Quantitation of Chronic and Acute Treatment Effects on Neuronal Network Activity Using Image and Signal Analysis

Upon maturation, primary neuronal cultures form an interconnected network based on neurite outgrowth and synaptogenesis in which spontaneous electrical activity arises. Measurement of network activity allows quantification of neuronal health and maturation. A fluorescent indicator was used to monitor secondary calcium influxes after the occurrence of action potentials, allowing us to examine activity of hippocampal cultures via confocal live cell imaging. Subsequently, nuclear staining with DAPI allows accurate cell segmentation. To analyze the calcium recording in a robust, observer-independent manner, we implemented an automated image- and signal-processing algorithm and validated it against a visual, interactive procedure. Both methods yielded similar results on the emergence of synchronized activity and allowed robust quantitative measurement of acute and chronic modulation of drugs on network activity. Both the number of days in vitro (DIV) and neutralization of nerve growth factor (NGF) have a significant effect on synchronous burst frequency and correlation. Acute effects are demonstrated using 5-HT (serotonin) and ethylene glycol tetra-acetic acid. Automated analysis allowed measuring additional features, such as peak decay times and bursting frequency of individual neurons. Based on neuronal cell cultures in 96-well plates and accurate calcium recordings, the analysis method allows development of an integrated high-content screening assay. Because molecular biological techniques can be applied to assess the influence of genes on network activity, it is applicable for neurotoxicity or neurotrophics screening as well as development of in vitro disease models via, for example, pharmacologic manipulation or RNAi.

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Early prediction of developing spontaneous activity in cultured neuronal networks

Scientific Reports ◽

10.1038/s41598-021-99538-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

David Cabrera-Garcia ◽

Davide Warm ◽

Pablo de la Fuente ◽

M. Teresa Fernández-Sánchez ◽

Antonello Novelli ◽

...

Keyword(s):

Machine Learning ◽

Spontaneous Activity ◽

Early Development ◽

Neuronal Network ◽

Multivariate Adaptive Regression Splines ◽

Network Activity ◽

Support Vector ◽

Neuronal Cultures ◽

Neuronal Network Activity

AbstractSynchronization and bursting activity are intrinsic electrophysiological properties of in vivo and in vitro neural networks. During early development, cortical cultures exhibit a wide repertoire of synchronous bursting dynamics whose characterization may help to understand the parameters governing the transition from immature to mature networks. Here we used machine learning techniques to characterize and predict the developing spontaneous activity in mouse cortical neurons on microelectrode arrays (MEAs) during the first three weeks in vitro. Network activity at three stages of early development was defined by 18 electrophysiological features of spikes, bursts, synchrony, and connectivity. The variability of neuronal network activity during early development was investigated by applying k-means and self-organizing map (SOM) clustering analysis to features of bursts and synchrony. These electrophysiological features were predicted at the third week in vitro with high accuracy from those at earlier times using three machine learning models: Multivariate Adaptive Regression Splines, Support Vector Machines, and Random Forest. Our results indicate that initial patterns of electrical activity during the first week in vitro may already predetermine the final development of the neuronal network activity. The methodological approach used here may be applied to explore the biological mechanisms underlying the complex dynamics of spontaneous activity in developing neuronal cultures.

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Clinically Approved Heterocyclics Act on a Mitochondrial Target and Reduce Stroke-induced Pathology

Journal of Experimental Medicine ◽

10.1084/jem.20032053 ◽

2004 ◽

Vol 200 (2) ◽

pp. 211-222 ◽

Cited By ~ 68

Author(s):

Irina G. Stavrovskaya ◽

Malini V. Narayanan ◽

Wenhua Zhang ◽

Boris F. Krasnikov ◽

Jill Heemskerk ◽

...

Keyword(s):

Cell Death ◽

Bioactive Compounds ◽

Mitochondrial Permeability Transition ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Glucose Deprivation ◽

Neuronal Cultures ◽

Primary Neuronal Cultures ◽

Structural Class

Substantial evidence indicates that mitochondria are a major checkpoint in several pathways leading to neuronal cell death, but discerning critical propagation stages from downstream consequences has been difficult. The mitochondrial permeability transition (mPT) may be critical in stroke-related injury. To address this hypothesis, identify potential therapeutics, and screen for new uses for established drugs with known toxicity, 1,040 FDA-approved drugs and other bioactive compounds were tested as potential mPT inhibitors. We report the identification of 28 structurally related drugs, including tricyclic antidepressants and antipsychotics, capable of delaying the mPT. Clinically achievable doses of one drug in this general structural class that inhibits mPT, promethazine, were protective in both in vitro and mouse models of stroke. Specifically, promethazine protected primary neuronal cultures subjected to oxygen-glucose deprivation and reduced infarct size and neurological impairment in mice subjected to middle cerebral artery occlusion/reperfusion. These results, in conjunction with new insights provided to older studies, (a) suggest a class of safe, tolerable drugs for stroke and neurodegeneration; (b) provide new tools for understanding mitochondrial roles in neuronal cell death; (c) demonstrate the clinical/experimental value of screening collections of bioactive compounds enriched in clinically available agents; and (d) provide discovery-based evidence that mPT is an essential, causative event in stroke-related injury.

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meaRtools: an R Package for the Analysis of Neuronal Networks Recorded on Microelectrode Arrays

10.1101/242065 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sahar Gelfman ◽

Quanli Wang ◽

Yi-Fan Lu ◽

Diana Hall ◽

Christopher D. Bostick ◽

...

Keyword(s):

Spike Train ◽

Neuronal Networks ◽

Microelectrode Arrays ◽

R Package ◽

Network Activity ◽

Neuronal Cultures ◽

Mixed Cell ◽

Spike Train Analysis ◽

Neuronal Network Activity

AbstractHere we present an open-source R package ‘meaRtools’ that provides a platform for analyzing neuronal networks recorded on Microelectrode Arrays (MEAs). Cultured neuronal networks monitored with MEAs are now being widely used to characterize in vitro models of neurological disorders and to evaluate pharmaceutical compounds. meaRtools provides core algorithms for MEA spike train analysis, feature extraction, statistical analysis and plotting of multiple MEA recordings with multiple genotypes and treatments. meaRtools functionality covers novel solutions for spike train analysis, including algorithms to assess electrode cross-correlation using the spike train tiling coefficient (STTC), mutual information, synchronized bursts and entropy within cultured wells. Also integrated is a solution to account for bursts variability originating from mixed-cell neuronal cultures. The package provides a statistical platform built specifically for MEA data that can combine multiple MEA recordings and compare extracted features between different genetic models or treatments. We demonstrate the utilization of meaRtools to successfully identify epilepsy-like phenotypes in neuronal networks from Celf4 knockout mice. The package is freely available under the GPL license (GPL>=3) and is updated frequently on the CRAN web-server repository. The package, along with full documentation can be downloaded from: https://cran.r-project.org/web/packages/meaRtools/.Author summaryCultured neuronal networks are widely used to study and characterize neuronal network activity. Among the many uses of neuronal cultures are the capabilities to evaluate neurotoxicity and the effects of pharmacological compounds on cellular physiology. Multi-well microelectrode arrays (MEAs) can collect high-throughput data from multiple neuronal cultures simultaneously, and thereby make possible hypotheses-driven inquiries into neurobiology and neuropharmacology. The analysis of MEA-derived information presents many computational challenges. High frequency data recorded simultaneously from hundreds of electrodes can be difficult to handle. The need to compare network activity across various drug treatments or genotypes recorded on the same plate from experiments lasting several weeks presents another challenge. These challenges inspired us to develop meaRtools; an MEA data analysis package that contains new methods to characterize network activity patterns, which are illustrated here using examples from a genetic mouse model of epilepsy. Among the highlights of meaRtools are novel algorithms designed to characterize neuronal activity dynamics and network properties such as bursting and synchronization, options to combine multiple recordings and use a robust statistical framework to draw appropriate statistical inferences, and finally data visualizations and plots. In summary, meaRtools provides a platform for the analyses of singular and longitudinal MEA experiments.

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The “Dark Side” of Endocannabinoids: A Neurotoxic Role for Anandamide

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200405000-00011 ◽

2004 ◽

Vol 24 (5) ◽

pp. 564-578 ◽

Cited By ~ 33

Author(s):

Ibolja Cernak ◽

Robert Vink ◽

JoAnne Natale ◽

Bogdan Stoica ◽

M. Lea Paul ◽

...

Keyword(s):

Cell Death ◽

Neuronal Cell ◽

Cell Loss ◽

Dark Side ◽

Neuronal Cultures ◽

Cb1 Receptors ◽

Central Administration ◽

Primary Neuronal Cultures ◽

Neuroprotective Effects

Endocannabinoids, including 2-arachidonoylglycerol and anandamide ( N-arachidonoylethanolamine; AEA), have neuroprotective effects in the brain through actions at CB1 receptors. However, AEA also binds to vanilloid (VR1) receptors and induces cell death in several cell lines. Here we show that anandamide causes neuronal cell death in vitro and exacerbates cell loss caused by stretch-induced axonal injury or trophic withdrawal in rat primary neuronal cultures. Administered intracerebroventricularly, AEA causes sustained cerebral edema, as reflected by diffusion-weighted magnetic resonance imaging, regional cell loss, and impairment in long-term cognitive function. These effects are mediated, in part, through VR1 as well as through calpain-dependent mechanisms, but not through CB1 receptors or caspases. Central administration of AEA also significantly upregulates genes involved in proinflammatory/microglial-related responses. Thus, anandamide produces neurotoxic effects both in vitro and in vivo through multiple mechanisms independent of the CB1 receptor.

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Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates

Biotechnology and Bioengineering ◽

10.1002/bit.23310 ◽

2011 ◽

Vol 109 (1) ◽

pp. 166-175 ◽

Cited By ~ 16

Author(s):

Emilia Biffi ◽

Andrea Menegon ◽

Francesco Piraino ◽

Alessandra Pedrocchi ◽

Gianfranco B. Fiore ◽

...

Keyword(s):

Network Activity ◽

Neuronal Cultures ◽

Primary Neuronal Cultures

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Difluoroboron β-diketonate polylactic acid oxygen nanosensors for intracellular neuronal imaging

Scientific Reports ◽

10.1038/s41598-020-80172-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Meng Zhuang ◽

Suchitra Joshi ◽

Huayu Sun ◽

Tamal Batabyal ◽

Cassandra L. Fraser ◽

...

Keyword(s):

Neuronal Activity ◽

Oxygen Sensing ◽

Brain Slices ◽

Neuronal Cell ◽

Quantum Yields ◽

Poly Lactic Acid ◽

Neuronal Cultures ◽

Primary Neuronal Cultures ◽

Non Invasive ◽

Mitotracker Red

AbstractCritical for metabolism, oxygen plays an essential role in maintaining the structure and function of neurons. Oxygen sensing is important in common neurological disorders such as strokes, seizures, or neonatal hypoxic–ischemic injuries, which result from an imbalance between metabolic demand and oxygen supply. Phosphorescence quenching by oxygen provides a non-invasive optical method to measure oxygen levels within cells and tissues. Difluoroboron β-diketonates are a family of luminophores with high quantum yields and tunable fluorescence and phosphorescence when embedded in certain rigid matrices such as poly (lactic acid) (PLA). Boron nanoparticles (BNPs) can be fabricated from dye-PLA materials for oxygen mapping in a variety of biological milieu. These dual-emissive nanoparticles have oxygen-insensitive fluorescence, oxygen-sensitive phosphorescence, and rigid matrix all in one, enabling real-time ratiometric oxygen sensing at micron-level spatial and millisecond-level temporal resolution. In this study, BNPs are applied in mouse brain slices to investigate oxygen distributions and neuronal activity. The optical properties and physical stability of BNPs in a biologically relevant buffer were stable. Primary neuronal cultures were labeled by BNPs and the mitochondria membrane probe MitoTracker Red FM. BNPs were taken up by neuronal cell bodies, at dendrites, and at synapses, and the localization of BNPs was consistent with that of MitoTracker Red FM. The brain slices were stained with the BNPs, and the BNPs did not significantly affect the electrophysiological properties of neurons. Oxygen maps were generated in living brain slices where oxygen is found to be mostly consumed by mitochondria near synapses. Finally, the BNPs exhibited excellent response when the conditions varied from normoxic to hypoxic and when the neuronal activity was increased by increasing K+ concentration. This work demonstrates the capability of BNPs as a non-invasive tool in oxygen sensing and could provide fundamental insight into neuronal mechanisms and excitability research.

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Dementia with Lewy bodies—associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions

Human Molecular Genetics ◽

10.1093/hmg/ddab036 ◽

2021 ◽

Author(s):

Maryna Psol ◽

Sofia Guerin Darvas ◽

Kristian Leite ◽

Sameehan U Mahajani ◽

Mathias Bähr ◽

...

Keyword(s):

Neuronal Network ◽

Dementia With Lewy Bodies ◽

Lewy Bodies ◽

Sporadic Case ◽

Network Activity ◽

Proteinase K ◽

Neuronal Network Activity ◽

Distinct Disease

Abstract ß-Synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson’s disease-related α-Synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in CNS neurons in vitro and in vivo, albeit at a slower pace as compared to α-Syn. Here we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of Dementia with Lewy Bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but does not aggravate neurodegeneration. ß-Syn WT, V70M and P123H formed proteinase K (PK) resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared to α-Syn. Under cell free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared to WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, that are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn, and thus likely to be directly involved into etiology of DLB. Over all, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.

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