Protein voltage sensor imaging in vivo

Summary

Principal Investigator: Mark J Schnitzer
Stanford Neuroscience
Title: "Protein voltage sensors: kilohertz imaging of neural dynamics in behaving animals"
BRAIN Category: Large-Scale Recording-Modulation - Optimization (RFA NS-14-008)

Dr. Schnitzer and his team have created a new system for developing optical voltage sensors, which will allow scientists to simultaneously record firing of large groups of neurons or electrical activity in precise locations inside of neurons, such as synapses.

Principal Investigator: Mark J Schnitzer
Stanford Neuroscience
Title: “Protein voltage sensors: kilohertz imaging of neural dynamics in behaving animals”
BRAIN Category: Large-Scale Recording-Modulation – Optimization (RFA NS-14-008)

Dr. Schnitzer and his team have created a new system for developing optical voltage sensors, which will allow scientists to simultaneously record firing of large groups of neurons or electrical activity in precise locations inside of neurons, such as synapses.

NIH Webpages

The diagrams show a hypothetical protein (orange oval) and the formation of an active site, which is due to a voltage-induced conformational change that is mediated by the defined regions of the protein (green and yellow cylinders). a | Charged amino acids may move within membranes in response to changes in voltage. The side groups of Asp and Arg are shown. b | Reorientation of an intrinsic residue dipole, such as Tyr, through changes in the field. c | An alpha-helix that is the length of the membrane (red to blue gradient) has a dipole moment that is equivalent to the length of the helix that separates plusminus0.5 electronic charges (e0); therefore, it can also reorientate when the field is changed. The oval that is attached to the alpha-helix corresponds to a fluorophore that is quenched on the left and unquenched on the right, which indicates a conformational change. d | A channel within the protein can redistribute ions (light blue circle) according to the direction of the field and initiate a conformational change. Alternatively, a conformational change may form a channel that confers voltage dependence to the process as the ions move in the channel. From Nature Reviews | Molecular Cell Biology 4/2008

The diagrams show a hypothetical protein (orange oval) and the formation of an active site, which is due to a voltage-induced conformational change that is mediated by the defined regions of the protein (green and yellow cylinders). a | Charged amino acids may move within membranes in response to changes in voltage. The side groups of Asp and Arg are shown. b | Reorientation of an intrinsic residue dipole, such as Tyr, through changes in the field. c | An alpha-helix that is the length of the membrane (red to blue gradient) has a dipole moment that is equivalent to the length of the helix that separates plusminus0.5 electronic charges (e0); therefore, it can also reorientate when the field is changed. The oval that is attached to the alpha-helix corresponds to a fluorophore that is quenched on the left and unquenched on the right, which indicates a conformational change. d | A channel within the protein can redistribute ions (light blue circle) according to the direction of the field and initiate a conformational change. Alternatively, a conformational change may form a channel that confers voltage dependence to the process as the ions move in the channel.
From Nature Reviews | Molecular Cell Biology 4/2008

Project Description

Attaining effective genetically encoded optical voltage-indicators has been a longstanding goal in neuroscience research and is a key near-term aim of the BRAIN Initiative. Unlike small molecule sensors or hybrids of fluorescent proteins with organic molecules, optical voltage-indicators that can be fully encoded genetically are readily combined with genetic tools and viral delivery methods that enable long-term expression and chronic imaging studies without addition of exogenous agents. Genetically encoded Ca2+-sensors offer similar targeting advantages, but Ca2+-imaging fails to reveal individual spikes in many neuron types, poorly captures sub- threshold membrane dynamics, and has insufficient temporal resolution to capture spike timing to better than ~50-100 ms. Voltage-indicators directly sense the membrane potential and promise faithful reporting of spike waveforms, spike bursts and sub-threshold dynamics, in cells targeted by their genetic class or connectivity. An ideal voltage-indicator would produce large fluorescence responses, to facilitate spike detection, and have millisecond-scale kinetics, to study synchrony and spike-timing aspects of neural coding. However, prior protein voltage-indicators have generally suffered performance-limiting tradeoffs between modest brightness, sluggish kinetics, and limited signaling dynamic range in response to action potentials. To date, no protein voltage-indicator combines the attributes needed for accurate reporting of voltage activity in behaving animals. However, if such a sensor emerged, this would likely have even greater impact on brain science than the surge in research enabled by recent advanced versions of the GCaMP Ca2+-indicator. This proposal seeks to create broad voltage-imaging capabilities and involves two Co-PDs who are highly experienced in fluorescence imaging of neural activity. Working collaboratively, we recently created two new classes of voltage-indicators, of distinct colors and voltage-sensing mechanisms, each of which has substantially superior signaling fidelity than earlier protein voltage-indicators while offering faster kinetics and higher brightness. Using thes two sensor types, we have imaged fast spike trains in cultured neurons and brain slices. Calculations using signal detection theory show our indicators are now on the brink of transitioning into a mainstay approach to monitor large numbers of individual neurons in behaving animals. To enact this, we will use novel massively parallel methods to screen variants of our protein indicators at 100-1000¿ greater throughput than screening methods used previously in the field. We will validate and iteratively optimize the resulting indicators in cultred neurons, mammalian brain slices, and behaving flies, nematodes and mice, by using signal detection theory to benchmark indicator performance. To accompany these voltage-indicators, we will also create imaging instrumentation custom-designed for high-speed (~1 kHz) voltage-imaging in awake head-restrained and freely behaving mice. If our work succeeds, it will be a game-changer for brain research, propelling studies of how cells and circuits function normally and go awry in disease.

Public Health Relevance Statement

Attaining a rich understanding of how neural circuits convey and process information will require new technologies for high-fidelity recordings of electrical activity, including high-frequency and sub-threshold events, in genetically defined ensembles of neurons. Having recently developed two new classes of genetically encoded optical voltage-indicators with fast kinetics and large dynamic range, here we will use novel massively high-throughput protein screening methods to further improve their signaling capabilities, which we will validate and iteratively optimize in behaving nematodes, fruit flies and mice. To accompany these voltage-indicators, we will also create imaging instrumentation custom-designed for high-speed (~1 kHz) voltage-imaging in awake head-restrained and freely behaving mice.

NIH Spending Category

Bioengineering; Neurosciences

Project Terms

Action Potentials; Animals; awake; Benchmarking; Brain; brain research; Cells; Chronic; Code; Color; Communities; Custom; Data; design; Detection; Disease; Drosophila genus; Event; experience; Fluorescence; fluorescence imaging; fly; Frequencies (time pattern); Genetic; Goals; Grant; Head; Hybrids; Image; imaging modality; improved; In Vitro; Individual; information processing; instrument; instrumentation; Interneurons; Kinetics; Knowledge; Label; Lead; Learning; Left; Libraries; Life; Lighting; Mammals; Membrane; Membrane Potentials; Methods; Metric; Microscope; millisecond; miniaturize; Monitor; Mus; mutant; Nature; Nematoda; neural circuit; Neurons; Neurosciences; Neurosciences Research; new technology; novel; Optics; Paper; Performance; Proteins; public health relevance; relating to nervous system; Reporting; Research; Resolution; Resource Sharing; response; Rodent; Science; screening; sensor; Signal Detection (Psychology); Signal Transduction; Slice; small molecule; Speed (motion); System; Testing; theories; Time; Tissues; tool; Training; Variant; Viral; voltage; Work

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