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Electrical Responses and Spontaneous Activity of Human iPS-Derived Neuronal Networks Characterized for 3-month Culture with 4096-Electrode Arrays.

ABSTRACT: The recent availability of human induced pluripotent stem cells (hiPSCs) holds great promise as a novel source of human-derived neurons for cell and tissue therapies as well as for in vitro drug screenings that might replace the use of animal models. However, there is still a considerable lack of knowledge on the functional properties of hiPSC-derived neuronal networks, thus limiting their application. Here, upon optimization of cell culture protocols, we demonstrate that both spontaneous and evoked electrical spiking activities of these networks can be characterized on-chip by taking advantage of the resolution provided by CMOS multielectrode arrays (CMOS-MEAs). These devices feature a large and closely-spaced array of 4096 simultaneously recording electrodes and multi-site on-chip electrical stimulation. Our results show that networks of human-derived neurons can respond to electrical stimulation with a physiological repertoire of spike waveforms after 3 months of cell culture, a period of time during which the network undergoes the expression of developing patterns of spontaneous spiking activity. To achieve this, we have investigated the impact on the network formation and on the emerging network-wide functional properties induced by different biochemical substrates, i.e., poly-dl-ornithine (PDLO), poly-l-ornithine (PLO), and polyethylenimine (PEI), that were used as adhesion promoters for the cell culture. Interestingly, we found that neuronal networks grown on PDLO coated substrates show significantly higher spontaneous firing activity, reliable responses to low-frequency electrical stimuli, and an appropriate level of PSD-95 that may denote a physiological neuronal maturation profile and synapse stabilization. However, our results also suggest that even 3-month culture might not be sufficient for human-derived neuronal network maturation. Taken together, our results highlight the tight relationship existing between substrate coatings and emerging network properties, i.e., spontaneous activity, responsiveness, synapse formation and maturation. Additionally, our results provide a baseline on the functional properties expressed over 3 months of network development for a commercially available line of hiPSC-derived neurons. This is a first step toward the development of functional pre-clinical assays to test pharmaceutical compounds on human-derived neuronal networks with CMOS-MEAs.

SUBMITTER: Amin H

PROVIDER: S-EPMC4811967 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCortical cultures grown long-term on multi-electrode arrays (MEAs) are frequently and extensively used as models of cortical networks in studies of neuronal firing activity, neuropharmacology, toxicology and mechanisms underlying synaptic plasticity. However, in contrast to the predominantly asynchronous neuronal firing activity exhibited by intact cortex, electrophysiological activity of mature cortical cultures is dominated by spontaneous epileptiform-like global burst events which hinders their effective use in network-level studies, particularly for neurally-controlled animat ('artificial animal') applications. Thus, the identification of culture features that can be exploited to produce neuronal activity more representative of that seen in vivo could increase the utility and relevance of studies that employ these preparations. Acetylcholine has a recognised neuromodulatory role affecting excitability, rhythmicity, plasticity and information flow in vivo although its endogenous production by cortical cultures and subsequent functional influence upon neuronal excitability remains unknown.ResultsConsequently, using MEA electrophysiological recording supported by immunohistochemical and RT-qPCR methods, we demonstrate for the first time, the presence of intrinsic cholinergic neurons and significant, endogenous cholinergic tone in cortical cultures with a characterisation of the muscarinic and nicotinic components that underlie modulation of spontaneous neuronal activity. We found that tonic muscarinic ACh receptor (mAChR) activation affects global excitability and burst event regularity in a culture age-dependent manner whilst, in contrast, tonic nicotinic ACh receptor (nAChR) activation can modulate burst duration and the proportion of spikes occurring within bursts in a spatio-temporal fashion.ConclusionsWe suggest that the presence of significant endogenous cholinergic tone in cortical cultures and the comparability of its modulatory effects to those seen in intact brain tissues support emerging, exploitable commonalities between in vivo and in vitro preparations. We conclude that experimental manipulation of endogenous cholinergic tone could offer a novel opportunity to improve the use of cortical cultures for studies of network-level mechanisms in a manner that remains largely consistent with its functional role.

Project description:BackgroundThe lack of understanding of fascicular organisation in peripheral nerves limits the potential of vagus nerve stimulation therapy. Two promising methods may be employed to identify the functional anatomy of fascicles within the nerve: fast neural electrical impedance tomography (EIT), and penetrating multi-electrode arrays (MEA). These could provide a means to image the compound action potential within fascicles in the nerve.New methodWe compared the ability to localise fascicle activity between silicon shanks (SS) and carbon fibre (CF) multi-electrode arrays and fast neural EIT, with micro-computed tomography (MicroCT) as an independent reference. Fast neural EIT in peripheral nerves was only recently developed and MEA technology has been used only sparingly in nerves and not for source localisation. Assessment was performed in rat sciatic nerves while evoking neural activity in the tibial and peroneal fascicles.ResultsRecorded compound action potentials were larger with CF compared to SS (∼700 μV vs ∼300 μV); however, background noise was greater (6.3 μV vs 1.7 μV) leading to lower SNR. Maximum spatial discrimination between Centres-of-Mass of fascicular activity was achieved by fast neural EIT (402 ± 30 μm) and CF MEA (414 ± 123 μm), with no statistical difference between MicroCT (625 ± 17 μm) and CF (p > 0.05) and between CF and EIT (p > 0.05). Compared to CF MEAs, SS MEAs had a lower discrimination power (103 ± 51 μm, p < 0.05).Comparison with existing methodsEIT and CF MEAs showed localisation power closest to MicroCT. Silicon MEAs adopted in this study failed to discriminate fascicle location. Re-design of probe geometry may improve results.ConclusionsNerve EIT is an accurate tool for assessment of fascicular position within nerves. Accuracy of EIT and CF MEA is similar to the reference method. We give technical recommendations for performing multi-electrode recordings in nerves.

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Electrical Responses and Spontaneous Activity of Human iPS-Derived Neuronal Networks Characterized for 3-month Culture with 4096-Electrode Arrays.

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