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Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities.

The retina contains neural circuits that carry out computations as complex as object motion sensing, pattern recognition, and position anticipation. Models of some of these circuits have been recently discovered. A remarkable outcome of these efforts is that all such models can be constructed out of a limited set of components such as linear filters, instantaneous nonlinearities, and feedback loops. The present study explores the consequences of assuming that these components can be used to construct models for all retinal circuits. I recorded extracellularly from several retinal ganglion cells while stimulating the photoreceptors with a movie rich in temporal and spatial frequencies. Then I wrote a computer program to fit their responses by searching through large spaces of anatomically reasonable models built from a small set of circuit components. The program considers the input and output of the retinal circuit and learns its behavior without over-fitting, as verified by running the final model against previously unseen data. In other words, the program learns how to imitate the behavior of a live neural circuit and predicts its responses to new stimuli. This technique resulted in new models of retinal circuits that outperform all existing ones when run on complex spatially structured stimuli. The fitted models demonstrate, for example, that for most cells the center--surround structure is achieved in two stages, and that for some cells feedback is more accurately described by two feedback loops rather than one. Moreover, the models are able to make predictions about the behavior of cells buried deep within the retina, and such predictions were verified by independent sharp-electrode recordings. I will present these results, together with a brief collection of ideas and methods for furthering these modeling efforts in the future. / Physics

D-Serine is regarded as an obligatory co-agonist required for the activation of NMDA-type glutamate receptors (NMDARs). In the retina D-serine and a second NMDAR coagonist, glycine, are present at similar concentration and the cells that produce and release them are in close apposition. This arrangement allows for an abundant supply of coagonists and under certain conditions the NMDAR coagonist binding site could be saturated. There is also evidence suggesting that D-serine can act in an inhibitory manner at AMPA/kainate-type glutamate receptors (GluRs). Glutamate receptor activation can lead to direct and indirect elevation of intracellular calcium (Ca2+) concentration ([Ca2+]i). Therefore, in this thesis, I predominantly used Ca2+ imaging techniques to study the effect of D-serine on GluR activation in the mammalian retina. I first describe a novel method I developed to load retinal cells with Ca2+ indicator dye using electroporation and show that retinas remain viable and responsive following electroporation. This technique was used to explore the excitatory role of D-serine at NMDARs and its potential inhibition of AMPA/kainate receptors using cultured retinal ganglion cells (RGCs) and isolated retina preparations. Using cultured RGCs I demonstrated that D-serine and glycine enhance NMDAR-mediated Ca2+ responses in a concentration-dependent manner and are equally effective as coagonists. In isolated retinas I showed that D-serine application enhanced NMDA-induced responses consistent with sub-saturating endogenous coagonist concentration. Degradation of endogenous D-serine reduced NMDAR-mediated Ca2+ responses supporting the contribution of this coagonist to NMDAR activation in the retina. Using imaging and two different electrophysiological approaches, I found that D-serine reduced AMPA/kainate receptor-mediated responses in cultured RGCs and isolated retinas at concentrations that are saturating at NMDARs. Antagonist experiments suggest that the majority of inhibition is due to D-serine acting on AMPA receptor activity. Degradation of endogenous D-serine enhanced AMPA/kainate-induced responses of some cells in isolated retina suggesting that, under these conditions, D-serine concentration may be sufficient to inhibit AMPA receptor activity. Overall, the work in this thesis illustrates the utility of electroporation as a method to load Ca2+-sensitive fluorescent dyes into retinal cells and highlights the potential role for D-serine as a modulator of ionotropic GluRs in the CNS.

Die vorliegende Arbeit befasst sich mit der Anwendbarkeit von transparenter Elektronik basierend auf oxidischen Halbleitern in Multielektrodenarrays zur Messung von neuronalen Signalen. Im ersten experimentellen Kapitel werden auf Zinkoxid basierende Bauelemente untersucht. Verschiedene Varianten von Feldeffekttransistoren (FETs) werden charakterisiert und ihre Eignung zur Detektion von Zellsignalen überprüft. Die Anwendbarkeit physikalischer Modelle zur Beschreibung von ZnO-basierten Metal-Halbleiter-FETs (MESFETs) wird behandelt. Weiterhin wird die Eignung von einfachen Inverterschaltungen zur Spannungsverstärkung diskutiert. Das zweite Kapitel thematisiert Rauschmessungen an unterschiedlichen ZnO-basierten Proben, darunter Dünnfilme, Mikronadeln, MESFETs und Inverter. Darauf aufbauend wird die Auswirkung des gemessenen Stromrauschens auf die Sensitivität der Bauelemente nachvollzogen und theoretisch modelliert. Im dritten Kapitel wird das Verhalten der Bauelemente im Kontakt mit Elektolyt beschrieben. Die Signalübertragung von Spannungsänderungen im Elektrolyt auf die Chipelektronik wird mit verschiedenen Messmethoden charakterisiert. Dabei kommt teilweise ein selbstgebauter Vorverstärker zum Einsatz, dessen Aufbau ebenfalls beschrieben wird. Die Stabilität der verwendeten Materialien in physiologischen Salzlösungen und ihre Biokompatibilität wird überprüft. Darüber hinaus werden FETs mit Elektrolytgate und Zinkzinnoxid-Kanal vorgestellt.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22335号 / 医博第4576号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 岩田 想, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The purpose of this study is to find out that if antibiotic streptomycin decreases neuronal network activity or affects the pharmacological responses. The experiments in this study were conducted via MEA (multi-electrode array) technology which records neuronal activity from devices that have multiple small electrodes, serve as neural interfaces connecting neurons to electronic circuitry. The result of this study shows that streptomycin lowered the spike production of neuronal network, and also, sensitization was seen when neuronal network pre-exposed to streptomycin.

Die vorliegende Arbeit befasst sich mit der Anwendbarkeit von transparenter Elektronik basierend auf oxidischen Halbleitern in Multielektrodenarrays zur Messung von neuronalen Signalen. Im ersten experimentellen Kapitel werden auf Zinkoxid basierende Bauelemente untersucht. Verschiedene Varianten von Feldeffekttransistoren (FETs) werden charakterisiert und ihre Eignung zur Detektion von Zellsignalen überprüft. Die Anwendbarkeit physikalischer Modelle zur Beschreibung von ZnO-basierten Metal-Halbleiter-FETs (MESFETs) wird behandelt. Weiterhin wird die Eignung von einfachen Inverterschaltungen zur Spannungsverstärkung diskutiert. Das zweite Kapitel thematisiert Rauschmessungen an unterschiedlichen ZnO-basierten Proben, darunter Dünnfilme, Mikronadeln, MESFETs und Inverter. Darauf aufbauend wird die Auswirkung des gemessenen Stromrauschens auf die Sensitivität der Bauelemente nachvollzogen und theoretisch modelliert. Im dritten Kapitel wird das Verhalten der Bauelemente im Kontakt mit Elektolyt beschrieben. Die Signalübertragung von Spannungsänderungen im Elektrolyt auf die Chipelektronik wird mit verschiedenen Messmethoden charakterisiert. Dabei kommt teilweise ein selbstgebauter Vorverstärker zum Einsatz, dessen Aufbau ebenfalls beschrieben wird. Die Stabilität der verwendeten Materialien in physiologischen Salzlösungen und ihre Biokompatibilität wird überprüft. Darüber hinaus werden FETs mit Elektrolytgate und Zinkzinnoxid-Kanal vorgestellt.:1. Introduction 2. Measurement Setup and Sample Fabrication 2.1. Device Fabrication 2.2. Measurement Methods 2.3. Current Amplifier with Offset Compensation 3. Oxide Semiconductor Based Devices 3.1. Theoretical Description 3.2. Thin Films 3.4. Simple Inverter 3.5. Test Circuit for Active Matrix Configurations 4. Noise 4.1. Noise Sources 4.2. Contributions from Measurement Setup 4.3. Homogenous ZnO Samples 4.4. ZnO Based Devices 5. Experiments in Electrolyte and with Cells 5.1. Cell-Transistor Coupling 5.2. Materials in Electrolytical and Biological Environment 5.3. Electrode Arrays with Field-Effect Transistors 5.4. Electrode Arrays with Simple Inverters 5.5. Electrode Arrays with Solution Gated Transistors 6. Conclusion and Outlook Appendices Bibliography Symbols and Abbreviations List of Own and Contributed Articles Acknowledgements

The study of cultured neuronal networks has opened up avenues for understanding the ion channels, receptor molecules, and synaptic plasticity that may form the basis of learning and memory. The hippocampal neurons prepared from Wistar rats and put in culture, show, after a few days, spontaneous activity with typical electrophysiological pattern ranging from stochastic spiking to synchronized bursting. Using a multi-electrode array (MEA) having 64 electrodes, the electrophysiological signals are acquired, and connectivity maps are constructed using correlation matrix to understand how the neurons in a network communicate during the burst. The response of the neuronal system to epilepsy caused by induced glutamate injury and subsequent exposure of the system to phenobarbital to form different connectivity networks is analyzed in this study. The correlation matrix of the neuronal network before and after administering glutamate as well as after administering phenobarbital is used to understand the neuronal and network level changes that take place in the system. In order to interface a neuronal network to a physical world, the major computations to be performed are noise removal, pattern recovery, pattern matching and clustering. These computations are to be performed in real time. The system should be able to identify a pattern and relate a physical task to the pattern in about 200-400 ms. Algorithms have been developed for the implementation of a real-time neuronal system on a multi-node digital processor system.

En el presente trabajo, utilizamos técnicas de microfabricación, simulaciones numéricas, experimentos de electrofisiología para explorar la viabilidad en me- jorar la interface ordenador-neurona a través de microcanales, y la biofísica para la generación de señales en los dispositivos con microcanales. También demos- tramos que los microcanales pueden ser usados como una técnica prometedora con alto rendimiento en el muestreo automático de canales iónicos a nivel subce- lular. Finalmente, se ha diseñado, fabricado y probado el micropozo-microcanal como modificación adicional a los arreglos de multielectrodos, permitiendo una alta ganancia en la relación señal/ ruido (en inglés Signal to Noise Ratio SNR), y el registro de múltiples-lugares en poblaciones de baja densidad de redes neu- ronales del hipocampo in vitro. Primero, demostramos que son de alto rendimiento los microcanales de bajo costo con interface neurona-electrodo, para el registro extracelular de la activi- dad neuronal con baja complexidad, por periodos estables de larga duración y con alta ganancia SNR. En seguida, se realiza un estudio mediante experimentos y simulaciones nu- méricas de la biofísica para la generación de las señales obtenidas de los dispositi- vos con microcanales. Basados en los resultados, racionalizamos y demostramos como es que la longitud del canal (siendo 200 μm) y la sección transversal del microcanal (siendo 12 μm2) canaliza a los potenciales de acción para estar dentro del rango de milivolts. A pesar del bajo grado de complexidad envuelto en la fabricación y aplicación, los dispositivos con microcanales otorgan una sola media de valor SNR de 101 76, lo cual es favorablemente comparable con la SNR que se obtiene de desarrollos recientes que emplean electrodos curados con CNT y Si-NWFETs. Más aún, nosotros demostramos que el microcanal es una técnica promete- dora para el alto rendimiento del muestro automático de canales iónicos a nivel subcelular: (1) Información experimental y simulaciones numéricas sugieren que las señales registradas sólo afectan los parches membranales localizados dentro del microcanal o alrededor de 100 μm de las entradas del microcanal. (2) La transferencia de masa de los componentes químicos en los microcanales fue ana- lizada por experimentos y simulaciones FEM. Los resultados muestran que los microcanales que contienen glía y tejido neuronal pueden funcionar como barre- ra de fluido/química. Los componentes químicos pueden ser solamente aplicados a diferentes compartimentos a nivel subcelular. Finalmente, basado en simulaciones numéricas y resultados experimentales, se propone que del micropozo-microcanal, obtenido de la modificación de MEA (MWMC-MEA), la longitud óptima del canal debe ser 0,3 mm y la posición 1 óptima del electrodo intracanal, hacia la entrada más cercana del microcanal, debe ser 0,1 mm. Nosotros fabricamos un prototipo de MWMC-MEA, cuyo hoyo pasante sobre las películas de Polydimethylsiloxane (PDMS) fue microtrabajado a través de la técnica de grabados reactivos de plasma de iones. La baja densidad del cultivo (57 neuronas /mm2) en el MWMC-MEAs permitió que las neuronas vivieran al menos 14 días, con lo que la señal neuronal con la máxima SNR obtenida fue de 142. 2 / In this present work, we used microfabrication techniques, numerical simulations, electrophysiological experiments to explore the feasibility of enhancing neuron-computer interfaces with microchannels and the biophysics of the signal generation in microchannel devices. We also demonstrate the microchannel can be used as a promising technique for high-throughput automatic ion-channel screening at subcellular level. Finally, a microwell-microchannel enhanced multielectrode array allowing high signal-to-noise ratio (SNR), multi-site recording from the low-density hippocampal neural network in vitro was designed, fabricated and tested. First, we demonstrate using microchannels as a low-cost neuron-electrode interface to support low-complexity, long-term-stable, high SNR extracellular recording of neural activity, with high-throughput potential. Next, the biophysics of the signal generation of microchannel devices was studied by experiments and numerical simulations. Based on the results, we demonstrate and rationalize how channels with a length of 200 μm and channel cross section of 12 μm2 yielded spike sizes in the millivolt range. Despite the low degree of complexity involved in their fabrication and use, microchannel devices provided a single-unit mean SNR of 101 76, which compares favourably with the SNR obtained from recent developments employing CNT-coated electrodes and Si-NWFETs. Moreover, we further demonstrate that the microchannel is a promising technique for high-throughput automatic ion-channel screening at subcellular level: (1) Experimental data and numerical simulations suggest that the recorded signals are only affected by the membrane patches located inside the microchannel or within 100 μm to the microchannel entrances. (2) The mass transfer of chemical compounds in microchannels was analyzed by experiments and FEM simulations. The results show that the microchannel threaded by glial and neural tissue can function as fluid/chemical barrier. Thus chemical compounds can be applied to different subcellular compartments exclusively. Finally, a microwell-microchannel enhanced MEA (MWMC-MEA), with the optimal channel length of 0.3 mm and the optimal intrachannel electrode position of 0.1 mm to the nearest channel entrance, was proposed based on numerical simulation and experiment results. We fabricated a prototype of the MWMCMEA, whose through-hole feature of Polydimethylsiloxane film (PDMS) was micromachined by reactive-ion etching. The low-density culture (57 neurons/mm2) were survived on the MWMC-MEAs for at least 14 days, from which the neuronal signal with the maximum SNR of 142 was obtained.