization. The peak and tail current densities of IKr at 0 mV were 1.0±0.3 and 1.1±0.2 pA/pF, respectively . Similarly, the peak and tail current densities of IKs at 40 mV were 0.6±0.1 and 0.5±0.1 pA/pF, respectively . In addition, inward rectifying potassium current (IK1) is activated during and following the repolarization phase to ensure the terminal repolarization and stable resting membrane potential. The peak IK1 current density and funny current (If) density at 130 mV were −2.5±0.6 pA/pF and −3.7±1.0 pA/pF, respectively .The presence of various voltage-gated ionic currents in hiPSC-CMs were confirmed using voltage clamp techniques. + dynamics under a microscope were chosen for simultaneous recording of APs and CaTs. APs were continuously recorded in gap-free mode, while CaTs were recorded in between for a minimum of 30 s. The majority of the patched cells (∼90%) were characterized as ventricular-like hiPSC-CMs. Only a minority of the patched cells were atrial-like (∼5%) or nodal-like (∼5%) hiPSC-CMs. Examples of APs and CaTs recorded from the same cells representing all three types of hiPSC-CMs are shown in 2+]i. Notably, the [Ca2+]i decreased to the minimum diastolic level after the repolarization phase of AP in hiPSC-CMs subtypes. Thus, the CaT at 90% decay (CaT90) was always longer than AP duration (APD) at 90% repolarization (APD90) irrespective of hiPSC-CMs subtypes. Our results showed that the average APD90 and CaT90 in ventricular-like hiPSC-CMs were 326.3±4.5 ms  and 790.7±13.7 ms , respectively. This implies that CaT90s were approximately 2.4 times longer than APD90s  and 552.8±18.3 ms , respectively, and thus CaT90s were approximately 2.3 times longer than APD90s  and 512.5±12.4 ms , respectively, therefore CaT90s were 2.6 times longer than APD90s  between these parameters, indicating that CaT parameters and AP parameters are interdependent. The END-2 differentiation technique produces a lower number of atrial-like and nodal-like hiPSC-CMs, thus only small number of those cell types were recorded in this study. Therefore, we did not perform correlation tests in atrial- and nodal-like hiPSC-CMs.Spontaneously beating hiPSC-CMs with visible Ca2n APD90s A,D. The n APD90s B,E. The n APD90s C,F. We pn APD90s . Our resm recording corresponded to [Ca2+]i elevation . Furthermore, we performed a correlation test  on those 33 DADs with similar observations in Vm and CaT between relative DAD amplitude (i.e. % of DAD amplitude with respect to APA) and the corresponding relative CaT amplitude (i.e. % amplitude of [Ca2+]i elevation with respect to CaT amplitude). The results demonstrated a positive correlation between these two parameters , implying that the amplitude of DADs was dependent on the amplitude of elevated [Ca2+]i. Also, we compared the amplitude of DADs with and without corresponding elevation in [Ca2+]i . We found that the average DAD amplitude  with corresponding elevation in [Ca2+]i was significantly higher than the average DAD amplitude  without corresponding [Ca2+]i elevation . In addition, phase 3 EADs were recorded during the simultaneous recording of Vm and CaT during baseline conditions  were occasionally observed . DADs welevation A,D. Howeons n=2, C,F and eCa,L is important for the spontaneous beating of hiPSC-CMs, 5 µM nimodipine was used  of cells showed prolongation of both APD and CaT, but there was no occurrence of EAD (n=12), meaning APs became slightly more triangular. In addition, the APD90 and CaT90 were 420.9±3.6 (n=12) and 891.7±8.7 (n=12) respectively; increasing by 72% and 77%, respectively, from their baseline values. Secondly, 40% (N=4/10) of cells showed prolongation of both APD and CaT duration and this eventually led to phase 2 EADs , respectively. Notably, the CaT90 was approximately 1.1 times longer than the APD90. Thus, the gaps between Vm and [Ca2+]i were closer during the terminal repolarization in phase 2 EAD episodes. Thirdly, 30% (N=3/10) of cells had depolarized MDP and increased in beating frequency, i.e. an oscillation configuration with or without the occurrence of phase 2 and phase 3 EADs (N=3) and beating frequency was increased by 110.6±43.1% (N=3). In these cases, the APD90s were increased by 41% and the average value was 355.2±3.7 (n=60). In contrast, the CaT90s were decreased by 2.4% and the average value was 547.3±6.9 (n=60). Although CaT90s were decreased from baseline, APD90s were still shorter than CaT90 in oscillation conditions. Moreover, the APD90/APD50 ratio was increased to 1.8±0.01 (n=60), indicating that AP shape became more triangular than in baseline conditions. Finally, 20% (N=2/10) of cells showed a cessation of beating, with minimal Vm and [Ca2+]i fluctuation (data not shown).Initially, simultaneous APs and CaTs were recorded in a normal extracellular solution. To test whether Iwas used A. This csed N=3, B. E-4031observed . Firstlye of EAD A,D. The e 2 EADs B,E. The e 3 EADs C,F. The m recordings are mostly characterized by elevation in [Ca2+]i; (4) blocking of IKr causes variable responses, including prolongation of both APD and CaT duration, occurrence of EAD and even cessation of beating. The AP recordings from hiPSC-CMs exhibit all phases of AP and current densities of different voltage-gated channels measured in the present study are comparable to earlier studies in hiPSC-CMs  voltage-gated ionic currents are functional; (2) there are strong correlations between AP parameters and CaT parameters; (3) DADs observed in ViPSC-CMs . The depfor CICR . However studies , indicatormal AP . Similarased CMs .2+ mapping using Fura-4F in the hiPSC-CMs monolayer, the CaT90s were also observed to be longer than APD90s (approximately 1.2 times) (2+ indicators: there was low-affinity ratiometric Fura-4F in previous studies as opposed to high-affinity non-ratiometric Fluo-4 as Ca2+ dye in our study. Ca2+ dye with high-affinity (Fluo-4) had significantly longer CaT duration than Ca2+ dye with low-affinity (Fura-4F)  . A possiFura-4F) .2+ release plays a role in the occurrence of both DADs and EADs  the [Ca2+]i amplitude got reduced by spatial averaging since the amplitude of DADs without [Ca2+]i elevation was significantly lower, (2) high-affinity Fluo-4 artificially prolongs the CaT duration, which overshadowed the DAD observed nearer to terminal repolarization of AP, or (3) the involvement of Ca2+-independent currents promoting depolarization of Vm, and possible mechanisms is still unknown. Consistent with a previous study , where CaT is no longer associated with Vm . Patients donating skin biopsies gave informed consent in Tays Heart Hospital, Tampere University Hospital, Finland. Two control hiPSC lines, UTA.04602.WT  and UTA.04511.WT  were used in this study. The UTA.04511.WT hiPSC line was generated using Sendai vectors, and UTA.04602.WT was generated by using pMX retroviral vectors without Cre-LoxP site. Both hiPSC lines were derived and cultured on mouse embryonic fibroblast (MEF) feeder cell layers  in culture medium containing knockout-DMEM (ko-DMEM) (Gibco) supplemented with 20% knockout serum replacement (ko-SR) (Gibco), 1% nonessential amino acids (NEAA) , 2 mM GlutaMax (Gibco), 50 U/ml penicillin/streptomycin (Lonza Group Ltd), 0.1 mM 2-mercaptoethanol (Gibco) and 4 ng/ml basic fibroblast growth factor (bFGF) . The characterization of these lines were found in previous study .2) as described earlier (Both hiPSC lines were differentiated into cardiomyocytes by co-culturing with Mitomycin C (Sigma-Aldrich) treated mouse visceral endodermal-like cells (END-2)   and RyR2  primary antibodies. The secondary antibodies were Alexa-Fluor-488-anti-goat-IgG, Alexa-Fluor-568-anti-rabbit-IgG (1:800) . Cells were mounted with Vectashield  containing DAPI for staining nuclei. The z-stack images were captured with Nikon A1 confocal laser-scanning microscope  using 60× oil immersion objectives . The images were further processed using ImageJ  and Adobe Photoshop CC 2017.Cardiomyocytes were dissociated onto 12 mm diameter coverslips. After 7 days, CMs were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with cTnT , I4) was ordered from MP Biomedicals . Stock solutions for extracellular solution were prepared in 1 M concentration and refrigerated until use. Extracellular solution was made freshly by diluting stock solution (in mM): 143 NaCl, 4.8 KCl, 1.8 CaCl2, 1.2 MgCl2, 5 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH). Intracellular solution was made and refrigerated (in mM): 132 KMeSO4, 4 EGTA, 20 KCl, 1 MgCl2 and 1 CaCl2 (pH adjusted to 7.2 with KOH). E-4031, nimodipine and chromanol 293B were dissolved in Milli-Q™ water (Millipore), ethanol (EtOH) and dimethyl sulfoxide (DMSO) respectively, and stored frozen. Cadmium chloride (CdCl2) and barium chloride (BaCl2) were dissolved in Milli-Q™ water and stored at +4°C. On the day of the experiments, the drugs were diluted to the final concentration in extracellular solution. 4-Aminopyridine (4-AP) was dissolved in the extracellular solution on the day of experiment. Amphotericin-B was first dissolved in DMSO, and this solution was added to intracellular solution to make final concentration of 0.24 mg/ml.All the drugs and chemicals for the experiments were purchased from Sigma-Aldrich unless otherwise specified. Potassium methanesulfonate . APs were recorded in gap-free mode in the current-clamp from the spontaneously beating hiPSC-CMs. Current-clamp recordings were digitally sampled at 20 kHz and filtered at 2 kHz using a low pass Bessel filter on the recording amplifier. Ca2+ imaging was performed using ANDOR iXon 885 EM-CCD camera  synchronized with a Polychrome V light source controlled by a real time control unit. Ca2+ kinetics of spontaneously beating CMs were imaged with an inverted Olympus IX70 microscope using UApo/340 0.75NA 20× air objective  and recorded with LiveAcquisition software , which was used to control light source and camera during recording. Fluo-4 was excited at 490 nm wavelength and the emission was recorded through Olympus U-MF2 Alexa 488 band-pass filter cube . The sampling interval was 20 ms. The pCLAMP software was configured to perform the synchronization between patch clamp and Ca2+ imaging system. When the Ca2+ imaging started, the synchronization pulse (SP) was sent from the imaging system to patch clamp system, thus SPs were recorded alongside with APs, and the SP stopped once the Ca2+ imaging stopped recording. The schematic diagram and video illustrating simultaneous recordings of patch clamp and CaT is shown in Fig. S3 and Movie 1.Cells on a coverslip were loaded with 4 μM Fluo-4 AM (Thermo Fisher Scientific) for 30 min in extracellular solution at 37°C. Extracellular solution was heated to 35-36°C with inline heater SH-27B controlled with a TC-324B controller unit . The perfusion was controlled by a gravity driven VC38 application system . Patch pipettes  were freshly prepared using PC-10 micropipette pullers and flame polished with MF-830 microforge . The patch electrodes had tip resistance of 2.0-3.0 MΩ with intracellular solution. AP recordings and CaNa was measured with ramp protocol, with a holding potential (HP) of −80 mV. First, the cells were depolarized to −120 mV for 50 ms and then ramped up to 40 mV with rate of 0.53 V/s. The ICa were measured in the presence of 3 mM 4-AP to block Ito, and an HP of −40 mV. To elicit the Ca2+ current, step-protocol from −60 mV to 70 mV with the step-size of 10 mV was used. The Ito was measured in the presence of 300 µM CaCl2 to block the Ca2+ current, and HP of −80 mV. Two-step protocol was used: first step was −50 mV for 50 ms to inactivate sodium channels, then to test potential of 500 ms duration from −50 mV to 70 mV with step-size of 10 mV. The IKr was measured as 1 µM E4031-sentitive current in the presence of 5 µM nimodipine and 10 µM chromanol to block ICa and IKs respectively. Using the HP of −40 mV, step protocol from −20 to 40 mV of 3 s with step size of 20 mV was used. The IKs was measured as 10 µM chromanol 293B-sentitive current in the presence of 5 µM nimodipine and 1 µM E-4031 to block ICa and IKr respectively. Using the HP of −40 mV, step protocol from 0 to 40 mV of 3 s with step-size of 20 mV was used. The IK1 and If were measured as 2 mM BaCl2 sensitive and insensitive current respectively in the presence of 300 µM CdCl2 and 3 mM 4-AP. From the holding potential of −40 mV, test potential from −140 mV to 0 mV with duration of 700 ms and step-size of 10 mV was used.All the voltage clamp experiments were performed at 35-36°C. The extracellular and intracellular solutions were similar to those used in the current clamp experiments, with the addition of specific ion channel blockers. The I2+ imaging analysis, a whole cell region of interest (ROI) was drawn. Acquired signal was normalized as ΔF/F0 using LiveAcquisition software (TILL Photonics). The Ca2+ traces were analyzed with Clampfit software to extract Ca2+ transient at 50% and 90% of repolarization (CaT50 and CaT90), and time-to-peak (time taken for CaT to reach its peak intensity). Each AP parameter was compared with its corresponding CaT parameter. Data from both hiPSC lines were combined. The hiPSC-CMs were categorized as ventricular-like and atrial-like CMs when they showed APD90/APD50<1.3 and APA>80mV; and APD90/APD50>1.35, respectively. Similarly, hiPSC-CMs were categorized as nodal-like CMs when they showed APD90/APD50>1.3 and APA<80mV with slower dV/dt. To check the correlation between two parameters, Pearson's correlation test was used. Differences between two groups were evaluated with student's unpaired t-test (two-tail). In this study, ‘N’ represents number of cells whereas ‘n’ represents number of AP or CaT used. P<0.05 was considered as statistically significant. Data is presented as mean±standard error of mean (s.e.m.).Recorded APs were analyzed with OriginTM 9.1  to extract APD50 and APD90. Voltage clamp data were analyzed using Clampfit software version 10.5 (Molecular Devices LLC). For Cam in hiPSC-CMs both during normal regular beating and in arrhythmic conditions. The simultaneous recording of Vm and CaT allows us to study the intracellular Ca2+ dynamics with respect to changes in Vm and vice versa. These experiments and models could be very helpful in understanding the correlation between Vm and CaT in more detail. Furthermore, hiPSC-CMs provide a safe and powerful tool to study the cardiac physiology and pathophysiology in vitro.This study demonstrates the interrelation between CaT and Vin vitro modeling of various genetic cardiac diseases, they have potential limitations because of their intrinsic differences compared to adult CMs. Some of the main ways that they do not fully resemble adult CMs are the lack of t-tubules and low expression of IK1. Furthermore, the majority of hiPSC-CMs differentiate into ventricular-like cardiomyocytes with our differentiation protocol, thus only limited atrial-like and nodal-like hiPSC-CMs are obtained. This hinders the ability to study the correlation between Vm and CaT in atrial and nodal cardiomycoytes. In addition, technical limitations exist in our measurement. During an AP measurement, the sampling rate was 20 kHz, which is enough to measure small changes in Vm. However, the frame-recording interval of 20 ms in Ca2+ imaging is not fast enough to capture small intracellular dynamics, especially during phase 0 of AP.Although hiPSC-CMs offer a robust platform for


The essential role of catecholaminergic effects on ventricular cardiomyocytes in this situation is well documented, but the importance of heart rate Sixteen CPVT1 patients performed a bicycle stress-test. Occurrence of VT triggers, i.e. premature ventricular complexes (PVC), depended on high heart rate, with individual thresholds. Atrial pacing above the individual PVC threshold in three patients did not induce PVCs.RyR2-R2474S (RyR2-RS) mutation, which exhibit exercise-induced VTs. While rapid pacing increased the number of Ca2+ waves in both RyR2-RS and wild-type (p<0.05), β-adrenoceptor (βAR) stimulation induced more Ca2+ waves in RyR2-RS (p<0.05). Notably, Ca2+ waves occurred despite decreased sarcoplasmic reticulum (SR) Ca2+ content in RyR2-RS (p<0.05), suggesting increased cytosolic RyR2 Ca2+ sensitivity.The underlying mechanism for the clinical observation was explored using cardiomyocytes from mice with the 2+ sensitivity was increased. Importantly, diastolic fluctuations in phosphorylation of RyR2 and SR Ca2+ content determined Ca2+ wave initiation. These factors were modulated towards increased propensity for arrhythmia initiation by increased pacing rates, but even more by βAR stimulation.A computational model of mouse ventricular cardiomyocyte electrophysiology reproduced the cellular CPVT1 phenotype when RyR2 CaIn CPVT1, VT propensity depends on individual heart rate thresholds for PVCs. Through converging data from clinical exercise stress-testing, cellular studies and computational modelling, we confirm the heart rate-independent pro-arrhythmic effects of βAR stimulation in CPVT1, but also identify an independent and synergistic contribution from effects of high heart rate. Patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) have an increased risk of sudden cardiac death due to ventricular arrhythmias. The mortality rate in untreated patients is 30–33% by the age of 35. Current In vivo, βAR stimulation increases heart rate,i) and SR Ca2+ content ([Ca2+]SR) during the last three seconds of a pacing protocol similar to the one used in the cellular experiments, while the right panel shows the same parameters in a 10 s post-pacing period after cessation of pacing. (B) Bar graphs of results from the post-pacing period: The left panel shows the frequency of Ca2+ waves in a 10 s period after 0.5 and 4 Hz pacing in presence and absence of ISO. Increased pacing frequency increased the frequency of Ca2+ waves in the post-pacing period in both RyR2-RS and WT, while ISO increased the Ca2+ wave frequency more in RyR2-RS. The right panel shows the time to occurrence of the first Ca2+ wave after cessation of pacing, i.e. Ca2+ wave latency. Increased pacing frequency decreased Ca2+ wave latency in a post-pacing period in both RyR-RS and WT, while ISO decreased the Ca2+ wave latency more in RyR2-RS.F.The effects of increased pacing frequency and stimulation of βAR on RyR-RS and WT observed in cellular experiments were reproduced in a computational model of mouse ventricular myocyte electrophysiology and ion homeostasis. The only adaptation needed to recapitulate the difference between RyR-RS and WT was increased luminal RyR2 Ca2+ sensitivity by 10% in RyR-RS. (A) The left panel shows modelled representation of whole-cell intracellular Ca(PDF)Click here for additional data file.


A point-biserial correlation revealed a strong association  between the presence of viable mites and degree of psoroptic lesions in the field trial.Mange is a common disease of rabbits globally, and knowledge of efficacy of drugs used in its treatment is critical for effective disease control. The current study evaluated the efficacy of three commonly used therapeutic agents in Kenya against mange. In a controlled laboratory trial, 20 adult rabbits were recruited for the study . The 16 mange-infested rabbits were randomly allocated into 4 treatment groups each consisting of 4 rabbits, while 4 mange-free rabbits formed the negative control group. Treatments were administered as follows: group 1 (G1) received two ivermectin injections at an interval of 14 days, group 2 (G2) was treated with a combination of carbaryl and liquid paraffin applied every other day up to the end of the experiment, group 3 (G3) was treated with liquid paraffin droplets applied daily until the lesion cleared, while group 4  received distilled water applied topically on their ears and group 5  was not treated with any preparation. The lesions were scored and sampled daily to check the viability of the mites. A field efficacy trial of the test compounds was performed using 105 mange-infested rabbits. The results revealed that all the test agents: ivermectin, liquid paraffin, carbaryl-water, and carbaryl-liquid paraffin combination were effective against mange, recording the lesion score of zero for psoroptic mange by day 21 in the laboratory and field trials. Lesion scores in the treated groups were significantly reduced ( Psoroptes cuniculi, Sarcoptes scabiei var. cuniculi, and Cheyletiella parasitovorax are the commonly reported mites infesting rabbits in Kenya [Psoroptes cuniculi is a large surface mite that causes psoroptic mange. Affected rabbits primarily present with clinical signs of intense pruritus, head shaking, erythema, and crusty, scaly, and scabby lesions on the inner side of the pinnae and on the external ear canal [Sarcoptes scabiei var cuniculi is a burrowing mite that causes sarcoptic mange characterized by extreme scratching, alopecia, crusts, scales, and erythema on the skin of the margins of pinna, lips, nose, legs, and areas adjoining the genitalia [Cheyletiella parasitovorax is a large, fast-moving, non-burrowing mite, which can be seen moving on the skin [Sarcoptes scabiei var. cuniculi and Cheyletiella parasitovorax have zoonotic potential [Skin conditions are among the most common diseases affecting pet, commercial, and laboratory rabbits in Kenya , 2. The ar canal . In extrar canal , 7. On tenitalia , 9. In cenitalia . Cheyletthe skin . Infestethe skin . Sarcoptotential .Sarcoptes scabiei species [Mange in rabbits has been treated effectively using chemotherapeutic and non-chemotherapeutic methods . Common  species  and side species  have bee species , it is p species .Randomized controlled laboratory and field trials were undertaken to determine the comparative efficacy of ivermectin, carbaryl, and liquid paraffin against mange of rabbits. In the laboratory trial, twenty adult rabbits of mixed breeds obtained from commercial rabbit farms which had never been treated with any antiparasitic drugs were used. Sixteen rabbits were naturally infested with psoroptic mange, while four were mange-free. The mange-infested rabbits were randomly allocated to 4 treatment groups: group 1 (G1), group 2 (G2), group 3 (G3), and group 4 (G4), each consisting of 4 rabbits. Group 5 (G5) composed of mange-free farmed rabbits served as the uninfected-untreated negative control group. In the field trial, 95 (91.3%) psoroptic-infested rabbits and 9 (8.7%) sarcoptic-infested rabbits were used. They were randomly allocated into 3 groups (treatment group 1 (TG1), treatment group 2 (TG2), and treatment group 3 (TG3)) each with 35 rabbits. All experimental rabbits were housed individually in widely separated cages to avoid direct contact in a house with adequate natural ventilation and lighting.The rabbits were examined for clinical signs and pathological lesions consistent with mange before and after commencement of treatment. The presence of live mites was confirmed by microscopic examination, and lesions examined included alopecia, ear scabs, erythema, crusts, dandruff, and exudation. Further examination after commencement of treatment was done at 3-day interval until day 21. Thereafter, the last examination was done 7 days after the last treatment (day 28). Rabbits with psoroptic mange were scored for severity of infestation using criteria described by Pan et al. , where 0Before recruitment into the study, deep and superficial skin scrapings and ear scab samples were collected from the rabbits to confirm sarcoptic and psoroptic mite infestation, respectively. Samples were then collected on day 0 (start of the treatment day) and afterwards on days 7, 14, and 21 during the treatment period, and last sampling was done on day 28 (one week after treatment termination) to assess response to treatment. During the field trial, additional sampling was done on day 56 to check for any relapse. Samples were placed in labelled sterlin tubes until the time of analysis. Each sample was divided into two portions where one was digested with 10% potassium hydroxide and examined under a light microscope (Germany Leica DM750 biomicroscope) for identification of the mites. The other portion was soaked in warm physiological saline (32°C), debris was filtered out, and then examined under a microscope for presence or absence of live mites as described by OIE . Viabiliμg/kg body weight on days 0 and 14 as recommended by the manufacturer for dogs. On day 0, G2 rabbits were treated topically with 5–8 drops (0.5 ml) of liquid paraffin  to wet the lesions. Thereafter, the wetted lesions were dusted with carbaryl  to cover the entire lesion. This treatment was repeated at 1-day interval until day 21. For G3 rabbits, 9 drops (0.6 ml) of liquid paraffin was applied topically on the lesions on a daily basis until day 21. The G4-infested rabbits were treated topically with 8 drops (0.5 ml) of distilled water applied daily until day 21 and therefore served as the positive control. The G5 non-infested healthy rabbits were not treated with any preparation and served as the negative control. All the rabbits in G4 (non-treated infested rabbits) received full dose of ivermectin injection at termination of the study in line with animal welfare requirements. Efficacy of the test compounds was based on elimination of sarcoptic mange lesions, reduction of lesion scores in psoroptic mange, and absence of viable mites on microscopic examination.The rabbits in G1 were subcutaneously injected with ivermectin  at 400 μg/kg body weight on day 0 and repeat injection administered after 14 days; rabbits in TG2 were dusted with carbaryl to cover the lesion after applying 5 drops of water to wet the lesion on day 0 and then after every 3 days for a period of 21 days; lastly, 9 drops of liquid paraffin were applied on the lesions on day 0 and then after every three days up to day 21 in TG3. Efficacy of test compounds was determined as described for the laboratory trial.Rabbits in TG1 were treated with subcutaneous injections of ivermectin at 400 p < 0.05. Kruskal–Wallis one-way analysis of variance in SPSS was conducted to examine the difference in lesion scores depending on the test agents administered. Dunn–Bonferroni test for pairwise comparisons was conducted post hoc to illustrate the significant differences between treatment groups. A point-biserial correlation was then run to check the relationship between the presence of viable mites and the extent of psoroptic mange lesion scores.Psoroptic lesions of the ears were scored in the laboratory and field trials. In the field trial, though each treatment group had 35 rabbits, only 29 rabbits with psoroptic mange lesions were scored. Data were analyzed using SPSS programme. Descriptive statistic results were presented as mean ± SEM, and significance levels itemized at Psoroptes cuniculi was recovered from the rabbits in the controlled laboratory trial, while P. cuniculi (a), Sarcoptes scabiei var cuniculi (b), and Cheyletiella parasitovorax (c) were recovered from the rabbits during the field trial. The mites recovered during the two trials are shown in rpb = 0.79, p < 0.05) between the presence of viable mites and the extent of psoroptic mange lesions assessed through lesion scores.Only p = 0.001) in psoroptic mange lesion scores 7 days (day 28) after termination of treatment between the positive control (infected not treated) group and the treated groups in the controlled laboratory trial. All the treated rabbits showed progressive recovery manifested by improved skin texture, healing of skin lesions, resolution of erythema, and fading of crusts. By day 21, all rabbits treated with liquid paraffin and carbaryl-liquid paraffin combination were negative for mites, while only one rabbit from the ivermectin treatment group had partially resolved clinical lesions (Ivermectin, liquid paraffin, carbaryl-liquid paraffin combination as used in the laboratory trial, and carbaryl-water combination used during the field study were all efficacious against psoroptic and sarcoptic mange. Efficacy was manifested by reduction of lesions in the course of treatment as presented in Tables  lesions , but the lesions . The relμg/kg after 14 day interval against local psoroptic and sarcoptic mite isolates. There was no relapse of mite infestation in groups of rabbits treated with ivermectin or carbaryl-water combination, during the field trial. Effectiveness of avermectins in controlling rabbit mange has been reported in previous studies for moxidectin [Similarly, findings of the present study confirmed efficacy of two injections of ivermectin administered at 400 xidectin , selamecxidectin , 13, 36,xidectin , and ivexidectin . Since iBased on the dosages and durations of treatment used in this study, ivermectin, liquid paraffin, and carbaryl were effective against sarcoptic and psoroptic mite-infested rabbits. The authors recommend adoption of liquid paraffin as a farmer's level interlude remedy for rabbit sarcoptic and psoroptic mange. Liquid paraffin has a wide safety margin and does not carry the risk of drug resistance. Furthermore, it is readily available, affordable, and easily administered at a farm level. Liquid paraffin also offers a break in the use of conventional synthetic treatments such as ivermectin which minimizes the development of drug resistance.


Two major sources are involved in the initiation of Ca2+-dependent signals: influx from the extracellular space and release from the intracellular Ca2+ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca2+ release from the stores under high spatiotemporal precision, we established a new method termed “organelle optogenetics.” That is, one of the light-sensitive cation channels , which is Ca2+-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGRER) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGRER co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGRER; (3) an increase of fluorometric Ca2+ was evoked by the optical stimulation (OS) in the cells expressing ChRGRER in a manner independent on the extracellular Ca2+ concentration ([Ca2+]o); (4) the ΔF/F0 was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and (5) the store-operated Ca2+ entry (SOCE) was induced by the OS in the ChRGRER-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca2+ from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca2+ dynamics under spatiotemporal precision.As one of the ubiquitous second messengers, the intracellular Ca The construct was verified by DNA sequencing.A cDNA fragment encoding ChRGR-Venus  relationship of the ChRGR photocurrent was assessed using the ND 7/23 cell—a hybrid cell lines derived from neonatal rat dorsal root ganglia neurons fused with the mouse neuroblastoma  according to the manufacturer's instructions. The medium was supplemented with 2.5 μM all-trans retinal at 6 h after transfection. Electrophysiological recordings were then conducted 24-48 h after the transfection. Successfully transfected cells were identified by the presence of Venus fluorescence.The current-voltage , which was supplemented with 20% fetal bovine serum , 100 units/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich). C2C12 myoblasts were grown to 80–90% confluence on a collagen-coated coverslip, transfected with plasmids using either Effectene or Lipofectamine 2000 (Invitrogen) and used for patch clamp and calcium imaging experiments.The C2C12 cell is a myoblast line derived from mouse skeletal muscle , which has been used as one of model systems of skeletal muscle development and differentiation. The cells were maintained for no more than fifteen passages and kept at 37°C with a 5% COI-V relationship of the ChRGR photocurrent was investigated as previously described : 120 N-methy-D-glucamine, 50 tetraethylammonium (TEA)-Cl, 10 EGTA, 50 HEPES, 5 MgCl2, 2.5 MgATP, adjusted to pH 8.4 with HCl. The low Ca2+ extracellular solution contained (in mmol): 100 N-methy-D-glucamine, 50 HEPES, 0.1 CaCl2, 46.9 MgCl2, 11 glucose, adjusted to pH 8.4 with HCl whereas the high Ca2+ solution contained (in mmol): 100 N-methy-D-glucamine, 50 HEPES, 10 CaCl2, 37 MgCl2, 11 glucose, adjusted to pH 8.4 with HCl. The directly measured liquid junction potential was −3.4 mV for either the low Ca2+ or the high Ca2+ external solution and was not compensated for. The photocurrent was evoked by a light from SpectraX light engine  at a wavelength  of 475 ± 28 nm. The power of light was directly measured under microscopy by a visible light-sensing thermopile  that was 1.3 mWmm−2 on the specimen.All experiments were conducted at room temperature (23 ± 2°C). The ER-Venus) were identified by Venus fluorescence using a conventional epifluorescence microscope , which was equipped with a 60× water-immersion objective lens  and a filter cube . Photocurrents were recorded as previously described : 138 NaCl, 3 KCl, 2.5 CaCl2, 1.25 MgCl2, 10 HEPES, 4 NaOH, and 11 glucose (pH 7.4 adjusted with HCl). The standard patch pipette solution contained (in mmol): 120 CsOH, 100 glutamic acid, 0.2 EGTA, 10 HEPES, 2.5 MgCl2, 3 MgATP, 0.3 Na2GTP, and 0.1 leupeptin (pH 7.4 adjusted with CsOH) for the voltage clamp. To test the optical responses, we used a cyan LED , which was regulated by a pulse generator  and pCLAMP 10.2 computer software. Its power density through an objective lens was 1.6 mWmm−2 when focused on the specimen.The myoblasts expressing either ChRGR-Venus or that with the ER retention signal  and mouse monoclonal anti-KDEL  diluted in 0.1% Triton X-100/PBS containing 5% goat serum, and then incubated for 2 h with the secondary antibodies, i.e., Alexa Fluor 488-conjugated goat anti-rat IgG (Invitrogen) and Alexa Fluor 555-conjugated goat anti-mouse IgG (Invitrogen), diluted at 1/200 in 0.1% Triton X-100/PBS containing 5% goat serum. The specimens were washed three times with PBS between treatments and mounted with ProLong Gold antifade mounting medium (Invitrogen). These procedures were performed at room temperature. Super-resolution images were taken under TCS SP8 STED 3X  with the STED (depletion) laser at 660 nm and white light laser. Alexa488 was excited at 488 nm and detected between 489 and 550 nm  and Alexa555 was excited at 561 nm and detected between 563 and 680 nm .ER and the red fluorescent Ca2+ probe R-CaMP1.07 /F0, where Ft is the fluorescence intensity at time t, and F0 is the average baseline fluorescence 1 s before the stimulation. The averaged ΔF/F0 within 300 ms during the stimulation was used as the magnitude of the Ca2+ transient. The imaging experiment was performed under superfusion with normal extracellular Tyrode's solution containing (in mmol): 138 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 4 NaOH, and 11 glucose (pH 7.4 adjusted with HCl), then switched to the Ca2+-free extracellular Tyrode's solution containing (in mmol): 138 NaCl, 3 KCl, 5 MgCl2, 10 HEPES, 4 NaOH, 1 Na2EGTA and 11 glucose (pH 7.4 adjusted with HCl).Cells were transfected with ChRGR2+ entry (SOCE) were investigated for the ChRGRER-expressing C2C12 myoblasts co-transfected with R-GECO1, a red-shifted fluorescent Ca2+-sensor : 138 NaCl, 3 KCl, 3.75 MgCl2, 10 HEPES, 4 NaOH, and 11 glucose (pH 7.4 adjusted with HCl), then switched to the standard Tyrode's solution containing (in mmol): 138 NaCl, 3 KCl, 2.5 CaCl2, 1.25 MgCl2, 10 HEPES, 4 NaOH and 11 glucose (pH 7.4 adjusted with HCl). In some experiments thapsigargin  was included in the Ca2+-free solution. For the store depletion experiments, images of R-GECO1 fluorescence were acquired using a laser-scanning confocal microscopy system  equipped with 16 × water-immersion objectives (0.8 NA). A fiber-coupled 451-nm laser source  was used for the optical stimulation. The free end of the optic fiber  was placed close to the C2C12 cell. The power of light was directly measured at the free end of the optic fiber, and was 18.7 μW. For the SOCE experiments, images of R-GECO1 fluorescence were acquired every 10 s on a FV1200 confocal laser scanning microscope (Olympus) equipped with a 40 × UPlanSApo objective lens using the 559 nm excitation and 590 nm emission long pass filter sets. The OS  were applied at 20 Hz with 10 ms duration and 100 pulses between imaging sequences. The store-operated Ca2+ entry (SOCE) was monitored using Ca2+ add-back protocol after treating with either OS or TG in a Ca2+-free Tyrode's solution. The ΔF/F0 after Ca2+ add-back was expressed by averaging 20 s of peak value after changing [Ca2+]o.The store depletion and the subsequent store-operated CaU-test for the unpaired data, the Wilcoxon signed rank test for the paired data, and the one-way Kruskal-Wallis test by ranks for multi-group data to determine statistical significance, unless stated otherwise. It was judged as statistically insignificant when P > 0.05.All data in the text and figures are expressed as the mean ± SEM and evaluated using the Mann-Whitney 2+ dynamics could be achieved by the specific targeting of light-sensitive actuators to the ER/SR membrane. Expanding the optogenetic toolbox enabled us to choose the optimal light-sensitive actuator depending on the experimental context . The expression patterns of ChRGR and the ChRGRER were visualized by Venus, which was tagged at their C-terminus  evoked a photocurrent of 428 ± 65 pA (n = 6) in the ChRGR-expressing cells  even with the same light stimulation  of blue LED light (4.5 mWmm−2 at 450 nm). In the standard extracellular milieu containing 2 mM Ca2+, the ChRGRER-expressing cells responded to the light by 7.1 ± 1.4% (n = 11) red fluorescence ΔF/F0 .To determine whether the ChRGR Figures . When th Figures . In fact2+ from the ER/SR induced Ca2+ entry through the plasma membrane and facilitated the subsequent uptake of Ca2+  by the coupling between STIM and Orai proteins  in a [Ca2+]o-free milieu, the entry of Ca2+ was evident from the [Ca2+]i increase upon the add-back of [Ca2+]o to 2.5 mM  whole cell patch clamp recording detected the photocurrent in the ChRGR-expressing C2C12 cells but not in the ChRGRER-expressing ones; (3) the fluorometric Ca2+ signal (ΔF/F0) was induced by the OS in the ChRGRER-expressing cells even in the absence of [Ca2+]o  the ΔF/F0 was sensitive to the SERCA inhibitor (thapsigargin). (5) the SOCE was induced by the OS in the ChRGRER-expressing cells. The organelle optogenetics  (Endo et al., 2+ have not been extensively studied because of technical limitations in differentiating between the two major sources of Ca2+ mobilization: the extracellular milieu and the internal Ca2+ stores. Therefore, the present optogenetic manipulation of ER/SR would represent a breakthrough in elaborating its physiological significance, such as the triggering of the sarcomere assembly (Ferrari et al., 2+ stores similarly have an important functional role in neurons; for example, somatodendritic signaling, synaptic transmission and plasticity (Simpson et al., 2+ in neuroscience.This paper demonstrated for the first time specific control of intracellular Cas Figure  would thTA, HI, TI, and HY conceived, designed, and performed the experiments, and analyzed the data. HI, TI, and HY contributed reagents, materials, and analysis tools. All authors drafted and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since little is currently known about NCX1 in the airway smooth muscle and its involvement in airway diseases, the present study was designed to investigate the expression and function of NCX1 in normal airway smooth muscle and its relevance to airway inflammation. Western blot analysis, tracheal smooth muscle contraction, and [Ca2+]cyt measurements were performed in mouse tracheal smooth muscle tissues and primary airway smooth muscle cell cultures. Additional studies were performed in a mouse model of allergic airway inflammation. Our data showed that NCX1 proteins were expressed in the human bronchial smooth muscle cells (HBSMCs), murine airway and whole lung. Carbachol raised [Ca2+]cyt in mouse tracheal smooth muscle cells and induced murine tracheal contraction, all of which were significantly attenuated by KB-R7943, a selective NCX inhibitor. Removal of extracellular Na+ increased [Ca2+]cyt in HBSMCs and mouse tracheal SMCs, which was dependent on extracellular Ca2+ and sensitive to KB-R7943. TNF-α treatment of HBSMCs significantly upregulated mRNA and protein expression of NCX1 and enhanced NCX activity. Finally, KB-R7943 abolished the airway hyperresponsiveness to methacholine in an ovalbumin-induced mouse model of allergic airway inflammation. Together, these findings indicate that NCX1 in airway smooth muscle may play an important role in the development of airway hyperresponsiveness, and downregulation or inhibition of NCX1 may serve as a potential therapeutic approach for asthma.Cytosolic free Ca Asthma is a major public health issue afflicting as many as 22 million people in the United States and approximately 300 million worldwide . The con2+]cyt) in airway smooth muscle cells is essential for airway contraction, secretion and remodeling; therefore abnormalities of [Ca2+]cyt homeostasis in airway cells may play a key role in the pathogenesis of asthma. Although multiple molecules and proteins control [Ca2+]cyt homeostasis, one of these is the plasma membrane Na+/Ca2+ exchanger (NCX). NCXs are actually a family of membrane transporters that can exchange Na+ and Ca2+ (or K+) in either direction depending on the transmembrane electrochemical gradient of Na+. Two sub-families of these exchange proteins have been described in mammalian cells (2+ movement is dependent only on Na+ (NCX1-3), and the other in which Ca2+ transport is also dependent on K+ (NCKX1-6). NCXs have been described in mammalian tissues for over three decades, but very little is currently known about the expression and function of these proteins in airway smooth muscle (or other cell types in the airway). Although NCX-mediated Ca2+ fluxes may contribute to enhanced [Ca2+]cyt regulation in airway inflammation , a major isoform of NCXs in human airway smooth muscle cells  the effect of the proinflammatory cytokine, TNF-α, on the expression and function of NCX1 proteins in primary cultures of HBSMCs in a cell model of allergic airway inflammation; (4) the role of NCX1 proteins on airway hyperresponsiveness in a mouse model of allergic airway inflammation. Some of these data have been published previously in an abstract form  of the following composition: NaCl 118 mM; KCl 4.7 mM; CaClIsometric force development was recorded in segments of tracheal rings (5 mm) by multichannel recorder, as previously described , 2000. B2 + 5% CO2. After adherent connective tissue was removed from the trachea, they were cut longitudinally and epithelial cells were also removed by mildly rubbing the luminal surface with a cotton swab. The tracheal tissues were washed three times with fresh PSS and then cut with fine scissors into small pieces (3 mm × 4 mm). These pieces were seeded onto P-100 plastic dishes , and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin G and 100 mg/ml streptomycin at 37°C in a humidified incubator equilibrated with 5% CO2 and 95% air. After 4–5 days in culture, the small pieces of tracheal tissue were removed and single cells were trypsinized and re-plated on 10-mm diameter circular, glass cover slips that were pre-coated with 1 mg/ml poly-D-lysine (Sigma).Primary cultures of murine tracheal smooth muscle cells were prepared from adult C57BL/6 mice. The isolated trachea was placed in cold PSS bubbled with 95% OThe collection and use of all human samples were approved by the Institute’s Human Ethics Committee of the First Hospital of Qinhuangdao and in accordance with the Declaration of Helsinki. Patients undergoing lobectomy or lung transplantation for lung cancer who had no evidence of asthma were the source of lung tissues for preparing primary cultures of HBSMCs. Lung tissues were removed from patients in the operating room, immediately placed in cold (4°C) saline, and taken to the laboratory for dissection. Bronchi were isolated from the lung tissues, and the smooth muscle segment was digested with 2.0 mg/ml collagenase and 0.5 mg/ml elastase at 37°C. The HBSMCs were cultured in smooth muscle growth medium , which consisted of smooth muscle basal medium, 5% FBS, 5 μg/ml insulin, 2 ng/ml human fibroblast growth factor, and 0.5 ng/ml human epidermal growth factor, for 5–7 days before each experiment.2) was used to compare cell growth rate. Cell viability was determined by using 0.45% trypan blue .Cell number was determined by a hemocytometer. The normalized cell number by size of the Petri dishes or cover slips  from the cells, as well as background fluorescence, was collected at room temperature (22°C) with the use ofa 40 × Nikon UV-Fluor objective and an intensified CCD camera (ICCD200). The fluorescence signals emitted from the cells were monitored continuously using a MetaFluor Imaging System  and were recorded in an IBM-compatible computer for later analysis. [Ca2+]cyt was calculated from Fura 2-AM fluorescent emission excited at 340 and 380 nm (F340/F380) using the ratio method based on the equation: [Ca2+]cyt = Kd × (Sf2/Sb2) × (R-Rmin)/(Rmax-R), where Kd (225 nM) is the dissociation constant for Ca2+, R is the measured fluorescence ratio, and Rmin and Rmax are minimal and maximal ratios, respectively , were combined with 2× Laemmli sample buffer. Samples were not boiled because this hydrophobic, membrane-bound protein was found to aggregate with boiling. Proteins were separated by electrophoresis on 4–15% SDS-PAGE and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose membranes were first incubated 30 min at room temperature in blocking buffer containing 2% non-fat dry milk in distilled water. Nitrocellulose membranes were incubated with R3F1 monoclonal antibody to NCX1  diluted in blocking buffer  overnight at 4°C and then rinsed for 1 h with a wash buffer containing 20 mM Tris, pH 7.5, 500 mM NaCl, and 1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated donkey anti-mouse IgG antibody for 30 min at room temperature and washed for 1 h with agitation, changing the wash buffer every 15 min. Protein bands were visualized with ECL Plus detection reagents , with NCX1 bands occurring at 120 and 70 kDa.Proteins were extracted from mouse heart, lung and tracheal tissues, or from HBSMC by homogenization on ice in 500 μl of lysis buffer containing: 20 mM Tris⋅HCl (pH 7.5), 150 mM NaCl, 1 mM disodium EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, and complete protease inhibitor cocktail . Equal amounts of protein, as determined by Lowry assay  according to the manufacturer’s instructions. To remove residual DNA, RNA samples were treated with RNAse-free DNAse and purity was confirmed by 260/280 ratios. RNA was reverse-transcribed using Superscript II . Real time PCR was performed in an M × 3000P real-time PCR system  using SYBR Green I dye as the detection format. A 16 μl reaction volume contained 16 ng of cDNA, 100 nM primers, using 1× of iQ SYBR Green super mix . The primers for human NCX1 were (forward) TGTGCATCTCAGCAATGTCA and (reverse) TTCCTCGAGCTCCAGATGTT. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control (housekeeping gene) to control for potential differences in RNA input. The primers for GAPDH were (forward) ACAGTCAGCCGCATCTTCTT and (reverse) TGGAAGATGGTGATGGGATT. Data were analyzed by determining the threshold cycle time (Ct) and normalizing NCX1 Ct to GAPDH Ct using the ΔΔCt method .C57BL/6 mice  6–8 weeks old were immunized and challenged over a 21 day protocol modified from a previously reported method . On days2O of PEEP. The proper placement of the endotracheal tube was verified by the generation of negative inspiratory airway pressures and expiratory gas output via the PEEP trap. The animals were then paralyzed with an intraperitoneal injection of 0.2 μg/gm pancuronium bromide . After a standard volume history and two total lung capacity maneuvers, small volume amplitude oscillations at a frequency of 0.9 Hz were applied at a constant volume to the airway opening for 16 s and respiratory system resistance (Rrs) was calculated with the FlexiVent 5.0 software package. The animals were then challenged with an ultrasonic aerosol of PBS containing 0, 3, 6, 12, and 24 mg/ml of methacholine for 10 s each. For 5 min intervals, measurements of airway resistance were again obtained using the forced oscillation technique every 30 s and Rrs calculated. The peak Rrs with each dose was used to generate a dose response curve.On day 21, anesthesia was induced with 0.5 mg/0.5 mg ketamine/xylazine  in 50 μl PBS and 3% Isoflurane . Mice were then intubated using a 20 ga, 1 inch intravenous catheter  guided by a PE10 stylet placed under direct visualization of the vocal cords. Mice were then placed on a mechanical ventilator , and ventilated quasi-sinusoidally at 150 breaths per minute, a tidal volume of 6 μl/g and 4 cm Hg for 10 min), and then fixed with acetone. Cytocentrifuge samples were then hydrated and incubated with FC Block  (1:100 diluted in permeabilization Buffer), at RT for 20 min. They were then treated with and Avidin/Biotin blocking kit  and then incubated with 1:200 dilution R3F1 primary or isotype control antibody at 4°C overnight, followed by a biotin-conjugated anti-mouse secondary antibody  for 1 h. Samples were then washed and incubated with Alexa fluor 488 labeled streptavidin  for 1 h. The samples were mounted in an aqueous mounting solution containing DAPI. The samples were examined with a Nikon i80 fluorescent microscope fitted with a low light digital camera and image analysis software.Human bronchial smooth muscle cells were grown to 70% confluence in T75 tissue culture flasks. These cells were trypsinized and cytocentrifuged , a muscarinic receptor agonist, induced two comparable phases of tracheal contraction in normal PSS  induced a transient increase in [Ca2+]cyt in mouse tracheal smooth muscle cells  and then perfused with same concentration of CCh. KB-R7943 markedly attenuated CCh-induced increase in [Ca2+]cyt  reverses the transmembrane Na+ gradient to favor Na+ extrusion and Ca2+ entry via NCX  also markedly attenuated the increase in [Ca2+]cyt via NCX  induced an increase in [Ca2+]cyt  the 0 Na+-induced increase in [Ca2+]cyt, indicating that the 0 Na+-induced rise in [Ca2+]cyt is due to inward transportation of Ca2+ which is caused by the Ca2+ entry model of NCX. Indeed, extracellular application of KB-R7943 (30 μM), a selective inhibitor of the Ca2+ entry model of NCX, abolished the increase in [Ca2+]cyt via NCX  treatment of HBSMCs with TNF-α  significantly enhanced the NCX activity. As shown in Figure  Figures . Thus, t2+ exit mode of NCX, i.e., the outward transportation of Ca2+, was also tested in HBSMCs. To isolate this mode of NCX, cyclopiazonic acid , a selective inhibitor of the sarco(endo)plasmic reticulum Ca2+ pump (SERCA), was used to prevent sequestration of Ca2+ into the sarcoplasmic reticulum Ca2+ stores and the plasma membrane Ca2+ pump (PMCA), or Ca2+/Mg2+ ATPase, was selectively inhibited by lanthanum  (2+]cyt in HBSMCs was raised by ionomycin (10 μM), a Ca2+ ionophore, the extrusion of elevated [Ca2+]cyt via NCX on the plasma membrane would be the major Ca2+ extrusion mechanism under these circumstances (3+ (or when both SERCA and PMCA were inhibited), the rate of Ca2+ decay represents activity of the Ca2+ exit mode of NCX. As shown in Figure 2+ decay was incomplete because only one of the Ca2+ extrusion mechanisms (via the Ca2+ exit mode of NCX) remained under these conditions. To test if the proinflammatory cytokine also enhances activity of the Ca2+ exit mode of NCX, HBSMCs were pretreated with TNF-α (20 ng/ml) for 24 h. TNF-α significantly increased the rate of Ca2+ decay in the presence of CPA and La3+  . Thus, wmstances . In the  Figures . These dn airway .To further study the role of NCX1 proteins in the airway, we used a well-described ovalbumin mouse model of allergic inflammation and airway hyperresponsiveness. To examine the functional relevance of upregulated expression and activity of NCX1 proteins enhanced in this model, we performed methacholine dose-response curves in control mice, mice immunized and challenged with ovalbumin and in the same mice pretreated with KB-R7943 . As illustrated in Figure 2+]cyt homeostasis in airway smooth muscle cells. Moreover, we show that there is a functional role for NCX1 in both mouse and human airway smooth muscle. Finally we show that the expression and activity of NCX1 proteins are enhanced by proinflammatory cytokine and that the airway hyperresponsiveness to methacholine can be substantially attenuated by KB-R7943, a selective inhibitor of the Ca2+ entry mode on NCX1. Our findings are in good agreement with previous reports on the functional expression of NCXs in the airway of animals plasmic reticulum (S/ER) stores and/or extruding Ca2+ across the plasma membrane. [Ca2+]cyt homeostasis, particularly in the loading state of the S/ER stores, is controlled by the activity of plasma membrane ion channels and exchangers  channels and NCXs  that reverses the transmembrane Na+ gradient for Na+ extrusion and Ca2+ entry via NCX . Under physiological conditions, opening of SOC/ROC results mainly in Na+ influx into the restricted plasma membrane-S/ER junctional space, causing membrane depolarization. Both the increase in Na+ and depolarization drive NCX into the Ca2+ entry mode of operation, further bringing Ca2+ into the cell. In airway smooth muscle cells, an increase in [Ca2+]cyt cannot only trigger Ca2+ release from the intracellular Ca2+ stores to induce airway smooth muscle contraction, but also may refill the empted intracellular stores with Ca2+. A growing line of evidence indicates the physical and functional couplings of TRPC3 and NCX1 proteins (Naeostasis . Blausteeostasis  and van eostasis  have recproteins , Since Tproteins  and NCX1proteins .In summary, we have confirmed the expression and function of NCX1 in normal human and murine airway smooth muscle cells. We have demonstrated that TNF-α treatment of human airway smooth muscle cells enhances the expression and activity of NCX1. We have also revealed that pharmacological inhibition of NCX1 attenuates murine airway hyperresponsiveness observed in an allergic model. We therefore suggest that NCX1 might be a relevant therapeutic target in inflammatory airway diseases, such as asthma.YW and HD designed the experiment and wrote and finalized the manuscript. JW conducted most experiments and data analysis. XM, BX, TZ, and HG conducted part of experiments. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pharmacological analysis revealed that both the Ca2+ signals and the nuclear NY translocation in astrocytes required NMDA and mGluR5 receptors and depended on extracellular Ca2+ entry via a store-operated mechanism. Our results provide a proof of principle that calcineurin in astrocytes may be activated in response to neuronal activity, thereby delineating a framework for investigating the role of astroglial CaN in the physiology of central nervous system.Astrocytes respond to neuronal activity by generating calcium signals which are implicated in the regulation of astroglial housekeeping functions and/or in modulation of synaptic transmission. We hypothesized that activity-induced calcium signals in astrocytes may activate calcineurin (CaN), a calcium/calmodulin-regulated protein phosphatase, implicated in neuropathology, but whose role in astroglial physiology remains unclear. We used a lentiviral vector expressing NFAT-EYFP (NY) fluorescent calcineurin sensor and a chemical protocol of LTP induction (cLTP) to show that, in mixed neuron-astrocytic hippocampal cultures, cLTP induced robust NY translocation into astrocyte nuclei and, hence, CaN activation. NY translocation was abolished by the CaN inhibitor FK506, and was not observed in pure astroglial cultures. Using Fura-2 single cell calcium imaging, we found sustained Ca Astrocytes are an abundant non-neuronal cellular type in the brain . They exCalcineurin (CaN) is a calcium/calmodulin-activated serine-threonine phosphatase, which is highly expressed in the brain . In neur2+ entry through the astroglial plasma membrane is required for this process to occur.The present work is designed as an in vitro proof of principle of astroglial CaN activation in response to neuronal activity. We show that long-term potentiation (LTP)-like neuronal activity robustly activates CaN in adjacent astrocytes and that store-operated Ca2+ ionophore widely used to activate CaN in different cellular types [2+-free Krebs-Ringer buffer (KRB) buffer supplemented with 2 mM Ca2+ , followed by washout of the cLTP cocktail and addition of KRB containing 1 mM Mg2+ and 2 mM Ca2+  fused with EYFP (NY) A, has bear types ,25,26,272+-free KRB , which consisted in application of N-methyl-d-aspartate  for 20 min in Mg2+-containing KRB [Alongside the cLTP protocol, we used other strategies to induce neuronal activity, namely bicuculline alone at 40 μM in Mgfree KRB B and KRBuculline C. Both pning KRB , indicatGiven that the cLTP protocol produced the most robust and fast translocation of NY in astrocytes, we further used it for detailed characterization of cLTP-like activity-induced astroglial CaN activation.2+-free KRB + Ca solution to wash out Mg2+ ions. Then, cLTP cocktail was applied and, after 4 min, it was changed to KRB + Ca + Mg solution. After registration, Ca2+ signals were analyzed separately in neurons and astrocytes. As shown in 2+-free KRB application induced a single Ca2+ spike, while cLTP cocktail induced a burst of Ca2+ spikes which lasted for the whole duration of the cLTP induction phase in neurons. After cLTP cocktail was washed-out, neurons either did not exhibit any Ca2+ activity , or generated single spikes  with a frequency of 0.32 ± 0.14 spikes/min. Example of an experiment without neuronal Ca2+ spikes in the cLTP development phase is shown in 2+ spikes. In astrocytes, robust Ca2+ signals were generated during the induction phase, which followed the neuronal burst of Ca2+ spikes and slightly decayed before removal of the cLTP cocktail. After the re-addition of KRB + Mg + Ca, the cultures were registered for longer time (25 min) and delayed Ca2+ transients were observed in a fraction of astrocytes. Closer examination of Fura-2 images revealed that only astrocytes which were juxtaposed to neuronal bodies or neuronal processed generated delayed Ca2+ signals  astrocyte Ca2+ signals during the induction phase were secondary to neuronal Ca2+ signals; and (2) neuronal Ca2+ signals during the induction phase are necessary for the astroglial Ca2+ signals to occur during the phase of cLTP development. Next, involvement of the metabotropic glutamate receptor, mGluR5, was investigated. 2+ signals and NY nuclear translocation. This result suggests the requirement of mGluR5 for the activity-induced CaN activation, although, in the current setting, it does not discriminate between neuronal and astroglial localization of mGluR5. Previously, we published that in rat hippocampal mixed neuron-astroglial culture neurons preferentially responded to NMDA while astrocytes responded to 3,5-Dihydroxyphenylglycine (DHPG) [Next, we used pharmacological inhibition to investigate the molecular mechanisms of neuronal activity-induced CaN activation in astrocytes. Caof FK506 B. Furtheof FK506 C or onlyof FK506 D or duriof FK506 E. When Mobserved E. When Mobserved C or cLTPobserved , neithere (DHPG) , a power2+ entry has been shown to mediate long lasting Ca2+ elevations in cultured astrocytes [2+ signals and CaN activation in astrocytes. However, Pyr10   astroglial Ca2+ signals and CaN activation required extracellular Ca2+ entry via the SOCE mechanism. In this report, we provide an in vitro proof of principle of activation of astroglial CaN by neuronal activation. The main findings are: (1) in mixed neuron-astroglial hippocampal primary cultures, cLTP induction protocol, which specifically stimulates neuronal activity, induced intracellular CaAlthough, to our knowledge, there is no data published to date that neuronal activity may result in CaN activation in astrocytes, it has been reported that the increase in neuronal activity is able to induce CaN activation and nuclear translocation of NFAT in pericytes in cortical slices . This la2+ elevations [2+ signals in astrocytes is extremely complex, and depends on the nature of Ca2+-related receptors/channels expressed in a particular subdomain of astroglial plasma membrane [Calcineurin is activated by a specific pattern of calcium signaling which is characterized by low and sustained (minutes) elevations of baseline cytosolic calcium levels , while tevations . Stimulaevations ,43,44,45evations ,47, in bevations ,49,50,51evations ,53. Mostevations ,39,44. Sevations ,54 to spmembrane . Accordi2+ entry in astrocytes via SOCE mechanism. SOCE is one of the fundamental mechanisms of astroglial Ca2+ signalling [2+ oscillations, refilling of the endoplasmic reticulum with Ca2+ [2+ oscillations in vivo in fine astroglial processes were shown to involve Ca2+ entry through the plasma membrane, probably through store-operated channels [2+ increase in astrocytes, but not in neurons [2+ transients in neurons [2+ entry in cultures astrocytes that was efficiently blocked by SOCE inhibitors [Our findings suggest that neuronal activity induces Cagnalling  and is iith Ca2+ , astroglith Ca2+  and astrith Ca2+ . Spontanchannels ,61. Concchannels , and is channels  astrocytchannels  and dire neurons  .In neurons, CaN is involved in long-term changes during neuronal plasticity, e.g., forebrain neuronal deletion of CaN specifically affects bidirectional synaptic plasticity and episodic-like working memory , and ina2+-free buffer [2+ withdrawal, which indicates that different strategies to facilitate neuronal activity may lead to CaN activation in astrocytes, although with different temporal pattern. The present report is a proof of principle in vitro study, and is not devoid of limitations, the two principal of which are: (1) the in vitro setup, which, obviously, only in part replicates the complex LTP phenomenon occurring in intact brain or even in brain slices; and (2) chemical instead of electrical LTP induction. We have consciously accepted these limitations for the following reasons. The in vitro setting proved to be simple and highly reproducible both in terms of lentiviral NY infection and in terms of cLTP and NY nuclear translocation. Thereby, it allowed us to rule out astrocytes as a primary target of cLTP protocol. Yet, we used hippocampal primary cultures and the effect may be different in cultures prepared from the other brain regions. Regarding cLTP induction, there are several protocols which are basically the modifications of two principal variants: the first consists in the application of a cocktail containing protein kinase A (PKA) activators, like forskolin and rolipram ,71,72, we buffer ,74,75,76e buffer  and, not2+ signals in astrocytes, as well as of CaN activation, may differ between ex vivo and in vivo preparations, and may also depend on age at which the preparation is made.An important question which remains unanswered in this work is the nature of the mediator which is released by neurons to induce the astroglial response. Assuming that astrocytic mGluR5 receptors are involved in astrocyte activation, it is plausible to speculate that the messenger is glutamate released during neuronal activity. This has been demonstrated in vitro  and in s2+ entry through the plasma membrane, possibly implicating astrocytic mGluR5 receptors. The Ca2+ entry occurs through Orai1-containing SOCE channels and results in activation of CaN inside the astrocytes, which, in turn, leads to activation and nuclear translocation of CaN sensor NY. CaN activation in astrocytes may lead to transcriptional remodeling and long-term changes analogous to what occur during neuronal plasticity. In this model, we provide a framework for future investigation of astroglial CaN activation during neuronal activity and plasticity in physiology and pathology of CNS.In summary, we propose a model  in whichC57Bl/6 mice have been purchased from Charles River Laboratories , Italy). The animals have been given food and water ad libitum; light/dark cycle has been automatically controlled in respect with the natural circadian rhythm and the temperature has been thermostatically regulated. Animals were managed in accordance with European directive 2010/63/UE and with Italian low D.l. 26/2014. The procedures were approved by the local animal-health and ethical committee  and were authorized by the national authority . g for 5 min. Tissue pellet was resuspended in HBSS supplemented with 10% FBS and dissociated by 30 strokes of a 1000 μL automatic pipette. After pipetting, the tissues were left for 5 min to allow sedimentation of un-dissociated tissue, and cell suspension was transferred to a new tube and centrifuged at 250× g for 5 min. Hippocampal cells were resuspended in Neurobasal-A medium supplemented with 2% B-27 and with 2 mg/mL glutamine, 10 U/mL penicillin and 100 µg/mL streptomycin, and plated onto Poly-l-lysine-coated coverslips (0.1 mg/mL). For NY translocation time-course, the cells were plated onto 13 mm round coverslips in 24 well plates, 2 × 104 cells per well. For Fura-2 imaging, the cells were spotted (2 × 104 cells per spot) on 24 mm round coverslips in 6 well plates. Half of medium was changed every 5 days. Cultures were used for treatments and experiments after 12 days in vitro (DIV12). At DIV12-DIV14 the neuron/astrocyte ratio was 1.29 ± 0.49 as was counted in 34 coverslips from at least 8 independent cultures.To prepare mixed neuron-astroglial hippocampal primary cultures, new-born mice (less than one day old) were used. Pups were sacrificed by decapitation and hippocampi were dissected in cold HBSS . After dissection, hippocampi were digested in 0.1% Trypsin  for 20 min in 37 °C water bath. Then, trypsin was neutralized by addition of Dulbecco’s Modified Eagle’s Medium  supplemented with 10% heat-inactivated fetal bovine serum  and tissue was spun at 300× g, 5 min), pellet was resuspended in complete medium and plated in 100 mm Falcon culture dishes, pretreated with 0.1 mg/mL Poly-l-lysine (Sigma). At sub-confluence (DIV5-10), cells were detached with trypsin and microglial cells were removed by magnetic-activated cell sorting (MACS) negative selection using anti-CD11b conjugated beads and MS magnetic columns . After MACS, astrocytes were counted and plated for experiments as described above. Virtually no microglial cells have been detected in purified astroglial cultures after MACS procedure as was assessed by immunostaining with anti-Iba1 antibody  [To prepare purified astroglial cultures, one to three-day old pups were sacrificed by decapitation, hippocampi were rapidly dissected and placed in cold HBSS. Tissue was digested in 0.25% trypsin , washed in complete culture medium ((DMEM), supplemented with 10% FBS, and with 2 mg/mL glutamine, 10 U/mL penicillin and 100 µg/mL streptomycin  and resuspended in cold HBSS supplemented with 10% FBS. After 30 strokes of dissociation with an automatic pipette, cell suspension was centrifuged (250× 9-19741) .2O to a final volume of 500 mL) overnight. On the next day the precipitate was concentrated by centrifugation (2800× g at 4 °C for 30 min) and resuspended in HBSS  third generation lentiviral vector was a kind gift from Prof. Alexander Flügel, Göttingen . The proDIV10-12 mixed neuron-astroglial cultures or 50% confluent purified astrocytes were transduced by adding (50 μL/mL) concentrated NY-expressing viral particles. Experiments were conducted 2–4 days after infection. For each condition and time-point, at least 2 coverslips were used  in three independent culture preparations .2+-free Krebs-Ringer buffer . First the cells were rinsed by Mg2+-free KRB along to wash out Mg2+ ions. Then, cLTP cocktail was applied for 4 min to induce LTP (cLTP induction phase). After this, the cLTP cocktail was changed to Mg2+- and Ca2+-containing KRB (KRB + Mg + Ca) and kept until the cells were fixed or imaged (cLTP development phase). At indicated time-points after cLTP induction, KRB + Mg + Ca was quickly removed and cells were fixed with 4% formalin in PBS ). Fixed cells were washed 3 times with PBS and mounted on microscope slides using SlowFade® Gold Antifade mountant .cLTP was induced by a cLTP cocktail containing 20 μM bicuculline, 1 μM strychnine, 200 μM glycine in MgFixed cells were imaged on a Leica DMI6000 epifluorescent microscope equipped with Polychrome V monochromator  and S Fluor ×40/1.3 objective . The cells were alternatively excited by 488 and 546 nm and emission light was filtered through 520/20 and 600/40 nm bandpass filters, respectively, and collected by a cooled CCD camera . The fluorescence signals were acquired and processed using MetaMorph software . To quantify NY translocation, 5 random fields were photographed from each coverslip, and the astrocytes with clear expression of NY sensor were used for analysis (3–15 astrocytes per field). For each cell two regions of interest (ROIs) were placed inside the nucleus (which was evidenced by mCherry-H2Bc expression) and two in different sides of the cytosol close to the nucleus. For each cell, the fluorescence intensity of two nuclear (Nuc) and two cytosolic (Cyt) ROIs, respectively, measured in green channel, was averaged and Nuc/Cyt ratio was obtained by dividing the resulting Nuc fluorescence to Cyt fluorescence. The data are expressed as mean ± SEM for each cell analyzed form three independent culture preparations e.g., .2+ imaging experiments, the cells were first loaded with 2 μM Fura-2-AM in presence of 0.02% of Pluronic-127 (both from Life Technologies) and 10 μM sulfinpyrazone (Sigma) for 20 min at RT. Fura-2 loaded cells were washed in KRB + Mg + Ca and allowed to de-esterify for 20 min before cLTP induction. For Fura-2 Ca2+ transients the ratio values were normalized using the formula ΔF/F0 . At least two coverslips for each of three independent culture preparation were imaged for each condition. In mixed neuron-astroglial cultures, astrocytes were recognized as flat polygonal or star-like cells, while neurons were recognized by round bodies with few processes located on upper focal plane above the astrocytes. The cells with uncertain morphology were not taken in consideration.After that, the coverslips were mounted into acquisition chamber and placed on the stage of a Leica DMI6000 epifluorescence microscope equipped with S Fluor ×40/1.3 objective. The probe was excited by alternate 340 and 380 nm using a Polychrome IV monochromator and the Fura-2 emission light was filtered through 520/20 bandpass filter and collected by a cooled CCD camera . The fluorescence signals were acquired and processed using MetaFluor software . To quantify the differences in the amplitudes of Ca2O) and DHPG  were from Tocris . Pyr3 , Pyr6  and Pyr10  were from Sigma.MK801 , MTEP , 2APB , Xestospongin C , U73122 , NMDA (Cat. 0114, stock 100 mM in Ht-test. Differences were considered significant at p < 0.05. Data are expressed as mean ± SEM.Statistical analysis was performed using GraphPad Prism software v.7. For analysis of Nuc/Cyt NY ratio  each dat