In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules  Howeverelopment . Mdm2 anelopment . Both prelopment . Mdm2 caelopment .The N-terminal p53 binding domains and the RING domains of Mdm2 and MdmX are highly conserved in evolution , as is tin vivo (A notable feature of RING-type E3s is their tendency to form homodimers and heterodimers that seems to be the prerequisite for the E3 activity for many RING finger ubiquitin ligases . The RINin vivo . A preci2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin G, and 50 μg/ml streptomycin sulfate .Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively. Cells were cultured at 37°C / 5% COMammalian expression plasmids coding for wild-type Mdm2, Mdm2 mutant Mdm2∆9, lacking the conserved C-terminal tail required for RING dimerization, Mdm2 mutant C464A, disrupting the RING domain structure and function, Myc-tagged MdmX, FLAG-tagged human wild-type p53 (pcDNA3-FLAG-p53), and the Mdm2/MdmX chimera (MDM2/MDMXRFD) have been described previously . GFP-MdmU2OS cells in 60-mm dishes were transfected with 0.1 μg FLAG-p53 and 1.4 μg Mdm2 mutant plasmid constructs using Effectene transfection reagent according to guidelines of the manufacturer (Qiagen). Each transfection mixture also contained 0.2 μg pEGFP-N1 (Clontech) to control for transfection efficiency, and empty plasmid pcDNA3.1 (Invitrogen) was used to bring the total amount of transfected DNA to 1.7 μg. Cells were washed with cold PBS 30 h post-transfection and lysed with 0.25 ml of SDS sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with anti-Flag M2 , anti-Mdm2 Ab-1 , and anti-GFP 7.1/13.1  antibodies. In some experiments, p53 was also detected using the anti-p53 monoclonal antibody DO-1 .U2OS cells grown in 60-mm dishes were transfected with 1 μg Myc-MdmX and 6 μg wild-type Mdm2 or Mdm2 mutant plasmid constructs using Lipofectamine 2000 transfection reagent (Invitrogen) and DMEM was changed 4 h later. Each transfection mixture also contained 0.1 μg pEGFP-N1 as a control for transfection efficiency. The empty plasmid pcDNA3.1 was used to bring the total amount of transfected DNA to 7.1 μg in cells transfected with MdmX expression plasmid alone. Cells were cultured for 30 h, washed with cold PBS, and lysed with 0.3 ml of SDS-PAGE sample buffer. Proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-Myc 9E10 , anti-Mdm2 Ab-1 , and anti-GFP 7.1/13.1  antibodies.™594-conjugated secondary antibodies (Jackson ImmunoResearch) in the blocking solution containing 1 μg/ml DAPI (Sigma-Aldrich). Cells were washed with PBS and mounted with VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories). Images were taken with the FluoView 500 confocal laser scanning fluorescence microscopes (Olympus).U2OS cells grown on coverslips were transfected with Myc-tagged MdmX or GFP-tagged MdmX (0.3 μg), and wild-type Mdm2 expression plasmids or the Mdm2/MdmX chimeric construct (1.2 μg) using Effectene transfection reagent (Qiagen) and the culture medium was changed 4 h later. The empty plasmid pcDNA3.1 was used to bring the total amount of transfected DNA to 1.5 μg in cells transfected with MdmX expression plasmid alone. Twenty-four hours post-transfection, cells were treated with 15 μM MG132 (Sigma-Aldrich) in DMEM for 3 h, washed with PBS, and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After fixation, cells were washed with PBS and permeabilized with PBS containing 0.2% Triton X-100 (Sigma-Aldrich) for 5 min. Cells were blocked with PBS containing 0.5% bovine serum albumin at room temperature for 30 min and then incubated for 2 h at room temperature with anti-Mdm2 mouse monoclonal antibody IF2  in the blocking solution or the anti-Mdm2 antibody together with anti-c-Myc rabbit polyclonal antibody . Cells were washed with PBS and incubated for 1 h at room temperature with FITC- or DyLightHEK293 cells were transfected with 6 μg DNA per 100-mm plate using Lipofectamine 2000 reagent (Invitrogen). Cells were treated with a proteasome inhibitor (10 μM MG132 in cell culture medium) 24–36 h post-transfection, 4 h later washed with PBS, and lysed in Triton X-100 lysis buffer  containing protease inhibitors . Lysates were pre-cleared with 50 μl of protein G-Sepharose (Millipore). Immunoprecipitations were performed at 4°C with 1–2 μg of an antibody bound to 50 μl of protein G-Sepharose for 1 h. Mdm2 was immunoprecipitated with anti-Mdm2 antibody IF2  and Myc-MdmX with anti-Myc antibody 9E10 . Immunoprecipitated proteins were washed with lysis buffer and resuspended in SDS-PAGE sample buffer. Proteins from whole-cell extracts and immunoprecipitations were resolved by SDS-PAGE and analyzed by Western blotting with anti-Mdm2 Ab-1  and anti-Myc 9E10  or anti-Myc A-14 .U2OS cells grown in 60-mm dishes were transiently transfected with FLAG-p53 (0.5 μg), hemagglutinin (HA)-ubiquitin (0.5 μg), and Mdm2 plasmid constructs (5 μg) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. Empty plasmid pcDNA3.1 (Invitrogen) was used to bring the total amount of transfected DNA to 6 μg. Cells were treated 24 h post-transfection with 15 μM MG132 for 3 h, washed with PBS, and lysed in 0.3 ml 0.5% SDS. Lysates were boiled for 5 min, vortexed, cooled down to room temperature, and diluted with 1 ml of Triton X-100 lysis buffer. p53 was immunoprecipitated using 1 μg of anti-p53 monoclonal antibody DO-1 on a rotating wheel at 4°C. After 1 h of incubation, 20 μl of protein G-Sepharose beads (Sigma-Aldrich) were added for 45 min. The immunoprecipitates were washed three times with lysis buffer and analyzed by Western blotting. Ubiquitylated p53 was detected using anti-HA-Peroxidase antibody ; FLAG-p53 and MDM2 levels in the input were detected using anti-FLAG antibody M2 (Sigma-Aldrich), anti-Mdm2 Ab-1 (Merck).http://decibel.fi.muni.cz/cozoid/). The 2VJF, 2HDP, and 5MNJ structural data have been used for the Mdm2/MdmX analysis and visualization. Structures were visualized and simulated using the PyMOL tool .The Mdm2 contact residues mediating its binding to the MdmX and UbcH5b partners have been identified using COZOID (COntact ZOne IDentifier) tool . This protein retains most of the Mdm2 sequence  but the C-terminal portion of Mdm2 including the RING domain was exchanged with the MdmX sequence [Interestingly, in immunoprecipitations, the interaction of two MdmX RINGs was observed only when the cysteine residue was introduced into both of them , suggestvia its C-terminal tail -ubiquitin complex suggested that despite their apparent structural similarity, the Mdm2 homodimer and Mdm2-MdmX heterodimer have differing abilities to transfer Ub due to their abilities to interact with E2~Ub complexes (~ indicates thioester bond). While Mdm2 RING could bind both UbcH5b and ubiquitin, MdmX did not bind the E2 enzyme and contacted only ubiquitin, bcH5b~Ub .In the present study, we used PCR-mediated site-directed mutagenesis of the Mdm2 RING domain in the context of full-length Mdm2 to identify residues that could be responsible for the apparent lack of the capacity to homodimerize and low intrinsic E3 activity of the MdmX RING. We mutated parts of the Mdm2 RING into the corresponding MdmX residues and observed a significant drop in the activity toward MdmX in mutants affecting E436, VI439–440, QG442–443, C449, T463, and RQP479–481. Of these mutants, 436K, 439–440SL, 442-442EK, and 479–481KKE could be inactive because they lost at least one residue predicted to participate in the interaction with E2. Surprisingly, however, mutants 439–440SL and 442-442EK were still capable of targeting p53 for degradation, indicating that they remained active in the form of the Mdm2-Mdm2 homodimer. Such a result might suggest that a different set of residues at the Mdm2/E2 interface might be required for E2 binding in Mdm2 homodimers and Mdm2-MdmX heterodimers. Importantly, our mutant 479–481KKE was inactive both in the form of heterodimer and homodimer, confirming the critical contribution of R479 to the ubiquitin ligase activity of Mdm2 .in vivo. When cysteine was introduced into the corresponding position in the MdmX RING, the mutant MdmX protein gained the ability to form homodimers. Our data are consistent with earlier studies in which the RING domain of MdmX was mutated to contain selected Mdm2 residues. Iyappan et al. turned MdmX into an active ubiquitin ligase by mutating N448 to cysteine and replacing residues 465–480 with the corresponding Mdm2 sequence (in vitro. They found that substitution of N448 for cysteine and K478 for arginine granted the MdmX RING domain the E3 activity. Based on subsequent structural analysis, the authors reasoned that C449 of Mdm2 is critical for the stability of the RING dimer structure, while R479 could play a role in recruiting and activating the ubiquitin E2 conjugating enzyme (Our study also identified the conserved cysteine 449 of Mdm2 and asparagine 448 of MdmX located at the RING-RING interface as critical determinants of the ability of Mdm proteins to form dimers sequence . Egorovag enzyme . The crig enzyme .in vivo, in line with the results of structural analyses indicating that cysteine and asparagine residues contact in the Mdm2-MdmX heterodimer, but two asparagine residues would clash, preventing MdmX homodimerization. However, in immunoprecipitations, we could detect MdmX homodimerization only when N448 was mutated to cysteine in both MdmX RING domains, suggesting that the context of the RING domain also plays a role. Moreover, the interaction between Mdm2 and MdmX RING domains also seemed unstable when the two critical residues were swapped, as we could not detect the binding of Mdm2 449N to MdmX 448C in the immunoprecipitation assay, despite the predicted compatibility of the two residues. Last but not least, the Mdm2 449N mutant still served as E3 ubiquitin ligase targeting p53 for degradation, again suggesting that the asparagine and cysteine residues had a differential impact on Mdm RING domain function depending on the structural context.Interestingly, we found that only one RING with the N448C exchange was required for MdmX RING homodimerization It seems that the tight mutual regulation of Mdm2, MdmX, and p53 activity in cells is enabled by many individual differences between the RINGs of Mdm2 and MdmX that participate in the control of RING dimerization and E2 binding.PK, IS, and AV performed experiments and prepared the figures. BV, PM, and JP analyzed the results and prepared the figures. SU conceived the study, performed experiments, analyzed the results, and wrote 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.


The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K+ channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K+ hotspots. We show how intracellular Ca2+ buffers affect Ca2+ waves and why the classical Ca2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca2+ imaging.Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K 2 + transients. Here, the authors develop a computational modelling environment for astrocytes, akin to the NEURON environment, called ASTRO.Astrocytes have gained increasing attention for their roles in regulating neural circuits via neurotransmitter uptake, K + buffering, and ability to signal via Ca Tri-dimensional (3D) reconstructions of astroglia using electron microscopy (EM) have long revealed a system of nanoscopic processes7 that pervade the entire cell expanse9. Deciphering cellular mechanisms that shape Ca2+-dependent signalling and physiological membrane currents in this sponge-like system has been a challenge.Astroglia have emerged as an essential contributor to neural circuit signalling in the brain. In addition to the well-established mechanisms of neurotransmitter uptake and extracellular K11. There have also been numerous attempts to simulate astroglial function, mainly from a reductionist standpoint (reviewed in refs. 13). Aimed at a specific question, such models would normally focus on kinetic reactions inside astroglia15, between astroglial and neuronal compartments17 or on astroglial influences in neuronal networks19. These studies have provided some important insights into the biophysical basis of astroglial physiology. However, their scope would normally exclude complex cell morphology, intracellular heterogeneities or the impact of Ca2+ buffering mechanisms on Ca2+ signal readout. Thus, integrating cellular functions of an astrocyte on multiple levels, in one realistic entity in silico, remains to be achieved.In contrast, cellular machineries underpinning neuronal physiology have been understood in great detail. This is partly because it has been possible to interpret electrophysiological and imaging observations in neurons using realistic biophysical cell models, such as those developed in the NEURON environment22. We have combined patch-clamp electrophysiology, two-photon excitation (2PE) imaging and 2PE spot-uncaging, fluorescence recovery from photobleaching (FRAP), astroglia-targeted viral transduction Ca2+ indicators in vivo, and quantitative correlational 3D EM to systematically document the multi-scale morphology and key physiological traits of these cells. Based on these empirical constrains, we have built a multi-compartmental 3D cell model fully integrated into the NEURON environment. The latter was equipped with additional functionalities relevant to astroglia, such as control of tissue volume filling and surface-to-volume ratios, options for extracellular glutamate application and K+ rises, endfoot and gap junctions menus, choice of fluorescence imaging conditions, etc.Our aim was therefore three-fold. Firstly, to develop a modelling approach that would recapitulate fine astroglial morphology while retaining full capabilities of biophysical simulations enabled by NEURON. We have therefore generated (MATLAB- and NEURON-based) algorithms and software that (a) use experimental data to recreate the space-filling architecture of astroglia, and (b) make this cell architecture NEURON-compatible. Our case study focused on the common type of hippocampal protoplasmic astroglia in area CA1, which has been amongst the main subjects of studies into synaptic plasticity and neuron-glia interactionsOur second objective was to implement this approach as a flexible simulation instrument—cell model builder—capable of recreating and probing various types of astroglia in silico. Thus, we have integrated our algorithms and software as a modelling tool ASTRO, which enables an investigator to generate morphological and functional astroglial features at various scales.+ dynamics, the range of intracellular Ca2+ buffering capacity, and how the classical molecular machinery of Ca2+ ‘puffs’ and ‘sparks’ could explain some Ca2+ imaging observations in astrocytes. Our findings suggest that ASTRO could be a valuable tool for physiological hypothesis testing and causal interpretation of experimental observations pertinent to astroglia.Finally, as a proof of concept, we explore our test-case astrocyte models  to reveal some important aspects of astroglial physiology that are inaccessible in experiments. We therefore evaluate key electrodynamic features of the astroglial membrane, basic aspects of intracellular K25. To elucidate this structure further, we used acute hippocampal slices, loaded individual astroglia in whole cell with the morphological tracer Alexa Fluor 594 (Methods), and imaged the cell expanse using two-photon excitation  which could be resolved in optical images  as a stem tree skeleton , plus a ‘transitional slab’ , with base area Ti  occupied by astroglial processes in the hippocampal neuropil ranges between 5 and 10% VF Fig. . We thus VF Fig.  and used−1 range36. The astrocyte fragments sampled in our experiments  approach: the fluorescence recovery rate in a laser-bleached cell region reflects diffusion speed of the fluorescent molecules. We therefore sought to gauge diffusion connectivity among astroglial compartments by bleaching dye molecules within a thin cylindrical volume (laser line-scan) across the astrocyte arbour Fig. ; Methodsn = 10; Fig. First, we confirmed that fluorescence fully recovered within 60 s of the FRAP cycle , which gave Gm = 0.69 ± 0.18 mS cm−2 41 which were represented, for the sake of simplicity, by a non-specific channel current maintaining resting Vm near −83…−85 mV. Thus, Kir4.1 should have little effect on the voltage spread profile, even though these channels largely control the cell resting membrane potential42. Indeed, when we added evenly distributed Kir4.1, their effect on voltage was detected only when their overall conductance exceeded its expected physiological range .Astrocyte membranes are enriched in potassium channels, in particular K43, scattering the transporters uniformly on the cell surface (at ~104 µm−2)44, and applying extracellular glutamate within small spherical areas  at quasi-random distances from the soma. An excellent match between the modelled somatic current and whole-cell recording data could be obtained  affects membrane potential across the cell, we first recorded from a CA1 astrocyte the response to two-photon spot-uncaging of glutamate (20 ms) at variable distances from the soma Fig. , traces.ned Fig. , plot byir4.1 . The ensuing local increase in intracellular K+ concentration [K+]in (from 110 to ~113 mM) dissipated over several seconds after [K+]out returned to 3 mM  and no contribution from Kir4.1 channels  increased Ri by ~30% , enabling current leak and diffusional flow of molecules across the astroglial syncytiumWe have incorporated basic gap junction options in the ASTRO menu, both as electric conductance and as a diffusion channel  and possibly ryanodine receptor-channels displaying a highly non-linear (sometimes bell-shaped) dependence between channel activity and Ca2+ concentration50. Recently, local Ca2+ transients  have been documented in astroglia lacking IP3 receptors52. In any such cases, intracellular Ca2+ signal propagation must be tightly controlled by the local Ca2+ buffering capacity53, the feature comprehensively explored in nerve cells.Astroglial Ca2+ dynamics including diffusion, buffering, and IP3 action (Methods), to which we have added two further IP3-dependent mechanisms described in the literature54. In the default configuration, four parameters control intracellular Ca2+ waves: resting IP3 concentration CIP3, resting Ca2+ concentration [Ca2+]rest, endogenous Ca2+ buffer concentration [B] and its affinity (dissociation constant) KD. Most studies constrain CIP3 within 0.5–3 µM55 whereas earlier we were able to measure [Ca2+]rest using time-resolved fluorescence microscopy (range 50–100 nM)56. In contrast, [B] and KD can vary widely across cell types and thus remain unknown.The NEURON environment by default incorporates biophysical mechanisms of Ca2+ waves over a wide  range of the above parameters. To dissect the basic effect of Ca2+ buffering on Ca2+ wave propagation, we compared wave dynamics with and without a small amount (10 µM) of mobile Ca2+ buffer added , which appears remarkably similar in their basic morphological features and territorial volumes to hippocampal astrocytes58.Because there is an ongoing debate on whether astroglial CaB] and KD could be assessed by matching the empirical Ca2+ wave dynamics to the modelled outcome. Spontaneous activity of astrocytes in vivo was recorded using a virus-transduced Ca2+ indicator expressed under a GFAP promoter: to minimize filtering effects of free-diffusing Ca2+ indicators, we used the plasma-membrane-tethered GCaMP6f (~50 ms fluorescence response time)59. The animals were anaesthetized, to limit bursts of sensory input-evoked prominent Ca2+ rises that could be mistaken for self-propagating Ca2+ waves. Gross morphology of astrocytes was monitored in the red channel using bolus-loading of sulforhodamine 101  appeared on average ~5.4 times a minute over a 160 µm x160 µm ROI . Intriguingly, this speed appears significantly lower than that of the stimulus-induced astroglial Ca2+ waves in brain slices or in culture . One possible explanation is that an exogenous stimulus in situ, such as agonist application, is likely to trigger a synchronous receptor response over the entire cell expanse. Similarly, in awake animals, we detected single-cell Ca2+ waves resembling those under anaesthesia, albeit at lower frequencies and magnitudes, in between prominent, region-wide Ca2+ elevations  near the soma . This simple formula captures the buffering properties of cortical astrocytes, also suggesting the lower-limit Ca2+ buffer concentration of ~170 µM .In the astrocyte model, experimentally observed Caoma Fig. . Althoug KD Fig.  reflecte2+ elevations had been the key indicator of astroglial activity. Recent advances in Ca2+ imaging revealed faster and more local Ca2+ signals prevalent in small astrocyte processes (reviewed in refs. 64). Any such observations rely however on high-affinity Ca2+ indicators, which provide only a crude reference to the underlying Ca2+ signals35. To translate recorded fluorescence into Ca2+ dynamics, one has to model Ca2+ entry, diffusion, and buffering (by endogenous proteins as well as Ca2+ indicators), as demonstrated in numerous studies of nerve and muscle cells. We therefore used ASTRO to apply a similar modelling strategy to CA1 astrocytes loaded with Fluo-4, which show robust, multi-scale spontaneous Ca2+ activity in acute slices . The key features emerging from these studies are the occurrence of Ca2+ micro-domains (0.5–5 µm apart) represented by clusters of stochastically activated Ca2+ receptor channels (including IP3 or ryanodine receptors), with the agonist-dependent mean opening time of 2–20 ms, peak amplitude of ~5 pA, and an inter-opening interval varying between 0 and 50 ms68. Stochastic activation of this system produces local Ca2+ ‘sparks’ (reflecting ryanodine-receptor clusters) or ‘puffs’ (reflecting IP3-receptor clusters), typically with an event frequency of 0.1–2 Hz69, sometimes leading to global Ca2+ rises. The kinetic properties of other potential Ca2+ sources in astroglia, such as mitochondria and other Ca2+ channels, remain to be established.While the precise mechanism underlying astroglial Ca2+ channels (IP3 receptor type) along several branches of the modelled astrocyte, 1–5 µm apart  was recorded at four arbitrarily selected points in the area of interest .We found that at an average interval between channel openings of ~3 ms within clusters, and ~7 s among clusters, the simulated fluorescence signals Fig.  were simdel Fig. . ConstraOne advantage of cell modelling is the possibility to predict theoretically the impact of a specific cellular or environmental feature on cell’s behaviour. This approach could also reveal whether certain feature combinations can make the modelled cell biophysically unstable. We therefore carried tests in which some key functional traits of modelled cells were monitored against changes in model parameters.2+ wave generation  slows down dye equilibration . Simulations mimicking somatic dialysis showed how reducing diffusivity  clusters, with the same total amount of the IP3 activity, prompts de novo Ca2+ events which could feature prominently in fluorescent recordings  to quantify such processes using an empirical 3D EM sample and (b) to transform them into NEURON-compatible (cylinder-compartment based) shapes with matching biophysical properties. The latter was to be verified using dedicated Monte Carlo simulation tests (for diffusion and electrodiffusion) incorporated in ASTRO. This procedure provided all the key statistics characterising nanoscopic astroglial processes in the model.The task of recreating astroglia in silico included three steps. The first step was to construct a ‘stem tree’ based on experimental data on main astrocyte branches that are readily identifiable in the optical microscope. This procedure is similar to the common 3D reconstructions of nerve cells using Thus, the third step was to populate the modelled stem tree with stochastically generated nanoscopic processes, in accord with their morphometric stats obtained as outlined above. The main experimental constraint here was the VF occupied by astroglial processes, which we and others could measure directly using either 2PE microscopy or 3D EM. Because neighbouring astrocytes do not overlap in tissue, local VF faithfully reflects the space-filling properties of individual astroglial cells. We could therefore stochastically generate individual nanoscopic processes on dendritic branches until their bulk matched the empirical VF distribution. This procedure was to complete the modelled cell architecture: the model could now be explored using NEURON simulation environment which we equipped with several additional functions specific to astroglia.+ intake via Kir4.1 generate only very small membrane depolarisation across the astrocyte. It appears that transient rises of extracellular K+ concentration prompt relatively small changes of intra-astroglial K+, which dissipate relatively quickly, within one cell, due to efficient K+ efflux through Kir4.1 channels. Our tests illustrated that relatively small changes in Ca2+ buffering properties might significantly influence the spread of regenerative intra-glial Ca2+ waves. The model also showed that the classical mechanisms of rapid Ca2+ sparks and hotspots described in other cell types could be consistent with the slow Ca2+ signals reported by common Ca2+-sensitive fluorescent indicators.In our case study, we obtained detailed morphological data on hippocampal astroglia in area CA1 using 3D EM and 2PE imaging, recreated the ‘typical’ cell in silico, and partly constrained its functional features by further experimental tests. Exploring the model has shed light on some traits of astroglial physiology that have not been attainable in experiments. Our simulations predict that local glutamate uptake or K2+ buffering, channel current density, molecular transport, etc.) and the physiological phenotype registered experimentally.Thus, the modelling approach presented here serves several general purposes. Firstly, to recapitulate complex astrocyte morphology at multiple scales, paving the way to interrogating the form-function relationship in astroglia. Secondly, to assess whether a certain interpretation of experimental observations in astroglia is biophysically plausible. Thirdly, to understand the microscopic spatiotemporal dynamics of ion currents, molecular fluxes, and chemical reactions that cannot be monitored or registered experimentally. Finally, to predict the relationships between specific cellular features  will require a dedicated study in which receptor kinetics and expression pattern are constrained, at least in part, by experimental tests.ASTRO can employ all synaptic and non-synaptic receptor mechanisms enabled by NEURON, thus enabling simulations with arbitrary patterns of network influences on the modelled cell. In addition, excitatory synaptic function could be mimicked using the glutamate uncaging ASTRO-NEURON menu. Nonetheless, exploring any receptor action specific , stored in the slicing solution at 34 °C for 15 min and transferred for storage in an extracellular solution containing (in mM): NaCl 125, NaHCO3 26, KCl 2.5, NaH2PO4 1.25, MgSO4 1.3, CaCl2 2 and glucose 16 (osmolarity 300–305 mOsm). All solutions were continuously bubbled with 95% O2/5% CO2. Slices were allowed to rest for at least 60 min before recordings started.Acute hippocampal transverse slices (350 μm thick) were prepared from P21–28 Sprague-Dawley rats, in full compliance with the national guidelines, the European Communities Council Directive of 24 November 1986 and the European Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes, with the protocols approved by the UK Home Office. Slices were prepared in an ice-cold slicing solution containing (in mM): NaCl 60, sucrose 105, NaHCO3 26, KCl 2.5, NaH2PO4 1.25, MgSO4 1.3, CaCl2 2 and glucose 16 (osmolarity 300–305 mOsm), continuously bubbled with 95% O2/5% CO2. Whole-cell recordings were obtained with patch pipettes (3–5 MΩ) with an intracellular solution containing (in mM): KCH3O3S 135, HEPES 10, Tris-phosphocreatine 10, MgCl2 4, Na2ATP 4, Na3GTP 0.4 . The cell-impermeable Ca2+ indicator OGB-1  was added to the internal solution. CA1 .  protoplasmic astrocytes located in the stratum radiatum were identified either by full visualisation in fluorescence mode or in a DIC mode by their small soma size, low resting potential (below  −80 mV) and low input resistance (<10 MΩ). Astrocytes were held in voltage clamp at their resting potential or in current clamp.Whole-cell patch-clamp recordings of stratum radiatum astroglia were performed in a submersion-type recording chamber. Slices were superfused with an extracellular solution containing (in mM): NaCl 125, NaHCOc was carried out using a classical voltage-step method in which (a) a brief voltage step ΔV is applied, (b) the area under the transient capacitance current after the end of the voltage step is measured, giving electric charge Q, and (c) membrane patch capacitance is estimated as c = Q/ΔV. Thus the patch area is evaluated from the ratio c / cm where specific membrane capacitance cm = 1 µF cm−2 is a common characteristic of astroglial membranes39. The steady-state current response to the voltage step was used to calculate the patch conductance, which was then normalized to the membrane area to obtain Gm.In some experiments, whole-cell patches were excised by pulling gently the patch pipette attached to the cell body until the patch was completely detached from the processes and its membrane sealed. Estimation of the patch capacitance 20,26,56. We used an Olympus FV1000 imaging system optically linked to a femtosecond pulse Ti-sapphire MaiTai laser (Newport Spectra-physics). Cells were imaged using an Olympus XLPlan N 25x water immersion objective. Fluorescence recordings were obtained in line-scan mode  at λ = 800 nm at an increased laser power of 15–20 mW under the objective to induce substantial bleaching of Alexa Fluor 594. Fluorescence was collected for 750–1000 ms, then a mechanical shutter was placed in front of the laser beam for 1–2 s to allow fluorescence recovery20,26,56.Astrocytes were filled via whole-cell patch clamp with 40–100 µM Alexa Fluor 594 for 15–20 min, as described previouslyWe used a combined two-photon uncaging and imaging microscope  powered by two Ti:Sapphire pulsed lasers  or,   a Femto2D microscope  coupled with two MaiTai lasers, and fully integrated with patch-clamp. Cells were imaged using an Olympus XLPlan N ×25 water immersion objective. The intensity of the imaging and uncaging laser beams under the objective was set to ~5 and 12–17 mW, respectively. Fluorescence recordings were normally carried out in line-scan mode (500 Hz); in FRAP experiments, laser power was increased to 15-20 mW during photobleaching epochs. 2+ transients in frame scan mode, 200 µM Fluo-4 (Invitrogen) and 100 µM Alexa Fluor 594 (Invitrogen) were added to the intracellular solution. 350–500 µm2 fields of view where imaged within the arbour of the patched astrocyte and the fluorescence emitted by Alexa Fluor 594 and Fluo-4 was collected at a rate of 3–5 Hz for 2–3 min. Time-dependent fluorescence transients were expressed as ΔG/R where G corresponds to the background-subtracted Fluo-4 fluorescence and R to the background-subtracted Alexa Fluor 594 fluorescence. Further details of the imaging methods were reported previously20,26,56. To record spontaneous CaFor MNI-glutamate uncaging, astrocytes were loaded with 100 µM Alexa Fluor 594 as a morphological marker. Astrocytes were held in voltage-clamp mode at their resting membrane potential . The MNI-glutamate (12.5 mM) was either puffed within the tissue from a glass pipette placed above the patched cell, or added to the bath at 2.5 mM. Glutamate was uncaged for 20 ms at different distances from the soma (5–25 µm).x2p = 800 nm). A z-stack of 2PE images was collected using 100 × 100 µm (512 × 512 pixel) individual frames containing the entire visible astrocyte structure, with a 0.5 µm z-step over 61 µm in depth. The image stack was stored (8-bit tiff format), individual images were corrected for the depth-dependent, quasi-exponential fluorescence signal decrease . Fluorescence background was subtracted (Fiji Image-Process), identifiable cell branches (>0.3–0.5 µm in diameter) were traced semi-automatically in individual 2D optical sections and reconstructed in 3D using Neurite Tracer . The data sets representing diameters of tubular compartments and their 3D co-ordinates (pairs of end points) were stores in SWC format. The Vaa3D software  was used to convert these data sets into NEURON compatible files providing 3D structure of the astroglia stem-tree .A stratum radiatum astrocyte was held in whole-cell mode, with Alexa Fluor 594 added to the intracellular solution  was administered and the animal was secured in a stereotaxic frame  and kept warm on a heating blanket. The scalp was shaved and disinfected using three washes of topical chlorhexidine. Upon loss of pedal withdrawal reflexes, a small midline incision was made to expose the skull. A craniotomy of ~1–2 mm diameter was performed over the right somatosensory cortical region using a high-speed hand drill . Stereotactic coordinates were +0.1 mm on the anterioposterior axis relative to bregma, and 2 mm lateral to midline. Once exposed, a warmed aCSF variant  was applied to the skull and cortical surface throughout the procedure.Animal procedures were conducted in accordance with the European Commission Directive (86/609/ EEC) and the United Kingdom (Scientific Procedures) Act (1986), with the protocols approved by the UK Home Office. Young male C57BL/6 mice (2–3 weeks of age) were anaesthetized using isoflurane . Subcutaneous analgesic  was pressure injected into the somatosensory cortex using a pulled glass micropipette stereotactically guided to a depth of 0.6 mm beneath the pial surface, at a rate of ~1 nL s−1. A given injection bolus contained between 0.25 and 0.5 × 1010 genomic copies, in a volume not exceeding 500 nL. After injection, pipettes were left in place for 5 min before retraction. The scalp was sutured with absorbable 7–0 sutures  and the animal was left to recover in a heated chamber. Meloxicam  was administered once daily for up to two days following surgery. After a 4–6-week AAV incubation period, animals were prepared for multiphoton imaging through a cranial window implantation as described below.AAV5 −1, respectively). Adequate anaesthesia was ensured by continuously checking for the loss of pedal withdrawal reflexes and anaesthesia was supplemented appropriately throughout the procedure . Body temperature was maintained at 37.0 ± 0.5 °C using a feedback rectal thermometer and heating blanket. The animal was secured in a stereotaxic frame and a craniotomy of ~2.5 mm diameter was carried out over the right somatosensory cortex, centred 0.2 mm caudal to bregma and ~2.5 mm laterally from the midline. Once exposed, the cortical surface was continuously superfused with warmed aCSF . Cortical astrocytes were labelled using multicell bolus loading of sulforhodamine 101 . SR101 (in cortex buffer vehicle) was pressure-injected through a pulled glass micropipette targeted to 2–3 injection sites within the transduced region, comprising a total volume of 500 nL. The cortical surface was covered with 1% agarose and a glass coverslip was placed on top. Using tissue adhesive , the coverslip was partially secured and a custom-built headplate fixed to the skull. A single cranial-mounted screw was inserted over the contralateral hemisphere and the entire assembly was then secured using dental cement. During imaging, the headplate was used to secure the animal under the objective on a custom-built stage.Following viral transduction of LckGCaMP6f as above, male C57BL/6 mice (7–9 weeks of age) were prepared for cranial window implantation and 2PE microscopy. Animals were anaesthetized using fentanyl, midazolam and medetomidine . Acquisitions were carried out using a wavelength of 920 nm and the mean laser power under the objective was kept at 20–35 mW. Cortical astrocytes were readily identified through SR101 labelling and verified for GCaMP6f expression by frame-scanning for calcium transient activity. Recordings were made at a depth between 50 and 250 μm from the cortical surface. XY time series (at 0.5–2 Hz with a pixel dwell time of 0.5–4 μs and pixel size of 0.248–1.59 μm) were taken in identified regions to measure spontaneous calcium activity.2O2 in PB for 20 min to eliminate blood background, washed with 0.1 M TBS  and incubated with ABC solution  for 30 min at room temperature. Next sections were washed with 0.1 M TB , pre-incubated with DAB  solution (10 mg DAB tablet + 40 ml TB) for 30 min at room temperature in dark and finally incubated with DAB + H2O2 solution (5 μl of 33% H2O2 + 25 ml of DAB solution) for 10–20 min at room temperature in the dark. The DAB stained sections was washed in PB, post-fixed in 2% osmium tetroxide and further processing and embedding protocols were essentially similar to those reported previously26. Briefly, the tissue was dehydrated in graded aqueous solutions of ethanol (40–100%) followed by 3 times in 100% acetone, embedded into a mixture of 50% epoxy resin  and 50% acetone for 30 min at room temperature, embedded in pure epoxy resin, and polymerized overnight at 80 °C. Sections in blocks were coded and all further analyses were carried out blind as to the experimental status of the tissue.In a subset of experiments we loaded an astrocyte with biocytin, and after the experiment the slices were rapidly fixed (by submersion) with 1.25% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M PB , to be kept overnight, submerged in 10% sucrose in PB for 10 min and then in 20% sucrose in PB for 30 min. The slices were consequentially freeze-thawed in liquid freon and liquid nitrogen for 3 s each to gently crack intracellular membranes and embedded in 1% low gelling temperature agarose in PB . Embedded slices were sectioned at 50 µm on a vibrating microtome . Sections (50 µm) sections were incubated in 1% Hhttp://synapses.clm.utexas.edu/). 3D reconstructions of DAB-stained astrocyte fragments and the adjoined to stained astrocytes dendritic spines (that host clearly identifiable excitatory synapses) were performed in Trace 1.6b software (http://synapses.clm.utexas.edu/). 3D reconstructions of selected astrocytic segments and dendritic spines were imported to 3D-Studio-Max 8 software for rendering of the reconstructed structures. However, ASTRO can upload other standard text-formatted files with 3D coordinates representing the cell-membrane ‘mesh’.Serial sections (60–70 nm thick) were cut with a Diatome diamond knife and systematically collected using Pioloform-coated slot copper grids . Sections were counterstained with 4% uranyl acetate, followed by lead citrate. Finally sections were imaged in stratum radiatum area of CA1 (hippocampus) using AMT XR60 12 megapixel camera in JEOL 1400 electron microscope. Serial sections were aligned as JPEG images using SEM align 1.26b  of 10–15% or less.The present study contained no longitudinal or multifactorial experimental designs. In electrophysiological or imaging experiments the main source of biological variance was either individual cells or individual preparations (the latter in case of field measurements in acute slices), as indicated. In accord with established practice, in the ex vivo tests we routinely used one cell per slice per animal, which thus constituted equivalent statistical units in the context of sampling, unless indicated otherwise. Statistical hypotheses pertinent to mean comparisons were tested using a standard two-tailed Stot represented by the cylinder-based shape approximations . The Kir4.1 potassium current has been incorporated in accordance with17  using a built-in Ca2+ diffusion algorithm described in the next section (potassium.mod), the FRAP mechanism incorporated the same algorithm plus a reaction-diffusion step (FRAP.mod), and K+ extrusion was modelled as a first-order pump . Gap junction mechanisms were enabled either as a (zero-order) current leak (gap.mod) or as a diffuse escape (gapCa.mod). Throughout these mechanisms, the respective kinetic parameters can be set using the relevant NEURON-enabled ASTRO menus, as described in the User Guide (https://github.com/LeonidSavtchenko/Astro/blob/master/ASTRO_User_Guide.pdf).Several channel current and diffusion-reaction mechanisms have been written specifically for the present model. The kinetics of glutamate transporter GLT-1 involving glutamate and ion fluxes has been incorporated in accordance with2+ homoeostasis and diffusion  are detailed in Chapter 9 of the NEURON Book10 , and can be found in the modified cadifus.mod file in the model installation. In brief, Ca2+ diffuses freely whereas buffer-bound Ca2+ (which has much lower diffusivity) is considered stationary, for the sake of simplicity. In individual cylindrical cell compartments, radial diffusion occurs through four concentric shells surrounding a cylindrical central core, and longitudinal diffusion is calculated using fluxes between the corresponding concentric compartments adjusted for the cross-section areas. The longitudinal and radial diffusion coefficient for Ca2+ was set to 0.3 µm2 ms−1, the basal level was set to 50 nM, and IP3 concentration at 0 unless specified otherwise.ASTRO simulation algorithms enabling intracellular Ca2+ homoeostasis mechanisms included the SERCA pump, SERCA channel and SERCA leak, the endogenous (stationary) and exogenous (Ca2+ indicator) mobile buffers, and a plasma membrane Ca2+ pump with the threshold mechanism (cadifus.mod). The kinetics of buffers can be modified using NEURON-enabled menus. The mechanistic details of Ca2+ SERCA pump were as described earlier71. The current model implementation assumes that IP3 is distributed uniformly across cell compartments, i.e. that diffusion equilibration of IP3 is fast compared to Ca2+ concentration transients in space or time.In addition to free diffusion, Cahttps://github.com/LeonidSavtchenko/Astro/blob/master/Manual). The current version of ASTRO can also be downloaded directly from https://github.com/LeonidSavtchenko/Astro. The (regularly updated) User Guide can be downloaded from the same location or found (current version) in the Detailed information on the installation and running of ASTRO can be found in the User’s Manual , or at least free-download MATLAB Runtime package (https://www.mathworks.com/priducts/compiler/mcr), and NEURON  installed under Windows 7 or Windows 10. Simuations using ready-made astrocyte models require NEURON installation only.  In brief, running ASTRO with de novo 3D EM reconstructions and model building, but without full-scale simulations of intracellular Ca2+ dynamics is highly resource-consuming and normally requires an additional Worker computer/cluster operating under Linux, with preinstalled NEURON  and MPI whereas the Host computer will require MATLAB (2013 version or later), NEURON (7.0 or later), and access to the Internet. In house, the Linux version with the parallel computations including intracellular diffusion simulation was routinely run using a 12-node in-house computer cluster72, taking advantage of the computational optimization routines developed by us earlier for compartmental models and Monte Carlo simulations72. Simulating full intracellular CaSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8


Chronic urticaria is characterized by recurrent hives, with or without angioedema, persisting for 6 weeks or longer. Although often suspected by patients, in fact specific allergen triggers are infrequent. In contrast, the condition may be associated with autoimmune and thyroid disorders. While some evidence suggests an association of chronic urticaria with vitamin D levels, measurement of vitamin D or supplementation is not a part of diagnostic or treatment recommendations.We present a case of a 14-year-old white boy with refractory chronic urticaria who experienced prompt remission with high-dose vitamin D repletion when vitamin D deficiency was identified as an incidental finding.In some patients, vitamin D may have a role in the pathophysiology and treatment of chronic urticaria; however, the cost-effectiveness of routine laboratory screening in chronic urticaria is unknown. Chronic urticaria affects approximately 1% of the population and is often a spontaneous phenomenon without a clearly identified allergen trigger. Treatments involve non-sedating antihistamines in escalating off-label doses, omalizumab, anti-inflammatory medications such as hydroxychloroquine, and, in some patients, immunosuppressants such as cyclosporine. We report a case of a 14-year-old white boy with refractory chronic autoimmune vitamin D-responsive urticaria.1 blockade at four times the approved Food and Drug Administration (FDA) dosing (loratadine 10 mg twice daily and cetirizine 10 mg twice daily), H2 blockade (ranitidine 150 mg twice daily), and use of a leukotriene modifier (montelukast 5 mg once daily). His urticaria was recalcitrant to conventional treatment. Allergen-specific immunoglobulin E (IgE) testing was negative to dust mites, cat, dog, roach, tree pollens, grass pollens, weeds, molds, latex, and galactose-a-1,3-galactose. Laboratory evaluation revealed an elevated serum tryptase , elevated anti-IgE receptor antibody (78% CD203c basophils), and normal thyroid-stimulating hormone (TSH). A low 25(OH)-D (vitamin D) at 23 ng/mL  was identified as an incidental finding. A trial of omalizumab (300 mg every 4 weeks) was initiated in January 2018 but failed to provide benefit after four doses, and symptoms progressed to diffuse urticaria and facial angioedema prompting emergency evaluation for the worsening flare of urticaria and angioedema  remain first-line treatment for chronic urticaria, with omalizumab and cyclosporine considerations for more difficult to treat patients. It is estimated that approximately 7–8% of patients with chronic spontaneous urticaria will be refractory to all current treatments [et al. described 356 adult patients between 2001 and 2009 who underwent screening laboratory investigations [Second-generation or third-generation Heatments . While tigations . While 1


Here, we demonstrate that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. We further showed that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCAA617T induced positive staircase and arrhythmia, while SERCAE442K accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect.The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca Indeed, the Bowditch effect is important to determine the cardiovascular response to exercise, constituting a major contributor to the intrinsic myocardial reserve2. A positive staircase effect occurs in normal individuals and it is believed that 40% of the increase in cardiac output (volume of blood pumped by the ventricle in one minute) depends on the relationship between strength and frequency3. In people with heart failure, the force-frequency relationship is null or inverse (negative staircase) and the heart is unable to meet the blood circulation requirements even under low-stress exercises5.The Bowditch effect or staircase phenomenon is the intrinsic property of the heart to either increase or decrease the force of contraction in response to an increment in pacing rate. It was described by Bowditch in 18712+ handling and mishandling in cardiac cells. This involves proteins that participate in the excitation-contraction coupling, such as the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). A direct correlation between the reduced levels of SR Ca2+ uptake, decreased SERCA expression, -a hallmark of cardiac failure-, and a depressed force-frequency response was reported in the failing myocardium6. Furthermore, mutations in SERCA2 account for a dominant skin disease , with the effects of several of these mutations being exacerbated by elevated ambient temperatures7. However, a causal relation between SERCA mutations and impaired cardiac activity, has not been established yet. The mutations SERCAE442K and SERCAA617T were identified in D. melanogaster genetic screenings and involve amino acids changes in the hinge region of the protein, thus disrupting pumping activity10. Here, we investigated the physiological impact of these mutations on heart activity in D. melanogaster, with special emphasis on the staircase phenomenon.The most important mechanisms that underlie the staircase phenomenon and its reversal are associated with CaD. melanogaster, by analyzing heart movements and cytosolic Ca2+ levels during each contraction cycle (Ca2+ transients). We used semi intact preparations  adults. Thus, the Drosophila heart behaves similar to what has been determined for small mammals, such as rats and mice, following a change in stimulation frequency13.To determine the type of staircase in 13. We measured the time to half of relaxation (t½), as an index of relaxation of both, twitch shortening and Ca2+ transients. Increasing stimulation frequency produced a minimal reduction in t½ of twitch shortening that only reached statistical significance at the highest stimulation frequency explored. This decrease was completely absent in the corresponding Ca2+ transient 16. Human homologous CaMKII is present in D. melanogaster17, and corresponding targets of phosphorylation should be study to understand the modest FDAR observed in twitch shortening in this model associated with the absence of FDAR in intracellular Ca2+ transients.Independently of the nature of the staircase, an increment in the frequency of stimulation induced an acceleration of relaxation (FDAR)ent Fig. . The mec2+ reuptake and is the main contributor to cardiac relaxation. Human SERCA2 and Drosophila SERCA (dSERCA) share 73% identity at the amino acid level and 96% of the 89 sites altered by mutations are conserved in humans and Drosophila  family pumps that are affected by dominant mutations characterized in non-cardiac pathologies10. On the other hand, SERCAE442K elicits a dominant phenotype of paralysis induced by heat shock, thus causing recessive lethality. Furthermore, SERCAE442K changes glutamic acid 442 to lysine9. This mutation has been proposed to influence the binding of ATP and the resulting conformational change of the protein. Corresponding flies exhibit paralysis induced by exposure of individuals at a restrictive temperature of 41 °C8. Presence of the E442K mutation has been reported to reduce heart rate in larvae, pupae and intact adult flies after heat shock, along with enlarged cardiac dimensions in adult hearts20.We analyzed two different conditional and dominant mutations that cause global disruption of SERCA activity. SERCAA617T exhibited a positive staircase, which was not observed in the control individuals. By contrast, SERCAE442K showed a considerably more accented negative staircase than control individuals  and the proportion of flies that exhibit ectopic beats at the different stimulation frequencies explored. Figure A617T mutation caused a significant increase in ectopic beats and in the arrhythmicity index, both, at the basal level and at the highest stimulation frequency explored  or the different preparations used (semi intact preparation vs. intact specimens). In contrast, our results agreed with data obtained from intact flies expressing SERCAE442K in cardiac tissue, which after heat shock did also not exhibit any significant change in heart rate20.We next explored the effects of increasing the stimulation frequency on heart rate variability and arrhythmia generation. To this purpose, we analyzed and compared the arrhythmicity indexred Fig. . Interesats Fig. . PreviouD. melanogaster shows a negative staircase. Possibly more important, our results further showed that distinct point mutations in SERCA directly affect the staircase pattern. Finally, the SERCAA617T mutation is associated with an enhanced propensity to arrhythmia. Thus, our data reveal a direct contribution of SERCA to the intrinsic mechanisms of heart regulation and emphasize the importance that a putative dysfunction may have on heart performance.The present study demonstrated that A617T mutant converts a hydrophobic amino acid into a potentially phosphorylatable one, whereas replacement of glutamic acid for lysine in SERCAE442K induces a significant alteration of the amino acid charge. Moreover, each mutation has a highly distinct impact on heart physiology. Whereas SERCAA617T produced a positive staircase, SERCAE442K accentuated the negative staircase observed CONTROL flies. Interestingly, both mutations per se produced a staircase pattern opposite to the one observed after heat shock activation of the phenotype.Although both SERCA mutations tested are described to cause disrupted SERCA, they produced different effects at the molecular level: Replacement of alanine by threonine in the SERCA2+ content of both species under basal conditions13. This seems not to be the case in fly hearts. No significant differences were found in basal SR Ca2+ contents in the different groups of flies, but in control flies after the heat shock. However control flies presented a similar negative staircase under both conditions. The cause of the increase in the SR Ca2+ content in control flies is not clear from these experiments but might be a consequence of mechanisms activated by heat shock that modify the activity of SERCA. These mechanisms would be counteracted in both heterozygous mutants by the altered SERCA molecules. Taken together these results strongly suggest that the basal SR Ca2+ content does not play a significant role in the staircase pattern of the fly. Of note, corresponding similarities in Ca2+ content have already been reported in neural tissue and cultured cells tissue10. The lack of changes in the basal RS Ca2+ content does not exclude the possibility of changes in the SR Ca2+ content during the positive and negative staircase observed in SERCAA617T and SERCAE442K respectively. In rat ventricular myocytes the negative staircase in intracellular Ca2+ transients was associated to a similar beat-dependent decrease in the duration of the action potential, manifested primarily as a gradual loss of the action potential plateau22. The negative staircase of human heart failure is furthermore associated to a decrease in L-type Ca2+ current23. Interestingly, gated voltage Ca2+ current measured in larval skeletal muscle in SERCAE442K mutants after heat shock, are significantly reduced8. A similar reduced Ca2+ entry due to action potential shortening in SERCAE442K mutants hearts might have contributed to the exacerbated negative staircase exhibited by these flies eventually decreasing the Ca2+ availability in the cytosol. The mutation per se, without heat shock, manifests the opposite pattern, emphasizing the functional importance of the putative conformational change induces by heat shock. Regarding the SERCAA617T mutation and to the best of our knowledge, there are not previous reports about a possible link between mutation and ionic currents. This might suggest that this amino acidic change does not modify the Ca2+ current in the same way as the SERCAE442K mutation does. SERCA is a pleiotropic gene with several phenotypes, i.e. paralysis and incoordination and a detailed mechanistic analysis of the independent point mutations is required for a clearer understanding of fly cardiac responses to an increment in heart rate. While such analyses are beyond the scope of this work, our data emphasize the importance of SERCA function to the response of the fly to heart rate increases.The mechanisms by which these mutations alter the staircase phenomenon remain unclear yet. However, previous experiments demonstrated that the negative staircase effect observed in rat and mouse models is at least in part explained by the rather high SR CaA617T mutants. The origin of this arrhythmic pattern is not apparent to us. Since our experiments were performed in decapitated individuals, the influence of the autonomic nervous system in these alterations seems rather unlikely. Recent models24 emphasize the presence of a tight interaction between Ca2+ cycling proteins, including SERCA and cardiac automaticity, making SERCA mutations good candidates to explain the alterations of cardiac rhythm described here.The results further indicate that ectopic beats as well as the arrhythmicity index are exacerbated in SERCAD. melanogaster shows a negative staircase and that distinct point mutations in SERCA directly affect the type of staircase occurring within the heart. Our data further reveal a direct contribution of SERCA to the intrinsic mechanisms of heart regulation and emphasize the physiological importance of a putative dysfunction. Overall, this work establishes a molecular link for understanding a key physiological event in cardiac function and provides a new model for further research in vertebrates and humans.In conclusion, the results presented here demonstrate that Fly stocks were amplified and maintained in vials at 28 °C, partially filled with a mixture of maize flour, glucose, agar and yeast supplemented with 10% antimycotic.Drosophila Stock Center. Canton-S , is a control line. The other two strains were conditional mutants for the Drosophila SERCA gene . The first strain, express the mutant protein SERCA [E444K]. This conditional mutant, named Ca-P60A[Kum170], (BDSC # 26700)9 holds the amino acid replacement E442K of the protein encoded by the Ca-P60A gene. Activation of this dominant mutation induces paralytic phenotype and is also recessively lethal. The second strain holds the protein SERCA [A617T] (BDSC # 58974)10, that is a conditional mutant strain carrying the amino acid change A617T of the protein encoded by the Ca-P60A gene. Mutation activation induces a Drosophila incoordination phenotype and is recessively lethal.Assays were made with different strains obtained from Bloomington 2+ . These flies were a gift from Dr. Mathew Wolf, from the University of Virginia, USA12. The handC-GFP line, expressing the reporter protein GFP in heart and pericardial cells was provided by Prof. Achim Paululat 11.Flies harboring the reporter system GCaMP3 specifically expressed in heart cells were capable of sensing increments of cytosolic CahandC-GFP reporter) or Ca2+ transients (using the UAS-GCaMP3 reporter). Mutant flies were crossed with handC-GFP reporter and mechanical activity was evaluated.Control flies were crossed to individuals carrying one of these reporters. We analyzed heterozygous individuals of F1 that possess one copy of the reporter system in order to obtain information on mechanical activity , and placed in a 60 mm Petri dish containing Vaseline and fixed by the dorsal region. The head and thorax were removed in this preparation; therefore neuronal influence on the cardiac activity was not significant. The middle ventral region of the abdomen was opened and the internal organs were removed. The preparation was submerged in oxygenated artificial hemolymph solution containing 5 mM KCl, 8 mM MgCl2, 2 mM CaCl2, 108 mM NaCl, 1 mM NaH2PO4, 5 mM HEPES,4 mM NaHCO3, 10 mM trehalose, 10 mM sucrose, pH 7.126. Calcium recordings and mechanical activity of these semi intact preparations were carried out using a Carl Zeiss LSM410 and LSM800 confocal microscopes.The dissection of adult hearts was performed as described Santalla handC driver, expressed in cardiomyocytes and pericardial cells. A pericardial cell or a cardiomyocyte was focused with a 100X objective. We then tracked the fluorescent signal of a cardiac or pericardial cell edge at one of the two sides of the heart. The movement of a single cell expressing the GFP reporter was followed with laser scanning by setting a line along the displacement of either a pericardial cell or a cardiomyocyte. Recordings were obtained during 10 sec (Supplementary Video 1) intervals.Mechanical activity was recorded from all flies harboring the GFP protein under control of the 2+ transient images. All functional parameters were measured after verification that each recording had followed all stimulation frequencies tested. Values expressed as change of fluorescence along time, were analyzed with LabChart software .The images obtained were processed with an algorithm for Anaconda developed in our laboratory to obtain sequentially in time the intensity of fluorescence of a pericardial/cardiac cell over a threshold value, and convert it in a digitalized image of cell displacement. A similar procedure was carried out to obtain a digitalized recording from Ca0/F0) and t1/2, defined as the time from maximum fluorescence until half of the relaxation. Heart period was defined as the interval between two consecutive peaks. Arrhythmicity index (AI) was calculated as the standard deviation of periods normalized by their average. Additionally, we counted ectopic beats, which were defined as events that occurred immediately after the maximum was reached and before the finalization of a relaxation phase of a beat.We measured fluorescence intensity (F-F2+ transients were registered by the fluorescence produced by Ca2+ binding to the GCaMP3 reporter. Semi intact preparations were visualized using a 5X objective and the laser was focused to stimulate a minimal central region of the conical chamber where the intensity of signal was higher than in other regions of the preparation. Recordings of 60 sec allowed us to obtain a measurable pattern of Ca2+ transients.Ca27. A customized chamber was designed to provide electrical field stimulation. Electrical pulses of 16 msec duration were applied. The intensity of the pulses varied to reach the excitability threshold at all the frequencies analyzed. The range of frequencies assayed was 1–6 Hz over the basal spontaneous frequency and the stimulation frequency was set according to the basal heart rate. From this basal rate, stimulation frequency was increased by 0.5 Hz steps. Supplementary Figure The semi intact preparation immersed in artificial hemolymph was electrically stimulated using a conducting device connected to an electro-stimulator and computer. Neutral pH was essential to complete the sequence of stimulation, in agreement with previous studies2+ load in basal conditions, a pulse of 10 mM of caffeine was applied. This compound produces the release of calcium content through the RyR2. The amplitude of the fluorescence induced by massive calcium release was calculated for all compared fly lines with and without heat shock.To estimate the sarcoplasmic reticulum CaOne way ANOVA andTwo way ANOVA followed by Tukey’s post hoc test, was utilized to compare differences among groups. Categorical data were expressed as percentages and compared with Fisher’s exact test. A p value < 0.05 was considered significant.Supplementary informationSupplementary video 1


Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease, associated with ventricular arrhythmias, fibrofatty replacement of the myocardial mass and an increased risk of sudden cardiac death (SCD). Malignant ventricular arrhythmias and SCD largely occur in the pre-clinical phase of the disease, before overt structural changes occur. To prevent or interfere with ACM disease progression, more insight in mechanisms related to electrical instability are needed. Currently, numerous studies are focused on the link between cardiac arrhythmias and metabolic disease. In line with that, a potential role of mitochondrial dysfunction in ACM pathology is unclear and mitochondrial biology in the ACM heart remains understudied. In this review, we explore mitochondrial dysfunction in relation to arrhythmogenesis, and postulate a link to typical hallmarks of ACM. Mitochondrial dysfunction depletes adenosine triphosphate (ATP) production and increases levels of reactive oxygen species in the heart. Both metabolic changes affect cardiac ion channel gating, electrical conduction, intracellular calcium handling, and fibrosis formation; all well-known aspects of ACM pathophysiology. ATP-mediated structural remodeling, apoptosis, and mitochondria-related alterations have already been shown in models of PKP2 dysfunction. Yet, the limited amount of experimental evidence in ACM models makes it difficult to determine whether mitochondrial dysfunction indeed precedes and/or accompanies ACM pathogenesis. Nevertheless, current experimental ACM models can be very useful in unraveling ACM-related mitochondrial biology and in testing potential therapeutic interventions. Plakophilin-2 (PKP2) associate with most cases of ACM in which a genetic cause can be found. Mutations in other genes that encode desmosomal proteins , have been linked to ACM as well (2+) homeostasis can be modified by the expression of PKP2   and phospholamban (PLN). Variants in RyR2 are classically linked to the inherited arrhythmogenic disease Catecholaminergic polymorphic ventricular tachycardia (CPVT), although RyR2 missense mutations have also been found in ACM patients (Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease, associated with ventricular arrhythmias and an increased risk of sudden cardiac death (SCD). SCD often occurs as the first clinical manifestation, in particular in athletes and young adults . The pre as well . Desmoso as well , b. Rece of PKP2 . Besidesr (RyR2) . This str (RyR2) , and it patients . Moreovepatients .2+ handling, activate Ca2+/calmodulin-dependent protein kinase II (CaMKII), and cause mitochondria-induced cell death and fibrosis  where a changed metabolic state and altered mitochondrial balance caused by elevated blood glucose levels, glucose fluctuation, and hypoglycemia can set the stage for cardiac arrhythmias . The altfibrosis . Apoptosfibrosis . Mitochofibrosis . Mitochoon pumps . Mitochoon pumps . In thisThe mechanical force required for cardiac contraction consumes large amounts of energy, which needs to be replenished via the mitochondria. The heart is capable of utilizing all classes of energy substrates for ATP production, including carbohydrates, lipids, amino acids, and ketone bodies . Lactate2+ (2+ levels tightly regulate enzymes of the tricarboxylic acid cycle (TCA) cycle and excitation-contraction coupling in the heart, thereby linking myocyte contraction and energy production (2+-ATPase (SERCA2a)  and an electrogenic Ca2+ uniporter  through voltage dependent anion channels (VDAC), large channels that are permanently permeable to Ca2+. VDAC permits Ca2+ flux into the intermembrane space and the mitochondrial Ca2+ uniporter (MCU) allows Ca2+ entry from the intermembrane space into the mitochondrial matrix. The mitochondrial [Ca2+] plays an important role in regulation of mitochondrial ATP production  and via ubiquitous H+/Ca2+ exchange. This means that the mitochondrial [Ca2+] is set by the dynamic balance between MCU Ca2+ entry and Ca2+ extrusion via the mNCX , collapses the membrane potential (ΔΨm) and activates apoptotic pathways (The two most important factors regulating the cardiac energy production are cellular concentrations of ADP and Ca2+ . Cytosoloduction . The heaSERCA2a) . About 1he heart . The cel cycling . Mitochoniporter . Ca2+ croduction . Ca2+ ththe mNCX . Under pntration . Howeverpathways .Under pathological conditions, the fuel metabolism of the heart shifts back from FA oxidation (FAO) to an increased reliance on glucose, like in the fetal heart , 2013. ICardiomyocyte excitability and electrical cell–cell coupling are critical factors for electrical activation and a synchronized contraction of the heart. Changes in ion channel composition, function, and localization increase the susceptibility toward arrhythmias . Multipl+ channels on the sarcolemma (sarcKATP)  (2+ influx via L-type Ca2+ channels (LTCC) during the action potential plateau phase and increases the time for Ca2+ extrusion via NCX  . These con (APD) . Shorten via NCX . As a coimpaired . Via thile state . As consle state  , peroxynitrite (ONOO–) and hydroxyl radicals (OH). They can damage proteins and lipids within the cell, activate intracellular signaling cascades and induce cellular dysfunction or cell death (2+ homeostasis (by inducing SR Ca2+ sparks and increasing cytosolic Ca2+ levels) and causes fibrosis formation . Overexpression of MiR-25 reduced the MCU protein levels, diminished H2O2 driven elevation of the mitochondrial Ca2+ levels and inactivated the mitochondrial apoptotic pathway  and highly sensitive to oxidative stress, as it is oxidized by increased ROS levels in the cytosol  and impairment of the cardiac contractile force   .2+ and Calmodulin facilitate CaMKII activation under physiological conditions, though CaMKII is also a substrate for its own monomers (2+ levels) CaMKII can target several ion channels and transcription factors, which coordinate cardiac electrical and mechanical mechanisms (2+ channel (CaV1.2), NaV1.5 and RyR2, thereby increasing cytosolic Ca2+ levels  and Na+ channels  -induced intracellular accumulation of Na+ and Ca2+ and its subsequent incidence of ventricular arrhythmias, hyper-contraction, and SCD  . Elevatehe heart . Overexpof Ca2+i . CaMKIIδ and SCD . This im2+ channel (LTCC) is a voltage gated Ca2+ channel that couples electrical activation, via an action potential, to contraction of the cardiomyocyte. The effects of ROS on LTCCs are controversial  is an antiporter membrane protein which mainly works in the forward mode, pumping 3 Na+ ions into the cardiomyocyte in exchange for 1 Ca2+ ion  to ventricular tachycardia (VT) and ventricular fibrillation (VF) . The intfunction . Examinacoupling . In this+ and Ca2+ ions and release K+ ions  ROS-induced fibrosis formation, it has been demonstrated that mitochondrial ROS also directly promotes fibroblast differentiation . PPARα is the predominant isoform in the heart, although PPARδ and PPARγ also regulate lipid metabolism . PPARγ ipoptosis . Kim et poptosis . In addipoptosis . After ppoptosis . These dse model . KEGG anse model . Moreovese model .2+ homeostasis in the cell, directly or indirectly via Cx43 hemichannals and mtCx43, tentatively serves as an important substrate in RV initiated electrical disturbances and structural remodeling in ACM patients. In murine PKP2cKO-RV cardiomyocytes, Cx43 hemichannels in the sarcolemma increase the membrane permeability and cause intracellular Ca2+ overload and ISO-induced ventricular arrhythmias. ROS can increase the expression of mtCx43 and close the proximity of sarcolemmal Cx43 hemichannels, mitochondria and mtCx43 might facilitate ATP and Ca2+ exchange that under pathological conditions can interfere with intracellular and mitochondrial Ca2+ dynamics. In addition, cellular ATP release and increased oxidative stress are important pro-fibrotic aspects in the (ACM) heart. Cx43 hemichannels in the perinexus enable release of ATP into the extracellular space, via mitochondria close residing at sides of cell–cell contact. ATP can directly activate P2X/Y channels and also convert into ADP, AMP, and adenosine. Adenosine subsequently activates pro-fibrotic signaling pathways (as TGF-β1 activation) via A2AR binding. Pannexin-1 has the same ATP-mediated pro-fibrotic factor capacity as Cx43 hemichannels. Interestingly, pannexin hemichannels are activated via enhanced cytosolic Ca2+ levels and SR Ca2+ release, which is increased by ROS-induced RyR2 oxidation. Furthermore, Nox4-derived ROS can induce fibrosis via TGF-β1 activation and fibroblast differentiation into myofibroblasts. The increased apoptosis and adipogenesis in ACM patients is likely caused by co-activation of PPARγ and PPARα, which points toward an altered metabolic state of the ACM heart.In conclusion, ACM is a progressive and predominantly RV disease, characterized by ventricular arrhythmias and fibrofatty infiltrates in the myocardium. Mitochondrial interference with the CaIn ACM patients, malignant ventricular arrhythmias and SCD largely occur in the pre-clinical phase of the disease, before overt structural changes. To prevent or interfere with the disease progression, more insight into the mechanisms related to electrical instability in ACM hearts is needed. At present, a large extent of research is focused on the link between cardiac arrhythmias and metabolic disease. Mitochondrial dysfunction can affect the electrical balance in cardiomyocytes and favor arrhythmogenesis. The role of mitochondrial dysfunction in ACM pathology is still unclear, and mitochondrial biology and structure in the ACM heart remains understudied. Here, we reviewed cardiac mitochondrial dysfunction, cardiac arrhythmias and the potential links to typical hallmarks of ACM.2+ overload (via for example mtCx43) increases ROS production, oxidizes RyR2 and subsequently induces SR Ca2+ leakage. This results in repolarization abnormalities, ectopic activity, conduction defects and eventually (re-entry based) cardiac arrhythmias. ROS oxidation also indirectly affects ion channels, transcription factors and (pathogenic) intracellular processes, through the multifunctional protein CaMKII. Appearance of fibrosis, apoptosis, and lipogenesis is highly related to mitochondrial (dys)function. Fibrosis formation can be induced via Cx43 hemichannel-mediated ATP release, SR Ca2+-driven Pannexin-1 activation and ROS-TGF-β1-Nox4 activated fibroblast differentiation. Whether some or all of these changes are present in cardiomyocytes of hearts deficient in desmosomal proteins remains to be determined. PPARγ and PPARα co-activation is a possible link between metabolic changes in PKP2-deficient patients, apoptosis and lipogenesis. Yet their relative contribution remains a subject of further investigation.Mitochondrial dysfunction reduces ATP production and increases ROS production in the heart. Upon insufficient ATP supply, the predominantly closed sarcKATP channels can become conductive. Open sarcKATP channels can impair electrical conduction in the heart, by reducing cardiomyocyte excitability. In addition, increased ROS production leads to abnormal opening of various ion channels and reduces the expression of gap junctions. Mitochondrial Ca2+) and alters mitochondrial activity and homeostasis. The cardiac stress responses upon mutations in ID-associated proteins likely activates several mechanisms related to mitochondrial dysfunction, such as inflammation and apoptosis. Postulated hypotheses are largely based upon findings in experimental PKP2 models, which are of course not representative for the entire ACM population, therefore extrapolations should be done with caution. Nevertheless, these models can be very useful in discovering ACM-related mitochondrial biology and testing of therapeutic targets, as for example anti-oxidant treatment or genetic repair.The limited amount of experimental evidence in ACM models makes it hard to determine whether mitochondrial dysfunction indeed precedes and/or accompanies ACM pathogenesis. Mutations in mitochondrial DNA can provoke mitochondrial dysfunction and activate the mechanisms we discussed. On the contrary, loss of ID integrity might also affect the intracellular ion homeostasis (e.g., CaCO and LB prepared the primary manuscript. MD and TV critically revised the manuscript. CO produced the figures.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.


As clinical-psychological scientists and practitioners increasingly work with diverse populations of traumatized people, it becomes increasingly important to attend to cultural models that influence the ways in which people understand and describe their responses to trauma. This paper focuses on potential uses of the concept of cultural script in this domain. Originally described by cognitive psychologists in the 1980s, scripts refer to specific behavioral and experiential sequences of elements such as thoughts, memories, attention patterns, bodily sensations, sleep abnormalities, emotions and affective expressions, motivation, coping attempts, and ritualized behaviors that are relevant to posttraumatic adjustment. We differentiate between experiences of traumatic stress that are scripted  versus unscripted. Further characteristics such as script tracks, the effect of script interruptions, and contextual fit of scripts with other cultural models are also described. We consider examples of traumatic stress associated with war and organized, sexualized violence from “Western” and “non-Western” world regions. The concluding part of this review describes a number of possibilities for methodological approaches to assessment of cultural scripts. Capturing central elements of the script(s) of trauma would aid psychological researchers and clinicians in understanding the experiences of trauma in cultural context, which could ultimately lead to better clinical service opportunities worldwide. We will draw on work in anthropology, transcultural psychiatry, and cultural and clinical psychology to: (1) introduce the concept of cultural scripts; (2) apply it to cultural models of responses to trauma; and (3) describe the potential impacts of cultural scripts on psychological outcomes. We will also consider ways in which this concept can be useful to clinical scientists and practitioners, describing some examples of cultural scripts of posttraumatic distress and offering a review of methodological tools that can be used to assess scripts of trauma in clinical research and practice.In this review, we will introduce literature on cultural models to a psychological audience with a goal of fostering more work on this topic. We will rely on a more specific term, Responses to trauma are inherently complex, even confusing, going far beyond the core symptoms described in the major classification systems for mental disorders . They caTaken together, all changes that are perceptible to a person and close others following a traumatic event, whether or not attributed to trauma, constitute a phenomenological field of responses to trauma. Typically, the sheer number of changes outstrips any succinct definition, making it challenging to track, understand, and describe what is happening. The phenomenological field is also difficult to study, requiring broad and systematic bottom-up assessment of all potential signs of distress . Not surprisingly, we know little about whether broader phenomenological fields differ across cultural contexts. One study of Mexican trauma survivors suggested that their reported symptoms were indeed much more complex than a typical diagnostic set of symptoms . ResearcIn contrast to broader sets of changes within the phenomenological field of a given traumatized individual, descriptions of traumatic stress that appear in the clinical literature are far more succinct e.g., , comprisEach of these filters is in part a cultural one. They are applied in accordance with what is culturally meaningful, yielding culturally patterned signatures of illness and wellness . Althougillness narratives , idioms of distress , and commonsense or explanatory models of distress and illness or symptom schema  suggesting relevance to trauma. More recently, the concept of idioms of resilience  has been introduced in medical anthropology with the goal of understanding the full range of complex representations of subjective consequences of trauma , intersubjective , and culturally shared  knowledge about trauma and assess gaps between these representations . CulturaCultural scripts are types of cultural models that capture dynamic sequences of elements. This concept originates in broader cognitive psychology work on schema and artificial intelligence  and drawtype of schema, one with a temporal organization of its elements. It captures information about causal chains of phenomena that occur and reoccur in the same or similar sequence. It is common for script representations to have “tracks”  proposed by omoiyari in Japan: grief and mourning in Japan). People may shift between alternative scripts  in others. One example is the script of Indian syndrome . This tesyndrome . The pheIn sum, the concept of scripts captures culturally shared ideas about mental illness that: (1) acknowledge that symptoms are represented as dynamic sequences rather than static sets; (2) exists as families of schematic sequences; (3) can be assessed as mental concepts and also as overt behaviors; (4) are subjective and culturally shared as well as intersubjective; (5) are represented as sets of alternatives rather than single models; (6) can affect psychological functioning and meaning-making regardless of script adherence; and (7) differ across cultures in terms of whether a particular domain of mental illness is scripted, unscripted or somewhere in-between, and also in the details of the available scripts. We will now consider in more detail how this concept can be applied to the domain of traumatic stress studies.karma from prior lives , and associated conceptual models . Let us start with the focal script itself. Cultural scripts of trauma sequelae typically include only a few symptoms that are widely believed to be associated with each other and are perceived to have a common cause in encounters with traumatic events. Unlike rigid social scripts , scripts of trauma are more loosely structured, or “sketchy” e.g., . They alives see . Naturalives see  – or, moives see . Other eLet us now introduce some of the sources of complexity that arises from the fact that cultural models are usually not homogeneous and that they form networks of associations that take meaning from each other. As with other scripts, cultural scripts of traumatic stress are best considered and assessed not as self-contained models, but as sets of alternatives and their associated families of models  creating a salient discrepancy with subjective, intersubjective, or culturally shared ideas of desirable social functioning . The perceived differences between what is and what one would ideally want, or what one feels that one ought to do or have are known to be associated with depression and anxiety, respectively . BecauseIn sum, culturally sensitive assessment of scripts of trauma-related changes would capture core elements of the script as well as complex conceptual structures that are co-activated when thinking about the traumatic events and their consequences. This process involves attention to: (1) the extent to which a domain is scripted; (2) if it is scripted, the key elements of the script and its script tracks (or distinct sets of predictions for possible course of events); and (3) alternative scripts of traumatic stress, and associated cultural models, with particular attention to scripts of ideal and normative functioning.baksbat: Historically, posttraumatic stress was described in the clinical literature as a syndrome, a categorical rather than dimensional construct . This coThe transdiagnostic nature of traumatic stress means that its symptoms do not naturally lend themselves to being understood as a discrete syndrome any more than human behavior lends itself to be understood through the lens of personality types. Scripts of trauma and posttraumatic changes are important not only because they help us understand what people know about trauma. They can have important psychological consequences. One key domain of interest is attention and memory. Cultural scripts of responses to trauma tend to structure both personal memories and cultural accounts of trauma and its consequences. Research on cognitive effects of scripts suggests that they frame interpretations of events and serve as “cognitive cuing structures” , activatScript-relevant information is processed and remembered as a package, meaning that elements that are missing are often filled in, even if erroneously , confirmataque ne nervios facilitates experience of symptoms consistent with PTSD  changes subjective and neural representations of bodily sensations . ScriptsWhen studying the psychological effects of scripts, it is particularly important to assess not just subjective scripts but also those that are culturally shared and intersubjective. Although both subjective and intersubjective beliefs can shape psychological functioning e.g., , the assRegardless of the degree of similarity to shared and intersubjective scripts, the posttraumatic symptoms experienced by a given individual are likely to be interpreted in reference to these scripts. When considered at the subjective level, many scripts represent a blend of knowledge and belief , combiniOne fruitful area of inquiry is to capture similarities and differences in these representations and cognitive and affective discrepancies between them . What doAlthough these effects have not yet been demonstrated with posttraumatic symptoms, evidence is accumulating for other indicators of distress, such as negative emotions, neuroticism, and shyness . ExperieIn sum, a concept of cultural scripts of traumatic stress can be of use to researchers in cultural and clinical psychology and clinicians. We now will review case examples from the literature, considering culturally universal and culturally specific elements of potential scripts from different cultural contexts.The following section provides potential examples of cultural scripts of traumatic stress using existing narrative accounts. It focuses on the most important types of severe traumatic events and their aftermath see . None ofSexualized violenceThe Stadtmann study points to a peculiarity of these traumatic stress scripts from a middle European country. Because no script was available to these survivors to make sense of what was happening to them while it was happening or in the immediate aftermath, their stories were constructed after a time lag between the traumatic experiences  and the formation of a script. In the meantime, these individuals suffered from unscripted distress and tried to cope with their daily life and appear well-functioning. Retrospectively, the patients tend to feel regret and anger due to not being able to express the real causes of their suffering. The script only became visible to the outside world and intersubjectively represented after their mental breakdowns. The costs of unscripted distress, including difficulties with explaining one’s suffering to oneself or others and seeking help, are highlighted by these narratives. Also salient is the way in which scripts of responses to sexualized violence reference other cultural scripts in the domain of self-construal, relationships, and optimal life course.betrayal trauma shun the term “trauma” when narrating their ongoing suffering, adopting other ways to describe their script  is involved. CSA has been and still is a particularly silenced experience for various reasons, e.g., the affected children having no concept of healthy sexual boundaries and no adequate outlets of expression, or the ways in which cunning perpetrators intimidate their victims and cover their tracks. Many survivors of CSA have not experienced violent assaults, but the sexualized violence took place in the context of a terrible abuse of trust between an adult and an often emotionally needy child . The immr script . Others r script . The usevia such metaphors. Intersubjective script elements mentioned in his account start with the estrangement from other people and inferred beliefs of others about the veterans. He writes about the “moral vertigo” as an ongoing disorientation of core beliefs and values. According to him, US soldiers alternately feel like heroes, embedded in good comradeship, and like garbage, hating themselves for their past and projecting this nihilism to their future. The latter observation had been described as “moral injury,” and is understood PTSD as the core script of US veterans’ trauma sequelae  as a common post-trauma “cultural syndrome”  versus focusing on commonalities. We know little about whether people tend to blend or differentiate these scripts. A primary argument against the idea of being too narrow is that, in addition to the psychiatric tradition, the clinical reality is that individuals with trauma-related disorders experience an average of 4.5 different traumatic events . This meThe descriptions considered here are derived from the researchers’ and writers’ summaries of many individual’s stories of responses to traumatic events. Although these summaries reflect cultural scripts, they make it impossible to cleanly differentiate cultural scripts of what typically happens from individual experiences of what happened to a particular person. Future research on cultural scripts of traumatic stress will need to assess scripts as separate from individual experiences. In the next section, we will describe several methodological approaches to doing so.Although clinical psychology has developed a rich array of tools for assessing the psychological impact of trauma, these tools tend to focus on the individual sufferer rather than the cultural context. Moreover, assessment is typically limited to affected individuals, such as those who directly experience symptoms of mental illness and/or their family members. The project of capturing cultural scripts of trauma in research and clinical practice would require cross-fertilization between clinical and counseling psychology and psychiatry and fields that study culture, including cultural psychology, anthropology, and sociology. A number of research approaches from these fields  of trauma, a researcher would need to include questions focusing on local understandings of: (1) traumatic events, including whether they are understood to be different from other significant stressors, such as bereavement; (2) factors that predispose someone to experience traumatic events or be vulnerable to their effects see ; (3) immAnother important dimension of beliefs about trauma can be added to interview or self-report assessments by explicitly asking participants to report on their personal opinions, beliefs and knowledge (subjective assessment) as well as those reflecting perspectives of their community members (intersubjective assessment). Any question can be framed to yield both types of responses . See, for example, the way in which the questions are framed in the Cultural Formulation Interview. Culturally shared beliefs can be estimated by averaging over subjective reports. When culturally shared and intersubjective beliefs are captured, researchers and clinicians can use the information to assess and address subjective-intersubjective and subjective-culturally shared discrepancies and adaptations to them. Although assessment of subjective and intersubjective beliefs side-by-side requires time and effort, there is much to be gained from incorporating both  into the methodological repertoire of clinical psychologists.Another approach from cultural psychology is assessment of cultural models portrayed in publicly available and widely shared cultural products, such as songs, books, religious texts, newspaper stories, greeting cards, and paintings . CulturaBridge to Terabithia; Cultural products portraying post-trauma outcomes may include songs , stories, books, newspaper articles, and online discussions. Even children’s books may be used , if necessary . As such, consensus analysis always comes in the wake of ethnographic and/or self-report studies, with prior data informing researchers about the sort of information that falls in a given domain in a given cultural context. In the domain of trauma, this approach can be used to examine relatively focused sets of questions, such as “is there a consensual model of typical bodily sensations/spiritual symptoms that are associated with trauma?” Informants in a consensus analysis study are provided a resulting set of questions and asked to rate these questions’ relevance or significance to the domain.The resulting dataset is then factor analyzed, with participants serving as variables and their responses as cases. This analytical approach requires small samples, with the number of participants commensurate to the number of questions. A key assumption here is that shared consensual models are so accessible that they can be detected using relatively few informants; however, some researchers have opted for larger groups of participants, randomly sampling smaller subsamples to verify consensus . Data thIn sum, methodological tools used to study culture can enrich the toolkit of clinical researchers. These tools include ethnographic and interview-driven approaches, studies of cultural products, situation sampling, and cultural consensus analysis. These approaches can complement existing ways of assessing traumatic stress, taking the field further.We have offered our readers a vision for integrating clinical and cultural approaches to traumatic stress, with a goal of fostering generative work in psychology. Although the field of culture and traumatic stress has developed a rich array of theoretical models and assessment tools, the addition of a cultural script approach would offer a way to capture complex and sequentially arranged models of traumatic stress that are subjectively understood and intersubjectively shared, pointing a way toward operationalizing and assessing complex cultural models of posttraumatic changes. This approach would allow clinical psychologists to better differentiate between scripted and unscripted forms of individual distress and attend to the possibility of alternative scripts. The cultural script approach to trauma encourages researchers and clinicians to consider psychological and social consequences of “scriptedness” and offers specific predictions for the impact of script activation on attending, remembering, experiencing, and communicating posttraumatic distress. Introduction of this concept can also help clinicians better understand and assess potential discrepancies between how one experiences distress and what is understood to be normative. Finally, given research on how scripted information propagates from person to person  characterizing families of scripts of traumatic stress and (2) understanding their clinical impacts and the subjective-intersubjective discrepancies generated by them. We have argued that cultural scripts aid survivors of trauma and care providers in making sense of the complex and potentially confusing phenomenological fields of potential symptoms. The concept of cultural scripts can similarly draw on the rich work on cultural models of distress and aid clinical psychologists in their efforts to develop parsimonious yet contextually sensitive models of traumatic stress.YC-D and AM were involved in conceptualizing, drafting, and finalizing the paper. AM initiated the project and coordinated all stages.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.