Two-Photon Excitation of Potentiometric Probes Enables Optical Recording of Action Potentials From Mammalian Nerve Terminals In Situ

Fisher, Joan M.; Barchi, Jonathan R.; Welle, Cristin G.; Kim, Gi-Ho; Kosterin, Paul; Obaid, A. L.; Yodh, Arjun G.; Contreras, Diego; Salzberg, Brian M.

doi:10.1152/jn.00929.2007

Cited by 63 publications

(42 citation statements)

References 40 publications

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“…Small-molecule voltage-sensitive dyes have been previously used under 2P illumination conditions (44). However, styryl dyes used in these studies typically have σ TPA values in the range of 5-10 GM, and maximal voltage sensitivity is only achieved by excitation at the very far edge of the 1P excitation spectrum (43), necessitating the loss of many incident photons and increasing phototoxicity.…”

Section: Discussionmentioning

confidence: 99%

Voltage-sensitive rhodol with enhanced two-photon brightness

Kulkarni

Kramer

Pourmandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using twophoton (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue. Neuronal membrane potential dynamics drive neurotransmitter release and are therefore responsible for the unique physiology associated with neurons at cellular, circuit, and organismal levels. Despite the central importance of proper neuronal firing to human health, an integrated understanding of neuronal activity in the context of larger brain circuits remains elusive, due in part to a lack of methods for interrogating membrane potential dynamics with sufficient spatial and temporal resolution.Traditional methods for monitoring membrane potential rely heavily on the use of invasive electrodes, through one of two methods. The first method, patch-clamp electrophysiology, uses a single electrode to make contact with or puncture a cell to record changes in membrane potential, sacrificing throughput and spatial resolution to achieve a comprehensive description of a single cellular electrophysiological profile. A second method uses multielectrode arrays (MEAs), in which patterned arrays of electrodes introduced to cells or tissues report on electrical changes. Spatial resolution of MEAs depends on the number and positioning of the electrodes within the array. Although throughput is improved relative to patch-clamp electrophysiology, the extracellularly recorded signals are typically less sensitive than whole-cell methods and can be an amalgamation of several cells, making deconvolution of recorded signals and precise correlation to specific cells difficult or impossible. Addi...

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Section: Discussionmentioning

confidence: 99%

Voltage-sensitive rhodol with enhanced two-photon brightness

Kulkarni

Kramer

Pourmandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…The results in Figure 5E are not fundamental but based on collection efficiency and fluorophore characteristics: increases in imaging efficiency will proportionally increase the number of neurons that can be imaged. Likewise, using potentiometric dyes that do not increase membrane capacitance enables higher fluorophore membrane densities and signal-to-noise ratios (Fisher et al, 2008). Nevertheless, imaging technology currently remains a significant limiting factor in detecting action potentials and other fast electrical activity in the intact brain.…”

Section: Imaging Technology As a Limiting Factormentioning

confidence: 99%

Rational Optimization and ImagingIn Vivoof a Genetically Encoded Optical Voltage Reporter

Sjulson¹,

Miesenböck²

2008

J. Neurosci.

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The hybrid voltage sensor (hVOS) combines membrane-targeted green fluorescent protein and the hydrophobic anion dipicrylamine (DPA) to provide a promising tool for optical recording of electrical activity from genetically defined populations of neurons. However, large fluorescence signals are obtained only at high DPA concentrations (Ͼ3 M) that increase membrane capacitance to a level that suppresses neural activity. Here, we develop a quantitative model of the sensor to guide its optimization and achieved an approximate threefold increase in fractional fluorescence change at a lower DPA concentration of 2 M. Using this optimized voltage reporter, we perform optical recordings of evoked activity in the Drosophila antennal lobe with millisecond temporal resolution but fail to detect action potentials, presumably because spike initiation and/or propagation are inhibited by the capacitive load added even at reduced DPA membrane densities. We evaluate strategies for potential further improvement of hVOS quantitatively and derive theoretical performance limits for optical voltage reporters in general.

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“…Unfortunately, only a small fraction of the SHG signal can be epi-collected. Therefore, to explore the possibility of optical recording action potential in multiple neurons simultaneously in vivo, we tested the e±ciency and sensitivity of a promising TPF VSD, di-3-ANEPPDHQ, 108 in random access modality. The microscope was modi¯ed to epi-collect TPF signal 109 (see Fig.…”

Section: Optical Recording Of Action Potentialsmentioning

confidence: 99%

Advanced Optical Techniques to Explore Brain Structure and Function

Silvestri

Mascaro

Lotti

et al. 2013

J. Innov. Opt. Health Sci.

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Understanding brain structure and function, and the complex relationships between them, is one of the grand challenges of contemporary sciences. Thanks to their°exibility, optical techniques could be the key to explore this complex network. In this manuscript, we brie°y review recent advancements in optical methods applied to three main issues: anatomy, plasticity and functionality. We describe novel implementations of light-sheet microscopy to resolve neuronal anatomy in whole¯xed brains with cellular resolution. Moving to living samples, we show how real-time dynamics of brain rewiring can be visualized through two-photon microscopy with the spatial resolution of single synaptic contacts. The plasticity of the injured brain can also be dissected through cutting-edge optical methods that speci¯cally ablate single neuronal processes. Finally, we report how nonlinear microscopy in combination with novel voltage sensitive dyes allow optical registrations of action potential across a population of neurons opening promising prospective in understanding brain functionality. The knowledge acquired from these complementary optical methods may provide a deeper comprehension of the brain and of its unique features.

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Two-Photon Excitation of Potentiometric Probes Enables Optical Recording of Action Potentials From Mammalian Nerve Terminals In Situ

Cited by 63 publications

References 40 publications

Voltage-sensitive rhodol with enhanced two-photon brightness

Voltage-sensitive rhodol with enhanced two-photon brightness

Rational Optimization and ImagingIn Vivoof a Genetically Encoded Optical Voltage Reporter

Advanced Optical Techniques to Explore Brain Structure and Function

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