Long-term Intracellular Recording of Optogenetically-induced Electrical Activities using Vertical Nanowire Multi Electrode Array

Abstract

Continuous recording of intracellular activities in single cells is required for deciphering rare, dynamic and heterogeneous cell responses, which are missed by population or brief single-cell recording. Even if the field of intracellular recording is constantly proceeding, several technical challenges are still remained to conquer this important approach. Here, we demonstrate long-term intracellular recording by combining a vertical nanowire multi electrode array (VNMEA) with optogenetic stimulation to minimally disrupt cell survival and functions during intracellular access and measurement. We synthesized small-diameter and high-aspect-ratio silicon nanowires to spontaneously penetrate into single cells, and used light to modulate the cell's responsiveness. The light-induced intra-and extracellular activities of individual optogenetically-modified cells were measured simultaneously, and each cell showed distinctly different measurement characteristics according to the cell-electrode configuration. Intracellular recordings were achieved continuously and reliably without signal interference and attenuation over 24 hours. The integration of two controllable techniques, vertically grown nanowire electrodes and optogenetics, expands the strategies for discovering the mechanisms for crucial physiological and dynamic processes in various types of cells.Critical cellular dynamics, including transcriptional change, protein synthesis, receptor replacement, and synaptic plasticity in neural cells, take place over time periods ranging from several hours to days 1 . A common approach for discerning these cellular mechanisms in the single cell level is to measure its intracellular electrical activity using sharp microelectrodes or patch-clamping. However, as intracellular recording using these conventional electrodes is achieved by tearing the cell membrane, disruption of cell integrity limits the recording duration to several hours 2 , providing only brief 'snapshots' of cellular dynamics during limited experimental sessions 3 . Thus, such intermittent recordings have limitations in tracking single-unit activity over timescales relevant for most developmental and learning processes, or for pharmaceutical drug screening over long periods.Three-dimensional micro/nanostructure electrodes have shown superior feasibility for the electrophysiological study of single cells by accessing the cell interior and recent technologies allow simultaneous observations of the intracellular activity of individual cells in neuronal populations with high temporal/spatial resolution 4-17 . However, despite their advantages of high-sensitivity and minimal invasiveness, the reported time durations of intracellular recording using micro/nanostructure-based electrodes have not exceeded 80 min 4 . These intracellular recordings have been demonstrated by employing one of two agents: (i) external poration force achieved by a burst of electrical (electroporation) or optical (optoporation) 4 pulses applied through electrodes to increase the permeability ...

Ultrastructural analysis of neuroimplant-parenchyma interfaces uncover remarkable neuroregeneration along-with barriers that limit the implant electrophysiological functions

“…The realization that the use of substrate integrated planar MEA technologies for extracellular recordings (Figure 1) inherently limits the qualities of in-vitro and in-vivo systems has prompted the development of new 3D in-vitro technologies to enable parallel, multisite intracellular recordings and stimulation from many individual cultured cells (neurons, cardiomyocytes and striated muscles). In principle, this family of in-vitro MEA technologies utilizes different forms of 3D vertical nano-structures (nano-pillars) that pierce the plasma membrane of cultured cells (by electroporation or spontaneously) in a way similar to classical sharp electrodes (Figure 1 and Tian et al, 2010;Angle and Schaefer, 2012;Duan et al, 2012;Gao et al, 2012;Robinson et al, 2012;Angle et al, 2014;Qing et al, 2014;Abbott et al, 2017;Dipalo et al, 2017;Liu et al, 2017;Abbott et al, 2018;Abbott et al, 2019;Mateus et al, 2019;Li et al, 2020;Teixeira et al, 2020;Yoo et al, 2020;Xu et al, 2021;Zhang et al, 2021). At the same time, a number of laboratories have developed the "IN-CELL" recording and stimulation configuration, in which micrometer-sized, extracellular gold mushroom-shaped microelectrodes (gMμEs) record attenuated synaptic and action potentials (Figure 1 and Spira et al, 2007;Hai et al, 2010b;a;Fendyur and Spira, 2012;Spira and Hai, 2013;Rabieh et al, 2016;Shmoel et al, 2016;Weidlich et al, 2017;McGuire et al, 2018;Spira et al, 2018;Mateus et al, 2019;Spira et al, 2019;…”

Super-Selective Reconstruction of Causal and Direct Connectivity With Application to in vitro iPSC Neuronal Networks

“…A limitation of the system is the inability to detect subthreshold events due to the extracellular nature of the multielectrode array recording, and thus to investigate the potential relationship between miniature excitatory and inhibitory postsynaptic currents and connectivity. Recently, the application of nanotechnology on neuronal electrophysiology has brought about a promising solution to overcome this limitation and to fabricate devices that are capable of detecting sub-threshold potential (Liu et al, 2017;Wei et al, 2018;Yoo et al, 2020). Although substantial engineering issues remain before the potential of nano-neural interfaces can be fully exploited (Wu et al, 2020), the future application of our algorithm to more sensitive recordings appear to be a promising approach for a more accurate understanding of network connectivity as a consequence of synaptic and extra-synaptic inputs as well as subthreshold potentials in both physiological and pathological conditions.…”

Gold‐Mushroom Microelectrode Arrays and the Quest for Intracellular‐Like Recordings: Perspectives and Outlooks

“…Adapted with permission. [ 81 ] Copyright 2020, Springer Nature. Neuronal studies: c) vertical nanowire multielectrode array.…”

“…Vertical‐type electrodes with high aspect ratio have been used as probes for large cells which have higher coupling probability and consequently form a tight seal with the electrode. [ 21,81,96,98 ] In contrast, neuronal cells have a much smaller body size reducing the probability of active coupling electrode sites, hindering the study of neuronal connections which is still a problem for GMμEAs. [ 7 ] Nevertheless, Kwon et al.…”