However, neuronal [Ca2+]mito dynamics in vivo and its regulation by brain activity are largely unknown. By performing two-photon Ca2+ imaging in the primary motor (M1) and visual cortexes (V1) of awake behaving mice, we find that discrete [Ca2+]mito transients occur synchronously over somatic and dendritic mitochondrial network, and couple with cytosolic calcium ([Ca2+]cyto) transients in a probabilistic, rather than deterministic manner. The amplitude, duration, and frequency of [Ca2+]cyto transients constitute important determinants of the coupling, and the coupling fidelity is greatly increased during treadmill running (in M1 neurons) and visual stimulation (in V1 neurons). Moreover, Ca2+/calmodulin kinase II is mechanistically involved in modulating the dynamic coupling process. Thus, activity-dependent dynamic [Ca2+]mito-to-[Ca2+]cyto coupling affords an important mechanism whereby [Ca2+]mito decodes brain activity for the regulation of mitochondrial bioenergetics to meet fluctuating neuronal energy demands as well as for neuronal information processing.Mitochondrial calcium ([Ca Mitochondrial calcium signaling plays an important role in neuronal bioenergetics yet its regulation by brain activity in vivo is not understood. Here, the authors show that mitochondrial and cytosolic calcium transients are dynamically coupled as a function of brain activity. Many lines of evidence show that increases of cytosolic Ca2+ ([Ca2+]cyto) concentration stimulate mitochondrial Ca2+ ([Ca2+]mito) uptake, resulting in [Ca2+]mito transients8. Such [Ca2+]mito activity depends on mitochondrial Ca2+ uniporter (MCU) and its accessory regulatory subunits12. [Ca2+]mito dynamics is thought to be essential in regulating fundamental cellular and organellar functions, including energy metabolism, cellular differentiation, cell death, and redox signaling15. In addition, [Ca2+]mito is critically involved in various neuronal-specific physiological and pathophysiological processes. For instance, it has been shown that [Ca2+]mito modulates synaptic vesicle endocytosis in central nerve terminals16. During long-term potentiation (LTP) induction, elevated [Ca2+]mito triggers a rapid burst of mitochondria fission in dendrites for LTP expression17. [Ca2+]mito in presynaptic terminals has been implicated in synaptic transmission and can be disturbed by Amyloid β, leading to increasing Ca2+ influx in a mouse model of Alzheimer’s disease21. Lower [Ca2+]mito loads occur early in Huntington’s diseases (HD) in both patient tissues and the brain of HD transgenic mice, which increase neuronal susceptibility to apoptotic stimuli22.As one of the most prominent neuronal organelles, mitochondria are ubiquitously found in cell body, dendrites, and axons, including synaptic terminals. They play multifunctional roles in bioenergetics, cell fate decision, aging, reactive oxygen species (ROS), and Ca2+]mito dynamics and its physiological regulation in the brain of awake behaving mammalians remain largely unknown. In the present study, we simultaneously visualized [Ca2+]cyto and [Ca2+]mito dynamics in excitatory neurons in the primary motor cortex (M1) or primary visual cortex (V1) of behaving mice, using two-photon microscopy (2PM) in conjunction with genetically-encoded Ca2+ indicators GCaMP6f23 and jRGECO1a24. Our results reveal a probabilistic, yet spatially coordinated, coupling of [Ca2+]mito to [Ca2+]cyto transients in vivo, its physiological modulation by brain activity, and the role of Ca2+/calmodulin kinase II (CaMKII) in enhancing the [Ca2+]mito-to-[Ca2+]cyto coupling fidelity.Despite these in vitro research progresses, neuronal mito handling26, and mitochondrial Ca2+ influx can also alter mitochondrial morphology27. To delineate neuronal mitochondrial morphology in situ, we performed 3-D electron microscopy (EM) to reconstruct the mitochondrial structure29 in dendrites  and somas  of M1 pyramidal neurons in the brains from adult wild type C57BL6 mice (n = 3). Mitochondria within 6 randomly selected dendrites and 5 somas were traced and analyzed. Serial EM images and 3D reconstruction revealed that the majority of mitochondria in dendrites exhibited an elongated linear structure with regular regions interconnected by thin tubules analogous to the mitochondrial nanotunnels reported previously32 .The shape, length, and network connectivity of mitochondria are diverse and highly dynamic, depending on cell type, subcellular locationy32 Fig. . This reity Fig. . The len2+]mito dynamics in pyramidal neurons in vivo, we fused genetically-encoded Ca2+ indicator GCaMP6f with the mitochondrial targeting sequence of human thioredoxin 2 (TXN2) for its mitochondrial targeting and retention. We packaged it into recombinant adeno-associated virus (AAV) with its expression under the control of the Ca2+/calmodulin-dependent protein kinase type II subunit alpha (CaMKIIα) promoter  , a mitochondrial membrane potential indicator  promoter . Three to four weeks after infection of AAV-CaMKIIα-mito-GCaMP6f and AAV-hsyn-jRGECO1a in L2/3 of M1, we performed two-photon, dual-color imaging of [Ca2+]mito and [Ca2+]cyto in somas (L2/3) and dendrites (L1), through glass cranial window in awake, head-fixed mice  extending well beyond a single mitochondrion, and somatic [Ca2+]mito transients encompassed the whole network of mitochondria within a soma .We found that individual GCaMP6f-reported [Capes Fig. : the reg2+]mito and [Ca2+]cyto transients were of distinctively different properties. The duration of [Ca2+]mito transients was more than 10-fold longer than that of [Ca2+]cyto transients: more than 75% of [Ca2+]mito transients lasted 60 s or longer   Fig. . The ave0%) Fig. . In bothnts Fig. . Linear tes Fig. .Fig. 3Dy2+]mito transients and particularly the [Ca2+]mito-to-[Ca2+]cyto coupling are modulated by neuronal activity, we first imaged Ca2+ activity in the primary motor cortex in mice running on a treadmill under the head-fixed condition , and returned towards their basal levels upon cessation of running  of [Ca2+]cyto transient could solely determine the coupling  cyclosporine A in L2/3 neurons. We found that these respiratory inhibitors and the mPTP inhibitor all decreased the [Ca2+]mito-to-[Ca2+]cyto coupling fidelity in L2/3 somas of M1 neurons, while their effects on the [Ca2+]mito and [Ca2+]cyto transients  were variable in an inhibitor-specific and context-sensitive manner. We found that robust [Ca2+]mito-to-[Ca2+]cyto coupling occurred at 13 Hz or higher rate of stimulation, but became probabilistic at lower rates of stimulation  autofluorescence , an inactive analog control , and control artificial cerebrospinal fluid (ACSF) separately on the primary motor cortex and imaged the region of interest 20 min after.In order to gain mechanistic insights into the coupling of [Ca2+]cyto transients were not significantly altered in either dendrites or somas by the CaMKII inhibitors during both pre-running and running stages  and somas ) when mice were at rest or during motor or visual tasks. They arose with [Ca2+]cyto transients acting as the trigger, and the overall [Ca2+]mito-to-[Ca2+]cyto coupling fidelity was highly regulated by physiological neuronal activity. Furthermore, we provided evidence that CaMKII signaling plays a central role in regulating the coupling between [Ca2+]cyto and [Ca2+]mito transients.Utilizing genetically-encoded Ca2+]cyto transients are of variable amplitudes and frequencies, and that not every [Ca2+]cyto transient triggers a companion [Ca2+]mito transient. In fact, the fidelity of [Ca2+]mito-to-[Ca2+]cyto coupling was merely 2–5% at rest, and rose to 11–26% during the treadmill running paradigm when the primary motor cortex was ~2-fold more active as judged by the [Ca2+]cyto transient frequency. As such, the [Ca2+]mito-to-[Ca2+]cyto coupling in pyramidal neurons occurs in a loose, probabilistic rather than deterministic manner. Moreover, the coupling fidelity is modulated over a broad range by physiological neuronal activities and electrical stimulation, suggesting that [Ca2+]mito signaling de-codes the intensity of brain activity.Specifically, in M1 and V1 cortical neurons of awake behaving mice, we found that [Ca2+]mito-to-[Ca2+]cyto coupling, it is critical to ensure sensitive measurements for both [Ca2+]mito and [Ca2+]cyto events. The detection of [Ca2+]mito response is challenged by multiple factors, including indicator affinity, non-linearity, and dynamic range (fluorescence for Ca2+-free and bound species), as well as the basal and peak Ca2+ levels, and mitochondrial fractional volume. We opted to use GCaMP6f and jRGECO1a, for dual-color measurement of [Ca2+] in mitochondria and cytoplasm because GCaMP6f exhibits high and comparable sensitivity in detecting [Ca2+] than jRGECO1a24. Even though alkalizing pH environment  would increase GFP fluorescence in a deprotonation-dependent and Ca2+-independent manner51, the dynamic range of GCaMP6f should be 3–4 times higher than that of jRGECO1a24. Despite technical limitations and uncertainties with the measurement of [Ca2+] in different cellular compartments, several lines of evidence suggest that the unfaithful [Ca2+]mito coupling to [Ca2+]cyto is likely a genuine physiological phenomenon. First, discrete [Ca2+]mito transients, with their sudden and abrupt rises and distinctive long durations, were clearly discernible either from the baseline or on top of an ongoing [Ca2+]mito transient. Even though [Ca2+]mito transients were detected with a more sensitive indicator than [Ca2+]cyto, the frequency of [Ca2+]mito events was much lower than that of [Ca2+]cyto. Second, CaMKII inhibition did not affect [Ca2+]cyto, but reduced the coupling fidelity between [Ca2+]cyto and [Ca2+]mito, suggesting the involvement of CaMKII activity in the coupling process. Third, consistent with the involvement of biochemical signaling between [Ca2+]cyto and [Ca2+]mito, conspicuous, yet highly variable latencies up to a few seconds were found in a significant portion of these coupled events. This observation further suggests that the onset of a [Ca2+]mito transient could reflect the probabilistic gating of some fast mitochondrial Ca2+ uptake mechanism (see below). In addition, our in vitro data show that the probabilistic nature of the [Ca2+]mito-to-[Ca2+]cyto coupling in cultured neurons could be masked by strong electrical field stimulation used in previous studies46.For the quantification of mito transient reflects cooperative opening of MCUs in the mitochondrial network, via a Ca2+/CaMKII-dependent mechanism. In this scenario, uncoupling between [Ca2+]mito and [Ca2+]cyto may reflect that MCUs are not in their open state. It is possible that the three types of [Ca2+]mito kinetics reflect the patterns of interplay of several Ca2+-regulatory mechanisms of the mitochondria, including the influx through MCU53, the efflux through antiporter 54, and the flickering opening of the still elusive mPTP that acts as a fast Ca2+ release channel55. For example, in coupled events, the regular kinetics of gradual decay of [Ca2+]mito could be due to the efflux of mitochondrial Ca2+ outweighing Ca2+ influx shortly after MCU opening. The plateaued kinetics may be manifested when Ca2+ influx rate equals Ca2+ efflux rate or when both efflux and influx are shut off. The staircase kinetics may require both sustained activation of MCU and tandem occurrence of [Ca2+]cyto transients.Mitochondrial Ca2+]mito and [Ca2+]cyto transients. First and foremost, there seems to be a global cytosolic signal that, when at a supra-threshold level, permits the cell-wide mitochondrial network for the manifestation of [Ca2+]mito-to-[Ca2+]cyto coupling. Second, the underlying permissive signal depends on the level of [Ca2+]cyto since the coupled [Ca2+]cyto transients were, on average, of greater amplitudes. In the staircase type of [Ca2+]mito transients, we observed faithful coupling during successive [Ca2+]cyto transients, suggesting the presence of a sustained supra-threshold signal that allows for repetitive activation of the MCU mechanism. Nonetheless, any single attribute of [Ca2+]cyto cannot be the sole determinant, as reflected by the complex pattern of the [Ca2+]mito-to-[Ca2+]cyto coupling and uncoupling. In addition, a [Ca2+]cyto frequency-dependent mechanism is likely involved: the [Ca2+]cyto activity in a 5-s window prior to the onset was significantly higher than the average level. These salient features, including the coupling latency, the dependence of the coupling fidelity on the frequency of electrical stimulation, and the positive correlation between the amplitudes of [Ca2+]mito and [Ca2+]cyto, were replicated in vitro. Thus, both attributes of ongoing [Ca2+]cyto transient and recent history of [Ca2+]cyto are determinants of the [Ca2+]mito-to-[Ca2+]cyto coupling. Functionally, since mitochondrial Ca2+ uptake stimulates energy metabolism through activating three key dehydrogenases of the tricarboxylic acid cycle56, decoding of neuronal activity by [Ca2+]mito signaling may provide a novel mechanism to satisfy the augmented ATP demand due to enhanced neuronal activity. Indeed, our preliminary experiments showed that the onset of [Ca2+]mito transients may reciprocally elevate [Ca2+]cyto signaling in vivo, and alter mitochondrial energy and redox states in cultured neurons.Our analyses uncovered a few possible determinants for the coupling between cyto to signal for [Ca2+]mito activation. We provide direct evidence that CaMKII is central for permitting the [Ca2+]mito-to-[Ca2+]cyto coupling and regulating its coupling fidelity. When we applied CaMKII inhibitors in brain in vivo50, the [Ca2+]mito transient frequency was markedly inhibited, and the activity-dependent enhancement of the coupling fidelity was also largely abolished, even though the [Ca2+]cyto transient frequency per se and its increase by exercise were unaffected in either dendrites or somas. Therefore, we propose that a supra-threshold level of CaMKII activity serves as the global signal permissive for the [Ca2+]mito-to-[Ca2+]cyto coupling. That the dynamic coupling depends on both [Ca2+]cyto transient amplitude and frequency is also in good agreement with known properties of CaMKII activation, which decodes nonlinearly the amplitude and frequency of cytosolic Ca2+ signal and retains a memory of recent Ca2+ history on the order of a few seconds57. CaMKII could directly facilitate MCU activity via the phosphorylation mechanism58. Alternatively, the activation of CaMKII elevates sarcoplasmic reticulum Ca2+ leak59, which has been shown to contribute to mitochondrial Ca2+ uptake60 presumably via local Ca2+ signaling at the mitochondrial-sarcoplasmic/endoplasmic reticulum junctions61. Notably, it has been shown that the activation of CaMKII over a prolonged time scale can repress MCU expression as an early intermediate gene regulator to prevent excitotoxic cell death48.CaMKII, as a multimeric Ca2+ dynamics and their coupling in the primary motor and visual cortical neurons of awake behaving mice, we show, for the first time, that the neuronal [Ca2+]mito-to-[Ca2+]cyto coupling fidelity in vivo is very low at rest. More importantly, it is highly regulated by physiological neuronal activity. Mechanistically, CaMKII signaling plays a central role in regulating the dynamic mitochondrial response to cytosolic Ca2+ signaling. That [Ca2+]mito signaling could decode neuronal activity of different intensities and temporal patterns provide a natural mechanism for the mitochondrial participation of neuronal information processing and for the dynamic regulation of neuronal bioenergetics in keeping with brain activity. Future investigations are warranted to define the effectors downstream of CaMKII, and the mechanisms for spatial coordination of [Ca2+]mito transients. Given the prominent and versatile roles of mitochondrial Ca2+ signaling in cell physiology and pathophysiology62, it is also possible that dysregulation of the [Ca2+]mito-to-[Ca2+]cyto coupling might underlie the initiation and progression of neuropsychiatric diseases from autism to Alzheimer’s disease. As such, targeting the [Ca2+]mito-to-[Ca2+]cyto coupling may provide a promising therapeutic strategy for combating these neuronal diseases.In summary, by investigating cytosolic and mitochondrial Ca63 . Preliminary observations were made in mito-GCaMP5 transgenic mice in C57BL6 background at Peking University. All experimental protocols were conducted in accordance with the institutional guidelines.Wild type C57BL6eij mice (without NNT deletion) were purchased from the Jackson Laboratory and housed in the animal facility at New York University School of Medicine. Mice were maintained at 22 ± 2 °C with a 12-h light: dark cycle . Food and water were available ad libitum. 3-week old mice were used for AAV experiments and imaged 3–4 weeks later. Cortical neurons were cultured from postnatal 1-day-old wild type C57BL6eij mouse pups65. Briefly, deeply anesthetized mice were perfused with 0.9% saline, followed by 4% paraformaldehyde  and 2.5% glutaraldehyde  in PBS. The brains were dissected and post-fixed with 4% PFA and 2.5% GA at 4 °C overnight. On the second day, coronal slices ~200 µm thick were cut on a vibratome (Leica VT1000S). The M1 cortex region was dissected and post-fixed in 2% OsO4 (Ted Pella) in 0.15 M sodium cacodylate (Biolink) buffer at room temperature  for 90 min. Then the staining buffer was replaced by 2.5% ferrocyanide (Sigma) in 0.15 M sodium cacodylate buffer (pH 7.4) at RT for 1.5 h. The M1 cortex samples were washed with 0.15 M sodium cacodylate buffer and then incubated with filtered thiocarbohydrazide  at 40 °C for 45 min, 2% OsO4 at RT for 1.5 h, and 1% uranyl acetate (Merck) aqueous solution at 4 °C overnight. On the third day, the M1 cortex samples were washed with ddH2O at RT and then incubated in the lead aspartate solution, which contains 0.033 g lead nitrate (Sigma) in 5 ml 0.03 M aspartic acid , at 50 °C for 2 h. The brain sections were dehydrated through a graded ethanol series  and pure acetone. Finally, the M1 cortex samples were embedded by epon-812 resin (SPI).Sample preparation was performed as followings66. For serial section images alignment, we utilized the non-linear image registration method which can guarantee the continuity of the reconstructed anatomical object across the sections67. In consideration of the different grayscale and contrast distributions, we adopted histogram equalization to weaken the noise and keep the consistency of raw images. To reconstruct mitochondria, dendrites and somas from EM images, we adopted an effective fully connected network to segment the mitochondria in 2-D electron microscopy images though deep learning method67. The network combined with Resnet50, PSPnet and Inception-like net67. Given an input image, we employed a pre-trained Resnet50 model which consisted of 4 residual blocks to extract the feature map. The first convolutional layers (kernel size 3 × 3 with a stride 2) in residual block 1 and 2 are used for down-sampling, while residual block 3 and 4 adopted dilated network strategy, which did not reduce image size. As a result, the output feature map size of Resnet50 model was only 1/8 of the input image. Next, the feature map was passed to the PSPnet. The inception-like structure was applied to up-sample and refined the per-pixel prediction. We also utilized a simple skip connection to conserve high-resolution feature information and propagate gradients back to the early layers. The training dataset consists of a stack of 6840 images with the resolution in 5 nm × 5 nm × 50 nm. The testing dataset is with 437 slices in L1 area of the M1 cortex region and 332 slices in L2/3 area of the M1 cortex region. The number of pixels are 8928 × 8928 and 12288 × 12288, respectively. The adopted deep learning network was implemented by using Keras deep learning library and Tensor Flow backend. During training process, the network was optimized by Adaptive Moment Estimation (Adam) with the following optimization hyper parameters: learning rate = 0.0001, exponential decay rates for moment estimates β_1 = 0.9, β_2 = 0.999 and epsilon = 10−8. Pixel-wise mean squared error was chosen as the loss function. In what follows, we applied Image J software to reconstruct the mitochondria. We imported the 2-D electron microscopy images which mitochondria were segmented into Image J software, and then displayed the 3-D reconstruction of mitochondria. Additionally, we utilized a 3-D connection method to determine connectivity of the mitochondrial network both in dendrites and somas.The prepared M1 cortex samples were serially re-sectioned into 50 nm-thick ultrathin sections using an ATUMtome  and were collected though Atlas 5  using SE with scanning EM To create a mitochondria-targeted GCaMP6f (mito-GCaMP6f), the GCaMP6f sequence was first subcloned from pGP-CMV-GCaMP6f  using primers 5’-ATTCGTTGGGGGATCCCCATGGTTCTCATCATCATC-3’ (forward) and 5’-TTAACAACAACAATTGTCACTTCGCTGTCATC-3’ (reverse), and then in-frame fused with the mitochondrial target sequence of human TXN2 by inserting it into Mito-DsRed plasmid  between BamHI and MfeI restriction sites. To obtain the AAV for expression of mito-GCaMP6f in vivo, mito-GCaMP6f was subcloned using primers 5’-GAGCGGTACCGGATCCTCTAGAGTCGACTCCGGAGCCACCATGGCTCAGCGACTT-3’ (forward) and 5’-GATTATCGATAAGCTTTTAACAACAACAATTG-3’ (reverse), and inserted into pAAV-CaMK2α-hChR2(H134R)-EYFP , replacing the hChR2(H134R)-EYFP sequence using BamHI and HindIII restriction sites.68. After two weeks of expression, AAVs were harvested and viral titration was done by quantifying vector genomes (vg) packaged into viral particles by quantitative PCR using primers 5’-GACTGAAGAGCAGATCGCAGAAT-3’ (forward) and 5’-TGTCAGGAACTCAGGGAAGTCG-3’ (reverse). The titer obtained was in the range of 1012 to 1013 vg ml−1. Genetically-encoded calcium indicator jRGECO1a were expressed with recombinant AAV under the human synapsin-1 (SYN1) promoter .The transfer plasmid, Rep/Cap, and the helper plasmid were co-transfected with pAAV- CaMKIIα-mito-GCaMP6f into HEK293 cells to produce AAV particles2+ indicators was allowed for 3–4 weeks. 24 h before imaging, a head holder  was attached on top of the skull with dental acrylic cement . An open-skull surgery was performed to put a glass window over the region of interest of the skull. The center of the region for Ca2+ imaging is in the primary motor cortex  or in the visual cortex . During an imaging session of motor cortex, the head of the mouse was restrained by attaching the head holder to a custom stage fitted with free-floating treadmill70. During an imaging session of visual cortex, the head of the mouse was restrained by attaching the head holder to a custom stage fitted without free-floating treadmill. Mice were kept on the stage for a maximum of 1 h at a time. L2/3 somas of pyramidal neurons , L5 somas of pyramidal neurons  and their projected dendrites in L1  were imaged by using an Olympus Fluoview 1000 two-photon microscope (tuned to 1020 nm) equipped with a Ti:Sapphire laser . Images were acquired with two GaAsP detectors with two-photon excitation at 1020 nm and dual-channel emission collection at 495–540 nm (for GCaMP6f) and 575–630 nm (for jRGECO1a). The average laser power on the tissue sample was 30–50 mW, and the frame rate was 2 and 1 Hz for imaging in L1 and L2/3 of the cortex respectively. All experiments were performed using a ×25 objective  immersed in an ACSF solution and a ×2.0 (soma) or ×3.0 (dendrites) digital zoom. For the exercise protocol, the total imaging stack of each trial consisted of 300 frames, and animals were forced to run forward on the treadmill  during the 101–200 frames. For the visual stimulation protocol, square-wave black/white and round-shaped drifting gratings  of 8 changing orientations were used as visual stimuli. The total imaging stack of each trial consisted of 300 frames, and animals were subjected to visual stimuli during the 101–200 frames, while calcium imaging was performed simultaneously. The center of the drifting grating on monitor to perform stimuli is 18 cm away from the mouse’s eyes, and refresh rate of the monitor is 75 Hz. Image acquisition was performed using FV10-ASW v.3.0 software and analyzed post hoc using NIH ImageJ software.For viral injection, AAV-CaMKIIα-mito-GCaMP6f and AAV-hsyn-jRGECO1a were mixed 1:1 in volume without dilution. A total of 0.1–0.2 μl of AAV viruses were injected  over 10–15 min into L2/3 or L5 of primary motor cortex or L2/3 of visual cortex using a glass microelectrode. Expression of Ca49, we estimated that the final effective concentration for CaMKII inhibitors and dimethylsulphoxide (DMSO) were reduced to ~10 μM and <0.1%, respectively, due to diffusion. All of the drugs were dissolved in DMSO firstly to get stock solution and then dilute in ACSF to the final working concentration for each drug, with the final DMSO concentration lower than 1% (v/v).ACSF containing 100 μM KN-93 , 100 μM KN-62 , 100 μM KN-92  was dumped on the surface of the imaging region on the primary motor cortex after removing the dura. After 20 min, a glass window was attached over the region of interest before imaging. In a previous study2+ and Mg2+ . The dissociated neurons were then placed onto poly-D-lysine  coated coverslips with density of 0.5–1 × 106 cells per ml. The cells are kept in neurobasal medium (Invitrogen) containing 2% B-27 supplement (Invitrogen), 1% GlutaMax (GIBCO), and 1% penicillin–streptomycin (GIBCO). The neurons were cultured at 37 °C in 5% CO2. For mitochondrial Ca2+ imaging, AAV-CaMKIIα-mito-GCaMP6f affection was performed on days in vitro (DIV) 7 for 5–7 days before imaging (0.5 μl virus with titer at 3.41 × 1013 v.g. per ml was added in each well of 24-well plate)  prior to imaging, at 5 μM for 15 min and then washed for 3 × 5 min. During imaging, cortical neurons were perfused in extracellular solution containing (in mM): NaCl 150, KCl 3, CaCl2 3, HEPES 10, MgCl2 1, glucose 8, pH 7.30, at the speed of 0.7 ml/min. Electrical field stimulation of about 8 V cm−1 was applied through a pair of platinum wires, using 5 ms square-wave voltage pulses delivered from high current capacity stimulators. Neurons were stimulated with electrical pulses at different frequencies (1–15 Hz) for 5 s. Images were acquired with an inverted confocal microscope (Zeiss LSM NLO 710) equipped with an oil-immersion objective  by excitation at 488 nm  and 543 nm , and emission at 490–520 nm (for GCaMP6f), 495–540 nm (for FAD), 560–690 nm (for Rhod2) and 560–650 nm (for Rhod4), respectively at imaging rate of 1.28 Hz.The brain cortex was isolated and digested by 0.25% Trypsin-EDTA (Gibco) and washed twice in ice-cold modified HBSS without CaMice were deeply anesthetized with an intraperitoneal injection of ketamine (100 mg per kg) and xylazine (15 mg per kg), and then perfused with 25 ml of 4% paraformaldehyde. Before slicing, brain was dissected out and post-fixed overnight in 4% paraformaldehyde. At the region of virus infection, coronal slices around 100 µm thick were cut on a vibratome by using Leica VT1000S. Fixed brain slices were rinsed with PBS three times and then permeabilized with 0.5% Triton X-100 and blocked with 10% normal goat serum. The slices were incubated with TOM20  overnight at 4 °C cold room. Next day, the slices were rinsed with PBS three times and then incubated with tetrarhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Santa Cruz) in dark place for 1 h at room temperature. Slices were rinsed three times with PBS and then mounted between two glass coverslips in Vectashield (Vector Laboratories) and sealed with dental wax. The immunohistochemical staining slices were visualized using Zeiss LSM710 confocal microscope at 488 nm (Mito-GCaMP6f) and 568 nm (TRITC) excitation, and 490–550 nm and >560 nm emission, respectively.2+]mito activity and the same ROIs in red channel for imaging [Ca2+]cyto were analyzed. ImageJ plugin was used to register all imaging stacks. The fluorescence time course of each dendritic segment or cell body was measured with ImageJ software by averaging all pixels within the ROIs mentioned above. The fluorescence change was calculated as ΔF/F0 = (F − F0)/F0 × 100%, in which F0 is the baseline fluorescence signal averaged over a 2-s period. The threshold for detecting a [Ca2+]cyto or [Ca2+]mito transient was set at more than three times the standard deviation of baseline fluorescence noise and duration greater than 1 s for mito-GCaMP6f, jRGECO1a, Rhod-2 and Rhod-4.Regions of interests (ROIs) corresponding to visually identifiable double-labeling of GCaMP6f  or Rhod-2  in mitochondria and jRGECO1a (in vivo two-photon imaging) or Rhod-4  in cytosol were selected for quantification. ROIs in green channel for imaging [Cat-test was used when the dataset passed the normality test, otherwise a nonparametric test (Mann–Whitney test) was chosen. For more than two groups, one-way ANOVA analysis  was performed, with a post hoc Dunn’s test or Sidak’s multiple comparisons test only if significant. Data are reported as mean ± SEM. A value of P < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism (version 7.0) and IBM SPSS 20. All datasets were tested for normality distribution first. For two group datasets, a two-tailed unpaired Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Source Data


In 2019 it is estimated that more than 21,000 new acute myeloid leukemia (AML) patients will be diagnosed in the United States, and nearly 11,000 are expected to die from the disease. AML is primarily diagnosed among the elderly (median 68 years old at diagnosis). Prognoses have significantly improved for younger patients, but as much as 70% of patients over 60 years old will die within a year of diagnosis. In this study, we conducted a reanalysis of 2,213 acute myeloid leukemia patients compared to 548 healthy individuals, using curated publicly available microarray gene expression data. We carried out an analysis of normalized batch corrected data, using a linear model that included considerations for disease, age, sex, and tissue. We identified 974 differentially expressed probe sets and 4 significant pathways associated with AML. Additionally, we identified 375 age- and 70 sex-related probe set expression signatures relevant to AML. Finally, we trained a k nearest neighbors model to classify AML and healthy subjects with 90.9% accuracy. Our findings provide a new reanalysis of public datasets, that enabled the identification of new gene sets relevant to AML that can potentially be used in future experiments and possible stratified disease diagnostics. AML is best characterized by terminal differentiation in normal blood cells and excessive production and release of cells at various stages of incomplete maturation (leukemia cells). As a result of this faster than normal, and uncontrolled growth of leukemia cells, healthy myeloid precursors involved in hematopoiesis are suppressed, and ultimately can soar to death within months from diagnosis if untreated6. AML accounts for 70% of myeloid leukemia and nearly 80% of acute leukemia cases, making it the most common form of both myeloid and acute leukemia7. The number of new AML cases is increasing each year – in 2019 alone, an estimated 21,450 new AML patients will be diagnosed, and nearly 10,920 are expected to die from the disease8.Acute myeloid leukemia (AML) is a heterogeneous malignant disease of the hematopoietic system myeloid cell lineage9, AML prognosis criteria for classification are highly dependent on the presence of chromosomal abnormalities, including chromosomal deletions, duplications, translocations, inversions, and gene fusions. AML is diagnosed predominantly through microscopic, cytogenetic, and molecular genetic analyses of patients’ blood, and/or bone marrow samples. Microscopic examination may be used to detect distinctive features (e.g. Auer rods) in cell morphology, cytogenetic analysis to identify chromosomal structural aberrations , inv(16), t, or t), and molecular genetic analysis to identify gene fusion , and mutations in genes frequently mutated in AML 12. Such cytogenetic and molecular genetic analyses are used to identify prognosis markers for classifying AML patients into three risk categories: favorable, intermediate, and unfavorable, currently based primarily on the European LeukemiaNet (ELN) 2017 classification10 . A large group of AML patients present normal karyotypes and lack chromosomal abnormalities13. These patients are classified as intermediate risk, and often have heterogeneous clinical outcome with standard therapy with risk of AML relapse14.According to the 2016 World Health Organization (WHO) newly revised myeloid neoplasms and acute leukemia classification system16. AML can occur in people of all ages but is primarily diagnosed among the elderly (>60 years old), with a median age of 68 years at diagnosis8. Recent advances in AML biology have expanded our understanding of its complex genetic landscape, and led to significant improvement in prognoses and therapeutic strategy for younger patients16. For elderly patients, prognoses remain grim and the main therapeutic strategy, remission induction therapy followed by an intensive consolidation phase (post-remission), had remained nearly unchanged over the past three decades17. More recently, however, new therapeutic agents have been approved for older AML patients5, and these include venetoclax (combined with decitabine or azacitidine)18, midostaurin (combined with standard chemotherapy)19, and gilteritinib20. It is expected that the new therapeutic agents will improve prognosis for older AML patients (where in the past up to 70% of AML patients aged 65 or older were reported to die within a year following diagnosis21). While it is apparent that the nature of AML changes with age, still little is known about the extent of these associations and how they vary with patient age23, and current indications from ELN and the National Comprehensive Cancer Network (NCCN) essentially consider age as a surrogate variable that is used only in conjunction with other treatment‐related mortality factors10. Taking into consideration age in the identification of changes in AML global gene expression may lead to improved early diagnosis and improvement in treatment approaches for elderly patients. To further complicate matters, AML has multiple driver mutations and competing clones that evolve over time, making it a very dynamic disease24.Additionally, AML prognosis worsens with age, and older patients respond less to current treatments, with poorer clinical outcomes compared to younger patients25, who analyzed information on 4,918 genes, and identified 25 genes reported across multiple studies, with potential prognostic features. In this study, we performed a comprehensive gene expression analysis of 2,213 AML patients and 548 healthy subjects, by re-analyzing publicly available gene expression microarray data from 37 curated studies  and identified disease-, age- and sex-related gene expression changes associated with AML. The differentially expressed gene sets were associated to signaling pathways relevant in AML, and also used to train and test a predictive model of AML or healthy status. We believe that our results may lead to improved AML early detection, and diagnostic testing with target genes, which collectively can potentially serve as age- and sex-dependent biomarkers for AML prognosis, as well as new treatment targets with mechanisms of action different from those used in conventional chemotherapy.Multiple gene expression analyses of AML have been carried out, 25 of which have been systematically compared by Miller and StamatoyannopoulosWe searched the Gene Expression Omnibus (GEO) public repository, based on our systematic workflow and inclusion criteria, Fig. 26. The trained models were cross-validated using our annotated preprocessed expression data, and were 96 ± 8% and 96.7 ± 4% accurate for sex and sample source predictions respectively  of 2,761 curated data were found to be missing sex annotation, and 737 arrays  were missing sample source annotation . To predict the missing sex and sample source annotations, we trained and validated a logistic regression (LR) classification model. The prediction of missing annotations for these arrays was essential in our study, to increase the sample size, and statistical power27. After this dataset-wise correction, the 5 covariate reference datasets were removed, and our expression data were clustered using principal component analysis (PCA), to visually examine the effect of covariate reference datasets on distributing the batch weight during batch correction , normalizing sets of unevenly sized datasets may lead to an unbalanced batch correction. We used 5 additional datasets as a reference set, which we refer to as “covariate” hereafter. Each of the covariate reference datasets included within-study healthy controls. All 5 datasets together consisted of a total 613 arrays  (Table 30 was performed according to a linear model (see Online Methods), including factors for age, sex, and sample source , as well as binary interactions thereof. To avoid assumptions on averaging over multiple probe sets corresponding to the same gene symbol, we analyzed probe sets with the linear model. We identified 974 statistically significant differentially expressed probe sets (DEPS) (corresponding to 964 unique gene symbols) for AML, based on post-hoc analyses (Tukey’s Honestly Significant Difference (HSD) tests implemented in R, with adjusted p-value < 0.01), in conjunction with a two-tailed 5% quantile selection31 based on the mean difference distribution between AML-healthy group comparisons across probe sets . The heatmap  with mean difference of −0.52 and adjusted p-value < 4.11 × 10−11 was the least DE gene. Figure Following batch correction, we performed an analysis of differential expression (DE) on the 34 curated datasets including 2,213 AML patients and 548 healthy controls. Analysis of Variance (ANOVA)map Fig.  shows th34 signaling pathways, and Gene Ontology (GO) terms36 on all 974 DEPS, using the Database for Annotation, Visualization and Integrated Discovery (DAVID)38. Four KEGG signaling pathways were identified as enriched , including Hematopoietic cell lineage, Cell cycle, p53 signaling pathway, and Transcriptional misregulation in cancer. The 4 KEGG signaling pathways are summarized in Table 39 adjusted p-values. These signaling pathways were associated with 56 DEPS, including 27 up- and 29 down-regulated DEPS , with 727 DE unique identities (335 up- and 392 down-regulated). GO terms included protein and microtubule binding for the molecular function (MF) category, inflammatory and immune responses, mitotic nuclear division, and cell proliferation response for the biological process (BP) category, and finally, cytoplasm, extracellular exosome, cytosol, extracellular space, integral component of plasma membrane immune response, and others, for the cellular component (CC) category EPS Fig.  - the heory Fig. . The com−7 .To characterize sex- and age-specific gene expression changes in AML patients compared to healthy individuals we conducted the following additional analyses detailed further below: (i) Analysis 2a: “Sex-relevance differential gene expression analysis and associated signaling pathways in AML”, and (ii) Analysis 2b: “Age-dependent differential gene expression analysis and associated signaling pathways in AML”. We used the same filtering criteria in both analyses as those used in Analysis 1 for identifying DEPS and signaling pathways between AML patients and healthy controls. In addition, DEPS were regarded as statistically significantly (up- or down-regulated) for each factor, sex and age, if they displayed p-value from Tukey’s HSD < 2.2 × 10−7), as listed in Supplementary Table We identified 266 DEPS that show sex differences between AML patients . This resulted in 43 unique DEPS, statistically significant for age, and for disease, and for interaction between age and disease in the linear model  were used for training, and testing was performed on the 5 covariate reference datasets, which included both AML and healthy subjects  algorithm in ClassificaIOts Table .n DEPS post sorting . We then iteratively trained and tested a KNN model on the top In the present study, we reanalyzed data aggregated from our curation of 34 publicly available microarray gene expression datasets covering 2,213 AML patients and 548 healthy individuals to identify changes in AML gene expression associated with disease state , sex-linked , and age-dependent (across age-groups compared to baseline). We performed 3 differential probe set (gene) expression and gene enrichment analyses, as discussed below.Gene expression analysis and associated signaling pathways of AML disease state compared to healthy individuals, was carried out to identify DEPS in AML disease state. The results from this analysis were then used as a baseline indicator for AML disease state. 974 DEPS (487 up- and 487 down-regulated) were identified as statistically significant differentially expressed between AML patients and healthy individuals  and showing high effect size (5% 2-tailed quantile selection). Among these, 6 genes are known to be involved in AML functional pathways, including 4 up-regulated, JUP, CCNA1, FLT3, PIK3R1, and 2 down-regulated, CD14, CEBPE. The top 10 up- and down-regulated genes from this analysis are listed in Table 43, in agreement with our findings. However, CRISP3’s direct role in AML is still under investigation. CRISP3 is a member of the cysteine-rich secretory protein CRISP family with major role in female and male reproductive tract, and is mainly expressed in salivary glands and bone marrow44. Recently, 80 genes were reported as “extracellular matrix specific genes” in leukemia, and CRISP3 was among the downregulated DE genes reported45. CRISP3 associations with AML merit further investigation.47. GO terms in the Biological process (BP) category included inflammatory and immune responses, and cell proliferation, which are expected as AML is characterized by terminal differentiation of normal blood cells, and excessive proliferation and release of abnormally differentiated myeloid cells, which affects many biological processes associated with the immune system. The four statistically significant KEGG pathways identified in the pathway enrichment analysis encompassed 56 DEPS  DDXs 1 Fig. . The rolAge-dependent gene expression analysis and associated signaling pathways in AML compared to healthy individuals, was carried out to identify common set of age-dependent gene expression and associated signaling pathways and to explore age-dependent trends in AML. The age-dependent analysis using ANOVA, identified 1,395 DEPS . To identify age-related DEPS in AML we overlapped the 1,395 DEPS to our findings of 974 DEPS in AML disease state  . The top 10 up and down DE age-associate genes in AML according to the mean difference values in seven age-groups are shown in Fig. 49. Also identified with age-specific DE, was ORM1, which in Analysis 1 was among the top-10 under-expressed genes, and was also among the 70 DE genes in analysis 2a. ORM1’s direct role in AML also merits further investigation, given ORM1 involvement in immunosuppression and inflammation50. Finally, we have identified 75 DEPS that show association with only one age-group, exclusively from all other age-groups, suggestive of potential age-specific differential gene expression signature.38 such as SOCS5 and SOCS6 , EBF1 (early B-cell Factor 1), CD160, TCL1A (T-cell leukemia/lymphoma 1A), VPREB3 (pre-B lymphocyte 3), KLF10 (Kruppel-like factor 10), NTM (neurotrimin), PLXNA4 (plexin A4), SLC25A21, SYT4 (synaptotagmin 4) and TCERG1 (transcription elongation regulator 1). While these genes/gene families have been associated with cancer52, their potential role in AML is still under and merits further investigation. These 43 DEPS with statistically significant age-disease interactions may be important in AML development, particularly for detecting early markers of AML, potentially identifying preleukemic conditions, and using these markers as treatment targets.We further wanted to assess the interaction of age with disease state. From the 375 unique DEPS which intersected between the age and disease analysis, 43 unique DEPS showed statistically significant interaction between age and disease in the linear model  with 96.1 ± 4% training accuracy. The model uses a minimal set of 10 DEPS . The feature set coincides with the top 10 down-regulated DEPS for Analysis 1 (disease), Table Our study identified multiple potentially significant DEPS, with age and sex related differences associated with AML. While our findings may generate further hypothesis-driven investigations, we need to also identify the study’s limitations: The primary limitation is that the analysis of AML and healthy subjects involved bone-marrow and blood samples respectively in each disease group. We tried to account for this utilizing tissue as an effect in our linear model, and including multiple interactions. Other limitations include an unbalanced AML/healthy ratio, as well as the lack of in-study healthy controls. To address these we attempted to account for batch effects using a dataset-wise iterative batch correction transformation, as discussed in the methods. Finally, we also included binary interactions between the factors in the analysis to account for interaction-related confounding effects. Additionally, the study is limited by the available data, particularly for prognostic utility. The low numbers of PB data, as well as the lack of healthy BM data do not allow for an equal-footing comparison of differences in AML between PB and BM cell gene expression signatures. Furthermore, the prognostic utility of the study is limited by the lack of uniformly reported or sparse clinical data, including progression/relapse-free survival, (in-)effective therapeutic intervention, such as bone marrow transplantation or pharmaceutical treatment, or mutational status. More uniform reporting of published metadata would greatly improve the utility of publicly available datasets. Additionally, more data would be necessary to fully evaluate mutational status and AML classifications. We anticipate that the use of sequencing data now being generated (e.g. RNA-sequencing data) as well as single cell level gene expression, will allow better concurrent determination of mutational status, global gene expression, and cell-type specific evaluation of patient samples.In summary, our study successfully integrated multiple datasets to perform an analysis of gene expression in AML, across multiple factors that included disease, sex and age considerations, and identified interesting genes, both known and not previously reported as differentially expressed in each factor. We identified 974 DEPS and 4 associated significant pathways involved in AML, and 70 sex- and 375 age-related DE signatures. Using the 10 of the 974 DEPS, a KNN model allowed AML classification with 90.9% accuracy. We hope that these findings may provide additional relevant targets for further experimental mechanistic studies, and to help identify new markers and therapeutic targets for AML.The generalized workflow consisted of five main steps: i) Curation of microarray gene expression data, ii) Preprocessing of raw data files followed by batch effect correction, iii) Predictions of missing annotations using supervised machine learning, iv) Differential gene expression analysis, and v) Pathway analysis, that included gene annotation, and finally gene expression-based prediction of AML Fig. .53 (https://www.ncbi.nlm.nih.gov/geo/). To facilitate speed of search and keep up-to-date with possible new and relevant datasets, as soon as they were released, a Python script was used that utilized functions from the Entrez Utilities from Biopython54. We used the script to navigate the GEO records, and downloaded microarray gene expression datasets up to October 2018. We additionally utilized Python packages, including Pandas, NumPy, and Matplotlib for data structure, numerical computing for data processing, and data visualization respectively. We used strict inclusion criteria to maintain consistency in each dataset selection, screening for availability of both raw and data annotation files provided, human samples used from untreated subjects, and that the sample source was from either BM or PB. Array platform was restricted to Affymetrix, which was found to have the most available data, and to avoid cross-platform normalization issues. Inclusion criteria and the data curation workflow are illustrated in Fig. The datasets used in this study were selected from the GEO public repository, maintained by the National Center for Biotechnology Information (NCBI)55 and GSE3764259, were originally conducted on two different Affymetrix array types , so each was separated into two subgroups and each subgroup was considered as individual dataset in our analysis, dataset GSE12417: (i) subgroup 1 included 73 BM and 5 PB samples, and (ii) subgroup 2 included 160 BM and 2 PB. For dataset GSE37642: (i) subgroup 1 included 140 BM and (ii) subgroup 2 included 422 BM samples  raw intensities; quantile normalization to obtain the same empirical distribution of intensities for each array; median polish summarization of probes into probe sets to estimate gene-level expression value: and logarithm base-2 transformations of gene expression values to facilitate data interpretation  and comparisons between arrays. Additionally, the expression data were reduced to 44,754 probe sets that overlapped across all datasets. Finally, within each dataset we standardized across all probe sets, by subtracting the mean and dividing by the standard deviation to obtain a Z-score.Figure 26. To address this, we used supervised machine learning classifiers to predict both sex and sample annotations. For all predictions, we used ClassificaIO41, a machine learning for classification graphical user interface, which we recently developed, that utilizes the scikit-learn machine learning package in Python63.From the curated datasets, 805 arrays  and 737 arrays  were missing sex or sample source annotations respectively. Without these metadata, we would have to discard the data, which in turn would limit the statistical power for the study, and our ability to correct for biases stemming from individual datasetsTo predict sex in pre-processed datasets, 1956 arrays , that included 44,754 probe sets and their annotated sex information, were used to train a logistic regression (LR) classification model, and to predict 805 sex annotations. Additionally, 2,024 arrays were used to train for sample source, with prediction performed on 737 arrays.63):random_state = None: specifies whether a seed should be used for the pseudorandom generator in selecting training and testing subsets.shuffle = True: determines that data will be shuffled before splitting for training and testing.penalty = l2: determines that an L2 norm should be used for penalization.multi_class = ovr: specifies that a binary problem is fit for each label.solver = liblinear: specifies that liblinear is the algorithm used in the optimization.max_iter = 100: maximum number of iterations for the solvers to converge.tol = 0.0001: tolerance for stopping criteria.fit_intercept = True: a constant (bias/intercept) is added to the decision function.intercept_scaling = 1.0: a “synthetic” feature with constant value equal to intercept_scaling is appended to the instance vector.Verbose = 0: turns off verbosity in evaluation.n_jobs = 1: number of CPU cores used.C = 1.0: inverse of regularization strength.dual = False: dual or primal formulation.warm_start = False: do not reuse the solution of the previous call to fit as initialization.class_weight = None: all classes assumed to have weight one.The supervised machine learning LR classifier we used had the following parameters , visualizing components in both 2 and 3 dimensions, to compare the clustering results for corrections , a is age (between 0 to 100 years), s is sex , t is sample source (BM or PB), and ε is a random error term, and colons represent interactions between factors. We note that the model includes sample source and its interactions to address comparisons involving different tissues in AML and healthy subjects (BM or PB respectively). The selection of using a linear model was based on having multiple factors to capture in the analysis, and also having a large number of samples (by integrating multiple datasets) – in that the Central Limit theorem allows for the assumptions for F-test to hold for ANOVA. We also evaluated fit residuals’ distribution for normality by plotting Quantile-Quantile (QQ) plots and density distributions.After the batch correction step, we performed differential gene expression analysis on the merged datasets , using the expression values for all 44,754 common probe sets. The effects of patients’ age, sex, and sample source, including their pairwise interactions were investigated using an analysis of variance (ANOVA)Based on the ANOVA we first identified statistically significant differences for the disease state factor . To identify statistically significant level differences  we then carried out post-hoc analyses for each statistically significant probe set using Tukey’s HSD tests implemented in R, . Finally, to focus on biological effects, we filtered the results to have mean difference values  in the <5% and/or >95% quantiles of the overall mean difference distribution across probe sets. The final set of the results are referred to as differentially expressed probesets (DEPS) with respect to the disease.38 for KEGG signaling pathways34 and GO functional annotation terms36. Pathways and terms identified were deemed statistically significant based on Benjamini-Hochberg adjusted p-value < 0.05.We carried out enrichment analysis (overrepresentation) for DEPS using the database DAVID41). All 34 datasets  were used as training data. Testing of the model was done independently of training on all 5 covariate datasets. The KNN model used the following parameters (please refer to scikit-learn documentation for further details63):random_state = None: specifies whether a seed should be used for the pseudorandom generator in selecting training and testing subsets.shuffle = True: determines whether or not data will be shuffled before splitting for training and testing.metric = minkowski and p = 2: define which metric to use. The minkowski metric is using the Minkowski distance of order p between two n-dimensional vectors weights = uniform, defines that uniform weights will be used so that all points in each neighborhood are weighted equally.metric_params = None: additional metric parameters (none used in this case).algorithm = auto: automatically determines the algorithm to use for computing nearest neighbors, can internally use a BallTree or KDTree or brute force algorithm.n_neighbors = 30: number of nearest neighbors to be used.leaf_size = 30: leaf size passed to BallTree or KDTree algorithms.n_jobs = 1: number of parallel jobs to run for neighbors search.To predict AML health status, normalized intensities from DEPS (with respect to disease) were used as features for training a k-nearest neighbor (KNN) model . We then iteratively trained and tested a KNN model on the top Supplementary InformationSupplementary TablesSupplementary File 1Supplementary File 2Supplementary File 3


Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca2+ channel Cacophony (Cac) and, in males in which all Cac channels are tagged, investigated the regulation of endogenous Ca2+ channels during homeostatic plasticity. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca2+ influx during PHD is achieved through functional adaptions to pre-existing Ca2+ channels. Thus, distinct mechanisms bidirectionally modulate presynaptic Ca2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.Neurons communicate through CaSIGNIFICANCE STATEMENT Presynaptic Ca2+ dynamics play an important role in establishing neurotransmitter release properties. Presynaptic Ca2+ influx is modulated during multiple forms of homeostatic plasticity at Drosophila neuromuscular junctions to stabilize synaptic communication. However, it remains unclear how this dynamic regulation is achieved. We used CRISPR gene editing to endogenously tag the sole Drosophila Ca2+ channel responsible for synchronized neurotransmitter release, and found that channel abundance is regulated during homeostatic potentiation, but not homeostatic depression. Through live imaging experiments during the adaptation to acute homeostatic challenge, we visualize the accumulation of endogenous Ca2+ channels at individual active zones within 10 min. We propose that differential regulation of Ca2+ channels confers broad capacity for tuning neurotransmitter release properties to maintain neural communication. AZ release properties are determined locally and can vary considerably within and between neuronal subtypes  where synaptic vesicles fuse to presynaptic membranes and release their contents following action potential-driven influx of Casubtypes . HoweverPr) is a defining property of neurotransmitter release that can be tuned during plasticity. Two key parameters have a large influence on Pr: the number of synaptic vesicles available for release and their individual probability of release. Vesicular release probability is heavily influenced by Ca2+ influx through presynaptic Ca2+ channels, which in turn is driven by the number, organization, and intrinsic properties of Ca2+ channels at AZs  provides a powerful model for investigating the establishment and modulation of release properties at individual AZs in vivo. At fly NMJs, single Drosophila glutamatergic motorneurons form hundreds of synapses with their postsynaptic muscle targets. These presynaptic motor terminals have many parallels to excitatory synapses in the mammalian CNS, including molecular composition, AZ ultrastructural organization, and neurotransmitter release properties , genetic or pharmacological disruption of postsynaptic glutamate receptors induces a compensatory increase in neurotransmitter release that maintains overall synaptic strength  to incorporate a fluorescent tag in all 15 known isoforms of Cac. Cac is the pore-forming subunit of the sole Ca2+ channel required for triggering synchronous neurotransmitter release in Drosophila : 1118w (RRID:BDSC_5905), vasa-Cas9 (RRID:BDSC_51324), piggyBac transposase (RRID:BDSC_8283), and OK371-Gal4 . Endogenously tagged Cac was generated using a scarless CRISPR/piggyBac-based approach . This site is in an exon common to all isoforms of Cac. piggyBac transposase was subsequently used for footprint-free removal of the marker, followed by molecular confirmation of precise tag incorporation.The following fly lines are available at the Bloomington RIIASP16  and UAS-AS-vGlut  were gen2+ levels at motor terminals was measured by forward filling of dextran-conjugated indicators  and a Ca2+-insensitive AlexaFluor 647 dextran  was applied to the cut nerve end for 15–45 min. Preparations were incubated in the dark for at least 3 h and rinsed every 30 min with fresh Schneider's insect medium. Thirty minutes before imaging, the Schneider's insect medium was replaced with hemolymph-like solution (HL6) containing 2.0 mm Ca2+ and supplemented with 7 mml-glutamic acid (N), 2–5 nonterminal boutons of a single NMJ were analyzed. Ca2+ levels are measured as the fluorescence ratio of rhod to AF647. Ten single action potentials were averaged into a single trace and used to calculate peak amplitude and the decay time constant (tau). Ca2+ imaging data were excluded from further analysis if they were collected from terminals where resting Ca2+ levels were assessed to be outliers. The bounds used to assess outliers were calculated as follows: the median absolute deviation (MAD) was calculated for resting Ca2+ levels of each group of terminals and the upper boundary was defined as 3× MAD above the median, and the lower boundary was defined as 3× MAD below the median .Cadicators . Male thmic acid . Analysie median . No furt2+-free hemolymph-like saline  filled with 3 m KCl. Recordings were performed on an Olympus BX61 WI microscope using a 40×/0.80 water-dipping objective, and acquired using an AxoClamp 900A amplifier, Digidata 1440A acquisition system, and pClamp 10.5 software (Molecular Devices). Electrophysiological sweeps were digitized at 10 kHz, and filtered at 1 kHz.Male third instar larvae were dissected in modified CaRin) and resting membrane potential (Vrest) were monitored during each experiment. Recordings were analyzed only if the Vrest was between −60 and −80 mV and Rin was ≥5 MΩ.Miniature excitatory junctional potentials (mEJPs) were recorded in the absence of any stimulation, and cut motor axons were stimulated to elicit EJPs. For each recording, at least 100 mEJPs were analyzed using Mini Analysis (Synaptosoft) to obtain a mean mEJP amplitude value. EJPs were stimulated with an ISO-Flex stimulus isolator (AMPI), with intensity adjusted for each cell to consistently elicit responses from both type Ib and Is motorneurons innervating the muscle segment. At least 20 consecutive EJPs were recorded for each cell and analyzed in pClamp to obtain mean amplitude. Quantal content was determined for each recording by calculating the ratio of mean EJP amplitude to mean mEJP amplitude and then averaging recordings across all NMJs for a given genotype. Muscle input resistance  diluted in HL3 containing 0.4 mm Ca2+ for 10 min at room temperature  or ProLong Diamond (ThermoFisher Scientific). The following antibodies were used at the indicated concentrations: mouse anti-Bruchpilot (Brp) at 1:100 , rabbit anti-GFP conjugated to AlexaFluor 488 at 1:500 , and anti-HRP conjugated to AlexaFluor 647 at 1:200–1:500 (Jackson ImmunoResearch). Species-specific Alexa Fluor 488 and 568 secondary antibodies  were used at 1:400–1:500.Male third instar larvae were dissected in ice-cold CasfGFP-N and Brp intensity at individual AZs, nonsynaptic structures including axons were removed from the images using freehand selection and fill. Z-stacks were flattened using the Maximum Intensity Z-projection function. Channels were separated, background subtracted, and noise was reduced using a light Gaussian filter (0.75 pixel σ radius). A threshold was applied to the Brp channel to remove irrelevant low intensity pixels and individual puncta were identified and segmented using the Find Maxima tool. The resulting segmented Brp image was used to create a binary mask that was also used to segment the CacsfGFP-N channel. Intensity data were collected using the Analyze Particles tool. The fluorescent intensity of each puncta is measured as the sum fluorescence calculated directly or as the product of average intensity and particle area.Confocal images were acquired on a Nikon A1R-Si+ with Plan-Apo 60× 1.40 NA and 100× APO 1.4 NA oil-immersion objectives or an Olympus FluoView FV1000 with Plan-Apo 60× (1.42 NA) oil-immersion objective. Image analyses and brightness and contrast adjustments were performed using the Fiji distribution of ImageJ . For anaTagRFP-N; PS-GCaMP male third instar larvae were dissected in ice-cold Ca2+-free HL3 and severed segmental nerves were drawn into a stimulating pipette. Preparations were then maintained in HL3 saline containing 1.5 mm Ca2+ and 25 mm Mg2+ for imaging. Type-Ib motor neuron terminals innervating muscle 6 in hemisegment A2 or A3 were first imaged using the galvanometer scanner to collect a CacTagRFP-NZ-stack. GCaMP5 was then imaged continuously in a single focal plane using the resonant scanner with full frame averaging for a final acquisition rate of 15.3 frames/s. Evoked postsynaptic Ca2+ transients were stimulated by applying a 0.4 ms pulse every second. The stimulus amplitude was adjusted to reliably recruit the Ib input. 94–100 stimulations were recorded for each experiment. CacTagRFP-N puncta location and intensity was analyzed in Fiji. Z-stacks were flattened using the Maximum Intensity Z-projection function. To identify individual CacTagRFP-N puncta, a mask was created using a Gaussian filter (σ radius = 2) and unsharp mask . A threshold was then applied to the mask to remove lower intensity pixels between puncta and individual puncta were segmented using the Find Maxima tool. By identifying large puncta with two local intensity maxima, the Find Maxima tool facilitates the identification and segmentation of closely spaced AZs. From each resulting image, a binary mask was then created and used to isolate puncta in the original Z-projection. Each punctum was then interrogated for intensity and X-Y coordinates using the Analyze Particles tool. GCaMP movies were processed using Nikon Elements software. Motion artifacts during acquisition were corrected by aligning all frames to the first frame of the movie. Baseline fluorescence was subtracted from every frame using a frame created from the average of 10 nonstimulus frames that also lacked spontaneous release events, and noise was reduced using the Nikon Elements Advanced Denoising function. The X-Y coordinates of postsynaptic Ca2+ transients were collected from all stimulation frames manually or automatically, with the two approaches yielding concordant results. For manual analysis, X-Y coordinates were obtained using hand selection of individual fluorescence peaks. Only peaks that persisted and decayed over subsequent frames were recorded. For automated analysis, all stimulation frames were thresholded to remove background noise and the Find Maxima tool in Fiji was used to identify event X-Y coordinates. Each postsynaptic event was then assigned to a CacTagRFP-N punctum through nearest neighbor analysis using Euclidean distance.Functional imaging experiments were performed using a Nikon A1R+ scanning confocal system built on a Nikon Eclipse FN1 microscope with a CFI Plan 100×W objective (Nikon). CacsfGFP-N during acute PHP was performed on a Nikon A1R-HD scanning confocal system built on a Nikon Eclipse FN1 microscope with a CFI75 Apochromat 25× 1.1 NA objective (Nikon). Third instar larvae were minimally dissected in Ca2+-free HL3 and one side of the body wall was carefully pinned down while avoiding any stretching of body wall muscles. Before treatment, Z-stacks of NMJ 6/7 were acquired using the resonant scanner with 4× averaging. Preparations were then exposed to 40 μm Philanthotoxin-433  or vehicle diluted in HL3 for 10 min. Because segmental nerves were left intact, experiments were performed in Ca2+-free HL3 to minimize muscle contractions and movement during imaging, a condition that does not affect PHP  was applied to the after treatment images to aid in AZ recognition and masking. After images were then segmented using freehand selection and fill to remove nonsynaptic structures. A threshold was applied to remove irrelevant low intensity pixels and individual AZ puncta were identified and segmented using the Find Maxima tool. The resulting segmented image was used to create a binary mask to segment the unfiltered before- and after-treatment background-subtracted maximum intensity projections. Intensity data were collected using the Analyze Particles tool and post-PhTx intensities were corrected for vehicle-only effects. For consistency, analysis was restricted to type-Ib AZs of NMJ 6/7.Live imaging of Cacfect PHP . Immediat test. Welch's correction was used in cases of unequal variance. The Mann–Whitney U test was used for single comparisons of non-normally distributed data. For multiple comparisons of normally distributed data, we performed ANOVA followed by Tukey's test. One-dimensional Pearson correlation coefficients (r) were used to compare intensity levels and release probability. Fisher's exact test was used to compare proportions. Paired t tests and Wilcoxon signed rank tests were used for comparison of normally and non-normally distributed repeated intensity measurements, respectively. Reported values are mean ± SEM unless otherwise stated. P values, statistical test used, and sample sizes are reported in each figure legend.Statistical analyses were conducted in GraphPad Prism 7, SigmaStat 3.5, and R. Single comparisons of normally distributed datasets, as determined by the D'Agostino–Pearson omnibus test, were conducted by Student's V2-family member in Drosophila and is homologous to mammalian N- and P/Q-type voltage-gated Ca2+ channels  cac is necessarily overexpressed, generally at high levels, under the control of GAL4 instead of endogenous regulatory elements; (2) only 1 of 15 annotated cac transcripts (corresponding to isoform cac-RC) is expressed and is likely not subject to RNA editing, a conserved mechanism for regulating ion channel function observed in endogenous cac (3) the C-terminal position of GFP likely obstructs a conserved PDZ-binding domain that mediates known interactions with synaptic proteins; and (4) expression of the single cac isoform was shown to rescue viability, but not flight, in null alleles, indicating channel function is not fully restored and/or properly regulated  using CRISPR-based gene editing (A). This insertion site also results in the in-frame translation of sfGFP within the longer N-terminal cytoplasmic domain of the single isoform (cac-RN) with an earlier start site, allowing us to follow the full complement of endogenously expressed Cac isoforms (B). sfGFP-Ncac flies are homozygous and hemizygous viable, emerge at expected frequencies, and are capable of effective flight. CacsfGFP-N localizes to AZs marked by the AZ cytomatrix protein Brp at presynaptic terminals of larval motorneurons with remarkable specificity (C). Endogenous Cac also localizes to the synaptic neuropils of the larval ventral ganglion and adult brain . In the adult brain, it was immediately apparent that endogenous Cac levels vary between brain regions. Most notably, we consistently observe higher Cac levels in the mushroom body. Therefore, incorporation of a fluorescent tag at the endogenous locus of Cac reports the expected gross localization of Ca2+ channels in the Drosophila nervous system along with new insights without obvious defects in viability or behavior.To overcome these limitations and establish a reagent for following all endogenous Ca editing A. This iisoforms B. cacsfGcificity C. Endogelt brain D,E. In t2+ levels via fluorescence imaging of a Ca2+-sensitive dye loaded into axon terminals in situ  or -Is  terminals on muscle fiber 6 . Similarly, there is no indication of a deficit in single action potential-mediated Ca2+ influx in sfGFP-Ncac on the basis of 1 Hz stimuli-evoked fluorescence transients . Finally, we measured the amplitude of the Ca2+ plateau during trains of action potentials and did not detect a significant change in sfGFP-Ncac relative to wild type . Thus, although we cannot rule out the possibility that tag incorporation could have subtle effects on Ca2+ channel function, we observe normal Ca2+ levels at motor terminals. To further interrogate the impact of endogenous tagging, we investigated synaptic transmission at sfGFP-Ncac NMJs. Consistent with normal Ca2+ influx, this analysis revealed no differences between control and sfGFP-Ncac in mEJP frequency, mEJP amplitude, or quantal content .To directly evaluate the function of endogenously tagged channels, we first assayed presynaptic Ca in situ . The usesfGFP-Ncac animals and found a mild decrease in bouton number at NMJ6/7 . At NMJ4, there is no difference in bouton number between sfGFP-Ncac and wild type . Similarly, AZ number per bouton is unaltered at sfGFP-Ncac NMJs . These analyses indicate that the incorporation of a fluorescent tag into the endogenous protein does not substantively disrupt Cac function in vivo.In addition to its role in neurotransmission, Cac promotes synapse formation . We assesfGFP-Ncac allowed us to interrogate endogenous Ca2+ channel levels and their relationship to single AZ function at the hundreds of individual synapses formed between a single motorneuron and postsynaptic muscle cell. Using Brp puncta to delineate single AZs, we found that Cac intensity per AZ varies considerably within a single motorneuron terminal, with most AZs exhibiting low-to-moderate levels and a smaller number expressing high Cac levels . This broad dynamic range in Ca2+ channel abundance could, in principle, underlie a similarly broad range of Ca2+ influx and Pr at individual AZs. Indeed, functional imaging studies at Drosophila NMJs have revealed significant heterogeneity in Pr between individual synapses of motorneuron–muscle pairs with the majority of AZs exhibiting low Pr and a small minority exhibiting high Pr, similar to the distribution we observe for Ca2+ channel levels . We then expressed postsynaptically targeted GCaMP5 . We observed significant heterogeneity in release probability, with a positively skewed distribution that matches observations from previous studies . Consistent with the previous finding that Brp levels positively correlate with Pr . This suggests that Brp levels largely predict Cac levels and that there is little additional predictive information in Brp measurements. However, this does not rule out the possibility that Brp levels also predict other parameters that influence Pr, such as readily releasable pool of synaptic vesicles (RRP) size, which may be coordinately regulated with the AZ cytomatrix and Ca2+ channels. Finally, we considered the possibility that high-Cac, high-Pr AZs represented the combined molecular content and output of two adjacent AZs mistakenly identified as single AZs in our analysis. Our imaging parameters provided lateral resolution of ∼250 nm, so more closely spaced AZs would not be resolved. A recent study used super-resolution microscopy to investigate the spacing between AZs and determined that fewer than 2.5% of Drosophila motor AZs are separated by less than 280 nm . Next, we investigated CacsfGFP-N levels in GluRIIA null mutants to determine whether endogenous Ca2+ channel levels are enhanced during chronic PHP expression. Consistent with the modulation of overexpressed Cac-GFP (2+ channel levels are indeed increased 36% in GluRIIA mutants (G–I). We also found that Brp levels were similarly increased ; a change that was previously reported and postulated to underlie the observed increase in RRP size . In agreement with Fig. 4G,H,J). To reconcile the difference between overexpressed and endogenous Cac, we repeated our analysis with Gal4-driven UAS-Cac-GFP (Cac-OE). As previously reported, we observed robust PHD expression and a decrease in overexpressed Cac-GFP levels, most notably at AZs of type Is boutons where n = 15 NMJs; vGlut-OE; Cac-OE: 0.61 ± 0.03, n = 24 NMJs, p < 0.0001, Mann–Whitney U test). In contrast, we observed no change in endogenous Cac levels at AZs of type Is boutons overexpressing vGlut , similar to our observations at type Ib AZs . This points to the use of the transgenic construct as the source of the discrepancy and highlights potential differences between the regulation of exogenous versus endogenous ion channels. Together, these findings suggest that increased Cac abundance contributes to increased Ca2+ influx and Pr during chronic PHP adaptation, but that diminished Ca2+ influx during PHD is likely achieved through functional modulation of channel gating properties. More generally, these results indicate that AZ reorganization during PHP involves the coordinated recruitment of new vesicles to the RRP and an increase in Ca2+ channel levels, whereas PHD occurs without apparent morphological reorganization of the AZ, changes to RRP size, or modulation of Ca2+ channel levels, highlighting the diversity of responses used by AZs to maintain stable synaptic communication.We next tested the model by investigating the modulation of endogenous Cac levels at NMJs overexpressing vGlut (VGlut-OE). In contrast to PHP, we did not observe a significant change in endogenous Can of PHD G,H,J. InDrosophila NMJ, PHP can be acutely induced through pharmacological inhibition of postsynaptic GluRIIA-containing glutamate receptors by the wasp venom PhTx. This triggers a potentiation of neurotransmitter release that precisely offsets the postsynaptic deficit within 10 min . We then treated partially dissected sfGFP-Ncac larvae with non-saturating concentrations of PhTx for 10 min and, immediately following PhTx treatment, fully dissected, fixed, and stained preparations for imaging of Cac and Brp. Strikingly, we observed a large increase in Cac levels (D–G). We also observed a significant increase in Brp levels across AZs  as previously reported . Paired analysis of single AZs demonstrated that Ca2+ channel levels were significantly increased relative to control across AZs with heterogeneous baseline states, although levels change little or decrease at a subset of AZs . These findings are further supported by our fixed imaging results, where we observed a significant rightward shift in the entire distributions of Cac and Brp following PhTx treatment . The smaller increase in Cac levels observed in our live imaging assays is likely due to the use of imaging conditions optimized for rapid acquisition and minimization of photobleaching. Given the heterogeneity in baseline Cac levels and Pr, we next investigated how the degree of Ca2+ channel accumulation might be influenced by the baseline state of an AZ. Cac content could be increased by addition of the same amount of Cac to all AZs  or by an amount proportional to baseline levels . We found that the data are well explained by a multiplicative model, indicating a proportional increase in Ca2+ channel levels across heterogeneous AZs (D). We next examined the population of AZs where Cac content is unchanged or decreased, and found that they were more frequently observed among AZs with lower baseline Cac levels (E). Although imaging constraints make it harder to detect proportional changes in Cac levels at low-content AZs, we observe a significant effect consistent with the conclusion that baseline Cac content influences the likelihood of Ca2+ channel accumulation at a particular AZ. Notably, we did not observe significant formation of new AZs in our live preparations, a finding we confirmed in our fixed samples (F). This indicates that potentiation of release occurs largely through the strengthening of existing AZs. Together our findings provide insight into how functional heterogeneity is established at single AZs and further modulated during homeostatic plasticity.We observe significant differences in baseline Cac levels and lication A. Pairedt of AZs B,C. Thesreatment G,H. The eous AZs D. We nexc levels E. Althou samples F. This i2+ channel accumulation at AZs contributes to the establishment and modulation of AZ-specific release properties to maintain stable communication. Endogenous tagging of Cac allowed us to track Ca2+ channels live and in fixed tissue without the potential artifacts associated with transgene overexpression. This approach revealed differences in the regulation of endogenous and exogenous Ca2+ channels, underlining the value of developing and validating reagents for following endogenous proteins in vivo.Diverse synaptic release properties enable complex communication and may broaden the capacity of circuits to communicate reliably and respond to changing inputs. We have investigated how the regulation of CaPr. This is consistent with previous studies in multiple systems linking endogenous Ca2+ channel levels at individual AZs to presynaptic release probability and efficacy, and a recent investigation of transgenically expressed Cac (2+ channel levels might be regulated to tune Pr during plasticity, so we investigated the modulation of endogenous Cac levels in several Drosophila models of presynaptic homeostatic plasticity. Previous studies have suggested that the bidirectional regulation of Ca2+ influx at synapses contributes to the modulation of presynaptic release observed during both PHP and PHD (2+ influx observed during the expression of PHP (2+ influx during PHP, leading to the model that modulation of presynaptic membrane potential might increase influx through Cac channels (2+ channel levels during development (2+ channel abundance is modulated during PHP remained an open question.The abundance of endogenous Cac at individual AZs of single motorneurons is heterogeneous and correlates with single-AZ ssed Cac . This st and PHD . A long-n of PHP . For exachannels . On the elopment , 2015. HHere, we demonstrate that Cac abundance is indeed enhanced during both the acute and chronic expression of PHP. This increase occurs in conjunction with the accumulation of Brp and enhancement of the RRP , pointin2+ influx during PHP, far less is known about how Ca2+ influx is regulated during PHD. One attractive idea was a reduction in AZ Ca2+ channel levels based on studies revealing reduced levels of transgenic UAS-Cac-GFP upon vGlut overexpression (sfGFP-Ncac, this observation indicates that a reduction in Cac abundance at AZs is not necessary to achieve PHD. We determined that the source of the discrepancy is the use of the transgene to report overexpressed versus endogenous Cac levels, demonstrating that exogenous and endogenous channels are regulated differently, at least during this form of PHD. This indicates that a mechanism other than modulation of Cac abundance drives PHD expression. Levels of Brp and RRP size are also unchanged during PHD (Drosophila photoreceptor synapses following prolonged light exposure (2+ influx. PHD signaling operates independently of PHP, and was recently proposed to function as a homeostat responsive to excess glutamate, not synaptic strength, raising the possibility that mechanisms distinct from those that have been elucidated for PHP may regulate presynaptic inhibition during PHD (In contrast to the many mechanisms proposed for modulating Capression . Howeverring PHD . Thus, texposure . In the ring PHD .sfGFP-N during acute PHP enabled the investigation of how baseline heterogeneity in Cac levels and Pr intersects with the homeostatic reorganization of AZs. Monitoring endogenous Cac at the same AZs before and after PhTx treatment, we observed the accumulation of Ca2+ channels across AZs with diverse baseline properties. As with PHP expression over chronic timescales, our findings leave open the possibility of multiple mechanisms acting simultaneously, perhaps to ensure precise tuning, and do not rule out additional modulation of channel function or indirect regulation of Ca2+ influx. In fact, a prevailing model posits rapid events that acutely modulate Pr followed by consolidation of the response for long-term homeostasis (2+ channel function and levels coupled with long-term restructuring of AZs provides an attractive mechanism for this model. We also found that Cac accumulation is proportional across low- and high-Pr AZs. Therefore, baseline heterogeneity in Cac levels is maintained following the expression of PHP. At mammalian excitatory synapses, proportional scaling of postsynaptic glutamate receptor levels stabilizes activity while maintaining synaptic weights (Drosophila NMJ. Notably, receptor scaling can occur globally or locally (Drosophila NMJ. A proportional increase in Cac levels could arise through homeostatic signaling from individual postsynaptic densities responding to similar decreases in quantal size; a strategy that would allow for both the remarkable synapse specificity and precision with which homeostatic modulation of neurotransmitter release operates.Finally, live imaging of Caceostasis , 2009. C weights . Our fin locally . A recen locally , demonst


Enhanced expression of STIM1 in cancer tissue has been associated with poor patient prognosis. Therefore, this study investigated the functional consequences of enhanced expression of STIM1 and Orai1 in a tumor-initiating subpopulation of Huh-7 hepatocellular carcinoma (HCC) cells that express epithelial cell adhesion molecule (EpCAM) and Prominin 1 (CD133).The endoplasmic reticulum (ER) Ca2+ monitoring, protein analyses, and real-time cell proliferation assays on EpCAM(+)CD133(+) subpopulation of tumor-initiating Huh-7 HCC cells expressing high levels of STIM1 and/or Orai1. Statistical significance between the means of two groups was evaluated using unpaired Student’s t-test.We performed qRT-PCR, intracellular Ca2+ release and proliferation rate of EpCAM(+)CD133(+) cells.Enhanced STIM1 expression significantly increased ER Ca2+-buffering capacity, which makes more Ca2+ available for the cytosolic events, on the other hand, possibly preventing Ca2+-dependent enzymatic activity in mitochondria whose Ca2+ uniporter requires much higher cytosolic Ca2+ levels.STIM1 overexpression may facilitate cancer cell survival by increasing ER Ca Hepatocellular carcinoma (HCC) appears to be the third leading cause of cancer-related deaths worldwide –11. The 2+ influx through Ca2+-release activated Ca2+ (CRAC) channels in non-excitable cells /[18S rRNA] × 100).FastStart DNA Master SYBR Green I kit was used in real-time qRT-PCR experiments performed . Primer sequences are shown in Table 4 cells seeded on 6-well plate by cOmplete Lysis-M, EDTA-free (Roche) according to the manufacturer’s instructions. Protein extracts, separated by SDS-PAGE were transferred onto PVDF membranes, then, incubated with antibodies targeted against STIM1 , Orai1  and β-actin  overnight at 4 °C. Membranes were incubated with secondary antibodies  for 1 h via shaking at room temperature. Protein bands were visualized in an infrared imager  based on the appropriate channel properties (680RD or 800CW) of secondary antibodies.Protein isolation was performed on 15 × 102+ levels were monitored via a front-surface spectrofluorometer (PTI QM8/2005) as described earlier . The proliferation rate at 48th h was significantly higher in EpCAM(−)CD133(−) cells comparable to that of EpCAM(+)CD133(+) CD133(+) Huh-7 cells. Comparable to the control, STIM1-OE cells at 72nd h showed the highest proliferation rate .The difference in multidrug resistance gene (MDR1) expression between tumor-initiating cells and tumor cell lines as well as the effects of STIM1 and Orai1 overexpression on MDR1 transcription in a number of experimental settings were investigated as increases in SOCE appeared to be associated with chemoresistance . MDR1 mR2+ homeostasis mechanism of non-excitable cells, SOCE appears to be operational in hepatocellular carcinogenesis [2+ regulation was investigated on Huh-7 TICs expressing cell surface antigens EpCAM and CD133 through monitoring intracellular Ca2+ dynamics (ER Ca2+ release and SOCE), proliferation and MDR1 expression responsible partly for drug resistance. High intracellular Ca2+ concentration comprises toxic and proapoptotic conditions for cells. Excessive Ca2+ is buffered by certain proteins  inside ER and by mitochondria. ER Ca2+ release and SOCE are significantly higher in EpCAM(+)CD133(+) cells comparable to that of EpCAM(−) CD133(−).In addition to being involved in intracellular Caogenesis . In this2+ release. As STIM1 has Ca2+ binding EF hand domains located on the intracellular part of ER [2+, leading to more Ca2+ available to be released from ER following SERCA blockade by CPA. STIM1 is the key initiating molecule in SOCE. After ER depletion, as a sensor of ER Ca2+ content, STIM1 accumulated in ER membrane closely located to PM with Orai1. At this ER and PM junctions, STIM1 interacts with Orai1 as a result SOCE is activated [Overexpression of STIM1 and Orai1 is shown in many cancer types like prostate cancer, breast cancer, glioblastoma and hepatocellular carcinoma . More sprt of ER , its ovectivated . Lower Ectivated . Higher ctivated . Similarctivated , 48, 49.ctivated , 47, 48.2+ buffer capacity of ER. Again, the significantly higher proliferation rate observed with STIM1-OE cells over that of EpCAM(+)CD133(+) cells overexpressing both STIM1 and Orai1 (present data) confirms the poor prognosis of several cancer types with overexpressed STIM1 [TICs tended to remain in a quiescence state . These Eed STIM1 –56.2+/calmodulin dependent protein kinase II (CaMKII), suggesting an interaction between Ca2+ and MDR mechanisms in liver cancer [Cancer cells show resistance to chemotherapeutic treatments. This may result from drug inactivation, changing drug targets, DNA damage repair, and efflux of drug from cells by ABC transporters . Becauser cancer .2+ buffering capacity due to higher levels of Ca2+-binding protein, STIM1. Furthermore, one may also speculate that increased ER Ca2+ buffering prevents Ca2+- dependent processes in mitochondria localized within the ER microenvironment by inhibiting Ca2+ uptake via low affinity/high capacity Ca2+ uniporter of mitochondria.Based on the higher proliferation rates observed in STIM1-overexpressing EpCAM(+)CD133(+) Huh7 cells compared to that of STIM1 + Orai1-OE constructs, one may conclude that HCC stem cells might undergo a phenotypical switch process from a quiescent to proliferative stage by increasing ER Ca


Saccharomyces cerevisiae. When DSB ends are occupied by KU, a central component of the NHEJ, the Mre11-Rad50-Xrs2 (MRX) nuclease complex (MRN in human), aided by the associated factors Sae2 (CTIP in human), initiates the resection process, inducing a nick close to the DSB ends. Then, starting from the nick, the nucleases Mre11, Exo1, Dna2, in cooperation with Sgs1 helicase (BLM in human), degrade DNA strand in both the directions, creating the 3′ ssDNA filament. Multiple levels of regulation of the break processing ensure faithful DSB repair, preventing chromosome rearrangements, and genome instability. Here we review the DSB resection process and its regulation in the context of chromatin. Particularly, we focus on proteins that limit DSB resection, acting as physical barriers toward nucleases and helicases. Moreover, we also take into consideration recent evidence regarding functional interplay between DSB repair and RNA molecules nearby the break site.In all the eukaryotic cells, nucleolytic processing (resection) of a double strand DNA break (DSB) is a key step to channel the repair of the lesion toward the homologous recombination, at the expenses of the non-homologous end joining (NHEJ). The coordinated action of several nucleases and helicases generates 3′ single strand (ss) DNA, which is covered by RPA and recombination factors. Molecular details of the process have been first dissected in the model organism  DSBs are classically defined as broken chromosomes, however uncapped telomere ends and reversed forks are bound and processed by the same factors. In this review we generally focus on broken chromosomes, although proteins, and mechanisms that we mention are active on whole types of DSBs.de novo telomere addition  or homology directed recombination (HDR). Both pathways are organized in distinct steps and sub-pathways, which involve the coordination of several factors and enzymes  of the DSB ends is a critical and finely regulated step to promote HDR over NHEJ. Indeed, the resection process generates an extended 3′-end ssDNA filament, which is then covered by RPA and the recombinase Rad51, depending on the sub-pathway , and Exo1 process a DSB whose 5′ ends are accessible. Alternatively, a DSB with blocked or chemically modified ends needs the activity of the MRX (MRN in human) complex to initiate the process . Indeed,The importance of regulating the DNA ends resection in DSB repair is underlined by the increasing list of factors participating in the reaction in human cells, including the oncosuppressor BRCA1  binds DNA ends in all the cell cycle phases, acting as a platform for the association of several factors involved in NHEJ  and its yeast ortholog Rad9 are important regulators of the DSB repair pathway choice  at the DSB is influenced by DNA:RNA hybrids?Other relevant questions regard the role and mechanisms of resection barriers at stall or collapsed replication forks. Indeed, transcription, and the DNA damage response are highly influenced by the chromatin architecture changes occurring during DNA replication.Remarkably, factors involved in DSB resection are deregulated in different cancers and genome instability syndromes, being also considered promising therapy targets. Indeed, the importance of all the factors involved in establishing and/or dampening resection barriers clearly emerged by treating tumor cells, which carry mutations in the BRCA1-axis, with the PARP1 inhibitor Olaparib. Notably, inactivation of the 53BP1-dependent resection barrier dramatically reduces the effectiveness of the treatment on BRCA1 defective cells, possibly leading to genome instability, poor prognosis, and cancer relapse (Lord and Ashworth, AP conceived the idea. All authors wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.