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.
Highlights
Rapid changes in membrane potential, or action potentials, underlie the physiology of the nervous system
We demonstrate the utility of optical spike and connectivity analysis (OSCA), a voltage-sensitive fluorophore-based imaging and analysis approach in dissociated hippocampal neurons, to (1) provide high fidelity tracking of action potentials across large numbers of neurons, (2) quantify hyperactivity following pharmacological and pathophysiological network modifications, (3) validate a statistical measure of functional connectivity (Spike-Time Tiling Co-efficient; Spike time tiling coefficients (STTC)), and (4) apply STTC to understand network organization in pharmacologically and pathophysiologically modified circuits
Together with frequency data showing no change to the number of unresponsive neurons (Figure 3E), the increase observed in STTC points to a mechanism where active neurons, under chronic exposure to Aβ1−42, increase the number or strength of existing connections in downstream neurons
Summary
Rapid changes in membrane potential, or action potentials, underlie the physiology of the nervous system. Action potential firing depends on both extrinsic and intrinsic factors, many of which are neuronal cell-type specific. Brain function arises not from the behavior of isolated cells but instead from the concerted action of neuronal ensembles, making connectivity between neurons a fundamental prerequisite of neural circuits. Studying neuronal connectivity throughout development increases our understanding of brain circuitry and function, and insights into the mechanisms of neuronal circuit construction may help shed light on how network activity breaks down in aging and disease. An outstanding challenge in these diseases is to define the mechanisms driving network dysfunction. Despite the importance of localizing the where and when of neuronal activity within the context of development, health, and disease, it has been difficult to capture a view of entire network behavior while maintaining cellular resolution
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