Endogenous cholinergic tone modulates spontaneous network level neuronal activity in primary cortical cultures grown on multi-electrode arrays

Background Cortical 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. Results Consequently, 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. Conclusions We 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.

Glimpsing regular lattice arrangements of primary rat hippocampal astrocytes on ultra-thin nodes of Parylene-C

In our seminal work, we reported how the biomaterial Parylene-C has the unique ability to coerce neurons and glial cells to migrate to and then grow in straight lines along serum coated rectangular parylene-C structures mounted on an oxidised silicon substrate. In this brief communication, we report how astrocyte cell bodies, from the dissociated postnatal rat hippocampus, can now to be successfully localised on an ultra-thin 13nm layer of parylene-C mounted on oxidised silicon (Figure 1). What is extremely interesting about this finding is that the astrocyte processes extended mainly in horizontal and vertical directions from the cell body thus creating a regular lattice network of individual cells. In addition, they comfortably extended a 50μm gap (equivalent to ~ 10 cell body diameters) to connect to adjacent astrocytes on neighbouring Parylene-C structures. This was found to occur repeatedly on circular geometries of 20μm diameter. In comparison to our previous work [1], we have decreased the thickness of the parylene-C structures by a factor of 10, to allow such technology to be able to be utilised for passive electrode design that requires extremely thin structures such as these. Thus, being able to culture astrocytes in regular lattice networks will pave the way for precise monitoring and stimulation of such ensembles via multi-electrode arrays, allowing a closer insight into their dynamic behaviour and their network properties.