scholarly journals A genetically encoded fluorescent sensor for in vivo imaging of GABA

2018 ◽  
Author(s):  
Jonathan S. Marvin ◽  
Yoshiteru Shimoda ◽  
Vincent Malgoire ◽  
Marco Leite ◽  
Takashi Kawashima ◽  
...  
Keyword(s):  
Neural Circuits ◽  
Fluorescent Sensor ◽  
Gaba Release ◽  
Library Screening ◽  
Spatiotemporal Resolution ◽  
Fluorescent Reporter ◽  
Inhibitory Neurotransmitter ◽  
Single Action ◽  
Gaba Content

AbstractCurrent techniques for monitoring GABA, the primary inhibitory neurotransmitter in vertebrates, cannot follow ephemeral transients in intact neural circuits. We applied the design principles used to create iGluSnFR, a fluorescent reporter of synaptic glutamate, to develop a GABA sensor using a protein derived from a previously unsequenced Pseudomonas fluorescens strain. Structure-guided mutagenesis and library screening led to a usable iGABASnFR (ΔF/Fmax ~ 2.5, Kd ~ 9 μM, good specificity, adequate kinetics). iGABASnFR is genetically encoded, detects single action potential-evoked GABA release events in culture, and produces readily detectable fluorescence increases in vivo in mice and zebrafish. iGABASnFR enabled tracking of: (1) mitochondrial GABA content and its modulation by an anticonvulsant; (2) swimming-evoked GABAergic transmission in zebrafish cerebellum; (3) GABA release events during inter-ictal spikes and seizures in awake mice; and (4) GABAergic tone decreases during isoflurane anesthesia. iGABASnFR will permit high spatiotemporal resolution of GABA signaling in intact preparations.

scholarly journals Dual-color volumetric imaging of neural activity of cortical columns

2018 ◽  
Author(s):  
Shuting Han ◽  
Weijian Yang ◽  
Rafael Yuste
Keyword(s):  
Neural Activity ◽  
High Speed ◽  
Neural Circuits ◽  
Emergent Properties ◽  
Spatiotemporal Resolution ◽  
Population Activity ◽  
Volumetric Imaging ◽  
Two Photon ◽  
Dual Color

To capture the emergent properties of neural circuits, high-speed volumetric imaging of neural activity at cellular resolution is desirable. But while conventional two-photon calcium imaging is a powerful tool to study population activity in vivo, it is restrained to two-dimensional planes. Expanding it to 3D while maintaining high spatiotemporal resolution appears necessary. Here, we developed a two-photon microscope with dual-color laser excitation that can image neural activity in a 3D volume. We imaged the neuronal activity of primary visual cortex from awake mice, spanning from L2 to L5 with 10 planes, at a rate of 10 vol/sec, and demonstrated volumetric imaging of L1 long-range PFC projections and L2/3 somatas. Using this method, we map visually-evoked neuronal ensembles in 3D, finding a lack of columnar structure in orientation responses and revealing functional correlations between cortical layers which differ from trial to trial and are missed in sequential imaging. We also reveal functional interactions between presynaptic L1 axons and postsynaptic L2/3 neurons. Volumetric two-photon imaging appears an ideal method for functional connectomics of neural circuits.

scholarly journals Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Nami Kitajima ◽  
Kenji Takikawa ◽  
Hiroshi Sekiya ◽  
Kaname Satoh ◽  
Daisuke Asanuma ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Fluorescent Sensor ◽  
Atp Release ◽  
Extracellular Atp ◽  
Cysteine Residue ◽  
Spatiotemporal Resolution ◽  
Extracellular Signaling ◽  
Ratiometric Measurement ◽  
Atp Binding Protein

Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

scholarly journals Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo

2017 ◽  
Author(s):  
Yusuke Hirabayashi ◽  
Juan Carlos Tapia ◽  
Franck Polleux
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Large Scale ◽  
Neural Circuits ◽  
Structural Connectivity ◽  
Synaptic Connections ◽  
General Applicability ◽  
Fluorescent Reporter ◽  
Scanning Electron

A challenging aspect of neuroscience revolves around mapping the synaptic connections within neural circuits (connectomics) over scales spanning several orders of magnitude (nanometers to meters). Despite significant improvements in serial section electron microscopy (SSEM) technologies, several major roadblocks have impaired its general applicability to mammalian neural circuits. In the present study, we introduce a new approach that circumvents these roadblocks by adapting a genetically-encoded ascorbate peroxidase (APEX2) as a fusion protein to a membrane-targeted fluorescent reporter (CAAX-Venus), and introduce it in single pyramidal neurons in vivo using extremely sparse in utero cortical electroporation (IUCE). This approach allows to perform Correlated Light-SSEM (CoLSSEM) on individual neurons, reconstructing their dendritic and axonal arborization in a targeted way via combination of high-resolution confocal microscopy, and subsequently imaging of its ultrastuctural features and synaptic connections with the ATUM-SEM (automated tape-collecting ultramicrotome - scanning electron microscopy) technology. Our method significantly improves the the feasibility of large-scale reconstructions of neurons within a circuit, and bridges the description of ultrastructural features of genetically-identified neurons with their functional and/or structural connectivity, one of the main goal of connectomics.

scholarly journals A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

2020 ◽  
Author(s):  
Ao Dong ◽  
Kaikai He ◽  
Barna Dudok ◽  
Jordan S Farrell ◽  
Wuqiang Guan ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Basolateral Amygdala ◽  
Fluorescent Sensor ◽  
Brain Slices ◽  
High Specificity ◽  
Cultured Neurons ◽  
Spatiotemporal Resolution ◽  
Wide Range

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

scholarly journals In vivo optogenetic stimulation of the primate retina activates the visual cortex after long-term transduction

2021 ◽  
Author(s):  
Antoine Chaffiol ◽  
Matthieu Provansal ◽  
Corentin Joffrois ◽  
Kévin Blaize ◽  
Guillaume Labernede ◽  
...  
Keyword(s):  
Visual Cortex ◽  
Information Transfer ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Spatiotemporal Resolution ◽  
Fluorescent Reporter ◽  
Optogenetic Stimulation ◽  
Stimulation Of

SummaryVarious therapeutic strategies for vision restoration have been developed, including retinal prostheses [1–4], stem cell transplantation [5–8] and optogenetic therapies [9,10,19,11–18]. In optogenetic therapy, the residual retinal neurons surviving the pathological degenerative process are rendered light-sensitive. Using this approach, we targeted the retinal ganglion cells (RGCs) through the in vivo expression of an ectopic light-sensitive ion channel, ChrimsonR [13] coupled to the fluorescent reporter tdTomato. The application of this strategy to blind patients [20] suffering from retinal dystrophies raises important concerns about the long-term functional expression of efficient signal transmission to higher brain centers (i.e. the visual cortex). We have previously shown that the transduced retina displays high spatiotemporal resolution ex vivo, compatible with the perception of highly dynamic visual scenes at light levels suitable for use in humans. Other studies have provided evidence of retinal activation in vivo [17]. Here, we demonstrate, in non-human primates, sustained functional efficacy ~20 months after delivery of an AAV2.7m8-ChrimsonR-tdTomato vector similar to that currently undergoing clinical evaluation. Our results reveal a persistence of expression in the perifovea, mediating information transfer to higher brain centers. Indeed, we recorded visually evoked potentials in the primary visual cortex of anesthetized animals in response to optogenetic retinal activation. We used an intravitreal injection of synaptic blockers to isolate the cortical component resulting from the in vivo optogenetic stimulation of primate RGCs. Our findings demonstrate the long-term functional efficacy of optogenetic retinal information transfer to the brain in vivo.

scholarly journals High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiang Lan Fan ◽  
Jose A. Rivera ◽  
Wei Sun ◽  
John Peterson ◽  
Henry Haeberle ◽  
...  
Keyword(s):  
Blood Flow ◽  
High Speed ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Fluorescence Signal ◽  
Imaging Method ◽  
Spatiotemporal Resolution ◽  
Two Photon

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

scholarly journals CRISPR/Cas9 mediated dual fluorescent reporter mice to study spatial bone genomics of osteoblasts and osteoclasts in the local in vivo environment

Bone Reports ◽  
2021 ◽  
Vol 14 ◽  
pp. 100871
Author(s):  
Dilara Yilmaz ◽  
Yannick Fischer ◽  
Sandra Zimmermann ◽  
Gaonhae Hwang ◽  
Ralph Müller ◽  
...  
Keyword(s):  
Fluorescent Reporter ◽  
Reporter Mice

scholarly journals Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

2021 ◽  
Vol 7 (8) ◽  
pp. eabd6167
Author(s):  
Capucine L. Grandjean ◽  
Zacarias Garcia ◽  
Fabrice Lemaître ◽  
Béatrice Bréart ◽  
Philippe Bousso
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Critical Path ◽  
B Cell Lymphoma ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Fluorescent Reporter ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Partial Depletion

Anti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies.

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