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 Oscillatory integration windows in neurons

2016 ◽  
Author(s):  
Nitin Gupta ◽  
Swikriti Saran Singh ◽  
Mark Stopfer
Keyword(s):  
Neural Circuits ◽  
Neural Circuit ◽  
Field Potential ◽  
Coincidence Detection ◽  
Uniform Structure ◽  
Detection Mechanism ◽  
Oscillation Frequencies ◽  
Local Field Potential Lfp ◽  
Oscillatory Cycle

AbstractOscillatory synchrony among neurons occurs in many species and brain areas, and has been proposed to help neural circuits process information. One hypothesis states that oscillatory input creates cyclic integration windows: specific times in each oscillatory cycle when postsynaptic neurons become especially responsive to inputs. With paired local field potential (LFP) and intracellular recordings and controlled stimulus manipulations we directly tested this idea in the locust olfactory system. We found that inputs arriving in Kenyon cells (KCs) sum most effectively in a preferred window of the oscillation cycle. With a computational model, we found that the non-uniform structure of noise in the membrane potential helps mediate this process. Further experiments performed in vivo demonstrated that integration windows can form in the absence of inhibition and at a broad range of oscillation frequencies. Our results reveal how a fundamental coincidence-detection mechanism in a neural circuit functions to decode temporally organized spiking.

scholarly journals Optogenetic activation of SST-positive interneurons restores hippocampal theta oscillation impairment induced by soluble amyloid beta oligomersin vivo

2018 ◽  
Author(s):  
Hyowon Chung ◽  
Kyerl Park ◽  
Hyun Jae Jang ◽  
Michael M Kohl ◽  
Jeehyun Kwag
Keyword(s):  
Learning And Memory ◽  
Peak Power ◽  
Pyramidal Neurons ◽  
Hippocampal Slices ◽  
Field Potential ◽  
Theta Oscillations ◽  
Ca1 Pyramidal Neurons ◽  
Theta Frequency ◽  
Hippocampal Theta

AbstractAbnormal accumulation of amyloid β oligomers (AβO) is a hallmark of Alzheimer’s disease (AD), which leads to learning and memory deficits. Hippocampal theta oscillations that are critical in spatial navigation, learning and memory are impaired in AD. Since GABAergic interneurons, such as somatostatin-positive (SST+) and parvalbumin-positive (PV+) interneurons, are believed to play key roles in the hippocampal oscillogenesis, we asked whether AβO selectively impairs these SST+ and PV+ interneurons. To selectively manipulate SST+ or PV+ interneuron activity in mice with AβO pathologyin vivo, we co-injected AβO and adeno-associated virus (AAV) for expressing floxed channelrhodopsin-2 (ChR2) into the hippocampus of SST-Cre or PV-Cre mice. Local field potential (LFP) recordingsin vivoin these AβO–injected mice showed a reduction in the peak power of theta oscillations and desynchronization of spikes from CA1 pyramidal neurons relative to theta oscillations compared to those in control mice. Optogenetic-activation of SST+ but not PV+ interneurons in AβO–injected mice fully restored the peak power of theta oscillations and resynchronized the theta spike phases to a level observed in control mice.In vitrowhole-cell voltage-clamp recordings in CA1 pyramidal neurons in hippocampal slices treated with AβO revealed that short-term plasticity of SST+ interneuron inhibitory inputs to CA1 pyramidal neurons at theta frequency were selectively disrupted while that of PV+ interneuron inputs were unaffected. Together, our results suggest that dysfunction in inputs from SST+ interneurons to CA1 pyramidal neurons may underlie the impairment of theta oscillations observed in AβO-injected micein vivo.Our findings identify SST+ interneurons as a target for restoring theta-frequency oscillations in early AD.

scholarly journals A high-speed, bright, red fluorescent voltage sensor to detect neural activity

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Connor Beck ◽  
Yiyang Gong
Keyword(s):  
Blue Light ◽  
Neural Activity ◽  
High Speed ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Fluorescent Sensors ◽  
Fast Kinetics ◽  
Spectral Separation ◽  
Green Fluorescent

Abstract Genetically encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy. GEVIs have demonstrated ultra-fast kinetics and high spike detection fidelity in vivo, but existing red-fluorescent voltage indicators fall short of the response and brightness achieved by green fluorescent protein-based sensors. Furthermore, red-fluorescent GEVIs suffer from incomplete spectral separation from green sensors and blue-light-activated optogenetic actuators. We have developed Ace-mScarlet, a red fluorescent GEVI that fuses Ace2N, a voltage-sensitive inhibitory rhodopsin, with mScarlet, a bright red fluorescent protein (FP). Through fluorescence resonance energy transfer (FRET), our sensor detects changes in membrane voltage with high sensitivity and brightness and has kinetics comparable to the fastest green fluorescent sensors. Ace-mScarlet’s red-shifted absorption and emission spectra facilitate virtually complete spectral separation when used in combination with green-fluorescent sensors or with blue-light-sensitive sensors and rhodopsins. This spectral separation enables both simultaneous imaging in two separate wavelength channels and high-fidelity voltage recordings during simultaneous optogenetic perturbation.

scholarly journals CRISPR-Cas9 mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

2021 ◽  
Author(s):  
Enrique Lin Shiao ◽  
Wolfgang G Pfeifer ◽  
Brian R Shy ◽  
Mohammad Saffari Doost ◽  
Evelyn Chen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetic Material ◽  
Specific Cell ◽  
Dna Nanostructures ◽  
Primary Cells ◽  
Cell Nuclei ◽  
Genomic Integration ◽  
Dna Nanostructure ◽  
Human Primary Cells

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000's of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei.

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 Rem2 regulates distinct homeostatic mechanisms in visual circuit plasticity

2017 ◽  
Author(s):  
Anna R. Moore ◽  
Sarah E. Richards ◽  
Katelyn Kenny ◽  
Leandro de Oliveira Royer ◽  
Urann Chan ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Neural Circuit ◽  
Ocular Dominance ◽  
Scaling Up ◽  
Homeostatic Plasticity ◽  
Intrinsic Excitability ◽  
Neuronal Membrane ◽  
List Type ◽  
Ocular Dominance Plasticity

SUMMARYActivity-regulated genes sculpt neural circuits in response to sensory experience. These calcium-sensitive genes generally fall into two categories: transcription factors and proteins that function at synapses. Yet little is known about activity-regulated, cytosolic proteins that transduce signals between the neuronal membrane and the nucleus. Using the visual system as a model, we investigated the role of the activity-regulated, non-canonical Ras-like GTPase Rem2 in vivo. We demonstrate that Rem2-/- mice fail to exhibit normal ocular dominance plasticity during the critical period. At the circuit level, cortical layer 2/3 neurons in Rem2-/- mice show deficits in both postsynaptic scaling up of excitatory synapses and misregulation of intrinsic excitability. Further, we reveal that Rem2 plays a novel, cell-autonomous role in regulating neuronal intrinsic excitability. Thus, Rem2 is a critical regulator of neural circuit function and distinct homeostatic plasticity mechanisms in vivo.HIGHLIGHTSRem2 is required in excitatory cortical neurons for normal ocular dominance plasticityRem2 regulates postsynaptic homoeostatic synaptic scaling upRem2 alters the intrinsic excitability of neurons in a cell-autonomous manner

scholarly journals In vivo visualization of uterine mast cells by two-photon microscopy

Reproduction ◽  
2014 ◽  
Vol 147 (6) ◽  
pp. 781-788 ◽  
Author(s):  
Franziska Schmerse ◽  
Katja Woidacki ◽  
Monika Riek-Burchardt ◽  
Peter Reichardt ◽  
Axel Roers ◽  
...  
Keyword(s):  
Mast Cells ◽  
Intravital Microscopy ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Specific Cell ◽  
Enhanced Yellow Fluorescent Protein ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Custom Made

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study ofin vivobehavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, theirin vivolocalization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCsin vivousing double-transgenic C57BL/6JMcpt5-Cre ROSA26-EYFPmice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1(Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs livein uteroduring the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFPmice offers a novel and powerfulin vivoapproach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

scholarly journals Comprehensive mapping of whisker-evoked responses reveals broad, sharply tuned thalamocortical input to layer 4 of barrel cortex

2011 ◽  
Vol 105 (5) ◽  
pp. 2421-2437 ◽  
Author(s):  
Noah C. Roy ◽  
Thomas Bessaih ◽  
Diego Contreras
Keyword(s):  
Cortical Neurons ◽  
Barrel Cortex ◽  
Current Source ◽  
Field Potential ◽  
Fundamental Information ◽  
Layer 4 ◽  
Density Analysis ◽  
Evoked Activity ◽  
Rats And Mice

Cortical neurons are organized in columns, distinguishable by their physiological properties and input-output organization. Columns are thought to be the fundamental information-processing modules of the cortex. The barrel cortex of rats and mice is an attractive model system for the study of cortical columns, because each column is defined by a layer 4 (L4) structure called a barrel, which can be clearly visualized. A great deal of information has been collected regarding the connectivity of neurons in barrel cortex, but the nature of the input to a given L4 barrel remains unclear. We measured this input by making comprehensive maps of whisker-evoked activity in L4 of rat barrel cortex using recordings of multiunit activity and current source density analysis of local field potential recordings of animals under light isoflurane anesthesia. We found that a large number of whiskers evoked a detectable response in each barrel (mean of 13 suprathreshold, 18 subthreshold) even after cortical activity was abolished by application of muscimol, a GABAA agonist. We confirmed these findings with intracellular recordings and single-unit extracellular recordings in vivo. This constitutes the first direct confirmation of the hypothesis that subcortical mechanisms mediate a substantial multiwhisker input to a given cortical barrel.

scholarly journals Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Yilai Shu ◽  
Yong Tao ◽  
Wenyan Li ◽  
Jun Shen ◽  
Zhengmin Wang ◽  
...  
Keyword(s):  
Inner Ear ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Sensory Epithelium ◽  
Specific Cell ◽  
Supporting Cells ◽  
Auditory Hair Cells ◽  
A Genome ◽  
Adenovirus Vectors

Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction.

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.

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