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REGULATION OF RETINAL ACTIVITY IN AN EX-VIVO GUINEA PIG MODEL BY EXPERIMENTAL CONDITIONS AND EFFECTS OF ISOFLURANE AND PROPOFOL ANESTHETICSWood, Leah M. 21 October 2010 (has links)
Electroretinoraphic signals (ERGs) are affected when recorded under isoflurane anesthesia in the operating room. We explored the effect of isoflurane and propofol in ex vivo guinea pig retinal preparations using a multielectrode array to record simultaneously ERGs and retinal ganglion cell (RGC) activity. The viability and light-response characteristics of the model were documented. In the presence of isoflurane, the ERG and RGC activity was reduced in a dose-dependent manner, even at sub-clinical doses; the OFF responses were consistently more affected. Propofol had minimal effects: at subclinical doses, a small excitation was measured while a concentration a hundred times stronger than the clinical concentration was required to measure a significant decline in EGR and RGC signals. This study confirms the usefulness of the guinea pig model to study clinically relevant retinal issues and shows that propofol is a better anesthetic to use in the operating room when retinal investigations are required.
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SHH signalling mediates astrocyte crosstalk with neurons to confer neuroprotectionUgbode, Christopher I., Smith, I., Whalley, B.J., Hirst, W.D., Rattray, Marcus 09 May 2017 (has links)
Yes / Sonic Hedgehog (SHH) is a glycoprotein associated with development that is also expressed in the adult CNS and released after brain injury. Since the SHH receptors PTCH1 (patched homolog-1) and SMO (Smoothened) are highly expressed on astrocytes, we hypothesised that SHH regulates astrocyte function. Primary mouse cortical astrocytes derived from embryonic (E15) Swiss mouse cortices, were treated with two chemically distinct agonists of the SHH pathway, which caused astrocytes to elongate and proliferate. These changes are accompanied by decreases in the major astrocyte glutamate transporter, GLT-1 and the astrocyte intermediate filament protein GFAP. Multi-site electrophysiological recordings revealed that the SHH agonist, SAG supressed neuronal firing in astrocyte-neuron co-cultures and this was abolished by the astrocyte metabolic inhibitor ethylfluoroacetate, revealing that SHH stimulation of metabolically-active astrocytes influences neuronal firing. Using 3D co-culture, MAP2 western blotting and immunohistochemistry, we show that SHH-stimulated astrocytes protect neurons from kainate induced cell death. Altogether the results show that SHH regulation of astrocyte function represents an endogenous neuroprotective mechanism. / BBSRC
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A Multielectrode Microcompartment Platform for Signal Transduction in the Nervous SystemRavula, Surendra Kumar 23 June 2006 (has links)
This dissertation presents the development of a multielectrode microcompartment platform for understanding signal transduction in the nervous system. The design and fabrication of the system and the characterization of the system for pharmacological and electrophysiological measurements of cultured neurons is presented in this work. The electrophysiological activity of cultured dorsal root ganglion (DRG) neurons and cortical neurons is shown on the MEA substrate. These recordings were measured and tied to the toxicological effects of the chemotherapeutic drug vincristine on DRGs.
Conventional electrophysiological recordings (via a patch micropipette) are made routinely to record action potentials and ion channel activity in neurons. Moreover, Campenot chambers (traditional compartmented culture systems) have been used for the last thirty years to study the selective application of drugs to neurons. Both of these techniques are useful and well established; however they have their limitations. For instance, Campenot chambers cannot be used very well for small processs-producing neurons, since the barriers are difficult to tranverse. Moreover, conventional patch recordings are labor-intensive, especially when more than one microelectrode needs to be positioned.
The developed system is composed of a two compartment divider, each compartment capable of housing axons or cell bodies. Underneath the divider, the substrate has 60 electrodes, arranged in several lines to accommodate several different neurite tracks. Neurons can be stimulated and their activity can be recorded in both of the compartments. The neurotoxin and chemotherapeutic drug vincristine was tested in the system on the DRGs. The drug caused length-dependent axonal degeneration in the DRGs when applied locally. Moreover, electrophysiological activity in both compartments showed that only the activity in the axonal compartment was affected, leading us to believe that the mechanism behind the degeneration is localized to the distal axon.
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Microstimulation and multicellular analysis: A neural interfacing system for spatiotemporal stimulationRoss, James 19 May 2008 (has links)
Willfully controlling the focus of an extracellular stimulus remains a significant challenge in the development of neural prosthetics and therapeutic devices. In part, this challenge is due to the vast set of complex interactions between the electric fields induced by the microelectrodes and the complex morphologies and dynamics of the neural tissue. Overcoming such issues to produce methodologies for targeted neural stimulation requires a system that is capable of (1) delivering precise, localized stimuli a function of the stimulating electrodes and (2) recording the locations and magnitudes of the resulting evoked responses a function of the cell geometry and membrane dynamics. In order to improve stimulus delivery, we developed microfabrication technologies that could specify the electrode geometry and electrical properties. Specifically, we developed a closed-loop electroplating strategy to monitor and control the morphology of surface coatings during deposition, and we implemented pulse-plating techniques as a means to produce robust, resilient microelectrodes that could withstand rigorous handling and harsh environments. In order to evaluate the responses evoked by these stimulating electrodes, we developed microscopy techniques and signal processing algorithms that could automatically identify and evaluate the electrical response of each individual neuron. Finally, by applying this simultaneous stimulation and optical recording system to the study of dissociated cortical cultures in multielectode arrays, we could evaluate the efficacy of excitatory and inhibitory waveforms. Although we found that the proximity of the electrode is a poor predictor of individual neural excitation thresholds, we have shown that it is possible to use inhibitory waveforms to globally reduce excitability in the vicinity of the electrode. Thus, the developed system was able to provide very high resolution insight into the complex set of interactions between the stimulating electrodes and populations of individual neurons.
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Role of spontaneous bursts in functional plasticity and spatiotemporal dynamics of dissociated cortical culturesMadhavan, Radhika 08 June 2007 (has links)
What changes in our brain when we learn? This is perhaps the most intriguing question of science in this century. In an attempt to learn more about the inner workings of neural circuitry, I studied cultured 2-dimensional networks of neurons on multi-electrode arrays (MEAs). MEAs are ideal tools for studying long-term neural ensemble activity because many individual cells can be studied continuously for months, through electrical stimulation and recording. One of the most prominent patterns of activity observed in these cultures is network-wide spontaneous bursting, during which most of the active electrodes in the culture show elevated firing rates. We view the persistence of spontaneous bursting in vitro as a sign of arrested development due to deafferentation. Substituting distributed electrical stimulation for afferent input transformed the activity in dissociated cultures from bursting to more dispersed spiking, reminiscent of activity in the adult brain. Burst suppression reduced the variability in neural responses making it easier to induce and detect functional plasticity caused by tetanic stimulation. This suggests that spontaneous bursts interfere with the effects of external stimulation and that a burst-free environment leads to more stable connections and predictable effects of tetanization. Moreover, our culture models continuously receive input stimulation in the form of background electrical stimulation, and so better resemble the intact brain than isolated (non-continuously stimulated) cultures. The proportion of GABAergic neurons in the cultures was significantly increased (p<1e-2, paired t-test) after burst-quieting for 2 days, suggesting that burst suppression operated through the homeostatic control of inhibitory neurotransmitter levels. We also studied the role of spontaneous bursts as potential carriers of information in the network by clustering these spatiotemporally diverse bursts. Spontaneous burst clusters were stable over hours and tetanic stimulation significantly reorganized the distribution of the clusters. In summary, this body of work explores the rules of network-level functional plasticity and provides the input (electrical stimulation) output (spatiotemporal patterns) mappings for behavioral studies in embodied hybrid systems. The results of this study may also have clinical implications in the development of sensory prostheses and treatment of diseases of aberrant network activity such as epilepsy.
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Implementação de um protocolo experimental para estudo de propriedades de resposta visual de neurônios do córtex visual primário (V1) em ratos utilizando matrizes de eletrodosFONTENELE NETO, Antonio Jorge 25 August 2015 (has links)
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Previous issue date: 2015-08-25 / CAPEs / O córtex visual primário (V1) é a região do córtex cerebral responsável pela primeira etapa de
processamento da informação visual capturada pela retina. Por ser uma das áreas corticais melhor
compreendidas, V1 constitui um dos principais paradigmas de compreensão do processamento
sensorial. Desde os anos 70 há uma extensa literatura que estuda propriedades de resposta de
neurônios de V1, principalmente com eletrodos individuais e utilizando-se como modelo animal
gatos e macacos. Tem-se conhecimento de onde partem seus principais inputs e quais estímulos
fazem os neurônios dispararem (grades senoidais com determinadas frequências espaciais e
temporais). Mais recentemente, com o uso de matrizes de eletrodos, se tornou possível a investigação
de propriedades coletivas da atividade e codificação neurais, que não eram possíveis de
serem desvendadas com eletrodos individuais. Além disso, no estado da arte tecnológico atual,
o uso do rato como modelo animal permite o registro da atividade neural com os animais em
comportamento livre (sem anestesia ou contenção). No entanto, pouco se sabe sobre especificidades
das propriedades de resposta dos neurônios do córtex visual do rato. Este trabalho teve por
objetivo desenvolver um aparato e um protocolo experimental no Laboratório de Neurociência
de Sistemas e Computacional adequado para estudo das propriedades de resposta de neurônios
de V1 de ratos usando matrizes de eletrodos. Finalmente, apresentamos resultados experimentais
onde caracterizamos respostas de neurônios de V1 a diferentes estímulos visuais (Funções de
Gabor ou Grades) seja em ruído denso ou rarefeito, variando as propriedades de frequências
temporal e espacial de estimulação, densidades de estímulos, velocidade, etc. Concluímos que
implementamos com sucesso a técnica experimental, que abre inúmeras perspectivas futuras de
pesquisas nesta linha no Departamento de Física da Universidade Federal de Pernambuco. / The primary visual cortex (V1) is the cerebral cortex region responsible for the first processing
step of the visual information captured by the retina. Being one of the most studied and well
described cortical sensory areas, V1 is one of the main paradigms for the study of sensory
processing. Since the 70s, there is a vast literature that studies properties of V1’s neurons,
specially using single electrodes and using cats and monkeys as animal models. The anatomical
conectivity of the visual pathway is known, from the retina to the lateral geniculate nucleus to
V1, as well as the main visual stimulations that make V1 neurons fire (sinusoidal gratings with
certain spatial and temporal frequencies). More recently, using multielectrode arrays, it became
possible to study coletive properties of the activity and neural codification, that could not be
unveiled with single electrodes. Furthermore with, the current state of the art in multielectrode
recordings it is possible to record the neural activity in frelly behaving rats (without anesthesia or
restraint). This represents an advantage in using the rat as animal model. However, little is known
about specificities of the V1 neurons response properties in the rat. The aim of this work is to
develop, in the Laboratório de Neurociência de Sistemas e Computacional, an apparatus and an
experimental protocol suitable for the study of visual response properties of V1’s neurons in rats,
using multielectrode array recordings. Finally, we present experimental results that characterize
the response of V1’s neurons with different visual stimuli (Gabor or Grating Functions), either in
dense os sparse noise modes, varying the spatial and temporal stimulation frequencies, stimulus
density, speed, etc. We conclude that the experimental technique was implemented successfully.
These results open important perspectives of future research on this field for the Departamento
de Física at the Universidade Federal de Pernambuco.
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Dynamics of embodied dissociated cortical cultures for the control of hybrid biological robots.Bakkum, Douglas James 14 November 2007 (has links)
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.
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Neuronal Plasma Membrane Disruption in Traumatic Brain InjuryPrado, Gustavo R. 12 July 2004 (has links)
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.
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An Active Microscaffold System with Fluid Delivery and Stimulation/Recording Functionalities for Culturing 3-D Neuronal NetworksRowe, Laura Elizabeth 08 March 2007 (has links)
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.
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Zarovnání excitabilních buněk na multielektrodových polích / Patterning of excitable cells on multi-electrode arraysSlavík, Jan January 2021 (has links)
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.
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