Rational Optimization and ImagingIn Vivoof a Genetically Encoded Optical Voltage Reporter

Abstract

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.

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

“…Direct measurement of transmembrane potential with fluorescent indicators would provide a more accurate account of the timing and location of neuronal activity. Despite the promise of fluorescent voltage-sensitive dyes (VSDs), previous classes of VSDs have each been hampered by some combination of insensitivity, slow kinetics (14)(15)(16), heavy capacitative loading (17)(18)(19)(20)(21), lack of genetic targetability, or phototoxicity. Two of the more widely used classes of VSDs, electrochromic and FRET dyes, illustrate the problems associated with developing fast and sensitive fluorescent VSDs.…”

Single-sweep voltage-sensitive dye imaging of interacting identified neurons

“…Thus, the optical signal and the recording device have to be fast and sensitive to small membrane potential changes; (5) in addition, the toxicity of the VSDs is often a problem, especially when high light levels are required. Also, since VSDs are often charged molecules they may change the electrical properties of the neurons (such as capacitance; Sjulson and Miesenbock, 2008), which is particularly unfortunate during long-term recordings; (6) only if simple ways to apply VSDs and to read the optical signal are available, researchers will be ready to pay the high prices for the microscope and camera equipment (about 10 times that of a good intracellular amplifier). It will also allow them to use these techniques themselves rather than having a separate expert in the lab.…”

Recordings from human myenteric neurons using voltage-sensitive dyes

“…We used Di-4-ANEPPDHQ which is less phototoxic, more water soluble and leads to larger fluorescence signals than Di-8-ANEPPS (Obaid et al, 2004) at concentrations of 7.5-75 M. Di-4-ANEPPS, which incorporates into the cell membrane faster than Di-8-ANEPPS (Fluhler et al, 1985;Fromherz and Lambacher, 1991;Bedlack et al, 1992) was used at 10 M. Another analogue of Di-8-ANEPPS is the more hydrophobic dye Di-8-ANEPPQ (Tsau et al, 1996) which was used at a concentration of 20 M. ANNINE-6Plus and Di-4-ANEPPDHQ were tested in addition with and without Pluronic F-127 (Invitrogen, Karlsruhe, Germany). To increase the signal-to-noise ratio for experiments in freshly dissected human myenteric plexus preparations we used DPA (dipicrylamine) as a resonance-energy-transfer acceptor as described elsewhere (Bradley et al, 2009;Sjulson and Miesenböck, 2008;Chanda et al, 2005a,b). DPA is a non-fluorescent hydrophobic anion, which localizes at the lipid-aqueous interface and can be used as a classical Förster-energy-transfer acceptor from variety of donor fluorophores (Chanda et al, 2005a).…”