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

Abstract

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.

Scheme 1.

Mechanisms of fluorescent voltage sensing. (A) Electrochromic VSDs sense voltage through the Stark effect, whereby the chromophore interacts directly with the electric field. Absorption of a photon significantly alters the excited state molecular dipole, which at hyperpolarizing potentials is stabilized (Left). At depolarizing potentials the charge shift inverted state is destabilized (Right). Changes in the energy levels of the chromophore result in small spectral shifts in the emission of the dye. (B) FRET-pair voltage sensors use lipophilic anions (red), which partition in a voltage-dependent fashion on the inner or outer leaflet of the membrane. Depolarization causes translocation of the anion, which can now quench the fluorescence of an immobilized fluorophore (green). (C) Molecular wire PeT VSDs depend upon the voltage-sensitive electron transfer from an electron-rich donor (orange) through a membrane-spanning molecular wire (black) to a fluorescent reporter (green). At hyperpolarizing potentials, the electric field is aligned antiparallel to the direction of electron transfer, resulting in efficient PeT and quenched fluorescence (Left). Depolarization aligns the electric field in the direction of PeT, decreasing the rate of electron transfer and increasing fluorescence (Right).

Scheme 2.

Synthesis of VF probes.

Fig. 1.

Characterization of VF sensors in HEK cells. (A) Confocal image of HEK 293 cells stained with 2 μM VF2.4.Cl. (Scale bar, 20 μm.) (B) Fractional changes in VF2.4.Cl fluorescence during a series of voltage steps to +100 or −100 from a holding potential of −60 mV (40-mV increments). (C) Fractional changes in VF2.4.Cl fluorescence from B plotted against membrane potential for voltage changes from a holding potential of −60 mV. Each datapoint represents three to four separate measurements. Error bars are SEM.

Fig. 2.

Characterization of the speed, wavelength sensitivity, and capacitance of the VF2 fluorescence response. (A) Rising edge of a 100-mV depolarizing step from −60 mV in HEK cells stained with VF2.4.Cl. (B) Falling edge of the same step. Black, solid trace is the integrated current measured electrophysiologically; red points are the optical recording. Time constants are calculated by fitting a monoexponential equation to each side of the step. Traces are the average of 100 sequential trials. (C) Voltage sensitivity vs. excitation wavelength. The normalized response of VF2.4.Cl to a 100 mV depolarization from −60 mV in HEK cells is plotted in red, and the excitation spectrum in HEK cells is the dotted black line. Error bars are SEM for n = 3 experiments. (D) Measurement of capacitative loading in leech Retzius cells. Traces show the normalized voltage decay following hyperpolarizing current injection into Retzius cell stained with 3× VF2.1.Cl (red trace), 3× oxonol 413 (black trace), or nothing (gray trace). (Inset) An expanded time scale revealing no difference between cells stained with VF2.1.Cl and control cells.

Fig. 3.

VF2 dyes resolve action potentials in neurons. (A) Rat hippocampal neurons stained with 2 μM VF2.4.Cl for 15 min show strong membrane staining. (Scale bar, 20 μm.) (B) VF2.4.Cl can detected evoked action potentials in rat hippocampal neurons in single trials. The black trace is the recorded electrophysiology signal. Individual points represent the optical signal from VF2.4.Cl captured with a high speed EMCCD camera at a rate of 2 kHz. (C) Optical imaging of spontaneous activity in leech Retzius cells using the dye VF2.1.Cl. Desheathed midbody leech ganglion stained with 200 nM VF2.1.Cl for 15 min. Pixels within the region of interest (red circle around a single Retzius cell body) were averaged in each frame to produce the optical trace. (Scale bar, 25 μm.) (D) Simultaneous optical and electrophysiological recording of spontaneous activity in cell from C. The red trace is the hi-pass filtered VF2.1.Cl signal, sampled at 50 Hz. The black trace is the electrophysiological recording, sampled at 10 kHz. The optical trace shows near-perfect matching of the subthreshold membrane potential and a clear detectable signal indicating action potentials. Action potentials have variable amplitudes in the optical traces because of the relatively slow optical sampling rate (SI Appendix, Fig. S4).