All-Optical Electrophysiology in hiPSC-Derived Neurons With Synthetic Voltage Sensors

Abstract

Voltage imaging and "all-optical electrophysiology" in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.

Keywords: BeRST-1; optogenetics; phenotyping; stem cells; voltage imaging.

FIGURE 1

Optimization of the imaging protocol in primary neurons. (A) Imaging Setup. The 635-nm and 473-nm laser beams were collimated and spatially filtered through a combination of lenses (L1–L4) and pinholes (PH1 and PH2). A shutter SH was used to block the 635-nm beam when not acquiring data; the 473-nm beam was modulated using an analog signal. The beams were combined by a dichroic mirror DM1 and focused onto the rear focal plane of the objective via a lens LS. The lens and mirror M5 were translated together in the plane orthogonal to the optical axis to offset the illumination enter the objective producing oblique illumination. Fluorescence was directed to a camera for detection of BeRST-1 or OGB1 signals through a dichroic mirror an emission filter. (B) Overlaid excitation (dashed line) and emission (solid line) spectra of BeRST-1 (red) (Huang et al., 2015) and OGB1 (blue, reproduced from Fisher Scientific) and action spectrum of CheRiff (green) (Hochbaum et al., 2014). (C) Comparison of BeRST-1 fluorescence profile with axial and oblique illumination (gray and black lines, respectively). Scale bar, 10 μm. (D) Primary neurons co-labeled with OGB1 (left) and BeRST-1 (right). Scale bar, 10 μm. Time-courses of spontaneous Ca²⁺ and voltage activity extracted from these two neurons are shown below the respective OGB1 and BeRST-1 images. (E) Segmentation of single-neuron ROIs. Scale bar, 10 μm. (F) Extraction of single-neuron voltage time-courses. The four traces in (F) correspond to four different neurons segmented in panel (E). (G) All-optical electrophysiology with BeRST-1 in CheRiff-expressing neurons. Left: Primary neurons stained with BeRST-1 (red) and expressing CheRiff-EGFP (green). Right: Voltage response to OG stimulation of varying frequency and duration. (H) High K⁺ increases spiking activity in primary neurons. Left: Voltage time-courses of three representative neurons in normal imaging buffer and after perfusion with 5 mM KCl. Right: Instantaneous firing rate and bursting rate at baseline (Base, n = 9 neurons), after 5 min of perfusion with 5 mM KCl (KCl, n = 9 neurons) and after washing off high K⁺ (Wash; n = 25). Error bars indicate mean ± SD; unpaired Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 2

Voltage imaging and all-optical electrophysiology in human neurons. (A) Monolayer cultures of human neurons. Left: Transmitted light image of cell culture after 8 weeks of differentiation. Right: cell culture after 10 weeks of differentiation immunostained for MAP2 and DAPI. Scale bars, 200 μm. (B) Ca²⁺ imaging in human neurons. Left: A representative FOV with OGB1-loaded neurons. Right: Color-coded Ca²⁺ time-courses from six ROIs corresponding to individual neurons. Scale bar, 200 μm. (C) Human neurons stained with OGB1 (left, green), BeRST-1 (middle, red), and overlay of the two signals (right). (D) Voltage imaging in human neurons with BeRST-1. Representative time-courses of spontaneous voltage activity in three hiPSC-derived control cell lines showing different levels of activity and distinct firing patterns. (E) Quantification of instantaneous firing rate and bursting rate across the three cell lines (n = 24 neurons in line WT4; n = 36 in line WT156; n = 41 in line WT83). Data are shown as mean ± SD; unpaired Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001). (F) AP waveforms for the three control cell lines (line WT4: n = 16 neurons, 383 APs; line WT156: n = 12, 2349 APs; line WT83: n = 8, 783 APs. (G) Distribution of the AP duration [full width at half maximal (FWHM) amplitude] for the APs shown in panel (E): line WT4, 14.8 ± 2.1 ms; line WT156, 12.3 ± 2.8 ms; line WT83, 12.2 ± 3.2 ms. Data are shown as mean ± SD. (H) Immunostaining of human neurons for MAP2 (red) and EGFP (green). The nuclei were counterstained with DAPI (blue). Scale bar, 350 μm. (I) OG stimulation in spontaneously active CheRiff-expressing control neurons (line WT4). The top trace shows activity evoked by stimulation with a continuous 500-ms OG stimulus; the two bottom traces show stimulation with 5-ms long light pulses of different frequencies (1 and 10 Hz). The timing of OG stimulation is indicated in blue. (J) Evoked depolarization and spiking in human neurons with low spontaneous activity. Left: A representative human neuron expressing CheRiff-EGFP (green) and stained with BeRST-1 (red). Scale bar, 15 μm. Right: spiking induced by 10-ms long OG stimulation of increasing frequency (1, 5, and 10 Hz). (K) Left: Post hoc immunolabeling with MAP2 (red), and EGFP (green); the nuclei are counterstained with DAPI (blue). The localization grid is visible in the zoomed-in image. Scale bar, 500 μm. Right: all-optical electrophysiology from one neuron prior to fixation. The timing of OG stimulation is indicated in blue.