The results showed that RC displayed a high response heterogeneity in a cell-dependent manner following serum stimulation. Most of these responses relied on inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release associated with Ca2+ influx, partly due to store-operated calcium entry. Our study further found that blocking the IP3 production, Ca2+ influx, or both did not prevent the activation of RC. Intra- or extracellular Ca2+ chelation did not impede RC activation. However, their migration potential depended on Ca2+ responses displayed upon stimulation, and Ca2+ blockers inhibited their movement. We conclude that the two major steps of muscle regeneration, namely the activation and migration of MuSC, differently rely on Ca2+ signals.Muscle regeneration is essential for proper muscle homeostasis and relies primarily on muscle stem cells (MuSC). MuSC are maintained quiescent in their niche and can be activated following muscle injury. Using an in vitro model of primary human quiescent MuSC , we analyzed their Ca It requires the presence of muscle stem cells (MuSC), formerly called satellite cells, which are nonproliferating cells maintained in a quiescent state and able to be activated, meaning they can re-enter the cell cycle in response to specific stimuli. MuSC activation occurs, for example, after muscle fiber injury  and is teristics ,9. Theseeristics . RC actieristics ,12.2+ signaling is essential for myotube differentiation [2+ entry (SOCE) was shown to be essential for muscle differentiation [2+ selective Orai channels by the endoplasmic/sarcoplasmic reticulum (ER/SR)-localized STIM proteins, which are activated upon Ca2+ store depletion [2+ signals in MuSC activation. Both Ca2+ release from the stores and Ca2+ influx have been described as sources of cytosolic Ca2+ elevation depending on the type of stimulation, namely release from the ER/SR through ryanodine receptors in amphibian larvae [2+ channels linked to mechanical stress [2+ response occurring in MuSC upon activation and its possible link with the fate of these cells is missing, and almost nothing is known about the role of Ca2+ signals in RC in humans.Cantiation ,14,15, mntiation , and thentiation . In partntiation ,14,17,18epletion . In addin larvae , influx l stress , or tranl stress  in murin2+ response of RC after serum stimulation and uncovered a great heterogeneity of Ca2+ signals, which relied on Ca2+ release and, to a variable extent, on Ca2+ entry. We then established whether the Ca2+ responses were causally linked to the fate of RC in terms of activation and migration. We found that their re-entry into the cell cycle occurred independently of any Ca2+ response, while the pattern of Ca2+ signals affected their migration during the 48 h following serum stimulation.In this work, we used an in vitro model of human quiescent RC to study the first steps of their activation. This model allows cell activation to be precisely distinguished from subsequent cell proliferation. We first analyzed the CaHuman primary myoblasts were isolated from semitendinosus muscle samples obtained after orthopedic surgery  on patients without known muscular diseases. All samples were collected anonymously after obtaining written consent and approval from the University of Geneva (protocol CCER no. PB_2016-01793 (12-259) and was accepted by the Swiss Regulatory Health Authorities and approved by the “Commission Cantonale d’Ethique de la Recherche” from the Geneva Cantonal Authorities, Switzerland). The purification of myoblasts was performed as previously described ). The cu® 488-conjugated goat antimouse IgG  and Alexa Fluor® 546-conjugated goat antirabbit IgG .Cells were fixed in PBS with 4% paraformaldehyde, permeabilized, and blocked in PBS containing 0.3% of Triton X-100 and 5% goat serum. Immunofluorescence was performed by overnight incubation at 4 °C with the following primary antibodies: mouse anti-α-actinin  and rabbit anti-MEF2c . Secondary antibodies were incubated for 75 min at room temperature (RT) with Alexa Fluor® Gold Antifade Reagent with DAPI . Images were acquired with a widefield AxioImager M2 microscope  through a 20× objective (EC Plan-Apochromat 20×/0.8).RC activation and myoblast proliferation were determined by EdU assay  based on the provider’s protocol. Nuclei were stained using ProLong2+ on activation and/or proliferation, cells were stimulated with GM supplemented by chemicals including the phospholipase C inhibitor U73122  or its inactivated control U73343 , the G-protein-coupled receptor αq inhibitor YM254890 , the inhibitor of Orai1 and -2 channels GSK7975a , EGTA (0.3 and 0.6 mM), Gd3+ (50 µM), and BAPTA-AM . In the same way, cells were stimulated with DM supplemented with endothelin-1 . To estimate the proportion of activated and nonactivated RCs, we quantified the proportion of DAPI+/MEF2c−/EdU+ cells compared to the DAPI+/MEF2c− cell population. Analysis of the immunofluorescence images was performed using ImageJ software.To assess effect of Ca2, 10 mM HEPES, 2 mM CaCl2, and 10 mM glucose, with pH adjusted at 7.45 with NaOH (Ca2+ medium). Following the incubation, cells were washed and kept for 10 min in the same medium to allow de-esterification of the dye. Calcium-free medium (CF medium) was composed of 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, and EGTA 1 mM, with pH adjusted at 7.45 with NaOH.Cells were loaded with Fura-2-AM  at room temperature and in the dark for 30 min in a medium containing 135 mM NaCl, 5 mM KCl, 1 mM MgCl2+ signals were recorded using a ZEISS Axio Observer A1 microscope equipped with a Lambda XL illumination system , with the excitation wavelengths switched between 340 nm  and 380 nm . Emission was collected through a 415 DRLP dichroic mirror and a 510WB40 filter  by a cooled 16-bit CMOS camera . Image acquisition was performed with the VisiWiew software , and the analysis was done with Fiji and MATLAB software. Cells were recorded for 30 min after stimulation, with a ratio taken every 2 s. RC were stimulated with CA medium containing 15% serum, and cells were treated with the same chemicals described previously. Stimulations with endothelin-1  and ATP  were performed in CA medium.Ratiometric images of Ca2+ was recorded using Cal520-AM , and nuclei were labeled with SPY650-DNA  to follow cell movement and allow their individual identification. After incubation in the dark at 37 °C in DM for 80 min, the cells were washed and kept for 20 min in DM to allow de-esterification of the Cal520-AM dye. Cells were stimulated with GM, and cytosolic Ca2+ concentration was measured for 10 min at a 20× magnification with a picture taken every 15 s. After this initial Ca2+ recording, pictures of the phase contrast and SPY650 of the same field were captured at a magnification of 10× for 48 h with one picture taken every 15 min. Experiments were performed on a ZEISS Axio Observer Z1 with definite focus 2, thermoregulated at 37 °C, and CO2 controlled. Timelapse recordings of the Ca2+ signals and tracking experiments were automatically registered using phase correlation after upsampling of the last frame of the Ca2+ acquisition to reach the same lateral resolution as the one used for the tracking experiment. This allowed the identification and classification of the tracked cells according to their Ca2+ response category. The tracking was performed automatically with Imaris 9.7.2  using the spot module under systematic user supervision and with a manual correction to minimize cell position errors. Thereafter, the tracking information (cell positions and time) was processed with MATLAB R2021a  to compute different features, such as the maximum speed and the mean displacement (average distance between two consecutive timepoints).To reduce UV phototoxicity, cytosolic Cap < 0.05, ** p < 0.01, ***p < 0.001, and **** p < 0.0001. For comparison of two populations, T-test or Mann–Whitney test were used depending on the normality of the data set. For comparison of more than two populations, ANOVA or Kruskal–Wallis test were used depending on the normality of the data set.Data are presented as mean ± SEM, and statistical differences were determined using the test specified in the legend of each figure, where * 2+ medium supplemented with 15% fetal calf serum (serum stimulation) and analyzed the Ca2+ response for 30 min. Serum stimulation triggered a strong Ca2+ response in RC, while no response was observed in myotubes -induced Ca2+ release by stimulating the cells in the presence of a PLC (phospholipase C) blocker (U73122) or a GPCRαq (G-protein-coupled receptor) inhibitor (YM254890) . Surprisingly, a significant proportion of cells (around 25%) did not respond with an initial Ca2+ elevation but started to display Ca2+ oscillations of rather large amplitude after 4–7 min , which was decreased by 79 and 72% after treatment with U73122 and YM254890, respectively  A–C. The ound 60% C. In cel 4–7 min B,C. To aectively D. From t2+ entry inhibition using GSK7975a, an inhibitor of Orai1 and -2 channels [2+ entry blocker Gd3+ . This led to a strong, but not full, inhibition of more than 90% of the Ca2+ response  to serum stimulation , which prevented intracellular Ca2+ signals by around 80% (data not shown). None of these maneuvers impacted the ability of RC to activate upon serum addition . Endothelin-1 receptor B (EDNRB) is expressed almost exclusively in the human reserve cells compared to myotubes and proliferating myoblasts . This treatment induced a robust Cativation . Howevertivation , showing2+ independency of RC activation, we investigated whether their migration potential upon serum activation was dependent on their patterns of Ca2+ responses. To this end, we first recorded the Ca2+ responses induced by serum addition before following the same cells for 48 h to evaluate their movement . This protocol enabled us to image the RC migration for 48 h without increasing the mortality of the cells but with a less precise Ca2+ response classification, thereby allowing us to only categorize the RC response as “no response”, “single Ca2+ transient”, and “oscillating pattern”. Cells were imaged for 48 h using phase contrast to differentiate the myotubes from the RC and using SPY650 staining to label the nuclei and track the cells . Accordingly, an increase in the maximum speed was also observed in RC with Ca2+ response   E,F. This2+ signals  cells. According to a previous study [2+ patterns were consistently observed between the experiments, while their proportion slightly varied between donors . Around 90% of the cells responded with an initial Ca2+ elevation due to IP3R-induced Ca2+ release. This was followed or not by Ca2+ oscillations associated with a phase of sustained Ca2+ elevation in some cells. The second phase of the response was absent in experiments performed in a Ca2+-free medium, confirming the necessity of Ca2+ entry to maintain Ca2+ oscillations [2+ entry is through the SOCE pathway. Indeed, the SOCE blocker GSK7975a [3+, a more general Ca2+ influx blocker, suppressed the plateau phase responses, and the AUC of the response was decreased by around 60% overall. However, surprisingly, neither GSK7975a nor Gd3+ prevented Ca2+ oscillations from taking place. Hence, it appears that Ca2+ entry occurs in part through the SOCE pathway but also through another route that remains to be determined. Potential candidates are the arachidonic-acid-regulated Ca2+-selective (ARC) channels [3), even though one would expect these channels to be blocked by Gd3+. Alternatively, it could be that only a very limited entry of Ca2+ (not prevented by the blockers we used) was sufficient to drive the oscillations. When both SOCE and Ca2+ release were inhibited, it resulted in a strong reduction in the Ca2+ response to serum, with a decrease in AUC of more than 90%. However, a peculiar response consisting of the absence of initial Ca2+ elevation but followed by delayed oscillations was more frequently observed after blocking IP3 production. We did not investigate this type of Ca2+ pattern further, but it might be due to the production of other second messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), acting on acidic compartments [2+ response is likely the consequence of using cell stimulation by serum, which contains hundreds of proteins that activate multiple pathways, with some resulting in Ca2+ signaling. Indeed, the Ca2+ response stimulated by endothelin-1 was fully abolished by blocking Ca2+ release and entry, without the appearance of delayed oscillations (data not shown). Hence, endothelin-1 triggers a “simpler” response compared to serum and strongly suggests that at least two intracellular signaling pathways are activated by serum, thereby leading to cytosolic Ca2+ elevation. While this complicates a comprehensive analysis of Ca2+ response, stimulation by serum better mimics the physiological situation where multiple factors activate MuSC concomitantly [The Caus study  and our llations ,28. One GSK7975a  and Gd3+channels  and TRP artments ,31. The mitantly .2+ response negatively impacted RC activation, even those that almost entirely prevented cytosolic Ca2+ elevation, such as the combination of YM254890 and GSK7975a or the incubation of cells with BAPTA-AM. These results strongly indicate an RC activation process largely independent of Ca2+ signals. Another argument in favor of the Ca2+ independency of the RC activation process is the stimulation by rndothelin-1, which induced a robust Ca2+ response. Even though the Ca2+ responses were similar to the one induced by serum, no activation of RC was induced, leading us to conclude that a rise in cytosolic Ca2+ concentration per se is insufficient to promote cell activation. These results imply that RC activation depends on other stimuli present in serum in addition to those inducing the Ca2+ response. In contrast, the proliferation of myoblasts was decreased when cytosolic Ca2+ fluctuations were prevented by the presence of BAPTA-AM or when a low extracellular Ca2+ medium was used, showing that cell proliferation is a Ca2+-dependent process, as reported on many different cellular systems [2+ signals is reminiscent of findings obtained in the 1970s showing that DNA synthesis of cancer cells  was largely insensitive to Ca2+ concentration and that cancer cells were able to grow in very low extracellular Ca2+ environment [2+ and pointed to a role of Ca2+ channels, but not necessarily Ca2+ entry, as being important for cell proliferation, at least in some cellular systems [2+ entry, and in particular SOCE, for cancer cell proliferation [0 phase and the initial DNA synthesis, takes place regardless of the Ca2+ concentration. Once this first step is achieved, the myoblasts then require the generation of Ca2+ signals for their proliferation. Thus, our in vitro approach using quiescent RC allows clear differentiation between activation and proliferation.In the control condition, serum stimulation promoted re-entry into the cell cycle of around 30% of RC measured after 24 h. Unexpectedly, none of the procedures that limited the Ca systems ,34,35,36 systems . It is tironment ,39,40. M systems . Howeverferation ,43. From2+ response of MuSC after stimulation. One study recorded the Ca2+ response of MuSC still attached to freshly isolated FDB (flexor digitorum brevis) fibers with a very low sampling rate (one point every 5 min) [2+ elevation that was prevented by the TRPC channel blocker SK&F 96365. This Ca2+ signal was associated with the transcription factor MyoD expression and the translocation of NFAT1 (nuclear factor of activated T-cells 1) and NFAT4 into the nucleus. Both events were blocked by SK&F 96365, leading to the conclusion that satellite cell activation by FGF2 (fibroblast growth factor 2) is a process dependent on Ca2+ signaling [2+ elevation of isolated satellite cells. Ca2+ elevation was due to the sequential activation of stretch-activated cation channels (blocked by GsMTx-4) followed by voltage-gated L-type Ca2+ channel opening (blocked by nifedipine). Increase in Ca2+ eventually stimulated NO (nitric oxide) production, which in turn led to MMP2  production by MuSC that releases HGF (hepatocyte growth factor) from the extracellular matrix. Finally, by binding to its receptor c-met, HGF activated MuSC [2+ entry, stimulation of the cells directly with HGF promoted MuSC activation independently of Ca2+ entry  . Cell stignaling . Anotherted MuSC . Interes2+ dependent, with Ca2+-dependent protease calpain notably acting at the rear of the cells, inducing proteolysis of talin [2+ gradient from rear to front is established in migrating cells [2+ release events occur at the front [2+ response with YM254890 and GSK7975a had a significant impact on the RC mobility, with both the speed and distance covered by the RC reduced during the 48 h of analysis. We measured the Ca2+ response of each RC for 10 min and followed the exact same cells for the next 48 h to determine a possible correlation between the pattern of Ca2+ responses and the migration capacity. We separated the cells into three categories, namely nonresponders, those displaying only a single Ca2+ transient, and those having an oscillatory response. In line with reduced movement in the presence of Ca2+ blockers, the nonresponding cells moved less and slower than the responding ones. Cells exhibiting only a single Ca2+ transient displayed a significantly higher movement during the initial 24 h compared to those showing Ca2+ oscillations. However, no significant difference was observed between these two populations in the last 24 h of recording. To note, even the cells that did not display a cytosolic Ca2+ elevation during the first 10 min of serum stimulation presented a higher migration capacity than cells treated with Ca2+ blockers. This could be explained by the almost complete prevention of any Ca2+ signals for 48 h in the presence of blockers, while the cells classified as nonresponders might have generated Ca2+ signals after the initial 10 min of the recording. Interestingly, irrespective of the type of Ca2+ response (even no response), the cells moved less overall during the initial 24 h of serum stimulation, with the movement accelerating after 24 h.MuSC activation is accompanied by cell migration in the repair site, an essential step toward muscle regeneration . Migratiof talin  and FAK of talin . It has ng cells . Ca2+ rehe front , while She front . We anal2+ responses elicited by in vitro MuSC upon serum stimulation. These responses displayed very heterogeneous patterns that were surprisingly not correlated to the ability of RC to exit the G0 phase and enter the cell cycle. In contrast, the pattern of Ca2+ responses influenced the migratory potential of these cells. Hence, we have revealed that the two important steps of muscle regeneration, namely the activation and migration of MuSC, differently rely on Ca2+ signals.In conclusion, we have provided, for the first time to our knowledge, detailed analysis of the Ca


Acute myeloid leukemia post cytotoxic therapy (AML-pCT) is a rare complication of cancer treatment in childhood. We retrospectively analyzed the data of 40 children with AML-pCT, treated from 2005 to 2020. The most common primary malignancies were acute lymphoblastic leukemia and brain tumors. The probabilities of overall (OS), event-free (EFS), and relapse-free survival (RFS) in whole cohort were 0.49 ± 0.08, 0.43 ± 0.08, and 0.64 ± 0.10, respectively. Significant improvements in outcomes were observed in patients treated from 2015 to 2022 (two induction cycles followed by stem cell transplantation—SCT in 69% of patients) compared to the period 2005–2014 (four induction cycles followed by SCT in 49% of patients). The probabilities of EFS and RFS increased from 0.30 ± 0.10 and 0.46 ± 0.11 to 0.67 ± 0.12 and 1.0, respectively. The poorest outcome was found in AML post brain tumor, mainly due to toxic deaths. Treatment results in the group of patients with AML-pCT treated from 2015–2022 were comparable to outcomes in de novo AML.p = 0.07) and RFS increased from 0.46 ± 0.11 to 1.0 (p = 0.01). The poorest outcome (OS and EFS 0.25 ± 0.20) was in AML post brain tumor, mainly due to deaths from toxicities. To conclude, treatment results achieved in patients with AML-pCT treated from 2015–2022, with two induction cycles followed by immediate SCT, were better than those reported by other authors, and comparable to the results in de novo AML.Acute P./myeloid leukemia post cytotoxic therapy (AML-pCT) is rare complication of cancer treatment in childhood. The objective of the study was to identify clinical characteristics and provide an analysis of the outcomes in pediatric AML-pCT. We retrospectively analyzed the data of 40 children with AML-pCT, treated from 2005 to 2020 within the Polish Pediatric Leukemia and Lymphoma Study Group. The most common primary malignancies were acute lymphoblastic leukemia (32.5%) and brain tumors (20%). The median latency period was 2.9 years (range: 0.7–12.9). Probabilities of overall (OS), event-free (EFS), and relapse-free survival (RFS) in the whole cohort were 0.49 ± 0.08, 0.43 ± 0.08, and 0.64 ± 0.10, respectively. Significant improvements in outcomes were observed in patients treated from 2015–2022 (two induction cycles followed by stem cell transplantation—SCT in 69% of patients) compared to 2005–2014 (four induction cycles followed by SCT in 49% of patients). The probability of EFS increased from 0.30 ± 0.10 to 0.67 ± 0.12 ( Secondary malignancies are a rare complication of cancer treatment in childhood. Acute myeloid leukemia post cytotoxic therapy (AML-pCT), formerly known as therapy-related AML, is the most common secondary malignancy in childhood . The latThe majority of AML-pCT in adults is associated with TP53 mutations . HoweverThe prognosis in AML-pCT is worse compared to de novo AML with a 5-year OS of 20–55% ,8,9. TheHere, we present a retrospective analysis of the patients with AML-pCT registered from 2005 to 2022 in the nationwide AML database of the Polish Pediatric Leukemia and Lymphoma Study Group (PPLLSG). The objective of the study was to determine the incidence of AML-pCT and describe the clinical characteristics and outcome of AML-pCT in children.9/L, platelets 100 × 109/L, and no evidence of extramedullary involvement). Early death was defined as a death before the assessment of response after two inductions (42nd–56th day of therapy). Probabilities of survival were calculated with the Kaplan–Meier method. Overall survival (OS) was calculated from the diagnosis of AML-pCT to death from any cause or the last follow-up. Event-free survival was calculated from the diagnosis of AML-pCT to the first event  or last follow-up. Failure to achieve morphological remission was considered an event on day 0. Relapse-free survival (RFS) was calculated as the time from the first remission to the relapse. Probabilities of survival were presented as decimal fractions with standard errors (SE). The sub-groups were compared with a log-rank test.There were 823 children with AML registered from January 2005 to June 2022 in the nationwide AML database of the PPLLSG . In 40 patients from the whole cohort (4.9%), preceding malignancy was reported. Those patients were enrolled to this retrospective study. We analyzed data concerning the diagnosis of underlying cancer, therapy of the primary cancer, family history, time interval between primary diagnosis and AML-pCT diagnosis (latency period), characteristics of AML-pCT , therapy of AML-pCT, and the treatment outcome. Most patient were treated according to current therapeutic protocols for AML. The AML-BFM 2004 Interim protocol (2005–2014) did not contain special recommendations for patients with AML-pCT, while both the AML-BFM 2012 Registry (2015–2019) and AML-BFM 2019 (2019–2022) recommended treatment with AIE and HAM, or Ida-FLA and FLA , dependiQuantitative variables were compared within subgroups with the Mann–Whitney U test or Kruskal–Wallis test with a post hoc test (for more than 2 variables) and qualitative variables with a chi-square test or Fisher’s exact test.All statistical analyses were performed with STATISTICA, version 13, TIBCO Software Inc, Palo Alto, CA, USA. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Jagiellonian University . Forty patients were enrolled onto the retrospective study, including twenty-four patients in the first period (2005–2014) and sixteen in the second period (2015–2022). The patients’ characteristics are summarized in Primary diseases included acute lymphoblastic leukemia , brain tumors , neuroblastoma , rhabdomyosarcoma , osteosarcoma (three patients), retinoblastoma , hemophagocytic lymphohistiocytosis , synovial sarcoma (one patient), juvenile myelomonocytic leukemia , nephroblastoma (one patient), immature teratoma (one patient), acute myeloid leukemia (one patient). In one patient, there were two malignancies preceding AML—synovial sarcoma and osteosarcoma. As a treatment of the first malignancy, all patients received chemotherapy. Fourteen patients also received radiotherapy and four patients received hematopoietic stem cell transplantation (SCT). Most patients were treated with multiagent chemotherapy for the primary disease. Thirty one children (77.5%) received both alkylating agents and topoisomerase II inhibitors, seven patients (17.5%) were treated with alkylating agents without topoisomerase II inhibitors, two children (5%) received only topoisomerase II inhibitors. Details of the chemotherapy used in the treatment of the primary disease are shown in The median latency period was 2.9 years (range: 0.7–12.9 years). No significant difference in latency period was found depending on primary disease, kind of chemotherapy , or use of radiotherapy or SCT in the therapy preceding AML. The details are presented in The AML-pCT characteristics are presented in A karyotype was available in 27 patients (67.5%). Analysis revealed abnormalities in the karyotype in 23 (85.2%) of those patients. A complex karyotype was found in five children (18.5%). Aneuploidies were revealed in fourteen patients (51.8%), including monosomy 7 in four children (14.8%), monosomy 5 and trisomy 8 together in one of them, monosomy Y in three (11.1%), trisomy 8 in two (7.4%), and trisomy 13, trisomy 11, monosomy 5, monosomy 6, monosomy 8, monosomy 17, and monosomy X in one patient each. Results of molecular diagnostics were available in 30 patients (75%) and revealed KMT2A rearrangements in nine patients (30%), including KMT2A::MLLT3 , KMT2A::MLLT1 , KMT2A::AFF1 ; RUNX1::RUNX1T1 fusion in four children (13.3%), and CBFβ::MYH11 fusion in one patient (3.3%). There was no FLT3 mutation found in our cohort. Results of genetics depending on primary cancer is presented in In the first period (2005—2014), 23/24 patients were treated according to standard AML protocols (AML-BFM 2004 Interim). One received Ida-FLA and FLA cycles, eleven children (46%) were transplanted, most of them (9/11) received four therapy cycles before SCT, and two patients had two cycles. In the second period (2015–2022), 14/16 patients were treated with standard chemotherapy , two children received individualized therapy, eleven patients (69%) were transplanted, most of them (10/11) received two chemotherapy cycles before SCT, and one received four cycles . n = 5), NBL, RMS, RBL ,and brain tumor (each n = 1). The median time from AML-pCT diagnosis to relapse was 10.4 (range: 1.2–27.6) months. There were five patients who relapsed after SCT. Four patients relapsed (44.4%) after achieving second remission, five (55.6%) died of the disease progression, and one patient died in second CR (CRII) because of transplantation related toxicities. Three patients remain in CRII. In one patient (first period), a third malignancy—diffuse large B-cell lymphoma (DLBCL)—occurred 60 months after the AML-pCT diagnosis. The child finally died in the course of the disease. His primary neoplasm was ALL, diagnosed at the age of 14. Treatment results in the whole cohort and two periods are presented on Thirty one patients (77.5%) achieved complete remission. There were two non-responders (5%), one from each period (2005–2014 and 2015–2022), and both died in the course of the disease progression. One of them was an almost 10-year-old boy, who died 51 months after a NBL diagnosis, with no genetic results available, and 10 months after the AML-pCT diagnosis. The second non-responder was an almost 18-year-old girl with AML-pCT recognized 25 months after an ALL diagnosis. Her karyotype was normal; no fusion gene was found in molecular diagnostics, a WT1 mutation was revealed without an FLT3 mutation. She died 2 months after the AML-pCT diagnosis. Early death occurred in five patients (12.5%), with four from the first period and one from the second, including three patients after brain tumors, one patient after ALL, and one after RMS treatment. In all of them, death was caused by severe infection and multiorgan failure. Two children (5.0%), one from each period, died in course of aplasia without complete remission more than 45 days from the start of therapy. There was one patient after brain tumor therapy and one after RMS who died as a result of severe infections. Three patients (7.5%) died in remission: one in the first period, two in the second, and all of them because of transplantation related toxicities. Of these, there was one patient who died after brain tumor, one after osteosarcoma, and one after immature teratoma. Relapse occurred in nine patients (22.5%), all from the first period. Primary malignancies in that group included ALL . There was trend to a better 3-year EFS in the second period . The probability of OS did not differ significantly between the groups , solid tumors , and brain tumors (n = 8). The poorest outcome was observed in AML secondary to brain tumors, with a significantly lower probability of OS (p = 0.023) and trend to lower EFS (p = 0.079). The probability of RFS did not differ significantly depending on the primary diagnosis. Survival curves are presented in We compared survival depending on the primary diagnosis. Three groups were distinguished: systemic diseases . No significant difference in OS, EFS, and RFS was found between patients who underwent SCT and those who did not .In this retrospective study, we identified 40 patients with AML-pCT in a nationwide registry from a 17.5-year period. Taking into account the average number of newly diagnosed children with malignancies in Poland per year—1100–1200 ,4,7,11. Acute lymphoblastic leukemia, osteosarcoma, Ewing sarcoma, Hodgkin and non-Hodgkin lymphomas, NBL, RBL, and brain tumors are the most frequent primary diseases preceding AML-pCT reported by other authors ,6,7,8. TThe median latency period in our study was 2.9 years, similar to that reported by other authors ,9,12,13.Cytogenetic analysis revealed an abnormal karyotype in 85.2% of the children in our cohort. A complex karyotype was found in 18.5% of the patients. Monosomy of chromosome 7 was identified in almost 15% of the children. The most frequent fusion genes identified in our cohort were: KMT2A rearrangements (30%) and RUNX1-RUNX1T1 13.3%). No FLT3-ITD was identified in our cohort. Similar genetic changes were described by other authors. In the study of Tsurusawa et al., chromosomal abnormalities were detected in 94% of patients, abnormalities of chromosomes 5 and/or 7 in 41%, and KMT2A abnormalities in 31% [.3%. No FIt was reported in previous studies that prior exposure to topoisomerase II inhibitors may be associated with translocations involving KMT2A rearrangements and RUNX1 translocations ,15,16, wThe treatment of AML-pCT is challenging, taking into account the previous cytotoxic therapy for primary disease, poor hematopoietic reserves, organ dysfunction, and possible colonization with antibiotic-resistant bacteria and fungi as a results of a chronic immunosuppression. In most patients, AML-like induction therapy followed by SCT is used ,8,12. Inn = 70 1993–2003) to 34 ± 7% , then up to 45 ± 9%  [Despite these efforts, outcomes in pediatric AML-pCT remains worse compared to de novo AML ,7,9,13. n = 17) and the 3-year OS in that group was 0.46, comparable to the mentioned study. We analyzed the influence of primary disease on outcome. We found the poorest outcome in AML secondary to brain tumors compared to systemic malignancies and solid tumors. The main cause of deaths in that group were toxicities. Similarly, the study of Waack et al. revealed that AML following brain tumors as the primary malignancy showed the worst prognosis . SchmiegMost authors agree that AML-type induction chemotherapy followed by SCT as a post-remission therapy is the most effective way of treating AML-pCT. A significant survival advantage for patients who underwent SCT was reported by many investigators. Brown et al. showed a 5-year survival of 52.4% in patients treated with SCT compared to 5.7% for those who did not have SCT . SchmiegThe major limitation of our study was the small patient numbers, and some missing data, especially concerning cytogenetics and molecular genetics. That precluded robust analyses and the comparison of subgroups. However, most of the previous studies included a similar number of even fewer patients ,11,12,13Considering that complications were the main cause of treatment failure in our group, it seems that therapy with sufficient efficacy but reduced toxicity, especially in patients heavily pretreated due to primary cancer, may be of key importance for improving outcomes in AML-pCT. The promising results of CPX-351, a fixed 5:1 molar ratio of liposomal cytarabine to daunorubicin, were obtained by Hu et al. The study comprised five pediatric patients with AML-pCT treated with CPX-351. All of them achieved CR, four were transplanted and remained in CR, and one relapsed and died of disease progression before SCT . CPX-351TP53-mutated cancer cells, and flotetuzumab, a bispecific antibody binding CD123 and CD3, anti-CD123 CAR-T [Research on the use of targeted therapies in pediatric AML may also contribute to improving the outcomes of AML-pCT. Unfortunately, due to the small number of children with AML-pCT and the significant heterogeneity of this disease, it is very difficult to conduct prospective studies in this group. Most studies concern adult patients. Gemtuzumab ozogamicin (GO) is the only antibody–drug conjugate approved by both the Food and Drug Administration and European Medicines Agency in newly diagnosed and refractory CD33-positive AML for patients aged 1 month or older. GO used in combination with standard chemotherapy could be offered for a patient with AML-pCT eligible for intensive chemotherapy. However, the phase 3 EORTC-GIMEMA AML-19 Trial including 73 adult patients with therapy-related or secondary AML showed that first-line monotherapy with low-dose GO, compared with best supportive care, significantly improved OS in older patients who were ineligible for intensive chemotherapy . There w23 CAR-T . Taking To conclude, treatment results achieved in patients with AML-pCT treated from 2015–2022 with two induction cycles followed by immediate SCT were better than those reported by other authors, and comparable to outcomes in de novo AML. As the important cause of the treatment failures were toxicities, it seems that novel, less toxic therapeutic agents together with intensive supportive care may play a crucial role in the improvement in the treatment results in AML-pCT in the future.


TP53-mutated acute myeloid leukemia (AML) represents one of the most informative examples of adverse risk AML. As the currently available therapies have not translated to meaningful advances in the survival of these patients, a clinical trial should be the recommendation for all newly diagnosed patients. CD47/SIRPα axis and TIM-3 inhibition appear to be some of the more promising strategies, but other agents with novel mechanisms of action are in development. We review the pathobiology of TP53-mutated AML, the possible heterogeneity among patients with this disease and how some of the novel and emerging therapies may fit into the treatment landscape in the hopefully not-so-distant future.TP53-mutated acute myeloid leukemia (AML) are insufficient, as they translate to a median overall of only 6–9 months, and less than 10% of patients undergoing the most aggressive treatments, such as intensive induction therapy and allogeneic hematopoietic stem cell transplantation, will be cured. The lack of clear differences in outcomes with different treatments precludes the designation of a standard of care. Recently, there has been growing attention on this critical area of need by way of better understanding the biology of TP53 alterations and the disparities in outcomes among patients in this molecular subgroup, reflected in the development and testing of agents with novel mechanisms of action. Promising preclinical and efficacy data exist for therapies that are directed at the p53 protein rendered dysfunctional via mutation or that inhibit the CD47/SIRPα axis or other immune checkpoints such as TIM-3. In this review, we discuss recently attractive and emerging therapeutic agents, their preclinical rationale and the available clinical data as a monotherapy or in combination with the currently accepted backbones in frontline and relapsed/refractory settings for patients with TP53-mutated AML.The currently available therapeutic options for patients with  TP53 is a critical tumor suppressor gene located on chromosome 17p13.1 that encodes the p53 protein, which, in response to cellular stress, including deoxyribonucleic acid (DNA) damage, increases in level and ultimately induces the transcription of the genes responsible for DNA damage repair and cell cycle arrest/apoptosis, among others ..TP53m-AMThe introduction of venetoclax-inclusive combinations represents a paradigm shift in the treatment of patients with AML who are not appropriate for intensive therapy. p < 0.001) ..TP53m-AMTP53m-AML. SRF231, a fully humanized anti-CD47 antibody, has demonstrated an ability to increase phagocytosis in AML cell lines, including the p53-null HL60 cell line, as well as in primary bone marrow samples from patients with AML . I. ITP53m-TP53m-AML is unknown, but the current options available to providers charged with the care of patients afflicted with this disease are inadequate. A meaningful effort is being put forth to develop and study agents, either as monotherapies or in combination with the currently available regimens, with novel or optimized mechanisms of action to address this critical gap. Clinical data for agents exploiting MDM2–p53 homeostasis, such as MDM2 and NEDD8-activating enzyme inhibitors, have recently been met with disappointment. However, among the most promising are agents that inhibit the CD47/SIRPα axis or other immune checkpoints, such as TIM-3. Agents hypothesized to “reactivate” mutant and dysfunctional p53 proteins are also in development. A better understanding of the mechanisms of TP53 alterations and the nuances that influence the amount of functional TP53 protein, such as higher fidelity and universal methods or copy number determination, are required to identify the variance within the molecular subgroup of TP53m-AML. The full profiling of TP53 alterations should occur in large databases and in larger clinical trials to fully understand their differences in behavior and where research efforts can be more efficiently directed. With a more sophisticated understanding of TP53 alteration biology and its determinants of the patient outcomes, future randomized trials employing novel agents could soon establish a true standard of care for TP53m-AML, a disease that, to date, has served as the chief example of poor-risk AML.The standard of care for the treatment of


Urbanization and aging populations are threatening the sustainability of rural development around the world. Improving the happiness of rural residents is closely related not only to rural development but also to the harmony and stability of a country. Sustainable development has become an important strategy for China’s rural areas. Although withdrawal from rural homesteads is an important issue in rural land policy, few researchers have examined the determinants of the subjective well-being of farmers following withdrawal. The current paper investigated 315 rural residents under three models of the “withdrawal from homestead” policy in Jinjiang City, Fujian Province, China. The application of the orderly probit model revealed how satisfaction with economic, social, environment, cultural, and policy factors impacted their subjective well-being. The pooled results showed that satisfaction with cultural and policy factors had no significant impact; however, the other aspects significantly promoted their subjective well-being. The empirical model with interaction terms indicated the significant positive impact of economic, environmental, and social factors on subjective well-being under the index replacement model, while only environment and social factors exerted a significant positive impact under the asset replacement and monetary compensation models. Corresponding policy implications are discussed. Urbanization and aging populations worldwide have increased the occurrence of rural hollowing, which refers to the neglect and vacancy of rural dwellings . WithdraIn the context of rapid urbanization in developing countries such as China or India, the livelihood of rural residents, which depends on access to productive land, is under threat. The impact of the transfer of agricultural land is an important index measuring whether transfer behavior brings positive benefits. Land-lost rural residents have become a significant focus of studies on happiness . More anThe close connection between WRH policy and sustainable rural development lies in activating existing land resources of farmers who have withdrawn and enhancing the life cycle of farmers’ houses by redeveloping or renovating existing houses which are not being used efficiently. Systematic re-planning of the countryside has been adopted as the model that will ensure the sustainable development of China through improving living environments and maximizing idle land resources. Rural residents face both favorable and unfavorable impacts after WRH. These impacts involve land resources, human resources, and personal wealth . FarmersAre rural residents happy after WRH? What aspects of SWB do they experience? As different WRH pilots are implemented in various parts of rural China, different WRH models have different practical effects, such as monetary compensation or asset allocation. Are there differences in SWB under different WRH models? These are the questions asked in this paper. Our results contribute to the literature on WRH and the life quality of rural residents, specifically in helping to understand the perception of residents of WRH policy. Rural residents represent a large group in China, and with current trends of urbanization, this group has an important role to play in realizing the integrated development of urban and rural areas . The ChiThe purpose of this study was to determine the perspective of rural residents in Jinjiang City, where China’s first WRH pilot was conducted. We aimed to explore the satisfaction of rural residents in different dimensions and how to increase SWB after WRH as well as further explore the impacts of different WRH modes. The remainder of this paper is arranged as follows: The pursuit of happiness is undertaken by individuals in every culture. Happiness has become an integral part of discussions in sociology, psychology, and economics . The meaIn recent decades, SWB has received increasing attention from the academic community. The representative “happiness paradox”  and the The main livelihood capital of farmers is land, and empirical studies have found that land acquisition prompts declining health and SWB . VariousSince the implementation of reform policy in 1978, the Chinese government has achieved considerable economic development but faces serious threats to sustainable rural development, including a shortage of infrastructure investment, poor industrial development, a weak economic foundation, a widening gap between the rich and the poor, and aging residents . AttemptRural communities must provide various services for rural people, such as hospitals, schools, and shops . Once abHypothesis 1 (H1).After WRH, the satisfaction of rural residents with economic, social, environmental, and cultural aspects as well as with policy (particularly regarding compensation) positively affects their SWB.Different WRH models are carried out in various parts of China, resulting in different impacts on the rural community. Jinjiang City, one of the earliest WRH pilots in China, has implemented three models: asset replacement, index replacement, and monetary compensation . In the Hypothesis 2 (H2).The satisfaction of residents with economic, society, environment, and cultural factors as well as with policy has differing impacts on their SWB under different WRH models. In particular, the impact of these factors under the index replacement model is more significant.Jinjiang City in Quanzhou, China, is one of 15 pilot cities for WRH policy. It is also the only selected pilot area in Fujian Province. Reform aimed at realizing the intensive use of rural-land resources was launched in 2015. This paper selected four villages representing different WRH policy models in Jinjiang: Qiekeng Village (under index replacement), Dapu Village (under index replacement), Guishan Village (under asset replacement), and Xibian Village (under monetary compensation). As an important representative of the index replacement model, Qiekeng Village was in the first batch of 8 pilot villages for WRH policy in 2015, and Dapu Village was selected for the second batch of policy demonstrations in 2016 based on the base of Qiekeng Village model. Guishan Village is also representative of the asset replacement model of the first batch of pilot villages. Xibian Village is a special case of WRH policy. This area is representative of monetized WRH because it received a large amount of funds donated by local overseas Chinese, so it is also included in the study area. A map of these study areas is presented in The farmers in these villages were the subjects of this study. WRH policy affects not only individual farmers, but also other co-residents; however, considering the operability of the survey, the main owner of the homestead was the object of the survey. Sampling criteria included the following two conditions: participation in the relevant activities of WRH policy and ownership of the property rights of the existing homestead. Considering the existing population size and limited research time, we applied stratified sampling to distribute 359 questionnaires, of which 100 questionnaires were distributed in Qiekeng Village and Dapu Village, 80 questionnaires were distributed in Guishan Village, and 79 questionnaires were distributed in Xibian Village. The survey was conducted from September 2020 to April 2021. A total of 315 valid questionnaires were recovered, representing a recovery rate of 89.75%, with a validity rate of 87.74%. We recovered 81 samples from Qiekeng Village and 80 samples from Dapu Village (representing the income index replacement model), 62 shares from Guishan Village (representing the asset replacement model), and 62 shares from Xibian Village (representing the monetary compensation model).This study designed a questionnaire in Chinese to measure SWB and its determinants after WRH. Measurement of all variables was as follows:SWB has been well-researched and is a moderately stable concept . There aSeveral studies have shown that the SWB of rural residents depends on their individual characteristics and their household status; it is also affected by their village environment ,49,50,51The impact of health and social capital on the SWB of rural residents has also been evidenced ,37. In oDrawing on the work of Kumar et al. , we measThe core aim of this study was to understand how differing WRH models affect the SWB of residents. Therefore, three dummy variables were used to represent the different models: dummyMode1 , dummyMode2 , and dummyMode3 . In order to further examine whether different models exert different impacts on different aspects of SWB, the dummy variables were multiplied by each of the satisfaction variables. The coefficient of the interaction term was examined to verify Hypothesis 2.i represents SWB, Xi represents household characteristics and satisfaction in each dimension, βi represents the coefficient of the independent variable, Dummymodel represents the dummy variables of the three models, and εL represents random error and is assumed to be normally distributed, representing other unobservable factors that affect SWB, with a mean value of 0 and a variance of 1.Originally proposed by Theodossiou  and LelkIn order to study differences under different WRH, we generated interaction terms to distinguish the slopes of different groups. We studied the promotion effects of different WRH models using the following equation:To increase the robustness of empirical results, Knight et al.  suggeste2 and 60 m2. Roughly half of the respondents (50.16%) reported themselves as “happy” following WRH, while 39.37% of the respondents rated their emotional state as “neutral”. In terms of satisfaction with the economic dimension after WRH, 46.98% of the respondents felt “satisfied”. The majority (72.7%) were also satisfied with the social dimension. In total, 53.33% and 66.03% of the respondents, respectively, expressed satisfaction with the environmental and cultural dimensions. However, 46.03% of the respondents indicated that their satisfaction level with regard to policy was “neutral”. This result is poor compared to the other dimensions.Before formally examining the results of the ordered probit model, we must perform correlation analysis among the independent variables see . We founNotes: All of the variables were counted and calculated by the authors.This study observed the impact of satisfaction with various aspects on SWB after WRH. In all models, household variables such as education level, HLAPC, income, health, and social capital all positively impacted SWB, with the exception of gender, which exerted an insignificant negative impact see . The pooWe further examined whether different WRH models promote the satisfaction of farmers and thus promote their SWB see . As abovIn order to check the robustness of the results, we introduced instrumental variables through a simple OLS model. The instrumental variable replaces the original policy and cultural variables with “overall satisfaction with implementation of WRH ”. This variable better reflects the farmers’ perception of overall village progress after WRH, meeting the requirements of “correlation” and “exogenousness” of instrumental variables. The first column of Although the SWB of land-lost farmers has been well-researched over the years ,11,12,21We found that the basic household characteristics of income, health, household living area per capita, and social capital have a significant positive impact on SWB in most cases. This echoes the literature, which offers evidence regarding the effects of income ,48, healThe pooled results indicated that satisfaction with economic, social, and environmental factors after WRH positively impacts SWB, which validates Hypothesis 1. However, in-depth analysis of different WRH models reveals that the impact of various aspects on SWB under different models varies, which supports Hypothesis 2. The significant positive impact of economic satisfaction on SWB was only evident in the index replacement model, which may be attributed to the village collective’s financial guarantee of the livelihoods of residents after reconstruction and reasonable compensation mechanisms . In thisSatisfaction with social and environmental factors has a significant positive impact on SWB in all models, which is in line with the argument that the improvement of public facilities and services is a necessary condition for improving SWB ,11,62. FViganó et al.  pointed As an important policy tool focused on the improvement of vacant rural land in China, WRH policy has a multi-dimensional impact on rural villages in terms of economic, social, and environmental factors. These aspects further impact the SWB of residents. This study surveyed 315 residents from four rural areas of Jinjiang City subjected to three WRH models. The pooled empirical results point out that satisfaction with economic, environmental, and social factors makes a significant positive impact on SWB. Interaction terms indicated that residents in the index replacement model experienced increased SWB from economic, environmental, and social factors, while residents in the asset replacement and monetary compensation model experienced positive impacts only in terms of environmental and social factors. Household characteristics such as income, health, household living area per capita, and social capital all positively impacted SWB, which supports the findings in the literature. However, the fact that satisfaction with cultural and policy factors had no impact reveals the shortcomings of current WRH policy.The results from this study provide evidence that the WRH policy can promote the SWB of residents from the perspective of sustainable development. They also have reference and policy implications for China’s comprehensive expansion of WRH policy and for other developing countries implementing similar policies. Although all three models reflect positive effects in terms of social and environmental factors on SWB, the index replacement model proved the most effective. This model not only implements the protection of property rights for resettlement houses, but it also avoids the expropriation of agricultural land, creating more security in terms of sustainable livelihoods. Although under the asset replacement model farmers receive houses in the same area, these cannot be traded within the existing legal system. Therefore, villagers who receive compensation should receive certification of house property rights. In addition, under the monetary compensation model, farmers can only receive a one-time monetary compensation, and it is necessary to make clear and reasonable regulations regarding the flow of funds to avoid the loss of farmers’ rights when compensation is not received. This study also suggests that whatever WRH model is implemented, farmers’ understanding and participation in the WRH policy should be strengthened. Moreover, local cultural elements and characteristics should be better integrated into planning and design.Despite its valuable contributions, this study is subject to several limitations. First, comparative studies on the differences in satisfaction over time and their impact on happiness are required. Second, due to limited resources, the sample size was restricted to 300 respondents. This could be expanded in future research to increase reliability and generalizability. Subsequent research could also use the natural experiment method and other means to further explore differences in SWB and their determinants in other rural locations under WRH policy.


Here, we used in situ electrophysiology and two-photon imaging of mitochondrial Ca2+, cytosolic Ca2+, and NAD(P)H to test the relevance of MCU activation to pyramidal neuron Ca2+ signalling and energy metabolism during action potential firing. We demonstrate that mitochondrial Ca2+ uptake by the MCU is tuned to enhanced firing rate and the strength of this relationship varied between neurons of discrete brain regions. MCU activation promoted electron transport chain activity and chemical reduction of NAD+ to NADH. Moreover, Ca2+ buffering by mitochondria attenuated cytosolic Ca2+ signals and thereby reduced the coupling between activity and the slow afterhyperpolarization, a ubiquitous regulator of excitability. Collectively, we demonstrate that the MCU is engaged by accelerated spike frequency to facilitate neuronal activity through simultaneous control of energy metabolism and excitability. As such, the MCU is situated to promote brain functions associated with high frequency signalling and may represent a target for controlling excessive neuronal activity.Neuronal activation is fundamental to information processing by the brain and requires mitochondrial energy metabolism. Mitochondrial Ca 2+ uptake to the regulation of neuronal bioenergetics and excitability in the brain is demonstrated using single-cell patch-clamp recording and two-photon imaging.The importance of MCU-mediated mitochondrial Ca In addition, these organelles influence reactive oxygen species production, apoptotic cascades, metabolite generation, and Ca2+ signalling9. Of particular interest to the relationship between neuronal activity and mitochondrial function is the uptake of cytosolic Ca2+ by the mitochondria. This process relies on the inner mitochondrial membrane potential and is mediated by the mitochondrial Ca2+ uniporter (MCU). The MCU is a Ca2+-selective ion channel that is normally closed, but opens in response to elevations of cytosolic Ca2+12. The Ca2+-dependent activation of the MCU is mediated by the regulatory subunits MICU1 and MICU2, which associate with the channel13. Physiological elevation of mitochondrial Ca2+ via the MCU can enhance oxidative phosphorylation by facilitating the activity of the ATP synthase and dehydrogenases in the tricarboxylic acid cycle (TCA)17. The uptake of Ca2+ by the mitochondria can also shape the magnitude and spatio-temporal dynamics of cytosolic Ca2+ and thereby control Ca2+-dependent signalling21. In synaptic terminals, these effects of the MCU control synaptic transmission and promote local ATP production24. Aside from its physiological role, excessive mitochondrial Ca2+ uptake can disrupt organelle function and may contribute to neuronal death during acute excitotoxicity and progressive degenerative disorders, such as Alzheimer’s and Parkinson’s disease29. This highlights the importance of examining the mechanisms which govern mitochondrial Ca2+ uptake and the associated implications for neuronal function in the brain.The activation of neurons and neuronal networks is fundamental to brain function and is critically dependent on ATP production by mitochondria2+ uptake in neurons. However, these experiments were often performed under pathological conditions and in cultured cells33. Moreover, previous measurements of mitochondrial Ca2+ in brain tissue were performed using Ca2+-sensitive dyes which do not selectively localize to the mitochondria and can have adverse effects35. Consequently, the functional relevance of mitochondrial Ca2+ uptake to neurons during information processing in the mammalian brain is unclear. Interestingly, it was recently shown that the MCU in excitatory neurons is required for sustaining fast neuronal network oscillations in rodent hippocampal tissue36. Moreover, in vivo imaging with a genetically encoded mitochondrial Ca2+ sensor revealed that sensory or motor evoked brain activation triggered robust mitochondrial Ca2+ elevations in the somatodendritic compartment of excitatory pyramidal neurons37. These studies point to a potentially important role for the MCU and mitochondrial Ca2+ signalling during heightened brain activity, but several outstanding issues remain unaddressed. For example, the relationship between single neuron activity and mitochondrial Ca2+ uptake has not been established. This may be important considering that signalling pattern determines metabolic demand, synaptic transmission and plasticity40. Additionally, discrete cell types and brain regions can exhibit distinct metabolic profiles, Ca2+ channels, and mitochondrial properties44, indicating that the relevance of mitochondrial Ca2+ uptake to specific neuronal populations may vary. Lastly, the functional importance of mitochondrial Ca2+ uptake in the somatodendritic compartment of excitatory neurons during brain activity is not certain, but it could influence numerous processes, including adaptive energy metabolism, excitability, synaptic plasticity, and gene expression47.MCU activation may have a critical role in governing information processing in the brain. Numerous studies have demonstrated functions for mitochondrial Ca2+ uptake in excitatory neurons of the cortex and hippocampus. Using a genetically encoded mitochondrial Ca2+ sensor, we demonstrate that the MCU activates in response to increasing action potential firing rate to initiate long-lasting mitochondrial Ca2+ elevations in the somatodendritic compartment of pyramidal neurons. By doing so, the MCU governed activity-dependent changes in mitochondrial energy metabolism and reduced the activation of the slow afterhyperpolarization (sAHP), which mediates negative feedback control of excitability. These results suggest that the MCU is engaged by changes in spike firing frequency to facilitate signal processing in pyramidal neurons of the brain.In the present study, we examined the functional relevance of the MCU during neuronal activation by determining the relationship between action potential firing and mitochondrial Ca2+ in pyramidal neurons, we drove the expression of mitoRGECO1.0 (mRGECO) in the rat brain by intracortical injection of adenoassociated virus (AAV)  and has red emission, which allows for it to be imaged simultaneously with green fluorescent sensors48. Following AAV injections, two-photon imaging of acute cortical and hippocampal brain slices showed the mitochondrial localization of mRGECO in the neuropil and pyramidal neuron cell bodies  which presumably occurred as a result of spontaneous neuronal signalling   Fig. . This prite Fig. . This ma2+ uptake into the mitochondria occurs primarily via the MCU, although other Ca2+ entry routes have been suggested55. To examine this, we tested the effect of Ru360, an MCU inhibitor56, on mitochondrial Ca2+ uptake during spike firing. As Ru360 has limited membrane permeability57, it was dissolved in the internal solution of the pipette and delivered to neurons via intracellular dialysis 61. Delivering an action potential train  to pyramidal neurons co-expressing the two sensors caused an initial rapid increase in cytosolic Ca2+ in the somatodendritic compartment followed by a latent slower rise in mitochondrial Ca2+ . During low-frequency action potential trains (1 & 5 Hz), no apparent changes in the somatic mitochondrial Ca2+ signals were detected throughout the duration of stimulus  (Kd = 12 µM)48. Action potential evoked mLaRGECO fluorescence changes were smaller and briefer in duration relative to those measured with the high-affinity mRGECO sensor  but can enter states of high-frequency action potential firing (10-50 Hz) during sensory processing or other cognitive taskslus Fig. . In cont 2+ Fig. . These r 2+ Fig. . The relsor Fig. . Moreoveude Fig. . Collect2+ channel subtypes, and suite of mitochondrial proteins43. Therefore, it may be possible for the relationship between neuronal activity and mitochondrial Ca2+ uptake to differ between distinct cell populations. To address this, we patch clamped mRGECO expressing hippocampal pyramidal neurons and compared the evoked responses to those measured in cortical neurons  that occur at theta frequency (~5 Hz) in CA1 neurons during rodent exploratory behaviour and learning processes66. When CA1 neurons were triggered to fire high-frequency bursts applied at theta frequency, somatodendritic mitochondrial Ca2+ increased within seconds after the stimulus onset and remained elevated above pre-stimulus baseline for an extended period of time after the train  to 50% recovery: 87.41 ± 20.64, n = 4, N = 3)  Fig. . This evons Fig. . Interestex Fig. . Consisttex Fig. . These d2+ uptake can depolarize the mitochondrial membrane potential, facilitate the activity of the electron transport chain, and promote the Ca2+-dependent activation of several key enzymes of the tricarboxylic acid cycle69. We therefore examined how MCU-dependent mitochondrial Ca2+ flux controlled action potential evoked metabolic responses in single pyramidal neurons using two-photon microscopy of intracellular nicotinamide adenine dinucleotide hydride (NADH) autofluorescence72. NADH is a coenzyme that is generated from the reduction of non-fluorescent NAD+ by the mitochondrial TCA cycle or via glycolysis in the cytosol73. NADH is oxidized to NAD+ at complex I of the electron transport chain in order to generate the proton gradient used to synthesize ATP73 H autofluorescence which became more prominent at higher action potential firing frequency H autofluorescence returned to pre-stimulus baseline shortly after the end of the stimulus H transients triggered by a 50-Hz train were influenced by 5 min of pre-exposure to the excitation laser at the illumination power used for imaging. Conditioning cells with the laser had no effect on the train-evoked NAD(P)H decrease or the long-lasting overshoot phase, suggesting that laser illumination had no influence on our results H autofluorescence in neuronal cell bodies that was distributed in a punctate pattern, similar to mitochondria labelled with mRGECO Fig. . We nextncy Fig. . This dilus Fig. . Converslus Fig. . This NAlts Fig. .77. Wash in of rotenone caused an elevation of neuronal autofluorescence in unstimulated neurons, consistent with a disruption of NADH oxidation in the presence of ongoing TCA cycle activity H transients H transients to rotenone (10 µM), which prevents NADH oxidation by complex I of the electron transport chainity Fig. 78. Subsents Fig. . This da2+ uptake. We next examined a role for mitochondrial Ca2+ uptake in generating these NAD(P)H signals by blocking the MCU with Ru360 dialysis. Under these conditions, applying a 50-Hz, 4-sec train produced a briefer train-evoked dip in NAD(P)H autofluorescence relative to controls H transients with increasing firing frequency paralleled the previously measured occurrence of mitochondrial Caols Fig. . Moreove2+ during high-frequency activity implies that mitochondrial Ca2+ uptake may shape the magnitude and temporal dynamics of cytosolic Ca2+ in the somatodendritic compartment9. We therefore assessed the extent to which MCU-dependent Ca2+ uptake influenced the relationship between cortical pyramidal neuron action potential firing frequency and evoked cytosolic Ca2+. This was accomplished using simultaneous whole-cell current-clamp recordings and measurement of cytosolic Ca2+ with the low-affinity Ca2+-sensitive dye Fluo5N. In control conditions there was an increase in the Ca2+ rise magnitude with progressively greater action potential firing frequencies  to inhibit the MCU. At a low action potential firing frequency of 5 Hz there was no apparent influence of Ru360 on evoked cytosolic Ca2+ dynamics relative to control conditions  with greater cytosolic Ca2+ and higher action potential firing frequency , and therefore neuronal excitability.The detected rise in mitochondrial Caies Fig. . The infons Fig. . Howeverncy Fig. . The sAHing Fig. . The pro2+ uptake and the sAHP, we next measured IsAHP in hippocampal CA1 pyramidal neurons, as it is well characterized in these cells46. Under whole-cell voltage-clamp, applying a series of progressively longer transient step depolarizations, to initiate voltage-gated Ca2+ influx, resulted in the serial enlargement of the IsAHP . Under these conditions, BAPTA effectively eliminated the depolarization-evoked IsAHP and largely prevented the enhancement of the current by Ru360  The degree of mitochondrial Ca2+ uptake is a function of spike firing frequency in pyramidal neurons; (2) Increasing spike frequencies coupled with greater MCU-dependent Ca2+ uptake led to adaptive mitochondrial energy metabolism; (3) The coupling between action potential firing and mitochondrial Ca2+ uptake varied between brain regions; (4) Ca2+ uptake by mitochondria provides a remarkable degree of cytoplasmic Ca2+ buffering in pyramidal neurons during high-frequency firing and thereby controlled sAHP magnitude. We propose that MCU activation facilitates pyramidal neuron signalling by enhancing energy metabolism and excitability during the brain processes associated with high-frequency action potential firing  to the resting mitochondrial Ca2+ concentration80, the lack of responsiveness at low activity levels likely reflects a true absence of Ca2+ uptake rather than unresponsiveness of the sensor. The apparent threshold level of action potential firing frequency required for mitochondrial Ca2+ uptake in cortical pyramidal neurons is consistent with the established relationship between cytosolic Ca2+ and MCU activation. This relationship shows a threshold and positive cooperativity, properties which are controlled by the Ca2+-sensitive MICU1 and MICU2 proteins43. The Ca2+ threshold of MCU activation likely also contributed to the observed temporal dissociation between the rise in cytosolic and mitochondrial Ca2+ levels during the onset of action potential firing. While the specific Ca2+ concentration required for MCU activation in pyramidal neurons was not determined in our study, it has been estimated to be ~1 µM in non-neuronal cells11. However, this threshold is likely to be lower in neurons, due to the abundance of MICU3, which enhances MCU activation43. Additionally, recent in vivo imaging of pyramidal neurons provided evidence that mitochondrial Ca2+ uptake evoked by brain activity is facilitated by CaMKII function, which is sensitive to Ca2+ signal frequency and duration81. Thus, such factors may govern the relationship between spike firing frequency and MCU activation, in addition to the absolute magnitude of cytosolic Ca2+ elevations.Using a genetically encoded mitochondrial Ca2+. Moreover, mitochondrial Ca2+ uptake by the MCU governed NAD(P)H transients during high-frequency signalling. The partial influence of the MCU on the activity evoked NAD(P)H dip is consistent with enhanced NADH oxidation by the electron transport chain as a result of Ca2+ entry dissipating the mitochondrial membrane potential78. As NADH oxidation was not eliminated by MCU blockade, electron transport chain activity is likely also facilitated by ADP produced by activity-dependent ATP consumption69. The MCU dependence of the latent NAD(P)H overshoot phase indicates that this channel is also critical for increasing the mitochondrial NADH/NAD+ ratio. This can occur by enhanced chemical reduction of NAD+ to NADH mediated by the Ca2+-dependent facilitation of TCA cycle dehydrogenases16. Measurements of activity dependent changes in NAD(P)H autofluorescence have been extensively used to monitor adaptive energy metabolism in brain tissue, but the importance of the MCU to these processes was not certain83. For example, evoked NAD(P)H transients in the hippocampus have been shown to either be independent of or only partially dependent on Ca2+ influx from the extracellular space83. Therefore, the present work clarifies the importance of mitochondrial Ca2+ entry via the MCU to metabolic adaptation in activated pyramidal neurons in situ. Our findings in cortical neurons are consistent with recent work in the hippocampal dentate gyrus, which showed that MCU knockdown partially disrupted the evoked mitochondrial NAD(P)H dip and strongly attenuated the delayed NAD(P)H overshoot phase75.Using single-cell imaging of intracellular NAD(P)H and electrophysiology we found that overt changes in adaptive energy metabolism increased in relation to spike firing frequency and paralleled the uptake of mitochondrial Ca2+ uptake was required to sustain ATP levels during activity, and thereby maintained synaptic vesicle endocytosis2. An analogous process likely occurs in the somatodendritic compartment in situ, as the NADH overshoot promoted by the MCU would be expected to contribute to oxidative phosphorylation14. As we found that substantial mitochondrial Ca2+ uptake only occurs during enhanced action potential firing rate, the MCU appears to serve as a conditional regulator of energy metabolism during periods of intense energy demand. This mirrors findings in cardiac tissue, where mitochondrial Ca2+ uptake is required for producing sufficient ATP to sustain action potential firing selectively during fight-or-flight accelerations in heart rate84. The functional consequence of MCU-dependent ATP production in the somatodendritic compartment is unclear. Enhanced mitochondrial metabolic activity could contribute to the production of ATP required for maintaining membrane ion gradients85 or supporting activity-dependent plasticity in response to high-frequency signalling86. Aside from controlling neuroenergetic adaptation, changes in mitochondrial Ca2+ evoked by high-frequency firing could also alter the production of reactive oxygen species or mitochondrial metabolites, which can serve as cellular signalling molecules87.In cultured neurons, direct measurements of presynaptic ATP revealed that mitochondrial Ca2+ uptake differs between discrete brain regions. The greater coupling between spike firing and MCU activation in hippocampal neurons relative to cortical neurons can be accounted for by various factors. One explanation is cell type-dependent variation in the properties of evoked cytosolic Ca2+ signals. For instance, discrete neuronal populations exhibit unique Ca2+ buffering capacities88 and the relative abundance of voltage-gated Ca2+ channel types can also vary, with some channel classes showing differential efficacy in triggering Ca2+ uptake by the mitochondria91. The differential coupling could also reflect regional differences in intrinsic mitochondrial properties, such as energy metabolism44 or the function of the MCU. For example, although the MCU complex is ubiquitously expressed, emerging evidence suggests that mitochondrial Ca2+ uptake in non-neuronal cells may vary substantially as a result of post-translational modification93 or changes in the relative expression of the MCU and the MICU regulatory subunits96. Interestingly, an analogous process may occur within the nervous system, as the expression profile of the MCU complex components is different between neurons and astrocytes as well as hippocampal subregions94. Our work provides evidence that is consistent with such variation in the function of the MCU complex between brain regions. Physiologically, the brain region-dependent variation in coupling between activity and mitochondrial Ca2+ uptake may represent a mechanism for cell-specific adaption of energy generation. This may be advantageous, as discrete neuronal populations can exhibit distinct energy demands during signalling97. In addition to its physiological role, excessive mitochondrial Ca2+ uptake can also contribute to metabolic dysfunction, reactive oxygen species generation, and neuronal death98. Therefore, heightened mitochondrial Ca2+ uptake in the hippocampal neurons could contribute to the sensitivity of this region to dysfunction in various pathologies, such as Alzheimer’s disease or ischemia99, in which mitochondrial dysfunction and Ca2+ overload have been implicated27.Our results provided evidence that the degree of coupling between neuronal activity and mitochondrial Ca2+ buffering by the mitochondria clamps cytosolic Ca2+ concentration and thereby shapes aspects of synaptic transmitter release in central synapses100. However, its functional relevance in other subcellular compartments of central neurons during signalling has not been well established101. Our work demonstrates a potent effect of mitochondrial Ca2+ uptake on cytosolic Ca2+ levels in the somatodendritic compartment, selectively during high-frequency action potential firing. By doing so, mitochondrial Ca2+ uptake strongly reduced the cytosolic Ca2+ available to activate the K+ current underlying the sAHP. Aside from the canonical Ca2+-dependent sAHP, disruption of intracellular ATP production by MCU blockade could conceivably lead to the activation of a KATP channel-mediated sAHP102. Nevertheless, this seems unlikely to have contributed to our results considering that increasing intracellular Ca2+ buffering prevented the enhancement of the sAHP seen during MCU disruption. The interplay between mitochondrial Ca2+ transport, ion channel modulation, and excitability has been demonstrated in olfactory sensory neurons, myenteric neurons, invertebrate neurons, and non-neuronal cells106. However, it has not been established whether this link between mitochondrial Ca2+ uptake and excitability also occurs in central neurons. Our results reveal that Ca2+ buffering by the mitochondria exerts control over neuronal excitability in the brain. By reducing the coupling between Ca2+ influx and the activation of the sAHP, mitochondrial Ca2+ uptake is poised to promote neuronal signalling by disrupting negative feedback control of spike firing and synaptic integration108. Such changes in the coupling between spike firing and activation of the sAHP can have meaningful consequences for information processing, as seen in the hippocampus during age-related learning disruption110.Prior studies have demonstrated that Ca2+ uptake in pyramidal neurons is tuned to enhanced action potential firing rate and promotes neuronal activity through a dual role in metabolic regulation and excitability control  to periods of high frequency action potential bursts (>50 Hz) occurring at theta frequency, and thereby act as place cells during exploratory behaviour63. Also, in the visual cortex, pyramidal neurons have little spontaneous activity (<1 Hz), but fire at ~10-20 Hz in response to the application of a visual stimulus39. Consistent with MCU activation during information processing in the brain, a recent in vivo study demonstrated that mitochondrial Ca2+ elevations occur in neurons of the motor and visual cortex in response to motor behaviour or visual stimulation, respectively37. Our results suggest that MCU activation may be engaged during such brain activity to sustain neuronal signalling through its influence on energy metabolism and excitability. Consistent with this, preventing mitochondrial Ca2+ uptake by the MCU has been found to disrupt spike firing rate increases in cortical neurons during sensory stimulation113. Moreover, the activation of fast neuronal network oscillations, which are associated with a multitude of cognitive processes, require high mitochondrial performance, and are prevented by MCU knockdown in excitatory neurons36. A similar property may also be important in fast-spiking GABAergic interneurons, as these cells exhibit comparatively higher frequency firing and more prolonged action potential firing114. As activation of the MCU is primarily engaged during high-frequency signalling, this pathway could also serve as a target for controlling excessive neuronal activity in various brain pathologies.Collectively, our in situ study demonstrates that mitochondrial Carol Fig. . This duSprague Dawley rats were used for this study and were purchased from Charles River Laboratories. Rat housing and experimental procedures were performed in accordance with Canadian Council on Animal Care (CCAC) regulations and protocols approved by the University of British Columbia Animal Care Committee. Animals were group housed, fed ad libitum, and kept on a 12/12 hr light/day cycle. Rats were 17-28 days old when experiments were conducted.3, 1 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 20 D-glucose, 2.4 sodium pyruvate, and 1.3 sodium L-ascorbate. The slicing solution was continuously bubbled with 95% O2 and 5% CO2. Brains were then cut with a vibratome (Leica VT1200S) into coronal or horizontal slices (400 µm thick) for cortical and hippocampal experiments, respectively. Brain slices were immediately transferred to a chamber containing artificial cerebral spinal fluid (aCSF), which was warmed to 32 °C and continuously oxygenated with 95% O2 and 5% CO2. aCSF contained (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 1.5 MgCl2, 1.25 NaH2PO4, and 10 D-glucose, pH 7.3–7.4, osmolarity 310 mOsm. A Ca2+-free ACSF solution was used to test the involvement of extracellular Ca2+ entry on mitochondrial Ca2+ signals, and contained (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO3, 3.5 MgCl2, 1.25 NaH2PO4, 10 D-glucose, 1 EGTA.Rats were anesthetized with isoflurane in an induction chamber and subsequently decapitated. The brains were then quickly removed and placed in ice-cold slicing solution containing the following (in mM): 120 N-methyl-D-glucamine, 2.5 KCl, 25 NaHCO13-1.5 × 1013 genome copies/mL) was then injected into the cortex at a rate of 600 nL/min using a motorized stereotaxic injector (Stoelting). Following injections, animals recovered from anesthesia and were then returned to the home cage. After 2-4 weeks, animals were sacrificed to prepare acute brain slices for experiments. AAVs were purchased from the Canadian Neurophotonics Platform Viral Vector Core Facility and Addgene: AAV9-synapsin-GCAMP6f ; AAV2/9-synapsin-mitoRGECO1.0 (Neurophotonics); AAV2/9-synapsin-mitoLaRGECO1.2 (Neurophotonics); AAV2/9-synapsin-mitoGFP (Neurophotonics).For AAV injections, neonatal rats, 1-3 days old, were anesthetized with isoflurane and placed in a stereotaxic frame. A glass micropipette attached to a Hamilton Syringe was then used to pierce the skull unilaterally at a region ~2/3 the distance between lambda and the eye. AAV solution : 0.02 Ru360 , 0.01 rotenone , 0.05 Alexa 488 hydrazide , 0.05 Alexa 594 hydrazide , 0.4 Fluo5N pentapotassium salt . All drugs or chemicals were dissolved into the internal solution of the patch pipette or the aCSF. DMSO was used as the vehicle for rotenone and was diluted to a maximal working concentration of 0.1%.Live brain slice imaging was performed using a two-photon laser-scanning microscope  equipped with a Zeiss water immersion objective lens . Excitation light was provided by a mode-locked Ti:Sapphire laser , which was pulsed at 80 MHz (140 fs per pulse). The excitation wavelength for each experiment was adjusted according to the fluorescent probes being imaged: mitoGFP & mRGECO, 1000 nm; NAD(P)H, 750 nm; Fluo5N & Alexa 594, 800 nm; mRGECO & Alexa 488, 1040 nm; mLaRGECO & Alexa 488, 1040 nm; mRGECO and GCAMP6f, 1040 nm. Emitted light was first passed through a 700 nm shortpass IR filter and then split with a 560 nm long-pass dichroic mirror (Chroma). Emitted light was then detected by two separate LSM BiG GaAsP detectors after passing through the appropriate emission filters (Chroma): green filter (520/60 nm) for MitoGFP, GCAMP6f, Fluo5N, Alexa488; blue filter for NAD(P)H (460/50 nm), red filter (630/75 nm) for mRGECO, mLaRGECO, Alexa594. Images were acquired using 8-16 line averaging and were processed and stored with Zen software (Zeiss).2/5% CO2 and warmed to 32 °C with a stage heater (Luigs & Neumann). Pyramidal neurons in the cortex (layer 3-5) and hippocampus (CA1 and CA3) were identified with transmitted light optics and an IR-1000 (DAGE-MTI) camera mounted to the LSM 7MP microscope. All neurons used for experiments were located >50 µm below the slice surface and had a resting membrane potential ranging from −55 to −70 mV. Neurons were patch-clamped using glass pipettes pulled from borosilicate glass capillaries (World Precision Instruments) with a P-97 Flaming/Brown Micropipette Puller (Sutter Instrument). Patch pipettes had a resistance of 3-8 MΩ and contained an intracellular solution comprised of the following (in mM): 110 K-Gluconate; 3 KCl; 10 Na-Gluconate; 2 MgCl2; 4 K2-ATP; 0.5 Na-GTP; 10 HEPES; 0.138 CaCl2; 0.4 EGTA; pH adjusted to 7.2 with KOH and osmolarity adjusted to 290 mOsm. For high Ca2+ buffering experiments, the internal solution contained (in mM): 70 K-Gluconate; 3 KCl; 10 Na-Gluconate; 2 MgCl2; 4 K2-ATP; 0.5 Na-GTP; 10 HEPES; 4.15 CaCl2; 10 K4-BAPTA; pH adjusted to 7.2 with KOH and osmolarity adjusted to 290 mOsm. The free Ca2+ concentration of both internal solutions was calculated with WEBMAXC Standard (UC Davis) to be between 70-170 nM. For some experiments, the internal solution was supplemented with fluorescent dyes or drugs at the following concentrations (in mM): 0.05 Alexa 594 hydrazide; 0.4 K5-Fluo5N; 0.05 Alexa 488 hydrazide; 0.02 Ru360. For all patch-clamp recordings, cells were dialyzed for a minimum of 15 min after whole-cell breakthrough before starting experiments. Neurons were confirmed to be pyramidal cells by morphological inspection, revealed by Alexa488 or Alexa594 fluorescence. Only neurons that possessed a pyramid-shaped cell body, large apical dendrite, and multiple basal dendrites were used in the study. Neuronal membrane voltage and current were recorded and controlled using a MultiClamp 800b amplifier, Digidata 1440 A digitizer, and Clampex 10.2 software . During voltage-clamp recordings, capacitance transients were compensated, and series resistance (8-20 MΩ) corrected to >40%. During whole-cell current clamp recordings, membrane voltage was maintained between −60 and −65 mV and series resistance was compensated using the bridge balance. Voltage offsets were nulled prior to seal formation and the membrane potentials presented were adjusted to correct for the liquid junction potential. Current and membrane voltage signals were low pass filtered by a built-in Bessel filter at 1 kHz and 3 kHz, respectively and digitized at 10 kHz.Following brain slice preparation and a 30-min recovery period, tissue was transferred to the microscope recording chamber and perfused with aCSF at 2–3 mL/min. aCSF was bubbled with 95% Oo = [(F1) − (Fo)] / (Fo). Where F1 and F0 represent the fluorescence at a given time point and the mean baseline fluorescence, respectively. Background fluorescence subtraction was not performed for these experiments as all regions neighboring the ROI contained meaningful fluorescence signal. Changes in Fluo5N fluorescence were determined similarly, but using background fluorescence subtraction: ΔF/Fo = [(F1 − B1) − (Fo − Bo)] / (Fo – Bo). B0 and B1 represent the background fluorescence during the baseline period and at a given time point during the time series recording, respectively. The ROIs used for measuring cytosolic Ca2+ in the soma with Fluo5N were drawn to exclude the nuclear fluorescence signal. For some data sets, fluorescence changes were also measured from ROIs placed over the proximal apical dendrite. Spontaneous mRGECO fluorescence transients were quantified by measuring the number and magnitude of fluorescence responses from a grid of 10 ×10 µm ROIs mapped onto each experiment time series. A spontaneous event was defined as a fluorescence increase in a given ROI that was >3 standard deviations above the mean baseline fluorescence. For analysis, peak or min ΔF/Fo responses were defined as the maximal changes in fluorescence after the stimulus onset time. The Ca2+ rise time was defined as the time from stimulus onset to half of the peak train-evoked ΔF/Fo. The coupling ratio between mitochondrial Ca2+ and cytosolic Ca2+ was calculated as the ratio of mRGECO ΔF/Fo and GCAMP ΔF/Fo at 2-sec post-stimulus onset. The rate of Ca2+ recovery in the mitochondria or cytosol was quantified by fitting the recovery phase with a monoexponential decay function or by measuring the time span from the end of the stimulus to the point at which ΔF/Fo was 50% of the peak fluorescence response. Images presented for publication were minimally spatially filtered and in some cases were also averaged across multiple time points. For some images, a mask of the neuronal outline was produced by forming a binary image from the intracellular Alexa594 fluorescence signal.All image analysis and image processing was performed using ImageJ (v 1.53). Prior to analysis, images were first processed to correct for translational movement using the correct 3D drift plugin on ImageJ. Activity-evoked fluorescence changes for mRGECO, mLaRGECO, GCAMP6f, and NAD(P)H were measured from a somatic region of interest (ROI) in pyramidal neurons and were defined as ΔF/FsAHP magnitude was determined by quantifying the post-depolarization area above prestimulus baseline (ms*pA/pF). This value equates to charge and was measured starting at 30 ms after the end of the step depolarization.The analysis of membrane voltage and the current was performed using Clampfit (v10.2). The sAHP duration was determined by measuring the time span between the end of the action potential train to the point of 50% recovery of the membrane voltage back to the pre-stimulus baseline. The Ip ≤ 0.05. The exact p-values are indicated in the figure legends.Statistics were performed using GraphPad Prism (v7). All summary data shown represent the mean ± standard error of mean. For each data set, the cell replicates and the animal replicates are described within the summary data and figure legends. The cell replicate number is represented by the ‘n’ value and the animal replicate number by the ‘N’ value. Unless otherwise stated, all presented summary data represent distinct samples. Outliers were identified using the ROUT test and removed prior to further statistical testing. The Shapiro-Wilk normality test was used to examine data sets for normality. If data were normal, a Student’s paired or unpaired t test was used to test for differences between two means, while a one-way ANOVA with a Dunnett’s multiple comparisons test was used to test for differences between multiple means and a control condition. If the data were not normally distributed, a Mann–Whitney U test was used for two means, whereas a Friedman test with Dunn’s multiple comparison test was used to compare multiple means to a control condition. A Welch’s t test was used to compare two data sets if the variances differed significantly. Tests were considered statistically significantly different at Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary DataSupplementary Data 1Supplementary Video 1Supplementary Video 2Reporting Summary