Toward the development of biocompatible surfaces for implantable electrode arrays and the creation of patterned neuronal networks, the impact of select biochemical substrates [poly-D-lysine (PDL), polyornithine (PO), polyethylenimine (PEI), and a basement membrane extract (BM)] on network morphology and spontaneous electrophysiological activity of dissociated hippocampal neurons was investigated. Cultured in serum-free Neurobasal medium at 100 000 cells/cm(2), neurons attached to each substrate. PDL, PO, and PEI induced little or no neuronal clustering and process fasciculation, whereas the addition of BM promoted these features. The ratios of somas to processes, and axons to dendrites, as determined by immunohistochemical staining and image analysis were comparable across all substrates. Spontaneous firing was recorded using planar multielectrode arrays (MEAs) at the third week in vitro for the two most divergent morphologies according to Euclidian cluster analysis, namely those induced by PO + BM and PEI. Mean spike amplitude, mean firing rate, median interspike interval (ISI), mean burst rate, and correlation index were analyzed and compared to morphological features. Synchronized bursting was highly correlated with neuronal clustering and process fasciculation. Spike amplitude was negatively correlated with thin branching which was most evident in neurons grown on PEI. These data indicate that factors, which influence adherence of neurons to surfaces, can profoundly impact both neuronal network morphology and electrophysiological activity in vitro.