scholarly journals Versatile live-cell activity analysis platform for characterization of neuronal dynamics at single-cell and network level

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyue Yuan ◽  
Manuel Schröter ◽  
Marie Engelene J. Obien ◽  
Michele Fiscella ◽  
Wei Gong ◽  
...  
Keyword(s):  
Neuronal Networks ◽  
Microelectrode Array ◽  
Cell Activity ◽  
Live Cell ◽  
High Signal ◽  
Neuronal Cultures ◽  
Label Free ◽  
Fluorescent Indicators ◽  
Analysis Platform

AbstractChronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Current labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescent indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended time scales. We report on a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode with 19,584 recording sites and (ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we develop reliable analysis tools, which drastically increase the throughput to infer axonal morphology and conduction speed.

scholarly journals Versatile live-cell activity analysis platform for characterization of neuronal dynamics at single-cell and network level

2020 ◽  
Author(s):  
Xinyue Yuan ◽  
Manuel Schröter ◽  
Marie Engelene J. Obien ◽  
Michele Fiscella ◽  
Wei Gong ◽  
...  
Keyword(s):  
Neuronal Networks ◽  
Microelectrode Array ◽  
Cell Activity ◽  
Live Cell ◽  
High Signal ◽  
Neuronal Cultures ◽  
Label Free ◽  
Analysis Platform

AbstractChronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Existing labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescence indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended times. We report on a novel dual-mode high-density microelectrode array, which can simultaneously record in i) full-frame mode with 19,584 recording sites and ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we developed reliable analysis tools with drastically increased throughput for extracting axonal morphology and conduction parameters.

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Label-free monitoring of 3D cortical neuronal growth in vitro using optical diffraction tomography

2021 ◽  
Author(s):  
Ariel J. Lee ◽  
DongJo Yoon ◽  
SeungYun Han ◽  
Herve Hugonnet ◽  
WeiSun Park ◽  
...  
Keyword(s):  
Optical Diffraction ◽  
Neuronal Cultures ◽  
Multiple Time ◽  
Diffraction Tomography ◽  
Label Free ◽  
Neuronal Growth ◽  
Monitoring Method ◽  
Optical Diffraction Tomography ◽  
Immunofluorescence Labeling

The highly complex central nervous systems of mammals are often studied using three-dimensional (3D) in vitro primary neuronal cultures. A coupled confocal microscopy and immunofluorescence labeling are widely utilized for visualizing the 3D structures of neurons. However, this requires fixation of the neurons and is not suitable for monitoring an identical sample at multiple time points. Thus, we propose a label-free monitoring method for 3D neuronal growth based on refractive index tomograms obtained by optical diffraction tomography. The 3D morphology of the neurons was clearly visualized, and the developmental processes of neurite outgrowth in 3D spaces were analyzed for individual neurons.

scholarly journals Directed effective connectivity and synaptic weights of in vitro neuronal cultures revealed from high-density multielectrode array recordings

2020 ◽  
Author(s):  
Chumin Sun ◽  
K.C. Lin ◽  
Yu-Ting Huang ◽  
Emily S.C. Ching ◽  
Pik-Yin Lai ◽  
...  
Keyword(s):  
Neuronal Networks ◽  
Spatial Scales ◽  
Effective Connectivity ◽  
Small World ◽  
Synaptic Strength ◽  
Multielectrode Array ◽  
Neuronal Cultures ◽  
Cross Covariance ◽  
Spatially Extended

AbstractStudying connectivity of neuronal cultures can provide insights for understanding brain networks but it is challenging to reveal neuronal connectivity from measurements. We apply a novel method that uses a theoretical relation between the time-lagged cross-covariance and the equal-time cross-covariance to reveal directed effective connectivity and synaptic weights of cortical neuron cultures at different days in vitro from multielectrode array recordings. Using a stochastic leaky-integrate-and-fire model, we show that the simulated spiking activity of the reconstructed networks can well capture the measured network bursts. The neuronal networks are found to be highly nonrandom with an over-representation of bidirectionally connections as compared to a random network of the same connection probability, with the fraction of inhibitory nodes comparable to the measured fractions of inhibitory neurons in various cortical regions in monkey, and have small-world topology with basic network measures comparable to those of the nematode C. elegans chemical synaptic network. Our analyses further reveal that (i) the excitatory and inhibitory incoming degrees have bimodal distributions the excitatory and inhibitory incoming degrees have bimodal distributions, which are that distributions that have been indicated to be optimal against both random failures and attacks in undirected networks; (ii) the distribution of the physical length of excitatory incoming links has two peaks indicating that excitatory signal is transmitted at two spatial scales, one localized to nearest nodes and the other spatially extended to nodes millimeters away, and the shortest links are mostly excitatory towards excitatory nodes and have larger synaptic weights on average; (iii) the average incoming and outgoing synaptic strength is non-Gaussian with long tails and, in particular, the distribution of outgoing synaptic strength of excitatory nodes with excitatory incoming synaptic strength is lognormal, similar to the measured excitatory postsynaptic potential in rat cortex.Author summaryTo understand how the brain processes signal and carries out its function, it is useful to know the connectivity of the underlying neuronal circuits. For large-scale neuronal networks, it is difficult to measure connectivity directly using electron microscopy techniques and methods that can estimate connectivity from electrophysiological recordings are thus highly desirable. Existing methods focus mainly on estimating functional connectivity, which is defined by statistical dependencies between neuronal activities but the relevant direct casual interactions are captured by effective connectivity. Here we apply a novel covariance-relation based method to estimate the directed effective connectivity and synaptic weights of cortical neuron cultures from recordings of multielectrode array of over 4000 electrodes taken at different days in vitro. The neuronal networks are found to be nonrandom, small-world, excitation/inhibition balanced as measured in monkey cortex, and with feeder hubs. Our analyses further suggest some form of specialisation of nodes in receiving excitatory and inhibitory signals and the transmission of excitatory signals at two spatial scales, one localized to nearest nodes and the other spatially extended to nodes millimeters away, and reveal that the distributions of the average incoming and outgoing synaptic strength are skewed with long tails.

Label-Free Quantitative In Vitro Live Cell Imaging with Digital Holographic Microscopy

2019 ◽  
Author(s):  
B. Kemper ◽  
A. Bauwens ◽  
D. Bettenworth ◽  
M. Götte ◽  
B. Greve ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Digital Holographic Microscopy ◽  
Label Free ◽  
Holographic Microscopy

scholarly journals Super-selective reconstruction of causal and direct connectivity with application to in-vitro iPSC neuronal networks

2020 ◽  
Author(s):  
Francesca Puppo ◽  
Deborah Pré ◽  
Anne Bang ◽  
Gabriel A. Silva
Keyword(s):  
Neuronal Network ◽  
Neural Development ◽  
Drug Testing ◽  
Neuronal Networks ◽  
Effective Connectivity ◽  
Functional Characterization ◽  
Neuronal Cultures ◽  
Causal Connection ◽  
Model Free

AbstractDespite advancements in the development of cell-based in-vitro neuronal network models, the lack of appropriate computational tools limits their analyses. Methods aimed at deciphering the effective connections between neurons from extracellular spike recordings would increase utility of in-vitro local neural circuits, especially for studies of human neural development and disease based on induced pluripotent stem cells (hiPSC). Current techniques allow statistical inference of functional couplings in the network but are fundamentally unable to correctly identify indirect and apparent connections between neurons, generating redundant maps with limited ability to model the causal dynamics of the network. In this paper, we describe a novel mathematically rigorous, model-free method to map effective - direct and causal - connectivity of neuronal networks from multi-electrode array data. The inference algorithm uses a combination of statistical and deterministic indicators which, first, enables identification of all existing functional links in the network and then, reconstructs the directed and causal connection diagram via a super-selective rule enabling highly accurate classification of direct, indirect and apparent links. Our method can be generally applied to the functional characterization of any in-vitro neuronal networks. Here, we show that, given its accuracy, it can offer important insights into the functional development of in-vitro iPSC-derived neuronal cultures by reconstructing their effective connectivity, thus facilitating future efforts to generate predictive models for neurological disorders, drug testing and neuronal network modeling.

scholarly journals Genetically Encoded Fluorescent Indicators for Imaging Brain Chemistry

Biosensors ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 116
Author(s):  
Xiaoke Bi ◽  
Connor Beck ◽  
Yiyang Gong
Keyword(s):  
Dynamic Range ◽  
Signal To Noise Ratio ◽  
Spatial Scales ◽  
High Signal ◽  
Spatiotemporal Resolution ◽  
Brain Chemistry ◽  
Fluorescent Indicators ◽  
Recent Developments

Genetically encoded fluorescent indicators, combined with optical imaging, enable the detection of physiologically or behaviorally relevant neural activity with high spatiotemporal resolution. Recent developments in protein engineering and screening strategies have improved the dynamic range, kinetics, and spectral properties of genetically encoded fluorescence indicators of brain chemistry. Such indicators have detected neurotransmitter and calcium dynamics with high signal-to-noise ratio at multiple temporal and spatial scales in vitro and in vivo. This review summarizes the current trends in these genetically encoded fluorescent indicators of neurotransmitters and calcium, focusing on their key metrics and in vivo applications.

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

2021 ◽  
Vol 15 ◽  
Author(s):  
Francesca Puppo ◽  
Deborah Pré ◽  
Anne G. Bang ◽  
Gabriel A. Silva
Keyword(s):  
Neural Development ◽  
Neuronal Networks ◽  
Functional Characterization ◽  
Electrode Array ◽  
Network Models ◽  
Neuronal Cultures ◽  
Causal Connection ◽  
Model Free ◽  
Rigorous Model

Despite advancements in the development of cell-based in-vitro neuronal network models, the lack of appropriate computational tools limits their analyses. Methods aimed at deciphering the effective connections between neurons from extracellular spike recordings would increase utility of in vitro local neural circuits, especially for studies of human neural development and disease based on induced pluripotent stem cells (hiPSC). Current techniques allow statistical inference of functional couplings in the network but are fundamentally unable to correctly identify indirect and apparent connections between neurons, generating redundant maps with limited ability to model the causal dynamics of the network. In this paper, we describe a novel mathematically rigorous, model-free method to map effective—direct and causal—connectivity of neuronal networks from multi-electrode array data. The inference algorithm uses a combination of statistical and deterministic indicators which, first, enables identification of all existing functional links in the network and then reconstructs the directed and causal connection diagram via a super-selective rule enabling highly accurate classification of direct, indirect, and apparent links. Our method can be generally applied to the functional characterization of any in vitro neuronal networks. Here, we show that, given its accuracy, it can offer important insights into the functional development of in vitro hiPSC-derived neuronal cultures.

scholarly journals In VitroStudies of Neuronal Networks and Synaptic Plasticity in Invertebrates and in Mammals Using Multielectrode Arrays

2015 ◽  
Vol 2015 ◽  
pp. 1-18 ◽  
Author(s):  
Paolo Massobrio ◽  
Jacopo Tessadori ◽  
Michela Chiappalone ◽  
Mirella Ghirardi
Keyword(s):  
Neuronal Networks ◽  
Microelectrode Array ◽  
Molecular Mechanisms ◽  
Multielectrode Arrays ◽  
Neural Connections ◽  
Brain Functions ◽  
Array Technology ◽  
Circuit Formation

Brain functions are strictly dependent on neural connections formed during development and modified during life. The cellular and molecular mechanisms underlying synaptogenesis and plastic changes involved in learning and memory have been analyzed in detail in simple animals such as invertebrates and in circuits of mammalian brains mainly by intracellular recordings of neuronal activity. In the last decades, the evolution of techniques such as microelectrode arrays (MEAs) that allow simultaneous, long-lasting, noninvasive, extracellular recordings from a large number of neurons has proven very useful to study long-term processes in neuronal networksin vivoandin vitro. In this work, we start off by briefly reviewing the microelectrode array technology and the optimization of the coupling between neurons and microtransducers to detect subthreshold synaptic signals. Then, we report MEA studies of circuit formation and activity in invertebrate models such asLymnaea,Aplysia, andHelix. In the following sections, we analyze plasticity and connectivity in cultures of mammalian dissociated neurons, focusing on spontaneous activity and electrical stimulation. We conclude by discussing plasticity in closed-loop experiments.

scholarly journals In vitro neuronal networks show bidirectional axonal conduction with antidromic action potentials effectively depolarizing the soma

2021 ◽  
Author(s):  
JC Mateus ◽  
CDF Lopes ◽  
M Aroso ◽  
AR Costa ◽  
A Gerós ◽  
...  
Keyword(s):  
Action Potential ◽  
Action Potentials ◽  
Neuronal Networks ◽  
Neuronal Cultures ◽  
Neuronal Function ◽  
Axonal Conduction ◽  
Technological Advances ◽  
Transduction Mechanisms ◽  
Or Gene

ABSTRACTRecent technological advances are revealing the complex physiology of the axon and challenging long-standing assumptions. Namely, while most action potential (AP) initiation occurs at the axon initial segment in central nervous system neurons, initiation in distal parts of the axon has been shown to occur in both physiological and pathological conditions. However, such ectopic action potential (EAP) activity has not been reported yet in studies using in vitro neuronal networks and its functional role, if exists, is still not clear. Here, we report the spontaneous occurrence of EAPs and effective antidromic conduction in hippocampal neuronal cultures. We also observe a significant fraction of bidirection axonal conduction in dorsal root ganglia neuronal cultures. We set out to investigate and characterize this antidromic propagation via a combination of microelectrode arrays, microfluidics, advanced data analysis and in silico studies. We show that EAPs and antidromic conduction can occur spontaneously, and also after distal axotomy or physiological changes in the axon biochemical environment. Importantly, EAPs may carry information (as orthodromic action potentials do) and can have a functional impact on the neuron, as they consistently depolarize the soma. Plasticity or gene transduction mechanisms triggered by soma depolarization can, therefore, be also affected by these antidromic action potentials/EAPs. Finally, we show that this bidirectional axonal conduction is asymmetrical, with antidromic conduction being slower than orthodromic. Via computational modeling, we show that the experimental difference can be explained by axonal morphology. Altogether, these findings have important implications for the study of neuronal function in vitro, reshaping completely our understanding on how information flows in neuronal cultures.

Sign in / Sign up

Export Citation Format

Share Document