Figure 1

Setup used to examine long-term effects of ACh applications on network synchrony in open-and closed-loop regimes. a) Schematic illustration of the experimental system used to record from neurons growing on MEA substrates, maintain their viability, and apply ACh pulses at predetermined rates or at rates adjusted online to maintain network synchrony at predefined levels. b) Schematic illustration of open- and closed-loop experimental protocols. Note that in open-loop experiments ACh application timings are predefined, whereas in closed-loop experiments, ACh application timings are determined online according to instantaneous Sync Ratio values calculated by the real-time controller (item 8 in a, see Additional file 3: Figure S3 for an explanation of the Sync Ratio measure). c) Preparatory and experimental phases. Each experiment was preceded by the following preparatory phases: Phase I, slow perfusion (at least 24 hours); Phase II, activation of continuous mixing system (12 to 24 hours); Phase III, addition of AChE (0.1 U/ml) to the MEA dish and perfusion media reservoir (30 minutes, see Additional file 7: Figure S6 for the importance of AChE addition); Phase IV, insertion of ACh application needle into the MEA dish; and Phase V, experiment (open or closed loop). Note that the experimental component added in each phase was in effect from that moment until the end of the experiment. See Methods for further details. ACh, acetylcholine; AChE, acetylcholine esterase; MEA, multielectrode array.