scholarly journals Wide-field imaging of cortical neuronal activity with red-shifted functional indicators during motor task execution

2018 ◽  
Author(s):  
Elena Montagni ◽  
Francesco Resta ◽  
Emilia Conti ◽  
Alessandro Scaglione ◽  
Maria Pasquini ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Cortical Neurons ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Motor Task ◽  
Free Calcium ◽  
Wide Field ◽  
Task Execution ◽  
Additional Advantage

AbstractIntracellular concentration of free calcium ions in neuronal populations can be longitudinally evaluated by using fluorescent protein indicators, called genetically encoded calcium indicators (GECIs). GECIs with long emission wavelengths are particularly attractive for deep tissue microscopy in vivo, and have the additional advantage of avoiding spectral overlap with commonly used neuronal actuators like Channelrhodopsin.Here we investigated the performances of selected red-shifted GECIs through an ex vivo characterization and in vivo imaging of cortical mouse activity during motor task execution. Cortical neurons were infected with adeno-associated virus (AAV) expressing one of the red GECI variants (jRCaMP1a, jRCaMP1b, jRGECO1a, jRGECO1b). First we characterized the transfection in terms of extension and intensity using wide-field fluorescence microscopy. Next, we used RCaMP1a to analyse the cortical neuronal activity during motor behaviour. To that end, wide-field fluorescent microscopy and a robotic device for motor control were combined for simultaneous recording of cortical neuronal-activity, force applied and forelimb position during task execution.Our results show that jRCaMP1a has sufficient sensitivity to monitor in vivo neuronal activity over multiple functional areas, and can be successfully used to perform longitudinal imaging in awake mice.

scholarly journals Wide-field imaging of cortical neuronal activity with red-shifted functional indicators during motor task execution

2018 ◽  
Vol 52 (7) ◽  
pp. 074001 ◽  
Author(s):  
Elena Montagni ◽  
Francesco Resta ◽  
Emilia Conti ◽  
Alessandro Scaglione ◽  
Maria Pasquini ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Motor Task ◽  
Wide Field ◽  
Task Execution ◽  
Field Imaging ◽  
Wide Field Imaging

scholarly journals Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

2019 ◽  
Author(s):  
Joshua M. Lorenz-Guertin ◽  
Matthew J. Bambino ◽  
Sabyasachi Das ◽  
Susan T. Weintraub ◽  
Tija C. Jacob
Keyword(s):  
Cortical Neurons ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adult Brain ◽  
Scaffolding Protein ◽  
Gamma Aminobutyric Acid ◽  
Inhibitory Neurotransmitter

Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting γ2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between γ2 and γ2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes.

scholarly journals Multi-plane, wide-field fluorescent microscopy for biodynamic imaging in vivo

2019 ◽  
Vol 10 (12) ◽  
pp. 6625 ◽  
Author(s):  
Ruheng Shi ◽  
Cheng Jin ◽  
Hao Xie ◽  
Yuanlong Zhang ◽  
Xinyang Li ◽  
...  
Keyword(s):  
Fluorescent Microscopy ◽  
Wide Field

scholarly journals TATA Binding Protein (TBP) Promoter Drives Ubiquitous Expression of Marker Transgene in the Adult Sea Anemone Nematostella vectensis

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1081 ◽  
Author(s):  
Yael Admoni ◽  
Itamar Kozlovski ◽  
Magda Lewandowska ◽  
Yehu Moran
Keyword(s):  
Fluorescent Protein ◽  
Binding Protein ◽  
Fluorescent Microscopy ◽  
Cell Types ◽  
Tata Binding Protein ◽  
Nematostella Vectensis ◽  
Sea Anemones ◽  
Animal Cells ◽  
Animal Evolution

Nematostella vectensis has emerged as one as the most established models of the phylum Cnidaria (sea anemones, corals, hydroids and jellyfish) for studying animal evolution. The availability of a reference genome and the relative ease of culturing and genetically manipulating this organism make it an attractive model for addressing questions regarding the evolution of venom, development, regeneration and other interesting understudied questions. We and others have previously reported the use of tissue-specific promoters for investigating the function of a tissue or a cell type of interest in vivo. However, to our knowledge, genetic regulators at the whole organism level have not been reported yet. Here we report the identification and utilization of a ubiquitous promoter to drive a wide and robust expression of the fluorescent protein mCherry. We generated animals containing a TATA binding protein (TBP) promoter upstream of the mCherry gene. Flow cytometry and fluorescent microscopy revealed expression of mCherry in diverse cell types, accounting for more than 90% of adult animal cells. Furthermore, we detected a stable mCherry expression at different life stages and throughout generations. This tool will expand the existing experimental toolbox to facilitate genetic engineering and functional studies at the whole organism level.

Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein

2012 ◽  
Vol 108 (8) ◽  
pp. 2323-2337 ◽  
Author(s):  
Walther Akemann ◽  
Hiroki Mutoh ◽  
Amélie Perron ◽  
Yun Kyung Park ◽  
Yuka Iwamoto ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Neural Circuit ◽  
Fluorescent Protein ◽  
Hippocampal Slices ◽  
Field Potential ◽  
Specific Cell ◽  
Fast Kinetics ◽  
Detection Range ◽  
Voltage Imaging

Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.

scholarly journals High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments

2018 ◽  
Author(s):  
Hod Dana ◽  
Yi Sun ◽  
Boaz Mohar ◽  
Brad Hulse ◽  
Jeremy P. Hasseman ◽  
...  
Keyword(s):  
Calcium Imaging ◽  
High Performance ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Neuronal Cell ◽  
Wide Field ◽  
Decay Kinetics ◽  
Calcium Indicators ◽  
Large Populations

AbstractCalcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics, and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and tracking large populations of neurons using 2-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

scholarly journals A Novel System for Monitoring in vivo Cell Signaling Pathways Involved in Early Embryonic Patterning

2021 ◽  
Author(s):  
◽  
Louise Rooney
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Cell Signalling ◽  
Original System ◽  
Signal Propagation ◽  
Mouse Embryonic Fibroblast ◽  
Embryonic Patterning ◽  
Transgenic Embryos

<p>Early developmental events, such as the arrangement of the head-tail axis, are fundamentally driven by cell signalling cascades. Such incidents are regulated in a highly complex manner by promoters and inhibitors at many levels of the cascade. This complexity makes it difficult to understand where and when certain signalling occurs, and what effects additional factors have on the signalling system. Nodal signalling, executed by intracellular Smad2/3 signal propagation, is thought to induce the anterior-posterior and head-tail patterning of the early mouse embryo. Target gene outputs of this signalling are fine-tuned by a vast array of modulators; TGBβ co-receptors, extracellular ligand and receptor inhibitors, DNA binding cofactors, and intracellular enhancers and inhibitors. The endogenous target genes of this system cannot be used as a measure of signalling as they themselves feedback on the original system and others, creating diverse signals. In this body of work, we have distilled the Nodal signalling cascade to a single variable by creating a fluorescent genetic reporter to semi-quantitatively measure Smad signalling during early embryonic development. Reporter constructs contain Smad binding elements, a minimal promoter and fluorescent protein elements. Various sensitivity Smad binding elements were created to respond to different thresholds of signalling. Fluorescent microscopy and flow cytometry were used to verify responsiveness of reporter constructs, tested first in a mouse embryonic fibroblast line and subsequently in transgenic embryos. This study will provide an understanding of how extracellular cues dictate gene expression during early embryonic formation. The knowledge acquired from this work may have implications in dairy cattle and human fertility.</p>

scholarly journals Effects of Acoustic Stimuli on Neuronal Activity in the Auditory Cortex of the Rat

2011 ◽  
pp. 687-693 ◽  
Author(s):  
Y. ZHANG ◽  
L. HAN ◽  
X. XIAO ◽  
B. HU ◽  
H. RUAN ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Auditory Cortex ◽  
Neuronal Activity ◽  
Cortical Neurons ◽  
Slow Wave Sleep ◽  
Sound Stimulus ◽  
Sound Stimulation ◽  
Acoustic Stimuli ◽  
Spontaneous Neuronal Activity

Spontaneous activity of cortical neurons exhibits alternative fluctuations of membrane potential consisting of phased depolarization called "up-state" and persistent hyperpolarization called "down-state" during slow wave sleep and anesthesia. Here, we examined the effects of sound stimuli (noise bursts) on neuronal activity by intracellular recording in vivo from the rat auditory cortex (AC). Noise bursts increased the average time in the up-state by 0.81±0.65 s (range, 0.27-1.74 s) related to a 10 s recording duration. The rise times of the spontaneous up-events averaged 69.41±18.04 ms (range, 40.10-119.21 ms), while those of the sound-evoked up-events were significantly shorter (p<0.001) averaging only 22.54±8.81 ms (range, 9.31-45.74 ms). Sound stimulation did not influence ongoing spontaneous up-events. Our data suggest that a sound stimulus does not interfere with ongoing spontaneous neuronal activity in auditory cortex but can evoke new depolarizations in addition to the spontaneous ones.

scholarly journals Parasite Burden Measurement in the Leishmania major Infected Mice by Using the Direct Fluorescent Microscopy, Limiting Dilu-tion Assay, and Real-Time PCR Analysis

2020 ◽  
Author(s):  
Sepideh HAGHDOUST ◽  
Mahdieh AZIZI ◽  
Mostafa HAJI MOLLA HOSEINI ◽  
Mojgan BANDEHPOUR ◽  
Mandana MOHSENI MASOOLEH ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Parasite Burden ◽  
Pcr Analysis ◽  
Green Fluorescent ◽  
Limiting Dilution

Background: We aimed to compare parasite burden in BALB/c mice, using three methods including the direct fluorescent microscopic using recombinant Leishmania major expressing an enhanced green fluorescent protein, limiting dilution assay, and real-time PCR technique. Methods: The current study was carried out in 2018, to induce stable enhanced green fluorescent protein (EGFP) production. Initially, the linearized DNA expression cassette (pLEXSY-egfp-sat2) was integrated into the ssu locus of L. major. The expression of EGFP in recombinant parasite was analyzed using direct fluorescent microscopy. Afterward, BALB/c mice were infected with the L. majorEGFP, and the infection was evaluated in the foot-pads and inguinal lymph-nodes using an in vivo imaging system. Subsequently, eight BALB/c mice were infected with L. majorEGFP, and the results of evaluating parasite burden by a SYBR-Green based real-time PCR analysis and the limiting dilution assays were compared to the results obtained from the direct fluorescent microscopy. Results: The results of the direct fluorescent microscopy showed that EGFP gene stably was expressed in parasites. Moreover, the in vivo imaging analysis of foot-pad lesions revealed that the infection caused by L. majorEGFP was progressing over time. Additionally, significant correlations were observed between the results of parasite burden assay using the direct fluorescent microscopy and either limiting dilution assay (r=0.976, P<0.0001) or quantitative real-time PCR assay (r=0.857, P<0.001). Conclusion: Ultimately, the utilization of the direct fluorescent microscopy by employing a stable EGFP-expressing L. major is a suitable substitution for the existing methods to quantify parasite burden.

Sign in / Sign up

Export Citation Format

Share Document