We examined neural network ontogeny using microelectrode array (MEA) recordings made in multiwell MEA (mwMEA) plates over the first 12 days in vitro (DIV). In primary cortical cultures, action potential spiking activity developed rapidly between DIV 5 and 12. Spiking was sporadic and unorganized at early DIV, and became progressively more organized with time, with bursting parameters, synchrony, and network bursting increasing between DIV 5 and 12. We selected 12 features to describe network activity; principal components analysis using these features demonstrated segregation of data by age at both the well and plate levels. Using random forest classifiers and support vector machines, we demonstrated that four features (coefficient of variation [CV] of within-burst interspike interval, CV of interburst interval, network spike rate, and burst rate) could predict the age of each well recording with >65% accuracy. When restricting the classification to a binary decision, accuracy improved to as high as 95%. Further, we present a novel resampling approach to determine the number of wells needed for comparing different treatments. Overall, these results demonstrate that network development on mwMEA plates is similar to development in single-well MEAs. The increased throughput of mwMEAs will facilitate screening drugs, chemicals, or disease states for effects on neurodevelopment.
