The influence of neuronal density on network activity: A methodological study

It is well 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. In this work, we present a methodological study to characterize the electrophysiological activity of neuronal cultures which were plated at different cell density and followed over maturation. We gathered data from 86 independent hippocampal cultures plated at six different neuronal densities, whose electrical activity was sampled by means of MicroElectrode Arrays (MEAs) from 4 to 32 days in vitro (DIV). Network activity was evaluated in terms of simple spiking, burst and network burst activities. We evaluated the distribution of the six density populations (ANOVA test) and we observed it was possible to merge our results in three categories (i.e. 900 cells/mm²-like cultures - sparse, 1800 cells/mm²-like cultures - medium, and 3600 cells/mm²-like cultures- dense). We observed that electrical descriptors are characterized by a functional peak during the initial maturation, followed by a stable phase (for sparse and medium density cultures) or by a decrease (for high dense neuronal cultures). These results do constitute an helpful reference/ benchmark for the planning of in vitro neuropharmacological and neurophysiological experiments with MEAs. Indeed, sparse cultures seem to be suitable for long lasting experiments (e.g. chronic modulatory effect of a drug treatment) while medium cultures are useful for experiments which require intense electrical activity (e.g. when the drug effect requires high firing rates, such as antiepileptics). Finally, cell dense cultures are more suitable for shorter experiments (e.g. rapid screening).