Main Menu (Mobile)- Block

Main Menu - Block

janelia7_blocks-janelia7_fake_breadcrumb | block
Koyama Lab / Publications
custom | custom

Filter

facetapi-Q2b17qCsTdECvJIqZJgYMaGsr8vANl1n | block
facetapi-W9JlIB1X0bjs93n1Alu3wHJQTTgDCBGe | block
facetapi-PV5lg7xuz68EAY8eakJzrcmwtdGEnxR0 | block
facetapi-021SKYQnqXW6ODq5W5dPAFEDBaEJubhN | block
general_search_page-panel_pane_1 | views_panes

46 Publications

Showing 1-10 of 46 results
Your Criteria:
    06/15/25 | RubyACRs Enable Red-Shifted Optogenetic Inhibition in Freely Behaving Drosophila
    Bushey D, Shiozaki H, Shuai Y, Zheng J, Jayaraman V, Hasseman JP, Kolb I, GENIE Project Team , Turner GC
    bioRxiv. 2025 jun 15:. doi: 10.1101/2025.06.13.659144

    Optogenetic activators with red-shifted excitation spectra, such as Chrimson, have significantly advanced Drosophila neuroscience. However, until recently, available optogenetic inhibitors required shorter activation wavelengths, which don’t penetrate tissue as effectively and are stronger visual stimuli to the animal, potentially confounding behavioral results. Here, we assess the efficacy of two newly identified anion-conducting channelrhodopsins with spectral sensitivities similar to Chrimson: A1ACR and HfACR (RubyACRs). Electrophysiology and functional imaging confirmed that RubyACRs effectively hyperpolarize neurons, with stronger and faster effects than the widely used inhibitor GtACR1. Activation of RubyACRs led to circuit-specific behavioral changes in three different neuronal groups. In glutamatergic motor neurons, activating RubyACRs suppressed adult locomotor activity. In PPL1-γ1pedc dopaminergic neurons, pairing odors with RubyACR activation during learning produced odor responses consistent with synaptic silencing. Finally, activation of RubyACRs in the pIP10 neuron suppressed pulse song during courtship. Together, these results demonstrate that RubyACRs are effective and reliable tools for neuronal inhibition in Drosophila, expanding the optogenetic toolkit for circuit dissection in freely behaving animals.

     

     

    Preprint: https://www.biorxiv.org/content/early/2025/06/15/2025.06.13.659144

    View Publication Page
    04/08/25 | Glutamate indicators with increased sensitivity and tailored deactivation rates
    Podgorski K, Aggarwal A, Negrean A, Chen Y, Iyer R, Reep D, Liu A, Palutla A, Xie M, Maclennan B, Hagihara K, Kinsey L, Sun J, Yao P, Zheng J, Tsang A, Tsegaye G, Zhang Y, Patel R, Hasseman J
    Research Square. 2025 Apr 8:. doi: 10.21203/rs.3.rs-6257403/v1

    Identifying the input-output operations of neurons requires measurements of synaptic transmission simultaneously at many of a neuron’s thousands of inputs in the intact brain. To facilitate this goal, we engineered and screened 3365 variants of the fluorescent protein glutamate indicator iGluSnFR3 in neuron culture, and selected variants in the mouse visual cortex. Two variants have high sensitivity, fast activation (< 2 ms) and deactivation times tailored for recording large populations of synapses (iGluSnFR4s, 153 ms) or rapid dynamics (iGluSnFR4f, 26 ms). By imaging action-potential evoked signals on axons and visually-evoked signals on dendritic spines, we show that iGluSnFR4s/4f primarily detect local synaptic glutamate with single-vesicle sensitivity. The indicators detect a wide range of naturalistic synaptic transmission, including in the vibrissal cortex layer 4 and in hippocampal CA1 dendrites. iGluSnFR4 increases the sensitivity and scale (4s) or speed (4f) of tracking information flow in neural networks in vivo.

    View Publication Page
    04/07/25 | Far-red fluorescent genetically encoded calcium ion indicators.
    Dalangin R, Jia BZ, Qi Y, Aggarwal A, Sakoi K, Drobizhev M, Molina RS, Patel R, Abdelfattah AS, Zheng J, Reep D, Hasseman JP, GENIE Project Team , Zhao Y, Wu J, Podgorski K, Tebo AG, Schreiter ER, Hughes TE, Terai T, Paquet M, Megason SG, Cohen AE, Shen Y, Campbell RE
    Nat Commun. 2025 Apr 07;16(1):3318. doi: 10.1038/s41467-025-58485-z

    Genetically encoded calcium ion (Ca) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm, respectively, display large responses to Ca in vitro (ΔF/F = 6 for FR-GECO1a, 18 for FR-GECO1c), are bright under both one-photon and two-photon illumination, and have high affinities (apparent K = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca. FR-GECOs offer sensitive and fast detection of single action potentials in neurons, and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators.

    Preprint: https://doi.org/10.1101/2020.11.12.380089

    View Publication Page
    03/27/25 | iGABASnFR2: Improved genetically encoded protein sensors of GABA
    Kolb I, Hasseman JP, Matsumoto A, Arthur BJ, Zhang Y, Tsang A, Reep D, Tsegaye G, Zheng J, Patel R, Looger LL, Marvin JS, Korff WL, Yonehara K, Turner GC
    bioRxiv. 2025 Mar 25:. doi: 10.1101/2025.03.25.644953

    Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis, and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.2-fold improved sensitivity and 20% faster kinetics, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance, enabling us to make the first measurements of direction selective GABA release in the retina and confirm a longstanding hypothesis for how sensitivity to motion arises in the visual system.

    View Publication Page
    03/20/25 | Glutamate indicators with increased sensitivity and tailored deactivation rates
    Aggarwal A, Negrean A, Chen Y, Iyer R, Reep D, Liu A, Palutla A, Xie ME, MacLennan BJ, Hagihara KM, Kinsey LW, Sun JL, Yao P, Zheng J, Tsang A, Tsegaye G, Zhang Y, Patel RH, Arthur BJ, Hiblot J, Leippe P, Tarnawski M, Marvin JS, Vevea JD, Turaga SC, Tebo AG, Carandini M, Rossi LF, Kleinfeld D, Konnerth A, Svoboda K, Turner GC, Hasseman J, Podgorski K
    bioRxiv. 2025 Mar 20:. doi: 10.1101/2025.03.20.643984

    Identifying the input-output operations of neurons requires measurements of synaptic transmission simultaneously at many of a neuron’s thousands of inputs in the intact brain. To facilitate this goal, we engineered and screened 3365 variants of the fluorescent protein glutamate indicator iGluSnFR3 in neuron culture, and selected variants in the mouse visual cortex. Two variants have high sensitivity, fast activation (< 2 ms) and deactivation times tailored for recording large populations of synapses (iGluSnFR4s, 153 ms) or rapid dynamics (iGluSnFR4f, 26 ms). By imaging action-potential evoked signals on axons and visually-evoked signals on dendritic spines, we show that iGluSnFR4s/4f primarily detect local synaptic glutamate with single-vesicle sensitivity. The indicators detect a wide range of naturalistic synaptic transmission, including in the vibrissal cortex layer 4 and in hippocampal CA1 dendrites. iGluSnFR4 increases the sensitivity and scale (4s) or speed (4f) of tracking information flow in neural networks in vivo.

    View Publication Page
    11/18/24 | Patch-walking: Coordinated multi-pipette patch clamp for efficiently finding synaptic connections
    Mighten C. Yip , Mercedes M. Gonzalez , Colby F. Lewallen , Corey R. Landry , Ilya Kolb , Bo Yang , William M. Stoy , Ming-fai Fong , Matthew JM Rowan , Edward S. Boyden , Craig R. Forest
    eLife. 2024 Nov 18;13:RP97399. doi: 10.7554/elife.97399

    Significant technical challenges exist when measuring synaptic connections between neurons in living brain tissue. The patch clamping technique, when used to probe for synaptic connections, is manually laborious and time-consuming. To improve its efficiency, we pursued another approach: instead of retracting all patch clamping electrodes after each recording attempt, we cleaned just one of them and reused it to obtain another recording while maintaining the others. With one new patch clamp recording attempt, many new connections can be probed. By placing one pipette in front of the others in this way, one can 'walk' across the mouse brain slice, termed 'patch-walking.' We performed 136 patch clamp attempts for two pipettes, achieving 71 successful whole cell recordings (52.2%). Of these, we probed 29 pairs (i.e. 58 bidirectional probed connections) averaging 91 μm intersomatic distance, finding three connections. Patch-walking yields 80-92% more probed connections, for experiments with 10-100 cells than the traditional synaptic connection searching method.

    View Publication Page
    11/15/24 | A novel rhodopsin-based voltage indicator for simultaneous two-photon optical recording with GCaMP in vivo
    Villette V, Yang S, Valenti R, Macklin JJ, Bradley J, Mathieu B, Lombardini A, Podgorski K, Dieudonné S, Schreiter ER, Abdelfattah AS
    bioRxiv. 2024 Nov 15:. doi: 10.1101/2024.11.15.623698

    Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells in vivo. However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed in vivo with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae Klebsormidium nitens. 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.

    View Publication Page
    03/22/24 | Visualization of Glutamatergic Neurotransmission in Diverse Model Organisms with Genetically Encoded Indicators
    Aggarwal A, Chan J, Waring AK, Negrean A, Marvin JS, Podgorski K, Looger LL, Kukley M
    New Technologies for Glutamate Interaction: Neurons and Glia;2780:3–34. doi: 10.1007/978-1-0716-3742-5_1

    Glutamate is the principal excitatory neurotransmitter, and occasionally subserves inhibitory roles, in the vertebrate nervous system. Glutamatergic synapses are dense in the vertebrate brain, at \textasciitilde1/μm3. Glutamate is released from and onto diverse components of the nervous system, including neurons, glia, and other cells. Methods for glutamate detection are critically important for understanding the function of synapses and neural circuits in normal physiology, development, and disease. Here we describe the development, optimization, and deployment of genetically encoded fluorescent glutamate indicators. We review the theoretical considerations governing glutamate sensor properties from first principles of synapse biology, microscopy, and protein structure-function relationships. We provide case studies of the state-of-the-art iGluSnFR glutamate sensor, encompassing design and optimization, mechanism of action, in vivo imaging, data analysis, and future directions. We include detailed protocols for iGluSnFR imaging in common preparations (bacteria, cell culture, and brain slices) and model organisms (worm, fly, fish, rodent).

    View Publication Page
    02/23/24 | Recording physiological history of cells with chemical labeling.
    Huppertz M, Wilhelm J, Grenier V, Schneider MW, Falt T, Porzberg N, Hausmann D, Hoffmann DC, Hai L, Tarnawski M, Pino G, Slanchev K, Kolb I, Acuna C, Fenk LM, Baier H, Hiblot J, Johnsson K
    Science. 2024 Feb 23;383(6685):890-897. doi: 10.1126/science.adg0812

    Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

    View Publication Page
    06/01/23 | Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.
    Aggarwal A, Liu R, Chen Y, Ralowicz AJ, Bergerson SJ, Tomaska F, Mohar B, Hanson TL, Hasseman JP, Reep D, Tsegaye G, Yao P, Ji X, Kloos M, Walpita D, Patel R, Mohr MA, Tillberg PW, GENIE Project Team , Looger LL, Marvin JS, Hoppa MB, Konnerth A, Kleinfeld D, Schreiter ER, Podgorski K
    Nature Methods. 2023 Jun 01;20(6):. doi: 10.1038/s41592-023-01863-6

    The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

    View Publication Page