Nevertheless, emerging insights, primarily derived from studies on human pathogenic bacteria, suggest that this ion also plays a pivotal role in prokaryotes. In this review, our primary focus will be on unraveling the intricate Ca2+ toolkit within prokaryotic organisms, with particular emphasis on its implications for plant growth-promoting rhizobacteria (PGPR). We undertook an in silico exploration to pinpoint and identify some of the proteins described in the existing literature, including prokaryotic Ca2+ channels, pumps, and exchangers that are responsible for regulating intracellular Calcium concentration ([Ca2+]i), along with the Calcium-binding proteins (CaBPs) that play a pivotal role in sensing and transducing this essential cation. These investigations were conducted in four distinct PGPR strains: Pseudomonas chlororaphis subsp. aurantiaca SMMP3, P. donghuensis SVBP6, Pseudomonas sp. BP01, and Methylobacterium sp. 2A, which have been isolated and characterized within our research laboratories. We also present preliminary experimental data to evaluate the influence of exogenous Ca2+ concentrations ([Ca2+]ex) on the growth dynamics of these strains.The role of Calcium ions (Ca Some paleontologists propose that 3.5 billion years ago, when life began, the concentration of free Ca2+ surrounding the first cells was somewhere in the range of 100 nM [2+ content in their cytoplasm and lived in a low Ca2+ environment (nanomolar range). Low Ca2+ in the cytosol of primeval cells is also compatible with energetics based around ATP and the usage of DNA/RNA for genetic encoding [2+] can lead to cell death since it will irreversibly damage mitochondria and can cause chromatin condensation, precipitation of phosphate, protein aggregation, and disruption of lipid membranes through the activation of proteases, nucleases, and phospholipases  in plant cells varies in very wide limits depending on its location. Under resting conditions in the cytoplasm, it is ~10−7 M, while in intracellular compartments, such as the endoplasmic reticulum (ER), mitochondria, chloroplasts, or the vacuole, [Ca2+] is 1–5 × 10−4 M. These organelles that can accumulate Ca2+ are referred to as Ca2+ stores. Extracellular [Ca2+] in the cell wall or apoplast is ~10−3 M [2+] in prokaryotes is tightly regulated in the 100 to 300 nM range ; unlike other complex molecules, Ca2+ cannot be chemically altered; thus, cells must chelate, compartmentalize, or extrude it. Eukaryotic and prokaryotic cells employ Ca2+ pumps and Ca2+ exchangers to keep their basal concentration at a very low (~20–200 nM) level.Cavised in ) and is vised in ). The in2+ signatures  are decoded byCaBPs. Hundreds of proteins harbor Ca2+ binding domains either to buffer or lower Ca2+ levels or to trigger cellular processes. A variety of proteins ranging from calmodulin to membrane channel voltage sensors or nuclear DNA-binding proteins present EF hand domains  that consist of a ~12 amino acid loop between two orthogonal α helices. The affinities of EF hand domains for Ca2+ vary ~100,000-fold depending on a variety of factors ranging from critical amino acids in the Ca2+ binding loop to sidechain packing in the protein core  changes. The CaBPs compete for Ca2+ and play a direct role in modulating Ca2+ transients and the resulting biochemical message. Upon Ca2+ binding, these proteins undergo a conformational change and are able to interact with the effector proteins that amplify the signal, allowing the cell to respond to the stimuli. Ca2+ can also play a role in regulating lipid synthesis and, consequently, membrane composition. It has been indicated that Ca2+ is involved in nucleoid structure and the function of proteins required for DNA replication contributes to the concept.CaBPs are the fastest players within the Ca2+ is very well studied and understood in eukaryotes, but the current knowledge, mostly based on human pathogenic bacteria, indicates that this ion is also a key player in prokaryotes. In this review, we will very briefly summarize the multifaceted role of Ca2+ in plant-microorganism interactions, and we will focus on the Ca2+ toolkit in prokaryotes, particularly in plant growth-promoting rhizobacteria (PGPR). Additionally, we determined the effect of exogenous Ca2+ concentrations on the growth of four PGPR strains that were isolated and characterized in our labs , control the rate of Ca2+ delivery to the xylem, and prevent the accumulation of toxic cations in the shoot  in such tiny cells i against large changes in external [Ca2+]. The evolution of the Ca2+-dependent system of the Eukaryotic Endoplasmic Reticulum using sequence-based homology searches was analyzed, and it was found that at least three protein families of the ER Ca2+-stores system have a clearly identifiable bacterial provenance, namely the P-type ATPase pump SERCA, sarcalumenin, and calmodulin and related EF-hand proteins  in the bacterial cell without any direct implication in these morphological processes ex in the media (2+ tolerance among our isolates; for example, P. donghuensis SVBP6 was the most affected by high [Ca2+]ex in solid and liquid media (2+-related proteins were not abundant within this strain (P. chlororaphis SMMP3, this reduction level was only noticeable when exposed to 100 mM Ca2+ (Pseudomonas sp. BP01 exhibited a reduction of less than 20% in the colony diameter up to [Ca2+]ex = 50 mM and a reduction of less than 40% at the highest [Ca2+] (Methylobacterium, high Ca2+ exerted a strong inhibition in growth and colony diameter (2+ toolkit and in the tolerance to high Ca2+ among PGPR strains, even from the same genus. As all the strains evaluated came from soil or rhizosphere samples and showed PGPR properties in vitro, these differences could play an important role in their interaction with plants and maybe should be considered for bioinput development.To test if these isolates could maintain Ca genomes . The addhe media C. Howeveid media A,C. Thiss strain . In P. c mM Ca2+ C. Convert [Ca2+] C. In thediameter B,C. Thisdiameter B. These 2+ in prokaryotes and highlight several topics related to its role in regulating protein function and cellular signaling networks in different physiological processes within PGPRs. However, a deeper exploration is required to unveil the role of this cation within the context of PGPR interactions with plants and other soil microorganisms. Most of the proteins reported to have demonstrated Ca2+ binding activity or function in prokaryotes were identified in human pathogenic bacteria. We used these proteins as a query for the in silico search of homologues in our PGPR isolates (2+ levels is a typical response in both pathogenic and mutually beneficial plant/microbe interactions. As observed in 2+ influx and extrusion systems and adopt several strategies in the course of evolution to proliferate in their ecological niches, employing to this end specific CaBP proteins. Recognizing its relevance, the prospect of modulating Ca2+ concentrations in bioformulations, soils, or rhizospheres emerges as an intriguing tool for enhancing the efficacy of microbiological candidates in the development of agricultural bioinputs.The results compiled in this review demonstrate the crucial significance of Caisolates , keeping


A 29-year-old male, hemodynamically stable, presented with chest pain radiating to the interscapular region, with no fever, cough, dyspnea, or other constitutional symptoms. He had right cervical lymphadenopathy on physical examination. Investigations revealed a 3.1 cm anterior mediastinal nodular mass, peripheral immature blood cells, and thrombocytopenia. Bone marrow core biopsy findings were consistent with acute myeloid leukemia (AML). The mediastinal mass was resected via robotic-assisted thoracoscopic surgery. Histopathology revealed involvement of the mediastinal adipose tissue with myeloid sarcoma. Molecular testing showed TP53 mutation, signifying a poor prognosis. The patient failed several lines of therapy and expired. This case demonstrates an atypical presentation of AML and emphasizes the criticality of early detection in individuals who do not exhibit the usual symptoms associated with the disease. The presence of immature cell lines in peripheral blood should prompt an investigation to determine bone marrow involvement in an otherwise healthy young adult. Acute myeloid leukemia (AML), although rare overall, is the second most common leukemia. Based on National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) data from 2016 to 2020 in the United States, AML has an age-adjusted annual incidence rate of 4.1 per 100,000 individuals and a mortality rate of 2.7 per 100,000 individuals [This report describes an unusual AML presentation in a young man whose only initial symptom was chest pain, which was later found to be secondary to an MS of the anterior mediastinum. In this patient, the presence of blasts on the complete blood count prompted a bone marrow core biopsy, which led to the diagnosis of AML.It is important to be aware of rare clinical presentations of AML as the early diagnosis and initiation of therapy impact prognosis and survival.A 29-year-old male with a past medical history of bipolar disorder, gastroesophageal reflux disease, and obesity presented with a two-day history of sore throat and chest discomfort to the emergency room. He had also noticed an enlarged lymph node on the right side of the neck for one day. COVID-19 testing was negative, and he was sent home on a course of cefalexin for a presumed upper respiratory tract infection. Over the next few days, he had worsening chest pain with radiation to the interscapular region, prompting him to present to the emergency room again. He denied fever, chills, weight loss, shortness of breath, fatigue, or night sweats. He was a non-smoker and had no family history of malignancy. On examination, he was afebrile and vital signs were hemodynamically stable. Physical examination was remarkable for right cervical lymphadenopathy.3 with abnormal differential , hemoglobin of 12.9 g/dL, and a platelet count of 63 K/mm3. A computed tomography (CT) scan of the chest with intravenous contrast showed a 3.1 cm anterior mediastinal nodular mass  expressing CD13 (dim), CD33, CD117 (dim), human leukocyte antigen (HLA)-DR, CD11b (subset), CD11c (subset), CD64 (dim), CD71 (dim), CD34 (dim), and myeloperoxidase (MPO) with aberrant expressions of CD4, CD56, and CD9 (dim). These findings were consistent with AML (non-M3 phenotype).The finding of mediastinal mass or adenopathy is unusual for AML. Given the location of the mediastinal mass being superiorly adjacent to the aorta, there was an additional concern that chemotherapy-induced tumor shrinkage could potentially cause mechanical stress and damage to the blood vessel wall. A robotic-assisted resection of the mediastinal mass via left thoracoscopy was done, along with subtotal thymectomy. Histopathology revealed a thymic mass and mediastinal adipose tissue involved with MS. Immunohistochemical studies revealed atypical cells positive for MPO, CD4, CD117 (weak), and CD34 . Concurrent flow cytometry revealed an abnormal immature myeloid population  expressing CD13 (dim), CD33, CD117 (dim), HLA-DR, CD64 (dim), CD71 (dim), CD34 (dim), and MPO with aberrant expressions of CD4, CD56, and CD9 (dim).2/day on days one to seven, and daunorubicin at 90 mg/m2/day on days one to three), which aligns with the current standard of care for AML [He received induction chemotherapy using a 7+3 regimen of cytarabine and daunorubicin  gene. Karyotype abnormalities included chromosome 3q rearrangement, loss of chromosome 16, 9-16 translocation, and deletion of 11q. The presence of TP53 mutation and complex karyotype abnormalities conferred poor prognosis, so the role of allogeneic hematopoietic stem cell transplantation (HSCT) following morphologic remission with chemotherapy was discussed with the patient.Bone marrow core biopsy on day 14 did not show any leukemic cells, but a repeat biopsy on day 21 showed 81% blasts on flow cytometry. The patient was ultimately transferred to an outside hospital for his primary refractory AML but failed several lines of therapy and expired.AML comprises 1% of all new cancer cases in the US. Although it has been reported in all ages, it is primarily seen in older adults with a mean age of 68 at the time of diagnosis. Approximately one in 10 newly diagnosed cases are persons younger than 35 years of age [The clinical presentation of AML is varied, with many symptoms attributed to pancytopenia. Pancytopenia presents as a result of the excessive proliferation of leukemic cells within the bone marrow, causing a decrease in the synthesis of normal cell lines. The decreased synthesis of red blood cells leads to anemia, which can present with weakness and pallor. Thrombocytopenia can present with signs and symptoms of hemorrhage, petechiae, purpura, epistaxis, and gum bleeding. In addition, a decrease in granulocytes due to the failure of differentiation of immature precursors predisposes patients to infections. Constitutional symptoms, such as weight loss, night sweats, and fatigue, can also be seen.The above-mentioned patient had symptoms of chest pain and was found to have an anterior mediastinal mass on the CT scan with a biopsy proving MS. Given the location of the mass, the initial differential diagnosis included germ cell tumor, thymoma, and lymphoma . Beta-huMS is a collection of myeloid blasts forming a tumor mass outside the bone marrow and blood. The World Health Organization describes it as a unique presentation of any AML subtype . CommonlGiven the rarity and unfavorable outcome associated with this disease, early and accurate diagnosis is important. The above patient was very unusual in his presentation for AML for two primary reasons: younger age (less than 10% of AML cases affect people under 35 years of age) and extramedullary disease (EMD) (seen in about 1% of AML patients) involving mediastinum .MS with concurrent AML is treated with intensive AML chemotherapy. Induction therapy commonly involves a combination of cytarabine and an anthracycline. In younger patients with unfavorable-risk AML, HSCT is recognized as the most effective strategy to minimize the risk of disease relapse after achieving remission. For patients with favorable-risk AML and a low risk of relapse, high-dose cytarabine is usually given during the consolidation phase . ManaginAML typically presents with symptoms related to low blood cell count, such as fever, fatigue, easy bruising, and recurrent infections. MS is a rare presentation of the disease and is associated with a poor prognosis. As the tumor mass can involve any extramedullary site, the clinical presentation is heterogeneous. Given the rarity and unfavorable outcome associated with this, early and accurate diagnosis is important. Moreover, the identification of immature cell lines in peripheral blood should raise suspicion of potential bone marrow involvement in an otherwise healthy young adult, warranting further investigation.


Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) exhibit the potential to repair the injured heart after myocardial infarction (MI) by promoting neovascularization and cardiomyocyte survival. However, because of the low cellular retention and poor engraftment efficacy, cell therapy of MI is partly mediated by exosomes secreted from the transplanted cells. In this study, we investigated whether exosomes secreted from hiPSC-ECs could become a promising acellular approach to repair the infarcted heart after MI and elucidated the underlying protective mechanism.2+ transient and cell shortening assessment, and Western blot, was used to determine the protective effects of hiPSC-EC exosomes on the infarcted heart. Furthermore, microRNA sequencing, luciferase activity assay, and microRNA gain–loss function experiments were performed to investigate the enriched microRNA and its role in exosome-mediated effects.The hiPSC-ECs were differentiated, and exosomes were isolated in vitro. Then, hiPSC-EC exosomes were delivered by intramyocardial injection in a murine MI model in vivo. Echocardiography, combined with hemodynamic measurement, histological examination, Ca2+ transients, increased ATP content, and improved cell survival to protect cardiomyocytes from oxygen–glucose deprivation injury. Congruously, hiPSC-EC exosome administration in vivo improved the myocardial contractile function and attenuated the harmful left ventricular remodeling after MI without increasing the frequency of arrhythmias. Mechanistically, the hiPSC-EC exosomes notably rescued the protein expression and function of the sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA-2a) and ryanodine receptor 2 (RyR-2) to maintain intracellular Ca2+ homeostasis and increase cardiomyocyte contraction after MI. The microRNA sequencing showed that miR-100-5p was the most abundant microRNA in exosomes. miR-100-5p could target protein phosphatase 1β (PP-1β) to enhance phospholamban (PLB) phosphorylation at Ser16 and subsequent SERCA activity, which contributes to the hiPSC-EC exosome-exerted cytoprotective effects on maintaining intracellular Ca2+ homeostasis and promoting cardiomyocyte survival.In vitro, the hiPSC-EC exosomes enhanced intracellular Ca2+ homeostasis to improve myocardial recovery after MI, which may provide an acellular therapeutic option for myocardial injury.The hiPSC-EC exosomes maintain cardiomyocyte CaThe online version contains supplementary material available at 10.1186/s13287-023-03462-w. Myocardial infarction (MI) causes irreversible cell death and may lead to heart failure (HF). It is the leading cause of morbidity and mortality worldwide –4. Cell 2+ homeostasis are a critical hallmark of a failing heart, as cardiomyocyte function depends on synchronized movements of Ca2+ cycling, which contributes to the heart rhythm and excitation–contraction coupling. There is cumulative evidence showing gross Ca2+ homeostasis disorders after MI, such as decreased Ca2+ transients, reduced sarcoplasmic reticulum (SR) Ca2+ content, and diminished SR Ca2+ uptake and sequestration. These abnormalities are attributed to the expression and function alterations of Ca2+-handling proteins and transporters. Ryanodine receptor 2 (RyR-2), which causes the release of Ca2+ from the SR, could be a potential therapeutic target in MI /dtmax/resting calcium, was significantly reversed by hiPSC-EC exosome administration in a stimulation frequency-dependent manner, which indicated that hiPSC-EC exosomes could rectify the impaired FFR and CaFR of cardiomyocytes in infarcted hearts.It has been extensively established that disrupted Caion (AF) , 13, 41.c tissue , 42, 43 c tissue . In line2+ levels may have implications for energy metabolism and cell apoptosis because the higher Ca2+ level will be taken up by mitochondria and damage its function [2+ homeostasis, energy metabolism, and cell apoptosis mediated by hiPSC-EC exosomes, which could ameliorate the Ca2+ cycling abnormalities to increase energy metabolism and diminish cardiomyocyte apoptosis. This is also supported by the evidence that gene transfer therapy to rescue SERCA-2a from failing hearts could restore its energy metabolism to a more efficient state [2+ level could excessively activate calpain, a well-conserved cysteine protease [2+ homeostasis modulation might be a molecular mechanism of hiPSC-EC exosome to ameliorate cellular energy metabolism, diminish cardiomyocyte apoptosis, and enhance cardiomyocyte contraction.Increasing intracellular Cafunction . The disfunction . Our resnt state . Meanwhiprotease . The hypprotease , which i2+-cycling protein targets, such as PLB [β is the major catalytic subunit of PP-1 in the heart that could elevate PLB phosphorylation at Ser16 to enhance Ca2+ transients and cell shortening [β to hiPSC-EC exosome-mediated Ca2+ modulation and cardioprotective effects. It is intriguing to observe that hiPSC-EC exosome administration inhibited PP-1β expression in the infarcted myocardium. This inhibition further increased the phosphorylation of PLB, which could remove its suppression on SERCA to maintain intracellular Ca2+ homeostasis. It has been shown that exosomes could be considered therapeutic cargoes to communicate between target cells and parental cells because exosomes are regarded as the harbor of nonsense oligonucleotides [β inhibitor meditating the PP-1–PLB cascade. Furthermore, the gain–loss function analysis demonstrated that miR-100-5p could contribute to hiPSC-EC exosome–provided cytoprotective effects by maintaining intracellular Ca2+ homeostasis and abrogating cardiomyocyte apoptosis.PP-1, the main abundant serine/threonine phosphatase, plays a pivotal role in regulating protein dephosphorylation and has multiple Cah as PLB . A previortening , 48. Intleotides –16. Ther2+ homeostasis disorders by rescuing the function and protein expression of SERCA-2a and RyR-2. However, our results only verified that miR-100-5p enriched in hiPSC-EC exosomes could reverse SERCA-2a function through the PP-1β–PLB axis signaling to partially favor cardiomyocyte protection. There is a need to elucidate the mechanism by which hiPSC-EC exosomes regulate RyR-2 function, as well as SERCA-2a and RyR-2 protein expression, in future.Undoubtedly, there are some limitations of the present study. The present study demonstrated that hiPSC-EC exosome administration eliminated Ca2+ homeostasis. Highly enriched miR-100-5p contributes to hiPSC-EC exosome-mediated beneficial effects via the PP-1β-PLB axis signaling pathway.Administration of hiPSC-EC exosomes can alleviate the ischemic myocardial injury after MI, thereby restoring heart function and suppressing harmful LV remodeling by modulating resident cardiomyocyte CaAdditional file 1. Supplementary figures.


The presynaptic sites of action leading to reduced electrically evoked increases in presynaptic Ca2+ concentration and Ca2+-dependent exocytosis are unknown. Endoplasmic reticulum (ER) of Ca2+ release via ryanodine receptor 1 (RyR1) and uptake by SERCA are essential for regulation intracellular Ca2+ and are potential targets for anesthetic action. Mutations in sarcoplasmic reticulum (SR) release channels mediate volatile anesthetic-induced malignant hyperthermia (MH), a potentially fatal pharmacogenetic condition characterized by unregulated Ca2+ release and muscle hypermetabolism. However, the impact of MH mutations on neuronal function are unknown. We used primary cultures of postnatal hippocampal neurons to analyze volatile anesthetic-induced changes in ER Ca2+ dynamics using a genetically encoded ER-targeted fluorescent Ca2+ sensor in both rat and mouse wild-type (WT) neurons and in mouse mutant neurons harboring the RYR1 T4826I MH-susceptibility mutation. The volatile anesthetic isoflurane reduced both baseline and electrical stimulation-evoked increases in ER Ca2+ concentration in neurons independent of its depression of presynaptic cytoplasmic Ca2+ concentrations. Isoflurane and sevoflurane, but not propofol, depressed depolarization-evoked increases in ER Ca2+ concentration significantly more in mouse RYR1 T4826I mutant neurons than in wild-type neurons. The RYR1 T4826I mutant neurons also showed markedly greater isoflurane-induced reductions in presynaptic cytosolic Ca2+ concentration and synaptic vesicle (SV) exocytosis. These findings implicate RyR1 as a molecular target for the effects of isoflurane on presynaptic Ca2+ handling.Volatile anesthetics reduce excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms which include inhibition of depolarization-evoked increases in presynaptic Ca While research on malignant hyperthermia has focused on skeletal muscle effects, much less is known about its neuronal effects. We identify neuronal endoplasmic reticulum (ER) Ca2+ regulation as a novel target for volatile anesthetic action and as a potential target in malignant hyperthermia. While depression of CNS electrical activity in vivo by anesthesia has been observed in another model of malignant hyperthermia, our study reveals fundamental presynaptic mechanisms of volatile anesthetics with implications for the development of more selective anesthetics and for prevention and treatment of malignant hyperthermia.Despite their essential clinical roles, the molecular and cellular mechanisms of action of general anesthetics are not fully understood. Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder that leads to dysregulation of intracellular Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle leading to hyperthermia, tachycardia, and muscle rigidity . Malignant hyperthermia is a hypermetabolic syndrome characterized by excessive intracellular Carigidity . While trigidity . However2+ influx into presynaptic terminals and Ca2+-dependent synaptic vesicle (SV) exocytosis by reducing neuronal excitability and Ca2+ entry. However, potential sites of action upstream of reduced Ca2+ entry and SV exocytosis are not fully understood.Volatile anesthetics modulate neurotransmission and communication between neuronal networks , includi2+ concentration modulates Ca2+ entry involving ER Ca2+ sensing proteins. For example, reduced ER Ca2+ concentration is linked to reduced presynaptic Ca2+ influx  in response to MH-triggering agents including volatile anesthetics to initiate the pathologic features of MH including muscle rigidity and hypermetabolism. We used mice with the well characterized T4826I-RYR1 MH-susceptibility mutation (2+ regulation in the presynaptic mechanisms of volatile anesthetic action and MH pathogenesis.Presynaptic endoplasmic reticulum (ER) Ca+ influx . Ryanodimutation  to invesAll animal procedures were performed in accordance with the Weill Cornell Medical College Institutional Animal Care and Use Committee regulations and conform to National Institutes of Health  Guidelines for the Care and Use of Animals as well as ARRIVE guidelines, where appropriate. We used both wild-type (WT) Sprague Dawley rats  and wild-type BALB/c mice . Mice homozygous for the MH mutation T4826I-RYR1 in a BALB/c background were purchased from the University of California, Davis  and bred for use. Homozygous T4826I-RYR1 mice were used, since 100% of homozygous T4826I-RYR1 mice develop fulminant MH. In contrast, only 17% of male heterozygous T4826I-RYR1 mice develop fulminant MH .m glucose, 0.1 g/l bovine transferrin , 0.25 g/l insulin, 0.3 g/l glutamine, 5–10% fetal bovine serum , 2% B-27 . Cultures were incubated at 37°C with 95% air/5% CO2 in a humidified incubator before imaging. Transfection was performed on day 6 or 7 in vitro  using Ca2+ phosphate-mediated gene transfer to transfect a low percentage of cells. Live-cell imaging was performed on DIV14–DIV19. For each experiment, neurons were derived from at least three separate culture preparations to minimize artifacts from small variations in culture conditions. The N in each figure corresponds to the number of cells recorded per treatment group.Bilateral hippocampi were dissected from postnatal rats or mice  and plated on poly-ornithine-coated coverslips. Neurons were maintained in culture medium containing of MEM , 30 mER-GCaMP6-150 , VAMP-mCherry, and synaptophysin-GCaMP6f (syn-GCaMP6) were gifts from Timothy Ryan (Weill Cornell Medicine). Synaptophysin-pHluorin (syn-pH) was a gift from Stephen Heinemann and Yongling Zhu .Experiments were performed using a Zeiss Axio Observer Z1 widefield fluorescence microscope with filter cubes and LEDs for eGFP and RFP illumination (Zeiss) and an Andor iXon1 EMCCD camera sampling at 10 Hz. Coverslips with attached cells were mounted in a custom closed-bath field stimulation perfusion chamber , with solutions and chamber maintained at 37.0 ± 0.2°C by an in-line solution heater and imaging chamber heater (Warner Instruments). Perfusion was at 1 ml/min using a custom system with a multibarrel closed syringe manifold (Warner Instruments). Only one imaging experiment was acquired per coverslip.2, 2.8 mM MgCl2, 25 mM HEPES buffered to pH 7.4, 30 mM glucose). The standard buffer for syn-pH imaging was Tyrode’s solution with 2 mM CaCl2 and 2 mM MgCl2. In experiments where external Ca2+ was decreased to study effects of reduced Ca2+, the standard buffer for imaging was Tyrode’s solution with 1 mM CaCl2 and 3 mM MgCl2. All perfusate solutions contained 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 μm D,L-2-amino-5-phosphonovaleric acid  to block recurrent excitation because of glutamatergic excitation. Neurons expressing syn-pH and syn-GCaMP6f were identified by their resting green fluorescence. ER-GCaMP6-150-expressing neurons were co-transfected with the presynaptic marker VAMP-mCherry to identify presynaptic boutons for selective quantification of presynaptic changes in fluorescence.Standard perfusion buffer for syn-GCaMP6 and ER-GCaMP6-160 imaging was Tyrode’s solution  trains of 20 AP. Cells transfected with syn-pH were stimulated with AP trains of 100 AP at 10 Hz for 10 s. Electrical stimulation was generated by field stimulation with a pulse generator , stimulus isolator  and platinum/iridium bath electrodes built into the imaging chamber to produce an electrical field of 10 V cmm (0.002% DMSO).Volatile anesthetic solutions were prepared daily from saturated stock solutions in Tyrode’s buffer. A 12 mM saturated stock isoflurane solution was diluted into an experimental solution equivalent to the clinically relevant dose of ∼1 minimum alveolar concentration  with the TimeSeries Analyzer plugin (rsb.info.nih.gov/ij/plugins/time-series.html) to measure fluorescence over time using the Background Correction plugin (https://imagej.nih.gov/ij/plugins/download/Background_Correction_.java) to compensate for between experiment variations in background fluorescence. Transfected boutons (∼20–50 boutons per neuron) were selected from control images before drug application and analyzed using 2-μm diameter regions of interest (ROIs). ROIs were selected based on their response to control stimulation in Tyrode’s buffer. Background corrected fluorescence changes were normalized to baseline fluorescence as ΔF/F0 = (F – F0)/F0. Baseline fluorescence (F0) was defined as the mean of 10 frames before stimulus onset, and peak fluorescence (F) was defined as the mean of the five consecutive frames with the highest values immediately following electrical stimulation. Boutons with a signal-to-noise ratio more than or equal to four were used in the analysis.Live-cell imaging recordings were analyzed using ImageJ  and Adobe Illustrator.Statistical significance was tested by paired or unpaired Student’s nificant . Dataset2+ efflux through ryanodine receptor 1 (RyR1), an endoplasmic reticulum (ER) Ca2+ efflux channel. Similar to human carriers, mice with the T4826I-RYR1 mutation are phenotypically normal unless challenged with an MH-triggering agent, which can produce skeletal muscle SR Ca2+ release and fulminant MH  mutation, we used homozygous knock-in mice with the homologous T4826I-RYR1 mutation . This muinant MH . We examocytosis .2+ (syn-GCaMP6f) and synaptic vesicle exocytosis (syn-pH) in both wild-type and T4826I-RYR1 mutant mouse neurons  mouse hippocampal neuron cultures were transfected with either syn-GCaMP6f or syn-pH for live-cell imaging. First, we measured presynaptic cytosolic Ca neurons . In cont neurons b–g.2+ and synaptic vesicle exocytosis  of action.We hypothesized that depression of presynaptic Caoflurane b. Howeve2+ in wild-type and T4826I-RYR1 mutant mouse hippocampal neurons to explore the impact of this MH-susceptibility mutation on anesthetic effects. Isoflurane produced a large reduction in the stimulation-evoked increase in ER Ca2+ concentration in T4826I-RYR1 mouse hippocampal neurons compared with wild-type neurons  compared with in wild-type neurons . In contrast, propofol, an intravenous anesthetic that is not a trigger of MH, reduced evoked ER Ca2+ concentration in wild-type neurons but did not potentiate the reduction in T4826I-RYR1 mutant neurons . The effects of isoflurane or sevoflurane on evoked increases in ER Ca2+ in T4826I-RYR1 neurons were significant compared with their effects on ER Ca2+ in wild-type neurons  in T4826I-RYR1 neurons . Hereditary ER dysfunction has been associated with cognitive dysfunction , inositol triphosphate receptor (IP3R), or RYR mediated mechanisms  are expressed in brain, however their roles in neuronal function have not been characterized. RyR expression has been detected in several hippocampal neuron compartments including in presynaptic terminals, dendritic spines, and somata . Howeverin vitro findings. Our results show that the T4826I-RYR1 mutation exacerbates isoflurane depression of stimulus-evoked increases in ER Ca2+, as well as of presynaptic cytosolic Ca2+ and SV exocytosis. This effect was specific to the volatile anesthetics sevoflurane and isoflurane, while propofol, a mechanistically distinct intravenous anesthetic, had no significant effect on ER Ca2+ in MH-susceptible neurons compared with wild-type neurons. This is consistent with the MH triggering activity of volatile anesthetics, which can be lethal in triggering MH, while propofol is safe for MH-susceptible patients (T4826I-RYR1 mutant mice are a suitable model for studying MH susceptibility: they have no overt phenotype in the absence of volatile anesthetics, as is typical in humans with this and other MH-susceptibility mutations. We used homozygous T4826I-RYR1 mice since 100% of homozygous T4826I-RYR1 mice develop fulminant MH while only 17% of male and no female heterozygous T4826I-RYR1 mice develop fulminant MH in response to halothane as a trigger . Such copatients . The mar2+ regulation.Our results imply that an MH episode might involve direct neurologic effects, including depression of synaptic transmission with possible short-term and long-term effects not yet investigated clinically . Heat-inTreatment of MH involves early administration of dantrolene, an RyR antagonist. Although dantrolene is lipid soluble with a molecular mass of 314, properties that generally predict blood-brain barrier permeability, it exhibits limited CNS penetration , and thein vivo. Our focus on presynaptic function might have overlooked other relevant mechanisms observed in intact neuronal circuits, including postsynaptic and glial contributions. We focused our studies on the hippocampus as a model given the large body of data available on its fundamental neurophysiology, sensitivity to general anesthetics, and critical role in anesthetic actions (2+ regulation are shared between many neuron types, however observations in hippocampal neurons might not translate to other neuronal types because of cell type-specific differences. Use of a single RyR1 mutation model for MH susceptibility from the many known human mutations (Use of primary dissociated neurons as an experimental model does not fully recapitulate cellular function and interactions  actions . The funutations  is a potutations .2+ has implications for the presynaptic mechanisms of volatile anesthetics. Future studies will examine the role of the STIM1 feedback loop in anesthetic mechanisms using knock-down models (Understanding the mechanisms of anesthetic effects on ER Can models . It is a2+ regulation and synaptic vesicle exocytosis in rodent hippocampal neurons. We identified and characterized anesthetic effects on intracellular Ca2+ regulation, specifically on stimulation-evoked changes in ER Ca2+ concentration, in hippocampal neurons from both wild-type and malignant hyperthermia-susceptible mice. We identified reduced baseline and stimulation-evoked increases in neuronal ER Ca2+ concentration as a novel mechanism of volatile anesthetic action. We also revealed a novel neuronal phenotype of MH susceptibility in response to the volatile anesthetics isoflurane and sevoflurane, which trigger MH. Finally, we provide functional evidence for a presynaptic role of RyR1 in synaptic transmission in the hippocampus. Taken together, our findings implicate RyR1 in presynaptic function and pave the way understanding and improving general anesthesia, and in treating those with MH-susceptibility mutations. Our results provide several important advances in elucidating the role of presynaptic ER Ca2+ mechanisms and RyR1 function in neuropharmacology and the actions of volatile anesthetics.In conclusion, we used optogenetic tools to study volatile anesthetic effects on presynaptic Ca


We generated transgenic mice for concomitant monitoring of OPCs Ca2+ signals and cell fate using 2-photon microscopy in the somatosensory cortex of awake-behaving mice. Ca2+ signals in OPCs mainly occur within processes and confine to Ca2+ microdomains. A subpopulation of OPCs enhances Ca2+ transients while mice engaged in exploratory locomotion. We found that OPCs responsive to locomotion preferentially differentiate into oligodendrocytes, and locomotion-non-responsive OPCs divide. Norepinephrine mediates locomotion-evoked Ca2+ increases in OPCs by activating α1 adrenergic receptors, and chemogenetic activation of OPCs or noradrenergic neurons promotes OPC differentiation. Hence, we uncovered that for fate decisions OPCs integrate Ca2+ signals, and norepinephrine is a potent regulator of OPC fate.Oligodendrocyte precursor cells (OPCs) generate oligodendrocytes, contributing to myelination and myelin repair. OPCs contact axons and respond to neuronal activity, but how the information relayed by the neuronal activity translates into OPC Ca How oligodendrocyte precursor cells (OPCs) integrate calcium signals and neuromodulatory cues to regulate fate is unclear. Here, the authors report that locomotion-induced norepinephrine release modulates OPC calcium dynamics and differentiation. Unlike neurons and astrocytes, OLs are continually produced throughout adulthood3 from a pool of oligodendrocyte precursor cells (OPCs) constituting 5-8% of all brain cells. Although myelination is thought to end in young adults, it remains incomplete in most brain areas and continues throughout the adulthood5 For example, about 20–40% of axons are unmyelinated in the corpus callosum6, and axonal segments of excitatory and inhibitory neurons exhibit discontinuous myelination in the cortex8. This partial myelination in the CNS opens up a window for the formation of new myelin sheaths on unmyelinated axonal segments. Newly layered myelin extends metabolic support to neurons9, fine-tunes conduction velocity of individual axons and consequently influences neural circuit function11. Hence, myelin remodeling is thought to be yet another form of long-term plasticity contributing to adaptive changes in brain function12.Oligodendrocytes (OLs) are myelinating cells in the central nervous system (CNS). They are post-mitotic cells and can survive for the entire lifespan of an organism14. The most common form of myelin remodeling originates from OPCs differentiating into new OLs, which then myelinate previously unmyelinated axonal segments13. Before OPCs differentiate into myelinating OLs, they integrate numerous cues in their local environment including neuronal activity and growth factors15. Rather than evolving unique signaling mechanisms, OPCs detect and respond to neuronal activity using the same pathways engaged by neurons during synaptic transmission. OPCs form direct synapses with unmyelinated axons17 and express a wide variety of synaptic and extra synaptic neurotransmitter receptors20, thereby allowing them to engage in rapid communication. Axon-OPCs synaptic junctions are rapidly lost as OPCs begin to differentiate into OLs20. This implies that, within the oligodendrocyte lineage, OPCs are uniquely endowed with the ability to sense and respond rapidly to electrical and chemical signals emanating from axons21.OLs can remodel myelin by extending and retracting existing sheaths, or by adding entirely new internodes22. In addition to the classical neurotransmitters such as glutamate and GABA, which act through local synaptic activity, OPCs in the adult brain might integrate extra-synaptic inputs from neuromodulators to guide their behavior. Indeed, several transcriptomics studies indicate that distinct subtypes of oligodendrocyte lineage cells (OLCs) express a wide variety of these neuromodulator receptors24 which can affect their proliferation and differentiation26. Notably, NE is a key neuromodulator involved in crucial biological processes including arousal, attention, memory formation, sleep/wake cycles and stress management27. NE has also been shown to regulate the proliferation and differentiation of neural progenitors29. Although NE imparts immense influence on the neuronal function, its effect on OPC fate and myelination is elusive.Neuromodulators including acetylcholine (ACh), norepinephrine (NE) and serotonin (5-HT) can influence brain-wide neuronal activity2+ as a second messenger. Ca2+ is a universal signaling ion, and intracellular changes in Ca2+ concentrations appear to modulate the fate of OPCs by regulating proliferation, differentiation and programmed cell death30. Recent single-cell RNA-sequencing studies on rodent brains have identified a dozen cell types constituting the oligodendrocyte lineage 23, each expressing a distinct repertoire of neurotransmitter and neuromodulator receptors which grants them a unique ability to interact with the neighboring cells. During development, OPCs can experience varying levels of local Ca2+ changes based on their location, level of synaptic and extra-synaptic connectivity and type of neurotransmitter receptor expressed, which in turn can influence their fate. In the larval zebrafish, it has been shown that, during development, the characteristics of Ca2+ signal experienced by OPCs can regulate their differentiation into OLs31 as well as the stability of newly formed myelin internodes33. Although these studies highlight the significance of Ca2+ signaling in regulating developmental OL generation and myelination, little is known about the characteristics of Ca2+ activity or the pathways that regulate Ca2+ signals in OPCs in the mature mammalian brain.Neuromodulators such as NE exert their influence on target cells through the activation of G-protein coupled receptors (GPCRs), and often involve Ca2+ signals in OPCs might encode information about their developmental stage and fate progression. Hence, to understand how Ca2+ signals regulate the fate of OPCs, we performed high-resolution imaging of intracellular Ca2+ transients across the entire oligodendrocyte lineage, monitored cell fate and chemogenetically manipulated OPC Ca2+ signals and neuronal activity. To simultaneously study the Ca2+ signals and map the fate of OPCs, we developed novel transgenic mouse lines expressing two variants of a genetically encoded Ca2+ sensor and a red fluorescent reporter in OPCs, and performed chronic 2-photon microscopy on awake behaving mice. We found that Ca2+ signals in OPCs mainly occur within processes, and that OPCs exhibit distinct Ca2+ signals while they proliferate or differentiate. We also found that NE release from neurons evokes intracellular Ca2+ increases in OPCs and promote their differentiation in oligodendrocytes.Like neurons, the source, location and amplitude of Ca2+ signals of OPCs while they proliferate or differentiate into oligodendrocytes (OLs), we took a transgenic approach and generated two distinct triple-transgenic mouse lines  in the soma as well in the processes of OPCs, we used a mouse line conditionally expressing a cytosolic variant of ultrafast genetically encoded green fluorescent Ca2+ sensor GCaMP6f34(TIGRE2-tTA-LSL-GCaMP6f)  in OPCs, and a Cre-reporter mouse line expressing cytosolic red fluorescent protein tdTomato36(Rosa26-LSL-tdTomato)  with tamoxifen to induce the expression of GCaMP6f and tdTomato reporters in OPCs  and several oligodendrocyte lineage cells (OLCs) specific markers  Fig. . To geneto) Fig. . At 3–4 PCs Fig. . 2–3 weeers Fig. . Our hisers Fig. . Howeverers Fig. . Both tders Fig.  and ASPAers Fig. , and rev37, myelin segments38, and axonal compartments of neurons39. Hence, to record Ca2+ transients in OPC processes, mature oligodendrocytes and axonal projections, we generated a novel ROSA26 targeted knock-in mouse line which can conditionally express a membrane anchored sensitive variant of GCaMP6 (Rosa26-LSL-mGCaMP6s) . 3-4 weeks old mice were injected with tamoxifen to induce expression of tdTomato and mGCaMP6s in OPCs . mHC permitted head-fixation and at the same time allowed mice to freely explore their cage . We observed that Ca2+ transients in OPCs mostly occur in the processes and remain restricted to small segments – we termed such spatially confined micrometer size (4.64 ± 3.33 µm2) regions in OPCs as Ca2+ microdomains (CaMs)  Fig. . BinarizaMs Fig. , thereby2+ activity, we tracked the location, trajectory, and locomotion speed of the mouse in real time   than during the resting phase , there was no change in the average amplitude of Ca2+ events (resting: 8.61 ± 1.17 z and active: 8.52 ± 1.31 z) between the resting and the active phases   during the resting phase  Fig. . ConversaM) Fig.  and eachses Fig. . During  s) Fig. . In addiher Fig. . On averase Fig. .2+ signals generated by OPCs, we imaged Ca2+ transients in individual OPCs twice on the same day at acquisition speeds of 5 Hz  constituted about 9.7% and 7.0% of all Ca2+ events during resting and active phase, respectively  increased slightly in response to locomotion, the change in frequency of ‘slower’ Ca2+ events (>0.6 s) accounted for the majority of the increase during locomotion  of OPCs and their Ca2+ activity at each stage of OPC fate. We took advantage of our NG2-GC6f;tdT mouse line, in which tdTomato and GCaMP6f are permanently expressed once OPCs undergo Cre-mediated recombination, even after they divide or differentiate. This implies that we could image cells across the many distinct stages of the OL lineage. Hence, to classify OLCs across developmental stages based on their distinct morphology, we performed immunohistochemistry on the brain sections from adult NG2-GC6f;tdT mice and labeled recombined cells with cell-type specific markers for OPCs (PDGFRα+), premyelinating oligodendrocytes  and mature OLs (ASPA+). We then acquired confocal z-stacks of the cytosolic tdTomato in each of those cell types  during active and resting phases (A/R ratio). When the Ca2+ events frequency increased to >150% in response to locomotion (i.e. A/R ratio of 1.5), cells were considered as locomotion-responsive    Fig. ; and (2)49) Fig. . We note49) Fig. . When OP2+ events frequency similar to that of OPCs, and exhibited significant increase in the frequency of Ca2+ transients in response to locomotion, albeit at reduced level compared to OPCs   45 with the conditional mGCaMP6s and tdTomato reporter mice . We imaged Ca2+ transients in mature OLs in the S1 cortex of awake Mog-mGC6s;tdT mice aged 8–10 weeks , and these signals were mostly restricted to the processes and myelin segments, as somatic Ca2+ transients were rarely seen    Fig. . AlthougOLs Fig. , black. 2+ signals showed that OPCs maintained very stable microdomain Ca2+ activity  and just as they were ready to differentiate   to this mode of activity likely undergo cell division.To systematically track the lineage progression of OPCs, we imaged tdTomato and GCaMP6f signals in individual OPCs in the S1 cortex every 2–4 days over a period of 2–3 weeks Fig. . A detaiity Fig. , Restingity Fig. . During ity Fig. , Active.nts Fig.  in responts Fig.  differennts Fig. . The popin) Fig. . Furtherin) Fig. . Taken t18. To study the effect of neuronal activity and ionotropic glutamate and GABA receptor activation on baseline and locomotion-induced Ca2+ signals in OPCs, we performed in vivo 2-photon Ca2+ imaging in the S1 cortex. We first imaged Ca2+ signals from a select population of OPCs (18 cells) . However, the number of activated CaMs (1.89 ± 1.78 CaMs/min) and the frequency of Ca2+ transients (3.79 ± 3.8 events/min) during spontaneous activity in acute brain slices . There were almost no changes in the number of CaMs (TTX: 1.92 ± 1.46 CaM/min) or the frequency of Ca2+ events (TTX: 3.64 ± 3.65 events/min), but a slight increase in the amplitude of Ca2+ events in the presence of TTX  and GABA receptor  antagonists respectively, suggesting spontaneous Ca2+ transients in OPCs are in part generated by the spontaneous release of glutamate and GABA , which constitute about 10% of the frequency of the baseline events seen in vivo, remains unknown, and can be possibly driven by activation of other cell-surface receptors and cell-intrinsic mechanisms.To further dissect the source of baseline Ca46. Since axonal projections of LC neurons in the cortex are very thin and profusely branched, cytosolic variants of GCaMPs don’t reach high enough concentration in axons for a reliable measurement of Ca2+ signals in vivo47. We therefore used a membrane anchored, sensitive variant of GCaMP6 (mGCaMP6s) to image Ca2+ signals in LC axons. We cross-bred Dbh-Cre mice48 with the mGCaMP6s mouse line to generate double transgenic mice   were also positive for tyrosine hydroxylase, an enzyme involved in NE synthesis and labeling adrenergic neurons in the cortex , which in turn release norepinephrine (NE) across several brain regions, including the cortex6s) Fig. . The immtex Fig. . Similartex Fig. . We fountex Fig. . Howevertex Fig. , the firses Fig. , and theses Fig. . NotablyDbh-mGC6s mice using antibodies against eGFP and PDGFRα, respectively 49. 3–4 days after DSP4 injection, we performed histological analysis to visualize LC fibers expressing mGCaMP6s, and found that almost two thirds (63.12%) of the LC projections in the cortex were successfully ablated  agonist, phenylephrine  in the presence of voltage gate sodium channel blocker TTX (0.5 µM), which blocks neuronal firing  , we bath-applied PE in the presence of a cocktail of drugs composed of a neuronal activity blocker (TTX) combined with either (1) ionotropic and metabotropic glutamate receptors antagonists   Fig. g. To furG) Figs.  or (2) GG) Figs.  antagoniPCs Fig. , suggest2+ transients in OPCs. Therefore, to confirm that indeed direct activation of α1-ARs in OPCs can mediate Ca2+ signaling, we isolated a pure OPC population using magnetic activated cell sorting (MACS) from the mouse cortex and cultured them for 2 weeks  evoked robust Ca2+ transients in CaMs and the soma of OPCs . To identify which α1-ARs subtypes are expressed by the OLCs, we performed simultaneous immunohistochemistry and single-RNA-molecule fluorescent in-situ hybridization (sm-FISH) in the S1 cortex of adult wildtype mice. We labeled OPCs, pmOL, and mature OLs using in-situ probes or antibody against CSPG4 (NG2), Enpp6, and ASPA respectively. All three α1-AR subtypes were labeled with their respective in-situ probes, and nuclei of all cells were labeled with DAPI , α1b  and α1d  antagonists when applied individually , but not for α1a and α1d α1-ARs  was bath-applied without α1-ARs antagonists , and signal through phospholipase C mediated production of IP3, downstream activation of IP3 receptors (IP3Rs) and release of Ca2+ from the internal stores55. At first, to confirm that indeed activation of hM3Dq DREADDs on OPCs increase intracellular Ca2+ signals, we co-expressed hM3Dq-mCherry and GCaMP7s in primary OPCs cultures. Then, we performed 2-photon Ca2+ imaging, and observed that a bath application of Clozapine N-Oxide  enhanced the frequency of Ca2+ transients 2-fold from the baseline   and control  littermates with tamoxifen to induce the expression of hM3Dq-mCitrine and tdTomato in OPCs  or CNO    and Dbh-Gq (Dbh-hM3Dq) mice for 5 days with CNO (1 mg/kg). Along with CNO, we treated control and Dbh-hM3Dq mice with BrdU for two weeks to label proliferating OPCs   when compared to the control (48.22 ± 11.38%) mice  Fig. . To studPCs Fig. . After tice Fig. , confirmice Fig. . This anice Fig.  in Dbh-hPCs Fig.  in Dbh-hly) Fig. . We did ice Fig. . In summ58. OPCs integrate several signaling pathways including growth factors and neuronal activity before they differentiate into myelinating OLs15. In this study, we discovered that OPCs and its lineage cells display a wide variety of Ca2+ signals, and that these signals can be modulated by NE signaling, which in turn influences the fate of OPCs , have previously been described in several cell types including neurons59 and astrocytes60. In OPCs, somatic Ca2+ transients were rarely seen and occurred only occasionally when mice engaged in intense locomotion activity  was dependent on the activation of ionotropic glutamate and GABA receptors , and Ca2+ efflux from mitochondria64.Ca2+ flux at OPC CaMs to be visualized as ‘fast’ Ca2+ transients remains an open question65. Of all the Ca2+ signals we recorded in OPCs, about 45% of Ca2+ signals in OPCs CaMs were of sub-second duration, about ~1.5% of all Ca2+ events were as ‘fast’ as 330 ms, and might represent EPSCs at OPC-axon synapses  of these events estimated with cytosolic GCaMP6f. Thus, we suggest that in OPCs ‘fast’ Ca2+ signals (330–600 ms) could be a mix of opening of calcium permeable AMPA (cpAMPA) and NMDA receptors, A-type voltage gated Ca2+ channels and even activation of various Gαq-GPCR coupled metabotropic receptors61.Whether excitatory post-synaptic currents (EPSCs) at OPC-axon synapses generate enough Ca2+ imaging studies revealed that OPCs exhibit distinct Ca2+ dynamics while undergoing fateful decision of cell division or differentiation  OPCs in larval zebrafish, and reported the existence of two distinct pools of OPCs based on the anatomical location and Ca2+ fluctuations31. OPCs with high baseline Ca2+ activity were located in neuronal soma-rich areas and those with low baseline Ca2+ activity were located in axo-dendritic rich areas of the SC. This study reported that OPCs with low baseline Ca2+ activity (in axon-rich areas) preferentially differentiated into OLs. While we identified two pools of OPCs within the cortical grey matter in the brain of adult mice, unlike in the zebrafish SC, both the OPC pools exhibited similar level of baseline activity31. However, both mouse OPC types exhibited distinct response to neuromodulatory signals i.e., one sub-type (LN OPCs) responded to locomotion and other sub-type (LNR OPCs) did not  integrate information encoded in localized Ca2+ transients to trigger myelin formation and fine-tune myelin internodes. However, further detailed analysis will be required to understand the role of pmOLs Ca2+ transients in regulating myelination in the adult brain. Remarkably, as OLs become fully mature, they undergo major physiological changes such as fluctuations in membrane potentials18, activation of K+ leak currents, expression of TRPA1 channels71 and downregulation of several Gq-coupled metabotropic receptors23. These changes potentially translate into the very low baseline Ca2+ transients, and the loss of locomotion evoked and NE-mediated Ca2+ signaling we observed  and early stage pmOLs (likely pmOL1 in this study) had higher expression of α1-ARs and IPted Fig. . Hence, ved Fig. , suggest72. In this study, we uncovered that locomotion-induced microdomain Ca2+ signals in OPCs is due to the activation of α1-ARs on OPCs. Several observations implied that the locomotion-induced Ca2+ signals in OPCs could be evoked by the action of NE  axonal projection from Dbh+ neurons criss-cross the entire cortex and make a close contact with OPC processes; and (3) ablation of LC fibers in the cortex, using the neurotoxin DSP-449, abolished locomotion-induced activation of CaMs in OPCs. Glutamatergic neurons release neurotransmitters within a few milliseconds once they are depolarized73. Hence, if glutamate or GABA release at the OPC-axon synapses would lead to locomotion induced Ca2+ transients, OPCs would have responded instantaneously. However, we observed a delay of about 4 s for OPCs to reach the maximal activation of CaMs from the instant the mouse engaged in locomotion  du75, and α1b ARs are expressed in both OPCs and OLs76. Although α1d-AR promoter-driven LacZ reporter expression shows these receptors are abundant in the cortex, a cell-type specific expression analysis has not yet been performed77. A recent study on single-cell RNA sequencing of OLCs showed that α1a and α1b ARs were expressed in various OLCs and that α1b-ARs was the most expressed sub-type23. However, none of these studies showed that α1-ARs are functionally active on OPCs, or explored their dynamic expression in OLCs. Our systematic single-RNA molecule fluorescent in-situ hybridization (smFISH), Ca2+ imaging and extensive α1-ARs pharmacology showed that about 50% of OPCs expressed various α1-AR sub-types, and that this proportion decreases as they differentiate into pmOL and OL  to confirm that OPCs indeed express α1-ARs and that their direct activation induces Ca2+ signals. Yet, there remains a possibility that some of the effect of NE on OPCs can be due to an indirect action of neurotransmitters and growth factors released by activation of other neural cells including neurons and astrocytes54 by NE. In the future, availability of more efficient OPC-specific Cre-driver mouse lines for gene deletion, and floxed mice for conditional deletion of Adra1b and Adra1d genes will allow us to perform a systematic deletion of all three α1-ARs in OPCs, and enable us to investigate the more definitive role of cell-autonomous α1-ARs mediated Ca2+ signaling in OPCs. In this study, we nonetheless highlight the role of neuromodulators such as NE in regulating Ca2+ signals and cell-fate of OPCs in adult brain in vivo, and shed light on distinct mechanisms for regulating OPC fate in an activity-dependent manner.Previous studies using transgenic mice expressing eGFP reporters under the control of promoters for α1a and α1b ARs showed that α1a ARs are expressed in OPCs but not in mature OLs OL Fig. . In this3. NE might be the missing link between the basal neuronal activity sensed by OPC and their fate. A brain wide volumetric release of NE inhibits random and spontaneous neuronal activity, strengthens neuronal connectivity, and potentiates sensory and motor functions78. Low NE levels are associated with sedation or sleep, whereas high levels of NE lead to arousal. A transcriptomics and histological analysis performed in mice during sleep and wake cycles suggested that the proliferation rate of OPCs doubled during sleep whereas wakefulness promoted the differentiation of OPCs79. Like neurons, we propose that NE enhances the gain of ongoing baseline activity at the axon-OPC synapses, thereby promoting the differentiation and maturation of OPCs into myelinating OLs. In line with this hypothesis, we observed that NE promoted differentiation of OPCs both in vitro and in vivo 82. This suggests that neuromodulators such as NE might promote the maturation of OPCs which are already primed to become differentiated.Despite the fact that all OPCs have axon-OPC synapses and that the synaptic activity at these junctions is known to guide their fate, it remains unknown why very few OPCs successfully differentiate, mature and myelinate in the adult brainivo Fig. . It is n83. Instead, we observed no change in the proliferation of OPCs in vivo in response to chemogenetically enhanced NE signaling  NE mediated activation of β-ARs suppresses proliferation and promotes differentiation of OPCs84 and (2) as OPCs progressed towards a more differentiated state (COPs and pmOLs) the activation of α1-ARs further promotes their maturation into myelinating OLs. It is well established that pmOLs are susceptible to death and that only a few survive in vivo85. We suggest that IP3R2+/α1-AR+ pmOLs, which integrate neuromodulatory cues, are more likely to mature into OLs23. Thus, pmOLs might represent a promising pool of cells which effectively respond to NE signaling, and contribute to the adaptive myelination and in myelin repair87.We also made an unusual observation - in general, OPC differentiation is associated with increased OPC proliferation events+ neurons in the LC were reported in the brains of individuals with multiple sclerosis (MS)88. Similar observations were made in the cortex and SC of a mouse model of demyelination , which had reduced NE levels90. In addition, studies on such mouse models indicate that increased levels of NE in the CNS can reduce disease severity91. NE could also be a connecting link between myelin and OLCs dysfunction seen during stress and depression92. For example, serotonin and norepinephrine reuptake inhibitors (SNRIs) such as venlafaxine, which enhance NE levels in the brain, promote myelination and improve cognitive function in mouse model demyelination93. Thus, enhanced NE levels through SNRIs or electrical stimulation of LC neuron e.g., using tDCS  might have the potential for improved remyelination in MS94.Although very little is known about the role of NE signaling on OPCs in vivo, reduced levels of NE and hypertrophy of TH2+ signals, which can be modulated by NE. NE signaling plays a pivotal role in regulating survival, proliferation and differentiation of OLC . All animal experiments were carried out in a strict compliance with German Animal Protection Law (TierSCHG) at the Heidelberg University, Germany.Both male and female mice were used for all experiments, and mice were randomly allocated to experimental groups. For ex vivo STOP cassette was placed upstream of the mGCaMP6s  to the first methionine (i.e. start ATG) of GCaMP6s sequence (termed mGCaMP6s). To enhance expression of mGCaMP6s, we used a strong ubiquitous CMV-βactin hybrid (CAG) promoter and placed the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) at the 3′ end of mGCaMP6s expression sequence. For inducible expression of mGCaMP6s, a loxP flanked 35 (Jax# 008538), Dbh-Cre48, GCaMP6f96, tdTomato reporter mouse lines36 (Jax# 007914), Mogi-Cre45 and hM3Dq-Cirtine chemogenetic effector mice56 (Jax# 026220) have been previously described  was prepared by dissolving tamoxifen  in sunflower seed oil . To ensure the tamoxifen was fully dissolved, the solution was sonicated at room temperature for approximately 5 min, then vortex for 30 s. This process was repeated until total dissolution was achieved and no crystals remained visible in the solution. The solution was then stored at 4 °C, protected from light, for a maximum of 5 days. Mice aged 4–6 weeks were injected intraperitoneally (i.p.) with tamoxifen at a dosage of 100 mg/kg of body weight once a day for 3 or 5 days, depending on the experiments. Each injection was performed at least 20 h after the previous one. All subsequent experiments were performed at least 2 weeks after the last tamoxifen injection.Clozapine-N-Oxide (CNO) solution was prepared by dissolving water-soluble CNO  in 0.9% saline at a concentration of 100 µg/mL. Mice were injected with CNO at a dosage of 1.0 mg/kg (i.p.) daily for 5 consecutive days. During the same period, mice were given 5-Bromo-2′-deoxyuridine (BrdU) . BrdU was dissolved in 0.9% saline at a concentration of 10 mg/mL. Mice were injected twice daily with BrdU at a dosage of 50 mg/kg (i.p.) for 5 consecutive days. For maximum labeling of proliferating cells, beginning on the day of the first BrdU injections, mice were also provided with a 1.0 mg/mL BrdU in 1% sucrose solution as drinking water.2+ imaging experiments were carried out at least 3 days after the DSP-4 injection.To deplete noradrenergic fibers, mice were injected with the noradrenergic neuron specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride  once at a dose of 50 mg/kg (i.p.). To ensure NE fibers were successfully depleted, subsequent histological and CaMice were deeply anesthetized by injecting pentobarbital (Narcoren) at 150 mg/kg (i.p.), and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (pH 7.4). Brains were carefully dissected out from the cranium and post-fixed in 4% PFA for 16 h (overnight) at 4 °C. The brains were then transferred to 1× phosphate-buffered saline (PBS) and 35 µm thick coronal sections were cut using a vibratome . Sections were collected in PBS containing 0.2% sodium azide and stored at 4 °C until further processed. Prior to immunostaining, the sections were permeabilized 0.3% Triton X-100 in 1× PBS for 10 min at room temperature (RT). To prevent non-specific binding of antibodies, the sections were then placed in a blocking buffer  in 1× PBS) for 1 hr at RT. Then, the sections were incubated overnight with the primary antibodies . Following the post-fixation overnight in 4% PFA at 4 °C, the brains were dehydrated by sequentially storing them in 10%, 20% and 30% sucrose in 1× PBS at 4 °C and until they sank to the bottom of the vial in each solution. Brains were then cut to fit a 7 mm × 7 mm × 5 mm (L × W × H) embedding mold, frozen in optimal cutting temperature compound  with dry ice and stored at −80 °C. The tissue was later sliced into 16 μm thick sections using a cryostat , and stored in an airtight container at −80 °C until further processed. In situ hybridizations was performed using the RNAscope Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics) for fixed frozen tissue according to the manufacturer’s instructions with the following probes: Mm-Adra1a-C2 (408611-C2), Mm-Adra1b-C2 (413561-C2), Mm-Adra1d (563571), Mm-Cspg4-C3 (404131-C3), and Mm-Enpp6-C4 (511021-C4). Fluorescent signals were then amplified using a TSA Plus Fluorescein kit . In addition, some sections were stained with DAPI and anti-ASPA antibodies (see Immunohistochemistry). All sections were mounted on SuperFrost Plus slides , and later imaged using a confocal microscope .smFISH confocal image stacks were imported into Image J for quantification. The nuclei of oligodendrocyte lineage cells were identified using a combination of DAPI and a cell-type specific marker , then carefully outlined. Puncta were manually counted on a plane-by-plane basis from the confocal z-stacks for optimal precision. Cells were only considered positive if the puncta were located directly on the nucleus or its perimeter.Images were acquired using a Leica SP8 Confocal microscope equipped with either a 40×  or 63×  objectives, with the pinhole set to 1 Airy Unit (AU). Images were acquired at a resolution of 1024 × 1024 pixels, with a system-optimized step size of 0.423 μm. Image size varied between 290.62 × 290.62 μm and 87.87 × 87.87 μm depending on the objective and magnification factor used. Images were then further processed using ImageJ, and presented as pseudocolored maximum intensity projections.97 plugin for ImageJ and imported them to MATLAB using a custom code. We then computed the area of the soma and its circularity using the MATLAB regionprops built-in function. Next, we calculated the distances between consecutive main branches as a proportion of the circumference of the soma and used standard deviation of these distances to estimate the spread of main processes around the OLCs somaThe morphology of OLCs was analyzed using the tdTomato channel on either in vivo 2-photon images (average intensity time-series projection of an entire recording) or confocal microscopy z-stack images. Importantly, confocal z-stacks were limited to only a few frames to ensure that the resulting analyzed image could be reliably compared to the 2-photon average time-series projections. Then, using ImageJ, we manually segmented the soma and traced the skeleton of each cell with the Simple Neurite Tracer (SNT)2PO4, 1.5 MgCl2, 0.5 CaCl2, 10 Dextrose, 20 Choline Bicarbonate (pH 7.4 and ~310 mOsm/L). Cortical slices were then incubated in artificial cerebrospinal fluid (ACSF) containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4, 26.2 NaHCO3 and 11 Dextrose (292–298 mOsm/L) for 30 min at 37 °C, then maintained at RT for the entire duration of the experiment. The NMDG cutting solution and the ACSF were continuously bubbled with carbogen (95% O2/5% CO2). All slice imaging experiments were performed at the RT.Mice were anesthetized with isoflurane and immediately decapitated with a pair of large scissors. Their brains were quickly dissected out and stuck on a 5 mm agar stage before being mounted on a vibratome (Leica VT1200S) equipped with a sapphire blade. 250 μm thick coronal cortical slices were cut in ice-cold N-methyl-D-glucamine (NMDG) based cutting solution containing (in mM): 135 NMDG, 1 KCl, 1.2 KH+ channel blocker TTX  to block neuronal firing of action potentials. Following drugs were used for pharmacological manipulations ; GABAB receptor antagonist—CGP55845 ; adrenergic α1 A receptor antagonist—RS17053 ; adrenergic α1B receptor antagonist –Chloroethylclonidine ; and adrenergic α1D receptors antagonist—BMY 7378 .All pharmacological experiments on the acute brain slices or cultured cells were performed by dissolving drugs directly in ACSF, and applying them through a fast perfusion system. Unless otherwise specified, all experiments were performed in the presence of a voltage-gated Na2+ concentration changes and morphology of OLCs were imaged on a custom-built 2-photon microscope  fitted with a 20× water-immersion objective , 8 kHz galvo/resonance scanner, piezo drive for fast z-scanning, and two GaAsP PMT detectors. 2-photon excitation for GCaMP6f, mGCaMP6s, jGCaMP8s and tdTomato at a single wavelength was achieved using a mode locked Ti:Sapphire pulsed laser  tuned at 940 nm. Images were acquired at a resolution of 512 × 512 pixels (85.5 × 85.5 μm) and 12-bit pixel depth, with each imaging session consisting of a 5-min recording of 920 frames at 3.1 Hz. Over the entire course of the imaging, the acute brain slices/cover slips were steadily perfused with oxygenated ASCF. Since it was difficult to identify OPCs expressing mGCaMP6s in acute brain slices, they were briefly exposed to 10 µM phenylephrine prior to each experiment, and responsive cells with OPC morphology were further imaged.Intracellular Ca2+ imaging the cover slips containing cultured OPCs were fixed in 4% PFA for 15 min at RT and rinsed 3 times with 1X PBS. Antigen retrieval was then performed by incubating coverslips in L.A.B solution (Polysciences 24310) for 10 min. We then performed immunohistochemical staining (see Immunohistochemistry for brain slices) with markers for OPCs and mature OLs on these cover slips to locate each imaged OLC and identify their cell-type .To validate the identity of cells in culture, after Ca2. Cells were kept in OPC medium composed of DMEM/F12 medium (Gibco 11330032) supplemented with SM1 (StemCell Technologies 05711), N2B (StemCell Technologies 07156), Penn/Strep , N-Acetyl-Cysteine , Forskolin , CNTF , PDGF-AA , and NT-3 . The medium was changed every other day until the cells reached optimal confluency of 60–80% for further experiments.Brain cortices from 4–5 days old C57BL/6N pups were dissociated into single cell suspensions using Neural Tissue Dissociation Kit (T) (Miltenyi Biotec) for magnetic activated cell-sorting (MACS) according to the manufacturer’s protocol. In order to eliminate astrocytes and obtain an enriched fraction of OPCs, cells were isolated from cortical suspensions by 2-step MACS during which anti-GLAST (ACSA-1) and anti-O4 microbeads (Miltenyi Biotec) were used sequentially. Isolated OPCs were seeded on poly-D-lysine (Gibco A3890401) coated glass coverslips at an approximate density of 30,000 cells/cm2+ imaging experiments were performed 2–3 days post transduction (see Time-lapse two-photon microscopy of ex vivo and in vitro preparations).We produced lentiviruses by co-transfecting GCaMP7s or Ubc-Caprese (cytoplasmic jGCaMP8s with an N-terminus fusion of mScarlet red fluorescent protein) expression vector with three packaging vectors  into HEK293 cells using polyethylenimine . The virus-containing media was then collected 24 h and 48 h post-transfection, and ultra-centrifuged at 25300 rpm for 2 h at 4 °C (SW 32 Ti Swinging-Bucket). The pelleted viral particles were resuspended in OptiMEM medium , aliquoted and stored at −80 °C until use. Custom-made mCherry-hM3Dq (pLV[Exp]-mCherry-UBC>hM3Dq) lentiviruses were purchased from VectorBuilder. One week (6–7 days) after the start of the culture, OPCs were transduced with the appropriate viruses. The CaImmunohistochemistry for brain slices) to evaluate and compare the efficiency of each differentiation protocol.Once the cover slips with OPCs reached 60–70% confluence, PDGF-AA was removed from culture medium. 48 h later, we began differentiating the OPC under four different conditions: (1) Control ; (2) 10 mM PE; (3) 40 ng/mL tri-iodo-thyronine  + 40 ng/mL thyroxine ; or (4) 10 mM PE + 40 ng/mL T3 + 40 ng/mL T4. Reagents were freshly supplemented every day to the cell culture medium. One week later, the cells were fixed, stained, imaged and quantified , and placed on a custom stereotaxic frame (RWD) fitted with a stereo microscope  and thermostat-controlled heating pad (RWD). Throughout the surgery, mice were maintained at 37 °C body temperature, and were administered dexamethasone  and 0.03 mg/kg buprenorphine sub-cutaneously (s.c.) to reduce inflammation and pain, respectively. Skin on the scalp was removed to expose the skull over the right cerebral hemisphere, where a 2 × 2 mm area  was marked using a scalpel. This perimeter was gently drilled to remove the bone, and a 2 × 2 mm piece of cover glass  was placed within the craniotomy and sealed to the skull using histoacrylamine glue . A helicopter headbar with a 5.2 mm circular opening  was attached using dental cement  mixed with cyanoacryl . After the surgery, mice were administered carprofen  every 12 h for two days to reduce pain and inflammation. Whenever possible, multiple mice were housed in the same cage with 1–2 wooden shelters, providing them with an enriched environment. Mice were allowed to recover from surgery for two weeks, before Mobile HomeCage training could begin.Mice aged 5–8 weeks old were implanted with chronic glass cranial windows12 vg/mL). 300–500 nL of the virus were injected 0.65 mm below the dorsal surface of the somatosensory cortex (see coordinates above) at a rate of 20 nL/min . To ensure optimal delivery of the virus to the targeted site, the pipette was withdrawn over 10 min after the injection was completed. Following the cortical virus injection, we implanted a cranial window over the injection site following the protocol in the above section. Finally, the anaesthesia was antagonized by i.p. injection of a mixture of atipazole , flumazenil  and naloxone  in a 1:1:6 ratio. After the surgery, mice were administered carprofen  every 12 h for two days to reduce pain and inflammation.C57BL/6 N mice aged 6–8 weeks were obtained from Janvier Labs and anesthetized by i.p. injection of a mixture of medetomidine , midazolam  and fentanyl  in a 2:3:8 ratio (110 μL/injection). Mice were then placed on a custom stereotaxic frame and were maintained at 37 °C body temperature (see previous section). Skin was gently removed to expose the skull and the injection site  was marked and drilled. Then, a borosilicate glass capillary  with a tip diameter of ~100 μm was front-filled with the ssAAV-9/2-hSyn1-ch1-GCaMP6f-WPRE-SV40p(A) virus , NeuroTar). The mHC consists of a solid aluminum platform coupled to an air compressor which allows a constant upwards flow of air through holes on its surface. The uniform air-cushion it creates is sufficient to support an ultralight carbon-fiber cage  with negligible friction. When mice were placed inside, they were able to easily displace the carbon-fiber cage and engage in unrestricted self-motivated exploration while still being head-fixed, enabling us to study CaTo ensure mice are comfortable being head-fixed in the mHC, mice were progressively acclimatized over the course of one week to remain head-fixed for about 60 min during awake 2-photon microscopy sessions in the mHC. On the first two days, the mice were introduced to the 2-photon imaging environment and extensively handled by the experimenter. The handling steps included occasional manual head-restriction to simulate imaging conditions inside the mHC. On the third day, mice were placed for ~15 min inside the carbon-fiber cage of mHC floated on the air-cushion, and could freely explore the imaging environment without head-fixation. Finally, over the next 4 days, the mice were head-fixed in mHC and placed under the 2-photon microscope for an increasing period of time (+10 min per day), replicating the exact conditions of an imaging session. By the end of the 7th day of habituation, mice were fully habituated and minimally stressed while head-fixated in mHC, and were ready for imaging session lasting between 45–60 min.2+ dynamics in OLCs with explorative behavior of mice. To ensure synchronization of behavioral and Ca2+ imaging data, image acquisition on the 2-photon microscope was initiated by a TTL pulse from the Mobile HomeCage motion tracking software (Tracker version 2.1.10.3). Images were acquired on a custom-built 2-photon microscope  with a 20× water-immersion objective . 2-photon excitation was achieved using a mode locked Ti:Sapphire pulsed laser  tuned at 940 nm (see Time-lapse two-photon microscopy in acute brain slices section for more details). Unless otherwise specified, image time-series were acquired at a rate of 5.1 Hz and a resolution of 512 × 512 pixels (85.5 × 85.5 μm) for a total of 10 min. Mice were kept head-fixed in the mHC for a maximum of one hour per imaging session.As previously mentioned, intravital 2-photon microscopy in awake mice was performed using the mHC setup, enabling us to correlate CaA Bicuculline 4 mg/kg) ionotropic receptor antagonists. Shortly after the injection ( ~ 40 min), the mice were placed back under the 2-photon microscope for another 1-h imaging session and the same cells imaged during pre-drug session were located and re-imaged.Following a one-hour pre-drug 2-photon imaging session, the mice were removed from the mHC and injected i.p. with a combination of glutamatergic  and GABAergic  using a custom code. Active periods were detected automatically and aligned with the 2-photon imaging data using a custom MATLAB code . Locomotion bouts were defined as the period during which the speed of the mouse was above threshold, which was set at one standard deviation above the mean speed of the animal during that session. Importantly, some mice displayed low levels of motor activity, significantly lowering the mean speed and standard deviation used to establish the threshold, which could lead to pseudo-bouts bouts being detected. Hence, bouts were excluded if they did not contain speeds above 20% of that of the maximum observed in that session. In addition, to account for residual locomotion-induced Ca99 (plugin for ImageJ). During registration, TurboReg transforms images into a 32-bit format. Therefore, after registration, images were reformatted to 16-bit. Surrounding background pixels that were not part of the cell (based on median-intensity projection of the image stack) including pericytes and larger blood vessels were cropped to accelerate analysis. For an automatic analysis of Ca2+ microdomain and Ca2+ signals in OLCs, our machine learning based algorithm CaSCaDe was modified accordingly for specific imaging datasets (see details below).To correct for movement artefacts in XY plane, 2-photon time-series image stacks were registered post hoc using automatic alignment algorithm TurboReg2+ microdomain map as described in our prior publication42. Prior to event detection, the traces from each Ca2+ microdomain were extracted and flattened using the MATLAB detrend function. Since awake in vivo imaging can lead to movement-induced changes in fluorescence that are independent of the Ca2+ concentration, we also extracted fluorescence signals from the static tdTomato channel using the same Ca2+ microdomain map. Because the fluorescence of tdTomato does not change as a function of Ca2+ concentration, any rapid changes in fluorescence observed in this channel can be interpreted as an artifact. To avoid including such artifacts in our analysis, we thus subtracted the tdTomato traces from those of GCaMP6f for microdomains. Ca2+ events were automatically detected using peak detection algorithm of CaSCaDe. Only Ca2+ events with amplitude more than 5 z-scores and duration at least 4 frames were considered. For each Ca2+ event, the beginning and ending points were determined using the point of maxima for each peak as a reference. Beginning of the peak was defined as the last point preceding the maximum that has a value below 1 z-score, and the ending is defined as a point where 1 z-score is reached for two consecutive frames, or falls within 10% of the starting point’s value. The resulting Ca2+ signals were classified based on our previously trained SVM classifier42.Using the average projection of the 2-photon image stack, we automatically generated a Ca2+ events. Next, peak detection was performed on the selected microdomains as described in the previous section. Since images acquired at 15 Hz are inherently noisy, an accurate estimation of the duration of Ca2+ event was not possible based on the method implemented to analyze images acquired at 5 Hz. To resolve this, the microdomain Ca2+ traces were passed through a 1 Hz lowpass filter. Then, start and end points of Ca2+ events were computed separately for both the original unfiltered and filtered traces. This method allowed for optimal detection of both fast events (using the unfiltered traces) and slow events (using the filtered traces) simultaneously. Hence, a combination of both binary detection signals (unfiltered and filtered) was computed to obtain the final duration of each event. Importantly, hyper-segmented events  were combined if they occurred less than 0.2 s apart. The resulting Ca2+ signals were also classified based on our SVM classifier42.To acquire image stack at a 15 Hz sampling frequency, the imaging was performed at 2 frame averaging and 256 × 256 pixels resolution, which resulted in higher pixel sizes (0.17 μm/pixel at 5 Hz vs 0.33 μm/pixel at 15 Hz) and increased background noise. Hence, CaSCaDe was modified as follows to accommodate images with lower signal to noise ratio. First, a filtering step was added to exclude inactive or highly noisy microdomains that did not exhibit clear (7+ z-score) Ca2+ time-series were acquired at a 15 Hz sampling frequency and 256 × 256 pixels resolution, as described in the Fast imaging (15 Hz) section above. To correct for movement artefacts in XY plane, 2-photon time-series image stacks were registered post hoc using the Flow Registration100 plugin for ImageJ. Using the resulting average intensity projection, we manually traced ROIs around the soma of every neuron present in the field of view, which were then used as input for CaSCaDe to extract the Ca2+ dynamics. Due to the rapid firing pattern of neurons, CaSCaDe would occasionally underestimate the number of Ca2+ signals if they occurred in quick succession. To circumvent this issue, the raw Ca2+ traces were also transformed (Δf/f) and used as an input in a spike inference algorithm from CASCADE 101. We combined this spike probability output (at a threshold of 0.2) with our own CaSCaDe output, allowing us to adequately segment consecutive peaks. Neurons were considered responsive if they produced a Ca2+ transient within 4 seconds of the initiation of the locomotion bout in at least 50% of the bouts.Neuronal Ca2+ microdomain map and to detect Ca2+ events.Due to high noise levels, the automatic segmentation on CaSCaDe resulted in spurious microdomain maps with hypersegmentation of ROIs. Using the average projection of the imaging stacks, noradrenergic fibers were manually identified and regions of interest (ROIs) delineated. Manual ROIs were imported into MATLAB as binary masks and used as a template for CaSCaDe to generate the Cap-values used for statistical analysis were derived from two-tailed tests. The statistical tests used for each comparison (and their significance level) are indicated in the figure legends. Data are shown in the figures as mean ± SEM and p < 0.05 was considered significant. N represents the number of animals and n the number of cells (physiology) or brains slices (histology).Statistical analyses were performed using GraphPad Prism (version 9.4.0). Each dataset was individually tested to assess the normality of its distribution using the D’Agostino-Pearson normality test. If both datasets passed the normality test, a two-tailed t-test was used to determine the statistical significance of the difference between two groups. Otherwise, a non-parametric test (Wilcoxon-matched pairs signed rank test) was used for paired comparisons. If three or more datasets were compared, we used one-way ANOVA with Tukey’s multiple comparisons test or Kruskall–Wallis test with Dunn’s correction to verify statistical significance between groups. All reported N = 3 mice).The images presented throughout the manuscript are taken directly from the dataset they are meant to represent, and the number of replicates for each experiment is indicated in the corresponding figures legends. In cases where qualitative characterization of the mouse lines Fig.  and otheAll the main and supplemental figure panels were assembled in Adobe Illustrator 2023, and cartoons were modified from graphical elements available at BioRender.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6Supplementary Video 7Supplementary Video 8Reporting SummarySource Data