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.

In Vivo Calcium Imaging of Neural Network Function

Physiology ◽  
2007 ◽  
Vol 22 (6) ◽  
pp. 358-365 ◽  
Author(s):  
Werner Göbel ◽  
Fritjof Helmchen
Keyword(s):  
Calcium Imaging ◽  
Activity Patterns ◽  
Network Activity ◽  
Network Function ◽  
Two Photon ◽  
Calcium Indicators ◽  
Recent Developments ◽  
In Vivo Calcium Imaging ◽  
Function Network

Spatiotemporal activity patterns in local neural networks are fundamental to brain function. Network activity can now be measured in vivo using two-photon imaging of cell populations that are labeled with fluorescent calcium indicators. In this review, we discuss basic aspects of in vivo calcium imaging and highlight recent developments that will help to uncover operating principles of neural circuits.

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 Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 343
Author(s):  
Elizabeth S. Li ◽  
Margaret S. Saha
Keyword(s):  
Synthetic Biology ◽  
Imaging Techniques ◽  
Detection Methods ◽  
Calcium Indicators ◽  
Model Animal ◽  
Calcium Dyes ◽  
Genetically Encoded Calcium Indicators ◽  
Synthetic Biology Approach

Since the 1970s, the emergence and expansion of novel methods for calcium ion (Ca2+) detection have found diverse applications in vitro and in vivo across a series of model animal systems. Matched with advances in fluorescence imaging techniques, the improvements in the functional range and stability of various calcium indicators have significantly enhanced more accurate study of intracellular Ca2+ dynamics and its effects on cell signaling, growth, differentiation, and regulation. Nonetheless, the current limitations broadly presented by organic calcium dyes, genetically encoded calcium indicators, and calcium-responsive nanoparticles suggest a potential path toward more rapid optimization by taking advantage of a synthetic biology approach. This engineering-oriented discipline applies principles of modularity and standardization to redesign and interrogate endogenous biological systems. This review will elucidate how novel synthetic biology technologies constructed for eukaryotic systems can offer a promising toolkit for interfacing with calcium signaling and overcoming barriers in order to accelerate the process of Ca2+ detection optimization.

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 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 Sensitive red protein calcium indicators for imaging neural activity

2016 ◽  
Author(s):  
Hod Dana ◽  
Boaz Mohar ◽  
Yi Sun ◽  
Sujatha Narayan ◽  
Andrew Gordus ◽  
...  
Keyword(s):  
Neural Activity ◽  
Emission Spectra ◽  
Tissue Imaging ◽  
C Elegans ◽  
Calcium Indicators ◽  
Large Populations ◽  
Consequent Reduction ◽  
Deep Tissue Imaging ◽  
Genetically Encoded Calcium Indicators

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

scholarly journals A deep learning framework for inference of single-trial neural population activity from calcium imaging with sub-frame temporal resolution

2021 ◽  
Author(s):  
Feng Zhu ◽  
Harrison A Grier ◽  
Raghav Tandon ◽  
Changjia Cai ◽  
Andrea Giovannucci ◽  
...  
Keyword(s):  
Deep Learning ◽  
Temporal Resolution ◽  
Calcium Imaging ◽  
Temporal Dynamics ◽  
Population Level ◽  
Neural Population ◽  
Single Trial ◽  
Population Activity ◽  
Temporal Precision ◽  
Network States

In many brain areas, neural populations act as a coordinated network whose state is tied to behavior on a moment-by-moment basis and millisecond timescale. Two-photon (2p) calcium imaging is a powerful tool to probe network-scale computation, as it can measure the activity of many individual neurons, monitor multiple layers simultaneously, and sample from identified cell types. However, estimating network states and dynamics from 2p measurements has proven challenging because of noise, inherent nonlinearities, and limitations on temporal resolution. Here we describe RADICaL, a deep learning method to overcome these limitations at the population level. RADICaL extends methods that exploit dynamics in spiking activity for application to deconvolved calcium signals, whose statistics and temporal dynamics are quite distinct from electrophysiologically-recorded spikes. It incorporates a novel network training strategy that exploits the timing of 2p sampling to recover network dynamics with high temporal precision. In synthetic tests, RADICaL infers network states more accurately than previous methods, particularly for high-frequency components. In real 2p recordings from sensorimotor areas in mice performing a "water grab" task, RADICaL infers network states with close correspondence to single-trial variations in behavior, and maintains high-quality inference even when neuronal populations are substantially reduced.

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