On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes.

In contrast to the extensive use of microelectrode array (MEA) technology in electrophysiological studies of cultured neurons and cardiac muscles, the vast field of skeletal muscle research has yet to adopt the technology. Here we demonstrate an empowering MEA technology for high quality, multisite, long-term electrophysiological recordings from cultured skeletal myotubes. Individual rat skeletal myotubes cultured on micrometer sized gold mushroom-shaped microelectrode (gMµE) based MEA tightly engulf the gMµEs, forming a high seal resistance between the myotubes and the gMµEs. As a consequence, spontaneous action potentials generated by the contracting myotubes are recorded as extracellular field potentials with amplitudes of up to 10 mV for over 14 days. Application of a 10 ms, 0.5-0.9 V voltage pulse through the gMµEs electroporated the myotube membrane, and transiently converted the extracellular to intracellular recording mode for 10-30 min. In a fraction of the cultures stable attenuated intracellular recordings were spontaneously produced. In these cases or after electroporation, subthreshold spontaneous potentials were also recorded. The introduction of the gMµE-MEA as a simple-to-use, high-quality electrophysiological tool together with the progress made in the use of cultured human myotubes opens up new venues for basic and clinical skeletal muscle research, preclinical drug screening, and personalized medicine.

Multisite electrophysiological recordings by self-assembled loose-patch-like junctions between cultured hippocampal neurons and mushroom-shaped microelectrodes.

Substrate integrated planar microelectrode arrays is the "gold standard" method for millisecond-resolution, long-term, large-scale, cell-noninvasive electrophysiological recordings from mammalian neuronal networks. Nevertheless, these devices suffer from drawbacks that are solved by spike-detecting, spike-sorting and signal-averaging techniques which rely on estimated parameters that require user supervision to correct errors, merge clusters and remove outliers. Here we show that primary rat hippocampal neurons grown on micrometer sized gold mushroom-shaped microelectrodes (gMµE) functionalized simply by poly-ethylene-imine/laminin undergo self-assembly processes to form loose patch-like hybrid structures. More than 90% of the hybrids formed in this way record monophasic positive action potentials (APs). Of these, 34.5% record APs with amplitudes above 300 µV and up to 5,085 µV. This self-assembled neuron-gMµE configuration improves the recording quality as compared to planar MEA. This study characterizes and analyzes the electrophysiological signaling repertoire generated by the neurons-gMµE configuration, and discusses prospects to further improve the technology.

A feasibility study of multi-site,intracellular recordings from mammalian neurons by extracellular gold mushroom-shaped microelectrodes.

The development of multi-electrode array platforms for large scale recording of neurons is at the forefront of neuro-engineering research efforts. Recently we demonstrated, at the proof-of-concept level, a breakthrough neuron-microelectrode interface in which cultured Aplysia neurons tightly engulf gold mushroom-shaped microelectrodes (gMµEs). While maintaining their extracellular position, the gMµEs record synaptic- and action-potentials with characteristic features of intracellular recordings. Here we examined the feasibility of using gMµEs for intracellular recordings from mammalian neurons. To that end we experimentally examined the innate size limits of cultured rat hippocampal neurons to engulf gMµEs and measured the width of the "extracellular" cleft formed between the neurons and the gold surface. Using the experimental results we next analyzed the expected range of gMµEs-neuron electrical coupling coefficients. We estimated that sufficient electrical coupling levels to record attenuated synaptic- and action-potentials can be reached using the gMµE-neuron configuration. The definition of the engulfment limits of the gMµEs caps diameter at ≤2-2.5 µm and the estimated electrical coupling coefficients from the simulations pave the way for rational development and application of the gMµE based concept for in-cell recordings from mammalian neurons.

Nanocrystalline diamond surfaces for adhesion and growth of primary neurons, conflicting results and rational explanation.

Using a variety of proliferating cell types, it was shown that the surface of nanocrystalline diamond (NCD) provides a permissive substrate for cell adhesion and development without the need of complex chemical functionalization prior to cell seeding. In an extensive series of experiments we found that, unlike proliferating cells, post-mitotic primary neurons do not adhere to bare NCD surfaces when cultured in defined medium. These observations raise questions on the potential use of bare NCD as an interfacing layer for neuronal devices. Nevertheless, we also found that classical chemical functionalization methods render the "hostile" bare NCD surfaces with adhesive properties that match those of classically functionalized substrates used extensively in biomedical research and applications. Based on the results, we propose a mechanism that accounts for the conflicting results; which on one hand claim that un-functionalized NCD provides a permissive substrate for cell adhesion and growth, while other reports demonstrate the opposite.