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 (OSCA) for high-throughput dissection of neuronal activity dynamics. 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 1–42 (Aβ 1–42), revealing modest changes to spiking frequency but profound changes to overall network connectivity. Finally, we use OSCA to track changes in neuronal connectivity during maturation in culture, 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.

Predictable Fluctuations in Excitatory Synaptic Strength Due to Natural Variation in Presynaptic Firing Rate

“…However, studying synapses from spikes has the disadvantage that it requires sufficient numbers of presynaptic spikes to accurately estimate efficacy. Advances in stimulation and recording techniques may make it possible to study fluctuations on faster timescales (Kwan and Dan, 2012;English et al, 2017;Walker et al, 2021;Hage et al, 2022), and systematic fluctuations in efficacy for inhibitory synapses may also occur (Reyes et al, 1998;Beierlein et al, 2003;Ma et al, 2012). Although disentangling the complex relationships between synaptic efficacy and external or internal variables in vivo is an ongoing challenge in systems neuroscience, our results here highlight the potentially important role of STP in driving slow fluctuations in synaptic efficacy.…”

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.…”

Controlling the bioelectrical properties of neurons with ferritin-based Magnetogenetics

“…We did not observe this instantaneous effect using the FeRIC system. To examine the consequences of the RF-induced membrane depolarization in a larger number of neurons, we conducted voltage imaging experiments with the voltage-sensitive fluorophore BeRST 1 ( 31, 32 ). We resolved the neuronal electrical activity on a millisecond time scale.…”

“…Rat hippocampal neurons were obtained as previously described ( 31, 32 ). Briefly, hippocampi were dissected from embryonic day 18 Sprague−Dawley rats (Charles River Laboratory) and maintained in HBSS (Gibco #14170112).…”