N = 34), in which multiple Go options enabled us to disentangle selective action learning from nonselective Pavlovian responses. Midfrontal theta-band (4 Hz–8 Hz) activity covaried with the level of Pavlovian conflict and was associated with reduced Pavlovian biases rather than reduced instrumental learning biases. Motor and lateral prefrontal regions synchronized to the midfrontal cortex, and these network dynamics predicted the reduction of Pavlovian biases over and above local, midfrontal theta activity. This work links midfrontal processing to detecting Pavlovian conflict and highlights the importance of network processing in reducing the impact of maladaptive, Pavlovian biases.Motivation exerts control over behavior by eliciting Pavlovian responses, which can either match or conflict with instrumental action. We can overcome maladaptive motivational influences putatively through frontal cognitive control. However, the neurocomputational mechanisms subserving this control are unclear; does control entail up-regulating instrumental systems, down-regulating Pavlovian systems, or both? We combined electroencephalography (EEG) recordings with a motivational Go/NoGo learning task ( The anticipation of reward and punishment are key drivers of behavior: we tend to take action for rewards while holding back in the face of punishment. This motivational bias might have an overall evolutionary advantage but can also work against us in specific situations. Here, we first asked whether this motivational bias relies on innate, automatic action tendencies or whether this bias might actually itself be learned. Secondly, we studied which brain processes reduce the impact of these motivational drives when they become dysfunctional. By comparing the actions of human participants to the predictions of several mathematical models, we showed that the motivational bias in action relies partly on automatic tendencies and partly on asymmetric learning from experience. We then observed that activity over the midfrontal cortex specifically increased as a function of how dysfunctional the automatic tendencies were. Additionally, this midfrontal cortex activity was functionally connected to the motor and lateral frontal cortices, which play a role in activating and inhibiting behavior. By incorporating this connectivity into the mathematical models, we showed that stronger midfrontal connectivity predicted reduced impact of dysfunctional automatic tendencies on behavior. We propose that the midfrontal cortex detects dysfunctional action tendencies and implements cognitive control by signaling across the network. Potential rewards and losses are key drivers of our actions, with consequences ranging from highly desirable  to unwanted  and even inconceivable . The valence of these outcomes is particularly well known to bias our actions: whereas anticipated rewards promote taking action, anticipated losses promote holding back from taking action –3. TheseThe well-established motivational biases in action ,8–13 ariThe motivational biasing of action is often adaptive but becomes maladaptive when these biases are incongruent with the environmental requirements. The midfrontal cortex is the key region that has been linked to reducing motivational biases when these biases become maladaptive ,6, whichHere, we propose that the midfrontal cortex is specifically implicated in reducing motivational biases by detecting and signaling conflict between the Pavlovian and instrumental systems, relying on similar neural mechanisms as evident in classic response conflict. Classically, the midfrontal cortex has been linked to detection of response conflict –26, signIn the current study, we employ a motivational Go/NoGo learning task with multiple Go response options  and concIn the motivational Go/NoGo learning task, 34 healthy subjects needed to learn the correct responses (Go left/Go right/NoGo) by trial and error in order to gain rewards (“win” cues) and avoid losses (“avoid” cues); see χ2(1) = 135.7, p < 0.001; χ2(1]) = 16.3, p < 0.001), independent of the Go/NoGo requirements (χ2(1) = 0.6, p = 0.427). This increase in Go responses was, by definition, incorrect for the NoGo cues and driven by both correct and incorrect Go responses for the Go cues (correct Go: χ2(1) = 10.8, p = 0.001; incorrect Go: χ21) = 5.1, p = 0.024). Moreover, the accuracy of Go responses did not significantly differ for Go-to-Win and Go-to-Avoid cues (χ2(1) = 1.6, p = 0.200). Thus, we observed a clear effect of motivational valence on Go responding, which was driven by both correct and incorrect Go responses.Over trials, subjects increased Go responding to the cues requiring Go versus NoGo responses  and feedback sensitivity parameter . Stepwise addition of the Go bias parameter , modeling a nonselective tendency towards Go responses, improved model fit . Addition of the Pavlovian response bias parameter  and instrumental learning bias parameter  further improved WAIC, indicating that Go versus NoGo responding was differentially influenced by cue and outcome valences beyond that explained by simple motor biases or instrumental learning alone. The model that best explained the task performance included both the Pavlovian response bias and instrumental learning bias parameter  is estimated based on computed action weights (w) using a softmax function . The action weights are determined by the learned instrumental Q values (b) and Pavlovian response bias (π) from, respectively, M2 and M3a onwards. Static Pavlovian values (V) were modeled once the first reward/punishment outcome had been experienced . Thus, subjects were more likely to repeat rewarded Go responses relative to rewarded NoGo responses. Conversely, punishment hampered the unlearning of NoGo responses . In other words, subjects were more likely to repeat punished NoGo responses relative to repeating punished Go responses. As reported previously, the Pavlovian bias parameter decreased when including the instrumental learning bias parameter , suggesting that variance corresponding to the instrumental learning bias would otherwise be attributed to the Pavlovian bias parameter. The impact of the Pavlovian response bias and instrumental learning bias on behavior has distinct dynamics, however [In the winning model, M3c, the Pavlovian bias estimates were positive at the group level (95.5% group-level samples > 0), indicating that “win” cues globally promoted Go responding while “avoid” cues globally suppressed Go responding. The instrumental learning bias estimates were positive as well (100% group-level samples > 0), indicating that reward enhanced the selective learning of Go responses  and outcome-driven  motivational biases, we set out to replicate the finding that midfrontal oscillatory activity in the theta frequency range (4 Hz–8 Hz) relates to reducing the motivational biasing of action . Moreovep = 0.024; −826 ms to −150ms; p = 0.002; We reasoned that if midfrontal oscillatory theta activity relates to modulating the Pavlovian response bias , midfront29 = 2.9, p = 0.007; see Recent theories suggest that the midfrontal cortex might be responsible for detection of conflict –26 and sTo assess whether the conflict-related midfrontal theta signal was indeed associated with reducing the motivational biasing of action, we extended our computational modeling approach. We employed the models developed by Cavanagh and colleagues  to test t) on the Pavlovian bias , as model evidence can only be directly compared when estimated over identical data. When allowing midfrontal theta power to scale the impact of the Pavlovian bias, model evidence greatly increased , relative to the refitted base model M3c  or the balance between the Pavlovian and instrumental contribution , although both models did better than base model M3c. Although the improvements in the R2 values might appear small, these improvements reflect the improvement for every subject on every trial. Additionally, the impact of the additional parameters in the EEG models is restricted because i) EEG data was rejected for trials with EEG artifacts and noise  and ii) the EEG parameters are only modeled in half the trials . Model evidence hardly increased relative to M3c when midfrontal theta power was allowed to modulate the instrumental learning bias . Thus, computational modeling indeed implicated the conflict-related midfrontal theta signals in modulation of the motivational biasing of action, for which the theta signals particularly scaled the Pavlovian response bias (M4a) rather than the instrumental learning bias (M4d).For model comparison, we refitted the winning base model M3c over the behavioral data of the subjects included in the EEG analyses . These negative estimates indicate that midfrontal theta power relates to weaker impact of the Pavlovian response tendencies such that higher levels of theta power are associated with better ability to resolve the Pavlovian conflict. Crucially, in model M4a, midfrontal theta power reduces the Pavlovian response tendencies only under conflict between the Pavlovian response tendencies and the instrumental action values. Modeling an effect of midfrontal theta power on all trials  greatly reduces model fit relative to M4a (ΔWAIC = +266), consistent with previous reports showing that midfrontal theta power relates to the decision threshold specifically on conflict trials [In the winning model, M4a, the impact of midfrontal theta power on the Pavlovian bias, as assessed by βt trials –31. Altop = 0.002; 0 + κ, punished NoGo: ε0− κ) relative to their unbiased counterparts . Alternatively, feedback-related midfrontal theta power might reflect the amount of instrumental learning that takes place. However, midfrontal theta power did not significantly differ from the conditions for which we observed relatively stronger  versus weaker  instrumental learning as observed at the behavioral level . Altogether, we observed well-established feedback-related modulations of midfrontal theta power [Although the cue-related midfrontal theta power was not linked to a modulation of the instrumental learning bias (M4d), theta power at the time of outcome, when reinforcement learning would occur, could in principle modulate the learning bias nonetheless. To this end, we next assessed whether feedback-related midfrontal theta power increased differentially following actions and outcomes that are consistent with instrumental learning biases. First, we determined the optimal time window for feedback-related theta power by contrasting nonpreferred outcomes  and preferred outcomes . Time-wise permutation testing using the a priori midfrontal channels revealed one time window during which midfrontal theta power was significantly higher for nonpreferred than preferred outcomes  or NoGo cues alone . The midfrontal–prefrontal phase synchrony did not show significant main effects of valence  or required action . Mirroring the midfrontal–prefrontal phase synchrony, phase synchrony between the midfrontal and nonexecuting motor sites also increased during incongruent cues  and required action . The motor synchrony significantly increased with motivational conflict during NoGo trials  but nonsignificantly during the Go trials . Taken together, we observed clear conflict-related changes in midfrontal theta phase synchrony with both the lateral prefrontal sites and the nonexecuting motor sites.Motivational congruency indeed modulated phase synchrony between the lateral prefrontal sites and the midfrontal cluster , which was, if anything, in the opposite direction . The absence of significant congruency effects for the Go responses seems to be in line with the cue-locked midfrontal theta power observations, in which the congruency effect also appears stronger for the NoGo than the Go cues , in which midfrontal–lateral prefrontal  and midfrontal–motor phase synchrony  modulated the Pavlovian bias. Although the trial-by-trial estimates of midfrontal theta power and ISPS correlated positively . In other words, while theta ISPS explained behavior over and above midfrontal theta power , midfrontal theta power barely explained any variance in addition to the phase synchrony. Thus, choices on incongruent trials were better explained when we allowed modulation of the Pavlovian response bias by intersite theta phase synchrony between the midfrontal and both task-relevant sites . Alternatively, target-dependent effects of ISPS, such as lateral prefrontal synchrony modulating the goal representations and motor synchrony modulating motor excitability, reduced model evidence  and motor (M5b) sites could also help to explain trial-by-trial choice variability related to reducing the Pavlovian response tendencies under motivational conflict. To this end, we adopted a similar modeling approach as above but using trial-by-trial synchrony estimates instead of power to scale the Pavlovian bias. Before doing so, we confirmed that the single-trial estimates replicate the trial-averaged results reported above . We thenevidence . See FigPFC; M5a) and midfrontal–motor  phase synchrony on the Pavlovian bias, have negative group-level estimates (both: 100% samples < 0). These negative estimates indicate that midfrontal synchrony with these task-relevant areas is related to weaker impact of the Pavlovian response tendencies, such that stronger connectivity is associated with better ability to resolve the Pavlovian conflict  in order to obtain rewards or avoid punishments. We replicated our previous finding that reward cues promoted actions nonselectively (Pavlovian response bias) while reward outcomes disproportionally facilitated credit assignment to selective actions  . ConversThe midfrontal cortex has long been linked to performance monitoring and the detection of conflict, realizing the need for control ,36–38 anThe classical performance monitoring theory proposed that the need for control is signaled to the dorsolateral PFC, which will then implement the control . More reIt has been proposed that the conflict-related intersite phase synchronization would have differential computational effects depending on the function of the target circuit; the lateral PFC might enhance the goal representations, whereas the motor cortex might increase the motor threshold . Here, wFinally, we have demonstrated increased midfrontal theta power for motivationally incongruent cues and linked trial-by-trial midfrontal theta power to the level of Pavlovian–instrumental conflict estimated from behavior. However, cognitive factors other than conflict might also play a role during motivational incongruency. Midfrontal theta power has been linked to the more general notion of “cognitive control” , includiTo summarize, in this study we replicate previous findings that i) the well-established motivational biasing of action arises partly from cue-based Pavlovian response biasing and partly from outcome-based instrumental learning biases  and thatAll procedures were approved by the local ethics committee (CMO/METC Arnhem Nijmegen: CMO2014/288) and performed in accordance with the Helsinki Declaration of 1975. Subjects signed informed consent prior to participation.Thirty-four healthy adults participated in the study . Exclusion criteria comprised a history of neurological or psychiatric disorders, use of psychotropic drugs, pregnancy, claustrophobia, and color blindness. EEG data of four subjects were excluded because of excessive muscle artifacts in the EEG signal (see EEG data acquisition and preprocessing). Subjects received a financial compensation or study credits upon completion of the experiment.We employed a motivational Go/NoGo learning task with multiple active response options  to dissoTrials start with presentation of a gem-shaped cue  followed by a fixation cross (700 ms) and feedback . Four cues are followed by reward or neutral outcomes (“win” cues) and four cues by punishment or neutral outcomes (“avoid” cues). During cue presentation, subjects make a button press with the left hand (Go left) or right hand (Go right) or withhold from responding (NoGo). Only one of these response options is considered correct per cue, and based on the response, subjects receive feedback. Correct responses are followed by a reward (“win” cues) and a neutral outcome (“avoid” cues) 80% of the time and by a neutral outcome (“win” cues) and a punishment (“avoid” cues) otherwise. For incorrect responses, these probabilities are reversed. Based on the observed outcomes, subjects need to learn the optimal responses by trial and error. Trials end with an intertrial interval (ITI) varying from 1,000 ms to 1,750 ms in steps of 250 ms.Subjects are informed that i) each cue can be followed by either reward or punishment, ii) each cue has one optimal response, iii) feedback is probabilistic, and iv) the rewards and punishments are converted to a monetary bonus upon completion of the study. The monetary bonus ranged from 0€–5€ . In contrast to our previous study , cue valEEG data were acquired at 500 Hz from 65 channels  placed according to the equidistant arrangement, under and above the left eye for vertical EOG and lateral to the eyes for horizontal EOG. The ground was placed on the forehead and the left mastoid was used for online referencing. EEG data are preprocessed and analyzed with the Fieldtrip software toolbox  in MATLAφ) were used to compute ISPS, which is thought to reflect intersite functional connectivity [N is the number of trials, i the complex operator, and j and k the seed and target channels. ISPS values are sensitive to the number of trials, and therefore we selected the same number of trials from each condition to compute ISPS. This trial selection was permuted 100 times, and ISPS values were averaged over permutations. Cells with fewer than 20 trials were discarded for the analysis, resulting in the exclusion of two subjects for this analysis. ISPS values can range from 0 (no phase synchrony) to 1  and were baseline transformed into percent signal change using a condition-averaged baseline .Cue- and response-locked time series were decomposed into their TF representations using wavelet convolution. Here, the time series are convolved with Morlet wavelets, i.e., sine waves convolved with a Gaussian model, based on multiplication in the frequency domain. The frequencies ranged from 1 Hz to 50 Hz in 39 logarithmically spaced steps, and the Gaussian width was fixed at four cycles. Wavelet convolution results in a complex signal from which power and phase are extracted for each TF point, down-sampled to 40 Hz. Condition-specific power values were averaged over trials and dB baseline corrected using a condition-averaged baseline condition (−250 ms to −50ms relative to cue onset). Phase angles  and Hilbert transformed. Power time series were extracted, z transformed, averaged over the time window, and t-weighted for the channels of interest . We then combined these post hoc significant channels using t-weighting based on the main contrast  and entered the t-weighted trial-wise data in the computational models. To emphasize, the t-weighting was only performed after establishing the effect of interest at the group level and thus did not bias our results. We performed the t-weighting at the group level rather than the subject level to reduce proneness to noise and enhance generalizability. To assess phase synchronization of the lateral PFC and motor cortex to the midfrontal cortex, we computed a t-weighted seed time series based on the significant midfrontal channels. We selected the following target clusters: i) eight dorsolateral prefrontal channels, including the dorsolateral peaks observed in the condition averaged data and extended laterally to surround the channels F5/6 in the 10–20 EEG montage , as scalp topographies can vary considerably between subjects and studies. Note that we recorded EEG with an equidistant channel arrangement instead of the more commonly used 10–20 arrangement. To assess midfrontal theta power, we selected two central channels corresponding to Cz and FCz in the 10–20 arrangement, as these channels showed a robust modulation of midfrontal theta power to response conflict in previous work ,47. We tchannels ,23,48); The behavioral data were analyzed in line with Swart and colleagues . We elabt tests, we assessed which of the three midfrontal channels contributed significantly to the resulting time window . Given that i) midfrontal theta power is known to strongly increase after errors [To assess whether midfrontal theta power is related to reduced Pavlovian response biases, we analyzed TF power for the midfrontal cluster and restricted the analysis to the frequency range of interest (4 Hz–8Hz) and to the response window, i.e., cue presentation. We employed a time-based permutation test (500 permutations) with the cue-locked, trial-averaged midfrontal theta power as dependent variable and the within-subject factor congruency (congruent versus incongruent); the Go-to-Win and NoGo-to-Avoid cues are considered to be motivationally congruent, as the Pavlovian response tendencies are in line with the instrumental requirements, whereas the NoGo-to-Win and Go-to-Avoid cues are considered to be motivationally incongruent. With post hoc r errors ,49 and iTo test whether midfrontal theta power might also be related to reduced instrumental learning biases, we assessed feedback-related midfrontal theta power, which has been linked to reinforcement learning ,18. In pcontra/Goipsi/NoGobilateral). For the significant valence × required response interaction, we then assessed whether this interaction could be explained by an effect of motivational congruency in the nonexecuting motor channels  versus executing motor channels .To assess phase synchronization of the lateral PFC and motor cortex to the midfrontal cortex, we assessed the t-weighted phase synchronization between the midfrontal cluster and the lateral prefrontal and motor clusters. We extracted the ISPS values for the significant time window of the cue-locked permutation test using correct trials. We analyzed ISPS using a repeated measures ANOVA with the factors valence (“win” versus “avoid” cue) × required action (Go versus NoGo) to assess whether ISPS increased during motivationally incongruent cues. For the motor channels, we reasoned that ISPS might be differentially affected in the “executing” motor cortex  relative to the “nonexecuting” motor cortex . To elaborate, midfrontal functional connectivity is thought to be activity dependent  such thaS1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.S5 Text(DOCX)Click here for additional data file.S6 Text(DOCX)Click here for additional data file.S7 TextISPS, intersite phase synchrony.(DOCX)Click here for additional data file.S8 TextISPS, intersite phase synchrony.(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Recent experimental studies have suggested that hypokalemia-induced arrhythmias are initiated by the reduced activity of the Na+/K+-ATPase (NKA), subsequently leading to Ca2+ overload, Ca2+/Calmodulin-dependent kinase II (CaMKII) activation, and development of afterdepolarizations. In this article, we review the current mechanistic evidence of hypokalemia-induced triggered arrhythmias and discuss how molecular changes in heart failure might lower the threshold for these arrhythmias. Finally, we discuss how recent insights into hypokalemia-induced arrhythmias could have potential implications for future antiarrhythmic treatment strategies.Routine use of diuretics and neurohumoral activation make hypokalemia (serum K Despite continuous improvements in therapies, long-term prognosis in heart failure (HF) remains poor, with overall 5-year mortality reaching 50%  < 3.5 mM   membrane as illustrated in Figure 2+ waves can occur due to overload of Ca2+ in the SR and/or reduced threshold for Ca2+ leak through the ryanodine receptors (RyRs) , leading to a depolarization of the resting membrane potential  or Iti derived from Ca2+ waves during the AP, are larger than the outward currents (mainly K+ currents) during late phases of the AP , polymorphic VT, ventricular fibrillation (VF), and ventricular ectopy  that uses the electrochemical gradient of Na+ and Ca2+ to exchange 3 Na+ for 1 Ca2+. This allows NKA to indirectly regulate cardiac Ca2+ fluxes through the regulation of [Na+]i. NKA is an ATP- and voltage-dependent ion transporter that exchanges 3 Na+ ions from the cytosol with 2 K+ ions from the extracellular compartment, leading to a net outward current   and by increasing reverse mode NCX activity, which mediates Ca2+ influx during a short time during the early phases of the AP  and have relatively small effects on NKAα1 (k0.5 = 1.5 mM)  and NKA inhibition (that increases Ca2+ levels).In addition to NKA inhibition, which reduces inward NCX current, hypokalemia also alters the NCX activity through hyperpolarization of the resting membrane potential. Hyperpolarization increases the inward NCX current and is associated with reduced intracellular Ca2+ overload in intact, beating hearts perfused with low K+. One study found that the Ca2+-induced K+ channel was active in hearts exposed to low 2+ levels, in addition to low 2+. In the latter study, both EADs and DADs appeared in rat hearts exposed to moderate hypokalemia, but only the EADs were followed by episodes of sustained ventricular arrhythmias  and increases ICa. Activation of INaL and ICa further amplifies the intracellular overload of Na+ and Ca2+, eventually activating CaMKII, in a detrimental, downward spiral, ultimately leading to afterdepolarizations and ventricular arrhythmias. Mathematical modeling supported this model, and both CaMKII inhibitors and inhibitor of INaL prevented development of VT/VF in hearts exposed to hypokalemia. Altogether, the collective data suggest that reduced NKAα2 activity initiates cellular Ca2+ overload in hypokalemia, which further leads to ventricular arrhythmias through a feed forward spiral where the activation of CaMKII amplifies intracellular Na+ and Ca2+ overload, ultimately leading to EADs  and structural remodeling (cardiac hypertrophy and fibrosis) are hallmarks of HF  primarily is due to higher TTX-sensitive Na+ influx and not due to reduced NKA activity  could cause sufficient rise in Na+ to cause Ca2+ overload and spontaneous SR Ca2+ release. In addition, one study found pronounced reduction in the NKAα2 isoform expression and function despite only minor changes in the NKAα1 isoform   preventing EADs/DADs, 2) shortening the APD, and (3) directly targeting the underlying mechanism. On the basis of the model for hypokalemia-induced ventricular arrhythmias in Figure  shorteniHF is also characterized by APD prolongation (Wang and Hill, Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


There is discussion about whether mitochondrial matrix Ca2+ dynamics always mirror the cytoplasmic changes and whether this happens in vivo in whole organisms. In this study, we characterized cytosolic and mitochondrial Ca2+ signals during spontaneous skeletal muscle contractions in zebrafish embryos expressing bioluminescent GFP-aequorin  and mitoGFP-aequorin . The Ca2+ transients measured with GA and mitoGA reflected contractions of the trunk observed by transmitted light. The mitochondrial uncoupler FCCP and the inhibitor of the mitochondrial calcium uniporter (MCU), DS16570511, abolished mitochondrial Ca2+ transients whereas they increased the frequency of cytosolic Ca2+ transients and muscle contractions, confirming the subcellular localization of mitoGA. Mitochondrial Ca2+ dynamics were also determined with mitoGA and were found to follow closely cytoplasmic changes, with a slower decay. Cytoplasmic Ca2+ kinetics and propagation along the trunk and tail were characterized with GA and with the genetically encoded fluorescent Ca2+ indicator, Twitch-4. Although fluorescence provided a better spatio-temporal resolution, GA was able to resolve the same kinetic parameters while allowing continuous measurements for hours.Mitochondria are believed to play an important role in shaping the intracellular Ca They can also be taken up by the mitochondrial Ca2+ uniporter (MCU) [2+, in turn, stimulates aerobic metabolism by activating the key enzymes of the Krebs cycle, the electron transport chain and oxidative phosphorylation [2+ into the sarcoplasmic reticulum by the SERCA ATPase). Increased ATP utilization lowers the ATP/ADP ratio, thus enhancing the rate of the respiratory chain (respiratory control). This functional interdependence between sarcoplasmic reticulum (SR) and mitochondria has a structural support, by the attachment of mitochondria to the Ca2+-release units in the SR [2+ microdomains in space that facilitate Ca2+ uptake by the MCU. According to one study, mitochondrial Ca2+ oscillates in synchrony with cytoplasmic Ca2+ [2+ have been studied mostly in myotubes and in isolated cells [In skeletal muscle, calcium ions (Caer (MCU) ,2,3, owier (MCU) . Mitochorylation . ATP is n the SR ,7. This mic Ca2+  to adjused cells . Howevered cells ,10,11.2+ levels during spontaneous contractions of the trunk and tail of live zebrafish embryo with bioluminescent probes. The zebrafish (Danio rerio) is an exceptional vertebrate animal model with a high degree of genetic homology with humans, rapid ex utero embryonic development, and a relatively low cost. Skeletal muscle contractions in zebrafish start at 17 h post-fertilization (hpf) and intensify at 23 hpf [The purpose of this study was to visualize cytoplasmic and mitochondrial Cat 23 hpf . Moreove2+ sensitive photoprotein aequorin [2+ associated with muscle contractions in zebrafish embryos during the segmentation period [2+ monitoring with aequorin (or FP-aequorin fusions) (see [2+ [2+ indicators (GECIs) that work by fluorescence are brighter and easier to detect than luminescent probes, but excitation light causes autofluorescence, photobleaching and possibly phototoxicity, limiting the total imaging time. Both types of genetically encoded probes can be targeted to cellular organelles.In our previous study, the Caaequorin  was moleaequorin ,15. We rn period . Ca2+ mons) (see  for a re(see [2+ . Since e2+ in vivo in zebrafish embryos. We imaged luminescence of GFP-aequorin (GA), expressed in the cytoplasm and trapped in the mitochondrial matrix (mitoGA) [f-coelenterazine. Microscopy and functional tests confirmed that mitoGA was correctly targeted to mitochondria. We found that mitochondrial Ca2+ mirrors the sarcoplasmic changes in Ca2+ during spontaneous contractions through muscle development. In addition, Ca2+ transients propagated along the tail. We tracked the kinetics of cytoplasmic and mitochondrial Ca2+ by bioluminescence, and cytoplasmic Ca2+ with the genetically encoded fluorescent Ca2+ indicator, Twitch-4 [In this study, we established a model to study mitochondrial Ca(mitoGA) . TransieTwitch-4 .2+ indicators GA (not tagged) [2+-sensing aequorin, their expression and subcellular location was verified by fluorescence microscopy. Non-injected embryos were used as controls for fluorescence and development. GA expression was detected at 2 hpf in 99% of embryos, whereas mitoGA expression was observed only after 4 hpf. At 27–28 hpf, 95% of the injected embryos were fluorescent.The luminescent Ca tagged)  and mito tagged)  were subThe somites were well delimited by fluorescence. The overall fluorescence of mitoGA tended to be less than that of GA, as expected, because the mitochondrial matrix compartment has less volume than bulk cytoplasm.For the goals of the study, it was crucial to ensure that mitoGA expression was restricted to mitochondria. We evaluated the expression of GA and mitoGA by confocal microscopy. Embryos expressing GA showed a fairly uniform labeling in skeletal muscle cells of the trunk, whereas the mitoGA embryos showed a filamentous staining, typical of mitochondria B. This sTo confirm the above conclusions, we performed colocalization experiments of GA and mitoGA with a mitochondrial chemical dye. The membrane potential dye TMRE is known to accumulate in respiring mitochondria, according to the Nernst equation, owing to its delocalized positive charge . We firsProteins endowed with mitochondrial targeting signals (like mitoGA), a sequence rich in basic residues, are synthesized in the cytoplasm and imported through the mitochondrial membranes into the matrix  and this2+ indicators in our experimental model, a test of maximum luminescence was performed on a microplate luminometer  was added to break the plasma membrane, bringing aequorin into contact with extracellular Ca2+ and releasing all counts. GA reconstituted with f-coelenterazine showed a >1000-fold increase in emission compared to either un-injected embryos, un-injected embryos incubated with f-coelenterazine, or GA-injected embryos not incubated with the substrate. mitoGA embryos incubated with f-coelenterazine emitted about 100-fold more counts than their controls. This proves that aequorin can be reconstituted in live zebrafish embryos by incubation with f-coelenterazine. Moreover, functional aequorin was quite stable over time, since total counts were similar at 10 and 27 hpf. Mitochondrially trapped GA provided about 10-fold less luminescence than cytosolic GA.To determine the functionality of the aequorin biosensors as Cainometer . Light r2+ signaling periods during the development of slow muscle cells within forming skeletal muscle in zebrafish embryos using transient ubiquitous expression of aequorin [2+ were found to occur between 17.5 and 19.5 hpf  and after 23 hpf (SP2), with a quiet period of about 3.5 h between them. The authors concluded that these temporal characteristics of Ca2+ signaling played a role in myocyte differentiation and myotome patterning in SP1. Both SP1 and SP2 periods correlated with muscle contractions, as was shown with Oregon green-1 dextran [2+, measured with mitoGA, would mirror the sarcoplasmic changes in Ca2+ during early muscle differentiation.Miller and coworkers have described two distinct Caaequorin  or transaequorin . These p dextran . In thisFor imaging experiments, a conventional fluorescence microscope adapted for luminescence was not sensitive enough to detect light emitted by GA or mitoGA, although images could be obtained at 1 Hz (1 s integration) in some embryos. To record luminescence images and increase the light gathering power, we constructed a low light microscope designed for bioluminescence and radioluminescence . With th2+ signaling, corresponding to the SP1 and SP2 periods mentioned above. These two Ca2+ signaling periods were also observed in mitochondria with the biosensor mitoGA  and compared their average frequencies. 2+ signals obtained by bioluminescence  and in mitochondria (mitoGA)  B. Thus, 2+ transients in the SP2 period were caused by neural input. Tricaine is a reversible general anaesthetic commonly used in manipulation of zebrafish, which blocks neural action potentials (blocking voltage-dependent Na+ channels) but does not directly cause paralysis in skeletal muscle at standard concentrations [2+ transient, suggesting that these Ca2+ spikes were caused by neural input.We examined whether Catrations . Embryos2+ uptake.The mitochondrial origin of the bioluminescence signals was demonstrated using two pharmacological inhibitors of mitochondria, the uncoupler FCCP and the recently described inhibitor of the MCU, DS16570511 , since mWe first checked the effect of 1 µM FCCP or 100 µM DS16570511 on contractions measured by transmitted light in control un-injected embryos during the SP2 period (24–28 hpf). Interestingly, after adding FCCP or DS16570511, the average frequency of contractions increased significantly by about 50% A. Since 2+ transients was measured by GA luminescence. The frequency of sarcoplasmic Ca2+ transients increased by both compounds, in agreement with the increase in the frequency of contractions. However, the amplitude of Ca2+ transients decreased (FCCP) or did not change significantly (DS16570511)  B. This iytes see .2+, both FCCP and DS16570511 decreased very significantly the frequency of mitochondrial Ca2+ transients (mitoGA luminescence)  C. The am2+ transients  did not change the frequency of contractions in control un-injected embryos , nor theansients . Furtherfor FCCP  and DS16for FCCP .2+ into the matrix [2+ measured with GA and mitoGA. Furthermore, DS16570511, a compound recently described to inhibit the MCU [The above results support the conclusion that mitoGA is located in mitochondria, since FCCP, which collapses the membrane potential used by the uniporter to take up Cae matrix , caused  the MCU , produce2+ transients with GA and with Twitch-4, a ratiometric fluorescent GECI [Next, we characterized the shape and kinetics of the cytoplasmic Caent GECI . To expr2+ rise (FRET/donor ratio images) during a spontaneous contraction of the trunk in a representative embryo. The Ca2+ rise was sharp and decayed in 1–2 s; the average kinetic parameters are shown in Twitch-4 was imaged at 33 Hz with a wide-field fluorescence microscope. To image at this speed, the donor and acceptor FRET images see  were acq2+ transients were also determined in GA-expressing embryos (2+ transient is shown. The kinetics of cytoplasmic Ca2+ spikes measured with GA (at 11.9 Hz) and Twitch-4 (at 33 Hz) were compared  [The kinetics of the Ca embryos B. A biolcompared C. Rise p is 1.04 , whereas1.04 [2+ . Thus, atrations , since ttrations , whereasn vitro)  is not v2+ transients with good time resolution (30 ms integration per image), whereas for GA, we used 84 ms integration per image  allowed identification of all Caractions . Howeverractions ; more ev2+ transients at various imaging sampling rates, to illustrate the limitations due to bioluminescence sampling. When imaging GA at a faster rate (30 Hz), only the largest Ca2+ peaks and the brightest portion of some transients were seen, and the kinetic parameters were artifactually shortened , nor in half-width of the Ca2+ transients . Due ton fusion . In contn fusion . In factn fusion ,39. Micr2+ in the cytoplasm, for mitoGA to give a reliable measure of mitochondrial Ca2+, it was important to confirm maximal localization within the matrix  and minimal mistargeting (the probe was not expressed in any other compartment) [2+ uptake supported that the biosensor was actually located within mitochondria; while contractions and cytosolic Ca2+ transients increased in frequency by these drugs, those in mitochondria decreased abruptly or disappeared  ,26,40. Iappeared . MitoGA 2+ rises occur spontaneously during aequorin reconstitution in zebrafish, leading to its consumption. Thus, if the burnout rate is faster than the reconstitution rate, aequorin will not be functional. Its luminescence is proportional to Ca2+ and to the amount of remaining functional photoprotein. We showed that after reconstitution of GA and mitoGA, embryos retained enough luminescence at 27 hpf to obtain a reliable signal for imaging experiments  to the bath . Howevereriments .2+ activity in the trunk of 17–25 hpf developing zebrafish (SP1 and SP2), with a quiet period in between [2+ signals were associated to excitation-contraction coupling of the skeletal muscle cells in the trunk and tail, resulting in coordinated movements at different developmental stages [2+ transients in long experiments (12 to 28 hpf) showed the same two periods of Ca2+ spikes with a similar frequency, although spikes were also observed in between SP1 and SP2 (2+ spikes and contractions at 24–28 hpf (0.02 Hz) increased to about 0.2 Hz when measured in short experiments (minutes)  . This hi2+ displayed the same distribution of Ca2+ signals, profiling SP1 and SP2. Moreover, the frequency of Ca2+ transients in cytoplasm and mitochondria was the same as that of contractions, measured by transmitted light  of 70 nM, and mitoYC2.60, with a K’d < 100 nM [2+ to a high concentration upon physiological stimulation, about 500 µM in chromaffin cells [2+ affinity in order to record mitochondrial Ca2+ peaks has been pointed out [2+ levels, whereas mitoGA  [The increase in the frequency of contractions and cytosolic Ca16570511  cannot be (CICR) . Blockin2+ transients with the bioluminescent sensors GA and mitoGA in prolonged experiments in developing zebrafish  [The Carin (GA) , Twitch-rin (GA)  (a kind rin (GA)  were clorin (GA) ,26 was uThe plasmids of the biosensors cloned in pCS2+ were linearized with the restriction enzymes NotI  or HindIII (Twitch-4), and in vitro mRNA synthesis was performed with the mMESSAGE mMACHINE SP6 kit  following the manufacturer’s protocol. mRNA resuspended in nuclease free water was quantified in a Nanodrop spectrophotometer and aliquots were made at 500 ng/µL and stored at −80 °C.Danio rerio) were kept in the Center for Animal Experimentation of the Albacete School of Medicine with a light/dark cycle of 14/10 h. Fertilized zebrafish eggs in synchronized stage were obtained following standard procedures and maintained in medium E3  at 28.5 °C. All animal procedures were carried out in compliance with UCLM Animal Experimentation Ethics Committee following national and EU regulations . All the experiments were conducted on zebrafish embryos and larvae of less than 120 hpf.Wild-type Tübingen zebrafish  in a horizontal puller  at 589 °C. Microinjection was performed manually on the blastodisc of one-cell stage eggs through the vegetal pole using a microinjection unit . The injection volume was 1–5 nL and the amount of mRNA was ~400 pg/egg. The injection solutions were diluted in 0.5% phenol red as an indicator of the injection volume. In some experiments, the mitochondrial chemical marker tetramethyl-ethylrhodamine  was injected together with mRNA of the Caescribed .f-coelenterazine  was prepared at 5 mM in methanol, 5 μL aliquots were prepared and frozen at −80 °C. To reconstitute functional aequorin, embryos injected with the mRNA of each biosensor were dechorionated at 4 hpf, transferred to a 24-well plate coated with agarose, and incubated (10 embryos per well) with 50 μM f-coelenterazine in E3 medium (28.5 °C in darkness) for 3–23 h, as indicated. To quantitatively determine the functional aequorin at different time points, total light emission from embryos of 7, 10 and 27 hpf was measured. Embryos were transferred to a 96-well opaque plate (1 embryo/well) in a microplate luminometer equipped with automatic injectors . A Fast Kinetics protocol was designed with a total reading time of 1 min per well . An injection volume of 50 µL 0.5% Triton X-100 was added per well  to trigger the release of luminescence.A stock of 2+ measurements.For the confocal and widefield fluorescence experiments, the embryos were dechorionated with fine forceps, embedded in 0.3% low melting point agarose in E3, preheated to 42 °C, and gelled on a 35-mm diameter glass bottom dish . Once solidified, E3 medium was added. For the bioluminescence experiments, 8-well glass slides (ibidi) were used, and the embryos were embedded in 0.15% low melting point agarose in E3 medium. Embryos over 24 hpf were anaesthetized with 0.03% (1.1 mM) Tricaine  in E3 for 3 min prior to mounting for confocal microscopy. Embryos were not anesthetized for in vivo CaAgar-embedded embryos were imaged in an inverse Axio Observer Zeiss LSM710 confocal system  configured to use a Zeiss BiG-2 GaAsP detector and two detection channels. A green channel for GA or mitoGA, with 488 nm excitation laser and a 500–550 nm emission filter and red channel for TMRE, with 561 nm excitation laser and a 565–615 nm emission filter. Images were obtained with a PlanApo 10×/0.45 NA, oil immersion PlanN 40×/1.30 NA or oil immersion PlanApo 63×/1.40 NA objectives. For colocalization experiments, embryos coinjected with TMRE and GA or mitoGA were dechorionated and agar-mounted for observation as indicated above. Control embryos expressing only TMRE or GA were used to ensure the absence of bleed through . Images Embryos injected with TMRE were dechorionated at 25 hpf and agar-mounted for observation under a confocal laser scanning microscope . A solution of 1 µM FCCP  in medium E3 was added to the plate and fluorescence images were recorded at 0, 5 and 15 min.f-coelenterazine in 0.5 mL of E3 medium (10 embryos per well). At 12 hpf (for overnight experiments) or at 24 hpf, embryos were embedded in 0.15% low melting point agarose and transferred to a 8-well glass slide (ibidi). Bioluminescence images were acquired with a custom-built microscope  was 5×. This microscope was equipped with a LED lamp for transmitted light. The microscope was housed in a light-tight box to maintain complete darkness during bioluminescence imaging. The embryos were maintained at 28 °C. The bioluminescence images were acquired continuously in 16 bits with 4 × 4 binning, 255 camera gain, at a rate of 1, 4.7, 11.9 or 30 Hz (frames/s). With this configuration, the resolution of the images was 12.8 µm × 12.8 µm/pixel and the total field of view (FOV) was 1638.4 µm × 1638.4 µm. Transmitted light images were acquired at 4.2 Hz to correlate the luminescence with the anatomical structures of the embryo, but they were not acquired simultaneously with luminescence.The procedure for aequorin reconstitution described above was slightly modified. Fluorescent embryos were selected at 12 hpf, dechorionated and incubated in the dark at 28.5 °C with 50 µM roscope, ) with anTo assess biosensor expression levels prior to bioluminescence experiments, fluorescence images of the embryos were acquired in a stereomicroscope  with a CCD camera  controlled with AquaCosmos2.6 (Hamamatsu Photonics). The microscope was equipped with a HC-YFP cube .Embryos basal contractions were registered for 15 min, observation was briefly interrupted to add 1 µM Carbonylcyanide-p-trifluoromethoxyphenylhydrazone , or 100 µM DS16570511  and resumed for 15 min.All the contractions shown were spontaneous, occurring naturally during development of skeletal muscle. No external stimulation was employed. Time-lapses of embryo trunk contractions were recorded in the Low Light Microscope configured for transmitted light (see Bioluminescent imaging above). Videos in TIFF format were analyzed and processed in Fiji-ImageJ (NIH) . For det2+ transients were differentiated from background by means of their location, intensity and size. Signals located over the embryo trunk, being larger than 9 adjacent pixels and brighter than a minimum intensity value, were considered a Ca2+ transient. The minimum intensity value was set to each frame taking into account a background analysis. To perform this analysis, two types of backgrounds were considered: general background (GB) and embryo background (EB). GB considers changes in detector noise over time; it was calculated on each individual frame by analyzing an artifact-free area in the medium surrounding the embryo. EB takes into account coelenterazine spontaneous bioluminescent emission; it was calculated once in each experiment by analyzing 15 different frames at times when no visible Ca2+ transients took place.Ca2+ transients. GB was calculated as GB = Mean pixel value in ROI2 + 2*S.D. (S.D. standard deviation). EB was calculated as:Image analysis and processing was automated through a FIJI macro (available upon request). The macro interacts with a user through a variety of graphical user interfaces that require: 1) drawing the region of the embryo to be analysed (ROI1), 2) moving the drawn region to an artifact-free area out of the embryo (ROI2) and 3) choosing 15 frames with no visible Ca2+ transients was calculated for each frame and imported into Clampfit for event analysis. For characterization of Ca2+ spike kinetics, data were analyzed with Clampfit 10.7 , and the following parameters were determined: frequency, time to peak, peak amplitude, rise time 10% to 90%, decay time 90% to 10%, half-width , and duration of the event.A final threshold was set to each frame adding GB and EB. The integrated density of CaWe acquired fluorescent images of the trunk and tail in 24–28 hpf embryos expressing the ratiometric fluorescent biosensor Twitch-4  with a wThe embryos were excited continuously with a LED source  using a 440AF21 nm bandpass filter  and a beamsplitter . Fluorescence passed through an image splitter with emission filters 483/32 nm and 542/27 nm , separated with beamsplitter 509-FDi01 (Semrock). A 10× air objective  and binning 4 × 4 were used. With this configuration, the resolution of the images was 2.9 µm × 2.9 µm/pixel and the total field of view (FOV) after image splitting was 742.4 µm (H) × 1484.8 µm (V). Transmission images were acquired to correlate fluorescence with the anatomical structures of the embryo.2+ transients.The FRET image corresponds to cpCitrine174 emission (542 nm channel) with 440 nm excitation, and the donor image corresponds to ECFP emission (483 nm channel) with 440 nm excitation. The ratio FRET/donor image was computed pixel by pixel. For ratio image processing, we used the custom-made software Ratioscope, programmed by Dr. Pierre Vincent  on IgorPro 8 , and program Clampfit 10.7 (Molecular Devices) for extracting the kinetic parameters of Capost-hoc test for paired comparisons. A two-tailed t-Student test was used for paired or independent samples, as indicated. Data are shown as the mean ± standard error of the mean (SEM) or standard deviation (S.D.), as indicated. Statistical significance was considered for * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data were analyzed in Graphpad Prism 6 .A one-way ANOVA test was used to compare groups of embryos where indicated with a Bonferroni


Ventricular myocytes isolated from iron-deficient animals produced smaller Ca2+ transients from an elevated diastolic baseline but had unchanged sarcoplasmic reticulum (SR) Ca2+ load, trigger L-type Ca2+ current, or cytoplasmic Ca2+ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message levels. The constancy of diastolic SR Ca2+ load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due to dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca2+ signaling and ejection fraction. Thus, 2 Ca2+-handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by i.v. iron supplementation.Iron deficiency is present in ~50% of heart failure (HF) patients. Large multicenter trials have shown that treatment of iron deficiency with i.v. iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet, and some received i.v. ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca Iron deficiency, a common comorbidity in heart failure, reduces the strength of cardiac contraction through effects on Ca2+-handling proteins crucial for normal cardiac function but typically deranged failing hearts. Iron deficiency is the most common medical disorder in the world , 2, and 2+ signals (2+-handling proteins in the ventricular myocyte include L-type voltage-gated Ca2+ channels (LCC) Na+/Ca2+ exchangers (NCX), ryanodine receptor channels (RyR2), and Ca2+ ATPase pumps at the sarcoplasmic reticulum (SERCA) and plasmalemma (PMCA). During electrical excitation, LCC conduct a current of Ca2+ ions, which triggers RyR2 channels at the sarcoplasmic reticulum (SR) to release stored Ca2+ ions, producing a cytoplasmic Ca2+ transient (CaT). This signal is terminated by Ca2+ reuptake into the SR by SERCA and extrusion from the cell by NCX and PMCA.Iron could affect cardiac function through myriad pathways. These include alterations in gene expression through iron- and oxygen-dependent enzymes, such as prolyl hydroxylases (PHDs) and lysine demethylases (KDMs), which regulate hypoxia-inducible factor (HIF) , 11 and  signals , but the2+ signaling in an otherwise healthy organism. For this purpose, we established iron deficiency in mice by restricting their dietary iron intake and monitored the ensuing changes in iron status and cardiac function. To explore the effects of subsequent iron repletion, some animals were supplemented i.v. with ferric carboxymaltose (FCM), a formulation used clinically in humans  by decreasing CaT amplitude. This systolic dysfunction relates to a downregulation of RyR2 channels and suppression of SERCA Ca2+ pumps, 2 proteins located at the SR membrane. I.v. iron supplementation restored normal CaT amplitude and EF. Thus, we propose that iron deficiency can lead to a loss-of-function of 2 Ca2+-handling proteins, commonly implicated in failing hearts  or saline (sham) were injected i.v. at 22 and 29 days of commencing the diet . Body man = 4). Mean red blood cell volume (MCV), measured by flow cytometry in blood samples diluted in HEPES-buffered salt solution, was reduced from of 61.0 ± 0.5 fl to 50.8 ± 0.2 fl (n = 4). In animals on the iron-deficient diet, serum ferritin, serum iron, and transferrin saturation were reduced to 47%, 44%, and 25% of control, respectively, and total serum transferrin was raised to 171% of control; these indices returned to normal levels after 2 injections of FCM . These histone marks are associated with activation (H3K36me3) and repression (H3K9me3) of transcription , a β-agonist, to interrogate Ca pathway . Under t pathway . The syseriments . As conferiments . These f2+ handling in conditions involving maladaptive hypertrophic growth  is not routinely measured in studies of Ca2+ signaling, although it can affect the amplitude and time course of CaTs  and FuraRed-AM (a ratiometric [Ca2+] reporter). A calibration curve for FuraRed is shown in 2+] produce fluorescence ratios in excess of 2, and over the range between diastolic and systolic [Ca2+], the FuraRed ratio increases near-linearly with cytoplasmic [Ca2+]; thus, this dye is suitable for recording Ca2+ dynamics in myocytes. Ratiometric imaging eliminates potential artefacts due to photobleaching or dye loss, which would otherwise overestimate βCa. To prevent Ca2+-induced Ca2+ release, the SR content was emptied with 10 mM caffeine, and to minimize sarcolemmal Ca2+ fluxes, myocytes were superfused in Na+-free/Ca2+-free solution (0Na0Ca). The measurement protocol  between different cells and conditions. Ratiometric imaging also allows transporter-generated Ca2+ fluxes to be plotted as Ca2+-activation curves, thus revealing any shifts in transporter kinetics. To optimize the dynamic range of FuraRed, imaging was performed in dual-excitation mode.In the absence of a change in trigger Ca2+ handling. In the first protocol  with HF  phosphorylation  and RyR2 activity reduced (by 47%). Simulations were run in the CellML environment (http://www.cellml.com) for a test period of 100 seconds until a steady state amplitude was attained. As shown in 2+ load. These simulations add further support to the proposed model of CaT remodeling in iron-deficiency anemia.Considering the numerous feedback loops operating between Ca release . Thus, tignaling  was run In vivo, hypoxia and iron are strongly interrelated, as shown here by the effect of iron deficiency on markers of cardiac tissue hypoxia  and by p2+-handling proteins were sufficient to evoke a meaningful effect on Ca2+ signals in cultured myocytes, resembling the phenotype of iron-deficient mice, CaTs were triggered by electrical pacing (2 Hz) in Fluo3-loaded cells , and resulted in an inadequate emptying of the ventricle during systole, as is characteristic of developing systolic dysfunction. Consistent with systolic dysfunction, myocytes isolated from iron-deficient mice fired smaller CaTs from an elevated diastolic level but reaching the same systolic peak as control animals, as determined using the ratiometeric Ca2+ dye FuraRed (0) disguises any differences in diastolic [Ca2+]. The size of the SR Ca2+ store in diastole was unchanged in iron deficiency  . The eff2+ signal occurred without a change in L-type Ca2+ current . Raising the Km of SERCA reduces Ca2+ sequestration into the SR, thus allowing baseline [Ca2+] in the cytoplasm to attain a higher level, as observed in myocytes from iron-deficient mice   . In ironmplitude . This ise course .2+ affinity relates to the 2-fold decrease in PLN phosphorylation at Thr17, a target of CaMKII . Since there are sex-related differences in baseline hematocrit and the cycle-dependent changes in body iron stores in females, male mice were used in this study. Age-matched controls received an iron-adjusted diet . Mice were given i.v. FCM or saline as sham control. Hemoglobin concentration in tail vein blood, collected using a 27-G needle, was monitored with HemoCue device (Radiometer). Protocols were randomized, but blinding was not implemented because the phenotype was identifiable upon bleeding. The inclusion criterion was completion of the dietary protocol . At the end of the protocol, animals were killed humanely by a Schedule I method or anesthetic overdose. Hearts were either snap-frozen in liquid nitrogen for protein measurements or used for Langendorff perfusion.MRI was performed using a 11.7 T (500 MHz) vertical bore system. Anesthesia was induced in 3% isoflurane and maintained in 2% isoflurane. Due to the small size of the rodent heart, high field strengths were applied to improve signal/noise ratios. Electrocardiogram (ECG) electrodes were inserted in the forepaws to provide information for gating, necessary for synchronizing acquisition steps for the relatively high cardiac rate in mice. Images were visualized using a Burker console running Paravision 2.1.1. Cardiac-triggered and respiration-gated cine-FLASH images were acquired, and for each animal, 10–11 contiguous 1-mm thick, true short-axis images were obtained, covering the entire heart. Each in vivo protocol was performed in approximately 45 minutes in the vertical position. EDV and ESV were rendered from Z-stacks and used to calculate stroke volume (EDV – ESV) and EF (SV/EDV).2, 5 HEPES, 0.4 Na2HPO4, 10 glucose, and 20 taurine . The heart was then digested with Tyrode perfusion solution supplemented with 0.1 mM CaCl2 (MilliporeSigma), 1 mM collagenase type II (Worthington) and 0.1 mM protease (MilliporeSigma). Following digestion, cells were dissociated by careful mechanical disruption and filtered through a 500-μm cell strainer. Enzymatic activity was quenched by adding 1% BSA (MilliporeSigma) in Tyrode perfusion buffer. Cells were washed once in 0.5 mM CaCl2 (Tyrode) and then once in 1 mM CaCl2 (Tyrode) prior to use.Excised hearts were cannulated at the aorta using a blunted 23-G needle, mounted on a Langendorff apparatus. The heart was first perfused with warm Tyrode perfusion solution containing (in mM): 130 NaCl, 5.6 KCl, 3.5 MgCl4, and 0.8 NaH2PO4. In fresh 1× ADS buffer, the ventricles were finely minced and digested with 0.45 mg/ml collagenase type A (Roche Diagnostics) and 1.25 mg/ml pancreatin (MilliporeSigma) for 5 minutes at 37°C with gentle stirring. The tissue was left to settle, supernatant was discarded, and the tissue was digested for a second time for 8 minutes at 37°C. Enzymatic activity was quenched using newborn calf serum (MilliporeSigma). Cells were pelleted at 1,250 rpm, resuspended in M1 medium: 80% DMEM  supplemented with 24 mM NaHCO3 and 20% M199 . Complete M1 medium was supplemented with 10% horse serum (MilliporeSigma) and 5% heat-inactivated newborn calf serum (Thermo Fisher Scientific). Cells were preplated on uncoated petri dishes for 2 hours at 37°C. The supernatant was collected and spun at 1,250 rpm for 5 minutes, and the pellet was resuspended in fresh M1 medium. To obtain lysates for Western blotting, cells were plated on fibronectin-coated 6-well plates at approximately 500,000 cells per well in M1 media. For imaging experiments, cells were plated on fibronectin-coated 4-well chambered coverslips  at approximately 60,000 cells/cm2 in M1 media. On day 1 after isolation, cells were serum starved (0%) and maintained in media containing 1:100 ITS . Forty-eight hours (western blotting) or 72 hours (imaging) after isolation, cells were treated for 16 or 24 hours with hypoxia in a chamber at 3% O2 or incubated in normoxia in the presence of 1 mM DMOG (MilliporeSigma), 50 μM DFO (MilliporeSigma), or no drug (control).Primary NRVMs were isolated from 1- to 2-day-old Sprague-Dawley rats (Charles River Laboratories). Pups were culled in accordance with a Schedule 1 method, and their hearts were excised and washed twice in ice-cold 1× ADS buffer containing (in mM): 106 NaCl, 5.3 KCl, 20 HEPES, 5 glucose, 0.4 MgSOCell lysate was prepared from crushed, frozen heart tissue using RIPA buffer or 8 M urea lysis buffer (for histone blots) supplemented with complete protease inhibitor. Protein lysate (100 μg) was loaded for SDS-PAGE. Membrane was blocked in primary antibody overnight at 4°C and then in HRP-conjugated secondary antibody. For immunofluorescence, isolated cardiomyocytes were plated on to laminin-coated plates and fixed in ice-cold 4% paraformaldehyde. Cells were permeabilized in 0.3% triton-X (MilliporeSigma), blocked first in primary antibody overnight at 4°C and then in secondary antibody and DAPI for nuclear staining. Cells were imaged on a Zeiss LSM 700 confocal microscope at 40× magnification. Densitometry was performed by numerically integrating below the intensity curve within a consistent frame, after offsetting the background. Fast Fourier transform was performed in ImageJ (NIH).4; TAAB Laboratories) in 1× PBS at 4°C for 2 hours with gentle rotation. Samples were rinsed 5 times in ddH2O, 15 minutes for each wash, and fixed in 0.2% uranyl acetate (Agar Scientific) overnight at 4°C in the dark. Samples were washed once in ddH2O for 10 minutes. For dehydration of specimen, samples were washed, for 15 minutes at 4°C, in 30%, 50%, 70%, 80%, 90%, and 95% EtOH. Samples were then incubated 3 times in 100% dry EtOH at 4°C — 30 minutes for each incubation step. For resin infiltration, samples were incubated in 2:1 100% dry EtOH/TAAB low viscosity (TLV) resin (TAAB Laboratories) for 2 hours, 1:1 100% dry EtOH/TAAB TLV resin for 3 hours, and 1:2 100% dry EtOH/TAAB TLV resin for 2 hours with gentle rotation. Samples were then incubated in 100% TAAB TLV resin for 48 hours at room temperature. During this time, the resin was changed every 8 hours. Tissue pieces were transferred to Beem capsules filled with fresh 100% TAAB TLV resin and polymerized overnight at 60°C. Samples were sectioned on the Leica UC7 ultramicrotome. Resin blocks were placed face up using a glass knife in preparation for ultra-thin sectioning. For ultra-thin sectioning, 90-nm thin sections were cut using a diamond knife (Diatome) and transferred to a 200 mesh Cu grid (TAAB Laboratories). Between 4 and 6 sections were cut per sample block. Prior to imaging, grids were poststained with Reynold’s lead citrate for 5 minutes, washed 3 times in ddH2O, and air-dried overnight. TEM imaging was performed at 120 kV on the FEI Tecnai 12, and images were acquired using the Gatan OneView CMOS camera with Digital Micrograph 3.0 software.Fresh samples were fixed in 2.5% glutaraldehyde (MilliporeSigma) made in 1× PBS, overnight at 4°C. The fixative was removed and replaced with a 0.25% glutaraldehyde solution and kept at 4°C until further processing. Tissues were rinsed 5 times in 1× PBS, 15 minutes for each wash, and were then fixed in 1% osmium tetroxide (OsOGapdh (SYBR green assays) or Actb (Taqman assays) were used as housekeeper genes. Taqman assays included Glut1 (Mm00441480), Glut4 (Mm00436615). The primers used were: Eno1: forward 5′-TAGGGTCCGGGCCTCGAT-3′, reverse 5′-TGTCTCGGTTACTAGGCCTGC-3′; Ryr2: forward 5′-CGAGGATGAGATCCAGTTCC-3′, reverse 5′-CAAATCCTTCTGCTGCCAAG-3′; Cacna1c: forward, 5′-AGCAAGAACCACTGCGGAT-3′, reverse 5′-GAAGAAATGCAGCAACAGCC-3′; Slc8a1: forward 5′-TTGAGGACACCTGTGGAGAG-3′, reverse 5′-GGGGCTCTCCAATCTCAAT-3′; and Atp2a2: forward 5′-GGGCAAAGTGTATCGACAGG-3′, reverse 5′-TCAGCAGGAACTTTGTCACC-3′.cDNA was reverse transcribed from total RNA using the Expand Reverse Transcriptase kit (Roche Diagnostics) and poly dT primers (Invitrogen), following manufacturer’s instructions. For quantitative PCR (qPCR), 25 μg of cDNA was used as a template, along with either the Taqman Fast Universal or PowerUp SYBR Green master mix (Thermo Fisher Scientific). 2+ dyes were used: Fluo3  for nonratiometric imaging (excitation 488 nm/emission >510 mm) and FuraRed  for ratiometric imaging, executed in either dual emission (excitation 488 nm/emission 600 ± 10 and 685 ± 25) or dual excitation mode . To measure cell size, myocytes were loaded with cSNARF1 , a bright dye (excitation 555 nm/emission 580–640nm). Photolysis of NP-EGTA  was performed using a 40 mW Ar-UV laser  . Fluo3 imaging (in line-scan mode) and cSNARF imaging (in xy mode) was performed on a Zeiss LSM 700 system. Dual-emission FuraRed imaging was performed on a Leica SP5 system. Dual-excitation FuraRed imaging was performed with a QImaging camera with a CoolLED pE-4000 LED light source. Cells were loaded with the compounds by AM-loading at room temperature for 5 minutes (Fluo3), 10 minutes , or 15 minutes (NP-EGTA). To measure electrically evoked CaTs, myocytes were superfused at 37°C with solution containing (in mM): 135 NaCl, 4.5 KCl, 20 HEPES, 1 CaCl2, 1 MgCl2, and 11 glucose (pH 7.4) at 37°C. Probenecid  was included to reduce the degree of dye loss due to extrusion. Caffeine (MilliporeSigma) was included at 10 mM in selected solutions. To elicit Ca2+ waves, superfusate Ca2+ was raised to 5 mM. To measure βCa, myocytes were superfused in 0Na0Ca containing (in mM): 135 N-methyl-D-glucamine (MilliporeSigma), 1 MgCl2, 0.5 EGTA, 4.5 KCl, 11 glucose, and 20 HEPES (pH 7.4) at 37°C.Two fluorescent Ca2+ imaging in monolayers was performed using Fluo-3 (excitation 488 nm/emission >510 nm). NRVMs were AM-loaded with Fluo-3  for 10 minutes in normal Tyrode (NT) at room temperature and then superfused in HEPES-buffered Tyrode at 37°C containing (in mM): 135 NaCl, 4.5 KCl, 1 MgCl2, 1 CaCl2, 20 HEPES, 11 glucose, and 1 probenecid; pH was adjusted to 7.4 at 37°C using 4.0 M NaOH. For cells treated under hypoxic conditions, superfusion was performed in NT bubbled with N2.Ca2, 3 CaCl2, 10 TEA-Cl, 5 Mg-ATP (MilliporeSigma), 1 Na2-GTP, 5 phosphocreatine, 5 BAPTA (MilliporeSigma), and 10 HEPES (pH 7.2) with CsOH at room temperature. To block K+ conductance, the superfusion solution contained (in mM): 135 NaCl, 5.4 CsCl, 1 MgCl2, 1.8 CaCl2, 5 4-aminopyridine (MilliporeSigma), 10.0 HEPES, and 11 glucose (pH 7.4) with NaOH at room temperature. Measurements were performed at room temperature using an Axopatch200B amplifier.Adult myocytes were voltage-clamped in whole-cell mode with a borosilicate glass micropipette (resistance 1–1.5 MΩ) containing an internal solution (in mM): 5 NaCl, 120 CsCl (MilliporeSigma), 1.MgCl2O. Inductively coupled plasma mass spectrometry (ICP-MS) was performed on the Perkin Elmer 6100DRC quadrupole system. For calibrations, 0, 0.5, 1, 10, 20, and 100 ng/g iron was spiked into a selected sample. An external iron standard  was measured to validate the calibration. Rhodium (1 ng/g) was also spiked into each sample as an internal standard. For each run, a sample of digested concentrated nitric acid was also submitted. Raw ICP-MS measurements were normalized to wet mass.Snap-frozen tissue (5–50 mg) was suspended in 1 ml concentrated nitric acid (MilliporeSigma), digested on a CEM SP-D80 Microwave Digestion System for 10 minutes at 180°C, and diluted to 2% acid in ultra-pure ddHThe ABX Pentra 400 was used to measure ferritin, transferrin, and iron in the serum of mice. Concentrations were automatically detected using the reagent cassettes (Horiba) Ferritin 2 CP), Transferrin CP, and Iron CP.v1.2 , rabbit-RyR2 , mouse-RyR2 , mouse-SERCA , rabbit-ser16-PLN , rabbit-thr16-PLN , mouse-PLN , rabbit-HIF1α , mouse-HIF2α , rabbit-H3K36me3 , rabbit-H3K9me3 , rabbit-H3K4me3 , rabbit-H3 , mouse-actin , goat anti–rabbit-HRP , goat anti–mouse-HRP , goat anti–rabbit-AlexaFluor 488 , and goat anti–mouse AlexaFluor 594 .Rabbit-Ca2+ studies, cells that did not properly respond to electrical pacing or caffeine  were excluded. No data point or dataset was deemed to be an outlier. The number of biological replicates for each experiment is specified in the figure legends, and exact numbers of animals/cells used per experimental condition can be found in t test was used for Western blots and qPCR measurements. A nested  1-way ANOVA was used to analyze other datasets  Act of 1986. All procedures relating to animals were approved by the UK Home Office under the Project License 30/3182 and the local Animal Care and Ethical Review Board .YJC, SLL, PAR, and PS designed research; YJC, KCP, AL, and AAL performed experiments; YJC, KCP, AL, AAL, and PS analyzed data; and YJC, SLL, PAR, and PS wrote the paper.


REC0)] or 60 min [recovery 60 (REC60)] following the cessation of stimulation. Using skinned fiber prepared from the superficial region, the ratio of force at 1 Hz to that at 50 Hz (low‐to‐high force ratio), the ratio of depolarization (depol)‐induced force to maximum Ca2+‐activated force (depol/max Ca2+ force ratio), the steepness of force‐Ca2+ concentration curves, and myofibrillar Ca2+ sensitivity were measured. At REC0, the low‐to‐high force ratio and depol/max Ca2+ force ratio decreased in stimulated muscles from both non‐ and thermal‐treated rats. At REC60, these two parameters remained depressed in non‐treated rats, whereas they reverted to resting levels in T‐treated rats. Thermal pretreatment exerted no effect on myofibrillar Ca2+ sensitivity. The present results reveal that thermal pretreatment can facilitate recovery of submaximum force after vigorous contraction, which is mediated via a quick return of Ca2+ release from the sarcoplasmic reticulum to resting levels.The aim of this study was to examine whether thermal pretreatment can accelerate recovery from prolonged low‐frequency force depression. The hindlimbs of thermal treated (T‐treated) rats were immersed in water heated to 42.0°C for 20 min . The thermal pretreatment was performed once a day for 5 days before fatiguing stimulation. Intact gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected immediately [recovery 0 ( It has been pointed out, however, that the results obtained from isolated muscles may not necessarily reflect changes in vivo  aged 10 weeks were used in this study. The animals were provided with water and standard chow  ad libitum and were individually housed in a cage in a thermally controlled room at 20–24°C with 12‐h light/dark cycle. An intraperitoneal injection of a mixture of medetomidine (0.4 mg kg‐body wt−1), midazolam (2.0 mg kg‐body wt−1), and butorphanol (2.5 mg kg body wt−1) was used for anesthesia. At the end of the experiments, the rats were euthanized with an overdose of pentobarbital sodium (200 mg kg body wt−1), followed by cervical dislocation.All experimental procedures used in this study were approved by the Animal Care Committee of Hiroshima University. Male Wistar rats (N = 16 for each group). Under anesthesia, the hindlimbs of T‐treated rats were immersed in water heated to 42.0°C for 20 min. Thermal pretreatment started 5 days prior to fatiguing stimulation (see below) and was performed once a day, five times in total. Non‐treated rats were anesthetized without thermal treatment.The rats were randomly divided a thermal treated (T‐treated) and a non‐treated group  muscles from the left hindlimb was electrically stimulated in vivo via the sciatic nerve, as described previously  Ca2+ activation solution was also similar to the K‐HDTA solution, but with all HDTA replaced with 49.5 mmol/L Ca‐EGTA and 0.5 mmol/L free EGTA to heavily buffer the free [Ca2+] at 10−4.7 mol/L. A similar strongly EGTA‐buffered solution containing 50 mmol/L EGTA (<10−9 mol/L) and no Ca‐EGTA was used as a relaxing solution. These two solutions were mixed in an appropriate ratio with free [Ca2+] in the range of 10−9–10−4.7 mol/L and used for force–[Ca2+] relationship analysis.All solutions for skinned fiber experiments were prepared as described in detail elsewhere ‐induced force was evaluated relative to max Ca2+‐activated force (depol/max Ca2+ force ratio). Previous studies  and 20 pulses at 50 Hz and then exposed to the max Ca2+ activation solution. Max Ca2+‐activated force was normalized to the cross‐sectional area of the fibers (specific max Ca2+ force). The cross‐sectional area was modeled as a circular profile if the diameter was similar along the fiber segment and was calculated from an average of three width measurements.The skinned fiber was placed in a small chamber containing ~500 2+ relationship was determined by exposing the skinned fiber to a sequence of solutions heavily buffered at progressively higher free [Ca2+] (10−9.0–10−4.7 mol/L). The force response produced at each [Ca2+] was expressed as a percentage of the max force. Using SigmaPlot 14 software , Hill coefficient (nH) and [Ca2+]50  were calculated. The steepness of force‐[Ca2+] curves and my‐Ca2+ sensitivity were evaluated on the basis of nH and [Ca2+]50, respectively.The force–CaαB‐crystallin, HSP25, and HSP70) present in rat skeletal muscles, HSP70  is highly susceptible to heat stress  are upregulated and interact with many proteins to help preserve and restore their function  of ice‐cold homogenizing buffer composed of 10 mmol/L Tris/HCl (pH 7.0), 150 mmol/L NaCl, 5 mmol/L NaF, 1 mmol/L Na‐orthovanadate, 1% (vol vol−1) Triton X‐100, 22 μmol/L leupeptin, and 14 μmol/L pepstatin and was homogenized on ice using a hand‐held homogenizer. The protein content was determined by the Bradford assay using bovine serum albumin as the standard (Bradford μg of proteins were applied to a 10% (mass vol−1) polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The membranes were incubated at room temperature with anti‐HSP70  and glyceraldehyde‐3‐phosphate dehydrogenase . The membranes were then incubated with secondary antibodies  at room temperature. Immunoreactive bands were visualized with chemiluminescence reagent  and evaluated using ImageJ software . The HSP70 amounts were evaluated relative to the band density of GAPDH.Frozen muscles were pulverized under liquid NBradford . A quantP < 0.05.Data are presented as means + SD. In this study, the effect of recovery time was not investigated. Two‐way ANOVA was used to determine the effects of thermal treatment and fatiguing stimulation. When significant differences were detected, Holm–Sidak post hoc test was performed. The level of significance was set at Intact GAS muscles were fatigued by repeated stimulation until the force was reduced to ~50% of the initial force. There were no significant differences in the time to fatigue among four groups examined ~90–120 sec). It has been shown previously that in skinned fibers, the ratio of twitch force (force at 1 Hz) to force at 50 Hz (low‐to‐high force ratio) in action potential‐induced forces can be utilized as an indicator of PLFFD  in stimulated fibers (Fig. 2+ sensitivity were observed between the corresponding fibers from non‐ and T‐treated rats. Inconsistent with the previous findings (Oishi et al. Neither fatiguing stimulation nor thermal treatment resulted in changes in specific max Ca60 Figs. E, and F.ers Fig. C and D. 2+ release.To our knowledge, no published study presently exists that shows an effective pretreatment strategy toward overcoming PLFFD. The present investigation, for the first time, provides evidence that thermal pretreatment can accelerate recovery from PLFFD. Skinned fiber analyses reveal that the beneficial effect of thermal treatment results from the faster recovery of impaired function of SR CaMany, if not all (Larkins et al. 2+ sensitivity and SR Ca2+ release, changes that can account for PLFFD (Bruton et al. 2+ release in fibers with low SOD activity, but not in fibers with high SOD activity, suggestive that superoxide has the deleterious influence on SR Ca2+ release and that reduced SR Ca2+ release observed in fatigued fibers (Fig. Different ROS are purported to have distinct effects on my‐Ca2+ release that occur immediately after fatiguing muscle contraction. However, contrary to our assumption, the present study showed that thermal pretreatment was unable to lessen these two parameters, but instead was able to facilitate recovery of them (Figs. 2+ release would be oxidized, which in turn results in reduced SR Ca2+ release. Cysteine and methionine are amino acids that contain sulfhydryl groups and are among the most sensitive to oxidation. Of the cysteine and methionine oxidation products, sulfenic and sulfinic acids and methionine sulfoxide can be reduced back to cysteine and methionine by specific enzymatic systems (Mary et al. Based on the inhibitory effect of thermal pretreatment on oxidative stress, which has been reported in previous study (Tamura et al. em Figs.  and 2. Tem Figs. , some pr2+ sensitivity seems likely to be variable depending on the cellular redox. Indeed, it has been demonstrated that my‐Ca2+ sensitivity is increased by the exposure of isolated fibers to a low concentration of hydrogen peroxide, while fibers treated with a high concentration display decreased my‐Ca2+ sensitivity (Andrade et al. 2+ sensitivity is one of the causes of PLFFD (Bruton et al. 2+ sensitivity found in isolated muscles may be ascribable to severe oxidative stress that rarely occurs in muscles in vivo (Dutka et al. 2+ sensitivity (Fig. S‐glutathionylated, which in turn brings about increased my‐Ca2+ sensitivity (Watanabe and Wada S‐glutathionylation of troponin I also occurs in exercising human skeletal muscles (Mollica et al. My‐Caity Fig. , which iity Fig. . Althoug2+ force and nH, but the depressions were reversible. It is equivocal as to whether thermal treatment in vivo causes the shift of the intracellular redox state to the oxidized side. However, our results of no changes in specific max Ca2+ force and nH suggest that if the shift occurs, the influence would disappear at least 24 h after thermal pretreatment.The impact of in vitro thermal treatment on the function of the contractile apparatus has been addressed by van der Peol and Stephenson . They fo2+ release function of SR to resting levels. It is well known that proper physical training not only improves physical performance, but also decreases the risk of developing chronic diseases such as cardiovascular diseases, hypertension, diabetes, and obesity (Kenney et al. In summary, we show that thermal pretreatment can accelerate restoration of submaximum force during a recovery period after vigorous contraction, which is mediated via a quick return of the CaThe authors declare no conflict of interest.


However, there is upcoming evidence that various ion channels might also initiate Ca2+ rises in glial cells by Ca2+ influx. We investigated AMPA receptor-mediated inward currents and Ca2+ transients in olfactory ensheathing cells (OECs), a specialized glial cell population in the olfactory bulb (OB), using whole-cell voltage-clamp recordings and confocal Ca2+ imaging. By immunohistochemistry we showed immunoreactivity to the AMPA receptor subunits GluA1, GluA2 and GluA4 in OECs, suggesting the presence of AMPA receptors in OECs. Kainate-induced inward currents were mediated exclusively by AMPA receptors, as they were sensitive to the specific AMPA receptor antagonist, GYKI53655. Moreover, kainate-induced inward currents were reduced by the selective Ca2+-permeable AMPA receptor inhibitor, NASPM, suggesting the presence of functional Ca2+-permeable AMPA receptors in OECs. Additionally, kainate application evoked Ca2+ transients in OECs which were abolished in the absence of extracellular Ca2+, indicating that Ca2+ influx via Ca2+-permeable AMPA receptors contribute to kainate-induced Ca2+ transients. However, kainate-induced Ca2+ transients were partly reduced upon Ca2+ store depletion, leading to the conclusion that Ca2+ influx via AMPA receptor channels is essential to trigger Ca2+ transients in OECs, whereas Ca2+ release from internal stores contributes in part to the kainate-evoked Ca2+ response. Endogenous glutamate release by OSN axons initiated Ca2+ transients in OECs, equally mediated by metabotropic receptors (glutamatergic and purinergic) and AMPA receptors, suggesting a prominent role for AMPA receptor mediated Ca2+ signaling in axon-OEC communication.Ca Animals were analyzed 7–12 days after the first injection. OBs were prepared as described previously -glucose, and 2.5 MgSO4 × 7 H2O). Standard artificial cerebrospinal fluid (ACSF) for experiments and storage of the preparations consisted of (molarities in mM): 120 NaCl, 2.5 KCl, 1 NaH2PO4x2H2O, 26 NaHCO3, 2.8 D-(+)-glucose, 1 MgCl2, and 2 CaCl2). Preparation solution and ACSF were continuously perfused with carbogen (95% O2 and 5% CO2) to maintain the pH of 7.4 and to supply oxygen.Mice of the GLAST-Cre strains  were kep strains . Animal eviously . Both OBS,3S,4S)-3-(Carboxymethyl)-4-(prop- 1-en-2-yl)pyrrolidine-2-carboxylic acid , (S)-α-Amino-3-hydroxy-5- methylisoxazole-4-propionic acid, 4--benzenamine hydrochloride , (E)-ethyl 1,1a,7,7a-tetrahydro-7-(hydroxyimino) cyclopropa[b]chromene-1a-carboxylate , 2-methyl- 6-(phenylethynyl) pyridine , 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate , 4-(2-triazolo triazin-5-ylamino]ethyl) pheno , (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine  and carbenoxolone disodium salt  were obtained from Abcam . The reagents D-2-amino-5-phosphonovaleric acid , N-[3-[[4-[(3-aminopropyl)amino]butyl] amino]propyl]-1-naphthaleneacetamide trihydrochloride (Naspm trihydrochloride antagonist of Ca2+-permeable AMPA receptors) and tetrodotoxin  were received from Alomone labs . All reagents were stored as stock solutions corresponding to the manufacturer’s instructions and added to ACSF directly before the experiment. Drugs were applied via the perfusion system driven by a peristaltic pump .The compounds . Slices were transferred into a recording chamber and continuously superfused with ACSF via the perfusion system. The experiments were performed at room temperature. Whole-cell voltage-clamp recordings  were performed on OECs, identified by tdTomato expression , and the distinct localization in the ONL  of GLAST-Cre the ONL . Recordi2+ imaging experiments, GLAST-CreERT2 × tdTomatofl/fl and NMRI mice were used. Whole OBs were glued onto coverslips, transferred into a recording chamber and the coverslip secured with a U-shaped platinum wire. For multi-cell bolus loading , followed by an incubation of 20 min. Changes in the cytosolic Ca2+ concentration in OECs were detected by the fluorescence of Fluo-8  using a confocal microscope . Images were acquired at a time rate of one frame every 3 s. A similar perfusion system and flow rate was used as described above. However, the experimental bath for Ca2+ imaging experiments was differently shaped  resulting in differences in the kinetics of wash-in and wash-out of drugs between the electrophysiological and Ca2+ imaging experiments. To analyze changes in cytosolic Ca2+ in single cell somata, regions of interest (ROIs) were defined using Nikon EZ-C1 3.90 software. OECs were identified by GLAST promoter-driven tdTomato expression in GLAST-CreERT2 × tdTomatofl/fl mice in most experiments. In wild type mice, OECs were identified by their distinct localization in the ONL. The changes in Ca2+ were recorded throughout the experiments as relative changes in Fluo-8 fluorescence (ΔF) with respect to the resting fluorescence, which was normalized to 100%. Quantification of the Ca2+ transients was achieved by calculating the amplitude of ΔF.For Ca loading , a glassn = x, where x is the number of analyzed cells. At least 3 animals were analyzed for each set of experiments. Statistical significance was estimated by comparing three means using Friedmann ANOVA and the Wilcoxon post hoc test for paired data, and for comparing two means using the Mann–Whitney U-Test, with the error probability p .All values are stated as mean values ± standard error of the mean. The number of experiments is given as ERT2 × tdTomatofl/fl mice (≥P28) was performed as described before , which form complex heteromeric cation channels, activated by glutamate or selective receptor agonists such as AMPA and kainate . The comfor Ca2+ . The OB for Ca2+  and immufor Ca2+ . We aimepression . OECs arpression , 1991. T to OECs . GluA1 i to OECs . Homogen to OECs . In the  to OECs . Additio to OECs . At highERT2 × tdTomatofl/fl mice. OECs were identified by GLAST promoter-driven tdTomato expression in the ONL , suggesting that the kainate-induced response is mainly mediated by AMPA receptors in OECs  , suggesting that OECs exhibit functional Ca2+-permeable AMPA receptors  . NASPM receptors .2+ transients in Bergmann glial cells of the cerebellum result from Ca2+ influx directly through the AMPA receptor channel (2+ imaging experiments to investigate the source of Ca2+ that fuels Ca2+ transients in OECs in response to kainate application. The Ca2+ indicator Fluo-8 AM was pressure injected into OB in-toto preparations of GLAST-CreERT2 × tdTomatofl/fl mice (2+ transients in OECs with a mean amplitude of 107.8 ± 5.3% ΔF. GYKI 53655 (100 μM), an AMPA receptor antagonist, greatly reduced kainate-evoked Ca2+ transients in OECs by 84.0 ± 6.1% of the control , showing that kainate-evoked Ca2+ transients are mediated by AMPA receptors . Kainate-evoked Ca2+ responses recovered after readdition of extracellular Ca2+, the average amplitude reached 122.0 ± 18.3% of the control (p = 0.16)  to inhibit Ca2+ uptake into Ca2+ stores via SERCA . Kainate-induced Ca2+ transients were significantly reduced by 57.6 ± 4.2% upon Ca2+ store depletion   . To esti × 10–8) . Our res in OECs .2+ rises in OECs  and recorded Ca2+ transients in Fluo-8-loaded OECs in wild type animals. Electrical stimulation of OSN axons induced Ca2+ transients in OECs with an amplitude of 51.8 ± 5.0% ΔF (n = 46). By eliminating purinergic (MRS2179), dopaminergic (SCH23390) and mGluR1-mediated Ca2+ transients (CPCCOEt) as well as possible indirect NMDA-mediated neuronal responses (D-APV), the mean amplitude of stimulation-induced Ca2+ transients in OECs were significantly reduced to 61.0 ± 9.3% of the control (p = 3.68 × 10–4). Additional inhibition of Ca2+-permeable AMPA receptors by the selective antagonist NASPM further reduced the mean amplitude to 30.6 ± 5.8 (p = 1.36 × 10–5) of the control. NBQX almost completely abolished stimulation-induced Ca2+ transients in OECs and the amplitude further decreased significantly to 13.01 ± 3.3% of the control (p = 0.002). Receptor inhibition was reversible and stimulation-induced Ca2+ transients in OECs recovered after wash out  and even a small Ca2+ increase generates a fast and large Ca2+ transient by CICR (2+ influx through AMPA receptors upon kainate application could induce CICR that boosts the Ca2+ increase and accounts for the fast rising kinetics of the Ca2+ signal as compared to the rising phase of the kainate-evoked membrane current. We conclude that Ca2+ influx is the initial Ca2+ signal that triggers subsequent internal Ca2+ release such as CICR, as shown by the entire suppression of the Ca2+ signal by external Ca2+ withdrawal but only partial reduction upon Ca2+ store depletion.While the presence of GluA2 in OECs argues against Carization . However signals . In addieceptors . Hence,  by CICR . Thus, C2+ signaling in OECs may serve different functions. In other glial cells such as astrocytes, increases in the cytosolic Ca2+ concentration has been reported to evoke release of so-called gliotransmitters that affect nearby neurons and synapses, but also release of vasoactive substances such as prostaglandins and arachidonic acid (2+ signals (2+ in OECs results in constriction of adjacent capillaries (2+ signals evoked by glutamate release from axons in the ONL and subsequent activation of Ca2+-permeable AMPA receptors may lead to vasoresponses that adjust blood flow to the metabolic demand upon action potential firing, a mechanism termed neurovascular coupling.Canic acid . OECs ennic acid  and respillaries . Hence, 2+ transients in OECs that are partially mediated by AMPA receptors. AMPA receptor-mediated Ca2+ transients were due to Ca2+ influx, most likely through Ca2+-permeable AMPA receptors themselves, and subsequent Ca2+ release from internal Ca2+ stores.In summary, our results show that release of glutamate from axons of OSN results in CaThe datasets generated for this study are available on request to the corresponding authors.The animal study was reviewed and approved by GZ G21305/591-00.33; Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany.AB and CL designed the experiments and wrote the manuscript. AB performed the experiments and analyzed the data.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.