Optically monitoring voltage in neurons by photo-inducedelectron transfer through molecular wires

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2012; 109(6):2114-9. DOI: 10.1073/pnas.1120694109
Source: PubMed


Fluorescence imaging is an attractive method for monitoring neuronal activity. A key challenge for optically monitoring voltage is development of sensors that can give large and fast responses to changes in transmembrane potential. We now present fluorescent sensors that detect voltage changes in neurons by modulation of photo-induced electron transfer (PeT) from an electron donor through a synthetic molecular wire to a fluorophore. These dyes give bigger responses to voltage than electrochromic dyes, yet have much faster kinetics and much less added capacitance than existing sensors based on hydrophobic anions or voltage-sensitive ion channels. These features enable single-trial detection of synaptic and action potentials in cultured hippocampal neurons and intact leech ganglia. Voltage-dependent PeT should be amenable to much further optimization, but the existing probes are already valuable indicators of neuronal activity.

Download full-text


Available from: John Y Lin,
91 Reads
  • Source
    • "While some groups have obtained action-potential data using these dyes, the technique is not widespread because of the specialized equipment needed and the low signal to noise in the best data (Tominaga et al., 2000; Tsutsui et al., 2001). Another, more sensitive dye-based approach involves detecting polarization changes in neurons by photo-induced electron transfer through a synthetic molecular wire to a dye (Miller et al., 2012). The speed of the electron transfer process makes this an ideal approach to monitoring fast voltage changes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS), which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanosecond time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested.
    Frontiers in Neuroscience 09/2015; 9:315. DOI:10.3389/fnins.2015.00315 · 3.66 Impact Factor
    • "The ability to tag a protein that undergoes a marked conformational change on ligand binding with a fluorescent protein that undergoes a concomitant spectral change, or tag a protein at each end with two compatible fluorophores that undergo a FRET change in response to ligand binding or a posttranslational modification, has permitted construction of in vivo biosensors that report responses in individual cells, such as an increase in intracellular calcium ion concentration (Akerboom et al. 2012; Tong et al. 2013) or activation of a particular protein kinase (Kunkel et al. 2007; Harvey et al. 2008; González-Vera 2012). Similarly , fusion of fluorescent labels to ion channels (Guerrero and Isacoff 2001; Mutoh et al. 2011) and photoinduced electron transfer to membrane-seeking chemical probes (Miller et al. 2012) provide sensitive readouts for voltage changes in individual neurons. Conversely, fluorescently tagged ion channels whose functional properties can be switched reversibly between an active and inactive state in response to laser illumination allow nondestructive manipulation of cell behavior by light of a particular wavelength (Gorostiza and Isacoff 2008; Kramer et al. 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: SUMMARY We have come a long way in the 55 years since Edmond Fischer and the late Edwin Krebs discovered that the activity of glycogen phosphorylase is regulated by reversible protein phosphorylation. Many of the fundamental molecular mechanisms that operate in biological signaling have since been characterized and the vast web of interconnected pathways that make up the cellular signaling network has been mapped in considerable detail. Nonetheless, it is important to consider how fast this field is still moving and the issues at the current boundaries of our understanding. One must also appreciate what experimental strategies have allowed us to attain our present level of knowledge. We summarize here some key issues (both conceptual and methodological), raise unresolved questions, discuss potential pitfalls, and highlight areas in which our understanding is still rudimentary. We hope these wide-ranging ruminations will be useful to investigators who carry studies of signal transduction forward during the rest of the 21st century.
    Cold Spring Harbor perspectives in biology 10/2014; 6(12). DOI:10.1101/cshperspect.a022913 · 8.68 Impact Factor
  • Source
    • "Recently, Kristan and colleagues developed a new type of fast VSD that combines the best properties of electrochromic and FRET dyes (Miller et al. 2012). This new fluorescent dye uses a process known as photon-induced electron transfer, and has been shown capable of resolving action potentials in both rat hippocampal neurons and leech neurons (Miller et al. 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Voltage-sensitive dye (VSD) imaging is a powerful technique that can provide, in single experiments, a large-scale view of network activity unobtainable with traditional sharp electrode recording methods. Here we review recent work using VSDs to study small networks and highlight several results from this approach. Topics covered include circuit mapping, network multifunctionality, the network basis of decision making, and the presence of variably participating neurons in networks. Analytical tools being developed and applied to large-scale VSD imaging data sets are discussed, and the future prospects for this exciting field are considered.
    Learning & memory (Cold Spring Harbor, N.Y.) 09/2014; 21(10):499-505. DOI:10.1101/lm.035964.114 · 3.66 Impact Factor
Show more