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.

This work characterized an in vitro 3-D neural co-culture model electrophysiologically via multi electrode arrays (MEAs), and morphologically via immunocytochemistry. Since MEA surface insulation SU-8 2000 can be used in neural micro- and multi- electrode arrays, this investigation first developed techniques to make SU-8 2000 cytocompatible. The in vitro 3-D neural co-culture model was then used to study viability and electrophysiological responses to physical injury as well as drugs known to affect network signaling. 1) SU-8 2000 cytotoxicity to neuronal cultures was linked to both poor adhesive properties and toxic components, such as solvents and photo acid generator elements. Surface treatments of oxygen plasma or parylene coating following optimal combinations of heat and isopropanol sonication showed improvement in SU-8 2000 cytocompatibility. 2) The 3-D neural networks within the 3-D co-cultures maintained considerable process outgrowth and complex 3-D structure. The cultures were viable up to three weeks in vitro with functional synaptic connections and spontaneous electrophysiological activity that was responsive to chemical modulation. This electrophysiological activity was modulated by synaptic inhibition. 3) Injury experiments demonstrated that both shear and compression loading significantly increased acute membrane permeability of cells in a strain rate dependent manner. Cell death correlated with higher membrane permeability, and shear resulted in more death than compression in these 3-D cultures. While techniques were developed for making a major micro-fabrication material cytocompatible, engineering the 3-D neural co-culture resulted in a more physiologically-representative neural tissue platform, allowing an increased understanding of structure-function relationships. Overall, this research established and characterized a neural culture system for the mechanistic study of cell growth, cell-cell and cell-matrix interactions, as well as the responses to chemical or mechanical perturbations. This is the first investigation of the network-level electrophysiological activity of 3-D dissociated cultures. This system can be used to model various pathological states in vitro, testing various reparative drugs; cell-, and tissue-engineering based strategies; as well as for pre-animal and pre-clinical testing of neural implants.

No description available.

It would be of great value to have a precise and non-damaging neuromodulation technique in the field of basic neuroscience research and for clinical treatment of neurological diseases. Infrared neural modulation (INM) is a new modulation modality developed in the last decade, which uses pulsed or continues infrared (IR) light with a wavelength of 1200 to 2200 nm to directly alter neural signals. INM includes both infrared neural stimulation (INS) and infrared neural inhibition (INI). INM is widely investigated for use on peripheral nerves, cochlear nerve fibers, cardiac cells, and the central nervous system. This technique holds the advantages of contact-free and high spatiotemporal precision compared to the traditional electrical stimulation. It does not depend on genetic modification and exogenous absorbers as other optical techniques, such as the optogenetic technique and the enhanced near-infrared neural stimulation (e-NIR). These advantages make INM a viable technique for research and clinical applications. The primary mechanism of the INM is believed to be a photothermal effect, where the IR laser energy absorbed by water leads to a rapid local temperature change. However, so far the details of the mechanism of action potential (AP) generation and inhibition remain elusive. Another issueis that the cells may be endangeredbythe heat exposure, consequently triggering a physiologicalmalfunction or even permanent damage.These concernshave hindered the transfer of the INM technique to the clinical therapy.Therefore, the general aim of this study was to improve the understanding of the details of how INM affects the cells. Laser parameters for safe and efficient stimulation were investigated on the basis of being useful for clinical applications. A tailored heating model and in vitro INM experiments on cortex neurons were used to reach this goal.The first paper was a feasibility study. A 1550nm laser with a beam spot diameter of around 6 mm was used to irradiate the rat cortex neurons, which were seeded on multi-electrode arrays (MEA) and formed well-connected networks. A heating model based on an estimated laser beam (standard Gaussian distribution) was used to simulate temperaturechanges. The damage signal ratio (DSR),based on the temperature,was calculated to predict the heat damage. The average spike rate of all the working electrodes from two MEAs was used to evaluate the degree of theinhibition of the neural networks. Results IVshowed that it is possible to use the 1550 nm laser to safely inhibit the neural network activity and that the degree of the INI is dependent on the power of the laser.The second paper wasan application and mechanism study. The aim of this study was to investigate the safety, efficiency, and cellular mechanism of INI. The same laser as in paper Iwas used in this study. A 20 X objective was used to decrease the beam spot diameteraround 240 μm. The measured laser profile (high order Gaussian beam) was used in the heating model to predict the temperature. The model was verified by local temperature measurements viamicropipette. The action potential rates, measured by the MEA electrodes, were quantified for different temperatures. Bicuculline was added to the cortex neuron cultures to induce hyperexcitation of the neural network. The results showed that the INI is temperature dependent and that the temperature needs to be less than 46 °C at 30 s laser irradiation for safe inhibition. The IR laser couldalso be used to inhibit the hyperexcitedactivity. The degree of inhibition, for the assessed subpopulation of neurons, was better correlated with the action potential amplitude than the width of it and INIcan be accomplished without inhibitory synapses / <p>QC 20180920</p><p></p>

La fibrilación auricular es una de las arritmias cardíacas más comunes observadas en la práctica clínica. Por lo tanto, es de vital importancia desarrollar nuevas tecnologías destinadas a diagnosticar y acabar con este tipo de arritmia, para mejorar la calidad de vida de los pacientes y reducir los costes de los sistemas nacionales de salud. En los últimos años ha aumentado el uso de las nuevas técnicas de mapeo auricular, basadas en sistemas multi-electrodo para mapear la actividad eléctrica en humanos. Dichas técnicas permiten localizar y ablacionar los impulsores de la fibrilación auricular, como son las fuentes focales o los rotores. Sin embargo, todavía existe incertidumbre sobre su precisión y los procedimientos experimentales para su análisis están limitados debido a su carácter invasivo. Por lo tanto, las simulaciones computacionales son una herramienta muy útil para superar estas limitaciones, al permitir reproducir con fidelidad las observaciones experimentales, dividir el problema bajo estudio en sub-estudios más simples, y realizar investigaciones preliminares imposibles de llevar a cabo en el práctica clínica. Esta tesis doctoral se centra en el análisis de la precisión de los sistemas de mapeo multi-electrodo a través de modelos y simulaciones computacionales. Para ello, desarrollamos modelos realistas multi-escala con el objetivo de simular actividad eléctrica auricular reentrante, en primer lugar en una lámina de tejido auricular, y en segundo lugar en las aurículas completas. Posteriormente, analizamos los efectos de las configuraciones geométricas multi-electrodo en la precisión de la localización de los rotores, mediante el uso de agrupaciones multi-electrodo con distancias inter-electrodo equidistantes, así como a través de catéteres de tipo basket con distancias inter-electrodo no equidistantes. Después de calcular los electrogramas unipolares intracavitarios, realizamos mapas de fase, detecciones de singularidad de fase para rastrear los rotores, y mapas de frecuencia dominantes. Finalmente, descubrimos que la precisión de los sistemas de mapeo multi-electrodo depende de su posición dentro de la cavidad auricular, de la distancia entre los electrodos y el tejido, de la distancia inter-electrodo, y de la contribución de las fuentes de campo lejano. Además, como consecuencia de estos factores que pueden afectar a la precisión de los sistemas de mapeo multi-electrodo, observamos la aparición de rotores falsos que podrían contribuir al fracaso de los procesos de ablación de la fibrilación auricular. / Atrial fibrillation is one of the most common cardiac arrhythmias seen in clinical practice. Therefore, it is of vital importance to develop new technologies aimed at diagnosing and terminating this kind of arrhythmia, to improve the quality of life of patients and to reduce costs to national health systems. In the last years, new atrial mapping techniques based on multi-electrode systems are increasingly being used to map the atrial electrical activity in humans and localise and target atrial fibrillation drivers in the form of focal sources or rotors. However, significant concerns remain about their accuracy and experimental approaches to analyse them are limited due to their invasive character. Therefore, computer simulations are a helpful tool to overcome these limitations since they can reproduce with fidelity experimental observations, permit to split the problem to treat into more simple substudies, and allow the possibility of performing preliminary investigations impossible to carry out in the clinical practice. This PhD thesis is focused on the analysis for accuracy of the multielectrode mapping systems through computational models and simulations. For this purpose, we developed realistic multiscale models in order to simulate atrial electrical reentrant activity, first in a sheet of atrial tissue and, then, in the whole atria. Then, we analysed the effects of the multi-electrode geometrical configurations on the accuracy of localizing rotors, by using multi-electrode arrays with equidistant inter-electrode distances, as well as multi-electrode basket catheters with non-equidistant inter-electrode distances. After computing the intracavitary unipolar electrograms, we performed phase maps, phase singularity detections to track rotors, and dominant frequency maps. We finally found out that the accuracy of multi-electrode mapping systems depends on their position inside the atrial cavity, the electrode-to-tissue distance, the inter-electrode distance, and the contribution of far field sources. Furthermore, as a consequence of these factors, false rotors might appear and could contribute to failure of atrial fibrillation ablation procedures. / La fibril·lació auricular és una de les arítmies cardíaques més comuns observades en la pràctica clínica. Per tant, és de vital importància desenvolupar noves tecnologies destinades a diagnosticar i acabar amb aquest tipus d'arítmia, per tal de millorar la qualitat de vida dels pacients i reduir els costos dels sistemes nacionals de salut. En els últims anys, ha augmentat l'ús de les noves tècniques de mapeig auricular, basades en sistemes multielèctrode per a mapejar l'activitat elèctrica auricular en humans. Aquestes tècniques permeten localitzar i ablacionar els impulsors de la fibril·lació auricular, com són les fonts focals o els rotors. No obstant això, encara hi ha incertesa sobre la seua precisió i els procediments experimentals per al seu anàlisi estan limitats a causa del seu caràcter invasiu. Per tant, les simulacions computacionals són una eina molt útil per a superar aquestes limitacions, en permetre reproduir amb fidelitat les observacions experimentals, dividir el problema sota estudi en subestudis més simples, i realitzar investigacions preliminars impossibles de dur a terme en el pràctica clínica. Aquesta tesi doctoral es centra en l'anàlisi de la precisió del sistemes de mapeig multielèctrode mitjançant els models i les simulacions computacionals. Per a això, desenvolupàrem models realistes multiescala per tal de simular activitat elèctrica auricular reentrant, en primer lloc en una làmina de teixit auricular, i en segon lloc a les aurícules completes. Posteriorment, analitzàrem els efectes de les configuracions geomètriques multielèctrode en la precisió de la localització dels rotors, mitjançant l'ús d'agrupacions multielèctrode amb distàncies interelèctrode equidistants, així com catèters de tipus basket amb distàncies interelèctrode no equidistants. Després de calcular els electrogrames unipolars intracavitaris, vam realitzar mapes de fase, deteccions de singularitat de fase per a rastrejar els rotors, i mapes de freqüència dominants. Finalment, vam descobrir que la precisió dels sistemes de mapeig multielèctrode depèn de la seua posició dins de la cavitat auricular, de la distància entre els elèctrodes i el teixit, de la distància interelèctrode, i de la contribució de les fonts de camp llunyà. A més, com a conseqüència d'aquests factors, es va observar l'aparició de rotors falsos que podrien contribuir al fracàs de l'ablació de la fibril·lació auricular. / Martínez Mateu, L. (2018). Mapping of the electrical activity of human atria. Multiscale modelling and simulations [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/104604

Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4^S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4^S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.

Parkinson’s Disease affects 10 million people worldwide, with 40% of patients developing an associated psychosis which has been identified by studies as the number one source of caretaker distress and is related to increased mortality. This is further complicated by the fact that typical antipsychotic drugs worsen many of the motor symptoms implicated in Parkinson’s Disease, with only one commercially available drug able to ameliorate both symptoms. This problem ushers the development of novel drugs to treat these symptoms, as first tested on research animals. Complicating matters, drug effectiveness on the degree of psychosis is hard to obtain in animals without a reliable biomarker. However, a hallmark of psychotic states is thought to be the reduced coordination between brain structures, through neuronal synchronization, as demonstrated by steady-state responses and is suggested to be a potential biomarker of psychosis. By building a MATLAB software we were able to analyze the degree of neural synchronization between structures, during an auditory steady-state response, in rats that had been unilaterally lesioned by the 6-Hydroxydopamine model of Parkinson’s Disease, before and after administration of the psychotomimetic drug MK801. These rats had been chronically implanted with 128-channel multi electrode array, enabling us to measure the strength of coherence between several limbic structures, associated with auditory processing, from the sampled local field potential, identifying the degree of synchronization in the animal brain. As our data demonstrate that coherence levels dropped in the psychotic drug state, for structures in both the healthy and the Parkinsonian hemisphere, we are able to further demonstrate the validity of coherence measures as a biomarker for psychosis. These results demonstrate that our software can be used as a tool to assess the therapeutic response of drugs developed, aimed at treating Parkinson’s associated psychosis. / Parkinsons sjukdom drabbar 10 miljoner världen över, där 40% av patienterna utvecklar en associerad psykos vilket har visats vara en av de största stressfaktorerna för deras vårdgivare och är även förknippat med en högre dödlighetsgrad. Denna situation förvärras av det faktum att de vanliga antipsykotiska drogerna kan förvärra många av de motoriska symptom som utgörs av Parkinsons sjukdom och det finns i dagsläget enbart en enda kommersiell drog som kan dämpa bägge symptom samtidigt. Detta problem frammanar vidare utveckling av nya läkemedel som kan behandla dessa symptom, som innebär att de först måste testas på försöksdjur. En komplikation som uppstår i relation till detta är svårigheten att utvärdera om läkemedel har någon terapeutisk effekt på de psykotiska tillstånden, enbart genom att observera försöksdjurens beteenden, och en pålitlig biomarkör krävs istället. En lösning kan dock finnas i det faktum att psykotiska tillstånd karaktäriseras av en reducerad förmåga för olika hjärnområden att koordinera genom neural synkronisering vilket demonstreras av ‘steady- state’ responser. Detta föreslår att ett mått på graden av koordineringsförmåga kan agera som en möjlig biomarkör för psykotiska tillstånd. Genom att konstruera ett MATLAB-program kunde vi analysera graden av synkronicitet mellan hjärnstrukturer, under den auditiva steady- state responsen i råttor som hade blivit ensidigt lesionerade genom 6-Hydroxiddopamin modellen av Parkinsons sjukdom, före och efter administration av den psykotomimetiska drogen MK801. Dessa råttor hade blivit kroniskt implanterade med 128 elektroder vilket möjliggjorde att vi kunde mäta styrkan i koherens i den lokala fält potentialen mellan limbiska strukturer, som är associerade med auditiv processering, vilket möjliggjorde identifiering av3dessa strukturers synkronicitet. Vår data demonstrerar att koherensen minskade under det psykotiska drogtillståndet för limbiska strukturer både i den intakta och den lesionerade hjärnhalvan. Detta är en vidare demonstration av att koherensnivåer kan agera som en biomarkör för det psykotiska tillståndet, liksom att vår mjukvara kan nyttjas som ett verktyg för att utvärdera nya läkemedels behandlingsförmåga på Parkinsons psykos.