Ryosuke Matsumura

Unidirectional signal propagation in primary neurons micropatterned at a single-cell resolution

The structure and connectivity of cultured neuronal networks can be controlled by using micropatterned surfaces. Here, we demonstrate that the direction of signal propagation can be precisely controlled at a single-cell resolution by growing primary neurons on micropatterns. To achieve this, we first examined the process by which axons develop and how synapses form in micropatterned primary neurons using immunocytochemistry. By aligning asymmetric micropatterns with a marginal gap, it was possible to pattern primary neurons with a directed polarization axis at the single-cell level. We then examined how synapses develop on micropatterned hippocampal neurons. Three types of micropatterns with different numbers of short paths for dendrite growth were compared. A normal development in synapse density was observed when micropatterns with three or more short paths were used. Finally, we performed double patch clamp recordings on micropatterned neurons to confirm that these synapses are indeed functional, and that the neuronal signal is transmitted unidirectionally in the intended orientation. This work provides a practical guideline for patterning single neurons to design functional neuronal networks in vitro with the direction of signal propagation being controlled.

An electrically resistive sheet of glial cells for amplifying signals of neuronal extracellular recordings

Electrical signals of neuronal cells can be recorded non-invasively and with a high degree of temporal resolution using multielectrode arrays (MEAs). However, signals that are recorded with these devices are small, usually 0.01%-0.1% of intracellular recordings. Here, we show that the amplitude of neuronal signals recorded with MEA devices can be amplified by covering neuronal networks with an electrically resistive sheet. The resistive sheet used in this study is a monolayer of glial cells, supportive cells in the brain. The glial cells were grown on a collagen-gel film that is permeable to oxygen and other nutrients. The impedance of the glial sheet was measured by electrochemical impedance spectroscopy, and equivalent circuit simulations were performed to theoretically investigate the effect of covering the neurons with such a resistive sheet. Finally, the effect of the resistive glial sheet was confirmed experimentally, showing a 6-fold increase in neuronal signals. This technique feasibly amplifies signals of MEA recordings.

Dependence and Homeostasis of Membrane Impedance on Cell Morphology in Cultured Hippocampal Neurons

The electrical impedance of cell membranes is important for excitable cells, such as neurons, because it strongly influences the amount of membrane potential change upon a flow of ionic current across the membrane. Here, we report on an investigation of how neuronal morphology affects membrane impedance of cultured hippocampal neurons. Microfabricated substrates with patterned scaffolding molecules were used to restrict the neurite growth of hippocampal neurons, and the impedance was measured via whole-cell patch-clamp recording under the inhibition of voltage-dependent ion channels. Membrane impedance was found to depend inversely on the dendrite length and soma area, as would be expected from the fact that its electrical property is equivalent to a parallel RC circuit. Moreover, we found that in biological neurons, the membrane impedance is homeostatically regulated to impede changes in the membrane area. The findings provide direct evidence on cell-autonomous regulation of neuronal impedance and pave the way towards elucidating the mechanism responsible for the resilience of biological neuronal networks.

Interaction of plasma-generated water cluster ions with chemically-modified Si surfaces investigated by infrared absorption spectroscopy

We have investigated the interaction of water cluster ions generated by discharge plasma, with chemically modified Si surfaces using infrared absorption spectroscopy in the multiple internal reflection geometry. We observe that water cluster ions readily adsorb on SiO2-covered Si surfaces to form waterdroplets. We demonstrate that positively- and negatively-charged cluster ions adsorb on the SiO2-covered Si surface in different manners, indicating ionic interaction of the waterdroplets with the negatively-charged SiO2surface.Waterdroplets formed on the protein-coatedsurface rupture the amide bond of the proteins, suggesting the function of protein decomposition of water cluster ions.