Multielectrode arrays may have several advantages compared to the traditional single macroelectrode brain electrical stimulation technique including less tissue damage due to implantation and the ability to deliver several spatio-temporal patterns of stimulation. Prior work on cell cultures has shown that multielectrode arrays are capable of completely stopping seizure-like spontaneous bursting events through a distributed asynchronous multi-site approach. In my studies, I used a similar approach for controlling seizures in a rat model of temporal lobe epilepsy. First, I developed a new method of electroplating in vivo microelectrode arrays for durably improving their impedance. I showed that microelectrode arrays electroplated through the new technique called sonicoplating, required the least amount of voltage in current controlled stimulation studies and also produced the least amplitude and duration of stimulation artifact compared to unplated, DC electroplated or pulse-plated microelectrodes. Second, using c-fos immunohistochemistry, I showed that 16-electrode sonicoplated microelectrode arrays can activate 5.9 times more neurons in the dorsal hippocampus compared to a single macroelectrodes while causing < 77% the tissue damage. Next, through open-loop multisite asynchronous microstimulation, I reduced seizure frequency by ~50% in the rodent model of temporal lobe epilepsy. Preliminary studies aimed at using the same stimulation protocol in closed-loop responsive and predictive seizure control did not stop seizures. Finally, through an internship at Medtronic Neuromodulation, I worked on developing and implementing a rapid algorithm prototyping research tool for closed-loop human deep brain stimulation applications.

The thesis presents a new paradigm for studying the importance of interactions between an organism and its environment using a combination of biology and technology: embodying cultured cortical neurons via robotics. From this platform, explanations of the emergent neural network properties leading to cognition are sought through detailed electrical observation of neural activity. By growing the networks of neurons and glia over multi-electrode arrays (MEA), which can be used to both stimulate and record the activity of multiple neurons in parallel over months, a long-term real-time 2-way communication with the neural network becomes possible. A better understanding of the processes leading to biological cognition can, in turn, facilitate progress in understanding neural pathologies, designing neural prosthetics, and creating fundamentally different types of artificial cognition. Here, methods were first developed to reliably induce and detect neural plasticity using MEAs. This knowledge was then applied to construct sensory-motor mappings and training algorithms that produced adaptive goal-directed behavior. To paraphrase the results, most any stimulation could induce neural plasticity, while the inclusion of temporal and/or spatial information about neural activity was needed to identify plasticity. Interestingly, the plasticity of action potential propagation in axons was observed. This is a notion counter to the dominant theories of neural plasticity that focus on synaptic efficacies and is suggestive of a vast and novel computational mechanism for learning and memory in the brain. Adaptive goal-directed behavior was achieved by using patterned training stimuli, contingent on behavioral performance, to sculpt the network into behaviorally appropriate functional states: network plasticity was not only induced, but could be customized. Clinically, understanding the relationships between electrical stimulation, neural activity, and the functional expression of neural plasticity could assist neuro-rehabilitation and the design of neuroprosthetics. In a broader context, the networks were also embodied with a robotic drawing machine exhibited in galleries throughout the world. This provided a forum to educate the public and critically discuss neuroscience, robotics, neural interfaces, cybernetics, bio-art, and the ethics of biotechnology.

No description available.

During a traumatic insult to the brain, tissue is subjected to large stresses at high rates which often surpass cellular thresholds leading to cell dysfunction or death. Cellular events that occur at the time of and immediately after an insult are poorly understood. Immediately following traumatic brain injury (TBI), the neuronal plasma membrane may become disrupted and potentiate detrimental pathways by allowing extracellular contents to gain access to the cytosol. In the current study, neuronal plasma membrane disruption was assessed in vivo following moderate unilateral controlled cortical impact in rats using a normally cell-impermeant fluorescent compound as a plasma membrane permeability marker. This fluorescent dye was injected into the cerebrospinal fluid and was allowed to diffuse into the brain. TBI caused a widespread acute disruption of neuronal membranes which was significantly different compared to uninjured brains. Affected cells were present in cortex and hippocampal regions. These findings were complemented by an in vitro model of TBI where membrane disruption was quantified and its mechanisms elucidated. Permeability marker(s) were added to neuronal cultures before the insult as indicators for increases in plasma membrane permeability. The percentage of cells containing the permeability marker was dependent on the molecular mass, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were also positively correlated with the rate of insult. Membrane disruption was transient, evidenced by a robust resealing within the first minute after the insult. In addition, membrane resealing was found to be dependent on extracellular Ca2+, as chelation of the ion abolished a significant amount of resealing. We have also investigated the effects of mechanically-induced plasma membrane disruptions on neuronal network electrical activity. We have developed a multielectrode array system that allows the study of electrical activity before, during, and after a traumatic insult to neurons. Endogenous electrical activity of neuronal cultures presented a heterogeneous response following mechanical insult. Moreover, spontaneous firing dysfunction induced by injury outlasted the presence of membrane disruptions. This study provides a multi-faceted approach to elucidate the role of neuronal plasma membrane disruptions in TBI and its functional consequences.

An Active Microscaffold System with Fluid Delivery and Stimulation/Recording Functionalities for Culturing 3-D Neuronal Networks Laura Elizabeth Rowe 215 Pages Directed by Dr. A. Bruno Frazier An active microscaffold system with fluid delivery and electrical stimulation/recording functionalities for 3-D neuronal culture studies is presented. The microscaffolds presented in this dissertation consist of an array of microfabricated towers with integrated microfluidic channels, fluid ports, and electrodes. The microfluidic channels and ports allow for perfusion of nutrients, gas exchange, and biochemical control of the extracellular environment throughout the 3-D culture, while the electrodes allow for active stimulation/recording of the 3-D neuronal network. In essence, the microscaffold serves as an artificial circulatory system to enable 3-D in vitro growth and proliferation of re-aggregate neuronal cell cultures. Increased cell survival on microscaffolds with nutrient perfusion at 14 and 21 days in vitro (DIV) is presented. Additionally, the microtower scaffold is built upon a substrate that is compatible with the Multi Channel Systems preamplifier setup to enable electrical stimulation/recording of the cultured network in a 3-D mutilelectrode array (MEA) environment. Impedance measurements on the functioning microtower electrodes were obtained. The overall goal of this research was to develop a BioMEMS technology to provide neuroscientists with a better investigative tool for studying 3-D in vitro neuronal networks than is currently available.

Práce se zabývá zarovnáváním excitabilních buněk na multielektrodových polích. Nejprve bylo analyzováno zarovnávání excitabilních buněk. Byly použity embryonální neurony z hippocampusu potkanů a HL-1 buňky, které jsou odvozeny z AT-1 linie nádorových myších atriálních kardiomyocytů. Zarovnávání bylo testováno na drážkovaných površích a na površích s materiály s různou buněčnou afinitou. Bylo prokázáno, že na drážkových površích se ve směru drážek zarovnávají neurony i HL-1 buňky, ale na površích s různou chemickou affinitou se zarovnávají pouze neurony. Dále byly vyrobeny vlastní multielektrodová pole, na těchto multielektrodových polích byly kultivovány HL-1 buňky a byl změřen a analyzován akčních potenciál HL-1 buněk. Cílem bylo prokázat, že je možné měřit akční potenciál na vyrobených multielektrodových polích. Pro zarovnání buněk na multielektrodovém poli bylo vyrobeno speciální multieletrodové pole s uniformním povrchem. Toto multielektrodové pole je nazýváno planární multielektrodové pole. Planární multielektrodové pole bylo vyrobeno speciálním vyrobním procesem. Vrstvy planárního multielektrodového pole byly deponovány na pomocný substrát v opačném pořadí. Pomocný substrátem pro depozici byla křemíková deska, na který byla nadeponována další pomocná vrstva zlata. Horní izolační vrstva planárního multielektrodové pole byla deponována jako první a nejspodnější vrstva substrátu byla nadeponována jako poslední. Planární multielektrodové pole i s pomocnou zlatou vrstvou bylo strhnuto s křemíku díky nízké adhezi zlata ke křemíku a planární multielektrodové pole se otočilo vzhůru nohama. Pomocná zlatá vrstva byla odstraněna mokrým leptadlem a tím bylo planární multielektrodové pole dokončeno. Na planárním multielektrodovém poli byly zarovnány HL-1 buňky do pruhů chemickou metodou pomocí kombinace otisku adhezní látky a následným potažení neotisklých ploch anti-adhezní látkou. Elektrofyziologické vlastnosti zarovnaných HL-1 buněk byly změřeny pomocí planárního multielektrodového pole. Tímto experimentem byla představena výrobní technologie pro výrobu planárních multielektrodových polí a toto planární multielektrodové pole bylo úspěšně testováno pro zarovnání HL-1 buněk na jeho povrchu kombinací otisku adhezní látky a potahování antiadhezivním činidlem.

<p>Persistent and maladaptive fear in the absence of a threat can be disruptive because it decreases an organism’s opportunity to seek life-sustaining substances. Learned safety signaling can suppress fear and encourage reward-seeking behavior, thus freeing the organism from fear induced immobilization. The infralimbic (IL) region of the prefrontal cortex is important for recalling fear extinction memories and for suppressing fear via learned safety signals. Neurons in the IL show an excitatory response to an extinguished fear cue. We thus hypothesized that neurons in the IL would encode safety by showing an excitatory response during active fear suppression to a learned safety signal. </p> <p>To assess global changes in IL activity, we monitored IL multi-unit activity to different cues while training animals in a fear-reward-safety discrimination task (Sangha, Chadick, & Janak, 2013). During the discrimination task, male rats learned that the reward cue predicted liquid sucrose, the fear cue predicted footshock and the joint presentation of both the fear and safety cues resulted in no footshock. We also counterbalanced the modality of fear and safety cues (auditory vs visual) with two separate groups of animals to control for potential sensory modality effects. Male rats showed high levels of freezing to the fear cue, and significantly reduced levels of freezing to the combined fear+safety cue. Male rats also showed high levels of port activity to the reward cue. There was no significant difference in the learning rate between the two counterbalanced conditions. </p> <p>Our multi-unit-data showed an increase in IL neuronal firing to the fear+safety cue across training sessions. This effect was consistent between the two counterbalanced conditions. We also examined single-unit activity from all animals that received light as the safety cue (n=8). This allowed us to examine the population response profile with a subset of the total animals. Although not statistically significant, our preliminary single-unit data demonstrated a decrease in the percentage of neurons that showed an inhibitory response to the fear+safety cue, but no change in the percentage of neurons that showed an excitatory response to the fear+safety cue. There was also no change in the magnitude of averaged firing rate in fear+safety excitatory or inhibitory neurons across training. Taken together, the decreased inhibition of single-unit activity in the IL may drive the increased excitation in multi-unit activity in the IL during behavioral fear suppression to a safety signal. </p>

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This dissertation aims to make a study and processing of two types of signs of neuronal electrical activity. The first was recorded from multielectrode arrays (MEA), in reference to spontaneous activity of neuronal groups grown in cultures. We analyzed two cultures considered inactive, meaning, cultures that after a few days in vitro, there was not a connection between neurons. It was applied two mathematical tools, autocorrelation and power spectral density, allowing an analysis of the signal in time domain and frequency, respectively, trying to verify whether the data recorded from these inactive cultures could be considered as noise from instrumentation. In fact, the results indicate that these signals have similar characteristics to white noise, which disturbs any computer analysis usually performed by researchers in computational neuroscience. Another source of signals used for this study were records of electroencephalography (EEG), collected on 20 patients from the Clinical Hospital of Uberlândia under prior consent. After a clinical examination by qualified professional, an analysis was performed using computational Fourier transform and power spectral density. Through amplitude graphics (Fourier transform) and spectral density, we have realized that the signal energy is concentrated around the frequencies more representative and, in some cases, there was presence of noise from power network. With the phase spectrum, it was possible to conclude that signals with similar frequencies have spectra of similar phase. / Esta dissertação tem como objetivo fazer um estudo e processamento de dois tipos de sinais de atividade elétrica neuronal. O primeiro foi registrado a partir de matrizes multieletrodos (MEA), referentes a atividade espontânea de grupos neuronais em cultura. Foram analisadas duas culturas consideradas como inativas, ou seja, culturas que após alguns dias in vitro, não se verificou a conexão entre os neurônios. Foram aplicadas duas ferramentas matemáticas, autocorrelação e densidade espectral de potência, permitindo uma análise do sinal no domínio do tempo e freqüência, respectivamente, procurando verificar se os dados registrados destas culturas inativas poderiam ser considerados como ruído de instrumentação. De fato, os resultados apontam que estes sinais apresentam características semelhantes ao ruído branco, o qual perturba qualquer análise informática normalmente realizada por pesquisadores em neurociência computacional. Outra fonte de sinais utilizados para este estudo foram registros de eletroencefalografia (EEG), coletados em 20 pacientes do Hospital de Clínicas de Uberlândia sob prévio consentimento destes. Após uma análise clínica por profissional qualificado, foi realizada uma análise computacional utilizando transformada de Fourier e densidade espectral de potência. Através dos gráficos de amplitude (transformada de Fourier) e densidade espectral, percebeu-se que a energia do sinal está concentrada em torno das freqüências mais representativas e, em alguns casos, observou-se presença de ruído de rede elétrica. Com o espectro de fase, foi possível concluir que sinais com freqüências semelhantes apresentam espectros de fase semelhantes. / Mestre em Ciências

Tous les réseaux de neurones immatures génèrent une activité dite « spontanée »qui persiste même en l’absence de toute afférence et est impliquée dans de nombreux processus développementaux. Cette activité apparaît in vitro sous formes de vagues calciques ou électriques pouvant se propager sur de grandes distances et embraser toute la préparation. Toutefois, sa dynamique a été assez peu étudiée jusqu’à présent. En vue de combler quelque peu cette lacune, nous avons utilisé des matrices de microélectrodes (MEA) pour caractériser l’activité rythmique spontanée dans la moelle épinière embryonnaire de souris, sur des préparations aigues et en culture incluant également le tronc cérébral.Les enregistrements MEA produisent des volumes de données très importants qui nécessitent des outils d’analyse performants et adaptés à l’information que l’on souhaite extraire. Nous avons donc développé des méthodes pour la détection, la classification et la cartographie des patrons spatiotemporels d’activité dans les données multicanaux. Notre approche cartographique utilise l’interpolation par splines et est orientée vers la production de cartes multimodales combinant l’activité électrique et des données anatomiques ou biochimiques (marquages). Ces méthodes d’analyse nous ont permis de décrire très précisément l’évolution de l’activité spontanée aux stades précoces (E12.5–E15.5). Nous avons également montré que, à E14.5, l’activité est initiée dans le bulbe, plus précisément dans une région riche en neurones 5-HT, suggérant un nouveau rôle des voies sérotoninergiques descendantes dans la maturation des réseaux spinaux.Enfin, nous avons analysé les mouvements embryonnaires à E14.5 et avons découvert des caractéristiques analogues à la dynamiques spatiotemporelle des activités intraspinales. / Immature neural networks generate a peculiar type of activity that persists even in the absence of electrical inputs and was termed for this reason “endogenous”or “spontaneous”. This activity is ubiquitous and was found involved in a wide range of developmental events. In vitro, it can be observed as calcium or electrical waves propagating over great distances, often invading the whole preparation,but its dynamics remain poorly described. In order to somewhat fill this gap,we used multielectrode arrays (MEAs) to characterise the spontaneous rhythmic activity in the mouse developing spinal cord, in both acute and cultured isolated hindbrain-spinal cord preparations.To extract relevant information from the massive amounts of data yielded by MEA recordings, adapted analysis tools are needed. Thus, we have developedmethods for the detection, classification and mapping of spatiotemporal patternsof activity in multichannel data. Our mapping approach is based on the thin plates pline interpolation and includes the possibility to combine maps of activity with anatomical or stained data for multimodal imaging.These methods allowed us to analyse in great detail the evolution of spontaneousactivity at early stages (E12.5–E15.5). In addition, we have localised theinitiation site of E14.5 activity in the medulla and shown that it matches a densemidline population of serotoninergic neurons, suggesting a new role for 5-HTpathways in the maturation of spinal networks. Finally, we have recorded andtracked spontaneous limb movements of E14.5 embryos and found that features of motility were consistent with patterns of spinal activity.

Cette thèse présente un système bioélectronique prometteur, l’Hynet. Ce Réseau Hybride (vivant-artificiel) est conçu pour l’étude du comportement à long terme des cellules électrogénératrices, comme les neurones et les cellules betas, en deux aspects : l’individuel et en réseau. Il est basé sur une boucle fermée et sur la communication en temps réel entre la culture cellulaire et une unité artificielle (Matériel, Logiciel). Le premier Hynet utilise des Matrices d’électrodes (MEA) commerciales qui limitent les performances spatiotemporelles du Hynet. Une nouvelle Matrice d’électrodes intelligente (iMEA) est développée. Ce nouveau circuit intégré, analogique et mixte, fournit une interface à forte densité, à forte échelle et adaptative avec la culture. Le nouveau système améliore le traitement des données en temps réel et une acquisition faible bruit du signal extracellulaire. / This thesis presents a promising new bioelectronics system, the Hynet. The Hynet is a Hybrid (living-artificial) Network, developed to study the long-term behavior of electrogenic cells (such as Neurons or Beta-cells), both individually and in a network. It is based on real-time closed-loop communication between a cell culture (bioware) and an artificial processing unit (hardware and software). In the first version of our Hynet, we use commercial Multielectrode Arrays (MEA) that limits its spatiotemporal performances. A new Intelligent Multielectrode Array (iMEA) is therefore developed. This new analog/mixed integrated circuit provides a large-scale, high-density, and adaptive interface with the Bioware, which improves the real-time data processing and the low-noise acquisition of the extracellular signal. / Esta dissertação de doutorado apresenta um sistema bioeletrônico auspicioso, o Hynet. Esta Rede Híbrida (viva e artificial), é concebida para o estudo do comportamento à longo prazo de células eletrogeneradoras (como neurônios ou células beta), em dois aspectos : individual e em redes. Ele é baseado na comunicação bidirecional, em laço fechado e em tempo real entre uma cultura celular (Bioware) e uma unidade artificial (Hardware ou Software). Um primeiro Hynet é apresentado, mas o uso de Matrizes de Eletrodos (MEA) comerciais limita a performance do sistema. Finalmente, uma nova Matriz de Eletrodos Inteligente (iMEA) é desenvolvida. Este novo circuito integrado fornece uma interface adaptativa, em alta densidade e grande escala, com o Bioware. O novo sistema melhora o processamento de dados em tempo real e a aquisição baixo ruído do sinal extracelular.