A new cross-correlation algorithm for the analysis of “in vitro” neuronal network activity aimed at pharmacological studies

Biffi, Emilia; Menegon, Andrea; Regalia, Giulia; Maida, Simona; Ferrigno, Giancarlo; Pedrocchi, Alessandra

doi:10.1016/j.jneumeth.2011.05.010

Cited by 13 publications

(11 citation statements)

References 38 publications

(76 reference statements)

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“…The chosen threshold is the standard used in our laboratory during MEA experiments with hippocampal dissociated cultures [14], [15], i.e. five times the basal noise.…”

Section: Methodsmentioning

confidence: 99%

The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study

Biffi

Regalia

Menegon³

et al. 2013

PLoS ONE

117

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It is known that cell density influences the maturation process of in vitro neuronal networks. Neuronal cultures plated with different cell densities differ in number of synapses per neuron and thus in single neuron synaptic transmission, which results in a density-dependent neuronal network activity. Although many authors provided detailed information about the effects of cell density on neuronal culture activity, a dedicated report of density and age influence on neuronal hippocampal culture activity has not yet been reported. Therefore, this work aims at providing reference data to researchers that set up an experimental study on hippocampal neuronal cultures, helping in planning and decoding the experiments. In this work, we analysed the effects of both neuronal density and culture age on functional attributes of maturing hippocampal cultures. We characterized the electrophysiological activity of neuronal cultures seeded at three different cell densities, recording their spontaneous electrical activity over maturation by means of MicroElectrode Arrays (MEAs). We had gather data from 86 independent hippocampal cultures to achieve solid statistic results, considering the high culture-to-culture variability. Network activity was evaluated in terms of simple spiking, burst and network burst features. We observed that electrical descriptors were characterized by a functional peak during maturation, followed by a stable phase (for sparse and medium density cultures) or by a decrease phase (for high dense neuronal cultures). Moreover, 900 cells/mm2 cultures showed characteristics suitable for long lasting experiments (e.g. chronic effect of drug treatments) while 1800 cells/mm2 cultures should be preferred for experiments that require intense electrical activity (e.g. to evaluate the effect of inhibitory molecules). Finally, cell cultures at 3600 cells/mm2 are more appropriate for experiments in which time saving is relevant (e.g. drug screenings). These results are intended to be a reference for the planning of in vitro neurophysiological and neuropharmacological experiments with MEAs.

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“…The chosen threshold is the standard used in our laboratory during MEA experiments with hippocampal dissociated cultures [14], [15], i.e. five times the basal noise.…”

Section: Methodsmentioning

confidence: 99%

The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study

Biffi

Regalia

Menegon³

et al. 2013

PLoS ONE

117

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“…We, thus, co-cultured sgRNA-K6 and control treated human neurons together with primary mouse astrocytes on microelectrode arrays (MEAs) to enable dynamic recordings over time. We monitored the activity of the whole neuronal cultures by quantifying the number of active channels, the average spike number, the average number of channels involved in network bursts (NB) and the NB frequency42. Increasing doses of XE991 (0.002, 2, 20 μM) in both mouse primary hippocampal and hPSC-derived neuronal cultures did not affect the parameters describing the structure of the neuronal network (n° of active channels, n° of channels in NB), while elicited a rapid increase in firing activity (spike number) and its synchronization (NB frequency) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Network bursts (NB), i.e. recurrent events of synchronized firing encompassing simultaneously in different electrodes, were detected when the product of the number of active channels and the number of spikes considered in 25 ms bins was higher than 9 and when the minimum inter-NB interval was 100 ms, as previously described42. The considered parameters included the number of active channels, the average spike number on the active channels, the average number of channels involved in NB and the NB frequency.…”

Section: Methodsmentioning

confidence: 94%

“…To analyze MEA data, an off-line spike detection was performed with MC_Rack Software (MCS GmbH) appointing a channel-specific threshold equal to 5-folds the standard deviation of the average noise amplitude during the first 500 ms of recording. The subsequent spike analysis was implemented in Matlab (The Mathworks, Natick, USA), as previously described42. In brief, MEA channels were considered active when they displayed a spike rate higher than 0.03 Hz.…”

Section: Methodsmentioning

confidence: 99%

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Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming

Rubio

Luoni

Giannelli

et al. 2016

Sci Rep

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The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

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“…Shuffled values were previously generated simulating random data sharing, with the corresponding experimental data, the number of spikes for each electrode, but with shuffled interspike interval distribution. 55,56 Both intra-and inter-compartment values were compared to shuffled ones by means of a Mann-Whitney test. As control, values of cross correlation were computed within the two standard MEAs and between them, i.e., on two completely separated cultures.…”

Section: Electrophysiological Evaluation Of Twin Networkmentioning

confidence: 99%

A microfluidic platform for controlled biochemical stimulation of twin neuronal networks

et al. 2012

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Spatially and temporally resolved delivery of soluble factors is a key feature for pharmacological applications. In this framework, microfluidics coupled to multisite electrophysiology offers great advantages in neuropharmacology and toxicology. In this work, a microfluidic device for biochemical stimulation of neuronal networks was developed. A micro-chamber for cell culturing, previously developed and tested for long term neuronal growth by our group, was provided with a thin wall, which partially divided the cell culture region in two sub-compartments. The device was reversibly coupled to a flat micro electrode array and used to culture primary neurons in the same microenvironment. We demonstrated that the two fluidically connected compartments were able to originate two parallel neuronal networks with similar electrophysiological activity but functionally independent. Furthermore, the device allowed to connect the outlet port to a syringe pump and to transform the static culture chamber in a perfused one. At 14 days invitro, sub-networks were independently stimulated with a test molecule, tetrodotoxin, a neurotoxin known to block action potentials, by means of continuous delivery. Electrical activity recordings proved the ability of the device configuration to selectively stimulate each neuronal network individually. The proposed microfluidic approach represents an innovative methodology to perform biological, pharmacological, and electrophysiological experiments on neuronal networks. Indeed, it allows for controlled delivery of substances to cells, and it overcomes the limitations due to standard drug stimulation techniques. Finally, the twin network configuration reduces biological variability, which has important outcomes on pharmacological and drug screening.

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A new cross-correlation algorithm for the analysis of “in vitro” neuronal network activity aimed at pharmacological studies

Cited by 13 publications

References 38 publications

The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study

The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming

A microfluidic platform for controlled biochemical stimulation of twin neuronal networks

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