Graph theory for the analysis of micro-electrode array recordings of human brain slices - framework and benchmarking

Micro-electrode array (MEA) recordings are widely used to characterize functional connectivity in neural cultures and have gained traction for the analysis of human brain slices. However, the impact of graph construction methodology on the resulting network topology has not been systematically quantified. Here, we benchmark three methods - shared spiking activity, Pearson cross-correlation, and the spike time tiling coefficient (STTC) - across 37 recordings from human cortical slice cultures classified into low, moderate, and high activity groups. We show that method choice alone produces large topological differences (Cohens d = 0.86-1.14 for clustering coefficient, d > 1.0 for node count), while higher-order features such as modularity remain stable. Each method exhibits a distinct sensitivity profile: shared spiking detects activity-dependent changes primarily through network size, correlation uniquely captures clustering differences, and STTC combines strong biological sensitivity with negligible parameter dependence across lag windows (all d < 0.1). Within shared spiking, z-score normalization dominates all other parameter choices (d > 1.0 versus bin size effects of d < 0.23), functioning as an implicit analytical null model that fundamentally reshapes the edge set rather than merely rescaling weights. Inter-method edge overlap is low (Jaccard index 0.08-0.45) and activity dependent, demonstrating that these methods identify substantially different connections from identical data. Our results reveal that methodological choices including construction method, threshold, and normalization introduce hidden degrees of freedom with effect sizes comparable to the biological signals being measured. We provide practical recommendations for parameter selection, reporting, and cross-method validation in MEA-based network neuroscience. Author SummaryWhen we record electrical activity from brain tissue using grids of electrodes, we can ask how different sites influence one another and map the tissue as a network of connections. Thanks to novel culturing methods, this approach is increasingly used to study human brain slices. However, deciding what is "connected" is not well defined. Researchers use several different methods, and it has never been clear how much this choice shapes the network they end up describing. Here we compared three widely used methods on 37 recordings from human cortical slices spanning a range of activity levels. We found that the method alone can change the apparent structure of the network as much as real biological differences do. The methods frequently disagreed about which connections exist and some technical choices, including normalization techniques, had surprisingly large effects. Because these hidden choices can rival the biological signal, we provide this benchmarking work with practical recommendations for selecting, reporting, and cross-checking methods, so that network studies of brain tissue become more transparent, comparable, and reproducible.