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

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/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.

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions

“…It is well established that plating primary neurons at different cell densities affects network activity and alters dendrites and spine significantly (31)(32)(33). We examined if the ␦-catenin lossinduced perturbation of spine architecture would be altered by changes in network activity.…”

Taurine Protected Against the Impairments of Neural Stem Cell Differentiated Neurons Induced by Oxygen–Glucose Deprivation

“…Noteworthy, cell density has an important effect on the differentiation of NSCs and followed maturation process of in vitro neuronal networks, as reported in another research using a model of hippocampal neurons [17]. Neuronal cultures at different cell densities have differences in the number of synapses per neuron, which results in a densitydependent neuronal network activity [17].…”

“…Worthy of mentioning, when NSCs were co-treated with 8 h of OGD and 20 mM taurine, the NSC density is at a comparable level to the control without treatment. Therefore, NSCs were co-treated with 8 h of OGD and 20 mM taurine for further study in their differentiation and the followed maturation without interference from differences in the cell density [17]. Worth of mentioning, when the cells were treated by 20 mM taurine after OGD induction, taurine successfully protected the cells from the impairments induced by OGD insult, as evidenced by the improved cell viability and proliferation compared to OGD group (Supplementary Fig.…”

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease

“…Similarly, the functional properties of emerging iPSC-based neuronal networks in vitro are highly changeable and subject to differentiation efficiency, cellular subtype specification, and maturation of the neurons, as guided by the input cues provided by researchers. Cultures derived from primary tissues have been shown to readily form functional networks in vitro [Biffi et al, 2013;Penn et al, 2016], and stem cell-based models have likewise been proven capable of exhibiting similar connectivity in culture [Ban et al, 2006]. However, the extent to which these networks are capable of modeling connectivity in the aged brain has yet to be fully characterized and it seems likely that in situ maturation of iPSC-derived neuronal populations would likely improve our ability to study age-associated neurodegeneration using such systems.…”