Academic Appointments

Honors & Awards

  • The Award for Young Investigator, Japanese Society for Neuroscience (07/01/2020)
  • Trainee Professional Development Awards, Society for Neuroscience (09/07/2017)
  • The Award for Young Investigator, Japanese Society for Neurochemistry (7/26/2019)
  • Selected Hot-Topics research, Society for Neuroscience (8/15/2015)

Professional Education

  • PhD, University of Tokyo, Neurochemistry (2013)


  • Masatoshi Inoue. "United States Patent US20180372762 CALCIUM INDICATOR POLYPEPTIDES AND METHODS OF USE THEREOF", Stanford University, Jun 21, 2017
  • Masatoshi Inoue. "United States Patent US20170152295 CALCIUM REPORTER GENE", Japan Science and Technology Agency, Jun 14, 2014

All Publications

  • A Flp-dependent G-CaMP9a transgenic mouse for neuronal imaging invivo. Cell reports methods Sakamoto, M., Inoue, M., Takeuchi, A., Kobari, S., Yokoyama, T., Horigane, S., Takemoto-Kimura, S., Abe, M., Sakimura, K., Kano, M., Kitamura, K., Fujii, H., Bito, H. 2022; 2 (2): 100168


    Genetically encoded calcium indicators (GECIs) are widely used to measure calcium transients in neuronal somata and processes, and their use enables the determination of action potential temporal series in a large population of neurons. Here, we generate a transgenic mouse line expressing a highly sensitive green GECI, G-CaMP9a, in a Flp-dependent manner in excitatory and inhibitory neuronal subpopulations downstream of a strong CAG promoter. Combining this reporter mouse with viral or mouse genetic Flp delivery methods produces a robust and stable G-CaMP9a expression in defined neuronal populations without detectable detrimental effects. Invivo two-photon imaging reveals spontaneous and sensory-evoked calcium transients in excitatory and inhibitory ensembles with cellular resolution. Our results show that this reporter line allows long-term, cell-type-specific investigation of neuronal activity with enhanced resolution in defined populations and facilitates dissecting complex dynamics of neural networks invivo.

    View details for DOI 10.1016/j.crmeth.2022.100168

    View details for PubMedID 35474964

  • Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine. Cell Kishi, K. E., Kim, Y. S., Fukuda, M., Inoue, M., Kusakizako, T., Wang, P. Y., Ramakrishnan, C., Byrne, E. F., Thadhani, E., Paggi, J. M., Matsui, T. E., Yamashita, K., Nagata, T., Konno, M., Quirin, S., Lo, M., Benster, T., Uemura, T., Liu, K., Shibata, M., Nomura, N., Iwata, S., Nureki, O., Dror, R. O., Inoue, K., Deisseroth, K., Kato, H. E. 1800


    ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0A resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.

    View details for DOI 10.1016/j.cell.2022.01.007

    View details for PubMedID 35114111

  • Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo. Neuroscience research Inoue, M. n. 2020


    Over the past two decades, genetically encoded calcium indicators (GECIs) have been used extensively to report intracellular calcium (Ca2+) dynamics in order to readout neuronal and network activity in living tissue. Single wavelength GECIs, such as GCaMP, have been widely adapted due to advances in dynamic range, sensitivity, and kinetics. Additionally, recent efforts in protein engineering have expanded the GECI color palette to enable direct optical interrogation of more complex circuit dynamics. Here, I discuss the engineering, application, and future directions of the most recently developed GECIs for in vivo neuroscience research.

    View details for DOI 10.1016/j.neures.2020.05.013

    View details for PubMedID 32531233

  • Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals. Neuron Fenno, L. E., Ramakrishnan, C. n., Kim, Y. S., Evans, K. E., Lo, M. n., Vesuna, S. n., Inoue, M. n., Cheung, K. Y., Yuen, E. n., Pichamoorthy, N. n., Hong, A. S., Deisseroth, K. n. 2020


    The resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g., multiple gene expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically targeted technology for writing in and reading out activity patterns for cells in living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically encoded Ca2+ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, we develop a comprehensive intersectional targeting toolbox including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply defined cell types.

    View details for DOI 10.1016/j.neuron.2020.06.003

    View details for PubMedID 32574559

  • Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics CELL Inoue, M., Takeuchi, A., Manita, S., Horigane, S., Sakamoto, M., Kawakami, R., Yamaguchi, K., Otomo, K., Yokoyama, H., Kim, R., Yokoyama, T., Takemoto-Kimura, S., Abe, M., Okamura, M., Kondo, Y., Quirin, S., Ramakrishnan, C., Imamura, T., Sakimura, K., Nemoto, T., Kano, M., Fujii, H., Deisseroth, K., Kitamura, K., Bito, H. 2019; 177 (5): 1346-+
  • Cortical layer-specific critical dynamics triggering perception. Science (New York, N.Y.) Marshel, J. H., Kim, Y. S., Machado, T. A., Quirin, S. n., Benson, B. n., Kadmon, J. n., Raja, C. n., Chibukhchyan, A. n., Ramakrishnan, C. n., Inoue, M. n., Shane, J. C., McKnight, D. J., Yoshizawa, S. n., Kato, H. E., Ganguli, S. n., Deisseroth, K. n. 2019


    Perceptual experiences may arise from neuronal activity patterns in mammalian neocortex. We probed mouse neocortex during visual discrimination using a red-shifted channelrhodopsin (ChRmine, discovered through structure-guided genome mining) alongside multiplexed multiphoton-holography (MultiSLM), achieving control of individually-specified neurons spanning large cortical volumes with millisecond precision. Stimulating a critical number of stimulus-orientation-selective neurons drove widespread recruitment of functionally-related neurons, a process enhanced by (but not requiring) orientation-discrimination task learning. Optogenetic targeting of orientation-selective ensembles elicited correct behavioral discrimination. Cortical layer specific-dynamics were apparent, as emergent neuronal activity asymmetrically propagated from layer-2/3 to layer-5, and smaller layer-5 ensembles were as effective as larger layer-2/3 ensembles in eliciting orientation discrimination behavior. Population dynamics emerging after optogenetic stimulation both correctly predicted behavior and resembled natural neural representations of visual stimuli.

    View details for DOI 10.1126/science.aaw5202

    View details for PubMedID 31320556

  • Kilohertz two-photon brain imaging in awake mice. Nature methods Zhang, T. n., Hernandez, O. n., Chrapkiewicz, R. n., Shai, A. n., Wagner, M. J., Zhang, Y. n., Wu, C. H., Li, J. Z., Inoue, M. n., Gong, Y. n., Ahanonu, B. n., Zeng, H. n., Bito, H. n., Schnitzer, M. J. 2019


    Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10-30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000-211,000 µm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.

    View details for DOI 10.1038/s41592-019-0597-2

    View details for PubMedID 31659327

  • Functional emergence of a column-like architecture in layer 5 of mouse somatosensory cortex in vivo. The journal of physiological sciences : JPS Koizumi, K., Inoue, M., Chowdhury, S., Bito, H., Yamanaka, A., Ishizuka, T., Yawo, H. 2019; 69 (1): 65-77


    To investigate how the functional architecture is organized in layer 5 (L5) of the somatosensory cortex of a mouse in vivo, the input-output relationship was investigated using an all-optical approach. The neural activity in L5 was optically recorded using a Ca2+ sensor, R-CaMP2, through a microprism inserted in the cortex under two-photon microscopy, while the L5 was regionally excited using optogenetics. The excitability was spread around the blue-light irradiated region, but the horizontal propagation was limited to within a certain distance (λ < 130 μm from the center of the illumination spot). When two regions were photostimulated with a short interval, the excitability of each cluster was reduced. Therefore, a column-like architecture had functionally emerged with reciprocal inhibition through a minimal number of synaptic relays. This could generate a synchronous output from a region of L5 with simultaneous enhancement of the signal-to-noise ratio by silencing of the neighboring regions.

    View details for DOI 10.1007/s12576-018-0618-4

    View details for PubMedID 29761270

  • Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics. Cell Inoue, M. n., Takeuchi, A. n., Manita, S. n., Horigane, S. I., Sakamoto, M. n., Kawakami, R. n., Yamaguchi, K. n., Otomo, K. n., Yokoyama, H. n., Kim, R. n., Yokoyama, T. n., Takemoto-Kimura, S. n., Abe, M. n., Okamura, M. n., Kondo, Y. n., Quirin, S. n., Ramakrishnan, C. n., Imamura, T. n., Sakimura, K. n., Nemoto, T. n., Kano, M. n., Fujii, H. n., Deisseroth, K. n., Kitamura, K. n., Bito, H. n. 2019


    To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

    View details for PubMedID 31080068

  • Calmodulin kinases: essential regulators in health and disease JOURNAL OF NEUROCHEMISTRY Takemoto-Kimura, S., Suzuki, K., Horigane, S., Kamijo, S., Inoue, M., Sakamoto, M., Fujii, H., Bito, H. 2017; 141 (6): 808-818


    Neuronal activity induces intracellular Ca2+ increase, which triggers activation of a series of Ca2+ -dependent signaling cascades. Among these, the multifunctional Ca2+ /calmodulin-dependent protein kinases (CaMKs, or calmodulin kinases) play key roles in neuronal transmission, synaptic plasticity, circuit development and cognition. The most investigated CaMKs for these roles in neuronal functions are CaMKI, CaMKII, CaMKIV and we will shed light on these neuronal CaMKs' functions in this review. Catalytically active members of CaMKs currently are CaMKI, CaMKII, CaMKIV and CaMKK. Although they all necessitate the binding of Ca2+ and calmodulin complex (Ca2+ /CaM) for releasing autoinhibition, each member of CaMK has distinct activation mechanisms-autophosphorylation mediated autonomy of multimeric CaMKII and CaMKK-dependent phosphoswitch-induced activation of CaMKI or CaMKIV. Furthermore, each CaMK shows distinct subcellular localization that underlies specific compartmentalized function in each activated neuron. In this review, we first summarize these molecular characteristics of each CaMK as to regulation and subcellular localization, and then describe each biological function. In the last section, we also focus on the emerging role of CaMKs in pathophysiological conditions by introducing the recent studies, especially focusing on drug addiction and depression, and discuss how dysfunctional CaMKs may contribute to the pathology of the neuropsychological disorders. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

    View details for DOI 10.1111/jnc.14020

    View details for Web of Science ID 000403898100003

    View details for PubMedID 28295333

  • Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice. Science translational medicine Selimbeyoglu, A. n., Kim, C. K., Inoue, M. n., Lee, S. Y., Hong, A. S., Kauvar, I. n., Ramakrishnan, C. n., Fenno, L. E., Davidson, T. J., Wright, M. n., Deisseroth, K. n. 2017; 9 (401)


    Alterations in the balance between neuronal excitation and inhibition (E:I balance) have been implicated in the neural circuit activity-based processes that contribute to autism phenotypes. We investigated whether acutely reducing E:I balance in mouse brain could correct deficits in social behavior. We used mice lacking the CNTNAP2 gene, which has been implicated in autism, and achieved a temporally precise reduction in E:I balance in the medial prefrontal cortex (mPFC) either by optogenetically increasing the excitability of inhibitory parvalbumin (PV) neurons or decreasing the excitability of excitatory pyramidal neurons. Surprisingly, both of these distinct, real-time, and reversible optogenetic modulations acutely rescued deficits in social behavior and hyperactivity in adult mice lacking CNTNAP2 Using fiber photometry, we discovered that native mPFC PV neuronal activity differed between CNTNAP2 knockout and wild-type mice. During social interactions with other mice, PV neuron activity increased in wild-type mice compared to interactions with a novel object, whereas this difference was not observed in CNTNAP2 knockout mice. Together, these results suggest that real-time modulation of E:I balance in the mouse prefrontal cortex can rescue social behavior deficits reminiscent of autism phenotypes.

    View details for PubMedID 28768803

  • Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain. Nature methods Kim, C. K., Yang, S. J., Pichamoorthy, N., Young, N. P., Kauvar, I., Jennings, J. H., Lerner, T. N., Berndt, A., Lee, S. Y., Ramakrishnan, C., Davidson, T. J., Inoue, M., Bito, H., Deisseroth, K. 2016; 13 (4): 325-328


    Real-time activity measurements from multiple specific cell populations and projections are likely to be important for understanding the brain as a dynamical system. Here we developed frame-projected independent-fiber photometry (FIP), which we used to record fluorescence activity signals from many brain regions simultaneously in freely behaving mice. We explored the versatility of the FIP microscope by quantifying real-time activity relationships among many brain regions during social behavior, simultaneously recording activity along multiple axonal pathways during sensory experience, performing simultaneous two-color activity recording, and applying optical perturbation tuned to elicit dynamics that match naturally occurring patterns observed during behavior.

    View details for DOI 10.1038/nmeth.3770

    View details for PubMedID 26878381

  • Rational design of a high-affinity, fast, red calcium indicator R-CaMP2 NATURE METHODS Inoue, M., Takeuchi, A., Horigane, S., Ohkura, M., Gengyo-Ando, K., Fujii, H., Kamijo, S., Takemoto-Kimura, S., Kano, M., Nakai, J., Kitamura, K., Bito, H. 2015; 12 (1): 64-70


    Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca(2+) transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20-40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.

    View details for DOI 10.1038/NMETH.3185

    View details for Web of Science ID 000347668600021

    View details for PubMedID 25419959

  • Neuroprotective Role of the Basic Leucine Zipper Transcription Factor NFIL3 in Models of Amyotrophic Lateral Sclerosis JOURNAL OF BIOLOGICAL CHEMISTRY Tamai, S., Imaizumi, K., Kurabayashi, N., Minh Dang Nguyen, Abe, T., Inoue, M., Fukada, Y., Sanada, K. 2014; 289 (3): 1629-1638


    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons. Here we show that the basic leucine zipper transcription factor NFIL3 (also called E4BP4) confers neuroprotection in models of ALS. NFIL3 is up-regulated in primary neurons challenged with neurotoxic insults and in a mouse model of ALS. Overexpression of NFIL3 attenuates excitotoxic neuronal damage and protects neurons against neurodegeneration in a cell-based ALS model. Conversely, reduction of NFIL3 exacerbates neuronal demise in adverse conditions. Transgenic neuronal expression of NFIL3 in ALS mice delays disease onset and attenuates motor axon and neuron degeneration. These results suggest that NFIL3 plays a neuroprotective role in neurons and constitutes a potential therapeutic target for neurodegeneration.

    View details for DOI 10.1074/jbc.M113.524389

    View details for Web of Science ID 000332401700036

    View details for PubMedID 24280221

    View details for PubMedCentralID PMC3894342

  • Nonlinear Decoding and Asymmetric Representation of Neuronal Input Information by CaMKII alpha and Calcineurin CELL REPORTS Fujii, H., Inoue, M., Okuno, H., Sano, Y., Takemoto-Kimura, S., Kitamura, K., Kano, M., Bito, H. 2013; 3 (4): 978-987


    How information encoded in glutamate release rates at individual synapses is converted into biochemical activation patterns of postsynaptic enzymes remains unexplored. To address this, we developed a dual fluorescence resonance energy transfer (FRET) imaging platform and recorded CaMKIIα and calcineurin activities in hippocampal neurons while varying glutamate uncaging frequencies. With little spine morphological change, 5 Hz spine glutamate uncaging strongly stimulated calcineurin, but not CaMKIIα. In contrast, 20 Hz spine glutamate uncaging, which induced spine growth, activated both CaMKIIα and calcineurin with distinct spatiotemporal kinetics. Higher temporal resolution recording in the soma revealed that CaMKIIα activity summed supralinearly and sensed both higher frequency and input number, thus acting as an input frequency/number decoder. In contrast, calcineurin activity summated sublinearly with increasing input number and showed little frequency dependence, thus functioning as an input number counter. These results provide evidence that CaMKIIα and calcineurin are fine-tuned to unique bandwidths and compute input variables in an asymmetric manner.

    View details for DOI 10.1016/j.celrep.2013.03.033

    View details for Web of Science ID 000321897100002

    View details for PubMedID 23602566

  • Synaptic activity-responsive element (SARE): A unique genomic structure with an unusual sensitivity to neuronal activity. Communicative & integrative biology Inoue, M., Yagishita-Kyo, N., Nonaka, M., Kawashima, T., Okuno, H., Bito, H. 2010; 3 (5): 443-6


    Formation of a new memory requires plasticity at the synaptic level. However, it has also been shown that the consolidation and the maintenance of such a new memory involve processes that necessitate active mRNA at the nucleus of the cell. How can robust changes in synaptic efficacy specifically drive new transcription and translation of new gene transcripts, and thus transform an otherwise transient plasticity into a long-lasting and stable one? In this article, we highlight the conceptual advance that was gained by the discovery of a potent Synaptic Activity-Responsive Element (SARE) found ∼7 kb upstream of the transcription initiation site of the neuronal immediate early gene Arc. The unique genomic structure of SARE, which contained adjacent and cooperative binding sites for three major activity-dependent transcription factors within a 100-bp locus, was associated with an unusual responsiveness to neuronal stimuli. Taken together, these findings shed light on a new class of transcriptional sensor with enhanced sensitivity to synaptic activity.

    View details for DOI 10.4161/cib.3.5.12287

    View details for PubMedID 21057636

    View details for PubMedCentralID PMC2974076