An experimental paradigm to investigate stimulation dependent activity in topologically constrained neuronal networks.

We present a stimulate and record paradigm to examine the behavior of multiple neuronal networks with controlled topology in vitro. Our approach enabled us to electrically induce and record neuronal activity from 60 independent networks in parallel over multiple weeks. We investigated the network performance of neuronal networks of primary hippocampal neurons until 29 days in vitro. We introduced a systematic stimulate and record protocol during which well-defined 4-node neural networks were stimulated electrically 4 times per second (4Hz) and their response was recorded over many days. We found that the network response pattern to a stimulus remained fairly stable for at least 12 h. Moreover, continuous stimulation over multiple weeks did not cause a significant change in the stimulation-induced mean spiking frequency of a circuit. We investigated the effect of stimulation amplitude and stimulation timing on the detailed network response. Finally, we could show that our setup can apply complex stimulation protocols with 125 different stimulation patterns. We used these patterns to perform basic addition tasks with the network, revealing the highly non-linear nature of biological networks' responses to complex stimuli.