New and improved GRAB fluorescent sensors for monitoring dopaminergic activity in vivo

The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed the first red fluorescent GPCR-activation–based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors following our previous studies. In response to extracellular DA, both the red and green GRABDA sensors have a large increase in fluorescence (ΔF/F0 values of 150% and 340%, respectively), with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, both the red and green GRABDA sensors readily resolve evoked DA release in mouse brain slices, detect compartmental DA release in live flies with single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Importantly, co-expressing red GRABDA with either green GRABDA or the calcium indicator GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo.

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A genetically-encoded fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice

10.1101/332528 ◽

2018 ◽

Author(s):

Fangmiao Sun ◽

Jianzhi Zeng ◽

Miao Jing ◽

Jingheng Zhou ◽

Jiesi Feng ◽

...

Keyword(s):

Sexual Behaviors ◽

Fluorescent Sensor ◽

Brain Slices ◽

Electrical Stimulus ◽

Model Organisms ◽

Gpcr Activation ◽

Molecular Specificity ◽

Central Monoamine ◽

Freely Behaving ◽

Da Release

AbstractDopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here we report the development of novel genetically-encoded GPCR-Activation-Based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA rises, GRABDA sensors exhibit large fluorescence increases (ΔF/F0∼90%) with sub-second kinetics, nanomolar to sub-micromolar affinities, and excellent molecular specificity. Importantly, GRABDA sensors can resolve a single-electrical-stimulus evoked DA release in mouse brain slices, and detect endogenous DA release in the intact brains of flies, fish, and mice. In freely-behaving mice, GRABDA sensors readily report optogenetically-elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA changes during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporal precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

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GPCR-Based Dopamine Sensors—A Detailed Guide to Inform Sensor Choice for In Vivo Imaging

International Journal of Molecular Sciences ◽

10.3390/ijms21218048 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8048

Author(s):

Marie A. Labouesse ◽

Reto B. Cola ◽

Tommaso Patriarchi

Keyword(s):

In Vivo Imaging ◽

Dynamic Range ◽

New Techniques ◽

Experimental Parameters ◽

Sensor Properties ◽

Molecular Specificity ◽

Freely Behaving ◽

The Brain ◽

Da Release

Understanding how dopamine (DA) encodes behavior depends on technologies that can reliably monitor DA release in freely-behaving animals. Recently, red and green genetically encoded sensors for DA (dLight, GRAB-DA) were developed and now provide the ability to track release dynamics at a subsecond resolution, with submicromolar affinity and high molecular specificity. Combined with rapid developments in in vivo imaging, these sensors have the potential to transform the field of DA sensing and DA-based drug discovery. When implementing these tools in the laboratory, it is important to consider there is not a ‘one-size-fits-all’ sensor. Sensor properties, most importantly their affinity and dynamic range, must be carefully chosen to match local DA levels. Molecular specificity, sensor kinetics, spectral properties, brightness, sensor scaffold and pharmacology can further influence sensor choice depending on the experimental question. In this review, we use DA as an example; we briefly summarize old and new techniques to monitor DA release, including DA biosensors. We then outline a map of DA heterogeneity across the brain and provide a guide for optimal sensor choice and implementation based on local DA levels and other experimental parameters. Altogether this review should act as a tool to guide DA sensor choice for end-users.

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Downregulation of CDK5 Signaling In the Dorsal Striatum Alters Striatal Microcircuits and Perturbs Circadian Behavior in Mice

10.21203/rs.3.rs-1183888/v1 ◽

2021 ◽

Author(s):

Hu Zhou ◽

Jingxin Zhang ◽

Huaxiang Shi ◽

Pengfei Li ◽

Xin Sui ◽

...

Keyword(s):

Circadian Rhythms ◽

Basolateral Amygdala ◽

Fluorescent Protein ◽

Critical Role ◽

Motor Impairment ◽

Dorsal Striatum ◽

Thalamic Nuclei ◽

Whole Cell Patch Clamp ◽

Green Fluorescent

Abstract Dysfunction of striatal dopaminergic circuits has been implicated in motor impairment as well as in Parkinson’s disease (PD)-related circadian perturbations that may represent an early prodromal marker of PD. Cyclin-dependent kinase 5 (CDK5) acts negatively on dopamine (DA) signaling in the striatum, suggesting a critical role in circadian and sleep disorders. Here, we used CRISPR/Cas9 gene editing to produce dorsal striatum (DS)-specific knockdown (KD) of the Cdk5 gene in mice (referred to as DS-CDK5-KD mice) to investigate its role in vivo. DS-CDK5-KD mice exhibited deficits in locomotor activity and disturbances in daily rest/activity cycles. Additionally, Golgi staining of neurons in the DS revealed that Cdk5 deletion caused a reduction in dendrite length and functional synapses, which was confirmed by significant downregulation of MAP2, PSD95 and synapsin I. Correlated with this, DS-CDK5-KD mice displayed reduced phosphorylation of Tau at Thr181. Furthermore, whole-cell patch-clamp recordings of green fluorescent protein (GFP)-tagged neurons in the striatum of DS-CDK5-KD mice revealed a decrease in the frequency of spontaneous inhibitory post-synaptic currents and an alteration of the excitatory/inhibitory synaptic balance. Notably, anterograde labeling showed that CDK5 knockdown in the DS disrupted long-range projections to the secondary motor cortex, dorsal and ventral thalamic nuclei, and the basolateral amygdala, which are involved in the regulation of motor and circadian rhythms in the brain. These findings support a critical role of CDK5 in the DS in maintaining the striatal neural circuitry underlying motor and circadian rhythms related to PD.

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Abstract 160: Contribution of IP3 Receptors in the Electromechanical Response of LV Myocytes to Gq-protein Coupled Receptor Agonists

Circulation Research ◽

10.1161/res.113.suppl_1.a160 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Sergio Signore ◽

Andrea Sorrentino ◽

Antonio Cannata ◽

Chiara Mangiaracina ◽

Mark Sundman ◽

...

Keyword(s):

Critical Role ◽

Resting Potential ◽

Ip3 Receptors ◽

Ip3 Receptor ◽

Electrical Stability ◽

Functional Responses ◽

Gpcr Activation ◽

Rna Targeting

Gq-protein coupled receptor (GPCR) stimulation promotes PLC function, generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). The latter may promote Ca2+ translocation from intracellular stores altering Ca2+ homeostasis in cardiomyocytes. The aim of this study was to establish whether GPCR agonists enhance IP3 receptor (IP3R) activity, affecting the electromechanical properties of LV myocytes. For this purpose, the functional responses of myocytes to GPCR agonists ATP or ET-1 were established. In field-stimulated cells, GPCR activators increased diastolic Ca2+, transient amplitude, and contractility; extra-systolic Ca2+ releases and aftercontractions were promoted. These responses were prevented by inhibition of PLC, or blockade of IP3Rs. Since DAG promotes PKC activity, the effects of GPCR stimulation were tested in the presence of the PKC inhibitor chelerythrine. This compound failed to abrogate the effects of ATP and ET-1, indicating that PKC-independent pathways play a critical role in mediating the observed cellular responses to GPCR stimulation. Additionally, an AAV9 vector carrying EGFP and sh-RNA targeting IP3R type-2 was employed in vivo to downregulate IP3Rs. GPCRs activation failed to increase Ca2+ transients and to induce extra-systolic Ca2+ elevations in EGFP-positive myocytes. Conversely, these responses were preserved in EGFP-negative cells. In patch-clamped myocytes, changes in Ca2+ transient properties following GPCR activation were accompanied by a decrease in resting potential, action potential (AP) prolongation, and emergence of arrhythmic events. Similar electrical disturbances were detected by direct activation of IP3R with IP3 dialysis, or by enhancing the affinity of the receptors to its ligand, with thimerosal. To establish whether Ca2+ mobilized from the sarcoplasmic reticulum (SR) to the cytoplasm via IP3Rs was responsible for the electrical alterations caused by GPCR agonists, experiments were performed in which SR Ca2+ was depleted, or cytosolic Ca2+ was buffered. Under these conditions, ATP and ET-1 failed to prolong the AP and to induce arrhythmias. In conclusion, the GPCR/IP3R axis regulates Ca2+ homeostasis, contractile performance and the electrical stability of LV myocytes.

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In Vivo Study of Trichoderma-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.3073-3081.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 3073-3081 ◽

Cited By ~ 97

Author(s):

Zexun Lu ◽

Riccardo Tombolini ◽

Sheridan Woo ◽

Susanne Zeilinger ◽

Matteo Lorito ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Plant Pathogens ◽

Fluorescent Protein ◽

Morphological Changes ◽

Critical Role ◽

Pythium Ultimum ◽

Confocal Scanning Laser Microscopy ◽

Green Fluorescent

ABSTRACT Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.

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Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein

International Journal of Molecular Sciences ◽

10.3390/ijms21051644 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1644 ◽

Cited By ~ 7

Author(s):

Oksana M. Subach ◽

Vladimir P. Sotskov ◽

Viktor V. Plusnin ◽

Anna M. Gruzdeva ◽

Natalia V. Barykina ◽

...

Keyword(s):

Calcium Ions ◽

Fluorescent Protein ◽

Fast Response ◽

Two Photon ◽

Calcium Indicator ◽

Peptide Pair ◽

Calcium Indicators ◽

Green Fluorescent

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.

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Three dimensional microelectrodes enable high signal and spatial resolution for neural seizure recordings in brain slices and freely behaving animals

Scientific Reports ◽

10.1038/s41598-021-01528-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

P. Wijdenes ◽

K. Haider ◽

C. Gavrilovici ◽

B. Gunning ◽

M. D. Wolff ◽

...

Keyword(s):

Spatial Resolution ◽

Signal To Noise Ratio ◽

Brain Activity ◽

Three Dimensional ◽

Brain Slices ◽

High Signal ◽

Diagnostic Potential ◽

Freely Behaving

AbstractNeural recordings made to date through various approaches—both in-vitro or in-vivo—lack high spatial resolution and a high signal-to-noise ratio (SNR) required for detailed understanding of brain function, synaptic plasticity, and dysfunction. These shortcomings in turn deter the ability to further design diagnostic, therapeutic strategies and the fabrication of neuro-modulatory devices with various feedback loop systems. We report here on the simulation and fabrication of fully configurable neural micro-electrodes that can be used for both in vitro and in vivo applications, with three-dimensional semi-insulated structures patterned onto custom, fine-pitch, high density arrays. These microelectrodes were interfaced with isolated brain slices as well as implanted in brains of freely behaving rats to demonstrate their ability to maintain a high SNR. Moreover, the electrodes enabled the detection of epileptiform events and high frequency oscillations in an epilepsy model thus offering a diagnostic potential for neurological disorders such as epilepsy. These microelectrodes provide unique opportunities to study brain activity under normal and various pathological conditions, both in-vivo and in in-vitro, thus furthering the ability to develop drug screening and neuromodulation systems that could accurately record and map the activity of large neural networks over an extended time period.

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A compact head-mounted endoscope for in vivo calcium imaging in freely-behaving mice

10.1101/252205 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alexander D. Jacob ◽

Adam I. Ramsaran ◽

Andrew J. Mocle ◽

Lina M. Tran ◽

Chen Yan ◽

...

Keyword(s):

Open Source ◽

Calcium Imaging ◽

Low Cost ◽

Population Level ◽

Calcium Transients ◽

Promising Tool ◽

Calcium Indicator ◽

Neuroscience Research ◽

Freely Behaving

AbstractMiniaturized fluorescence microscopes for imaging calcium transients are a promising tool for investigating the relationship between behaviour and population-level neuronal activity in rodents. However, commercially available miniature microscopes may be costly, and, because they are closed-source, may not be easily modified based on particular experimental requirements. Here, we describe how to build and use a low-cost compact head-mounted endoscope (CHEndoscope) system for in vivo calcium imaging. The CHEndoscope uses an implanted gradient index (GRIN) lens along with the genetically encoded calcium indicator GCaMP6 to image calcium transients from hundreds of neurons simultaneously in awake behaving mice. This system is affordable, open-source, and flexible, permitting modification depending on the particular experiment. This Unit describes in detail the assembly, surgical implantation, data collection, and processing of calcium signals using the CHEndoscope system. The aim of this open framework model is to provide an accessible set of miniaturized calcium imaging tools for the neuroscience research community.Significance StatementThe ability to image calcium transients in awake, behaving rodents using miniature microscopes opens exciting and novel avenues for gaining insights into how information is encoded in neural circuits. The development of this tool has already had a significant impact on neuroscience research. The cost of commercial systems, however, may be prohibitive for many laboratories. Here, we describe an affordable, open-source compact head-mounted endoscope (CHEndoscope) system for performing in vivo calcium imaging in freely-behaving mice. CHEndoscopes may be manufactured by individual laboratories at relatively minor cost. Our hope is that greater availability of affordable, open-source tools (such as the one presented here) will accelerate the pace of discoveries in systems neuroscience.

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