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

TL;DR

This study develops and validates a robust framework for optogenetically induced long-term potentiation (LTP) in cortical networks cultured on microelectrode arrays. By systematically optimizing a low-frequency widefield test-stimulus to minimize ChR2 desensitization, the authors establish a reliable probe of network excitability, then pair it with spatially confined tetanic optical stimulation to elicit and quantify LTP. They introduce PSTH-based metrics, including an efficacy measure and a Potentiation Index, to capture electrode- and network-level plasticity and demonstrate lasting potentiation across multiple experiments. The approach provides a reproducible methodology for studying activity-dependent plasticity in optogenetically driven in vitro networks, with implications for mechanistic insights into learning-like processes in simplified systems, and the data and code are openly shared ($$data$$ and $$code$$).

Abstract

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 (<= 0.2 Hz) preserves stable evoked responses, whereas higher frequencies induce a pronounced sigmoid-like decay in firing rate. Following tetanic stimulation, a subset of electrodes exhibits robust and long-lasting potentiation, persisting for several hours. Significance. This work provides a systematic methodology for studying activity-dependent plasticity in optogenetically driven neuronal networks.

Figures (4)

• Figure 1: (A): Configuration of the 60 recording electrodes in the MEA chip 60MEA200/30iR-Ti (MCS). The numbering of the MEA electrodes arranged in the 8×8 grid follows the conventional scheme used for square arrays. In this system, the first digit identifies the column number, while the second digit corresponds to the row number. This systematic notation allows for an unambiguous localization of each electrode within the array and ensures consistency between experimental setups and data analysis. Position 15 is occupied by a larger internal reference electrode. (B): Acquisition of YFP image to confirm the expression of ChR2 in the culture. (C): Stimulation protocol for LTP experiments. In this representation the test-stimulus is chosen of frequency $0.2$ Hz ($\sim$ 17 min of stimulation, see table \ref{['tab1']}). During the ‘Recalling phase' the test-stimulus is repeated at time-steps 0, 30, 60, 90, 120, 180 (min) after the conclusion of the tetanic stimulation. The blue box shows the time windows $T_r$ and $T_b$ for the analysis of the spike rate (clarified in section \ref{['sec:WF_results']}), whilst the orange box depicts the stimulation time-pattern used for the tetanus.
• Figure 2: Main results for the test-stimulus. (A)-(B): Examples of spike rate's evolution for the frequencies $0.2$ Hz and $2.0$ Hz for the same electrode (window $T_r$). On the x-axis, $t/T$ is the fraction between the stimulation time and the pulse period $T$ and corresponds to the identification number of each of the total 200 pulses. (C): Evolution of the average decay rate $k$ of the fitting sigmoid, as a function of the stimulation frequency $f_0$ used. (D): Evolution of the average firing rate decline $\Delta$, as a function of the stimulation frequency $f_0$ used. The data corresponding to the frequencies $0.1$ and $0.2$ Hz are omitted, since sigmoidal fitting becomes difficult for the almost flat behavior.
• Figure 3: $\Delta A$ as a function of time for a single LTP-dataset. The plot shows the evolution of $\Delta A$ at different time steps for one of the 7 datasets. The x-axis shows all the 6 post-tetanus times plus the additional ‘baseline time' $-37$ min (this is the moment at which Control 2 stimulation started). Values are plotted in grey, red, or blue according to the classification explained in the text. The two electrodes chosen for tetanic stimulation are highlighted in yellow. The $S$ value for each electrode is indicated by a green dotted line (in subplots where the $S$ level falls outside the y-axis range defined by the data points, the line is not displayed).
• Figure 4: (A): The plot shows the mean value of $\Delta A$ computed across all electrodes classified as red and blue, at the different times after the LTP induction protocol. The additional data at -$37$ min correspond to $\Delta A$ evaluated for Control 2, to give a baseline reference. The yellow arrow indicates the moment of the start of the tetanic stimulation. (B): Average Potentiation Index (PI) as a function of time post-LTP, computed across the 7 independent datasets. Each point represents the mean PI value at a given time, with error bars indicating the standard deviation across experiments.