Development of in-vitro µ-channel devices for continous long-term monitoring of neuron circuit development

In this thesis various methods are presented towards long-term electrophysiological monitoring of in-vitro neuron cultures in µ-channel devices. A new µ-channel device has been developed. The StarPoM device offers multiple culture chambers connected with µ-channels allowing to study communication between neuron populations. For its fabrication an advanced multi level SU-8 soft-lithography master was developed that can mold µ-channels and culture wells simultaneously. The problem of aligning features across a thick SU-8 layer has been solved by integrating a chrome mask into the substrate and then using backside exposure through the chrome mask. A long-term monitoring of neuron electrophysiological activity has been conducted continuously during 14 days in the StarPoM device. For the analysis of the recorded dataset a new software tool-chain has been created with the goal of high processing performance. The two most advanced components - O1Plot and ISI viewer - offer high performance visualization of time series data with event or interval annotation and visualization of inter-spike interval histograms for fast discovery of correlations between spike units on a device. The analysis of the 14 day recording revealed that signals can be recorded from day 4/5 onwards. While maximum spike amplitudes in kept rising during the 14 days and reached up to 3.16 mV, the average spike amplitudes reached their maximum of 0.1-0.3 mV within 6 to 8 days and then kept the amplitudes stable. To better understand the biophysics of signal generation in µ-channels, the influence of µ-channel length on signal amplitude was studied. A model based on the passive cable theory was developed showing that spike amplitude rises with channel length for µ-channels < 250 µm. In longer µ-channels, further growth of spike amplitude is inhibited by cancellation of positive and negative spike phase. Also, clogging of the µ-channel entrances by cells and debris helps to enhance signal amplification

Closed-loop approaches for innovative neuroprostheses

The goal of this thesis is to study new ways to interact with the nervous system in case of damage or pathology. In particular, I focused my effort towards the development of innovative, closed-loop stimulation protocols in various scenarios: in vitro, ex vivo, in vivo

Anesthetic isoflurane influences baseline firing and disrupts chemosensitivity of 5-HT and GABA raphe neurons

Thesis (M.S.) University of Alaska Fairbanks, 2014General anesthetics are widely used in clinical and scientific contexts, but their molecular mechanisms, and how these mechanisms give rise to the state of anesthesia, are poorly understood. We investigated the influence of the volatile anesthetic isoflurane on serotonin (5-hydroxytryptamine; 5-HT) and γ-aminobutyric acid (GABA) synthesizing raphé neurons. These cell types have been proposed as central chemoreceptors, cells that sense changes in arterial CO₂/pH and stimulate respiratory output to regain homeostasis. We tested the hypotheses that isoflurane inhibits 5-HT neuron baseline firing, enhances GABA neuron baseline firing, and disrupts the chemosensitivity of both neuron types. We performed extracellular recordings in the medullary raphé using the unanesthetized in situ perfused brainstem preparation. Subsets of neurons were labeled with biotinamide using the juxtacellular labeling method and immunohistochemically identified by neurotransmitter phenotype. Results indicated that isoflurane inhibited action potential discharge in 5-HT neurons. Isoflurane inhibited action potential discharge in a subset of CO₂-inhibited putative GABA neurons and enhanced action potential discharge in a different subset of these neurons. Isoflurane disrupted the chemosensitivity of both 5-HT and GABA neurons. Disruption of 5-HT and GABA neuron chemosensitivity by isoflurane may contribute to the blunted hypercapnic ventilatory response that is a secondary effect of general anesthesia.1. Introduction -- 1.1 Introduction to general anesthetics -- 1.2 Secondary effects of general anesthetics -- 1.3 Theories of general anesthetic mechanisms of action -- 1.4 Working hypothesis describing central depression in anesthesia -- 1.5 Target populations in the medullary raphé of the brainstem -- 1.6 Introduction to the 5-HT system -- 1.7 Pathology associated with raphé 5-HT neurons -- 1.8 Role of medullary raphé 5-HT neurons in central respiratory control -- 1.9 Identity of 5-HT neurons as central chemoreceptors is controversial -- 1.10 Role of medullary raphé GABA neurons in central respiratory control -- 1.11 Specific aims -- 1.12 Experimental approach -- 1.13 Major findings -- 1.14 Literature cited -- 1.15 Figures -- 2. Isoflurane dose-dependently inhibits medullary raphé 5-HT neurons in situ -- 3. The response of CO2-inhibited neurons to isoflurane: Evidence for a heterogeneous population of medullary raphé GABA neurons -- 4. Conclusion -- Appendices

Microchannel enhanced neuron-computer interface: design, fabrication, biophysics of signal generation, signal strength optimization, and its applications to ion-channel screening and basic neuroscience research

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. 2In 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

Low-dimensional representations of neural time-series data with applications to peripheral nerve decoding

Bioelectronic medicines, implanted devices that influence physiological states by peripheral neuromodulation, have promise as a new way of treating diverse conditions from rheumatism to diabetes. We here explore ways of creating nerve-based feedback for the implanted systems to act in a dynamically adapting closed loop. In a first empirical component, we carried out decoding studies on in vivo recordings of cat and rat bladder afferents. In a low-resolution data-set, we selected informative frequency bands of the neural activity using information theory to then relate to bladder pressure. In a second high-resolution dataset, we analysed the population code for bladder pressure, again using information theory, and proposed an informed decoding approach that promises enhanced robustness and automatic re-calibration by creating a low-dimensional population vector. Coming from a different direction of more general time-series analysis, we embedded a set of peripheral nerve recordings in a space of main firing characteristics by dimensionality reduction in a high-dimensional feature-space and automatically proposed single efficiently implementable estimators for each identified characteristic. For bioelectronic medicines, this feature-based pre-processing method enables an online signal characterisation of low-resolution data where spike sorting is impossible but simple power-measures discard informative structure. Analyses were based on surrogate data from a self-developed and flexibly adaptable computer model that we made publicly available. The wider utility of two feature-based analysis methods developed in this work was demonstrated on a variety of datasets from across science and industry. (1) Our feature-based generation of interpretable low-dimensional embeddings for unknown time-series datasets answers a need for simplifying and harvesting the growing body of sequential data that characterises modern science. (2) We propose an additional, supervised pipeline to tailor feature subsets to collections of classification problems. On a literature standard library of time-series classification tasks, we distilled 22 generically useful estimators and made them easily accessible.Open Acces

Multi-electrode array recording and data analysis methods for molluscan central nervous systems

In this work the use of the central nervous system (CNS) of the aquatic snail Lymnaea stagnalis on planar multi-electrode arrays (MEAs) was developed and analysis methods for the data generated were created. A variety of different combinations of configurations of tissue from the Lymnaea CNS were explored to determine the signal characteristics that could be recorded by sixty channel MEAs. In particular, the suitability of the semi-intact system consisting of the lips, oesophagus, CNS, and associated nerve connectives was developed for use on the planar MEA. The recording target area of the dorsal surface of the buccal ganglia was selected as being the most promising for study and recordings of its component cells during fictive feeding behaviour stimulated by sucrose were made. The data produced by this type of experimentation is very high volume and so its analysis required the development of a custom set of software tools. The goal of this tool set is to find the signal from individual neurons in the data streams of the electrodes of a planar MEA, to estimate their position, and then to predict their causal connectivity. To produce such an analysis techniques for noise filtration, neural spike detection, and group detection of bursts of spikes were created to pre-process electrode data streams. The Kohonen self-organising map (SOM) algorithm was adapted for the purpose of separating detected spikes into data streams representing the spike output of individual cells found in the target system. A significant addition to SOM algorithm was developed by the concurrent use of triangulation methods based on current source density analysis to predict the position of individual cells based on their spike output on more than one electrode. The likely functional connectivity of individual neurons identified by the SOM technique were analysed through the use of a statistical causality method known as Granger causality/causal connectivity. This technique was used to produce a map of the likely connectivity between neural sources

Advances in Bioengineering

The technological approach and the high level of innovation make bioengineering extremely dynamic and this forces researchers to continuous updating. It involves the publication of the results of the latest scientific research. This book covers a wide range of aspects and issues related to advances in bioengineering research with a particular focus on innovative technologies and applications. The book consists of 13 scientific contributions divided in four sections: Materials Science; Biosensors. Electronics and Telemetry; Light Therapy; Computing and Analysis Techniques

Neuroelectronic and Nanophotonic Devices Based on Nanocoaxial Arrays

Thesis advisor: Michael J. NaughtonThesis advisor: Michael J. BurnsRecent progress in the study of the brain has been greatly facilitated by the development of new measurement tools capable of minimally-invasive, robust coupling to neuronal assemblies. Two prominent examples are the microelectrode array, which enables electrical signals from large numbers of neurons to be detected and spatiotemporally correlated, and optogenetics, which enables the electrical activity of cells to be controlled with light. In the former case, high spatial density is desirable but, as electrode arrays evolve toward higher density and thus smaller pitch, electrical crosstalk increases. In the latter, finer control over light input is desirable, to enable improved studies of neuroelectronic pathways emanating from specific cell stimulation. Herein, we introduce a coaxial electrode architecture that is uniquely suited to address these issues, as it can simultaneously be utilized as an optical waveguide and a shielded electrode in dense arrays.Thesis (PhD) — Boston College, 2017.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Physics

INTEGRATION OF CMOS TECHNOLOGY INTO LAB-ON-CHIP SYSTEMS APPLIED TO THE DEVELOPMENT OF A BIOELECTRONIC NOSE

This work addresses the development of a lab-on-a-chip (LOC) system for olfactory sensing. The method of sensing employed is cell-based, utilizing living cells to sense stimuli that are otherwise not easily sensed using conventional transduction techniques. Cells have evolved over millions of years to be exquisitely sensitive to their environment, with certain types of cells producing electrical signals in response to stimuli. The core device that is introduced here is comprised of living olfactory sensory neurons (OSNs) on top of a complementary metal-oxide-semiconductor (CMOS) integrated circuit (IC). This hybrid bioelectronic approach to sensing leverages the sensitivity of OSNs with the electronic signal processing capability of modern ICs. Intimately combining electronics with biology presents a number of unique challenges to integration that arise from the disparate requirements of the two separate domains. Fundamentally the obstacles arise from the facts that electronic devices are designed to work in dry environments while biology requires not only a wet environment, but also one that is precisely controlled and non-toxic. Design and modeling of such heterogeneously integrated systems is complicated by the lack of tools that can address the multiple domains and techniques required for integration, namely IC design, fluidics, packaging, and microfabrication, and cell culture. There also arises the issue of how to handle the vast amount of data that can be generated by such systems, and specifically how to efficiently identify signals of interest and communicate them off-chip. The primary contributions of this work are the development of a new packaging scheme for integration of CMOS ICs into fluidic LOC systems, a methodology for cross-coupled multi-domain iterative modeling of heterogeneously integrated systems, demonstration of a proof-of-concept bioelectronic olfactory sensor, and a novel event-based technique to minimize the bandwidth required to communicate the information contained in bio-potential signals produced by dense arrays of electrically active cells