A Chemogenetic Approach for the Optical Monitoring of Voltage in Neurons

Abstract Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for the interrogation of the cellular and molecular logic of the nervous system. Herein, a semisynthetic tethered voltage indicator (STeVI1) based upon nile red is described that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash‐free imaging and faithfully detects supra‐ and sub‐threshold activity in neurons.

Dorsal root ganglia (DRG) neurons were isolated from 3-8-week-old mice and enzymatically dissociated as described previously 2 and plated onto glass bottom MatTek dishes coated with poly-Llysine and laminin. Three to six hours after plating the medium was changed and supplemented with NGF (50ng/ml) and AAV particles, typically at 3x10 11 -1x10 12 vg/ml. Culture medium was refreshed every day, and neurons were labeled and imaged 3 to 6 days after. AAV1/2 serotype targeted primarily neurons, with negligible targeting of fibroblasts, satellite or Schwann glial cells.

Cell labeling protocol
Cells were washed with serum-free medium immediately before labeling. For labeling with NR12S compound, cells were labeled with 300-500 nM of NR12S for 7 min at room temperature directly in extracellular bath solution. SNAP-derivatized compounds at 0.5-1 µM were incubated in culture medium (DMEM + 10% FBS) for 30 minutes at 37°C. ACP-derivatized compounds at 1-3 µM were incubated at room temperature in serum-free medium with 1 µM of SFP synthase and 10 mM of MgCl2. For control experiments, cells were first pretreated with saturating concentration of CoA (2 mg/ml, with1 µM of SFP synthase and 10 mM of MgCl2) and then labelled with ACP-derivatized compounds as usual. Cells then were imaged directly or after washing with extracellular bath solution.

Epifluorescence imaging of labeled cells
Imaging was performed on Zeiss Axioobserver A1 manual microscope with Axiocam MR and Andor Zyla 4.2P camera. The light source was a 100 W Mercury short arc lamp (OSRAM), and excitation and emission filters for imaging Nile Red compounds were BP 540-552 nm (RFP), BP360-540 nm (Rhodamine) and LP590 nm (RFP), BP570-640 nm (Rhodamine) respectively.

Confocal imaging of live cells and neurons
Confocal images of live cells were performed on a Leica SP5 inverted microscope with resonant scanner using an oil-immersion 40x objective with NA 1.25. An argon laser line 488 nm or solid state 561 nm were used for dye excitation. Emission was collected with a hybrid HyD detector at various wavelengths. Emission spectra were measured and constructed with the in-built lambda-scan mode in the 500-700 nm window, with 5 to 10 nm steps and 5 to 10 nm bandwidth, excitation was performed at 488 nm.

Simultaneous electrophysiology and fluorescence imaging of HEK cells and neurons
Simultaneous optical and electrophysiological recordings were performed on a standard patch clamp setup (HEKA, EPC 10Usb) mounted on the inverted microscope (Zeiss Axioobserver A1). All experiments were performed at room temperature (22C).
Fluorescence excitation was delivered using a 100 W Mercury short arc lamp (OSRAM) through BP 540-552 nm (RFP) filter or BP360-540 nm (Rhodamine) filters. Fluorescence emission was passed through a LP590 nm (RFP) or BP570-640 nm (Rhodamine) filter, and recorded using a Zyla 4.2 Plus (Andor) sCMOS Camera operated with NIS-Elements Ar (Nikon). Voltage sensitivity was found to be similar for the data obtained with these two filters in HEK293T cells, and a Rhodamine filter was used for recordings in DRG neuronal cultures. The field of view was cropped to obtain the desired imaging speed (from 20 fps to 770 fps).
An oil-immersion 40x objective with NA 1.3 was used for imaging HEK293T cells and cultured neurons. Excitation light was delivered at 12 and 28 mW/mm 2 power densities measured with an optical power meter at the imaging plane.

Data analysis
Fluorescence images were analysed in NIS-Elements Ar and ImageJ by manually defining the regions of interest (ROI) and calculating the mean unweighted mean of pixel values within this region. The cell-free region was used for calculating background fluorescence, which was subtracted from the cell fluorescence. When required in the videos, photobleaching was corrected using an exponential decay function.
For analysis of compound fluorogenicity, images of labeled cells without wash were taken, and signal-to-noise ratios were calculated by dividing the background-subtracted mean intensity of the cell membranes by the standard deviation of the background region of interest. Images from several independent labelings were used.
To determine voltage sensitivity, %F/F values were generated by subtracting baseline fluorescence at holding potential from fluorescence during the voltage step and plotted against voltage. Voltage sensitivity of different substrates was then compared as %F/F per 100 mV depolarization according to the slope of linear fit of the fractional fluorescence change vs membrane voltage or from a -60 mV to +40 mV voltage step. Rise time and decay time of fluorescence response to individual voltage steps were determined by fitting exponential function to the data points. Signal-to-noise ratios for action potentials detected by fluorescence were computed as the ratio of action potential peak amplitude to the standard deviation of fluorescence baseline prior to any depolarization. Exported imaging and electrophysiology data were then analysed using Prism (Graphpad) and custom-written Python scripts. Final data is presented as mean ± SEM if not indicated differently. Values were calculated for multiple action potentials for each neuron, and then averaged, at least three independent cultures and rAAV transductions were used. Statistical tests were performed in SigmaPlot (Systat Software, Inc).

Chemical synthesis
All chemical reagents and anhydrous solvents for synthesis were purchased from commercial suppliers (Sigma-Aldrich, Fluka, Acros) and were used without further purification or distillation. The composition of mixed solvents is given by the volume ratio (v/v). 1 H magnetic resonance (NMR) spectra were recorded on a Bruker DPX 400 (400 MHz) or Bruker AVANCE III 400 Nanobay (400 MHz) with chemical shifts (δ) reported in ppm relative to the solvent residual signals of CD3OD (3.31 ppm for 1 H) or DMSO-d6 (2.50 ppm for 1 H). Coupling constants are reported in Hz. High resolution mass spectra (HRMS) were measured on a Micromass Q-TOF Ultima spectrometer with electrospray ionization (ESI) or LTQ Orbitrap ELITE ETD (Thermo fisher). Preparative RP-HPLC was performed on a Dionex system equipped with an UVD 170U UV-Vis detector for product visualization on a Waters SunFire™ Prep C18 OBD™ 5 µm 10×150 mm Column (Buffer A: 0.1% TFA in H2O Buffer B: acetonitrile. Typical gradient was from 0% to 100% B within 30 min with 4 ml/min flow.) NR12S was synthesized as described previously 3,4 . SI Scheme 1. Structures of SNAP-targeted Nile Red derivatives with reactive BG moiety, PEG repeats n=11 and charged groups (no charge, positive and negative) -compounds Nr001, Nr002, Nr003. Compounds were synthesized as described previously 4,5 . CoA-TMR and CoA-ATTO532 were synthesized and purified as described previously 6 using tetramethylrhodamine maleimide (Molecular probes) and Atto532 maleimide (Atto Tec). SI Scheme 2. Synthesis of ACP-targeted Nile Red derivatives (CoA-PEGn-Nile Red, n=5, n=11)compounds 6 and 7, as described below.

Nile Red-PEG11-COOH 3
Fmoc-PEG11-COOH (10.0 mg, 12 µmol) was dissolved in 0.5 ml MeCN/piperidine 9:1 and the solution was stirred for 20 min at rt. The solvents were evaporated, the redidue was coevaporated 3x with MeCN and dried under high vacuum. Separately, Nile Red-C6-COOH* (5.4 mg, 12 µmol) was dissolved in 0.25 ml DMSO, treated with DIPEA (4.1 µl, 24 µmol) and TSTU (3.6 mg, 12 µmol). The solution was incubated for 30 min at r.t. and was transferred in the vial that contained the above deprotected PEG linker. After 1h, the reaction was subjected to RP-HPLC and the product was lyophilized. Yield: 4.7 mg (37%). 1  SI Figure 2. Emission spectrum of NR12S measured from live membranes of HEK293T cells (a) and DRG neurons (b) using scanning confocal microscope. Cells were labeled at 500 nM for 7 mins at room temperature. Intensities were normalized to the maximum, mean ± SEM from n=15 cells. Nile Red fluorescence was excited with Argon laser, 488 line, and emission was collected in the 500-700 nm range, with 5 nm steps, 5 nm bandwidth (a) and 10 nm steps, 10 nm bandwidth (b  Table 2. Characteristics of fluorescent GEVI, VSD and hybrid voltage indicators performance tested on neurons. GEVI are color-coded in orange, VSD in magenta, hybrid in blue-green.
Where possible orange/red/far red shifted dyes were chosen. Main groups of indicators are presented, but many are omitted.
Notes: a Genetically targeted, b Labeling was toxic for neurons, c significant capacitative loading, d specialized infrared camera needed for detection. e Hysteresis in the voltage sensitivity. Linearity expressed as + (linear in small range of membrane voltage), ++ linear over entire physiological range of membrane voltage)