A Frequency-Optimized Optogenetic Study of Network-Level Potentiation in Cortical Cultures on Microelectrode Arrays

Objective. Long-term potentiation (LTP) is a fundamental mechanism underlying learning and memory, yet its investigation at the network level in vitro remains challenging, particularly when optogenetic stimulation is used. The objective of this work is to develop a robust experimental and analytical framework for inducing and quantifying optogenetically driven LTP in neuronal cultures recorded with microelectrode arrays (MEAs). Approach. We first systematically investigate the effect of widefield optogenetic stimulation frequency on evoked neuronal activity, to identify a test-stimulus that reliably probes network responses without inducing activity modulation. By analyzing spike-rate dynamics during repeated stimulation, we characterize frequency-dependent response adaptation consistent with Channelrhodopsin-2 photocycle kinetics. Based on these results, an optimized low-frequency test-stimulus is selected and combined with a spatially confined tetanic optogenetic stimulation to induce LTP. Network responses are quantified using post-stimulus time histograms and a normalized efficacy metric, enabling electrode-wise and network-level analysis of plasticity. Main results. Low-frequency optical stimulation (