scholarly journals Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

Science ◽  
2019 ◽  
Vol 365 (6454) ◽  
pp. 699-704 ◽  
Author(s):  
Ahmed S. Abdelfattah ◽  
Takashi Kawashima ◽  
Amrita Singh ◽  
Ondrej Novak ◽  
Hui Liu ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Spike Timing ◽  
Synthetic Dyes ◽  
Single Trial ◽  
Voltage Imaging ◽  
Larval Zebrafish ◽  
Minute Period ◽  
Mouse Cortex ◽  
Subthreshold Voltage

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

scholarly journals Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

2018 ◽  
Author(s):  
Ahmed S. Abdelfattah ◽  
Takashi Kawashima ◽  
Amrita Singh ◽  
Ondrej Novak ◽  
Hui Liu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Fluorescent Proteins ◽  
Spike Timing ◽  
Synthetic Dyes ◽  
Single Trial ◽  
Voltage Imaging ◽  
Larval Zebrafish ◽  
Mouse Cortex ◽  
Subthreshold Voltage

AbstractImaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, ‘Voltron’, that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 minutes of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

scholarly journals Syngap1 regulates experience-dependent cortical ensemble plasticity by promoting in vivo excitatory synapse strengthening

2021 ◽  
Vol 118 (34) ◽  
pp. e2100579118
Author(s):  
Nerea Llamosas ◽  
Sheldon D. Michaelson ◽  
Thomas Vaissiere ◽  
Camilo Rojas ◽  
Courtney A. Miller ◽  
...  
Keyword(s):  
Significant Proportion ◽  
Spike Timing ◽  
Excitatory Synapse ◽  
Cortical Networks ◽  
Risk Genes ◽  
Highly Active ◽  
Synapse Plasticity ◽  
Behavioral Abnormalities ◽  
Mouse Cortex

A significant proportion of autism risk genes regulate synapse function, including plasticity, which is believed to contribute to behavioral abnormalities. However, it remains unclear how impaired synapse plasticity contributes to network-level processes linked to adaptive behaviors, such as experience-dependent ensemble plasticity. We found that Syngap1, a major autism risk gene, promoted measures of experience-dependent excitatory synapse strengthening in the mouse cortex, including spike-timing–dependent glutamatergic synaptic potentiation and presynaptic bouton formation. Synaptic depression and bouton elimination were normal in Syngap1 mice. Within cortical networks, Syngap1 promoted experience-dependent increases in somatic neural activity in weakly active neurons. In contrast, plastic changes to highly active neurons from the same ensemble that paradoxically weaken with experience were unaffected. Thus, experience-dependent excitatory synapse strengthening mediated by Syngap1 shapes neuron-specific plasticity within cortical ensembles. We propose that other genes regulate neuron-specific weakening within ensembles, and together, these processes function to redistribute activity within cortical networks during experience.

scholarly journals Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

2021 ◽  
Author(s):  
Ahmed S Abdelfattah ◽  
Jihong Zheng ◽  
Daniel Reep ◽  
Getahun Tsegaye ◽  
Arthur Tsang ◽  
...  
Keyword(s):  
Saturation Mutagenesis ◽  
Voltage Sensitivity ◽  
Voltage Imaging ◽  
Neuronal Populations ◽  
Lower Baseline ◽  
Transmembrane Voltage ◽  
Automated Patch Clamp ◽  
Subthreshold Voltage ◽  
Voltage Indicator

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased ΔF/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

scholarly journals Changing the responses of cortical neurons from sub- to suprathreshold using single spikes in vivo

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Verena Pawlak ◽  
David S Greenberg ◽  
Henning Sprekeler ◽  
Wulfram Gerstner ◽  
Jason ND Kerr
Keyword(s):  
Cortical Neurons ◽  
Time Course ◽  
Visual Stimulation ◽  
Spike Timing ◽  
Sensory Cortex ◽  
Sensory Coding ◽  
Computational Simulations ◽  
Subthreshold Voltage ◽  
Stimulus Features

Action Potential (APs) patterns of sensory cortex neurons encode a variety of stimulus features, but how can a neuron change the feature to which it responds? Here, we show that in vivo a spike-timing-dependent plasticity (STDP) protocol—consisting of pairing a postsynaptic AP with visually driven presynaptic inputs—modifies a neurons' AP-response in a bidirectional way that depends on the relative AP-timing during pairing. Whereas postsynaptic APs repeatedly following presynaptic activation can convert subthreshold into suprathreshold responses, APs repeatedly preceding presynaptic activation reduce AP responses to visual stimulation. These changes were paralleled by restructuring of the neurons response to surround stimulus locations and membrane-potential time-course. Computational simulations could reproduce the observed subthreshold voltage changes only when presynaptic temporal jitter was included. Together this shows that STDP rules can modify output patterns of sensory neurons and the timing of single-APs plays a crucial role in sensory coding and plasticity.

scholarly journals A positively tuned voltage indicator reveals electrical correlates of calcium activity in the brain

2021 ◽  
Author(s):  
Stephen Wenceslao Evans ◽  
Dongqing Shi ◽  
Mariya Chavarha ◽  
Mark Houston Plitt ◽  
Jiannis Taxidis ◽  
...  
Keyword(s):  
Temporal Resolution ◽  
Calcium Imaging ◽  
Photon Detection ◽  
Temporal Precision ◽  
Voltage Imaging ◽  
Calcium Indicators ◽  
Subthreshold Voltage ◽  
Relationship Of ◽  
Genetically Encoded Calcium Indicators

Neuronal activity is routinely recorded in vivo using genetically encoded calcium indicators (GECIs) and 2-photon microscopy, but calcium imaging is poorly sensitive for single voltage spikes under typical population imaging conditions, lacks temporal precision, and does not report subthreshold voltage changes. Genetically encoded voltage indicators (GEVIs) offer better temporal resolution and subthreshold sensitivity, but 2-photon detection of single spikes in vivo using GEVIs has required specialized imaging equipment. Here, we report ASAP4b and ASAP4e, two GEVIs that brighten in response to membrane depolarization, inverting the fluorescence-voltage relationship of previous ASAP-family GEVIs. ASAP4b and ASAP4e feature 180% and 210% fluorescence increases to 100-mV depolarizations, respectively, as well as modestly prolonged deactivation and high photostability. We demonstrate single-trial detection of spikes and oscillations in vivo with standard 1 and 2-photon imaging systems, and confirm improved temporal resolution in comparison to calcium imaging on the same equipment. Thus, ASAP4b and ASAP4e GEVIs extend the uses of existing imaging equipment to include multi-unit voltage imaging in vivo.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

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