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

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          Abstract

          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/mm 2 cultures showed characteristics suitable for long lasting experiments (e.g. chronic effect of drug treatments) while 1800 cells/mm 2 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/mm 2 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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          Most cited references 11

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          Developing networks play a similar melody.

          During development, when synapses start to be established, a primitive form of network-driven activity provides most of the synaptic activity. This pattern enables a high degree of synchrony in immature neurons in spite of the small number of functional synapses and could participate in activity-dependent growth and synapse formation. Relying on the giant depolarizing potentials that provide most of the synaptic activity in the developing hippocampus, this article reviews the common properties and generating mechanisms of these patterns, and particularly the role of the early depolarizing action of GABA(A) and glycine receptors and the sequential expression of GABA and glutamate synapses. Patterns similar to giant depolarizing potentials have been observed in a wide range of structures and species suggesting that there is a temporal template throughout evolution that constitutes an essential step in the formation of functional networks.
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            Dissociated cortical networks show spontaneously correlated activity patterns during in vitro development.

            In vitro cultured neuronal networks coupled to microelectrode arrays (MEAs) constitute a valuable experimental model for studying changes in the neuronal dynamics at different stages of development. After a few days in culture, neurons start to connect each other with functionally active synapses, forming a random network and displaying spontaneous electrophysiological activity. The patterns of collective rhythmic activity change in time spontaneously during in vitro development. Such activity-dependent modifications play a key role in the maturation of the network and reflect changes in the synaptic efficacy, fact widely recognized as a cellular basis of learning, memory and developmental plasticity. Getting advantage from the possibilities offered by the MEAs, the aim of our study is to analyze and characterize the natural changes in dynamics of the electrophysiological activity at different ages of the culture, identifying peculiar steps of the spontaneous evolution of the network. The main finding is that between the second and the third week of culture, the network completely changes its electrophysiological patterns, both in terms of spiking and bursting activity and in terms of cross-correlation between pairs of active channels. Then the maturation process can be characterized by two main phases: modulation and shaping in the synaptic functional connectivity of the network (within the first and second week) and general moderate correlated activity, spread over the entire network, with connections properly formed and stabilized (within the fourth and fifth week).
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              Investigating neuronal activity with planar microelectrode arrays: achievements and new perspectives.

              Neuronal networks underlie memory storage and information processing in the human brain, and ultimately participate in what Eccles referred to as "the creation of consciousness". Moreover, as physiological dysfunctions of neurons almost always translate into serious health issues, the study of the dynamics of neuronal networks has become a major avenue of research, as well as their response to pharmacological tampering. Planar microelectrode arrays represent a unique tool to investigate such dynamics and interferences, as they allow one to observe the activity of neuronal networks spread in both space and time. We will here review the major results obtained with microelectrode arrays and give an overview of the latest technological developments in the field, including our own efforts to develop the potential of this already powerful technology.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                27 December 2013
                : 8
                : 12
                Affiliations
                [1 ]Neuroengineering and Medical Robotics Laboratory, Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
                [2 ]Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, Milan, Italy
                Imperial College London, United Kingdom
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: EB GR AM. Performed the experiments: EB GR. Analyzed the data: EB GR. Contributed reagents/materials/analysis tools: GF AM AP. Wrote the paper: EB GR AP AM GF. Designed the software used in analysis: EB AM.

                [¤]

                Current address: Bioengineering Laboratory, Scientific Institute, IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy

                Article
                PONE-D-13-35615
                10.1371/journal.pone.0083899
                3873984
                24386305

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 16
                Funding
                The authors have no support or funding to report.
                Categories
                Research Article
                Biology
                Anatomy and Physiology
                Cell Physiology
                Electrophysiology
                Molecular Cell Biology
                Cell Growth
                Neuroscience
                Neurophysiology
                Central Nervous System
                Cellular Neuroscience
                Developmental Neuroscience
                Engineering
                Signal Processing
                Medicine
                Anatomy and Physiology
                Cell Physiology
                Electrophysiology

                Uncategorized

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