In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.Intracellular Ca Intracellular Ca2+ levels are affected by influx from extracellular fluid and release from intracellular stores such as the endoplasmic reticulum (ER). Along with Ca2+ influx, Ca2+ release is essential for a wide range of cellular responses, including muscle contraction, hormone secretion, and immune responses4. Therefore, functional maintenance of ER Ca2+ stores is essential for most eukaryotic cells7. In the central nervous systems, Ca2+ release is involved in various physiological events, such as excitation and transmitter release8. Therefore, stable Ca2+ release in response to stimuli is critical for proper functioning of the nervous system. However, the regulatory mechanisms behind Ca2+ release have not been fully understood in neurons in the central nervous system (CNS). Specifically, signaling molecules involved in functional maintenance of Ca2+ stores in CNS neurons are yet to be ascertained.Ca2+-store maintenance, we aim to explore the involvement of signaling adaptor proteins9 because previous studies have reported the possible binding of adaptor proteins to ER proteins that regulate intracellular Ca2+ release systems in non-neuronal cells. For example, in skeletal and cardiac muscle cells, insulin receptor substrate (IRS)-1 and IRS-2 have been shown to bind to sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-1 and SERCA-2, respectively10. Another adaptor protein, α-fodrin, also binds SERCA-2 in astrocytes11. The signaling adaptor Shc emerged at the dawn of metazoan evolution12. In mammals, at least for mouse and human genomes, four Shc loci Shc1, Shc2, Shc3 and Shc4 are present and encode the proteins ShcA, ShcB , ShcC  and ShcD, respectively14. ShcA is ubiquitously expressed throughout most tissues, but not in the mature brain. In the brain, ShcA expression gradually diminishes during development16, and ShcB and ShcC expression becomes dominant in mature neurons17. ShcB expression is more strongly confined to the nervous system, and ShcC appears to be exclusively expressed in neuronal cells21. In human and rat CNS, the expression profiles of ShcB and ShcC mRNAs overlap considerably, although there are some reports of distinct localization; in the adult rat brain, the level of ShcC mRNA is highest in the thalamus, whereas that of ShcB is highest in the hippocampus19. ShcD also shows relatively widespread distribution in the brain and spinal cord of adult rats, with prominent levels of expression throughout the olfactory bulb, as well as in sub-structures of the cerebellum and hippocampus, including the subgranular zone22. A genetic deletion of ShcA results in embryonic lethality due to malformation of the cardiovascular system23, whereas genetic deletion mutants for ShcB and/or ShcC, which have high expression in the nervous system, are viable17. ShcC null mice appear not to show gross anatomical abnormalities, but ShcB-deficient mice exhibit a loss of peptidergic and nonpeptidergic sensory neurons. In addition, mice lacking both ShcB and ShcC exhibit a significant additional loss of neurons within the superior cervical ganglia. Therefore, it is assumed that ShcB and ShcC take over the roles of phosphotyrosine (pTyr)-adaptor, at least partially, in mature neurons19. However, the functional significance of ShcB or ShcC is not fully understood, even though they retain similar pTyr-binding domains, i.e., phosphotyrosine-binding (PTB) and Src-homology 2 (SH2) domains for signal outputs, along with collagen-homology (CH)1 and CH2 domains26.Of the various candidates thought to contribute to ER Ca2+ store in CNS neurons through the regulation of SERCA activity, particularly in cerebellar Purkinje cells (PCs) in mice.Here, we show that ShcB is essential for the functional maintenance of Ca27. In line with this report, we observed a substantial ShcB-immunohistochemical signal in cerebellar PCs, and a weaker signal in the granular layer of adult wild-type (WT) mice but not of ShcB-knockout (ShcB-KO) mice  21–30 min after the LTD-inducing stimulus  was significantly lower than the level before CJS  at parallel fiber (PF)-PC synapses, because many previous studies indicate that cerebellar LTD is the cellular basis for cerebellar motor learningCerebellar dysfunction may also result from abnormal cerebellar morphology and/or synaptic transmission. However, we found no abnormality in the morphology and basic electrophysiological properties of excitatory synapses in the mature ShcB-KO cerebellum. Cerebellar lobule formation and the three-layer structure of the cerebellar cortex were normal in ShcB-KO mice Fig. D,E. Pair31  is essential for the induction of cerebellar LTD. In cerebellar slices from WT mice, bath application of (S)-3,5-dihydroxyphenylglycine (DHPG), a selective agonist for mGluR1, decreased the PF-EPSC amplitude and increased the paired-pulse ratio, suggesting a decrease in the release probability of PF2+ signals are also involved in the induction of various types of synaptic plasticity, including cerebellar LTD32. Application of a depolarization pulse elicited a transient increase in Ca2+ levels in WT PCs , the Ca2+ transient is indicated to be Ca2+ influx from the extracellular medium mediated by P/Q-type Ca2+ channels  induced a prominent increase in Ca2+ levels in WT PCs  , because pipette application of heparin (4 mg/ml), an IP3R blocker, abolished the DHPG-induced Ca2+ elevation  in PCs. Bath application of caffeine, a broad agonist for RyRs, induced a clear increase in intracellular Ca2+ levels in WT PCs  was diminished in ShcB-KO PCs 37, is also observed in cerebellar PCs  and RyR1 are the most dominant subtypes expressed in PCs39, and are essential for the induction of cerebellar LTD34. However, there were no significant differences in relative mRNA content  are balanced, and the intracellular Ca2+ levels are kept constant. Thus, in WT PCs, acute inhibition of SERCA by a specific inhibitor, thapsigargin, results in a continuous increase in intracellular Ca2+ levels , a ubiquitous store of Ca2+ in various types of cells, was analyzed using an ER tracker  or only mRFP were injected into the cerebellar cortex of the mutant mice. Approximately 8 weeks later, Ca2+ release was analyzed from PCs in a virus-injected area of ShcB-KO cerebellar slices to examine functional recovery of ER Ca2+ store . In non-infected PCs or PCs expressing only mRFP, bath application of nitric oxide (NO) did not elicit Ca2+ elevation  in PCs. When SERCA activity is not impaired, the spontaneous Ca2+ release is balanced by Ca2+ uptake by SERCA and the content of ER Ca2+ store is maintained homeostatically. However, in case where SERCA activity is reduced to about 50% of the normal level, spontaneous Ca2+ release might exceed Ca2+ uptake by SERCA, depleting the Ca2+ stores.In the ShcB-KO cerebellum, the ER CaPCs Fig. D–F, wherPCs Fig. E. Theref44. In the heart, it is well-established that the activity of SERCA2a is regulated by phospholamban (PLN) in the cardiac ER45. When the cytosolic Ca2+ concentration is low, dephosphorylated PLN interacts with SERCA2a and inhibits pump activity, but this inhibitory effect is alleviated when PLN is phosphorylated as a downstream effect of high cytosolic Ca2+ concentrations. Sarcolipin (SLN), a homologue of PLN, is also well known to bind and inhibit SERCA2a in skeletal muscle46. Because the expression of PLN or SLN in CNS neurons has not been previously reported, the protein(s) regulating SERCA activity in these neurons are yet to be determined. In the present study, however, the SERCA-dependent component of Ca2+ release was impaired in ShcB-KO PCs and rescued by ectopic expression of ShcB , with a 12 h light/dark cycle (lights on at 8:00 A.M.). Food and tap water were available ad libitum.All experiments were carried out according to the guidelines and approved by the Animal Welfare Committees of Nagasaki University, Kyoto University, Tokushima Bunri University, University of Toyama, Hokkaido University and Kitasato University. Efforts were made to minimize animal suffering and to reduce the number of animals used. WT and ShcB-KO mice at 8–12 weeks of age were used for all analyses. ShcB-KO mice were generated as described previously17. Antibodies against IP3Rs (sc-28613), RyRs (sc-8170) and SERCA2 (sc-8095) were purchased from Santa Cruz Biotechnology . Fluorescence images were obtained with the confocal imaging system Micro Radiance .Mice were pentobarbital-anesthetized and perfused with 4% paraformaldehyde in phosphate-buffered saline. Then, the brains were removed, postfixed for 24 h in 4% paraformaldehyde, and cryoprotected in 30% sucrose. Cerebellar sections (15 µm) were cut, mounted on slides and processed for Nissl staining or immunohistochemical staining. Staining with hematoxylin and Nissl staining were performed according to standard procedures. For immunochemical staining, the sections were permeabilized with 0.2% Triton X-100, blocked with 5% BSA, incubated with antibody overnight at 4 °C, and incubated further with Alexa Fluor 488-conjugated secondary antibody for 1 h at 37 °C. Antibody against ShcB was prepared as described previously51. Mice were placed on the stationary rod (diameter = 9 cm) of a rotating-rod device  for up to 120 s until they habituated to the experimental environment. The mice were then carefully placed on the rotating rod (9 rpm), and the time they remained on the rod was measured for each trial. The maximum retention time was 120 s.In the rotor-rod test, as with other behavioral tests including eyeblink conditioning and those described in supplemental information, 8- to 12-week-old WT- and ShcB-KO mice were used. The ability for motor coordination was examined with rotor-rod analysis52. For delay eyeblink conditioning, mice were surgically implanted with four Teflon-coated stainless-steel wires  under the left eyelid for recording EMG activity and delivering electric shock. A periorbital shock (100 Hz square pulses for 100 ms) was applied as unconditioned stimulus, and a tone  was used as the conditioning stimulus; the conditioning stimulus preceded and co-terminated with the unconditioned stimulus  with their forelimbs (2-grips test) or forelimbs and hindlimbs (4-grips test). Each mouse performed three trials for force measurement. The highest force value applied to the metal bar was recorded55. The time to falloff, with a 60-s cutoff time, was measured to evaluate the prehensile reflex. Three trials were performed for each mouse with a 20-min interval.Each mouse was placed on the center of a 0.5 cm grid at the bottom of a 28 cm × 36 cm box. The mouse was supported by the experimenter until it grabbed the grid with both its fore- and hind-paws. The grid was then inverted so the mouse was hanging upside-down. The duration of time that the animal remained on the grid was recorded. Only mice capable of hanging onto the grid for at least 1 s were included in analysis56. Each box could be divided by a sliding door (10 × 5 cm high). The test was started by placing a mouse in the black Plexiglas box. The time spent in the transparent and black Plexiglas boxes was measured for 10 min using digital counters with infrared sensors . The data were used to calculate the percentage of time spent in the light box: (time spent in transparent Plexiglas box/time spent in transparent and black Plexiglas boxes) × 100.In light and dark box test, as well as other behavioral tests described below, WT- and ShcB-KO mice at 12-week old were used. The light and dark box consisted of two compartments: a transparent Plexiglas box with a white Plexiglas floor and a black Plexiglas box with a black Plexiglas floor (both 15 × 15 × 15 cm)56.The elevated plus-maze consisted of two open arms (25 × 8 × 0.5 cm) and two closed arms (25 × 8 × 20 cm) emanating from a central platform (8 × 8 cm) to form a plus shape. The entire apparatus was elevated at a height of 50 cm above floor level. The test was started by placing a mouse on the central platform of the apparatus facing an open arm. The time spent in the open and closed arms was measured manually for 5 min. The data were used to calculate the percentage of time spent in the open arms: (time spent in open arms/ time spent in open and closed arms) × 10016. Mouse was allowed to freely explore for 10 min, its path of movement tracked with a video camera fixed on the ceiling of the room and stored in a computer system . The time spent in a concentric circular area (40 cm in diameter) of the open field was analyzed using the stored data.The open field test was started by placing a mouse at the edge of the wall of a circular open field (120 cm in diameter × 25 cm high)57.In the forced swimming test, a mouse was placed in a transparent glass cylinder (8 cm in diameter × 20 cm high), which contained water at 25 °C at a depth of 8 cm, and was forced to swim for 10 min. The duration of immobility was measured every 1 min using digital counters with infrared sensors , as described previously58. Whole-cell recordings were obtained from visually identified PCs under an upright microscope  using a 40 × water-immersion objective at room temperature (23–25 °C), except for the experiments using dantrolene (36 ± 1 °C). The resistance of patch pipettes was 2.5–4.0 MΩ when filled with an intracellular solution composed of (in mM) 120 K-gluconate, 5 KCl, 5 NaCl, 1 EGTA, 4 ATP, 0.4 GTP and 10 HEPES . For the voltage-clamp recording of CF-EPSCs, a pipette solution with the following composition was used (in mM) 60 CsCl, 10 Cs D-gluconate, 20 TEA-Cl, 20 BAPTA, 4 MgCl2, 4 ATP, 0.4 GTP and 30 HEPES (pH 7.3). The standard bathing solution was composed of (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 1.25 NaH2PO4, 26 NaHCO3 and 20 glucose, which was bubbled continuously with a mixture of 95% O2 and 5% CO2. Bicuculline (10 μM) was always present in the saline to block spontaneous inhibitory postsynaptic currents. Square pulses were applied for focal stimulation  through a glass pipette with a 5 to 10 μm-diameter tip and filled with the standard bath solution. The membrane potentials were held at − 90 to − 80 mV for recording PF-EPSCs, and at − 20 to − 10 mV for recording CF-EPSCs, after the compensation for liquid junction potential. Ionic currents were recorded using a patch-clamp amplifier . Stimulation and on-line data acquisition were performed using PULSE software (HEKA) on a Macintosh computer. Signals were filtered at 3 kHz and digitized at 20 kHz. The fitting of the decay phases of EPSCs was performed with PULSE-FIT software (HEKA).WT and ShcB-KO mice were sacrificed by cervical dislocation under deep anesthesia with diethyl ether. The cerebellum was excised, and parasagittal cerebellar slices (250 μm thick) were prepared from the vermis59. The PF-EPSC amplitude was normalized by the mean value observed for 10 min before the CJS.For the LTD experiments, the intensity of the stimulus was adjusted to evoke PF-EPSCs whose initial amplitudes were between 80 and 150 pA. After obtaining a stable initial recording for at least 10 min, a conjunctive stimulus was applied to induce LTD. The conjunctive stimulation protocol is composed of 300 single PF stimuli in conjunction with depolarization pulses  repeated at 1 Hz. Series resistance and membrane resistance were monitored throughout the experiments, and the data were discarded if either of these resistances varied by more than 10%. The data were also discarded if the slope of the PF-EPSC amplitude averaged every minute during the initial recording for 10 min was larger than 2% or if the amplitude did not stabilize within 20 min after the onset of the whole-cell conditions2+ imaging in PCs, a calcium-sensitive dye, Oregon Green BAPTA-1 (100 μM), was introduced into the cells through the patch pipette, and the concentration of EGTA in the pipette solution was decreased to 0.5 mM. Five to nine sequential confocal images (excitation at 488 nm) obtained at 3–4 µm z-axis intervals were acquired every 0.8 s using an upright microscope  equipped with a confocal scanning unit and an argon laser , and were projected onto a plane to obtain images of dendrites at 10-s intervals. Nitric-oxide (NO) induced Ca2+ release (NICR) was induced by bath application of an NO donor, NOC7 (300 μM), as previously described36. Values are normalized with F0, the average value before drug application.For intracellular CaThe morphology and distribution of the rough endoplasmic reticulum (ER) were analyzed by ER staining. Cerebellar slices were incubated with 1 μM ER-tracker red (Thermo Fisher Scientific) dissolved in the standard bathing solution for 30 min. Five to nine sequential confocal images were obtained and projected onto a plane to obtain images of PCs.2+-ATPase (SERCA2) were quantified by real-time PCR, according to the protocol in our previous study37. Total RNA preparations from 5 mice in each genotype were used as templates for cDNA synthesis . The mRNA content was analyzed using a real-time PCR system according to the manufacturer’s instructions . The cycle threshold (Ct) was determined from the cDNA amplification curve as an index for relative mRNA content in each reaction.Relative contents of type 1 inositol 1,4,5-tris phosphate receptor (IP3R1), type 1 ryanodine receptor (RyR1) and type 2 sarco/endoplasmic reticulum Cag for 10 min, and the supernatant was further centrifuged at 10,000×g for 15 min. The supernatant was centrifuged again at 165,000×g for 120 min, and we used the pellet as cerebellar microsomal fraction. For western blot analysis, the total protein in the cerebellar lysate or microsomal fraction was separated by SDS-PAGE and blotted onto a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with primary antibodies ; anti-SERCA2 , anti-neuronal nitric oxide synthase ; anti-immunoglobulin heavy chain-binding protein  and anti-β-actin ) and proteins were detected by HRP-conjugated secondary antibodies using the ECL detection kit . Because molecular weights of ShcB and BiP signals are close, the membranes were incubated with anti-BiP after deprobe of anti-ShcB for 40 min at 50 °C in the solution containing 2% SDS, 62.5 mM Tris–HCl (pH 8.0) and 100 mM 2-mercaptoethanol.Mouse brains were homogenized in a lysis buffer: 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM NaF, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor mixture  to obtain cerebellar lysate. In some experiments, the cerebellum was homogenized in homogenize buffer . The homogenate was centrifuged at 1000×Proteins in cerebellar lysate, prepared as described above, were precipitated with acetone at -20 °C, and the pellet was resuspended in lysis buffer containing 25 mM Tris–HCl (pH 7.5), 100 mM NaCl, 2 μM EDTA, 0.05% (v/v) TritonX-100, protease inhibitor cocktails and calpain inhibitor I. For coimmunoprecipitations, 2 μl of anti-SERCA2 polyclonal antibody  was added to the lysate and allowed to bind to the antibody by shaking at 4 °C overnight, before addition of 40 μl preequilibrated Protein A/G sepharose (abcam). After shaking at 4 °C for another 1 h, beads were washed 3 times in lysis buffer and bound protein was eluted at 95 °C for 5 min in 50 μl of SDS-loading buffer. The signal for ShcB protein was detected by western blot analysis, as described above.SERCA activity was analyzed using the EnzChek Phosphate Assay Kit . Briefly, the homogenates from the cerebellum and kidney were incubated with ATP for 60 min at 36 °C with or without thapsigargin, an SERCA inhibitor. Then, the concentration of the enzymatic product by ATPase, inorganic phosphate, was analyzed by the concentration of the ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine product converted from the 2-amino-6-mercapto-7-methylpurine riboside (MESG) substrate by the purine nucleoside phosphorylase (PNP) in the presence of inorganic phosphate. SERCA activity was determined by subtraction of inorganic phosphate production with thapsigargin from that without thapsigargin.14 was cloned into CSII-CMV-MCS-IRES2-mRFP1 to generate the transgene encoding ShcB-IRES-mRFP1. The vector plasmid carrying the transgene for ShcB-IRES-mRFP1 or mRFP1 in addition to packaging plasmids, cCAG-HIVgp and pCMV-VSV-G-RSV-Rev, was cotransfected into 293 T cells to prepare the lentiviral vectors. After the incubation for 48 h at 37℃, the medium was centrifuged at 50,000 g for 2 h to precipitate virus particles. The lentiviral titer was approximately 1 × 109/ ml. For in vivo infection with viral particles, ShcB-KO mice (4-weeks old) were anesthetized with pentobarbital, and through an incision in the scalp, a small piece of the occipital bone and the dura covering the surface of cerebellar lobule VII were removed. Then, solutions containing viral particles carrying the transgenes were injected into lobule VII of the cerebellar cortex with a microglass needle (tip diameter of 20–40 μm) attached to a manipulator59. A volume of approximately 1 μl was delivered unilaterally within 5–10 min by air pressure. After the injection, the scalp was sutured. Approximately 8 weeks after the injection, Ca2+ release was analyzed from PCs in a virus-injected area of ShcB-KO cerebellar slices. The vector plasmids (CSII-CMV-MCS-IRES2-mRFP1 and CSII-CMV-mRFP1), packaging plasmids (cCAG-HIVgp and pCMV-VSV-G-RSV-Rev) and 293 T cells were provided from RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan.Mouse ShcB cDNAp < 0.05.We tested statistical significance using Student’s t-test or one-way ANOVA, followed by Dunnett’s test. Values are presented as mean ± SEM unless otherwise stated. Differences were considered significant when Supplementary file1


ITPR3, encoding inositol 1,4,5‐trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot‐Marie‐Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot‐Marie‐Tooth disease gene.2+ imaging.Whole‐exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease‐causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and CaITPR3 p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified a de novo ITPR3 variant p.Arg2524Cys. Altered Ca2+‐transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant‐negative effect on inositol 1,4,5‐trisphosphate receptor type 3 function.Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygous ITPR3 is a disease‐causing gene for CMT and indicates altered Ca2+ homeostasis in disease pathogenesis.Together with two previously identified variants, our report adds further evidence that  ITPR3, which encodes the inositol 1,4,5‐trisphosphate (IP3) receptor (IP3R) type 3, were recently suggested as potential causes of CMT.Charcot‐Marie‐Tooth disease (CMT) is a group of hereditary neuropathies, characterized by progressive distal sensory and motor impairment, which affects 1:2500 individuals.3R isoforms: IP3R1, IP3R2, and IP3R3. They are homologous in sequence but differ in physiological functions and tissue expression.3 is produced after activation of G protein coupled receptors (GPCR), and binds the tetrameric IP3Rs, which release Ca2+ from ER into cytoplasm.2+ concentration has several downstream effects on the cell.3R signaling in neurons is underscored by the defects in IP3R1 leading to ataxia or Gillespie syndrome.3Rs is disrupted by inactivating variants in the genes ERLIN1, ERLIN2, and RNF170, causing hereditary spastic paraplegia and other neurodegenerative diseases.3R3 itself has been implicated in apoptosis control, while alterations in its activity and/or expression levels drive oncogenesis and impact the survival of malignant cells.Humans have three IPITPR3 with CMT. We introduce a CMT family with autosomal dominant mutation and one case with de novo mutation in ITPR3. In addition, we provide functional evidence of altered Ca2+ dynamics in patient fibroblasts.In this study, we provide confirmatory evidence of the association of °C in 5 % CO2.Individuals P1, P2, P3, and P4 gave written informed consent and the ethics review board of HUS Helsinki University Hospital approved the study. Control fibroblast cells were from anonymous donors, who consented to use of the cells in scientific research. The fibroblast cells were collected from skin biopsy and cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% FBS (Life Technologies), 1% penicillin/streptomycin (Life Technologies), 1% L‐glutamine (Life Technologies), and 0.2% uridine (Sigma). Cells were incubated at 37 Exome sequencing for the Finnish family was performed as described previously.3R1(CT‐1), IP3R2(NT‐2) , IP3R3 (BD‐Transduction #610312), and β‐tubulin . Anti‐rabbit and anti‐mouse (Jackson Immunoresearch#111‐035‐144 and #115‐035‐146) secondary antibodies were used. Detection was done by WesternBright™ ECL‐spray (Thermo Scientific) and imaging with Molecular Imager ChemiDoc XRS + with ImageLab (Bio‐Rad).Fibroblasts were lysed in RIPA buffer  and an aliquot containing 10 µg of total protein was boiled, separated in 4‐20% Criterion™TGX™ gels, and transferred to 0.2 µm nitrocellulose using Trans‐Blot Turbo . Blocking was done with 10% milk in 0.1% TBS‐T. Antibodies were: IPITPR1, ITPR2, ITPR3, and GAPDH (Table ITPR3 siRNA knockdown experiments were conducted as describedITPR3 ON‐TARGETplus SMARTpool siRNA (Dharmacon #L‐006209‐00‐0005) and ON‐TARGETplus Non‐targeting siRNA (Dharmacon #D‐001810‐01‐05).RNA was extracted by NucleoSpin® RNA extraction kit (Macherey‐Nagel #740955) and reverse transcribed by Maxima first strand cDNA synthesis kit (Thermo Fischer). RT‐qPCR was performed using DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific) with specific primers for DH Table  using Bi2+ imaging by two different methods in two different laboratories: non‐ratiometric manual Ca2+ assay using ATP stimulation (performed in University of Helsinki), and ratiometric automated Ca2+ assay using ionomycin, thapsigargin, and bradykinin stimulation (performed in KU Leuven).We performed fibroblast Ca2, 1.2 MgCl2, 10 HEPES, pH 7.4) and incubated for 50 min in dark in room temperature with 5 µg/ml Fluo‐4 AM Ca2+ indicator. After incubation, cells were washed three times with HBSS. For imaging, coverslips were placed on MatTek glass bottom dish (MatTek #P35G‐1.5‐14‐C) and imaged with Zeiss Axio Observer Z1 inverted phase contrast fluorescence microscope. During experiment, cells were perfused with Multichannel systems PPS2 Peristaltic perfusion system. Before every experiment, cells were allowed to rest for 5 min in the stage under HBSS perfusion. The excitation light was filtered through 494 nm band pass filter and the emission light passed through a 506 nm band pass filter. Emission wavelength was captured by Photometrics Prime BSI sCMOS Camera with ZEISS ZEN 3.1 (blue edition) imaging software. Acquisition protocol lasted a total of 12 minutes under constant perfusion at a rate of 2 ml/min. The cells were first perfused with HBSS containing 1.8 mM Ca2+ for 3 min and then with HBSS solution containing no Ca2+ (0‐Ca2+ HBSS) for another 3 min. Then Ca2+ release from ER was evoked by perfusion with 0‐Ca2+ HBSS containing 80 µmol/L adenosine 5’‐triphosphate (ATP) magnesium salt (Sigma‐Aldrich #A9187) for 2 min. After ATP‐evoked Ca2+ response, perfusion solution was changed back to 0‐Ca2+ HBSS for 2 min and finally back to 1.8 mmol/L Ca2+ containing HBSS solution for another 2 min to induce store‐operated Ca2+ entry (SOCE) as a positive control.Cells were washed two times with Hank’s Balanced Salt Solution (HBSS)  was selected from the first 0‐ Ca2+ period, from frames with stable intensity values. Relative intensities were calculated by first subtracting the baseline value from each frame and then dividing it by the baseline value [ΔFt/F0=(Ft‐F0)/F0]. To analyze the kinetics of the Ca2+ response peaks, we created an R‐script, which allows automatic analysis of the peaks. The area under the curve (AUC), peak amplitude and time to peak were measured from each cell, and the averages per coverslip were calculated. The scripts used are available online .The results were analyzed using MatLab (MATLAB R2019b) and RStudio (version 1.2.5033). Regions of interests (cells) were masked from each experiment and the mean pixel intensity was measured at each time point (frame) using a modified version of the previously described MatLab script.2+ levels of fibroblasts seeded in 96‐well plates (Greiner) were monitored using ratiometric fluorescent Ca2+ indicator dye Fura‐2 AM . They were loaded with the Fura2‐AM (1 µmol/L) at RT for 30 min in a modified Krebs solution , and 1.5 CaCl2). After loading, cells were rested for 30 min at RT in the absence of Fura‐2 AM to allow complete dye de‐esterification before proceeding to analysis on a FlexStation 3 microplate reader . The Ca2+ indicator was alternately excited at 340 and 380 nm and emission of fluorescence at 510 nm recorded. EGTA was added after 30 seconds in all conditions at a final concentration of 3 mmol/L. After another 60 seconds, cells were exposed to stimuli prepared in Ca2+‐free modified Krebs solution containing 3 mmol/L EGTA and Ca2+ transients were monitored for 6 min. Ionomycin and the irreversible SERCA‐inhibitor thapsigargin were both added at a final concentration of 10 µmol/L and bradykinin at a final concentration of 50 nmol/L. All traces are shown as the ratio of both emission wavelengths F340/F380 and were smoothened using a running average of 5. For quantification purposes, a baseline value was determined for each measurement as mean fluorescence between 30 sec and 90 sec. Fluorescence ratio F340/F380 was then normalized to the baseline values, and AUC of the peak, the peak amplitude  and the time to the peak were measured.Cytosolic CaThe proband of the Finnish family (P1) first came to neurologic investigations at age 38. His first symptoms had started slightly before age 30 with weakened foot dorsiflexion and tendency to foot drop. Around age 33 he also started to experience increased clumsiness in his hands. He was last evaluated at age 64. His symptoms have been slowly progressive with increasing difficulty in walking in rough terrain. He uses supportive insoles but has remained ambulant without external aids. He has severe muscle atrophy in his lower legs, hammer toes, pes cavus, and thenar muscle atrophy Fig. . SensatiThe proband had two brothers Fig. , one of Nerve conduction studies (NCS) of P1, P2, and P3 were consistent with demyelinating neuropathy, which were graded at least mild. The reduction in NCV was clearly less than is typical for CMT1A Table . Muscle The proband of the Ashkenazi Jewish family (P5) was developing normally until 4 years of age when he began falling. An evaluation at that time was concerning for pes cavus with hammertoes, and a motor only NCS showed a small tibial CMAP (0.5 mV) with a demyelinating range conduction velocity (20 m/sec). The median CMAP was normal (4.0 mV) but the NCV was 35 m/sec. No temporal dispersion or conduction block was present in either nerve. He was diagnosed with a demyelinating CMT and over the intervening decade he experienced progressive loss of leg strength and sensation in a symmetric distal to proximal gradient. After this evaluation he was not evaluated by a neurologist until 16 years of age when he established care at Columbia University Irving Medical Center (CUIMC).On neurological examination at age 16, he had pes planus with complete loss of muscle below the knees, and atrophy of proximal leg and hand intrinsic muscles. There was no movement at the ankles, anti‐gravity strength at the knees, and mild weakness of hip flexion and the hand intrinsics. Temperature, pin prick, and vibration were severely reduced at the toes and ankles, but remarkably, joint position sense was preserved. All reflexes were absent. Otherwise, general examination, cognitive evaluation, and neurological examination were normal.The proband was the product of a consanguineous union (parents are second cousins). His father is known to have flat feet and mildly reduced distal sensation in his early 40’s but no demonstrable weakness. His mother’s examination was normal. Of the proband’s 11 full siblings, none are suspected by the family to have similar symptoms but have not been formally tested.Repeat EMG/NCS showed absent motor and sensory responses in the legs Table . The medPMP22 duplication, MFN2 and GJB1 mutations. After this we performed exome sequencing on P1 and P3. We filtered the exome data for (1) shared nonsynonymous changes (excluding in frame insertions/deletions), (2) absence in SNP database (dbSNP), (3) prevalence ≤ 10‐5 in the Finnish subpopulation of gnomAD (v2.1.1), and (4) CADD scoreITPR3 (NM_002224.4) was of interest. We confirmed the segregation of the variant with disease in the family by Sanger sequencing  without other coding or copy number variants in those genes. A clinical exome was reported as negative. Trio sequence data were subsequently analyzed with an updated version of the Institute for Genomic Medicine’s established trio sequencing frameworkde novo missense variant in exon 55 of the ITPR3 gene . The variant is absent from the gnomAD V2.1.1 database, has a CADD score of 32.In P5, a chromosomal analysis and SNP microarray were within normal limits though the SNP microarray showed long contiguous runs of homozygosity consistent with the history of consanguinity. A CMT gene panel that included sequencing and deletion/duplication analysis of 42 genes identified single missense variants of uncertain significance in each of two autosomal recessive CMT genes . The affected amino acids are highly conserved . p.Arg2524 is located in the transmembrane domain (TMD) and lies in the channel pore   Fig. . This ex2+ homeostasis in fibroblasts from P1 and P2 and healthy controls. The response to GPCR agonist ATP, which results in IP3 signaling and thus opening of IP3Rs, was analyzed by manual non‐ratiometric assay in the absence of extracellular Ca2+. The ATP‐evoked Ca2+ release was altered in both patient fibroblasts. P1 fibroblasts had statistically significant decrease in AUC, while P2 fibroblasts had increased time to peak , which is the most common form of CMT.This study provides genetic and functional evidence for 3R3, shortly after the ligand‐binding domain,3R3 activity, for example, through interfering with allosteric regulators.3R3 function, which also influences disease severity.Both of the identified mutations affect highly conserved residues. Being located in the central, modulatory region of IP2+ flux demonstrate altered Ca2+ homeostasis in p.Val615Met patient cells. The primary fibroblasts express ITPR3 and thus are a useful tool to study the effects of the mutation under physiological conditions. The weakness of this model is that it does not account for possible neuron‐ or Schwann cell specific effects of the mutation. In addition, the results may be influenced by other genetic differences between control and patient cells in addition to the ITPR3 mutation. Treatment with ITPR3 siRNA was used to confirm that IP3R3 is active in fibroblasts under normal conditions and to model the effect of loss‐of‐function of the channel. We found altered GPCR agonist responses in both patient lines. The slowed response in P2 fibroblasts was similar but less pronounced than in siRNA‐treated cells. In addition, the P1 cells showed a decreased amplitude of the Ca2+ response to ATP. The differences in the Ca2+ responses between the two patient cell lines may be related to reduction in the amounts of IP3R2 and IP3R3 in P2 cells, which were compensated by ITPR3 mRNA upregulation in P1 cells. In addition, the difference in sex or other possible genetic differences may in part account for the differences between P1 and P2 fibroblasts. Based on these results, the p.Val615Met variant may produce a dominant negative effect on channel function. The effect appears to be subtle, which is consistent with late onset and slowly progressive nature of our patients’ phenotype. As ionomycin and thapsigargin‐induced Ca2+ release tended to be lower in patient fibroblasts, it cannot be excluded that IP3R3 p.Val615Met is leaky, that is, has an increased likelihood of being open compared with the wild type situation, thereby lowering steady state ER Ca2+ levels and thus dampening Ca2+ release in the cytosol upon agonist exposure.Our measurements of Ca3R3 in peripheral nerve maintenance. This is supported by its localization in paranodal regions of rat Schwann cells, where its proximity to another CMT gene product, gap junction protein beta‐1 (GJB1), may allow swift propagation of Ca2+ signals from cell to cell.2+ flux could contribute to altered axonal Ca2+ microdomains that disturb mitochondrial transport, as has been suggested for dominant mutations in the plasma membrane cation channel transient receptor potential cation channel, subfamily V, member 4 (TRPV4)3 signaling pathway, for example, FIG4 and SBF2, cause demyelinating CMT,3R3 has important implications for regulation of mitochondrial function and cell death and survival by participating in Ca2+ transfer between ER and mitochondria,MFN2).3R3 defects may decrease mitochondrial Ca2+ and predispose to defective bioenergetic or ER membrane function, which have been found in other forms of CMT.Our results suggest an important role of IPITPR3 is a disease gene for CMT. Additional studies, ideally in neuronal or animal models, will be needed to elucidate the effects of the disease variants on IP3R3 function and evaluate the potential of targeting Ca2+ flux as a therapeutic target in CMT.In conclusion, our results provide further evidence that J.R., S.M., M.A., G.B., S.H‐B., Ad.L., C.R., H.T., and E.Y. contributed to the conception and design of the study. J.R., S.M., M.A., J.T., J.N., A.P., and E.Y performed the experiments. J.R., A.R.P., E.M.P., J.K., An.L., J.N., G.B., and M.B.H contributed to the acquisition and analysis of data. J.R. and E.Y. wrote the manuscript with input from all authors.The authors report no competing interests.Table S1. Sequencing primers used for Sanger sequencing ITPR3 DNA and cDNA.Table S2. Primers used for quantitative reverse transcription PCR of ITPR1, ITPR2, ITPR3, and GAPDH.Table S3. Filtering of exome sequencing data of P1 and P3 left nine variants. The variants were analyzed further in silico. The variants not found in gnomAD were Sanger sequenced in all family members. The analysis left ITPR3 as a gene of interest.Click here for additional data file.


Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca2+ uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca2+ activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca2+/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na+ or Ca2+ extrusion, and in mouse dentate granule cells, Ca2+ creates the majority of this demand.When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca This, in turn, accelerates Na+ and Ca2+ pumping and leads to higher ATP consumption, which is replenished by oxidative phosphorylation (OXPHOS) and glycolysis 2+ is a strong candidate for coordinating the fast-metabolic responses to increased neuronal activity. Calcium handling is metabolically expensive and compartmentalized, involving several transporters at the plasma membrane and the endoplasmic reticulum. In addition, Ca2+ uptake into the mitochondria can directly dissipate the mitochondrial membrane potential H autofluorescence imaging, and extracellular O+ ratio in the cytosol using Peredox  can potentially influence the NADH pool in the other (e.g. cytosol) due to their connection via the malate-aspartate shuttle (MAS), whose components are highly expressed in neurons . Pyruvate is transported into the mitochondria via the mitochondrial pyruvate carrier (MPC) , where iWe first confirmed that MPC inhibition leads to cytosolic pyruvate accumulation by expressing the pyruvate-sensitive FRET sensor Pyronic  in the cStarving the mitochondria of pyruvate should also hamper the activity of the TCA cycle, thus diminishing NADH production in the mitochondria. To test this hypothesis, we assessed the effect of UK5099 on the NADH dynamics in the mitochondria of stimulated neurons.H+) by Ca2+ and/or ADP uptake into the matrix H signals in response to electrical stimulation of DGC axons, consisting of a prompt and brief (1–2 s) negative deflection (‘dip’), followed by an ‘overshoot’ that can last several minutes . The dipe matrix . The subtrix , to obtain a direct readout of sensor occupancy  that is independent of sensor expression level (+ ratio in the cytosol (NADHCYT)  . This reNADHCYT) . The cytCYT transients: baseline Peredox lifetime decreased by 3.4 ± 1.6% (CYT transients (expressed as the ΔPeredox/ΔRCaMP lifetime ratio) was attenuated by almost 40% H autofluorescence signals and the NADHmembrane  and actimembrane .2+ on the different phases of the NAD(P)H response, yielding contradictory results. Experiments using Ca2+-free extracellular solutions have been particularly hard to reconcile, with reports showing either a complete elimination of both phases of the NAD(P)H transient (2+ can contribute to both the dip and the overshoot of the NAD(P)H signal, although not necessarily to the same extent.Many studies have explored the effects of Caransient  or the pransient . We hypo2+ uniporter (MCU), an ion channel located at the inner mitochondrial membrane that constitutes the dominant path for mitochondrial Ca2+ uptake  by expressing Cre recombinase under the Dock10 promoter , in DGCsl Ca2+]  and 2. N+  and 2.chondria , or fastchondria .2+ influx in MCU-KD neurons leads to smaller mitochondrial NADH transients. Indeed, the prominent NAD(P)H overshoots observed in DGCs from control fl/ΔMcu mice (~2.1—4.8% for 25—200 pulse stimulations) were strongly attenuated in MCU-KD neurons from fl/ΔMcu Dock10Cre mice (2+ entry into the mitochondria via remnant MCU. The strong effect of Ca2+ deprivation on the NAD(P)H overshoot together with a delayed recovery from the dip H overshoot , which promotes the oxidation of NADH by Complex I (seen as the dip in autofluorescence). Consistent with the changes observed in the NAD(P)H dip, the amplitude of the transient decrease in the tissue [O2] was only diminished by MCU-KD when the slices were stimulated with 25 pulses, while the O2 decrease was delayed but not diminished in amplitude when using a higher number of pulses H dip remained unaffected in neurons lacking MCU, except for a small ~19% decrease when stimulated with 25 pulses . This suf pulses .2+ transient in the MCU-KD also produced some decrease in the magnitude of NADHCYT transient H autofluorescence signal, robust NADH increases were consistently elicited in the cytosol of stimulated neurons, especially when NADH re-oxidation through LDH was prevented.In any case, our experiments show that despite the strong effect of knocking out CYT responses, reflecting a temporary increase in aerobic glycolysis, correlates very closely with the cytosolic Ca2+ transient elicited during stimulation Second, we applied 100 µM of the cell-permeable Ca73 ± 11% .  and, just as with previous manipulations, the NADHCYT dropped by 73 ± 11% from the initial responses , is a major contributor to the glycolytic response in stimulated neurons but it is not strictly required.Overall, these results indicate that Ca2+/calmodulin axis, which has many downstream targets that promote glucose utilization , in the continuous presence of the LDH inhibitor GSK-2837808A to maximize the glycolytic NADHCYT transients. Then we applied α-pompilidotoxin, a toxin that slows down voltage-gated Na+ channel inactivation H transients, which is not recovered by a similar manipulation, even if a higher concentration of α-pompilidotoxin was applied , and in some cases decreased the transients even below pre-α-pompilidotoxin levels  gene in DGCs, which nearly abolished the Ca2+ entry into neuronal mitochondria, as seen also by 2+ entry caused a strong attenuation of the reductive phase of the NAD(P)H autofluorescence signal , consistent with a key role for mitochondrial Ca2+ in activating several dehydrogenases in the TCA cycle . This suggests that the direct contribution of mitochondrial Caoduction , so it i2+ from the extracellular solution recapitulated the effects seen with Mcu knockdown: the dip of the NAD(P)H signal was only decreased by ~21% while the overshoot was almost abolished, indicating again that Ca2+ influx is essential for the excess in NADH production in the mitochondria. Using a similar manipulation, 2+-independent in certain experimental circumstances, possibly produced by rapid pyruvate uptake into the mitochondria upon stimulation, rather than TCA stimulation by Ca2+ (Removing Ca by Ca2+ .2+ due to knockdown of MCU and the still-increased cytosolic Ca2+ will contribute to increased MAS function (Although the NAD(P)H overshoot has been universally observed in metabolic studies with neuronal stimulation, it is apparently not essential for maintenance of oxidative phosphorylation. We found that oxygen utilization, a sensitive indicator of flux through OXPHOS , was onlfunction .2+ stimulation may also contribute to production of mitochondrial NADH. For instance, we find that in contrast to the NAD(P)H overshoot, the late reductive phase of increased FADH2 (seen as a prolonged decrease in FAD+ autofluorescence) is preserved after MCU knockdown or when the MPC is blocked . In fact, the amplitude of the NADHCYT transient relative to the Ca2+ spike was even higher when the Ca2+/CaM complex was inhibited, indicating that this signaling pathway is not responsible for the glycolytic response to acute stimulation.We pharmacologically inhibited the Carm PMCA1 . However2+ and AMPK, a protein kinase that promotes glucose utilization   and driv2+ signaling: we were able to elicit NADHCYT transients by increasing Na+ influx, in the absence of extracellular Ca2+. Calcium is not strictly necessary for producing these glycolytic transients; the activity of the ATP-consuming ion pumps is sufficient, specifically the Na+,K+-ATPase  in the case of the Na+-only transients. Under these conditions, the Na+ pump activity is also necessary, as the Na+-driven glycolytic responses were reversed by the sodium pump inhibitor, strophanthidin.Our experiments provide evidence that neuronal glycolysis can indeed be driven by ATP hydrolysis rather than by CaCYT transients suggests that the NKA isoform α3 could mediate the coupling between neuronal activity and glycolysis. This isoform is more expressed in neurons than in any other cell type in the brain  to block the NADHhe brain , and conhe brain . Howeveractivity , or with2+ accounts for most of the total ion pumping after neuronal excitation. It is possible that Ca2+ extrusion is better coupled to glycolysis than Na+ extrusion (2+ ATPases (PMCAs) and glycolytic enzymes at the plasma membrane , as well as in different cell-types in the brain (e.g. interneurons and astrocytes).In summary, our work provides novel evidence on how neurons cope with moment-to-moment energy demands during bouts of action potentials, highlighting the different roles of Ca2+/calmodulin signaling pathway. Calmidazolium, a drug with similar effects on the Ca2+/calmodulin complex, was obtained from Cayman Chemical . The L-type calcium channel blocker isradipine and the NMDA-glutamate receptor inhibitor D-AP5 were obtained from Abcam . Stock solutions of MgCl2 (1M) were purchased from Teknova . dorsomorphin dihydrochloride (Compound C) and GSK-2837808A were obtained from Tocris , EGTA-AM from Anaspec Inc  and α-pompilidotoxin from Alomone Labs .All reagents were purchased from Sigma‐Aldrich , unless otherwise specified. The synaptic blocker NBQX was obtained from Toronto Research Chemicals . The MPC blocker UK5099 was purchased either from Tocris Bioscience  or Santa Cruz . Another drug from Santa Cruz was E6-berbamine, an inhibitor of the CaWe prepared stock solutions of calmidazolium (100 mM), E6-berbamine (33 mM), EGTA-AM (100 mM), GSK-2837808A (10 mM), isradipine (50 mM), strophanthidin (500 mM), and UK5099 (20 mM) in DMSO. The final concentration of DMSO in the experiments was kept ≤0.04%, except for EGTA-AM (0.1%), for which control experiments with 0.1% DMSO-only solution were performed to rule out interferences from the organic solvent in the recordings.2+ as a non-selective blocker of high voltage activated Ca2+ channels . Animals were housed in a barrier facility in individually ventilated cages with ad libitum access to standard chow diet (PicoLab 5053). All experiments were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. The Harvard Medical Area Standing Committee on Animals approved all procedures involving animals .Brain slice experiments were performed using brains of male and female wild-type mice , Mcu gene in DGCs by crossing conditional knockout fl/flMcu mice . Finally, the crossing of male wt/ΔMcu mice with female fl/fl Dock10CreMcu mice produced a 25% offspring of each experimental genotype: fl/ΔMcu and fl/Δ Dock10CreMcu (with Mcu selectively deleted in DGCs). Other combinations from the above-mentioned genotypes were able to produce experimental mice . In all crossings, the Dock10Cre transgene was present in the female parent.We selectively inactivated the /fl mice  with a C/fl mice . Male Mc/fl mice  were cro/fl mice  to generbination . Male McWe also worked with adult mice  instead of juveniles to permit more complete knockdown in the face of the late, postnatal expression of the Dock-10 promoter  and the We created an AAV plasmid that targets the sensor RCaMP1h into the mitochondrial matrix by inserting a portion of the precursor of the mitochondrial protein COX8 in the N-terminus of the fluorescent protein, as previously reported for GFP  and GCaMA 101 bp EcoRI-BamHI DNA fragment from pZac2.1-CaMKII-mito-GCaMP6s (obtained from Dr. Shinghua Ding), containing the mitochondrial leader sequence, was subcloned into the backbone of EcoRI-BamHI DNA fragment (5569 bp) of AAV.Syn.RCaMP1h.WPRE.SV40 (obtained from Dr. Loren Looger), using T4 DNA ligase.GAATTGGATCCACCATGGTGAGCAAGGGCGAGGAGAC 3’ and Reverse Primer: 5’ GGGCGAATTCGGAGCCTGC 3’). The amplicon was double digested with BamHI-EcoRI and was subcloned, using T4 DNA ligase, into the backbone of BamHI-EcoRI DNA fragment (6211 bp) of AAV.CAG.Pyronic.WPRE.SV40.We cloned the pyruvate sensor Pyronic into an AAV plasmid with the CAG promoter. The Pyronic insert, 2272 bp BamHI-HindIII DNA fragment, from Pyronic/pcDNA3.1(-)  were used for biosensor expression in brain tissue. For the expression of Peredox in the hippocampus, we used the AAV8 serotype  and the universal promoter CAG . For exp2+ sensor RCaMP1h, we used the AAV9 serotype and the neuron-specific promoter synapsin. For 2p-FLIM experiments, we used viral batches from three different suppliers: the Penn Vector Core, University of Pennsylvania, PA, the Viral Core Facility from Children Hospital in Boston, MA, and the Center for Genomics and System Biology, New York University, Abu Dhabi, UAE (kindly provided by Dr. G. Fishell and Dr. J Dimidschstein). For Ca2+ imaging in autofluorescence experiments, AAV9.Syn.RCaMP1h.WPRE.SV40 was produced in our laboratory using a protocol reported elsewhere  and the pups were intracranially injected with 150 nl of the AAV mix, twice per hemisphere, at the following coordinates with respect to lambda: (i) 0 mm in the anterior-posterior direction, ±1.9 mm in the medial-lateral axis, and −2.0 mm in the dorsal-ventral direction (ii) 0 mm in the anterior-posterior direction, ±2.0 mm in the medial-lateral axis and −2.3 mm in the dorsal-ventral direction. Viral injections were delivered at a rate of 50 nl/min using an UltraMicroPump III  microinjector. After injections, we waited ~2 min before gently pulling out the pipette, as a precaution to avoid spilling virus outside the target area. The pups recovered on a heat pad (covered by a paper towel) and/or under a heat lamp, before returning them to their cages. As a post-surgery care, one subcutaneous injection of ketoprofen (10 mg/kg) was delivered for up to 3 days. Acute brain slices were suitable for imaging from 2 weeks to 4 months post-injection.Alternatively, some intracranial injections were performed in adult mice . Mice were administered dexamethasone sodium phosphate (8 mg/kg) by an intramuscular injection to the hind leg, 1–2 hr before the surgery. Animals were anesthetized with isoflurane . Following confirmation of anesthesia, the mice were placed on a heated pad to maintain the body temperature at 37°C. Local anesthetics (10 mg/kg lidocaine and 2.5 mg/kg bupivacaine) were injected subcutaneously at the incision site, and the analgesic ketoprofen (10 mg/kg) and 0.5 ml of sterile 0.9% NaCl solution were also injected subcutaneously prior to the surgery. A small incision was performed in the skin to expose the skull, and a small hole was drilled over the desired area on the right hemisphere. The stereotactic coordinates with respect to lambda, for a single 2 µl viral injection, were the following: 3.39 mm in the anterior-posterior direction, −2.2 mm in the medial-lateral axis, and −2.4 mm in the dorsal-ventral direction. At the end of the surgery, a subcutaneous injection of buprenorphine SR (0.75 mg/kg) was delivered. We waited at least 2 weeks post-injection for experiments in acute hippocampal slices.14 gc/ml, and 1.45 × 1013 gc/ml for AAV9.Syn.mito-RCaMP1h.WPRE.SV40 (BCH). The viral titers in the Peredox:RCaMP1h mix varied depending on the AAV providers (in parenthesis): 2.05 × 1012 gc/ml of AAV8.CAG.Peredox.WPRE.SV40 (UPenn) and 1.87 × 1010 gc/ml of AAV9.Syn.RCaMP1h.WPRE.SV40 (Abu Dhabi), 4.1 × 1012 gc/ml of AAV8.CAG.Peredox.WPRE.SV40 (UPenn) and 2.55 × 1014 gc/ml of AAV9.Syn.RCaMP1h.WPRE.SV40 (BCH), or 3.08 × 1012 gc/ml of AAV8.CAG.Peredox.WPRE.SV40 (UPenn) and 2.01 × 1013 gc/ml of AAV9.Syn.RCaMP1h.WPRE.SV40 (UPenn).For 2p-FLIM experiments, the viral titer (genome copies/ml) used for injecting AAV8.CAG.Pyronic.WPRE.SV40 was 1.64 × 1013 gc/ml for AAV9.Syn.RCaMP1h.WPRE.SV40 (laboratory-made), and 4.83 × 1012 gc/ml for AAV9.Syn.mito-RCaMP1h.WPRE.SV40 (BCH). Several dilutions were tested until the bleedthrough of RCaMP fluorescence into the green channel was negligible.For autofluorescence experiments, the viral stocks were diluted in sterile 0.9% NaCl solution prior to the injections, to achieve the following titers: 1.45 × 102PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 75 sucrose, and 25 D-glucose (335–340 mOsm/kg). A different slicing solution was used for 2–4 months-old adult mice .Mice between 14 and 24 days-old were anesthetized with isoflurane, decapitated, and the brain was placed in ice-cold slicing solution containing (in mM): 87 NaCl, 2.5 KCl, 1.25 NaHult mice , consistBrains were glued by the dorsal side on a specimen tube holder and embedded in warm PBS with 2% low-melting agarose. The agarose was quickly congealed using a chilling block. The specimen tube was inserted in a chamber containing the same slicing solution (previously oxygenated) and horizontal slices were cut at a thickness of 275 µm using a Compresstome slicer . Alternatively, brains were glued by the dorsal side in a chamber containing the same slicing solution and horizontal slices were cut at a thickness of 275 µm using a vibrating slicer .2PO4, 26 NaHCO3, 2 CaCl2, 1 MgCl2, and 10 D-glucose (~290 mOsm/kg). All solutions were continuously bubbled with a mix of 95% O2 and 5% CO2, for adequate oxygenation and pH buffering around 7.4. Slices were incubated at 37°C for 35 min and then at room temperature for at least 30 min before the experiments, which were executed in the next 5 hr after slicing.Slices were immediately transferred to a chamber filled with artificial cerebrospinal fluid (ACSF) at 37°C, containing (in mM): 120 NaCl, 2.5 KCl, 1 NaH2+-free ACSF contained (in mM): 120 NaCl, 2.5 KCl, 1 NaH2PO4, 26 NaHCO3, 1 EGTA, 4.38 MgCl2 (~4.1 free) and 10 D-glucose. In these experiments, the control solution was modified to include EGTA, as follows (in mM): 120 NaCl, 2.5 KCl, 1 NaH2PO4, 26 NaHCO3, 1 EGTA, 3 CaCl2 (~2 free as in regular ACSF), 1 MgCl2, and 10 D-glucose. The concentrations for divalent ions in the presence of EGTA were estimated using Chelator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-TS.htm). An alternative, simpler, nominal zero Ca2+ ACSF was also tested (in mM): 120 NaCl, 2.5 KCl, 1 NaH2PO4, 26 NaHCO3, 4.1 MgCl2, and 10 D-glucose. The regular 2 mM CaCl2 was substituted by 3.1 free MgCl2 to preserve the charge screening effect of divalent ions on the membrane surface  or custom-made heaters. To prevent degassing in perfusion line, solutions were preheated at 38°C in a waterless bead bath  for 2p-FLIM experiments, or in water bath (VWR) for autofluorescence experiments.DGCs were stimulated with a concentric bipolar electrode CBBEC75  placed in the hilus. During the experiments, the ACSF contained NBQX (5 µM), D-AP5 (25 µM), and picrotoxin (100 µM) to block synaptic activity. Stimulation was delivered in trains of 25–200 brief (0.1 ms) pulses at a frequency of 50 Hz, using an A360 stimulus isolation unit . The stimulation intensity was adjusted to reliably evoke spike activity, which typically ranged from 750—1500 µA for antidromic stimulation .Lifetime imaging data were acquired with a modified Thorlabs Bergamo II microscope , with hybrid photodetectors R11322U-40 ; the light source was a Chameleon Vision-S tunable Ti-Sapphire mode-locked laser . The objective lens used for brain slice imaging was an Olympus LUMPLFLN 60x/W (NA 1.0). An excitation wavelength of 790 nm was used for the Peredox and RCaMP sensors. Fluorescence emission light was split with an FF562-Di03 dichroic mirror and bandpass filtered for green (FF01-525/50) and red (FF01-641/75) channels . For the Pyronic sensor, excitation was at 850 nm, and emission light was split with an FF506-Di03 dichroic mirror and bandpass filtered for CFP (FF01-475/35) and YFP (FF01-542/27) channels. The photodetector signals and laser sync signals were preamplified and then digitized at 1.25 gigasamples per second using a field programmable gate array board .Laboratory-built firmware and software performed time-correlated single photon counting to determine the arrival time of each photon relative to the laser pulse; the distribution of these arrival times indicates the fluorescence lifetime . LifetimImage analysis was performed using MATLAB software developed in our laboratory. Regions of interest (ROIs) were defined around individual cells, and photon statistics were calculated for all pixels within the ROI. Typical ROIs encompassed 100–900 pixels, in images of 128 × 128 pixels acquired at a scanning rate of 2 ms per line. Lifetime values were calculated as a standardized ‘tau8’ value, which minimizes the variability of the fits by restricting the averaging to the approximate time window of the actual data . Most daBleedthrough of green Peredox fluorescence into the red RCaMP optical channel was corrected using the ratio of measured red to green fluorescence observed when only Peredox was expressed, approximately 6.0% . SimilarNeurons with sudden and irreversible increases in the baseline lifetime of RCaMP or Peredox were excluded from the analysis.2+ biosensor, RCaMP1h or mito-RCaMP1h) were visualized with an Olympus BX51WI upright microscope using an LUMPlanFl/IR 60x/0.90W objective. The excitation light was delivered by an AURA light engine  at 365, 480 and 575 nm to excite NAD(P)H, FAD+ and the red Ca2+ biosensor, respectively. The times of exposure were 100 ms (excitation at 365 nm), 50—85 ms (excitation at 480 nm), and 5—85 ms (excitation at 575 ms). The fluorescence emission light was split with an FF395/495/610-Di01 dichroic mirror and bandpass filtered with an FF01-425/527/685 filter . Images were collected with a CCD camera , at a rate of 1 frame every two seconds in non-stimulated conditions, alternating the excitation wavelengths in each frame. During the fast response to stimulation, the acquisition rate was temporarily increased to six frames per second, for a total duration of 10 s (starting 2 s prior to each stimulation), and then returned to the initial lower frequency acquisition rate. Image acquisition and analysis was performed using laboratory-built software written in MATLAB . This software communicated with an electrophysiology setup by sending an external trigger to a protocol written in pClamp 10 , which in turn controlled a A360 stimulus isolation unit  via a Digidata 1321A digitizer . In pilot experiments, some recordings of local field potentials in the DGC layer were collected and amplified via a Multiclamp 700B  to confirm the stimulation, but they were not performed routinely in subsequent experiments.Autofluorescence signals in brain slices  per pixel was calculated in each ROI (A.U./px), as well as the relative fluorescence intensity changes (ΔF/F).Unlike many stimulation paradigms used for recording autofluorescence signals in brain slices , ours pr2, and an anoxic solution of 0.1 M sodium ascorbate and 0.1M NaOH (following the manufacturer instructions). Subsequent calibrations were performed at least once a week using two points: zero (anoxic solution) and 20% O2 in ACSF, both at 33–34°C. For routine calibrations, the pre-polarization times ranged between 1 and 2 hr, or until the signal was stable for 10 min. The microelectrode was connected to an OXY-Meter amplifier , and the signal acquired with the Sensor Trace software  at a rate of 1 Hz. The microsensor tip was inserted ~140 µm into the slice, in the DGC layer, and the signal was calibrated and expressed as [O2] in µM. The data was exported to an Excel file, and then analyzed offline using laboratory-built software written in MATLAB . Interpretation of the O2 changes in terms of ATP synthesis are made with the assumption that the proton leak across the mitochondrial membrane (independent of ATP synthesis and Ca2+ pumping) is unchanged.These measurements were executed simultaneously with the imaging of the autofluorescence signals. The Clark type oxygen glass microsensors, with tip diameters of 10 or 25 µm , were pre-polarized overnight for the initial calibration, which was performed in ACSF saturated with 20% or 95% O2 recordings, where the standard deviation is calculated using the number of slices.Statistical analyses were performed using GraphPad InStat v3.06 . Data were tested for normality with a Kolmogorov-Smirnov test. If the data did not fulfill all the assumptions for parametric tests , an equivalent non-parametric test was used. For comparisons of two populations with normal distributions but different SDs, a Welch’s t-test was used. The alternatives used in this study were: the Mann-Whitney test (for unpaired comparisons between two groups), the Wilcoxon matched pairs test (similar but for paired comparisons), the Kruskal-Wallis test (for multiple comparisons) and the Friedman test for repeated measures, with a Dunn’s post test. The selected tests and post-tests, as well as the descriptive statistics, are indicated in the figure legends and throughout the manuscript. Values are expressed as mean ± SD for individual neurons, except for autofluorescence experiments and OGraphics were constructed using Origin 9.1  and Microsoft Excel . Datasets were represented as box plots comprising the 25—75% (Q2—Q3) interquartile range, with whiskers expanding to the lower (Q1) and upper (Q4) quartiles of the distribution (5—95%). The median of the distribution is represented by a horizontal line inside the box, and the mean is represented by a cross symbol (×). In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:2+ in mitochondrial responses, which is a matter of debate. Using acute hippocampal slices as an experimental model, an appropriate ensemble of genetic and pharmacological tools, and NADH- and Ca2+-indicators as readouts of metabolic activity and activation status, respectively, these authors found that NADH signals in the cytosol occur independently of those in mitochondria. Using mitochondrial Ca2+ uniporter (MCU)-knockout mice, they further demonstrated a direct role for mitochondrial Ca2+ in driving TCA metabolism.In this manuscript, Carlos Manlio Diaz-Garcia et al. investigate potential coordination between responses of energy-regenerating pathways in the cytoplasm (glycolysis) and mitochondria (TCA/OXPHOS) during neuronal activation, focusing on the nature of the trigger for glycolytic activation, which is unknown, and the role of CaDecision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: James Hurley (University of Washington) (Reviewer #2).Thank you for submitting your article "The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:2+ in mitochondrial responses, which is a matter of debate. Using acute hippocampal slices as an experimental model, an appropriate ensemble of genetic and pharmacological tools, and NADH- and Ca2+-indicators as readouts of metabolic activity and activation status, respectively, these authors found that NADH signals in the cytosol occur independently of those in mitochondria. Using mitochondrial Ca2+ uniporter (MCU)-knockout mice, they further demonstrated a direct role for mitochondrial Ca2+ in driving TCA metabolism. However, they found no evidence for a role for Ca2+-dependent signaling in driving glycolysis in the cytoplasm. Instead, they propose that the trigger for glycolytic activation in response to neuronal activity is enhanced energy demand resulting from the ATP-dependent operation of surface membrane Ca2+ (and Na+) extrusion mechanisms. On the basis of these basic relationships, they conclude that Ca2+ plays distinct roles in the rapid control of neuronal glycolysis and the mitochondrial TCA cycle.In this manuscript, Carlos Manlio Diaz-Garcia et al. investigate potential coordination between responses of energy-regenerating pathways in the cytoplasm (glycolysis) and mitochondria (TCA/OXPHOS) during neuronal activation, focusing on the nature of the trigger for glycolytic activation, which is unknown, and the role of Ca2+ is important in TCA cycle activation. They are also consistent with the conclusion that the energy required to pump Ca2+ and Na+ out of the cell ultimately drives glycolysis.The results convincingly demonstrate that energetic responses to neuronal activation in the cytoplasm and mitochondria are independent of each other and that mitochondrial CaEssential revisions:2+ signal so that should be stated in the legend and the exact time of the train of pulses is not shown so it's timing relative to the RCAMP signal should be indicated. Also, there should be a very brief description of the meaning of fluorescence lifetime. I was familiar with this measurement from previous Yellen reports but some readers may not be.1) Figure 1B legend. Please provide a more complete description of this experiment. The bottom trace shows the Ca2+ increase in response to stimulation seems to be about the same with and without MCU, but without MCU the influx is not sustained. (I think they discuss later that this may be a non-MCU mechanism of Ca2+ influx but some comment should be made about this here). Also, the initial baseline (AU per px) is higher in the MCU knockdown neurons – is that just variation or does it reflect a higher resting Ca2+ concentration in the matrix?2) Subsection “Calcium entry into mitochondria is required for strong activation of TCA metabolism”. Based on the results in Figure 2A the loss of MCU seems to be having a specific effect. The magnitude of the initial Ca2+ is measured as AU/px not in lifetime, but in panel C the cytosolic Ca is reported in lifetime units. Please comment on why this was done and how that affects the significance and quality of the data and interpretation of the different types of experiments.3) In Figure 2A Ca4) In Figure 2B – 50% decay times are reported. Is that 50% of the initial spike of Ca? Or is it from a curve fitting to what appears to be a normal exponential decay curve that occurs following the spike. In other words is the decay of mitochondrial Ca biphasic and if so shouldn't both two time constants and their relative contributions be reported?5) In the MCU knockdown mitochondrial NADP(H) signals in Figure 2A the oxidative transients seem smaller than in the controls. That does not seem representative based on Figure 2—figure supplement 3. To avoid confusion would it be possible to use a more representative experiment for Figure 2A?2+. This is mentioned briefly later in the Discussion but it seems like it could be a significant factor in these studies and in their interpretation.6) Subsection “Calcium entry into mitochondria is required for strong activation of TCA metabolism”. I think there should also be some discussion of AGC1 activity being stimulated directly by cytosolic Ca+ or Ca2+ pumping triggers aerobic glycolysis”. Adding strophanthidin in the zero Ca2+ shows that ATP consumption stimulates GAPDH activity. However, wouldn't it be also worthwhile to determine if strophanthidin also blocks the cytosolic NADH increase triggered by stimulation when Ca2+ is present? In other words can the authors address whether Ca2+ does or does not contribute to accelerated glycolysis (independently of increased energy demand)?7) Subsection “Energy demand from Na2+ regulation of AGC1 (i.e. enhanced MAS activity) also could contribute to elevated NADH in the matrix. (this is a reiteration of my comment #6).8) Discussion. Please address here whether or not Ca9) Discussion: Is the "rapid dip" blocked by strophanthidin?2+ is little more than a bystander in the regulation of glycolysis. The "distinct roles" formulation of the paper needs to be walked back.10) The data presented show that Ca11) A scheme showing the biochemical linkages and cell-biological relationships  under consideration is necessary. Readers should not be made to forage through biochemistry texts and online resources to make sense of the basic questions a paper is attempting to address.2+ entry  are not only speculative but are potentially provocative.12) It is important to know the extent of MCU protein depletion; but it is acknowledged that this may be technically difficult. Have all the options been exhausted? Because the extent of MCU depletion is not known, references to possible non-uniporter routes of mitochondrial Ca13) The NADHcyto transient: There was a decrease in the NADHcyt transient with MCU depletion that could not be explained by (greater) flux through LDH because it was not "rescued" by the LDH inhibitor. It was stated in the text that the decrease could reflect increased MAS activity; this would constitute a compensation for loss of MCU.14) NAD(P)H dip: There was a small decrease in the amplitude of the NAD(P)H autofluorescence dip in MCU kd slices, and the kinetics of the dip were altered; these changes, while small, were significant .2 signal, in both the amplitude and kinetics .15) Likewise, there were small but significant changes in the concomitant OFAD+ undershoot during the NAD(P)H overshoot: In MCU kd slices, there was less of a decrease in the FAD+ undershoot compared to decrease in the NAD(P)H overshoot. What does the FAD+ undershoot reflect: PDH activity (the E3 subunit: FADH2 -> FAD+), SDH activity (FAD+ -> FADH2) or the glycerol-3-P-dehydrogenase (FADH2 -> FAD+)? Though the latter describes the NAD(P)H overshoot phase, the fact that there is a different effect on the NAD(P)H overshoot and FAD+ undershoot suggests that the FAD+ signal does not simply reflect TCA FAD+ -> FADH2 (at SDH), and that what contributes to the FAD+ signal may differ between Ctrl and MCU kd. The latter point would support\ that metabolic fluxes in response to stimuli can be different in MCU kd slices.16) FAD+ undershoot in MCU kd slices likely does not reflect flux through PDH or through SDH (since otherwise less of an attenuation of the NAD(P)H signal would be expected). So what does the FAD+ undershoot reflect in MCU kd? Increased glycerol-3-P-dehydrogenase activity? If yes, then there can be substantial ROS associated with that activity (see Quinlan 2013 PMID: 24024165), which could provoke additional changes.2) and the FAD+ undershoot seems to be deemphasized. At least in the text of Results, a fuller description of the changes in the dip and undershoot would be needed.17) Discussion: focus is on emphasizing the effect of MCU kd on the NAD(P)H overshoot and the changes to the dip (NAD(P)H and O18) This dataset might be the first actual evidence that metabolism compensates/responds/is not neutral to MCU loss (beyond the surprising observations in other studies that MCU loss appears to not impact metabolism). Acknowledging this evidence that metabolism is not neutral to MCU loss seems important in light of observations from several studies that MCU loss appears to not alter metabolism, which can lead to the conclusion that MCU plays no role in metabolism.2+; it would be worth pointing this out, and that the higher cyto Ca2+ during the pulses may lead to greater activation of MAS. The same would be the case for glycerol-3-P-dehydrogenase if it were to play a role.19) MAS depends on AGC1/2  which contain EF-hands that face the IMS and are therefore theoretically exposed to cytosolic Ca20) Referring to the NAD(P)H overshoot as TCA-refilling may be incorrect, and prematurely elevates an observation into an interpretation. That there is an NAD(P)H overshoot suggests that stimulation is followed by a phase in which the mitochondrial matrix is in a relatively reduced state. This may reflect a disproportionate NADH generation by PDH and TCA cycle relative to NADH oxidation by the electron transport chain. The overshoot could reflect a shift in oxidation state of the NADPH pool.2+ the different phases of NAD(P)H response to stimulation: That there are "contradictory results" is "merely" mentioned in Results and in Discussion. Has the current study been designed in any way that attempts to better understand the role of mito Ca2+ in the NAD(P)H response?21) About the mixed results in the literature on the effects of mitochondrial Ca22) Discussion: "We found that oxygen utilization,.…, was not altered by MCU knockdown". This does not seem to be accurate based on the data of Figure 2—figure supplement 3.2+ itself may not be necessary if.… Ca2+ extrusion". Metabolic response to Ca2+ extrusion and the use of/requirement for Ca2+ as part of that metabolic response can be independent.The sentence "More importantly, Ca Essential revisions:2+ signal so that should be stated in the legend and the exact time of the train of pulses is not shown so it's timing relative to the RCAMP signal should be indicated.1) Figure 1B legend. Please provide a more complete description of this experiment. The bottom trace shows the Ca2+ signals, and we have now shown the stimulation times with arrows in the figure.We have modified the legend to have a more complete description of the experiment, including the RCaMP CaAlso, there should be a very brief description of the meaning of fluorescence lifetime. I was familiar with this measurement from previous Yellen reports but some readers may not be.Thank you for this suggestion. A brief description of fluorescence lifetime has been included in the Results.2+ increase in response to stimulation seems to be about the same with and without MCU, but without MCU the influx is not sustained. (I think they discuss later that this may be a non-MCU mechanism of Ca2+ influx but some comment should be made about this here). Also, the initial baseline (AU per px) is higher in the MCU knockdown neurons – is that just variation or does it reflect a higher resting Ca2+ concentration in the matrix?2) Subsection “Calcium entry into mitochondria is required for strong activation of TCA metabolism”. Based on the results in Figure 2A the loss of MCU seems to be having a specific effect. The magnitude of the initial Ca2+ signals to better accord with the reviewers’ correct understanding of the effect of MCU-KD. Instead of “decay time”, we now report area-under-the-curve for the mitochondrial Ca2+ signals, which gives an integrated measure of both the amplitude and the sustained time course.We have changed the analysis and presentation of the mitochondrial Ca2+] and show that this is in fact lower in the MCU-KD neurons, as might be expected. We now describe this result in the Results.We have also performed additional experiments using two-photon fluorescence lifetime measurements of the RCaMP signal in MCU-KD and control neurons . These give results that are completely consistent with the widefield fluorescence data, but they also give a more reliable indication of baseline mitochondrial  were performed with a widefield fluorescence microscope (one-photon) that does not provide lifetime measurements. As explained in our response to #2 above, we now provide additional RCaMP lifetime measurements in Figure 3—figure supplement 2; these are quite consistent with the conclusions based on the widefield experiments.4) In Figure 2B – 50% decay times are reported. Is that 50% of the initial spike of Ca? Or is it from a curve fitting to what appears to be a normal exponential decay curve that occurs following the spike. In other words is the decay of mitochondrial Ca biphasic and if so shouldn't both two time constants and their relative contributions be reported?2+ signals by providing an integrated measurement of the area under the curve. A detailed study of the kinetics of mitochondrial Ca2+ handling is beyond the scope of our experiments.As explained in our response to #2 above, we now avoid any confusion about kinetics of the mitochondrial Ca5) In the MCU knockdown mitochondrial NADP(H) signals in Figure 2A the oxidative transients seem smaller than in the controls. That does not seem representative based on Figure 2—figure supplement 3. To avoid confusion would it be possible to use a more representative experiment for Figure 2A?Thank you for pointing this out. We have now included a more representative experiment.2+. This is mentioned briefly later in the Discussion but it seems like it could be a significant factor in these studies and in their interpretation.6) Subsection “Calcium entry into mitochondria is required for strong activation of TCA metabolism”. I think there should also be some discussion of AGC1 activity being stimulated directly by cytosolic Ca2+. Nevertheless, MCU KD has a much larger effect on matrix Ca2+ than on cytosolic Ca2+, so the matrix-side effect is likely the more important one to mention here (as we do).We have now included a paragraph in the Results, on the possible compensation by MAS (or the glycerol-P shuttle), especially during the activity-associated rise in cytosolic Ca2+ stimulation of AGC1 and of upregulation of MAS in the MCU KD.In the Discussion, we consider the possible contributions of Ca+ or Ca2+ pumping triggers aerobic glycolysis”. Adding strophanthidin in the zero Ca2+ shows that ATP consumption stimulates GAPDH activity. However, wouldn't it be also worthwhile to determine if strophanthidin also blocks the cytosolic NADH increase triggered by stimulation when Ca2+ is present? In other words can the authors address whether Ca2+ does or does not contribute to accelerated glycolysis (independently of increased energy demand)?7) Subsection “Energy demand from Na+/K+ pump is not necessarily sufficient to prevent ATP hydrolysis associated with Ca2+ extrusion (through other pathways), and because Na+/K+ pump inhibition leads rapidly to cell death by swelling under normal ionic conditions. Strophanthidin also causes severe dysregulation of Ca2+ and Na+, even at low doses. Our future experiments are targeted to address this general conceptual question.This is a complicated question, because blockade of the Na2+ regulation of AGC1 (i.e. enhanced MAS activity) also could contribute to elevated NADH in the matrix. (this is a reiteration of my comment #6).8) Discussion. Please address here whether or not CaAs mentioned in #6, we have discussed this possibility in the Discussion.9) Discussion: Is the "rapid dip" blocked by strophanthidin?As mentioned in #6, we have discussed this possibility in the Discussion.2+ is little more than a bystander in the regulation of glycolysis. The "distinct roles" formulation of the paper needs to be walked back.10) The data presented show that Ca2+ influx makes the NADHcyt transients nearly disappear. Ca2+ influx plays a major role, but not specifically as a signaling molecule. We have added a better explanation of its apparent role in energy demand, as a surprisingly large contributor (Results).We would not say it is a “bystander”, as eliminating Ca11) A scheme showing the biochemical linkages and cell-biological relationships  under consideration is necessary. Readers should not be made to forage through biochemistry texts and online resources to make sense of the basic questions a paper is attempting to address.We are happy to provide a scheme of the relevant biochemical processes and their compartmentation, now shown in Figure 1; .2+ entry  are not only speculative but are potentially provocative.12) It is important to know the extent of MCU protein depletion; but it is acknowledged that this may be technically difficult. Have all the options been exhausted? Because the extent of MCU depletion is not known, references to possible non-uniporter routes of mitochondrial CaThank you for pointing this out. We know the MCU knockdown is quite variable and may be incomplete; we have revised this sentence to mention that remnant MCU may be responsible in the Results. Additional explanations for the initial peak in mitoRCaMP are also included in the Results.13) The NADHcyto transient: There was a decrease in the NADHcyt transient with MCU depletion that could not be explained by (greater) flux through LDH because it was not "rescued" by the LDH inhibitor. It was stated in the text that the decrease could reflect increased MAS activity; this would constitute a compensation for loss of MCU.As mentioned in #6, we have added a local explanation (Results) about the possibility of increased/compensatory expression of the mitochondrial shuttles.14) NAD(P)H dip: There was a small decrease in the amplitude of the NAD(P)H autofluorescence dip in MCU kd slices, and the kinetics of the dip were altered; these changes, while small, were significant .In the Results, we mentioned that the dip of MCU-KD mice decreases when slices are stimulated with 25 pulses. In the figure legend of now Figure 3—figure supplement 3A, we now mention that the change in the kinetics of the dip likely reflects less overlap between the oxidative and reductive phases of the NAD(P)H signal due to the attenuation of the overshoot.2 signal, in both the amplitude and kinetics .15) Likewise, there were small but significant changes in the concomitant O2 signal in the Results section. In the Discussion section, we now say that the O2 dip is slightly diminished or delayed by MCU knockdown (depending on the duration of the stimulus).We have emphasized the changes in the OFAD+ undershoot during the NAD(P)H overshoot: In MCU kd slices, there was less of a decrease in the FAD+ undershoot compared to decrease in the NAD(P)H overshoot. What does the FAD+ undershoot reflect: PDH activity (the E3 subunit: FADH2 -> FAD+), SDH activity (FAD+ -> FADH2) or the glycerol-3-P-dehydrogenase (FADH2 -> FAD+)? Though the latter describes the NAD(P)H overshoot phase, the fact that there is a different effect on the NAD(P)H overshoot and FAD+ undershoot suggests that the FAD+ signal does not simply reflect TCA FAD+ -> FADH2 (at SDH), and that what contributes to the FAD+ signal may differ between Ctrl and MCU kd. The latter point would support\ that metabolic fluxes in response to stimuli can be different in MCU kd slices.This is an interesting point. We have now commented on the possibility of the glycerol-P shuttle contributing to the transfer of reducing equivalents into the mitochondria (please see #6). However, we also mention the possibility of glutamate and glutamine sustaining flux through part of the TCA cycle involving SDH, by entering at the α-ketoglutarate level and exiting as oxaloacetate (Discussion).16) FAD+ undershoot in MCU kd slices likely does not reflect flux through PDH or through SDH (since otherwise less of an attenuation of the NAD(P)H signal would be expected). So what does the FAD+ undershoot reflect in MCU kd? Increased glycerol-3-P-dehydrogenase activity? If yes, then there can be substantial ROS associated with that activity (see Quinlan 2013 PMID: 24024165), which could provoke additional changes.As commented in #15, there is still the possibility of SDH involvement if glutamate becomes a substrate for the TCA cycle. We have mentioned that the glycerol-P shuttle may also compensate for the reduced TCA cycle activity due to MCU loss (see #6). The relative contributions of these potential pathways, and their consequences for ROS production, are relevant topics for further investigation but are beyond the scope of the present study.2) and the FAD+ undershoot seems to be deemphasized. At least in the text of Results, a fuller description of the changes in the dip and undershoot would be needed.17) Discussion: focus is on emphasizing the effect of MCU kd on the NAD(P)H overshoot and the changes to the dip (NAD(P)H and O+ undershoot in the Results.We now mention the unexpected findings on the less attenuated FAD18) This dataset might be the first actual evidence that metabolism compensates/responds/is not neutral to MCU loss (beyond the surprising observations in other studies that MCU loss appears to not impact metabolism). Acknowledging this evidence that metabolism is not neutral to MCU loss seems important in light of observations from several studies that MCU loss appears to not alter metabolism, which can lead to the conclusion that MCU plays no role in metabolism.Thank you for this observation. We hope that the current version of the manuscript emphasizes more the impact of MCU loss on neuronal metabolism.2+; it would be worth pointing this out, and that the higher cyto Ca2+ during the pulses may lead to greater activation of MAS. The same would be the case for glycerol-3-P-dehydrogenase if it were to play a role.19) MAS depends on AGC1/2  which contain EF-hands that face the IMS and are therefore theoretically exposed to cytosolic CaWe have addressed this possibility in the previous answers  Referring to the NAD(P)H overshoot as TCA-refilling may be incorrect, and prematurely elevates an observation into an interpretation. That there is an NAD(P)H overshoot suggests that stimulation is followed by a phase in which the mitochondrial matrix is in a relatively reduced state. This may reflect a disproportionate NADH generation by PDH and TCA cycle relative to NADH oxidation by the electron transport chain.2+]MITO in activating dehydrogenases in the TCA cycle to resupply mitochondrial NADH after neuronal stimulation.” We think this statement agrees with the last sentence of the comment, as indeed we think that the overshoot arises from the imbalance between the “resupply” of NADH encouraged by Ca and the oxidation by the ETC spurred by ATP demand.We cannot find the statement on the NAD(P)H overshoot acting as a TCA-refilling event, but perhaps this comment refers to the statement about “a major role for CYT may not be necessary for the metabolic transient, although this increase apparently accounts for a surprisingly large part of the metabolic transient. It may be that the fast glycolytic response is simply reactive to the energy demand resulting from Ca2+ extrusion, and that this is a large fraction of the total energy demand produced by activity.”This sentence has been rewritten and now states that: “More importantly, activity-induced increases in [Ca