Optical recording of action potentials in mammalian neurons using a microbial rhodopsin

Abstract

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately tenfold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio >10. Arch(D95N) lacked endogenous proton pumping and had 50% greater sensitivity than wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits and electrophysiology in systems for which electrode-based techniques are challenging.

Figure 1

Arch is a fluorescent voltage indicator. (a) Model of Arch as a voltage sensor, in which pH and membrane potential can both alter the protonation of the Schiff base. The cuvettes contain intact E. coli expressing Arch. (b) Absorption (solid line) and fluorescence emission (dashed line) spectra of Arch at neutral and high pH. (c) Fluorescence of Arch as a function of membrane potential. The fluorescence was divided by its value at −150 mV. (d) Dynamic response of Arch to steps in membrane potential between −70 mV and +30 mV. The overshoots on the rising and falling edges were an artifact of electronic compensation circuitry. The smaller amplitude compared to (c) is because background subtraction was not performed in (d). Data averaged over 20 cycles. Inset: Step response occurred in less than the 500 µs resolution of the imaging system. (e) Top: HEK cell expressing Arch, visualized via Arch fluorescence. Bottom: pixel-weight matrix showing regions of voltage-dependent fluorescence. Scale bar 10 µm.

Figure 2

Optical recording of action potentials with Arch. (a) Cultured rat hippocampal neuron imaged via fluorescence of Arch (composite of two fields of view). Scale bar 10 µm. (b) Left: Low-magnification image of neuron in (a). Right: Whole-field fluorescence (red) during a single-trial recording at 500 frames s⁻¹. The fluorescence was scaled to overlay on the electrical recording (blue). (c) Left: Pixel-by-pixel map of cross-correlation between whole-field and single-pixel intensities (red) overlaid on the average fluorescence (cyan). Note that the process extending to the top left of the cell body does not appear in the red channel; it is electrically decoupled from the cell. Right: Pixel-weighted fluorescence (red) and electrical recording (blue). (d) Left: Pixel-by-pixel map of cross-correlation between electrical recording and single-pixel intensities (red) overlaid on the average fluorescence (cyan). Right: Pixel-weighted fluorescence (red) and electrical recording (blue). Scale bar in (b) – (d) 50 µm. (e) Sub-cellular localization of an action potential. Left: regions of interest indicated by colored polygons. Right: time-course of an action potential averaged over 98 events in the regions indicated with the corresponding colors. The top black trace is the electrical recording. Optical recordings appear broadened due to the finite (2 ms) exposure time of the camera. The white arrow indicates a small protrusion that has a substantially delayed AP relative to the rest of the cell. Vertical scale on fluorescence traces is arbitrary. Scale bar 10 µm. (f) Gallery of single-trial recordings of action potentials recorded at a frame rate of 2 kHz. The pixel weight matrix was determined from the accompanying electrophysiology recording. Top right: Averaged spike response for 269 events in a single cell, showing voltage (blue) and fluorescence (red). (g) Identification of processes associated with a single target neuron in a dense culture. Left: Time-average Arch fluorescence of multiple transfected neurons. Right: Membrane potential was modulated by whole-cell voltage clamp. Responsive pixels were identified via cross-correlation of pixel intensity and applied voltage (red). Scale bar 10 µm.

Figure 3

Arch(D95N) shows voltage-dependent fluorescence but no photocurrent. (a) Photocurrents in Arch and Arch(D95N), expressed in HEK cells clamped at V = 0. Cells were illuminated with pulses of light (λ = 640 nm; I = 1,800 W cm⁻²). (b) Fluorescence of Arch(D95N) as a function of membrane potential.. Inset: map of voltage sensitivity. Scale bar 5 µm. (c) Dynamic response of Arch(D95N) to steps in membrane potential between −70 mV and +30 mV. Data averaged over 20 cycles. Inset: Step response comprised a component faster than 500 µs (20% of the response) and a component with a time constant of 41 ms. (d) Response of Arch(D95N) to 10 mV steps in membrane potential.

Figure 4

Optical recording of action potentials with Arch(D95N). (a) Electrically recorded membrane potential of a neuron expressing Arch, subjected to pulses of current injection and laser illumination. Red bars indicate laser illumination. (b) Same as (a) in a neuron expressing Arch(D95N). (c) Neuron expressing Arch(D95N), showing Arch(D95N) fluorescence (cyan), and regions of voltage-dependent fluorescence (red). Scale bar 10 µm. (d) Single-trial recording of whole-cell membrane potential (blue) and weighted Arch(D95N) fluorescence (red) during a train of action potentials.

Figure 5

Optical indicators of membrane potential classified by speed and sensitivity. Green marks represent indicators based on fusions of GFP homologues to membrane proteins. Pink marks represent indicators based on microbial rhodopsins. Blue diamonds represent organic dyes and hybrid dye-protein indicators. Extended bars denote indicators where two time constants have been reported. The Proteorhodopsin Optical Proton Sensor (PROPS) is homologous to Arch(D95N), but only functions in bacteria. The speeds of most organic dyes are not known precisely; however they respond in less than 500 µs.