Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative <em>In Vitro</em> Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

University of Genova

Radboud university medical center

Radboudumc

Radboud University

In vitro neuronal cultures exhibit spontaneous electrophysiological activity that can be modulated by chemical stimulation and can be monitored over time by using Micro-Electrode Arrays (MEAs), devices composed by a glass substrate and metal electrodes. Dissociated networks respond to transmitters, their blockers and many other pharmacological substances, including neurotoxic compounds. In this paper we present results related to the effects, both acute (i.e. 1 hour after the treatment) and chronic (3 days after the treatment), of increasing glutamatergic transmission induced by the application of rising concentrations of glutamate and its agonists (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid - AMPA, N-methyl-D-aspartate - NMDA and AMPA together with cyclothiazide - CTZ). Increase of available glutamate was obtained in two ways: 1) by direct application of exogenous glutamate and 2) by inhibiting the clearance of the endogenously released glutamate through DL-threo-β-benzyloxyaspartate (TBOA). Our findings show that fine modulations (i.e. low concentrations of drug) of the excitatory synaptic transmission are reflected in the electrophysiological activation of the network, while intervention leading to excessive direct stimulation of glutamatergic pathways (i.e. medium and high concentrations of drug) results in the abolishment of the electrophysiological activity and eventually cell death. The results obtained by means of the MEA recordings have been compared to the analysis of cell viability to confirm the excitotoxic effect of the applied drug. In conclusion, our study demonstrates that MEA-coupled cortical networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic transmission and might provide sensitive endpoints to detect acute and chronic neurotoxic effects of chemicals and drugs for predictive toxicity testing.

Cortical cultures coupled to micro-electrode arrays: a novel approach to perform in vitro excitotoxicity testing.

Hippocampal networks exhibit spontaneous electrophysiological activity that can be modulated by pharmacological manipulation and can be monitored over time using Micro-Electrode Arrays (MEAs), devices composed by a glass substrate and metal electrodes. The typical mode of activity of these dissociated cultures is the network-wide bursting pattern, which, if properly chemically modulated, can recall the ictal events of the epileptic phenotypes and is well-suited to study the effects of antiepileptic compounds. In this paper, we analyzed the changes induced by Carbamazepine (CBZ) and Valproate (VPA) on mature networks of hippocampal neurons in "control" condition (i.e., in the culturing medium) and upon treatment with the pro-convulsant bicuculline (BIC). We found that, in both control and BIC-treated networks, high doses (100 μM-1 mM) of CBZ almost completely suppressed the spiking and bursting activity of hippocampal neurons. On the contrary, VPA never completely abolish the electrophysiological activity in both experimental designs. Interestingly, VPA cultures pre-treated with BIC showed dual effects. In fact, in some cultures, at low VPA concentrations (100 nM-1 μM), we observed decreased firing/bursting levels, which returned to values comparable to BIC-evoked activity at high VPA concentrations (100 μM-1 mM). In other cultures, VPA reduced BIC-evoked activity in a concentration-independent manner. In conclusion, our study demonstrates that MEA-coupled hippocampal networks are responsive to chemical manipulations and, upon proper pharmacological modulation, might provide model systems to detect acute pharmacological effects of antiepileptic drugs.

Effects of antiepileptic drugs on hippocampal neurons coupled to micro-electrode arrays.

Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These networks are usually grown onto specific rigid substrates (also with embedded electrodes) and lack of most of the constituents of the in-vivo like environment: cell morphology, cell-to-cell interaction and neuritic outgrowth in all directions. Cells in a brain region develop in a 3D space and interact with a complex multi-cellular environment and extracellular matrix. Under this perspective, 3D networks coupled to micro-transducer arrays, represent a new and powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we present a new experimental paradigm constituted by 3D hippocampal networks coupled to Micro-Electrode-Arrays (MEAs) and we show how the features of the recorded network dynamics differ from the corresponding 2D network model. Further development of the proposed 3D in-vitro model by adding embedded functionalized scaffolds might open new prospects for manipulating, stimulating and recording the neuronal activity to elucidate neurophysiological mechanisms and to design bio-hybrid microsystems.

Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology.

Homeostatic plasticity, a form of synaptic plasticity, maintains the fine balance between overall excitation and inhibition in developing and mature neuronal networks. Although the synaptic mechanisms of homeostatic plasticity are well characterized, the associated transcriptional program remains poorly understood. We show that the Kleefstra-syndrome-associated protein EHMT1 plays a critical and cell-autonomous role in synaptic scaling by responding to attenuated neuronal firing or sensory drive. Chronic activity deprivation increased the amount of neuronal dimethylated H3 at lysine 9 (H3K9me2), the catalytic product of EHMT1 and an epigenetic marker for gene repression. Genetic knockdown and pharmacological blockade of EHMT1 or EHMT2 prevented the increase of H3K9me2 and synaptic scaling up. Furthermore, BDNF repression was preceded by EHMT1/2-mediated H3K9me2 deposition at the Bdnf promoter during synaptic scaling up, both in vitro and in vivo. Our findings suggest that H3K9me2-mediated changes in chromatin structure govern a repressive program that controls synaptic scaling.

Histone Methylation by the Kleefstra Syndrome Protein EHMT1 Mediates Homeostatic Synaptic Scaling.

Heterozygous mutations or deletions in the human Euchromatin histone methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a neurodevelopmental disorder that is characterized by autistic-like features and severe intellectual disability (ID). Neurodevelopmental disorders including ID and autism may be related to deficits in activity-dependent wiring of brain circuits during development. Although Kleefstra syndrome has been associated with dendritic and synaptic defects in mice and Drosophila, little is known about the role of EHMT1 in the development of cortical neuronal networks. Here we used micro-electrode arrays and whole-cell patch-clamp recordings to investigate the impact of EHMT1 deficiency at the network and single cell level. We show that EHMT1 deficiency impaired neural network activity during the transition from uncorrelated background action potential firing to synchronized network bursting. Spontaneous bursting and excitatory synaptic currents were transiently reduced, whereas miniature excitatory postsynaptic currents were not affected. Finally, we show that loss of function of EHMT1 ultimately resulted in less regular network bursting patterns later in development. These data suggest that the developmental impairments observed in EHMT1-deficient networks may result in a temporal misalignment between activity-dependent developmental processes thereby contributing to the pathophysiology of Kleefstra syndrome.

Monica Frega

Department of Informatics,
Bioengineering,
Robotics and System Engineering (DIBRIS),
Department of Informatics, Bioengineering, Robotics and System Engineering (DIBRIS)

University of Genova

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Monica Frega

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Informatics,Bioengineering,Robotics and System Engineering (DIBRIS),Department of Informatics, Bioengineering, Robotics and System Engineering (DIBRIS)

University of Genova

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Department of Informatics,
Bioengineering,
Robotics and System Engineering (DIBRIS),
Department of Informatics, Bioengineering, Robotics and System Engineering (DIBRIS)