We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular. At the  few mGy ,8. Thus,Among the different types of radiation induced DNA lesions, a primary role in cell killing is attributed to DSB. Specifically, the number of residual DSB observed late after irradiation, rather than the number of initial induced lesions, has been found to give indications concerning cell inactivation probability –13. For A consistent description of DNA damage and processing in an extensive dose range is also needed for the implementation of radiobiological models which can be used in treatment planning. The Giant LOop Binary LEsion model (GLOBLE) is a radiobiological model able to predict biological effects resulting from photon irradiation . The modγH2AX assay. However, since such studies would imply the use of high doses in order to be sensitive to such effects, some practical issues have to be considered.In the framework of the validation of our modelling approach, we recently presented a DSB kinetic rejoining model based on the GLOBLE, which was able to consistently reproduce the dose dependence of DSB rejoining as measured by means of filter elution techniques after photon irradiation in a dose range between 10 and 200 Gy . This shγH2AX signal suggest that it should rather be considered as an indirect marker of DSB presence [γH2AX fluorescence signal needs about 30–60 min to reach a maximum in the fluorescence intensity, before decaying over time [γH2AX foci, being characterized by a finite spatial extension [In fact, while the physical methods of detection allow the quantification of DNA fragments after irradiation (which can be linked to the number of induced DSB), the spatio-temporal characteristics of the ver time . This isxtension ,31.γH2AX foci size in the scoring of DSB have been considered in the past, especially concerning high LET radiation, in experimental [The implications of the finite rimental ,33 and mrimental ,35 studiAdditionally, mechanisms have been recently proposed to describe the possibility of a three-dimensional accumulation of repair factors around the DSB, which would be responsible for a further extension of the phosphorylated region. In particular, according to the work presented by Muñoz et al , this woγH2AX fluorescence signal intensity after X-rays irradiation in an extended dose range, and compare them with a modelling analysis inspired by the GLOBLE approach. The aim was to analyse the dose dependence in DNA repair after low LET irradiation, thus contributing to the validation of the basic assumptions on which the GLOBLE model is built. At the same time, the use of the γH2AX assay at high doses, initially motivated by our interest in investigating DSB kinetics of rejoining, revealed to be an interesting aspect on itself for the study of basic model parameters. Thus, full dose response curves were measured with flow cytometry, complemented by parallel confocal microscopy imaging, and then compared to model predictions. The influence of the spatial extension of the phosphorylated regions and of the DSB induction yield is widely considered and discussed. The potential role which can be attributed to 3D phosphorylation mechanisms is also taken into account. These aspects are found of general relevance for the interpretation and the understanding of experimental data obtained by means of the γH2AX fluorescence assay. The kinetics of the γH2AX signal has been investigated up to 24 h after irradiation with increasing X-rays doses up to 500 Gy, and a GLOBLE-based kinetic model is proposed to describe the observed signal. The gradually slowed-down disappearing of the γH2AX signal at high doses is well described by the model. Together with our previous studies, this is supportive for the relevance of damage clustering at the micrometre scale in higher-order chromatin structures, in order to understand the biological effects induced by ionizing radiation.Here we present new experimental data, obtained by means of flow cytometry measurements of the 2) at 37°C in incubators, where the atmosphere was composed by 95% air and 5% CO2. Cells were kept in exponential growth by regular sub-culturing.Chinese Hamster Ovary cells  were cultivated using Ham's F12 medium (Biochrom) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penycilin/Streptomycin; a solution of 0.05% trypsin/0.02% EDTA was employed for trypsinization. Human skin fibroblasts  were grown in EMEM with EBSS medium (Lonza), where 15% FBS and 1% Glutamine were added. A solution of 0.05% trypsin/0.1% EDTA was used for trypsinization. The cells were grown in culture flasks (25 or 75 cmγH2AX kinetics at high doses (> 8 Gy) shown in Two different irradiation setups were employed. Flow cytometry experiments were conducted by using an x-ray tube working at 250 kV and 16 mA. A dose rate of 7 Gy/min was employed. Due to the need to reach high doses, a different x-ray tube working at 35 kV and 80 mA was adopted to perform experiments at high doses. This allows reaching a dose rate of about 100 Gy/min. Despite the different voltages, according to literature data no considerable difference is expected among the DNA damage patterns induced by the two x-ray qualities employed . In ordeγH2AX molecules. Cells were resuspended in 100 μl of primary antibody  and incubated for 1 h at room temperature. Therefore, the samples were washed, the cells were resuspended in 100 μl of secondary antibody solution 2 conjugate, Invitrogen, Karlsruhe, Germany) and incubated in the dark for 45 min at room temperature. Finally, 1 ml of DAPI solution (1:1000 in PBS) was added to obtain DNA staining as well. The fluorescence intensity of the samples was then measured at the flow cytometer after an incubation of at least 15 min in the dark at room temperature. In order to allow more accurate comparisons, samples belonging to the same experiments were stained and measured on the same day.Cells were trypsinized 1 h after irradiation, fixed with a solution of 2% Paraformaldehyde (PFA) in Phosphate Buffer Solution (PBS), and permeabilized with 1 ml of Triton X solution 0.5% in PBS. 1ml of 0.4% BSA in PBS was added as blocking agent, and cells were kept at 4°C for a maximum of 7 days. After washing, a double antibody staining was used to detect the presence of γH2AX mean fluorescence intensity of G1 cells was quantified. The analysis has been restricted to G1 cells in order to minimize the bias introduced by spontaneous foci which can be measured in the S and G2 phases [A PAS III cytometer produced by Partec was used in this study. The analysis of flow cytometry data was performed with the FloMax software, delivered together with the instrument. For each sample, the 2 phases . The bacFor the immunocytochemical staining experiments, cells were fixed in 2% PFA and permeabilized as described previously . The sam3. The higher-order chromatin organization is taken into account by considering the presence of Giant-Loops, also called domains here, each one involving about 2 Mbp of DNA [loop and domain will be used with the same meaning.The modelling work presented in this paper is based on the approach adopted in the GLOBLE for the description of radiation induced DNA damage . The tarp of DNA ,25  and clustered (cDSB) DSB according to whether only one or more than one DSB are simultaneously present in the same domain, respectively. Since the cDSB produce the loss of integrity of the loop structure, and they may even result in the loss of DNA fragments, they are considered to be more severe lesions compared to the iDSB.hit domain each domain where at least one DSB is scored. Then, we make the hypothesis that each hit domain can be associated to a loop where H2AX phosphorylation takes place following DSB induction. This is supported by literature data, suggesting a link between chromatin loops and H2AX histone phosphorylation [In our model we define as rylation ,40. We aD can be defined as follows:NL indicates the total number of domains and αDSB represents the DSB induction yield. In line with the standard parameters adopted in the GLOBLE, NL is chosen equal to 3000 while αDSB is initially set to a value of 30 DSB per Gy and cell nucleus [ni) and by cDSB (nc) at a given dose is then calculated with the following equations:nT results from the sum of the corresponding ni and nc:γH2AX fluorescence intensity. We use a normalization factor xfl representing the average fluorescence per hit domain, so that the fluorescence signal is given by:In order to predict the numbers of induced iDSB and cDSB after photon irradiation, the Poisson distribution is adopted ,27. The  nucleus ,41. Impo nucleus –44. The activated domains ;If the domain has an iDSB or a cDSB, an integer random number next of the 26 surrounding voxels are randomly activated to simulate the 3D extension of the phosphorylation starting from the hit domain;After the two random processes have been completed, the algorithm examines the following voxel, until all the voxels have been considered.A further step is introduced in the model in order to take into account the possibility that H2AX phosphorylation can extend to domains surrounding the hit ones and where no lesion is induced, which we define here as ains see . We assuIn order to increase calculation accuracy, the random process was repeated 500 times for each considered dose, and an average quantity was then computed. At this stage, we believe that the assumption of a uniform probability distribution defining the final extension of the phosphorylated region represents a reasonable approach for a first step approximation since no detailed experimental evidence is available. The same applies to the selection of which neighbouring domains are phosphorylated, assuming that there is no preferential direction for the spatial spreading. At the same time, we are aware of the fact that the choice of cubic domains is a simplistic approach. However, we think that in first instance this is a reasonable assumption to test the soundness of the model. Moreover, it is the genomic content of the domain rather than its exact geometry which is the most relevant aspect in GLOBLE. Therefore we do not exclude that more realistic geometry could improve the model, but we are confident that this would represent a second order effect which might be analysed in further studies. Starting from an initial average number of hit domains, the final outcome of the calculations is thus the average expected number of phosphorylated domains, including both directly hit or randomly activated domains.γH2AX signal kinetics, the approach recently presented in Tommasino et al [γH2AX signal over time is not strictly related to the DSB rejoining but more generally reflects ongoing repair processes [γH2AX signal kinetics is thus the following:ni and nc are calculated with the GLOBLE, and τfast and τslow refer to the half-lives of the fast and slow components of damage processing respectively. In addition, we need to introduce one more parameter in order to establish quantitative comparisons between the predicted frequencies of iDSB and cDSB and the measured signal. For this reason, the effective fluorescence per hit domain is considered by the term xfl. When the expression is used to fit experimental data as measured after irradiation with different doses or different radiation qualities, the three half-lives are considered as global fit parameters. The effective fluorescence is instead used as single-curve fit parameter. The frequencies of induced iDSB and cDSB represent input parameters of the model and their relative fractions define the complexity of the induced damage pattern, thus determining a faster or slower disappearing of the signal. The fit is based on a χ2 minimization on a linear scale, and error bars are considered. This model will be applied to the description of the γH2AX kinetics after irradiation with different doses of X-rays.Aiming at the description of the no et al  for the no et al ,46, a derocesses . The matThe dose response curve measured 1 h after irradiation, up to a dose of 140 Gy is shown in In addition, in Dedicated tests have been performed, including variation of antibodies concentration and the use of antigen retrieval techniques, in order to exclude that the observed saturation results from technical artefacts. However, we observed no indications pointing at technical issues as explanation for the saturation behaviour.γH2AX distribution pattern in cells irradiated with high doses of X-rays. The resulting microscopy pictures are shown in γH2AX fluorescence patterns obtained after irradiation of confluent AGD cells with 10, 50, 200 and 400 Gy of X-rays are shown. We realize that while after 10 Gy it is still possible to observe a heterogeneous distribution of foci, although their high number hinders an accurate counting, after 50 Gy the γH2AX seems to be distributed almost in the whole nucleus. However, the fluorescence is not homogeneous and we can distinguish spots of higher and lower intensity resulting in a more or less pronounced granularity of the staining, according to the specific cell under examination. A different fluorescence pattern is observed in the samples irradiated with very high doses, namely 200 and 400 Gy in this specific case, showing a much more homogeneous distribution of γH2AX. This suggests that, in line with the low residual slope observed in the saturation region with flow cytometry, there is still room for further H2AX phosphorylation at such high doses.A parallel microscopy analysis was performed, aimed at the qualitative investigation of the These results are further supported by the direct comparison of flow cytometry data and integral fluorescence intensity as extracted from microscopy pictures which is shown in The GLOBLE model was used to calculate the expected number of hit domains for a given photon dose , deviations from the measured points get smaller at high doses. On the contrary, in the case of CHO cells even the assumption of a large domain size is not enough to reproduce the initial steep increase of fluorescence which is measured in the experiments, although we can again observe a partial agreement for doses above 25 Gy if a DNA content comprised between 6 and 10 Mbp per domain is assumed.The same approach is adopted for the analysis of model calculations of hit domains as function of the dose, by using the predicted values corresponding to 140 Gy as normalization factors. Thus, a qualitative comparison can be directly established between experimental and modelling results. Predicted curves are calculated for different combinations of model parameters. In In From the comparison shown in activated domains  and the model results (lines) are reported in the upper panel of xfl parameters resulting from the fit. However, the global chi-square was only slightly higher when the simple Poisson statistics was employed.The kinetics of the The fit results are in good agreement with the data, and produce half-life values in line with what is found in the literature ,51. ConcIn the figure we can observe that for increasing doses the maximum fluorescence intensity increases and is reached at a later time point, while the signal persists for a longer time. The comparison of the kinetics can be better appreciated when looking at γH2AX induction and processing in an extensive dose range was initially motivated by the interest in investigating DSB repair kinetics after increasing doses of photon radiation. In fact, this represents an important end point to analyse the relevance of micrometre scale damage clustering, which is a key aspect of GLOBLE. However, preliminary experiments showed the onset of a saturation tendency at doses around 20 Gy in the γH2AX dose response curves as measured with flow cytometry  were employed to perform the experiments and where no clear dependence on the dose was observed ,57. ThesγH2AX signal kinetics as measured with flow cytometry. Specifically, after the normalization has been performed, clear dose dependence can be observed in the fit curves, which according to our approach is entirely due to DSB clustering at the micrometre scale. The quite extreme dose values employed are justified by the need to obtain a significant difference in the fraction of induced cDSB in order to be sensitive to the gradually delayed kinetics. In fact, cDSB represent only about 10% of the induced damage at 25 Gy, while the fraction increases up to about 95% at 500 Gy. Importantly, the best fit results are obtained when iDSB and cDSB are calculated assuming the standard domain size of 2 Mbp and including the 3D spreading mechanism. On the contrary, as shown in In The concept of the enhanced severity of cDSB compared to iDSB has been discussed in more detail also in the recent paper by Hufnagl et al. . AlthougA homogeneous distribution of DNA inside the nucleus is assumed in the GLOBLE. However, in experiments performed with low doses of low LET radiation, the fraction of DSB which is slowly repaired has been associated to the damage induced in the heterochromatic regions of the cell nucleus . In suchγH2AX dose response curve as measured with flow cytometry was investigated in the past by MacPhail et al [γH2AX also appear in publications with different aims, but they are usually restricted to low doses (up to few Gy) and therefore not comparable with what we want to discuss here [The il et al  up to a uss here –66.γH2AX dose response curve, the results of our modelling analysis suggest that a good agreement to experimental data can be obtained following two different approaches. For instance, γH2AX data as shown here.With respect to the saturation observed in the Concerning the relevance of the 3D spreading mechanisms, we extracted from the model calculations the Z projections of hit domains only and of all phosphorylated domains, as obtained when the random expansion algorithm is considered. The comparison between the two projections indicates that the overlap of neighbouring foci could affect the scoring of single foci. An example picture where the exposure to 1 Gy of photon radiation was simulated is shown in A deeper investigation of the potential confounding factors in microscopy measurements related to the mechanisms of H2AX phosphorylation is beyond the scope of this work. However, similar conclusions indicating a loss of accuracy in the foci counting when the delivered dose exceeds few Gy, can be found in published works as well ,8. This All these aspects pooled together could finally contribute to determine an underscoring of the actual number of induced DSB, even at low doses which are of interest for instance in radiation protection. This observation is in line with the results of the modelling work presented by Ponomarev et al. and mentioned in the Introduction . In theiγH2AX was employed, this approach is expected to partially solve the problems due to the kinetics of protein recruitment at DSB sites and to proximity effects (e.g. overlap of close-by foci). Moreover, the authors show that the DSB yield drops to about 30 DSB/Gy at 1 Gy, as the result of clustering of DSB into repair centres. This is actually in line with our conclusions. Nevertheless, these results contrast in a way with the value of 20–40 DSB/Gy, mainly resulting from gel elution experiments, which has been considered for many years as well established in the community [In contrast to the choice of the domain size, the DSB induction yield is expected to be less dependent on the cell line under consideration . Our resommunity ,49. HoweγH2AX signal for increasing doses, based on DSB clustering at the micrometre scale in the context of higher-order chromatin structures. These results, combined with recent works published by our group, support the relevance of the micrometre scale for the description and the interpretation of radiation induced biological effects. Furthermore, the outcome of our analysis on γH2AX dose response curves is of interest concerning the use and the interpretation of γH2AX data. In fact, when discussing the strong saturation observed in the measured signal, we showed that controversial interpretation of γH2AX data may arise not only as a consequence of the frequently discussed technical issues, but also due to an incomplete understanding of the phosphorylation process. This observation might even extend to other markers involved in the DNA damage and repair processes. We found out that a substantial role might be attributed to 3D mechanisms of H2AX phosphorylation. More direct experimental support would be needed to confirm the importance of this kind of processes.With our model based on the GLOBLE approach, we were able to consistently describe the delayed disappearing of the S1 FigPanel A: microscopy pictures showing the γH2AX staining pattern as observed at the confocal microscope for different doses 1 h after irradiation of AGD cells with X-rays ; the “sum slices” Z-projection obtained with the ImageJ software is shown. Larger dark areas in the DNA staining which are free of γH2AX fluorescence represent nucleoli . Panel B(EPS)Click here for additional data file.S2 FigThe expected number of hit domains is calculated by assuming a fixed DSB induction rate equal to 30 DSB/Gy and by changing the domain size in a range from 2 to 10 Mbp (A); calculations are also shown, where the expected number of hit domains is simulated by assuming a fixed number of domains  and by changing the DSB induction rate inside a range comprised between 15 and 75 DSB/Gy (B).(EPS)Click here for additional data file.S3 FigExperimental data are the same as shown in (EPS)Click here for additional data file.S4 FigExperimental data are the same as shown in (EPS)Click here for additional data file.S1 TableFit parameters resulting from the application of the γH2AX kinetic model to the experimental data shown in (DOC)Click here for additional data file.S2 TableFit parameters resulting from the application of the γH2AX kinetic model to the experimental data shown in (DOC)Click here for additional data file.


The occurrence of LTD involves the activation of a positive feedback loop formed by protein kinase C, phospholipase A2, and the extracellular signal-regulated protein kinase pathway, and its expression comprises the reduction of the population of synaptic AMPA receptors. Recently, a stochastic computational model of these signalling processes demonstrated that, in single synapses, LTD is probabilistic and bistable. Here, we expanded this model to simulate LTP, which requires protein phosphatases and the increase in the population of synaptic AMPA receptors. Our results indicated that, in single synapses, while LTD is bistable, LTP is gradual. Ca2+ induced both processes stochastically. The magnitudes of the Ca2+ signals and the states of the signalling network regulated the likelihood of LTP and LTD and defined dynamic macroscopic Ca2+ thresholds for the synaptic modifications in populations of synapses according to an inverse Bienenstock, Cooper and Munro (BCM) rule or a sigmoidal function. In conclusion, our model presents a unifying mechanism that explains the macroscopic properties of LTP and LTD from their dynamics in single synapses.Long-term depression (LTD) and long-term potentiation (LTP) of granule-Purkinje cell synapses are persistent synaptic alterations induced by high and low rises of the intracellular calcium ion concentration ([Ca The additional components of the model included Calmodulin (CaM), Ca2+/Calmodulin protein kinase II α (αCaMKII)31612+ In this work, we have expanded extensively the previous stochastic computational model of Ca2+ transients promoted both processes in a stochastic manner. Nevertheless, the intensity of Ca2+ signals used to induce synaptic plasticity modulated the likelihood of LTP and LTD occurrences in single synapses. In addition, alterations in the components of the signaling network regulated the effects of Ca2+ signals on the induction of LTP and LTD. In consequence, our results indicated the existence of dynamic Ca2+ thresholds for the occurrence of macroscopic synaptic modifications according to the inverse BMC rule proposed for cerebellar plasticity2+ transients used as input signals, we obtained the macroscopic sigmoidal relationship observed between the amplitudes of Ca2+ signals and the corresponding levels of depressionOur results showed that, in single synapses, LTD is an all-or-none process, but LTP is graded. Ca2, and ERK pathway, which we expanded and updated from a previous version32+ elevations transiently activate PKC and PLA22 and sustains its activity after the return of [Ca2+] to its basal level2 produces arachidonic acid (AA), a PKC co-factor that activates it synergistically with Ca2+ or alone in high concentrationssyn by PKC disrupted these interactions526syn. We set the basal percentage of AMPARsyn as 100%.The computational model presented in this work simulated LTD and LTP in a single Purkinje cell dendritic spine, which encloses the signalling machinery of the glutamatergic synapse182-ERK pathway. However, to simulate LTP we had to include CaN in the model  and high amplitudes (>0.5 μmol.L−1), respectively22+ pulses tested, including high amplitude signals that typically induce LTD in Purkinje cells12After the implementation of the model, we used Ca2-ERK3313Cerebellar LTD requires the activation of the feedback loop PKC-PLA2+/CaM and its subsequent autophosphorylation. The autophosphorylation of αCaMKII modulated its affinity for Ca2+/CaM and produced an autonomous state that sustained its partial activity2+/CaM for secondsαCaMKII has several putative targets during synaptic plasticity, including Raf34342+ elevations without affecting LTP induction for low Ca2+ transients with prolonged durations (10 s and 20 s) (2+ rises necessary to induce LTP in the model (0.15–0.4 μmol.L−1) was similar to experimental estimations (0.1–0.3 μmol.L−1)2+ pulses of 1 s, which was consequent to the mechanisms of activation of CaN simulatedαCaMKII inclusion restored the occurrence of macroscopic LTD induced with high Cand 20 s) . The ran2+ and Ca2+/CaM2+], two high affinity Ca2+-binding sites of the regulatory subunit CNB are constantly filled28392+-binding sites of CNB292+ promotes a conformational change that enables the binding of Ca2+/CaM to CNA and the exposure of its catalytic site39402+/CaM in absence of CNB422+ to CNB prior to the binding of Ca2+/CaM to CNA28292+ to CNB occurred with slow rate constants2+ pulses of 1 s. Consequently, the kinetic aspects of CaN activation constrained the durations of the Ca2+ transientsCaN is a heterodimer activated by Ca2+], to a state of high kinase activities consequent to the activation of the positive feedback loop. Thus, processes that favour the activation of the loop caused LTD in the model. For instance, a pulse of active MEK promoted LTD2-ERK. Moreover, partial blockages of PP1 and PP2A promoted the induction of LTD for a LTP protocol , for the entire range of Ca2+ amplitudes tested. However, LTD predominated as the synaptic modification obtained in the model for stimulations with high amplitude Ca2+ transients (>0.8 μmol.L−1)  . In cont signals .2+ on LTP and LTD inductions. Thus, we verified the changes of AMPARsyn as functions of the peak amplitudes of Ca2+ transients for simulations performed without αCaMKII. The results showed that the absence of αCaMKII increased the range of Ca2+ amplitudes that induced LTP and decreased the occurrence of LTD , LTP (PLTP) and failure of plasticity (PFailure) for Ca2+ pulses with different amplitudes and durations. Our results showed that PLTD increased with the increment of the durations and peak amplitudes of the Ca2+ signals used to trigger plasticity (LTP was low for short Ca2+ pulses (1 s). For Ca2+ signals of 10 s, PLTP was low for weak Ca2+ rises, increased for amplitudes ranging from 1–3.5 μmol.L−1 and dropped for higher concentrations while PLTD increased progressively with the increment of the amplitudes of the Ca2+ signals , and decreased progressively with the increment of the amplitudes of the pulses, which caused the increase of PLTD  predicted in the classical BCM rule2+ thresholds for synaptic modifications consistent with the inverse BMC ruleThe modulatory role of Ca and LTP . These r2+ rises, but failed to observe LTP2+] investigated in this work varied from approximately 0.5 to 6 μmol.L−1 2+ transients lower than 0.5 μmol.L−1 (θm = 0.37 μmol.L−1), which is consistent with other experimental results2+ pulses below from 0.5 μmol.L−1 from our analyses, we obtained the same sigmoid function observed experimentally2+ rise and the occurrence of opposite forms of synaptic plasticity.Interestingly, a previous work reported the existence of a macroscopic sigmoidal relationship between the magnitudes of LTD and the levels of Ca2+ thresholds for LTP and LTD. To investigate this aspect of the model, we varied the concentrations of some of its components and verified their impacts on the macroscopic Ca2+ thresholds for the induction of LTP and LTD  resulted in stronger LTP and affected θm  had no effect on the Ca2+ thresholds for LTP and LTD, but an increase of [PP1] (1 μmol.L−1) altered θm  (−1) suppressed LTP and promoted a sigmoidal relation between the amplitudes of the Ca2+ signals and the magnitudes of LTD (−1) and reduced concentration of PP2A   in comparison to the control model   . In conts of LTD . We verimol.L−1) . Additiomol.L−1)  exhibitemol.L−1) . TherefoLTP, PLTD and PFailure that changed with the amplitudes of the Ca2+ signals used to induce plasticity . As a result, the balance between PFailure and PLTP  were attributed to the stochasticity of the model and treated as failures of synaptic plasticity induction.To calculate the probabilities of LTP and LTD induction in How to cite this article: Antunes, G. et al. Stochastic Induction of Long-Term Potentiation and Long-Term Depression. Sci. Rep. 6, 30899; doi: 10.1038/srep30899 (2016).


The welfare of pet rabbits is an area of growing interest in Europe and the UK. This study analyses questionnaire results from a diverse population of 1254 rabbit owners from three different geographical areas in England with the aim of providing an accurate representation of how pet rabbits are currently housed and cared for and key aspects of their health and welfare.Rabbits were kept in a variety of different housing types, the most common being a traditional hutch/cage (59%). Although the majority had additional exercise areas, access was often unpredictable, or ill-timed, which may compromise welfare. Only 41.9% of owners kept their rabbit with conspecifics, limiting their ability to engage in social behaviour. Of those rabbits housed with a companion, although many were reported to be amicable and to engage in positive interactions, over a quarter were reported to fight at least occasionally (25.3%), whilst 22.7% guarded resources and 27.1% avoided one another. Whilst low levels of some of these behaviours may be a normal part of social interaction, the relatively high levels reported here suggest that not all cohabiting pairs of rabbits are compatible, which is potentially a significant welfare issue.Although the vast majority of owners fed hay for over 10% this was less than daily. Pelleted foods were very popular (71.4% at least daily) compared to commercial muesli mixes (32.6%). As in previous studies, dental problems were commonly reported (12.2% of rabbits); however, so were eye problems (12.9%), digestive problems (11.5%) and parasites (11.3%). A large proportion of rabbits (58%) were thought to be fearful of loud noises, and 61% were not reported as calm when handled by their owner, which may be a significant concern for this species.This study has confirmed and expanded on previous findings: many pet rabbits were found to be in good health, had compatible companions and were provided with enriched living areas. However, it also found numerous welfare issues that affect large numbers of pet rabbits. We suggest further studies are required exploring the accuracy of owner reports (which possibly under-report many problems) and prioritising the issues raised here. Rabbits are common pets in many western European countries . They ar2, the minimum cage area recommended for laboratory and farmed rabbits  the PDSA  conductre needs . While tre needs , 5, the The present study aimed to provide a representative point sample of the current state of care and welfare of English pet rabbits, against which subsequent surveys could be compared, and change assessed.A questionnaire was devised consisting of over seventy questions and was presented in three different forms: a) written form, distributed via pet care outlets, veterinary surgeries and schools; b) verbally usually over the telephone, and c) via the internet, recruited in multiple ways. Diversity of recruitment methods aimed to overcome some of the sampling bias inherent in past surveys. A prize draw was used to incentivise owners to take part.Owners were asked about the way they housed and cared for their rabbits, collecting information on many factors hypothesised to affect their welfare, including housing type and size, exercise and grazing provision, husbandry and cleaning regimes, diet, companionship and veterinary care .Increasingly, animal welfare is measured in terms of outcome- rather than input- based measures (e.g. ). TherefPast estimates of longevity have been based on the age of rabbits at the time of surveys and have raised concerns about potentially low life expectancy (e.g. 2.2 years in ). To avoSection A Respondent - gender, age, details of any children, number of rabbits currently owned, main carer;Section B Rabbit - name, sex, neuter status, age and who it was obtained for;Section C Companionship - whether rabbit lives with another animal, length of cohabitation and frequency with which they show specific social behaviours;Section D Housing - size and details of main living space, any attached and separate runs; information on the length, height and also the area of the home enclosure. The diversity of accommodation in which rabbits are kept has made some past survey findings difficult to interpret. Therefore we categorised the main living space as the area to which the rabbit had permanent access, but we also asked questions about any "separate" and "attached" runs to which the rabbit had intermittent access, with and without the necessity to be moved.Section E Husbandry - exercise and cleaning routines, in summer and winter;Section F Diet - frequency of feeding 12 different foodstuffs, and whether rabbit exhibits selective feeding;Section G Health - whether ten specific symptoms of ill health had ever, occasionally, or often been observed; whether the rabbit suffered from 15 common veterinary complaints, currently or in the last year, and whether it required veterinary treatment. Estimates of current teeth and nail condition.Section H Behaviour - owner’s opinion of how the rabbit reacts to a number of situations, including handling by themselves, children and other adults, approaching their living space, and five potentially fear-provoking stimuli e.g. loud noises and open spaces, and similarly the rabbits’ most common activity.The questionnaire included 76 questions divided into eight sections:To avoid bias created by owners of multiple rabbits choosing to give information about all or the healthiest or most interesting rabbit, respondents were instructed to answer only about the rabbit whose name came first alphabetically. They were then given the opportunity to provide extra details on the social groupings of all additional rabbits they owned, and the age at death of their most recently deceased rabbit.Many of the questions included component parts, hence a maximum of 326 variables were collected from each respondent.The same questionnaire was presented via three forms; a printed version, one delivered verbally during telephone interviews and an internet version. Each included the same questions presented in the same order, but the formatting was slightly modified for the internet software system available.South West – centring around Bristol and North Somerset;North West – centring around Manchester and the Wirral;Eastern – centring around Norwich and Eastern Norfolk.All three questionnaires were distributed in rural and urban locations within three target regions:A total of 1419 written questionnaires, accompanied by prepaid return envelopes and advertising posters were placed in 82 different prominent locations, in veterinary surgeries, pet shops, RSPCA  clinics, rehoming centres, and other pet-related outlets. Regular checks were made at each outlet, to replenish supplies. Questionnaires were also given to rabbit-owning families in four schools, and to friends and family of the research team. In total, 343 owners responded, giving a response rate of 24.2%.Veterinary surgeries in each target area were contacted, and ten (including both surgeries located within pet stores and independent practices) agreed to contact their rabbit-owning clients to recruit survey respondents. In addition, sign-up lists were placed in three PDSA clinics and all rabbit-presenting clients were asked to participate. Recruitment lists were displayed at a very large rabbit show, postcards handed out at an education stall in Manchester and by RSPCA inspectors on routine calls and were sent to owners who had recently acquired a rabbit from a re-homing centre. In total, 484 owners volunteered. They were contacted by telephone at a convenient time, and 335 interviews taking between 5 and 80 minutes, were conducted (mean = 27.8 ± 9.1 mins).The survey was linked to the University of Bristol homepage and publicised in each key region via radio, newspapers, fliers at agricultural shows, local shops, libraries and community centres, emails to staff of several companies and to members of the Rabbit Welfare Association and Fund; alerts on University of Bristol payslips, postings on Facebook and twitter pages, money-saving websites, and general pet and rabbit forums. A total of 1490 rabbit owners responded.Responses from the internet questionnaire were downloaded and exported to Excel, telephone and written responses were entered into the same spreadsheet. All those respondents from outside the key areas were excluded. Descriptive analysis was carried out using SPSS v19.Research was approved by the approved the University of Bristol ethics committee and was performed in accordance with the Declaration of Helsinki.Between January and July 2011, 2169 completed surveys were returned  in the age range 30–49 (56.5%). At the time, 48.3% had no children in the household, whilst 45.6% had children permanently and 6.1% visited at least once every two weeks. More respondents had girls, either under (63.2%) or over ten (54.5%), than boys of the same ages (58.6 and 40.4%).For those 877 owners who stated how many rabbits they owned, answers ranged from one to 37, two rabbits was most common (44.4%) and only 3.3% of owners owned more than six. Most were cared for by the person answering the questionnaire (53.1%), whilst 24.7% of respondents shared care with another adult and 16.4% with a child.In total, 58.5% of the rabbits were male, 40.9% female and 0.2% of unknown sex (0.4% of owners failed to answer). A reported 64.9% of males and 51% of females  were neutered, for 0.5% the neuter status was unknown. Rabbits ranged in age from 2 months to 12. 7 years, (mean = 3.2 ± 2.3 years). The mean length of ownership was 2.9 (±2.2) years.The most common breed/type were Lops, whilst Lionheads, Netherland dwarfs and mixed breeds were also popular Figure . A totalThe most popular source of rabbits was a pet shop or garden centre (39.1%), followed by rehoming centre (17.6%), breeder (15.8%), friend or neighbour (15%), advert (4%), whilst 2.1% were found as strays. Most rabbits were obtained for the respondents (49%), but 27.6% were mainly for a child.The majority of owners described their rabbit’s main living space as a hutch or cage (59.1%), whilst 29.9% were described as house rabbits, although most of these (82%) had a cage/hutch, but 5.5% of rabbits lived in the house, without any cage at all . When the number of animals in the enclosure was taken into consideration, the average space was 1.27 m2 per animal , but 8.2% of the population (95 rabbits) were provided with less than 0.54 m2.Most hutches/cages were on a single level (53.5%), 42.3% were over two and 4.2% had three or more levels. The surface area of the main hutch or cage, , ranged from 0.1 mSix cages were recorded as over 10 m tall (presumably roofless). When these were removed the median height was 0.9 m , but for 8.3% of the rabbits their main living space (with permanent run included only if they had constant access to it) was less than 0.5 m high throughout and for 42.1% it was less than 0.75 m. Within their home cage, 86.7% were reported to have a separate area for denning/sleeping, 71.1% had shelters, tunnels or boxes, 65.9% platform(s), and 59.2% had continual access to toys.2. Of these 42.1% (23.5% of population) had continual access; 43.3% were shut away at night and for 8.8%, run use varied with the weather.The vast majority of rabbits (97.5%) had some exercise area outside their home-cage, but the frequency and duration of access varied greatly. In total, 43.4% had an attached run (area they could reach without being moved by the owner); the average area being 198.59 (±406.3) m2, and whilst inside, 84.6% of rabbits had access to shelter, tunnel or box; 87.9% had grass to eat and 74% had ground to dig.Separate runs were more popular; 62.9% of rabbits having access in summer, most commonly every day , and for 4–8 hours (30.9%). However for 42.2% of rabbits, the frequency of access differed with the season; in winter only 49.9% had any access, with occasional (16.7%) 1–2 hour sessions being most frequently reported (11.1%). The mean run size was 30.56 (±90.63) mA reported 62.2% of rabbits were allowed to run loose in the garden, although only 50% in winter. For 22.8%, access was daily, or several times daily 8%; for the remainder, sessions were less predictable. Free running in the house was reported by 59% of owners.Although 43.5% of rabbits were described as living alone, only 41.9% lived with other rabbit(s), whilst others lived with guinea pigs (4.3%), cats, dogs, quail, chickens, chameleons and even kune kune pigs. Rabbits living with conspecific companions were reported to show a variety of social behaviours  or more (27.2%), and took the soiled material out daily (50.2%) or more than once a week (27.2%). However 2.6% reported never thoroughly cleaning their rabbit’s living space. When cleaning, 45.2% sometimes (29.7%) or always (13.8%) left bedding inside, most used disinfectant occasionally (42.2%) or always (41.3%).Most rabbits were handled at least weekly Table , but lesThe most popular food types were root vegetables  and hay , a small proportion were unsure (2.9% and 0.4% respectively), and some admitted that they were possibly  or definitely  overgrown.Of the presented list of clinical signs, indicative of underlying health issues, dirty bottoms were the most common; being reported as occurring at least occasionally by 30.1% owners Table . SwollenOf the range of presented veterinary conditions Table , digestiIn-cage behaviour Most rabbits were described as spending most time resting alone  when people approached their living area, but ignoring (25.2%), retreating (10.2%) or hiding (5.4%) were also common (some owners reported multiple responses). However, 1.7% of rabbits were thought likely to move towards the owner in an aggressive manner, and 2.6% to thump their hind legs or vocalise (2.2%).When rating their own confidence at handling their rabbit 73% of owners scored 5 (very confident), 13% scored 4, 8% 3, 4% 2 and 2% scored 1, .In total 39% of rabbits were described as calm when handled, but 61% of handled rabbits showed various signs of fear, Figure ; only 25Overall 830 owners provided data on the age at death of their last rabbit, which ranged from one month to 12 years with a mean of 5.6 (±0.1) years.The survey shows the diversity in ways rabbits are kept and cared for and highlights a number of potential welfare issues in the English pet rabbit population. By recruiting in diverse ways and targeting less keen owners, we aimed to reach a more diverse sample of rabbit owners than have some past detailed surveys (e.g. ). Our saOur survey shows that although the traditional hutch or cage remains the main living space of most English pet rabbits, rabbits are kept in diverse ways, including in Wendy houses, rooms and sheds. Patterns of access to living space also vary greatly between individuals. It is therefore important to understand how design and complexity, combined with size, affect welfare if guidance regarding optimal housing design is to be offered to rabbit owners, and this should be the focus of future research.2 space available. It has been demonstrated that cages of 0.88 m2 limit the behaviours which rabbits can exhibit [The results also show that although the majority of rabbits have additional exercise areas, less than a quarter of them are given continual access to these areas. We propose that the value of additional access areas varies with the timing and predictability of access, since unpredictable, uncontrollable routines have been shown to compromise welfare in numerous species (e.g. ). Patter exhibit  and 27.5 exhibit ), many oPrevious research has shown that rabbits sit and rear more in pens 75 cm high or with no ceilings, and the average height needed to fully ‘rear-up’ is 52.6 cm . PlatforLess than half of rabbits (42%) in this survey, and an even lower proportion of house-rabbits, lived with a conspecific. This figure is slightly higher than the recent PDSA  survey When grazing opportunities are limited, it is recommended that rabbit diets should consist primarily of high fibre forage, dried grass and hay. This is known to maintain intestinal physiology , to aid Whilst this survey suggests that pet rabbits on average live longer than was previously reported (5.6 compared to 4.2 years in ), morbidOwners reported behaviours which were categorised as potentially indicative of “positive” welfare in the vast majority of rabbits, but with varying frequencies, for example although binkying was reported in 88%, only a quarter of rabbits were seen to do this many times per day. Behaviours like binkying, rearing up and lying stretched out may be prevented in some living environments (e.g. due to inadequate space or height) and they may also be under-reported in specific environments where owners are less likely to watch their rabbits, e.g. in runs compared to indoors. Potentially negative behaviours such as thumping hind limbs, gnawing housing, grunting and digging on hard surfaces were also common, which could be cause for concern. The relationship between the occurrence of these behavioural indicators and the rabbits’ environment and care warrant further research.http://www.bio.miami.edu/hare/firstrabbit.html). Hence we would recommend that future surveys make this distinction between “picking up” and “handling”.Within this sample, although most owners reported “picking up and handling” their rabbit at least weekly, a small minority of rabbits (2.2%) were never picked up and handled. It is however plausible that some of these were handled and stroked whilst on the floor, which can be less stressful to the rabbit and is generally good practice  or other adults (75%). Combined with the facts that 27% of owners do not describe themselves as “very confident” when handling their rabbit, yet the majority of rabbits are handled at least weekly; this could potentially represent a significant source of stress and suggests that appropriate handling protocols are essential to ensure this is not aversive, and that fearful animals do not become further sensitised to handling. The value of early positive handling has been demonstrated for laboratory rabbits (e.g. ) but furOver half of the pet rabbits were reported to be fearful of loud noises, suggesting that celebrations, for example involving fireworks, could be a significant welfare concern for this species, especially as they are more likely to be housed outdoors than other domestic pets. Research into ways to best habituate rabbits to and treat developed fears of loud noises would be very valuable.Finally, it is worth noting that unlike in Mullan and Main , most raThis large-scale survey gives us up-to-date in-depth information on the ways in which pet rabbits are housed and cared for in England.Owner-reported data presented here suggest that whilst some rabbits have good health, compatible companions and are provided with enriched living areas, large numbers of rabbits experience potential stressors and welfare issues on a daily basis. The accuracy of the owners’ reports is yet to be tested, and it is likely that some of these issues are even more prevalent than this survey has suggested. Many rabbits are kept in ways which do not meet common recommendations (e.g. Wales ; NortherThis study has identified a large number of areas in which further research is required, for example prioritisation of the range of welfare issues highlighted by this survey, and to confirm postulated associations between aspects or housing and husbandry and rabbit welfare status. Future research also needs to focus on overcoming common issues, such as determining optimal protocols for habituating rabbits to human handling and to loud noises, which have been shown to be common causes of fear.


Epithelioid angiomyolipoma (E-AML), accounting for 8% of renal angiomyolipoma, is usually associated with tuberous sclerosis (TS) and demonstrates aggressive behavior. E-AML is macroscopically seen as a large infiltrative necrotic tumor with occasional extension into renal vein and/or inferior vena cava. However, without history of TS, renal sinus and venous invasion E-AML would be a challenging diagnosis, which may lead radiologists to misinterpret it as a renal cell carcinoma (RCC). In this case presentation, we aimed to report cross-sectional imaging findings of two cases diagnosed as E-AML and pathological correlation of these aforementioned masses mimicking RCC. Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer that covers more than 90% of all renal malignancies . AccordiExcept for RV and/or IVC invasion, renal sinus fat invasion as a prognostic significance can be also seen in a RCC . These tA 53-year-old woman with left flank pain that started 1 year before was admitted to urology department of our institution. Her physical examination was normal except for left-sided costovertebral angle tenderness. Complete blood count and blood biochemistry tests were in normal limits, but microscopic hematuria was detected in urinalysis. She initially underwent ultrasonography with the preliminary diagnosis of kidney stone. Upon detection of a solid mass at the mid-portion of left kidney, a CT examination was planned, but she underwent MRI because of iodinated contrast media allergy that she experienced before. On MRI, a 7 cm kidney mass with infiltrative characteristics, originating from central part of the left kidney with the involvement of the pelvicalyceal system, was revealed Figures . FollowiA 30-year-old woman with recurrent right-sided fullness that started 6 months ago was admitted to internal medicine department of our institution. Neither physical examination nor blood test was remarkable. She was referred to radiology department for ultrasonography. Right kidney enlargement and a solid mass with heterogeneous echogenicity were detected during ultrasonography. Upon detection of right-sided kidney mass, a biphasic CT scan was performed. CT revealed a kidney mass measuring 8.5 cm with involvement of RV and IVC at the central part of the right kidney . FollowiAngiomyolipoma (AML), constituting %1 of all surgically removed kidney masses, is the most common benign mesenchymal neoplasm of the kidney. AML is composed of variable degree of mature adipose tissue, smooth muscle, and blood vessels , 7. ThesOn the other hand, E-AML  which accounts for 8% of renal angiomyolipomas, is usually associated with tuberous sclerosis (TS) and demonstrates aggressive behavior . This exE-AMLs are regarded as potentially malignant lesions and as the tumor enlarges, the more likely it will spread . Nese etOn imaging, E-AML is seen as a large mass, which may contain hemorrhage and necrosis. E-AML is even larger than fat poor AML with a mean diameter of 7 cm. Small amount of fat can be seen on CT and/or MRI. Contrary to classic AML, E-AML is prevalently seen as a hyperattenuating lesion on unenhanced CT examination and it is presented as a hypointense lesion on T2-weighted images due to epithelioid muscle ingredient , 15. TsuThere are three major subtypes of RCC: clear cell (70%), papillary (10%), and chromophobe (5%). Clear cell RCCs are typically seen as hypervascular tumors avidly enhancing on contrast-enhanced studies including CT, MRI, and angiography. On the other hand, papillary RCCs appear as hypovascular and homogeneous on CT and MRI. Another interesting feature of papillary RCC is that it may rarely contain macroscopic fat . ChromopAlthough the detection of fat is a well-known imaging feature of classic AML, an important diagnostic issue is that some rare types of AMLs may contain few or no fatty tissue . Since malignancies can also demonstrate these imaging characteristics , informiSurgical resection is the main treatment option of E-AML. However, mammalian target of rapamycin (mTOR) inhibitors, such as temsirolimus or sirolimus, are reported to be medical management option for E-AML , 19.In conclusion, lack of macroscopic fat, renal sinus invasion, and RV-IVC extension and without given the history of TS, E-AML is a challenging diagnosis on cross-sectional imaging. If pathological and immunohistochemical analysis confirm this diagnosis, a full body scan is necessary because of the metastatic potential of this rare type of AML.


There is growing interest in understanding calcium dynamics in dendrites, both experimentally and computationally. Many processes influence these dynamics, but in dendrites there is a strong contribution of morphology because the peak calcium levels are strongly determined by the surface to volume ratio (SVR) of each branch, which is inversely related to branch diameter. In this study we explore the predicted variance of dendritic calcium concentrations due to local changes in dendrite diameter and how this is affected by the modeling approach used. We investigate this in a model of dendritic calcium spiking in different reconstructions of cerebellar Purkinje cells and in morphological analysis of neocortical and hippocampal pyramidal neurons. We report that many published models neglect diameter-dependent effects on calcium concentration and show how to implement this correctly in the NEURON simulator, both for phenomenological pool based models and for implementations using radial 1D diffusion. More detailed modeling requires simulation of 3D diffusion and we demonstrate that this does not dissipate the local concentration variance due to changes of dendritic diameter. In many cases 1D diffusion of models of calcium buffering give a good approximation provided an increased morphological resolution is implemented. Ca2+ entering through voltage-gated Ca2+ channels (VGCC) and ligand-gated channels gives rise to cytosolic Ca2+, which in turn controls Ca2+-activated K+ (KCa) channels during dendritic Ca2+ spikes . This is because Ca2+ influx scales with membrane surface while the change in Ca2+ concentration due to diffusion and buffering strongly depends on the volume. This results in larger amplitude transients expected in small diameter dendrites because they have a large SVR. Considering each dendritic segment as a cylinder, SVR is inversely proportional to the diameter of the cylinder. Therefore, even in the absence of intracellular Ca2+ mechanisms  and diffusion, changes in dendritic diameter across the dendrite will result in spatially variable Ca2+ levels. Moreover, because Ca2+ buffering and diffusion are also affected by geometry, dendrite diameters can also affect the decay time constants of Ca2+ transients . An additional Purkinje cell morphology (PC 2) used in this study was provided by Ede Rancz and Michael Häusser, UCL, London, UK. Considering the small sample size of available PC neurons (11 cells) and their large variability in dendritic diameters, we decided to obtain an additional morphology (PC 1) with carefully reconstructed diameters.Dendritic morphologies of 10 Purkinje cells (PC 3–12) used in this study were obtained from the NeuroMorpho database  was filled with intracellular solution containing potassium gluconate 140 mM, NaCl 10 mM, HEPES 10 mM, EGTA 0.2 mM, MgATP 4 mM, NaGTP 0.4 mM, Phosphocreatine 10 mM and 50 μM Alexa 594 (Invitrogen) with pH 7.3 and 300 mOsm.All procedures for the care of animals were according to the Science Council of Japan Guidelines for Proper Conduct of Animal Experiments, and also the guideline approved by OIST Graduate University Animal Resources Section. A 4-week old mouse was anesthetized with isofluorane and decapitated. The cerebellum was removed from the skull and immediately collected into a vial containing ice cold carbogenated ACSF: NaCl 125 mM, KCl 2.5 mM, NaHhttp://www.mbfbioscience.com/neurolucida). A different reconstruction of the same Purkinje cell has previously been used in Anwar et al.  with a Ti:sapphire laser , GaAsP photomultiplier tubes, and a 25x water  objective lens was used to acquire a 3D image stack of the Alexa-filled Purkinje cell with a z-step size of 0.25 μm and an xy field of view of 1024 × 1024 pixels. Next, the acquired 3D image stack was deconvoluted using AutoQuantX2 software (Media Cybernetics) using a theoretical point spread function (1–5 iterations) based on specifications from the image acquisition parameters. Later, the dendrite of Purkinje cell was reconstructed with Neurolucida, MBF Bioscience,     .The detailed model of spontaneous Ca2+ was modeled using the following Ca2+ buffering mechanisms.Intracellular Ca2+ pool was modeled asThe exponential decaying Ca2+]i is intracellular Ca2+ concentration, [Ca2+]0 is Ca2+ concentration at rest and is 45 nM, ICa(t) is total Ca2+ current per unit area through VGCC, F is the Faraday's constant, deq is the equivalent depth of a submembrane shell to define the volume for effective Ca2+ concentration, and β is the decay time constant. The values for depth (d) and β, 0.169 μm and 6.86 ms−1 respectively, were obtained from a past study , uses a mechanism widely used in multi-compartment modeling studies using NEURON . The second definition (SPnew) used in this study computed an equivalent depth (deq) for each submembrane shell, which gives the correct volume (see details in Results) when used in the mechanism described by (1):Two different definitions for dwhere diam is the diameter of each compartment.2+ dynamics model used in this study was obtained from our previous study  of the outer shell and the inner most shell is then:where Shells is the number of radial shells, diam is diameter of the compartment and d is depth of the outer radial shell, which was 0.1 μm. The discretization of the compartment volume into radial shells, where the depth of inner radial shells is twice the depth of outer radial shell, resulted in a varying depth of all shells, depending on the diameter of the compartment. The depth . Note that to model radial diffusion with a variable number of shells, a separate mechanism with a unique configuration of shells for every compartment with a different diameter needs to be created in NEURON.Here dhttp://cubit.sandia.gov).To allow 3D diffusion in the stochastic reaction-diffusion simulator STEPS  recorded during a Ca2+ spike was applied to each compartment in the NEURON simulations to evoke a constant shape of the spike. Two types of compartmentalization approaches were used in these simulations. Firstly, using a single compartment per dendritic section . Secondly, each of the dendritic section was split into multiple (1–22) sections , where each section consisted of adjacent traced points on the dendrite.A uniform current (in mA/cm2+ influx in STEPS was implemented using first-order surface based Ca2+ influx reaction (X 𠆒 X + Ca2+), where “X” channels  were distributed uniformly over the surface triangles  of the mesh. At each time point, the influx rate per channel was updated based on the Ca2+ influx profile . Due to high rate and uniformity of influx, effects of stochasticity were negligible. The results of STEPS simulations in this study are reported as the mean computed over 10 trials.Ca2+ dynamics in models of different complexity. Figure 2+ concentrations  in three different models of a spontaneous burst of Ca2+ spikes  also uses a simple pool but implemented differently; it results in strong Ca2+ gradients with higher concentrations in thin dendritic branches as expected from the SVR. Finally we simulated a detailed Ca2+ dynamics models with buffers and radial 1D diffusion ; Figure new) is defined as:Many multi-compartment dendritic models use a single pool model of Caf denotes the volume of a full compartment, Volc denotes the volume of the core, d is the depth of submembrane shell, diam is the diameter of compartment and L is its length. Using such a representation of submembrane shell, SVRnew equalswhere Volnew is less dependent on diameter than the SVR for the complete volume (1/diam), but, as shown in Figure new still increases for smaller diameters.where SA is the surface area of the compartment. Note that SVR2+ shell models implemented in NEURON use an incorrect volume for the submembrane shell , the volume of a submembrane shell is defined as:However, we noticed that most of the Cal Figure . In thosoldUsing such a representation of submembrane shell, gives SVRold is constant and independent of the dendrite diameter. As a result, we observe changes in peak amplitudes of the simulated Ca2+ transients using SPnew in compartments with different diameters  of submembrane shells is usually taken constant for variable diameter compartments, SVRs Figure  while th2+]. In Figure old (compared to SPnew) depends on the values of d and β used. The errors in peak Ca2+ were computed using β-values of 0.02 ms−1  and may reach up to 80% for 0.1 μm diameter compartments  and decay time constants (β) can be tuned to approximate the desired behavior of intracellular .If one wants to vary the number of shells modeled depending on compartment diameter, which is the correct solution, a separate NEURON mechanism has to be created for each specific number of shells that is required. Some authors have therefore decided to use a fixed number of shells with variable depth of each shell  may vary between d − 0.25 d and d + 0.25 d. In Figure 2+ influx conversion to Ca2+ concentration using the variable depth scheme (range of d1 due to discretization: 0.075–0.125 μm). The larger predicted errors in these cases are associated with small diameters, where small changes of diam will result in bigger changes in d1 Equation (4) and submembrane shell volume.The next question is then how to compute shell depth as the depth of at least one shell has to vary to fit the total exactly to a variable compartment diameter. As already mentioned, in the standard NEURON implementation  and Figure FD show very similar peak amplitudes and decay time constants for different diameter compartments   are limited to combinations where a compartment with an extremely small diameter (≤0.3 μm) is connected to one with large diameters. For DM and DMFD, this region expands to all compartments with diameter less than or equal to 1 μm that are connected to ones with larger diameters. Therefore, the detailed Ca2+ dynamics models are more sensitive to changes in dendritic diameter compared to pool based models, which explains the differences observed in Figure FD, due to the use of variable depth submembrane shells in DM, overall these maps are quite similar to each other.How do the different Ca) Figure . As explmeter ≤0. μm is co2+ diffusion requires a variable number of concentric shells that scales with compartment diameter, but that the Ca2+ dynamics are less sensitive to the actual scheme used to compute the depth of these shells.We conclude from Figures new) or only radial 1D diffusion (DM and DMFD). In this case, would the predicted large Ca2+ transients disappear in the presence of 3D diffusion? Also, what happens if dendritic diameter varies more smoothly than possible in a model using electrical compartmentalization? To address both issues, we used the STEPS simulator , dendrites were thought to be passive structures, whose main function was to transfer and sum information from presynaptic to postsynaptic neurons  decay time constants . Another important determinant of Ca2+ dynamics is inhomogeneous distribution of Ca2+ buffers in dendrites of a given neuron, as well as their properties, causing competitive binding of Ca2+ to available Ca2+ buffers and Ca2+ pumps . Also, this will require more detailed description of Ca2+ related mechanisms .Although morphological reconstructions obtained using electron microscopy (EM) capture dendrites much more precisely, because of rapid fluctuations in dendritic surface those reconstructions are not suitable for compartment based models. The proper use of EM reconstructions in modeling Ca2+ in the NEURON simulator Equations (8) and (9). The conversion of influx to intracellular Ca2+ concentration in these models is incorrect, which is due to the use of an inaccurate volume of the submembrane shell  and longitudinal (from one compartment to neighboring compartment) diffusion. Radial diffusion requires virtual submembrane shells  that ensures a fixed depth of the submembrane shell. It should be noted, however, that these differences may be larger in models of other neurons because the buffering capacity of PCs is exceptionally high .More detailed Cas Figure  unless as Figure  with a mNeither of the issues just mentioned are relevant for the GENESIS simulator. Both submembrane pools  will be an adequate approximation. It is then important to implement a variable number of submembrane shells, with larger number of shells in larger diameter compartments, and best using a fixed depth of the submembrane shell [DM2+ buffering (e.g., Schmidt et al., new, Figure Finally, we do not recommend the use of simple pool models, unless good data on the properties of CaThe 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 Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate [Ca2+]i homeostasis. Calmodulin (CaM) senses [Ca2+]i changes and relays Ca2+ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM1234 (a Ca2+-binding deficient mutant) in HEK293T cells and measured the increase in [Ca2+]i contributed by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM1234. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.Changes in intracellular Ca At the plasma membrane, Ca2+-pumps and Na+ gradient-dependent Ca2+ transporters are the two main pathways for exporting Ca2+ out of cells when [Ca2+]i is elevated. In addition, the direction of the Na+ gradient-dependent Ca2+ transporters can be reversed to transport Ca2+ into the cytosol according to the electrochemical gradients of coupled ions i by the previously reported protocol i increases were significantly reduced to 694 ± 77  and 462 ± 67 (n = 32 p < 0.001) nM in cells expressing NCX1.1ΔCaMS and NCX1.3ΔCaMS, respectively  and 1049 ± 108 (n = 37) nM, respectively. However, CaM1234, but not CaM, significantly enhanced the exchange activity of NCX1.1ΔCaMS to 899 ± 90  nM.To characterize the importance of CaM in regulating exchange activity, we co-expressed the NCX1 splice variants with wild-type CaM or the Ca2+]i response , but CaM1234 significantly attenuated the response to 701 ± 75  nM. NCX1.3ΔCaMS had a lower exchange activity than wild type . However, overexpression of either CaM or CaM1234 induced a significant decrease in exchange activity to 281 ± 59  or 201 ± 32 nM , respectively. These results indicate that the CaMS motif is required for both NCX1.1 and NCX1.3 to maintain exchange activity and that CaM has differential effects on NCX1.1 and NCX1.3.For NCX1.3, CaM overexpression did not change the [Ca2+-chelating ability of the CBD2 motif [2+]i; however, NCX1D had a smaller [Ca2+]i elevation than that of NCX1.4. The average [Ca2+]i change in cells expressing NCX1.4 was 850 ± 87 nM (n = 15); CaM and CaM1234 co-expression did not affect [Ca2+]i, with [Ca2+]i reaching 790 ± 119 (n = 12) and 836 ± 81 (n = 43) nM, respectively. In cells expressing NCX1D, the average [Ca2+]i elevation was 387 ± 61 nM , which was significantly less than that in cells expressing NCX1.4. CaM co-expression did not affect the exchange activity of NCX1D , but CaM1234 overexpression enhanced the [Ca2+]i changes to 707 ± 101 nM . These results suggest that exons A and B support exchange activity and are involved in the CaM-mediated modulation of NCX1 activity.The difference between NCX1.1 and 1.3 are the mutually exclusive exons A and B, which are important for the CaD2 motif ,14. To cF1A and NCX1.1V5A had lower [Ca2+]i elevation levels compared with the other two mutants. The averages show that mutants NCX1.1F1A and NCX1.1V5A had a significantly reduced change in [Ca2+]i to 559 ± 74  and 566 ± 90  nM, respectively, compared with NCX1.1  and 428 ± 48 (n = 37) nM, respectively, or NCX1.1V5A, with [Ca2+]i levels of 503 ± 73 (n = 43) and 539 ± 49 nM (n = 44), respectively.To further characterize the importance of the CaMS in CaM-mediated regulation, we mutated the 4 conserved a.a. residues  in the C2+]i changes in cells expressing NCX1.1L8D and NCX1.1L14D were 973 ± 123 (n = 33) and 1178 ± 108 (n = 39) nM, respectively, which are comparable to the changes in cells expressing NCX1.1. CaM co-expression significantly reduced the [Ca2+]i changes in cells expressing NCX1.1L8D and NCX1.1L14D to 641 ± 90  and 523 ± 101  nM, respectively. CaM1234 co-expression did not affect the [Ca2+]i changes in cells expressing NCX1.1L8D and NCX1.1L14D (820 ± 182 (n = 26) and 984 ± 85 (n = 61) nM). These data reveal that the first two conserved a.a. in NCX1.1 are important to support exchange activity; the 3rd and 4th a.a. mutations make the exchanger sensitive to CaM overexpression.The [CaV5A and NCX1.3L8D had lower [Ca2+]i elevations than those of NCX1.3F1A and NCX1.3L14D. The average [Ca2+]i changes in cells expressing NCX1.3F1A and NCX1.3L14D were 1160 ± 158 (n = 16) and 829 ± 90 (n = 49) nM, respectively, similar to that of NCX1.3  and 587 ± 59  nM, respectively, compared with the wild type  by CaM and significantly increased to 1013 ± 142 nM  by CaM1234. These results demonstrate that these conserved a.a. residues in the CaMS have differential effects on CaM-mediated regulation.The 4 conserved a.a. residues of the CaMS in NCX1.3 were individually mutated as F1A, V5A, L8D, and L14D to characterize the roles of these a.a. residues in modulating NCX1.3 activity . Immunosild type . CaM and2+-sensing protein that regulates a variety of physiological activities by interacting with different proteins i levels i levels requires further investigation.The Caectively ; for CaMectively . In addi complex ,36. Therrd and 4th conserved a.a. residues did not affect exchange activity, these mutations may weaken the loop complex, and the binding of overexpressed CaM, but not CaM1234, destabilizes the loop complex to reduce exchange activity. In summary, the first 2 conserved a.a. residues of CaMS in NCX1.1 may play a dominant role in stabilizing the structure to maintain exchange activity; the 3rd and 4th conserved a.a. residues may be involved in the access of CaM to this loop complex.Our results reveal that each conserved a.a. in the CaMS contributes differentially to the exchange activities of NCX1.1 and NCX1.3. For NCX1.1, mutating the first 2 conserved a.a. residues in the CaMS of NCX1.1 may directly destabilize the loop complex and down-regulate exchange activity. Although mutations in the 3st and 4th conserved a.a. residues in the CaMS of NCX1.3 does not affect exchange activity, suggesting that these mutations may not affect CaM binding. In contrast, the 2nd and 3rd conserved a.a. residues are the main residues involved in determining structural stability and CaM binding; therefore, mutations in these two residues decreased exchange activity. Since CaM1234 does not bind to the cytosolic loop, it is not clear how CaM1234 overexpression rescues the exchange activity of NCX1.3L8D. It is possible that CaM1234 transiently interacts with the loop complex and interferes with the interaction between endogenous CaM and the loop complex. How the CaMS interacts with other domains to modulate CaM binding will require further studies on the structure.For the NCX1.3, which contains exon B, CaM binding may be required to stabilize the loop complex and support exchange activity. Mutating the 12+ is highly cooperative, related to the binding of Ca2+ to the CBD motifs, but exhibits slow activation kinetics, with a 20–50 msec lag-phase to reach maximal exchange activity i levels during excitation may help tune NCX1 activity. The lag in reaching maximal exchange activity may be due to the time required to form a stable loop complex among these Ca2+-binding motifs. Therefore, CaM is involved in regulating not only structural stability but also temporal control. In addition, NCX isoforms are extremely sensitive to mild cytosolic acidification; a pH decrease from 7.2 to 6.9 results in nearly 90% inactivation of NCX, called "proton block" i, exon A, but not B, releases the Na+-dependent inactivation of exchange activity i and support various physiological functions [2+]i levels to regulate Ca2+ homeostasis. However, it is not clear whether CaM plays the same role in modulating forward exchange activity, and this question needs to be studied further.The activation of ionotropic receptors in excitable cells allows the influx of Naunctions ,42. Ther2+ homeostasis [2+]i changes [2+]i elevations will regulate NCX activity accordingly to modulate Ca2+ homeostasis. NCX isoforms are involved in heart disease and brain damage, and this interaction may provide new insights for the selective pharmacological targeting of NCX isoforms.NCX isoforms expressed in different tissues may contribute differentially in modulating Caeostasis ,43,44. I changes , our resS1 FigHEK293T cells expressing NCX1.1CL or NCX1.3CL were fixed and permeabilized by triton-X100; then the antibody against V5 epitope tagged at the C-terminals of these loop proteins was applied to visualize the expressed proteins. Scale bar: 10 μm.(TIF)Click here for additional data file.S2 FigThe NCX1 splice variants NCX1.1 (A) and NCX1.3 (B) with or without CaMS, NCX1.1ΔCaMS (C) and NCX1.3ΔCaMS (D), were coexpressed with GFP-GPI (Green) in HEK293T cells. After permeabilization, the NCX variants and mutants were immunostained (Red), and images were taken via confocal microscopy. The arrows indicate the region used to plot the line intensity profile. Scale bar: 10 μm.(TIF)Click here for additional data file.S3 Figst, 5th, 8th, and 14th a.a. residues at the CaMS of the A. NCX1.1 and B. NCX1.3 were independently mutated from F, V, L, and L, to A (F1A), A (V5A), D (L8D), and D (L14D), respectively. The images (upper row) shows the merged fluorescence of cells expressing various mutants stained with phalloidin (Red), DAPI (Blue), and antibody against V5 epitope (Green). The lower rows plotted the intensity profiles indicate the red and green fluorescence intensity along the arrows in the corresponding merged images. Scale bar: 10 μm.The conserved 1(TIF)Click here for additional data file.


DMPK gene. Expanded CTG repeats are transcribed into RNA and make an aggregate with a splicing regulator, MBNL1, in the nucleus, which is called the nuclear foci. The nuclear foci sequestrates and downregulates availability of MBNL1. Symptomatic treatments are available for DM1, but no rational therapy is available. In this study, we found that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone (PBZ), upregulated the expression of MBNL1 in C2C12 myoblasts as well as in the HSALR mouse model for DM1. In the DM1 mice model, PBZ ameliorated aberrant splicing of Clcn1, Nfix, and Rpn2. PBZ increased expression of skeletal muscle chloride channel, decreased abnormal central nuclei of muscle fibers, and improved wheel-running activity in HSALR mice. We found that the effect of PBZ was conferred by two distinct mechanisms. First, PBZ suppressed methylation of an enhancer region in Mbnl1 intron 1, and enhanced transcription of Mbnl1 mRNA. Second, PBZ attenuated binding of MBNL1 to abnormally expanded CUG repeats in cellulo and in vitro. Our studies suggest that PBZ is a potent therapeutic agent for DM1 that upregulates availability of MBNL1.Myotonic dystrophy type 1 (DM1) is caused by abnormal expansion of CTG repeats in the 3′ untranslated region of the  DMPK) on chromosome 19. Unaffected individuals have 5–30 copies of CTG repeats, whereas DM1 patients carry 50 to several thousand copies2469Myotonic dystrophy type 1 or dystrophia myotonica type 1 (DM1), is the most common form of adult-onset muscular dystrophy. The disease is dominantly inherited and affects multiple tissues, exhibiting symptoms including muscle hyperexcitability (myotonia), progressive muscle wasting, cardiac conduction defects, cardiomyopathy, insulin resistance, and neuropsychiatric disordersHSALR mice model for DM1, that PBZ reduces the number of central nuclei, partially normalizes aberrant splicing of Clcn1 encoding the skeletal muscle chloride channel, and increases expression of the chloride channel. PBZ enhances transcription of Mbnl1 mRNA, through demethylation of an enhancer region in Mbnl1 intron 1. In addition, PBZ suppresses binding of MBNL1 to CUG RNA in cellulo as well as in vitro.Using the drug repositioning strategy, in which a drug used for a specific disease is applied to another unrelated disease, we found that phenylbutazone (PBZ) upregulates the expression of MBNL1 in C2C12 mouse myoblasts. We identified, in the skeletal muscle of Mbnl1 mRNA 1.3-fold. We observed that PBZ upregulated Mbnl1 mRNA in C2C12 cells in a dose-dependent manner up to 1.9-fold  muscles , and found that PBZ significantly reduced the number of muscle fibers with central nuclei . These results suggest that PBZ inhibits the interaction between MBNL1 and CUG RNA foci, and ameliorates sequestration of MBNL1 to the RNA foci.In DM1, MBNL1 colocalizes with CUG RNA foci. The sequestration of MBNL1 to CUG RNA foci leads to a loss-of-function of the proteinALR mice . We founCHRNA1 intron 3 that we reported previouslyTo analyze whether PBZ directly inhibits MBNL1-CUG RNA interaction, we performed an electrophoretic mobility shift assay (EMSA) experiment using the recombinant MBNL1 protein and an RNA probe carrying ten copies of CUG repeats. As a control, we examined the interaction between polypyrimidine tract binding protein 1 (PTBP1) and an RNA probe carrying the polypyrimidine tract of HSALR mice muscle. Since DNA methylation is deeply involved in the regulation of gene expression during myogenic differentiation25Mbnl1 locus. We first analyzed the effect of the 5-aza(deoxy)cytidine (5-AC), a compound known to inhibit DNA methylation, on Mbnl1 expression. RT-PCR analysis showed that 5-AC treatment upregulated expression of Mbnl1 mRNA in C2C12 cells  analysis of C2C12 cells (GSE22077)Mbnl1 locus in C2C12 cells: MeR1, MeR2, and MeR3 are located upstream of exon1, in intron 1, and in exon 2, respectively  with an antibody against RNA polymerase II (RNAP II) to see the effect of PBZ on accumulation of RNAP II in this region  at exons 1 and 2 with two distinct promoters located upstream of exons 1 and 2, respectivelyMbnl1 in mouse has three transcriptional start sites (TSSs) at exons 1, 2, and 1′  was added. RNA was extracted at 24 h after treatment. Gene expression of Mbnl1 was estimated by real-time RT-PCR in the LightCycler 480 (Roche Applied Science) using the SYBR Premix ExTaq (Takara Bio).In order to screen for a drug that upregulates expression of MBNL1, C2C12 mouse myoblasts in a 12-well dish were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) containing 50 μg/mL gentamycin, and incubated at 37 °C with 5% COMbnl1 was estimated as stated above.To examine the dose-responsive upregulation of MBNL1 by PBZ  in differentiated C2C12 myotubes, C2C12 myoblasts were grown to 90% confluency in DMEM with 10% FBS, and then changed to differentiation medium containing 2% horse serum with variable concentrations of PBZ in DMEM (day 0). PBZ was dissolved in dimethyl sulfoxide (DMSO) to make a 972 mM solution, and then added to the culture medium at final concentrations of 50 to 972 μM. We also added DMSO to the medium to make the final concentration of DMSO in the medium 0.1% in all the experiments including controls without PBZ. Thus, the effect of PBZ in cultured cells was analyzed in the presence of 0.1% DMSO and was always compared to control cells cultured in 0.1% DMSO. Cells were serially harvested on days 1, 3, and 5, and total RNA was extracted with the TRIzol reagent (Thermo Fisher Scientific). Gene expression of HSALR transgenic mice that express the human α-skeletal actin mRNA containing (CUG)250 in the 3′ UTR were kindly provided by Dr. Charles A. Thornton at University of Rochestern = 5 in each group): (i) untreated HSALR mice, (ii) PBZ-treated HSALR mice, and (iii) untreated wild-type FVB/N mice. In n = 3 in each group): (i) untreated wild-type FVB/N mice and (ii) PBZ-treated wild-type FVB/N mice. At eight weeks after birth, HSALR mice started to take 16.7 mg/kg/day of PBZ, which is about three times more than that taken by patients as an NSAID (300 mg/day). Body weights were measured every week and the calculated amount of PBZ was directly dissolved in drinking water assuming that each mouse drank 3–5 mL of water every day. Mice were sacrificed twelve weeks after initiation of treatment.All mouse studies were approved by the Animal Care and Use Committee of the Nagoya University Graduate School of Medicine, and were performed in accordance with the relevant guidelines. Each mouse was kept in an individual cage with a counter-equipped running wheel  for 48 h to record the number of wheel rotations. The counter sensed rotations in both directions. Each mouse was moved to the running wheel cage every 14 days. We measured voluntary activities from 4 to 8 weeks of age without treatment, and from 8 to 20 weeks of age with PBZ treatment.In Vivo Research Instruments), according to the manufacturer’s protocol. Mice were allowed to grasp a wire mesh with their forelimbs and hindlimbs, and then pulled steadily by their tails horizontally until they lost their grip. Measurements were performed 3 times using untreated wild-type FVB/N mice, untreated HSALR mice, and PBZ-treated HSALR miceThe peak grip force was measured using a grip strength meter (Bioseb - Quadriceps muscles of three group mouse were snap-frozen in isopentane chilled with liquid nitrogen. Quadriceps muscles were sliced at 10 μm with a cryostat. Hematoxylin and eosin (H&E) staining was done according to the standard procedures.Frozen sections (10 μm) of quadriceps muscle were permeabilized with acetone on ice for 10 min and dried. Then, the sections were blocked in 5% horse serum for 1 h at 4 °C, rinsed with phosphate-buffered saline (PBS) for 5 min three times. The sections were immunostained with an antibody against chloride channel-1  and then with anti-rat FITC . Nuclei were visualized by staining with diamidino-2-phenylindole .http://imagej.nih.gov/ij/). We then estimated the ratio of exon inclusion by dividing the signal intensity of the upper band by the sum of signal intensities of two bands.Total RNA was extracted from mouse TA muscles and quadriceps muscles at 12 weeks after initiation of PBZ treatment using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using a random primer (Thermo Fisher Scientific) and ReverTraAce (Toyobo), and PCR amplifications were performed using GoTaq (Promega) for 30–35 cycles. The primer sequences used for RT-PCR are shown in Gapdh and Actb (β-actin) mRNAs were used as the housekeeping genes. We then normalized the expression level of a specific gene by that of the housekeeping gene. PCR primers are shown in Real-time RT-PCR was performed with the LightCycler 480 (Roche Life Science) using the SYBR Premix ExTaq (Takara Bio). In situ hybridization was performed essentially as described elsewhere6The number of nuclear foci was counted in six randomly selected visual fields of a section of quadriceps muscle of six mice. Association of MBNL1 with nuclear foci was automatically analyzed with the IN Cell Analyzer 6000 Cell Imaging system .DMPK; polypyrimidine tract RNA probe, 5′-UUUCUCCUUUUCUGUGGGUGGACAGGGUGACAUGGUA-3′ arising from CHRNA12. The signals of gel-shifted RNA probe were detected by the streptavidin-horseradish peroxidase conjugate  using the Image Quant LAS 4000 mini .The RNA probes with biotin labeling at the 5′-end were obtained from Hokkaido System Science. The sequences of RNA probes used in this study were: CUG repeat RNA probe, 5′-GCCUGCUGCUGCUGCUGCUGCUGCUGCUGCUGGC-3′ simulating abnormally expanding 3′ UTR of C2C12 cells were treated with 972 μM (300 ng/μl) PBZ on differentiation day 0, and incubated for 72 h. Then, ChIP assays were performed as described previouslyt-test, one-way ANOVA followed by Tukey’s post-hoc test, or two-way repeated measures ANOVA followed by Bonferroni’s post-hoc test.All data are presented as the mean and SD. Statistical difference was estimated either by Student’s How to cite this article: Chen, G. et al. Phenylbutazone induces expression of MBNL1 and suppresses formation of MBNL1-CUG RNA foci in a mouse model of myotonic dystrophy. Sci. Rep. 6, 25317; doi: 10.1038/srep25317 (2016).