Changes in axon fluorescence during activity: Molecular probes of membrane potential

Abstract

Summary. The fluorescence of dyes added to squid giant axons was studied during action potentials and voltage-clamp steps. One goal was to find fluorescence changes related to the increases in membrane conductance that underlie propagation. A second goal was to find large changes in fluorescence that would allow optical monitoring of membrane potential in neurons and other cells. Attempts were made to measure fluorescence changes using over 300 different fluorescent molecules and positive results were obtained with more than half of these. No evidence was found that would relate any of the fluorescence changes to the increases in membrane conductance that accompany depolarization; most, instead, were correlated with the changes in membrane potential. The fluorescence changes of several dyes were relatively large; the largest changes during an action potential were 10 -3 of the resting intensity. They could be measured with a signal-to-noise ratio of better than 10:1 in a single sweep.Changes in the fluorescence of molecules added to axons have been studied in an effort to learn about alterations in membrane structure that occur during excitation (Tasaki, Carnay &Watanabe, 1969;Cohen, Landowne, Shrivastav &Ritchie, 1970; Conti, Tasaki &Wanke, 1971). Although it was suggested that ANS and TNS fluorescence changes were related to increases in membrane conductance, our experiments (Davila, Cohen, Salzberg &Shrivastav, 1974) indicated that these fluorescence changes were, in fact, potential dependent. With the hope that other dyes would provide information about the structural basis of the conductance

Imaging Spatiotemporal Dynamics of Surround Inhibition in the Barrels Somatosensory Cortex

“…It is well established that the VSD signal measures membrane-potential changes in vitro and in vivo (Cohen et al, 1974;Petersen and Sakmann, 2001;Petersen et al, 2003) Fig. 23) (Sterkin, Lampl, Ferster, Glaser, Grinvald, and Arieli, unpublished observations).…”

Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

“…In the meantime, more than 1000 dyes have been tested for their ability to indicate membrane potential changes [3]. The potential-sensitive probes split mainly into three classes of polymethine dyes; merocyanines, cyanines and oxonols.…”

Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by covalent attachment of polyethylene glycol

“…5,7,10 The voltage sensor component of the dye should lie in the membrane where the electric field and charge movements are most significant during electrical activity. Molecular design features include (1) extended conjugation for long wavelength absorption while maintaining photostability; (2) various nitrogenheterocycles contributing to the long wavelength absorption and providing spectral sensitivity to voltage changes (3) hydrophobic groups for stable partitioning into the lipid bilayer; and (4) a hydrophilic group making the dye membrane impermeant and facilitating tissue staining.…”

“…6,8,9 Styryl dyes are a class of fluorescent VSDs widely used to measure membrane potentials because of their ability to follow voltage changes on a millisecond timescale. 3,6,[10][11][12] Their structures consist of an electron-rich aminophenyl group linked to a quaternized nitrogen heterocycle by a conjugated polymethine chain. Many styryl dyes, including the series of RH and JPW dyes, have been synthesized with a range of excitation and emission wavelengths and a variety of voltage responses.…”