Optical spike detection and connectivity analysis with a far-red voltage-sensitive fluorophore reveals changes to network connectivity in development and disease

Abstract

The ability to optically record dynamics of neuronal membrane potential promises to revolutionize our understanding of neurobiology. In this study, we show that the far-red voltage sensitive fluorophore, Berkeley Red Sensor of Transmembrane potential −1, or BeRST 1, can be used to monitor neuronal membrane potential changes across dozens of neurons at a sampling rate of 500 Hz. Notably, voltage imaging with BeRST 1 can be implemented with affordable, commercially available illumination sources, optics, and detectors. BeRST 1 is well-tolerated in cultures of rat hippocampal neurons and provides exceptional optical recording fidelity, as judged by dual fluorescence imaging and patch-clamp electrophysiology. We developed a semi-automated spike-picking program to reduce user bias when calling action potentials and used this in conjunction with BeRST 1 to develop an optical spike and connectivity analysis workflow (OSCA) for high-throughput dissection of neuronal activity dynamics in development and disease. The high temporal resolution of BeRST 1 enables dissection of firing rate changes in response to acute, pharmacological interventions with commonly used inhibitors like gabazine and picrotoxin. Over longer periods of time, BeRST 1 also tracks chronic perturbations to neurons exposed to amyloid beta (Aβ1-42), revealing modest changes to spiking frequency but profound changes to overall network connectivity. Finally, we use OSCA to track changes in neuronal connectivity during development, providing a functional readout of network assembly. We envision that use of BeRST 1 and OSCA described here will be of use to the broad neuroscience community.Significance StatementOptical methods to visualize membrane potential dynamics provide a powerful complement to Ca2+ imaging, patch clamp electrophysiology, and multi-electrode array recordings. However, modern voltage imaging strategies often require complicated optics, custom-built microscopes, or genetic manipulations that are impractical outside of a subset of model organisms. Here, we describe the use of Berkeley Red Sensor of Transmembrane potential, or BeRST 1, a far-red voltage-sensitive fluorophore that can directly visualize membrane potential changes with millisecond resolution across dozens of neurons. Using only commercially available components, voltage imaging with BeRST 1 reveals profound changes in neuronal connectivity during development, exposes changes to firing rate during acute pharmacological perturbation, and illuminates substantial increases in network connectivity in response to chronic exposure to amyloid beta.

Imaging Spontaneous Neuronal Activity with Voltage‐Sensitive Dyes

“…Thus, BeRST 1 displays high signal‐to‐noise ratios when measuring spontaneous voltage changes in dissociated culture. BeRST 1's properties enabled the imaging of spontaneous activity from up to 25 neurons simultaneously and the investigation of excitability and connectivity in bulk dissociated culture (Walker, Raliski, & Karbasi, et al., 2020) and microisland cultures (Walker, Raliski, & Nguyen, et al., 2020). However, one challenge that arose during these studies was the difficulty of processing large amounts of spontaneous activity imaging data.…”

Imaging Voltage in Complete Neuronal Networks Within Patterned Microislands Reveals Preferential Wiring of Excitatory Hippocampal Neurons

“…In microisland culture, action potential frequency in microislands increases significantly between 8 and 12 DIV (p < 0.0001, see details in Figure 3 caption), before decreasing back to the level initially observed at 8 DIV (p < 0.0001, Figures 3A,B). Bulk cultures show the same significant increase from DIV 8 to DIV 12, followed by a drop back to initial levels at DIV 15 (Supplementary Figure S2; Walker et al, 2021). We speculate that initial bouts of synapse formation at early time points in development lead to an excess of connections, initially "overshooting" a homeostatic set-point, which is restored following synaptic pruning and remodeling (Goda and Davis, 2003;Südhof, 2018).…”

“…Voltage imaging is a powerful technique which can be used to simultaneously record the functional signatures of multiple neurons with high sensitivity and temporal resolution ( Huang et al, 2015 ; Liu and Miller, 2020 ; Walker et al, 2021 ). Previously, we recorded small areas of neuronal networks cultured on 12 mm diameter coverslips ( Walker et al, 2021 ). The imaging field of view (FOV) comprised an area of approximately 120 × 650 μm 2 , or 0.086 mm 2 .…”

All-optical electrophysiology in hiPSC-derived neurons with synthetic voltage sensors

“…This limitation, however, can be mitigated by fixation and post-hoc immunolabeling, as long as the fixation and labeling procedure does not distort the cellular network allowing for registration with live images. Post hoc labeling and image registration have been performed on cell cultures in the past (Hanson et al 2010, Walker et al 2020b but not in the context of voltage imaging in human neurons. As a proof of principle, we plated neurons in imaging dishes with an imprinted grid.…”

“…Moreover, this bias may differ across the patient and control cell lines confounding experimental results. Here, we sought to establish an alternative protocol that uses synthetic voltage sensors (Miller 2016, Peterka et al 2011, Walker et al 2020a, Walker et al 2020b that can be easily delivered to cell cultures mitigating the problem of cell death due to introduction of large or multiple expression vectors. We demonstrated the functionality of this method by measuring spontaneous and OG-induced activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.…”