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Matthews, Brian. "Micromachined planar patch-clamp system for electrophysiology research." Diss., Restricted to subscribing institutions, 2006. http://proquest.umi.com/pqdweb?did=1188879521&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Squire-Pollard, Laura G. "A patch-clamp study of membrane ion channels in exocrine acinar cells." Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316552.
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Dargan, Sheila Louise. "Patch-clamp studies of single type-1 Ins(1,4,5)P3 receptor channels." Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393131.
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Mansell, Steven A. "The characterisation of ion channels in human spermatozoa by whole cell patch clamp electrophysiology." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/5fc0b4d5-ac64-474d-9cf3-5a123fa665cb.
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Kodandaramaiah, Suhasa Bangalore. "Robotics for in vivo whole cell patch clamping." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/51932.
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Whole-cell patch clamp electrophysiology of neurons in vivo enables the recording of electrical events in cells with great precision, and supports a wide diversity of morphological and molecular analysis experiments important for the understanding of single-cell and network functions in the intact brain. However, high levels of skill are required in order to perform in vivo patching, and the process is time-consuming and painstaking. Robotic systems for in vivo patching would not only empower a great number of neuroscientists to perform such experiments, but would also open up fundamentally new kinds of experiment enabled by the resultant high throughput and scalability. We discovered that in vivo blind whole cell patch clamp electrophysiology could be implemented as a straightforward algorithm and developed an automated robotic system that was capable of performing this algorithm. We validated the performance of the robot in both the cortex and hippocampus of anesthetized mice. The robot achieves yields, cell recording qualities, and operational speeds that are comparable to, or exceed, those of experienced human investigators. Building upon this framework, we developed a multichannel version of “autopatcher” robot capable establishing whole cell patch clamp recordings from pairs and triplets of neurons in the cortex simultaneously. These algorithms can be generalized to control arbitrarily large number of electrodes and the high yield, throughput and automation of complex set of tasks results in a practical solution for conducting patch clamp recordings in potentially dozens of interconnected neurons in vivo.
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Appenrodt, Peter. "Single-channel recordings of potassium channels from guinea-pig inner hair cells." Thesis, University of Sussex, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390054.
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Tessier, Christian. "Dissecting Kinetic Differences in Acetylcholine Receptors Incorporating an Ancestral Subunit." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/38868.
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At the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) convert chemical stimuli into electrical signals. They are heteropentameric membrane protein complexes assembled from four evolutionary related subunits (two α subunits, and one each of the β-, δ-, and ε-subunits), arranged around a central ion-conducting pore, which is regulated by the neurotransmitter acetylcholine. Understanding how the binding of acetylcholine leads to channel opening is of fundamental importance. While it is known that channel opening results from a global conformational change involving the cooperative action of all five subunits, how the subunits achieve this cooperativity is unclear. Our hypothesis is that this subunit cooperation is maintained through coevolution of the subunits, and thus studies of subunit coevolution can provide insight into subunit cooperativity. Using an ancestral reconstruction approach, combined with single-molecule patch clamp electrophysiology, we have begun dissecting the mechanistic consequences of preventing coevolution of the acetylcholine receptor β-subunit. This approach has allowed us to identify new amino acid determinants of acetylcholine receptor function.
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FLANNERY, RICHARD JOHN. "CLUSTERING OF CYCLIC-NUCLEOTIDE-GATED CHANNELS IN OLFACTORY CILIA." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1136913935.
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Immonen, E. V. (Esa-Ville). "In vitro electrophysiology of photoreceptors of two nocturnal insect species, Periplaneta americana and Gryllus bimaculatus." Doctoral thesis, University of Oulu, 2014. http://urn.fi/urn:isbn:9789526206479.
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Abstract
In dim light, reliable coding of visual information becomes compromised, unless the sensitivity of the visual system to light is improved by structural and functional adaptations. Thus far, many adaptations for night vision in the compound eyes of nocturnal insects have been described, but little is known about the mechanisms underlying the electrochemical signalling in their photoreceptors.
In this thesis, whole-cell patch-clamp and mathematical modelling are utilised to study basic electrical properties and ionic currents in photoreceptors of two nocturnal insects, the American cockroach Periplaneta americana and the field cricket Gryllus bimaculatus.
Photoreceptors in both species showed large input resistance, membrane capacitance and phototransduction gain (large single photon responses) compared with most studied diurnal insects, providing improved sensitivity to light. The photoreceptors also expressed two voltage-sensitive outward currents: a transient current and a sustained current. The cricket photoreceptor expressed a dominating transient current, which is a typical characteristic for insects adapted for slow vision in dim light. By contrast, in the majority of cockroach photoreceptors the sustained current dominated, which is more common among fast diurnal species. Model simulations indicated that the sustained current is necessary for improved photoreceptor dynamics. Examination of light-induced currents suggested that the functional variability in cockroach photoreceptors is in part derived from variations in the total area of the photosensitive membrane. Recordings of light-induced currents also revealed that the cockroach light-gated channels are only moderately Ca2+-selective and that the polarisation-sensitive photoreceptors of the cricket may utilise phototransduction machinery in some details different from that in regular photoreceptors. Furthermore, the dynamics and information transfer rates of polarisation-sensitive photoreceptors in the cricket were clearly inferior to their regular counterparts, suggesting that they are not necessary for image formation.
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Malezieux, Meryl. "Dynamique intracellulaire des cellules pyramidales de CA3 dans l'hippocampe pendant les états de veille." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0317/document.
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Les états de veille sont composés d’états cérébraux distincts, corrélés avec différents comportements et caractérisés par des oscillations spécifiques observables dans le potentiel de champ local (Local Field Potential, LFP). Bien que les différents états cérébraux et leur signature dans le LFP aient été caractérisés, les mécanismes cellulaires sous-jacents restent à ce jour peu connus. Des changements des propriétés de neurones uniques seraient corrélés avec, et pourraient participer à la génération de ces changements d’états cérébraux. L’activité coordonnée et synchronisée de neurones facilite certains processus cognitifs tels que la mémoire. L’hippocampe joue un rôle essentiel dans les mémoires spatiale et épisodique, et dans l’hippocampe, CA3 est important pour la formation d’associations facilitant l’encodage rapide de la mémoire. De plus, les informations provenant du cortex entorhinal, du gyrus denté, et de CA3 même sont comparées et intégrées dans CA3 avant d’être transmises à CA1. Lors de périodes de repos, le LFP hippocampique présente une activité large et irrégulière (Large Irregular Activity, LIA), ponctuée par des oscillations plus rapides, les sharp-wave ripples, jouant un rôle dans la consolidation de la mémoire. Lors de périodes exploratoires, le LFP hippocampique oscille aux fréquences theta (6-12 Hz) et gamma (30-100 Hz). Les cellules pyramidales (CP) de CA3 jouent un rôle important dans chacun de ces états ; elles sont nécessaires pour les sharp wave lors de périodes de repos, et les oscillations gamma lors de comportements exploratoires. Dans le but d’étudier les modulations intracellulaires des CP de CA3, nous avons réalisé des enregistrements de patch-clamp en configuration cellule entière chez l’animal éveillé. Nous avons associé ces enregistrements avec des mesures du diamètre pupillaire et de la vitesse de locomotion de l’animal, ainsi qu’avec l’enregistrement de l’activité oscillatoire du LFP dans l’hippocampe. Nos résultats montrent que certaines CP de CA3 sont sensibles à la modulation intracellulaire lors de différents rythmes hippocampiques, et ont tendance à diminuer leur potentiel de membrane moyen, leur excitabilité, leur variance et leur décharge de potentiel d’action lors des oscillations theta par rapport aux périodes de LIA. De futures études permettront de déterminer si ces changements sont dus à des changements d’entrées synaptiques et/ou de neuromodulateurs. Ces modulations pourraient jouer un rôle dans l’émergence des rythmes oscillatoires du LFP, et permettre à CA3 de réaliser différentes fonctions mnésiques à différents momentsWakefulness is comprised of distinct brain states, correlated with different behaviors and characterized by specific oscillatory patterns in the local field potential (LFP). While much work has characterized different brain states and their LFP signatures, the underlying cellular mechanisms are less known. Changes in single cell properties are thought to correlate with and possibly result in these changes in brain state. Synchronized and coordinated activity among distributed neurons supports cognitive processes such as memory. The hippocampus is essential for spatial and episodic memory, and within the hippocampus, area CA3 is important for rapid encoding of one-trial memory. Additionally, CA3 is the site where information from the entorhinal cortex, dentate gyrus, and CA3 itself is compared and integrated before output to CA1. During quiet wakefulness, the hippocampal LFP displays large irregular activity (LIA) punctuated by sharp-wave ripples, which play a role in memory consolidation. During exploratory behaviors, hippocampal LFP oscillates at both theta and gamma frequencies. CA3 pyramidal cells (PCs) play an important role in each of these brain states; they are necessary for both sharp waves during quiet wakefulness and for gamma oscillations during exploratory behavior. We explored the changes that occur in the intracellular dynamics of CA3 PCs during changes in brain state, by using whole-cell patch-clamp recordings from CA3 PCs in awake head-fixed mice. We combined those recordings with measurements of pupil diameter, treadmill running speed and LFP recordings of oscillatory activity. Our findings show that some CA3 PCs are prone to intracellular modulation during brain rhythms, and tend to decrease their average membrane potential, excitability, variance and output firing during theta as compared to LIA. Future studies will demonstrate whether these effects are due to changes in synaptic and/or neuromodulatory inputs. This modulation at the single-cell level in CA3 could play a role in the emergence of oscillations, and underlie the ability of CA3 to perform different memory functions during different brain states
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Barnes, Margaret. "A patch and voltage clamp investigation of the response of the C1 neurone of Helix aspersa to 5-hydroxytryptamine." Thesis, University of St Andrews, 1987. http://hdl.handle.net/10023/14118.
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Application of 5-hydroxytryptamine induces a voltage-dependent inward current in voltage clamped C1 neurones of Helix aspersa. This response has been shown to be the result of a decrease in K conductance and was studied using patch clamp and voltage clamp techniques. Single channel K currents were recorded from cell-attached patches of the C1 neurone. Two sizes of unitary outward currents were commonly observed. The I-V relationships of both these unitary currents could be fitted by the Goldman-Hodgkin-Katz equation for a K current, having slope conductances of around 14pS and 54pS at +10mV, patch potential. Experiments, altering the K concentration in the patch pipette, or on the outer surface of isolated outside-out patches, suggested that these unitary currents were due to the flow of K+ ions. Application of 5-hydroxytryptamine onto the C1 neurone, from out with the patch pipette, reduced the activity of the larger K channels, recorded in the cell-attached patch. Both Ca-dependent, and Ca-independent K channels were observed on isolated inside-out membrane patches. It was unclear which of these types of channel corresponded to the 5-hydroxytryptamine sensitive channel in the cell-attached patch. Voltage clamp experiments also gave confusing results regarding the Ca-dependency of the 5-hydroxytryptamine response. However, in some C1 neurones 5-hydroxytryptamine caused a flattening of the "N" shaped I-V relationship, suggesting a decrease in the Ca-dependent outward current. The possibility that more than one type of K current was suppressed by 5-hydroxytryptamine was considered. The effect of phosphodiesterase inhibitors was consistent with a mediation of the 5-hydroxytryptamine response by cyclic nucleotides. Injection of cAMP induced an inward current in the C1 neurone. Single channel outward currents, which reversed at -50mV, were recorded from the A neurone. The activity of these channels was increased by 5-hydroxytryptamine, but their ionic nature was uncertain. Unitary outward currents of the M neurone were also recorded.
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Gao, Lei. "Regulation of GABAA Receptors by Protein Kinase C and Hypoxia in Human NT2-N Neurons." University of Toledo Health Science Campus / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=mco1130337640.
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Grimm, Christiane. "Electrophysiological characterization of the microbial rhodopsins ReaChR and KR2 and their optogenetic potential." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20317.
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Mikrobielle Rhodopsine sind lichtsensitive Proteine, die von Mikroorganismen exprimiert werden um Licht wahrzunehmen oder dessen Energie zu nutzen. Ionen-transportierende mikrobielle Rhodopsine begründeten das Feld der Optogenetik. Hier erlauben sie transmembrane Ionenflüsse lichtsensitiv zu machen und neuronale Aktivität mit Licht zu steuern. Eine zielführende Nutzung beruht auf ihrer molekularen Charakterisierung, um sie dem Experiment anzupassen und es sinnvoll zu entwerfen. Teil I der Arbeit beschäftigt sich mit dem rotverschobenen Kanalrhodopsin ReaChR. Obwohl es mit breitem, nicht gaussförmigen Aktionsspektrum mit maximalen Strömen um 600 nm publiziert wurde, zeigte das Blitzlichtspektrum hier maximale Aktivität bei 535 nm ohne Besonderheiten. Mit steigender Intensität und längeren Pulsen verbreiterte sich das Spektrum; sehr ähnlich zum publizierten Spektrum. Dieses einzigartige Verhalten wird durch sekundäre Photochemie erklärt, welche zu einem komplexen Photozyklus mit lichtinduzierten Übergangen führt. Mutationen an Schlüsselpositionen wurden genutzt, um ReaChR über die publizierten Daten hinaus zu charakterisieren und neue Eigenschaften zu generieren. In Teil II wurde die auswärtsgerichtete Natriumpumpe KR2 elektrophysiologisch charakterisiert, was zuvor von schlechter Membranständigkeit in Säugetierzellen verhindert wurde. Ein verbessertes KR2 mit höherer Membranständigkeit und 60-fach größeren Photoströmen erlaubte Selektivitätsmessungen, welche zeigten, dass der Strom von Natriumionen getragen wird, wohingegen nichts auf Protonentransport hindeutete. Bei ausreichender Substratkonzentration war der Strom anders als bei Chlorid- oder Protonenpumpen von der Membranspannung unabhängig. Die Expression in Mausneuronen ermöglichte die reversible Unterdrückung von Aktionspotentialen mit Licht, wobei der Ausstrom von Kationen einen komplementären Weg zur neuronale Aktivitätsunterdrückung bietet, wenn etablierte Werkzeuge schlecht oder nicht funktionieren.Microbial rhodopsins are photosensitive proteins utilized by fungi, algae, and prokaryotes to sense light or harness its' energy. Ion transporting microbial rhodopsins initiated the field of optogenetics, where they are applied to render transmembrane ion fluxes light sensitive and control neuronal activity with light. Part I of the thesis focused on the electrophysiological characterization of the red-shifted channelrhodopsin ReaChR. Although published with a broad, non-Gaussian shaped action spectrum peaking around 600 nm, the flash action spectra of ReaChR recorded here had a maximum at 535 nm without peculiarities. Increasing intensities and prolonging illumination broadened the spectrum, which finally peaked around 600 nm. This unique behavior stems from pronounced secondary photochemistry leading to a complex photocycle with various light-induced transitions especially under constant illumination. Mutations at key positions like the central gate, DC-pair or counter ions were employed to characterize the properties of ReaChR beyond published data and engineer new features. In part II an electrophysiological characterization of the outward Na+ pump KR2 was pursued, which was hindered by poor membrane targeting in mammalian cells before. Engineering of eKR2 improved membrane targeting and lead to 60-fold larger photocurrents than in the wild type. Selectivity measurements revealed that the stationary photocurrent is primarily carried by sodium with no evidence for proton transport. At sufficient substrate concentration stationary photocurrents were independent of the membrane voltage distinguishing eKR2 from proton and chloride pumps. Finally, eKR2 reliably and reversibly inhibited action potential firing already at 0.5 mW/mm2 green illumination in cultured hippocampal mouse neurons. Inhibiting action potential firing through cation extrusion poses a complementary way of neuronal silencing in contexts where established tools are unfavorable or even impossible to use.
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Arnoult, Christophe. "Canaux ioniques et premières étapes du développement embryonnaire : études électrophysiologiques par la technique du patch-clamp de l'ovocyte du Tunicier Ciona intestinalis." Université Joseph Fourier (Grenoble ; 1971-2015), 1994. http://www.theses.fr/1994GRE10085.
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Nous avons etudie les variations de permeabilite ionique et de surface membranaire, affectant les ovocytes dans les premieres etapes du developpement embryonnaire. Ces etudes ont ete realisees sur des ovocytes d'ascidie ciona intestinalis, en utilisant la technique du patch-clamp epaulee par quelques cliches realises en microscopie electronique. Avant fecondation l'ovocyte de ciona possede deux courants calciques et un courant potassique. Differents inhibiteurs des courants calciques ont ete testes. Le courant calcique a haut seuil subit une diminution d'intensite transitoire pendant l'achevement de la division 1 de la meiose. Le courant calcique a bas seuil d'activation est par contre definitivement inhibe au temps de la premiere mitose. Apres fecondation deux courants entrant sont actives par hyperpolarisation. Un de ces courants post-fecondation est calcique. Le cadmium, le gadolinium et le cobalt inhibent ces courants. Ces deux courants disparaissent a la premiere mitose. En presence de cytochalasine b, la surface membranaire d'un ovocyte non feconde mesuree electriquement diminue alors que le diametre de l'ovocyte reste stable. Ce resultat est en accord avec la presence dans les ovocytes de reseaux membranaires internes connectes avec la membrane plasmique. Entre la fecondation et la premiere mitose la surface membranaire oscille selon trois cycles d'insertion et de reprise de membrane synchrones avec les deux cycles de meiose et la premiere mitose. En presence d'emetine, ces cycles sont detruits et la surface de l'uf augmente considerablement. La cytochalasine b inhibe la reprise membranaire de premier cycle. Le nocodazole est sans effet. Ces variations de surface ne sont pas correlees avec des changements de densites de microvillosites. Les mecanismes de base a l'origine de ces variations de surface seraient ainsi differents d'un cycle a l'autre. Ainsi en reponse aux profonds changements metaboliques lies au demarrage du developpement embryonnaire, les permeabilites membranaires sont fortement modulees, ces changements s'accompagnant de modifications de la surface membranaire
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Zachar, Peter C. "A Comparative Study of Neuroepithelial Cells and O2 Sensitivity in the Gills of Goldfish (Carrasius auratus) and Zebrafish (Danio rerio)." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30343.
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Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.
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Balu, Ramani. "Intrinsic and Synaptic Properties of Olfactory Bulb Neurons and Their Relation to Olfactory Sensory Processing." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1173540900.
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Hill, Jacqueline Suzanne. ""Mechanisms of Adrenal Medullary Excitation Under the Acute Sympathetic Stress Response"." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1341591989.
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Chiang, Elizabeth C. "INVESTIGATION OF THE ELECTROPHYSIOLOGICAL PROPERTIES OF THE MAJOR CELL TYPES IN THE RAT OLFACTORY TUBERCLE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1196707801.
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Assal, Reda. "Méthodes de production et étude électrophysiologique de canaux ioniques : application à la pannexine1 humaine et au canal mécanosensible bactérien MscL." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA11T093.
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La production hétérologue des protéines membranaires reste difficile, peut-être parce que l’insertion dans la membrane de la cellule hôte constitue une étape limitante de la production. Afin de tourner cette difficulté, deux modes de synthèse ont été envisagés: la synthèse de protéines dans un système a-cellulaire, en l’absence de membrane mais en présence de détergent, ou l’adressage forcé de la protéine vers les corps d’inclusion dans le cas d’une expression plus classique en bactérie entière. La réalisation des deux stratégies repose sur l’utilisation de protéines de fusion possédant une séquence d’entraînement en amont du gène d’intérêt, soit qu’elles améliorent la traduction du transcrit en limitant le repliement spatial de ce dernier, soit qu’elles favorisent la production de la protéine d’intérêt en corps d’inclusion. La porine OmpX et le peptide T7 ont été choisis en cas d’expression dans les systèmes bactériens. La protéine SUMO est utilisée pour la production dans un lysat eucaryote. Les différentes approches ont été testées sur la production de la pannexine1 humaine (Px1).Si les séquences d’entraînement OmpX et le peptide T7 sont correctement produites in vitro, aucune des deux, en revanche, ne favorise la production de la Px1. Seul l’entraîneur SUMO est efficace. En effet, nous avons observé que cette protéine augmente la production de la Px1 dans un lysat eucaryote de germe de blé. Par ailleurs OmpX, connue pour être largement produite in vivo dans les corps d’inclusion, n’entraîne pas la localisation de la Px1 dans ces structures. Contre toute attente, l’étiquette T7 dirige la Px1 dans les corps d’inclusion. L’étude électrophysiologique de la Px1 a donc été effectuée à partir de la protéine produite in vivo (T7his-Px1) après renaturation, ou produite sous forme soluble in vitro (his6-Px1) dans le lysat eucaryote. Dans le cas de la protéine T7his-Px1 renaturée, une activité canal qui rappelle celle qui est observée après expression dans l’ovocyte de Xénope, a été détectée en patch-clamp, mais dans trois cas seulement. Dans le cas de la protéine his6-Px1, aucune activité canal n’est clairement détectée. Dans une deuxième partie de ce travail on examine le rôle de la boucle périplasmique dans la sensibilité à la pression du MscL, un canal mécanosensible bactérien devenu un système modèle dans l’étude de la mécanosensibilité. Presque toutes les études fonctionnelles sur ce canal ont été réalisées sur le canal de E.coli, alors que la structure a été obtenue à partir de l’homologue de M. tuberculosis. Une étude fonctionnelle a montré que le MscL de M. tuberculosis est difficile à ouvrir : son ouverture requiert l’application d’une pression double de celle qui est nécessaire chez E.coli. Les deux homologues diffèrent principalement par la longueur de leur unique boucle périplasmique. De manière à examiner le rôle de la boucle, on a comparé l’activité du canal MscL de E.coli, celle du canal de M. tuberculosis et celle d’une protéine chimère constituée de la protéine de M. tuberculosis dans laquelle la boucle a été changée pour celle de la protéine de E.coli. De manière inattendue, nous avons constaté que les canaux de E.coli et de M. tuberculosis ont la même sensibilité à la pression. La protéine chimère n’avait pas d’activité canal. Si ce travail ne permet pas de conclure quant au rôle de la boucle, il montre sans ambigüité que contrairement à ce qui a été rapporté les canaux MscL de E.coli et de M. tuberculosis ne diffèrent pas sensiblement sur le plan fonctionnelThe production of heterologous membrane protein is notoriously difficult; this might be due to the fact that insertion of the protein in the membrane host is a limiting step. To by-pass this difficulty, two modes of synthesis were tested: 1) production in a cell-free system devoid of biological membrane but supplemented with detergent or liposomes, 2) production in bacteria, with targeting of the membrane protein to inclusion bodies. Both strategies were tested for the production of the human pannexin 1 channel (Px1). The gene coding the protein was fused with an “enhancer” sequence resulting in the addition of a peptide or short protein at the N terminus of the protein of interest. This enhancer sequence which is well produced in vitro or in vivo is supposed to facilitate the translation of the protein of interest. Three enhancer sequences were chosen: 1) the small porin OmpX of E. coli, which, in addition, should target the protein to inclusion bodies when the protein is expressed in bacteria 2) a peptide of phage T7 for expression in E.coli lysate or E.coli cells 3) the small protein SUMO for production in a wheat germ cell-free system. In a bacterial cell-free system, neither OmpX nor T7 promoted Px1 production. Px1 is only produced when the SUMO enhancer sequence is used in the wheat germ system. In bacteria, OmpX, known to form inclusions bodies did not promote the targeting of the fusion protein to inclusion bodies. Unexpectedly, the peptide T7 was able to do it.Px1 obtained from inclusion bodies (T7his-Px1) was renatured and reconstituted in liposomes. Similarly his6-Px1 produced in wheat germ system was reconstituted in liposomes. Both preparations were used for electrophysiological studies (patch-clamp and planar bilayers). With the refolded T7his-Px1, channel activity reminiscent of that observed with Px1 expressed in Xenope oocyte (Bao et al., 2004) could be detected, but only in three cases. In the case of his6-Px1, no clear channel activity could be observed. The second part of this work deals with the involvement of the periplasmic loop of the bacterial mechanosensitive channel MscL in its sensitivity to pressure. Mscl has become a model system for the investigation of mechanosensisity. Nearly all functional studies have been performed on MscL from E.coli while the structure of the protein has been obtained from the Mycobacterium tuberculosis homologue. In one functional study it was shown that MscL from M. tuberculosis is extremely difficult to open, gating at twice the pressure needed for E.coli MscL The periplasmic loop is the most variable sequence between the two homologues, being longer in E.coli than in M. tuberculosis. In order to assess the role of the periplamic loop in the sensitivity to pressure, we compared the activity of the E.coli and M. tuberculosis MscL and of a chimeric protein made of the M. tuberculosis protein in which the periplasmic loop has been exchanged for that of the E. coli channel. Unexpectedly, M. tuberculosis and E .coli MscL were observed to gate at a similar applied pressure. The chimeric protein had no functional activity. In conclusion, this study does not allow any conclusion as to the role of the loop in the sensitivity to pressure, but it shows clearly that, in contrast to the results of a previous study, there is no functional difference between E. coli and M. tuberculosis MscL
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Louiset, Estelle. "Implication de l'activité électrique des cellules mélanotropes de grenouille dans les processus de couplage stimulus-sécrétion : étude par la technique de patch-clamp." Rouen, 1989. http://www.theses.fr/1989ROUES033.
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Après avoir caractérisé les propriétés électriques des cellules mélanotropes de grenouille en culture primaire, il a été, dans le cadre d'une étude couplage stimulus-sécrétion, exploré les effets de 3 signaux neuroendocriniens: la thyrolibérine (TRH), l'acide gamma-aminobutyrique (GABA) et l'acétylcholine (ACH), en utilisant les techniques de patch clamp et de perfusion
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Negm, Ahmed. "Étude du rôle des canaux ASIC3 dans l'hypersensibilité à la douleur associée à une alimentation riche en lipides." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://theses.univ-cotedazur.fr/2019AZUR4046.
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L'obésité est un facteur de risque majeur pour de nombreux troubles métaboliques graves. Elle touche 13% de l'ensemble de la population adulte dans le monde, ce qui en fait un domaine de recherche important. L'obésité se caractérise par un indice de masse corporelle accru qui résulte d'un déséquilibre entre l'apport calorique et les dépenses énergétique. Cela peut être dû à une consommation accrue d'aliments riches en énergie, comme les aliments riches en graisses, qui correspondent à l'alimentation occidentale, en parallèle avec un faible niveau d’activité physique. Il est maintenant bien accepté que l'obésité induit une inflammation systémique chronique de bas grade, qui implique les adipokines et les cytokines libérées par l'intestin et le tissu adipeux. Cette inflammation de bas grade s'étend à d'autres tissus, entraînant des dysfonctionnements métaboliques systémiques. De plus, il a été démontré que l'obésité est corrélée à des douleurs chroniques, indépendamment des autres composantes du syndrome métabolique. Il n'est pas encore clairement établi comment cette douleur chronique est initiée et quels en sont les mécanismes. Notre étude s’est concentrée sur l'étude de l'effet de l'obésité sur l'activité des neurones sensoriels périphériques et la perception de la douleur, et la caractérisation des mécanismes cellulaires sous-jacents qui impliquent les canaux ioniques sensibles à l’acidose ASIC3. Méthodologie. Nous avons utilisé une alimentation riche en matières grasses composée essentiellement d'acides gras saturés pour induire l'obésité chez des souris juvéniles. Nous testons les seuils de perception des douleurs thermiques, mécaniques et chimiques chez les souris obèses à l'aide de tests de chaleur radiante Hargreaves, de von Frey dynamique et de formaline. Les approches électrophysiologiques, y compris les techniques de patch-clamp et l'enregistrement sur le nerf saphène, nous ont permis d'étudier l'effet l'obésité induite par un régime riche en graisses saturée sur l'excitabilité des neurones sensoriels périphériques. Résultats. Après 8 semaines d'alimentation riche en graisses (HFD), nous avons pu observer que les souris deviennent obèses. Ces souris sont prédiabétiques. Elles ont développé une dérégulation de l'homéostasie du glucose par rapport aux souris maigres nourries en régime standard. De plus, les souris obèses présentent une hypersensibilité thermique une fois l'obésité bien établie, alors que les autres modalités sensorielles ne sont pas affectées. Nous avons observé une surexpression des cytokines inflammatoires chez des souris obèses non seulement dans le tissu adipeux mais aussi dans d'autres tissus impliqués dans la voie de la douleur (c'est-à-dire les ganglions radiculaires dorsaux et la moelle épinière). Le régime alimentaire riche en lipides a provoqué une dyslipidémie avec une concentration accrue de plusieurs espèces de lipides dans le sérum des souris obèses, dont le lysophosphatidylcholine. Nous montrons que le sérum de souris obèses active directement les canaux ASIC3 et potentialise les réponses à une acidification modérée (pH 7). L'obésité augmente la réponse des fibres C mechano et thermosensibles cutanées à la chaleur. L’absence des canaux ASIC3 chez les souris knockout protège ces souris de l'hyperalgésie à la chaleur. Conclusions. Nos expériences ont mis en lumière l'impact de l'inflammation chronique de bas grade et de la dysrégulation métabolique induite par une alimentation riche en graisses, induisant l’obésité sur le système nerveux périphérique et la douleur. Nous démontrons le rôle des canaux ASIC3 dans sur l’excitation des neurones sensorielles et l’hyperalgésie thermique. Nos résultats donnent une portée clinique intéressante et suggèrent que l'hypersensibilité thermique associée à l'obésité induite par les lipides pourrait être traitée pharmacologiquement en bloquant ASIC3Obesity is a major risk factor for many serious disorders. It affects 13% of the whole adult population worldwide making it an important field for research. Obesity is characterized by an increased body mass index resulting from an energy imbalance between caloric intake and expenditure. This can be caused by an increased consumption of energy-dense foods such as food rich in fat, which corresponds to occidental diet. It is now well accepted that obesity induces chronic systemic low-grade inflammation, which is mediated by Adipokines and cytokines released from the gut and adipose tissue. This low-grade inflammation extends to other tissue leading to systemic metabolic dysfunctions. In addition, obesity was shown to be correlated to chronic pain regardless of other components of the metabolic syndrome. It is not yet clear how this chronic pain is initiated and what mechanisms are involved. Our study focuses on investigating the effect of obesity on peripheral sensory neurons activity and pain perception, followed by deciphering the underlying cellular and molecular mechanisms that involve the Acid Sensing Ion Channels ASIC3. Methods. Mice were fed with a high-fat diet composed of saturated fatty acids to induce obesity. We are using pain behavioral tests to measure the thermal, mechanical and chemical perception in obese mice using radiant heat Hargreaves test, dynamic von Frey, and formalin tests. Electrophysiological approaches including patch-clamp techniques and skin-saphenous nerve recording preparation allowed us to study the effect of high-fat diet and obesity on peripheral sensory neurons excitability, while qPCR and Immunohistochemistry chemistry were used in investigating the changes in pro-inflammatory factors expression. Results. After 8 weeks of high-fat diet (HFD), we observe that mice become obese. These mice developed a deregulation of glucose homeostasis compared to lean mice fed on standard regime. In addition, obese mice showed a long-lasting thermal hypersensitivity once the obesity was well established, while other sensory modalities were not affected. We found an overexpression of the inflammatory cytokines in obese mice not only in the adipose tissue but also in other tissues involved in the pain pathway (i.e. Dorsal root ganglions and spinal cord). In addition, the lipid rich diet induced dyslipidemia with increased concentration of several lipid species in the serum of obese mice. Delivering the serum from obese mice to recombinant ASIC3 channels directly activated the channels and potentiated the channels responses to moderate acidification (pH 7). Obesity led to increased firing of heat sensitive C-fibers. The genetic deletion of ASIC3 channels in ASIC3 knockout mice protected these mice from thermal hypersensitivity. Conclusions. Our experiments shed light on the impact of the chronic low-grade inflammation and metabolic dysregulation induced by fat-rich diet on the peripheral nervous system and pain, and on the role of ASIC3 channels in these conditions. Our results give an interesting clinical scope and suggest that the thermal hypersensitivity associated with lipid induced obesity could be treated pharmacologically by blocking ASIC3
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Wroblewska, Natalia. "Role of the ventromedial hypothalamus in control of innate defensive behaviours." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276036.
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Our senses are constantly bombarded with information. How does the brain integrate such a variety of inputs to generate appropriate behaviours? Innate defensive behaviours are a good model to address this question. They are essential for animal survival and the brain circuits that control them are highly conserved across species. Moreover, the sensory inputs and behavioural outputs can be well defined and reliably reproduced in the lab. This allows us to study function of the individual components of the circuit controlling these behaviours. Ventromedial hypothalamus (VMH) is a key brain region for controlling responses to predators; it has been shown that inactivating the VMH can reduce defensive behaviours. Interestingly, activating the VMH output neurons (SF1+ cells) can produce a variety of different behaviours, from immobility to escape, depending on the intensity of activation. During my PhD I used a variety of approaches to address the question of the function of the VMH in control of defensive behaviours. At first I hypothesised that the VMH might act as a centre responsible for choosing an appropriate behavioural response according to the stimulus. I set to investigate how different activation levels of SF1+ neurons can produce such different behavioural outputs, and how this activity is modulated in vivo in response to predator stimuli. I began the project by quantifying mouse defensive behaviours in response to olfactory and auditory predator cues, as well as to the optogenetic activation of SF1+ neurons. I then questioned whether there was heterogeneity within the population of SF1+ neurons, which could explain their ability to trigger different behaviours. I performed patch clamp recordings from acute brain slices and conducted a study of the electrophysiological properties of SF1+ neurons. I next investigated how SF1+ neurons integrate excitatory inputs from the medial amygdala, a region which receives olfactory inputs from the accessory olfactory bulb. By combining optogenetics with slice electrophysiology and behavioural assessment, I described the physiology and relevance of this connection. Finally, I investigated in vivo activity in the VMH in response to predator cues by performing calcium imaging of the VMH neurons in freely moving mice. By presenting different sensory stimuli, I addressed the question of heterogeneity of the input pattern to the VMH neurons and the relationship between the VMH activity and the behavioural output. Taken all together, the results of this project have led to a hypothesis whereby the function of the VMH is to facilitate rather than directly control the choice of an appropriate behavioural response.
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Werner, Sara Jane. "Long-Term Opiate-Induced Adaptations in Lateral Paracapsular Neurons of the Basolateral Amygdala." BYU ScholarsArchive, 2020. https://scholarsarchive.byu.edu/etd/8977.
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Increases in basolateral amygdala (BLA) activity drive avoidance-seeking behavior that may be associated with stress induced drug seeking. Activity of BLA pyramidal neurons is regulated by local and paracapsular gamma aminobutyric acid (GABA) interneurons. The lateral paracapsular interneurons (LPCs) border the external capsule, receive dense cortical/thalamic input and provide feed-forward inhibition onto BLA principle neurons. The GABAergic LPCs also express high concentrations of g-protein coupled µ-opioid receptors (MORs). Therefore, the effects of opiates on LPC activity and local GABA release were examined. Fluorescently double labeled LPCs were observed in glutamate decarboxylase (GAD) 65-mcherry/GAD67-green fluorescent protein (GFP) transgenic mice. Whole-cell electrophysiology experiments demonstrated that acute exposure to [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; a synthetic selective MOR agonist), reduced LPC firing and spontaneous inhibitory postsynaptic current (sIPSC) frequency in LPCs, with no apparent effect on spontaneous excitatory currents (sEPSCs). Current injection induced firing in LPC neurons, but less effectively than in saline controls. Morphine-exposed mice (10mg/kg/day, across 5 days, 1-2 days off) had increased sIPSCs compared to saline-injected controls, as well as enhanced adenylyl cyclase (AC) activity. Together these data show that LPC neurons are a highly sensitive targets for opiate-induced inhibition, and that long-term opiate exposure results in impaired LPC excitability, possibly contributing to anxiety observed during opiate withdrawal.
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Ramahaleo, Tiana. "Conductances ioniques, élasticité et perméabilité osmotique de cellules racinaires de colza (brassica napus)." Rouen, 1996. http://www.theses.fr/1996ROUES012.
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La racine est un organe essentiel pour l'absorption des ions et de l'eau chez les plantes supérieures. Dans le but de comprendre les processus impliqués dans le transport des ions au niveau de cet organe, des protoplastes et des vacuoles de cellules racinaires de colza ont été étudiés par la technique du patch clamp. Les courants macroscopiques majeurs détectés sur ces matériels sont des courants qui quittent le cytoplasme. Les courants vacuolaires observes présentent les caractéristiques des courants SV (Slow Vacuolar channels). Aussi bien sur les fragments de membrane du plasmalemme que sur le tonoplaste, des courants activés par la variation de la tension mécanique membranaire ont été mis en évidence. Nous avons également caractérisé les propriétés mécaniques des membranes qui contrôlent l'activation des canaux mécanosensibles sur les protoplastes et les vacuoles par l'élastimètre de Mitchison et Swann. Les valeurs du module d'élasticité K obtenues sur les protoplastes et les vacuoles sont semblables (88 et 50 milli-newton par mètre respectivement). Si le gadolinium, un inhibiteur des canaux mécanosensibles ne semble pas affecter l'élasticité du protoplaste, il paraît en revanche augmenter la rigidité du tonoplaste (96 milli-newton par mètre). Enfin, nous avons mis au point une technique simple de transfert d'un protoplaste isolé d'un milieu isotonique vers un milieu hypertonique (gradient osmotique : 0. 2 osmol). Elle permet de mesurer la vitesse initiale de plasmolyse des protoplastes et d'en déduire la valeur du coefficient de perméabilité osmotique, Pf(14 - 40 x 10#-3 centimètre par seconde). Des valeurs de Pf semblables ont été obtenues lors de transfert de vacuoles de betterave rouge (Pf = 7 - 25 x 10#-3 centimètre par seconde). Ces fortes valeurs de Pf pourraient signifier la présence d'aquaporines dans nos membranes.
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Chai, Shin Luen Chai. "Novel Genetic Modifiers in a Monogenic Cardiac Arrhythmia." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1516618028568975.
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Guerringue, Yannick. "Caractérisation du canal mécanosensible RMA et recherche de sa contribution à la mécanotransduction racinaire chez Arabidopsis thaliana." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS105.
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A la membrane plasmique des cellules végétales se concentrent des contraintes mécaniques générées par la pression de turgescence, la croissance des tissus adjacents ou des stimulations extérieures comme celles produites par le vent. Certaines protéines membranaires comme les canaux mécanosensibles sont activés par ces contraintes mécaniques. Ces canaux ioniques sont des protéines transmembranaires qui forment un pore dont l'ouverture dépend de la tension mécanique dans la membrane. Ils assurent ainsi la transduction directe d'une stimulation mécanique en transport ionique, activant des voies de signalisation cellulaires permettant de répondre à ces contraintes. L'activité d'un canal mécanosensible perméant au calcium a récemment été mise en évidence à la membrane plasmique de la plante modèle Arabidopsis thaliana par la technique de patch clamp. Ce canal mécanosensible, dont l'activité dépend de la protéine DEK1, reste non identifié et a été nommé RMA (Rapid Mechanically Activated). Dans le cadre de cette thèse, l'activation de RMA a été analysée en fonction de la tension membranaire et du temps, ce qui a permis de proposer un modèle d'activation du canal. De plus, différents mutants ont été analysés (Piezo, OSCA) pour trouver l'identité moléculaire de RMA. En parallèle, la participation de RMA à la signalisation calcique a été étudiée par l'expression de la sonde calcique R-GECO dans des plantules d'Arabidopsis. Ces plantules ont été cultivées dans des chambres microfluidiques, de telle sorte que leur racine principale grandisse dans un canal dont on puisse contrôler la composition du milieu liquide et la vitesse du flux. Les racines ont été soumises à des chocs osmotiques ou à une pression et les signaux calciques déclenchés ont été enregistrés. Les observations recueillies aux échelles moléculaire et racinaire sont mises en relation afin de proposer une vue intégrée du fonctionnement du canal RMAThe plant plasma membrane is subjected to mechanical stress generated by the turgor pressure, the development of the adjacent tissues or external mechanical cues such as wind. Transmembrane proteins, called mechanosensitive channels, permeate ions through the membrane when activated by an increase in the membrane mechanical tension. These nanosensors of mechanical cues directly transduce changes in tension into electrical potential variation, rapidly triggering cell mechanotransduction signaling pathways. The activity of a native mechanosensitive channel permeating calcium was recently recorded at the plasma membrane of the model plant Arabidopsis thaliana with the patch-clamp technique. This mechanosensitive channel, which is dependent on the DEK1 protein, is still not identified and was called Rapid Mechanically Activated (RMA). In the context of this PhD, the dynamics of gating of the RMA mechanosensitive channel were characterised over time and pressure and an activation model was proposed. Moreover, mutant plants knocked-out for genes encoding putative calcium mechanosensitive channels (Piezo, OSCAs) were analysed in order to find out its molecular identity. In parallel, the involvement of RMA in mechanically-induced calcium signaling in roots was investigated using the calcium sensor R-GECO expressed in Arabidopsis seedlings. These seedlings were grown in microponic chips in such way that their root grew in a channel of controlled liquid medium and controlled flow. Roots were subjected either to osmotic shock or to squeezing and calcium signals were recorded. The link between the observations obtained at the molecular and the root scales is discussed in order to give an integrated view of the function of RMA mechanosensitive channel
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Queyroy, Alain. "Principe d'étude de la cinétique coopérative de canaux chlore de cellules endothéliales humaines, à l'aide de diagrammes des transformations." Université Joseph Fourier (Grenoble), 1994. http://www.theses.fr/1994GRE10181.
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A l'aide de la technique du patch-champ, nous avons mis en evidence des canaux chlore de grande conductance (112,5 ps) sur des cellules endotheliales de veines ombilicales humaines. Pour etudier la cinetique de ces canaux qui se presentaient par groupes, nous avons utilise une methode originale d'analyse. Tout d'abord, nous avons developpe un filtre numerique appele lisseur adaptatif pondere (lap) qui ne presente pas de temps de montee et permet de reduire considerablement l'amplitude du bruit sans alterer les signaux utiles meme lorsque ceux-ci sont brefs et contigus. Ensuite, nous avons mis au point une representation graphique de la liste des evenements issue du signal. Cette representation appelee diagramme des transformations (dt) definit des empreintes cinetiques ou figurent la nature des evenements observes, leur proportion, et les temps de sejour moyens avant leur survenue. L'interpretation de telles empreintes nous a conduit a proposer un modele dimerique cooperatif et bistable pour decrire le comportement des canaux chlore observes. Cette hypothese a ete validee a l'aide de signaux simules selon des processus cooperatifs et markoviens, les signaux simules presentant le meme type d'empreintes cinetiques que les signaux reels. Une etude systematique du principe des dt nous a conduit a proposer la realisation de sequences animees d'empreintes (cinedt) a partir de signaux de tres longues durees. Ces cinedt devraient permettre de suivre l'evolution de cinetiques complexes de groupes canalaires sans necessiter de modelisation prealable. En outre, ils devraient constituer, une aide pour determiner rapidement les periodes stationnaires d'un signal ou il y a lieu d'effectuer une modelisation precise. Sur le plan technique le lap est tres performant malgre sa simplicite, par ailleurs les dt et cinedt ouvrent la porte a l'etude de la cooperativite inter-canalaire au niveau unitaire de simples groupes de canaux. La methode d'etude de la cooperativite que nous avons developpee devrait permettre de mieux comprendre un phenomene qui, du point de vue biologique, est susceptible de jouer un role fondamental dans les mecanismes de regulation de la physiologie cellulaire. Mots clefs: canaux ioniques cooperativite cellule endotheliale patch-clamp filtre numerique adaptatif diagramme des transformations
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Mangin, Jean-Marie. "Caractérisation fonctionnelle des sous-types du récepteur à la glycine exprimés au cours du développement du système nerveux dans un contexte non-synaptique." Paris 6, 2003. http://www.theses.fr/2003PA066205.
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Bessaïh, Thomas. "Excitabilité thalamique normale et pathologique." Paris 6, 2007. http://www.theses.fr/2007PA066290.
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Durant ma thèse, je me suis intéressé aux mécanismes cellulaires sous-jacents aux activités normales et pathologiques des neurones thalamocorticaux. Par des approches électrophysiologiques in-vitro, dans un modèle génétique reconnu d’épilepsie de type absence, j’ai étudié le rôle de l’inhibition GABAergique dans la pathophysiologie de cette maladie. J’ai mis en évidence des modifications de cette transmission circonscrites au noyau réticulé du thalamus. Leur implémentation dans un modèle théorique de ce noyau entraîne l’apparition d’une fréquence de résonance dans la gamme des fréquences des décharges pointe-onde observées in-vivo. Par la suite, j’ai étudié l’impact d’une régulation des canaux calciques de type T sur l’intégration synaptique des neurones thalamocorticaux. Mes résultats montrent qu’elle joue un rôle fondamental dans le traitement de l’information générée à l’intérieur de la boucle thalamocorticale mais ne modifie pas la réponse du réseau aux entrées sensorielles.
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Lado, Wudu E. "The Neural Substrate of Sex Pheromone Signalling in Male Goldfish (Carassius auratus)." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23460.
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The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.
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Michon, Francois-Xavier. "Electrophysiologie de l’hippocampe in vivo pendant le comportement : étude de l'impact de la locomotion sur le potentiel de membrane des cellules pyramidales de CA1 de l'hippocampe chez la souris naviguant dans un environnement virtuel." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0476/document.
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La locomotion spontanée a une forte influence sur l’état du réseau hippocampique et joue un rôle crucial lors de l’intégration de l'information spatiale. Différents états d'attention ou de comportement au cours de l'éveil peuvent modifier la réponse des neurones aux stimuli sensoriels ainsi que les performances dans les tâches associées. Au cours du mouvement (mov.) le potentiel de champ local de l’hippocampe est caractérisé par des oscillations de fréquence thêta et les cellules pyramidales (CPs) présentent une décharge spécifique à la localisation de l'animal dans un environnement donné. Cependant, les déterminants intracellulaires liés à l'activation des cellules pyramidales de CA1 pendant du mov. sont peu connus. Dans ce travail de thèse, nous avons enregistré le potentiel de membrane (Vm) des CPs de CA1 chez des souris qui alternaient spontanément entre des périodes de mov. et des périodes d’immobilité lors d’une tâche de navigation spatiale virtuelle. Nous avons trouvé une modulation opposée du Vm entre les CPs de CA1 qui déchargeaient de manière régulière par rapport à celles qui déchargeaient en bouffées de potentiels d’action. Les cellules qui déchargeaient de manière régulière étaient plus dépolarisées et déchargeaient plus pendant le mov.comparé à l’immobilité. Les cellules déchargeant en bouffées de potentiels d’action, préférentiellement inhibées pendant les sharp wave-ripples, étaient hyperpolarisées de façon dépendante à la vitesse pendant le mov.. Cette inhibition dépendante de la vitesse pourrait permettre d’augmenter le rapport signal sur bruit afin de coder l’information spatiale de manière plus efficace pendant le movSpontaneous locomotion strongly influences the state of the hippocampal network and is critically important for spatial information coding. In neocortex, different attentional or behavioral states during arousal can modify neurons responses to sensorial stimuli and associated task performance. During locomotion, the local field potential of the hippocampus is characterized by theta frequency oscillations (5-12 Hz) and the pyramidal neurons present a specific discharge to the localization of the animal in environments. However, the intracellular determinants of CA1 pyramidal cells activation during locomotion are poorly understood. Here we recorded the membrane potential of CA1 pyramidal cells (PCs) while non-overtrained mice spontaneously alternated between periods of movement and immobility during a virtual spatial navigation task. We found opposite membrane polarization between bursting and regular firing CA1 PCs during movement. Regular firing CA1 PCs were more depolarized and fired at higher frequency during movement compared to immobility while bursting CA1 PCs, preferentially inhibited during sharp wave ripples, were hyperpolarized during movement in a speed dependent manner. This speed-dependent suppression of a subpopulation of CA1 PCs could enhance signal to noise ratio for efficient spatial coding during locomotion
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Cao, William Sam. "Characterization and application of human pluripotent stem cell-derived neurons to evaluate the risk of developmental neurotoxicity with antiepileptic drugs in vitro." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/131.
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The risks of damage to the developing nervous system of many chemicals are not known because these studies often require costly and time-consuming multi-generational animal experiments. Pluripotent stem cell-based systems can facilitate developmental neurotoxicity studies because disturbances in nervous system development can be modeled in vitro. In this study, neurons derived from embryonal carcinoma (EC) and induced pluripotent stem (iPS) cells, were first characterized to establish their suitability for developmental neurotoxicity studies. The EC stem cell line, TERA2.cl.SP-12, was differentiated into neurons that expressed voltage-gated sodium and potassium channels as well as ionotropic GABA and glutamate receptors. These cells could also fire action potentials when stimulated electronically. However spontaneous action potentials were not observed. In contrast, pre-differentiated neurons derived from iPS cells fired evoked and spontaneous action potentials. Furthermore, iPS cell-derived neurons also expressed a wide array of functional voltage- and ligand-gated ion channels. Antiepileptic drugs (AEDs) are associated with developmental neurotoxicity. These agents can cause congenital malformations, cognitive deficits and behavioral impairment in children as a result of in utero exposure. The impact of four major AEDs, namely phenobarbital, valproic acid, carbamazepine and lamotrigine, on cell viability, cell cycle and differentiation of TERA2.cl.SP-12 into neurons was studied. All AEDs tested reduced differentiating stem cell viability. Valproic acid and carbamazepine increased apoptosis and reduced cell proliferation. A brief exposure to phenobarbital, valproic acid and lamotrigine at the start of differentiation impaired the subsequent generation of neurons. Additionally, the effect of transient exposure to phenobarbital and carbamazepine on neuronal maturation of iPS-derived neurons was investigated. Exposure to both AEDs resulted in diminished membrane potentials and reduced the proportion of cells that were able to fire action potentials spontaneously in culture. The data from these studies suggest that impairments in proliferation, differentiation and maturation of neurons derived from human stem cells may be sensitive indicators of neurodevelopmental disruption by these drugs that can result from in utero exposure. Furthermore, these findings suggest that the use of human pluripotent stem cells and neurons derived from them can reduce the time, cost and the number of animals used in toxicological research.
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Moussaud, Simon. "Etude de l'implication des cellules microgliales et de l'α-synucleine dans la maladie neurodégénérative de Parkinson." Phd thesis, Université de Bourgogne, 2011. http://tel.archives-ouvertes.fr/tel-00668186.
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Les maladies neurodégénératives liées à l'âge, telle celle de Parkinson, sont un problème majeur de santé publique. Cependant, la maladie de Parkinson reste incurable et les traitements sont très limités. En effet, les causes de la maladie restent encore mal comprises et la recherche se concentre sur ses mécanismes moléculaires. Dans cette étude, nous nous sommes intéressés à deux phénomènes anormaux se produisant dans la maladie de Parkinson : l'agrégation de l'α-synucléine et l'activation des cellules microgliales. Pour étudier la polymérisation de l'α-synucléine, nous avons établi de nouvelles méthodes permettant la production in vitro de différents types d'oligomères d'α-synucléine. Grâce à des méthodes biophysiques de pointe, nous avons caractérisé ces différents oligomères à l'échelle moléculaire. Puis nous avons étudié leurs effets toxiques sur les neurones. Ensuite, nous nous sommes intéressés à l'activation des microglies et en particulier à leurs canaux potassiques et aux changements liés au vieillissement. Nous avons identifié les canaux Kv1.3 et Kir2.1 et montré qu'ils étaient impliqués dans l'activation des microglies. En parallèle, nous avons établi une méthode originale qui permet l'isolation et la culture de microglies primaires issues de cerveaux adultes. En comparaison à celles de nouveaux-nés, les microglies adultes montrent des différences subtiles mais cruciales qui soutiennent l'hypothèse de changements liés au vieillissement. Globalement, nos résultats suggèrent qu'il est possible de développer de nouvelles approches thérapeutiques contre la maladie de Parkinson en modulant l'action des microglies ou en bloquant l'oligomérisation de l' α-synucléine.
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Pons, Bennaceur Alexandre. "Les mécanismes antiépileptiques de l’AppCH2ppA dans la sclérose tubéreuse de Bourneville." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0303.
Full textAbstract:
La Sclérose Tubéreuse de Bourneville est une pathologie génétique rare qui se caractérise par la survenue de crises épileptiques précoces à l’origine du développement de nombreux troubles neurologiques tels que des symptômes autistiques ou des retards mentaux. Les épilepsies retrouvées dans la Sclérose Tubéreuse de Bourneville sont souvent résistantes aux traitements pharmacologiques disponibles soulevant la nécessité de trouver de nouvelles approches médicamenteuses plus efficaces pour traiter les patients. Dans cette étude nous avons mis en évidence que l’AppCH2ppA est une molécule efficace pour bloquer la survenue des crises épileptiques dans un modèle de souris pour la Sclérose Tubéreuse de Bourneville ainsi que sur des résections chirurgicales de tissu provenant de patients humains atteints par la Sclérose Tubéreuse de Bourneville. Nous avons montré que les propriétés antiépileptiques de l’AppCH2ppA s’appuient sur une libération autocrine d’adénosine par les neurones de la couche IV du cortex somatosensoriel et d’une activation consécutive des récepteurs à l’adénosine de type A1. Cette activation a lieu spécifiquement au niveau du compartiment postsynaptique et est responsable d’une activation de conductances potassiques et d’une diminution de l’excitabilité des neurones. L’administration d’AppCH2ppA n’est associé à aucun effet secondaire notables sur la santé des souris. Ainsi l’AppCH2ppA semble être un outil thérapeutique prometteur et peu risqué qui stimule des mécanismes antiépileptiques endogènes naturellement sollicités par le cerveau et efficaces pour stopper et limiter la survenue des crises épileptiquesTuberous Sclerosis Complex (TSC) is a rare genetic disease characterized by the presence of epilepsies that appear early and in the life of patients and are responsible for the development of several neurological disorders such as autistic symptoms or mental retardations.In TSC, epileptic seizures often resist to pharmacological approaches raising the importance to find new molecules to treat more efficiently the patients.In this study we showed that AppCH2ppA is an effective molecule to block the onset of epileptic seizures in a mouse model for Tuberous Sclerosis as well as on human patients tissues.We have shown that AppCH2ppA nduce an autocrine release of adenosine by the spiny stellate cells present in the layer IV of the somatosensory cortex. This release is responsible for a subsequent activation of adenosine A1 receptors that occur specifically in the postsynaptic compartment of neurons and is responsible for an activation of potassium channels and a decrease of the excitability of neurons. The administration of AppCH2ppA is not associated with any significant side effects on mouse health. Thus, AppCH2ppA appears to be a promising and low-risk therapeutic tool that stimulates an endogenous antiepileptic pathway that is naturally used in the brain and that is efficient to stop and limit the appearance of epileptic seizures
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Zeitler, Alexandre. "Traitement d'une douleur neuropathique par la modulation pharmacologique du complexe basolatéral de l'amygdale." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ115/document.
Full textAbstract:
L’amygdale est une structure du système nerveux central impliquée dans l’intégration des émotions comme la peur et l’anxiété. Des études ont également montré que l’amygdale peut moduler de façon positive ou négative la douleur par le biais des projections de son noyau de sortie, le noyau central de l’amygdale (CeA), sur les structures impliquées dans les contrôles nociceptifs descendants. Le complexe basolatéral de l’amygdale (BLA), placé en amont du noyau central, est intimement connecté à ce dernier et peut ainsi réguler son fonctionnement. Les données obtenues au cours de ma thèse ont montré l’existence d’un contrôle tonique amygdalien de la nociception et de la douleur, directement dépendant de l’équilibre entre l’excitation et l’inhibition au sein de la structure. Ainsi la modulation de l’une ou l’autre des neurotransmission influence directement la sortie douloureuse, chez des animaux sains ou neuropathiques. Par ailleurs, nous nous sommes également intéressés à l’étude précise du mode d’action d’une molécule anxiolytique non benzodiazépinique, l’étifoxine (EFX), et à son effet sur le comportement douloureux. Les résultats des injections d’EFX dans le BLA indiquent qu’elle induit une action analgésique chez les animaux neuropathiques, mais ne modifie pas les seuils de sensibilité des animaux sains. Cette action analgésique est dépendante de l’effet indirect neurostéroïdogène de l’EFX. Ceci, associé au fait que l’EFX ne présente pas ou peu d’effets secondaires, en fait une molécule particulièrement intéressante pour un traitement alternatif des douleurs neuropathiques. Dans une dernière partie, nous avons cherché à observer les mécanismes en jeu au niveau cellulaire lorsque l’EFX est appliquée sur les neurones du BLA. Ainsi, nous avons montré que l’EFX potentialise l’inhibition au sein de la structure, via trois mécanismes indépendants ; l’augmentation de la fréquence des courants post-synaptiques inhibiteurs miniatures (mCPSIs), l’augmentation de leur amplitude, et l’augmentation de leur constante de déactivation. Ces deux derniers effets sont dépendants de l’action neurostéroïdogène de l’EFX, tandis que l’effet sur la fréquence des mCPSIs est du à l’action de l’EFX sur le récepteur GABAA. Les résultats de ma thèse ont ainsi permis de montrer l’existence d’un contrôle tonique amygdalien de la douleur, dans le cadre d’animaux neuropathiques, mais également de la nociception, chez des animaux sains. Par ailleurs, la place du complexe basolatéral de l’amygdale, souvent peu prise en compte dans les études sur la douleur, a été redéfinie. Ce complexe doit être pris en considération dans le circuit de la douleur et son rôle de pilote du noyau central de l’amygdale ne doit pas être négligéThe amygdala is a major control center of the emotions, but also integrates sensory, especially nociceptive information. Cortical afferents to the amygdala largely target its basolateral complex. The basolateral amygdala (BLA) then projects to the central amygdala nucleus, which in turn projects densely to the periaqueductal gray and thus can drive a behavioural output via the spinal cord. Data obtained during my thesis have shown that the balance between excitation and inhibition in the BLA triggers an tonic control of pain. Therefore modulating one of the neurotransmission directly influences the pain threshold of control or nociceptive mice. My thesis work also focused on the functioning of an anxiolytic and non benzodiazepinic drug ; Etifoxin (EFX). This molecule as a positive modulator of GABAA receptors and indirectly by increasing the synthesis of neurosteroids, also known as strong modulator of these receptors. In our team, we already showed that EFX has anti-nociceptive effects when injected intraperitonealy in rats. Here we wanted to determine the action of EFX on pain descending control drive by BLA. We showed that EFX infusion in the BLA seems to be anti-nociceptive, inducing a recover of the pre-cuff mechanical threshold level. We also used a patch-clamp approach to study directly in vitro the modulation of the inhibitory synaptic transmission produced by EFX. We showed that EFX potentiate the inhibition in BLA neurons via different and complementary mechanisms. These potentiating effects are mostly dependent of a neurosteroidogenesis increase
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Campelo, Tiago. "De la diffusion latérale des récepteurs AMPA à la perception des whiskers : un nouveau modèle de cartographie corticale." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0171.
Full textAbstract:
Les champs récepteurs corticaux se réorganisent en réponse aux changements de l'environnement. Par exemple, suite à une lésion périphérique, les modalités sensorielles préservées gagnent de l'espace cortical au détriment de celles lésées. L'étude du cortex somatosensoriel en tonneau des rongeurs a fourni des données importantes pour la compréhension des mécanismes synaptiques à l'origine de cette réorganisation corticale. En condition normale, les neurones de chaque colonne corticale répondent préférentiellement à la stimulation d'une seule vibrisse principale ("Principal Whisker, PW"). Au contraire, suite à l'amputation de l'ensemble des vibrisses sauf une ("Single Whisker Experience, SWE"), les neurones des colonnes associées aux vibrisses amputées répondent à la stimulation de la vibrisse conservée, à l'origine du renforcement et de l'expansion des représentations corticales des vibrisses conservées. Bien que des preuves indirectes aient révélées un rôle de la potentialisation à long terme ("Long-Term Potentiation, LTP") de synapses préexistantes dans la modification des cartes corticales, probablement via une augmentation du nombre des récepteurs AMPA (AMPARs) aux synapses, un lien direct entre la LTP, la réorganisation des cartes corticales, et l'adaptation des comportements sensori-moteurs suite à une altération des entrées sensorielles n'a pas encore été démontré. L'objectif de cette thèse a donc été de mettre en évidence cette relation de façon expérimentale et en condition physiologique. Pour cela, nous avons mis au point une stratégie in vivo combinant des enregistrements électrophysiologiques, de l'imagerie biphotonique et l'analyse du comportement d'exploration chez la souris contrôle ("Full Whisker Experience, FWE) et amputée de certaines vibrisses (SWE). Nous avons d'abord confirmé que la stimulation rythmique de la PW ("Rhytmic Whisker Swtimulation, RWS") renforce les synapses excitatrices (RWS-LTP) in vivo des souris anesthésiées FWE. Au contraire des souris FWE, les neurones pyramidaux des souris SWE présentent une augmentation de l'excitabilité neuronale et une absence de RWS-LTP, indiquant ainsi que les synapses corticales associées à la vibrisse intacte ont été potentialisées en réponse au protocole SWE. Pour mieux comprendre l'implication de la RWS-LTP dans la réorganisation des cartes corticales et l'adaptation des comportements sensori-moteurs, nous avons développé une nouvelle approche pour manipuler la LTP in vivo grâce à l'immobilisation des AMPARs par des anticorps extracellulaires ("cross-linking"). En effet, notre équipe a montré précédemment que le cross-linking des AMPARs empêche la LTP in vitro. Par ailleurs, une accumulation des AMPARs au niveau post-synaptique a été démontrée in vivo par imagerie biphotonique au cours d'une stimulation RWS, suggérant un rôle de la mobilité de ces récepteurs dans cette RWS-LTP. Au cours de cette thèse, nous avons démontré que le cross-linking des AMPARs in vivo bloque également l'expression de la RWS-LTP, mais sans affecter la transmission synaptique basale, ni l'induction de la RWS-LTP, indiquant ainsi que la mobilité des AMPARs est également fondamental pour l'expression de la LTP in vivo. De façon importante, le cross-linking des AMPARs de façon chronique, au cours du SWE, permet non seulement de rétablir la RWS-LTP et l'excitabilité neuronale, et donc de bloquer la réorganisation corticale, mais aussi de modifier les capacités de récupération sensori-motrices des souris amputées. Dans l'ensemble, nos données démontrent pour la première fois un rôle critique et direct de la RWS-LTP dans le réarrangement des circuits en réponse à l'amputation de certaines vibrisses. La réorganisation des cartes corticales serait ainsi assurée par le renforcement de la transmission synaptique, et constituerait alors un mécanisme compensatoire pour optimiser le comportement sensorimoteur de l'animal lors de l'altération des entrées sensoriellesNeuronal receptive fields in the cerebral cortex change in response to peripheral injury, with active modalities gaining cortical space at the expense of less active ones. Experiments on the mouse whisker-to-barrel cortex system provided important evidences about the synaptic mechanisms driving this cortical remapping. Under normal conditions, neurons in each barrel-column have receptive fields that are strongly tuned towards one principal whisker (PW). However, trimming all the whiskers except one (single-whisker experience, SWE) causes layer (L) 2/3 pyramidal neurons located in the deprived and spared-related columns to increase their response towards the spared input. This results in a strengthening and expansion of the spared whisker representation within the barrel sensory map. Indirect evidences suggest that these cortical alterations might depend on the activity-dependent potentiation of pre-existing excitatory synapses (LTP), likely through increased levels of postsynaptic AMPA receptors (AMPARs). However, a clear link between LTP, cortical remapping, and the adaptation of sensorimotor skills following altered sensory experience has not yet convincingly been demonstrated. Here, we combined in vivo whole-cell recordings, 2-Photon calcium imaging and a whisker-dependent behavior protocol to directly demonstrate this relationship. It has been described that rhythmic whisker stimulation potentiates cortical synapses (RWS-LTP) in vivo. An accumulation of postsynaptic AMPARs during similar sensory stimulation was also reported by imaging evidences. Our data demonstrates that this potentiation is occluded by SWE, suggesting that cortical synapses are already potentiated by this trimming protocol. This is translated into an increased neuronal excitability in the spared column and sensorimotor recovery by the spared whisker. To better understand the implication of LTP in cortical remapping, we developed a novel approach to manipulate LTP in vivo without affecting overall circuit properties. Our team showed previously that the blockage of AMPARs synaptic recruitment by extracellular antibody cross-linking prevents LTP in vitro. Here, we report that in vivo cross-linking of AMPARs blocks the expression but not the induction of RWS-LTP, suggesting that the synaptic recruitment of AMPARs is fundamental for in vivo LTP as well. Moreover, chronic AMPAR cross-linking during SWE reverts RWS-LTP occlusion and the increased neuronal excitability caused by whisker trimming. As consequence, the sensorimotor performance by the spared whisker is permanently impaired by the blockage of cortical remapping. Altogether, these evidences led us to define a critical role for synaptic LTP on circuit re-arrangement after whisker trimming. Our data shows that LTP-driven cortical remapping is a compensatory mechanism to optimize animal’s sensorimotor behavior upon altered sensory experience
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Delgado, Zabalza Lorena. "Electrophysiological characterization of neuronal diversity in the substantia nigra pars reticulata in control and parkinsonian mice." Thesis, Bordeaux, 2020. http://www.theses.fr/2020BORD0052.
Full textAbstract:
La substance noire réticulée (SNr) est la principale structure de sortie des ganglions de la base (BG), un réseau sous-cortical contrôlant l'élaboration des programmes moteurs ainsi que des fonctions d'apprentissage cognitives et associatives. L'identification de types de cellules distincts dans le BG a joué un rôle clé pour comprendre les propriétés et les fonctions de ce circuit. Des études récentes suggèrent que la SNr est composé de plusieurs types de cellules mais jusqu'à présent cette diversité neuronale n'a jamais été prise en compte concernant le fonctionnement normal et pathologique de ce noyau, notamment dans la maladie de Parkinson (MP). En combinant des approches immunohistochimiques et électrophysiologiques chez la lignée de souris PVCre::Ai9T, nous avons démontré que les neurones de la SNr exprimant la protéine parvalbumine (PV+) ont des propriétés anatomiques et électrophysiologiques différentes de celles des neurones ne l’exprimant pas (PV-). Notre analyse anatomique montre que les neurones PV+ et PV- sont présents à proportion égale dans la SNr, mais avec une distribution distincte, les neurones PV+ étant enrichis dans la partie latérale de la SNr alors que les neurones PV- sont présents dans la partie médiale de ce noyau. Nos enregistrements électrophysiologiques in vitro ont révélé que les neurones PV+ ont une activité électrique plus élevée que les cellules PV-. De plus, nos données indiquent que la perte de dopamine (DA) et le traitement à la L-DOPA induisent une réduction profonde de l'excitabilité des neurones PV+ de la SNr dans un modèle murin de MP (la souris 6-OHDA) sans modifier l'activité des neurones PV-.Il est bien connu que l'activité des neurones de la SNr est contrôlée par les afférences GABAergiques des neurones striataux (STR-SNr) de la voie directe (dSPN) et du globus pallidus (GP-SNr). Nous avons effectué une manipulation optogénétique de ces deux voies inhibitrices et montré que ces deux populations neuronales sont innervées de manière équivalente par le STR et le GP. Nos résultats ont également révélé que l'inhibition striatale était plus efficace que l’inhibition pallidale pour réduire l'activité des deux sous-types de neurones SNr. De plus, nous avons observé que les2synapses STR-SNr et GP-SNr affichent la même plasticité à court terme sur les neurones PV+ et PV- de la SNr. Enfin, nous avons montré que la transmission GABAergique est affectée de manière différentielle sur les cellules PV+ et PV- suite à une déplétion DAergique. D'une part, les neurones PV+ sont plus sensibles à l'inhibition striatale que les cellules PV- après une déplétion en DA. D’autre part, l’inhibition pallidale est réduite sur les neurones PV+ et augmentée sur les PV- après la perte de DA, suggérant un déséquilibre de l'inhibition pallidale entre ces deux populations SNr.Il est aussi connu que les niveaux extracellulaires de GABA sont élevés dans la SNr chez les modèles rongeurs de MP, ce qui suggère que les neurones de la SNr pourraient être inhibés de manière permanente par une inhibition dite tonique. Ainsi, Nous avons caractérisé la transmission extrasynaptique GABAergique dans la SNr des souris témoins et 6-OHDA. Nos enregistrements de patch-clamp ont révélés que les neurones PV- présentent une inhibition tonique plus importante que les cellules PV+ chez les souris témoins. La présence et l'implication des sous-unités δ et α5 dans les récepteurs GABAA extrasynaptiques ont également été étudiées, révélant une présence et un effet majeurs des sous-unités α5 sur les neurones PV. Cependant, contrairement à ce qui était attendu, la déplétion chronique en DA ne provoque aucun changement de l'inhibition tonique ni dans les cellules PV+ ni dans les neurones PV- de la SNr.Nos résultats mettent en évidence l'importance de différencier les populations cellulaires de la SNr pour une meilleure connaissance du fonctionnement des BG en situation physiologique et physiopathologique tel que dans la MPThe substantia nigra pars reticulate (SNr) is the main output structure of the basal ganglia (BG), a subcortical network controlling the elaboration of motor programs as well as cognitive and associative learning functions. The identification of distinct cell-types within the BG has played a key role for understanding the properties and functions of this circuit. Recent studies suggest that the SNr is composed of several cell types but until now this neuronal diversity has never been taken into consideration regarding normal and pathological functioning of this nucleus, particularly in Parkinson’s disease (PD). By combining immunohistochemical and electrophysiological approaches in the PVCre::Ai9T mouse line, we have demonstrated that SNr neurons expressing the protein parvalbumin (PV+) exhibit different anatomical and electrophysiological properties than non PV-expressing (PV-) neurons. Our anatomical analysis reveal that PV+ and PV- neurons are present in equal proportion in the SNr, but with a distinct distribution, PV+ being enriched in the lateral part of the SNr, while PV- are found in the medial portion of the nucleus. In vitro electrophysiological recordings from identified PV+ and PV- neurons in the SNr also revealed that PV+ neurons fired at relatively higher rates than PV- cells. Additionally, our data revealed that DA loss and subsequent L-DOPA treatment induce a profound reduction of the excitability of PV+ SNr neurons in a 6-OHDA mouse model of PD while activity of PV- remains unchanged by these treatments.It is well known that the activity of SNr neurons is controlled by GABAergic inputs from striatal dSPN and the GP. We performed optogenetic manipulation of STR-SNr and GP-SNr inputs in order to determine whether PV+ and PV- SNr neurons received equivalent inputs from these two nuclei. We tested the impact of STR-SNr or GP-SNr activation on the activity of SNr neurons in cell-attached configuration and then switched to whole-cell voltage-clamp to characterize short-term plasticity of these synapses. Our results show that both PV+ and PV- SNr neurons are innervated by the STR and the GP. They also revealed that inhibition from dSPN was more powerful to silence activity of both subtypes of SNr neurons. Indeed, we observed that both STR-SNr and GP-SNr synapses displayed short-term depression in PV+ and PV- SNr neurons. DA loss affected GABA transmission in a different manner in PV+ and PV- SNr cells. On one hand, PV+ neurons were more sensible to striatal synaptic inhibition than PV- cells after DA depletion. On the other hand, PV-GP inputs were reduced on PV+ neurons and increased in PV- cells after DA loss suggesting a disequilibrium in pallidal inhibition between these two SNr populations.Furthermore, considering that rodent models of PD have shown elevated extracellular levels of GABA in the SNr which can exert a tonic extrasynaptic inhibition on SNr neurons, we decided to characterize GABAergic extrasynaptic transmission in the SNr of control and 6-OHDA lesioned mice. We studied GABAA mediated tonic inhibition by performing whole-cell patch-clamp recordings of PV+ and PV- SNr neurons in acute slices. We observed that PV- SNr neurons displayed larger GABAA receptor-mediated tonic currents than PV+ cells in the SNr of control mice. The presence and involvement of δ and/or α5 extrasynaptic subunits in GABAA receptors mediating this type of transmission was also studied, revealing a major presence and effect of α5-subunits on PV- neurons probably mediating the tonic currents observed in these neurons. However, contrary to expected, chronic DA-depletion did not trigger any increase in tonic inhibition neither in PV+ cells nor in PV- SNr neurons.All these findings highlight the importance of differentiating cell populations in the SNr to a better knowledge of the BG circuit in normal and pathological states such as in PD
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Shekter, Lee Russell. "G protein regulation of human, neuronal, calcium channels /." 1999. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:9943114.
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Erickson, Andelain Kristiseter. "Role of Voltage-gated Sodium Channel Isoforms in Electrophysiological Properties of Neurons Innervating the Viscera in Mice." Thesis, 2019. http://hdl.handle.net/2440/120764.
Full textAbstract:
Chronic visceral pain is a poorly managed symptom of functional and inflammatory gastrointestinal disorders and there is a lack of suitable analgesics that are efficacious without gastrointestinal side effects. Voltage-gated sodium (Nav) channels regulate action potential generation and cell membrane excitability in sensory neurons and are implicated in several enhanced pain and loss-of-pain phenotypes in humans. Pharmacological modulation of Nav channels has been investigated as a therapeutic strategy for the past two decades in a range of pain modalities, including somatic, neuropathic, and more recently - visceral pain. In this thesis, gene transcripts for the nine Nav channel isoforms (Nav1.1-Nav1.9) were detected in dorsal root ganglia (DRG) neurons retrogradely labeled from the colon and bladder in mice, and the contribution of different isoforms to active electrophysiological properties in these neurons was evaluated using Nav-selective modulators. An evaluation of electrophysiological properties of colon-innervating DRG neurons from healthy and chronic visceral hypersensitivity (CVH) mice was also conducted and did not provide sufficient support for a model-related phenotype in vitro. In the pharmacological part of this thesis, it was found that inhibition of tetrodotoxin-sensitive Nav channels (Nav1.1-Nav1.4, Nav1.6 and Nav1.7) effectively altered electrophysiological responses in colon-innervating and bladder-innervating neurons, and furthermore reduced bladder afferent responses to distension and nociceptive signaling to the spinal cord. Electrophysiological responses in colon-innervating DRG neurons were also modulated by less selective Nav modulators, such as veratridine, which targets all Nav channel isoforms, and more selective Nav modulators, such as Hs1a, which targets Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7; OD1, which targets Nav1.4, Nav1.6, and Nav1.7; ICA-121341, which targets Nav1.1-Nav1.3; A-803467, which targets Nav1.8; and Compound B, which targets Nav1.1. inhibition of Nav1.1 using Compound B was furthermore shown to be effective in reducing pain responses to colorectal distension in CVH mice.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2019
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Elies, Jacobo, M. L. Dallas, J. P. Boyle, J. L. Scragg, A. Duke, D. S. Steele, and C. Peers. "Inhibition of the cardiac Na+ channel Nav1.5 by carbon monoxide." 2014. http://hdl.handle.net/10454/12221.
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YesSublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, L-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor Nω-nitro-L-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state.
This work was supported by the British Heart Foundation
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Huang, Helena Hung-Yin. "Odour Processing By Principal Neurons of the Piriform Cortex In Vivo." Phd thesis, 2015. http://hdl.handle.net/1885/101987.
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The piriform cortex (PC) is important for the cortical processing
of olfactory information. The PC is widely viewed as a pattern
recognition device whose primary function is the formation of
‘odour objects’, that is, the perception of complex odours
comprising many chemical components as single olfactory percepts
(e.g. the scent of a rose), a task that is heavily dependent on
the intracortical associational network. Two types of
glutamatergic principal neurons are present in approximately
equal numbers in layer 2 of the PC: semilunar (SL) and
superficial pyramidal (SP) cells. Despite a long-standing
misconception that the principal neurons of the PC are a
functionally homogenous population, recent work has shown that SL
and SP cells differ significantly in their connectivity and in
vitro electrophysiology. Specifically, emerging data has
indicated that intracortical connectivity increases with
increasingly deep somatic locations, suggesting that, in response
to olfactory stimulation, cortical computations shift from
afferent (sensory) processing to associative processing down the
PC superficial-to-deep axis. In light of the important
differences between the two main classes of principal neurons, we
hypothesised that SL and SP cells contribute differentially to
the cortical processing of olfactory sensory information.
In this thesis, we examined the odour responses of layer 2
principal neurons in the anterior PC using whole-cell patch clamp
electrophysiology. We show that odour responses are highly
variable and richly nuanced in vivo; however, they can be broadly
separated into three basic categories: excitation, inhibition and
unresponsiveness. Our current clamp data revealed that an
intrinsic property, the spike after-hyperpolarisation (AHP),
correlates strongly with the somatic laminar depth, suggesting
that the AHP could be used to identify neurons (SL or SP cells)
recorded in vivo. Importantly, current clamp data indicate that
olfactory excitatory tuning correlates strongly with the AHP in
vivo, such that putative SP cells (characterised by a small AHP)
are more broadly excited by odours than putative SL cells
(characterised by a large AHP), presumably due to a difference in
intracortical connectivity. These results further suggest that,
rather than assuming a sharp dichotomy of neuronal phenotypes,
SL-like cells gradually transition into SP-like cells along the
cortical superficial-to-deep axis.
Voltage clamp data indicate that principal neurons are under a
substantial level of tonic inhibition in the absence of odour. We
show that spontaneous and odour-evoked inhibition dominates and
scales with excitation under physiological conditions. These
results suggest that local inhibition contributes to the reported
sparse odour coding in the cortex.
Overall, these data are consistent with our proposed model of the
anterior PC excitatory network, which posits that 1) principal
neuron phenotype transitions smoothly across the cortical
superficial-to-deep axis and 2) principal neurons become
increasingly incorporated into the local intracortical
microcircuits with increasingly deep somatic location, as a
result, 3) neuronal excitatory tuning becomes progressively
broader with increasing somatic depth. These results suggest that
SL cells may be important for decorrelating overlapping input
patterns and hence preserving the salient olfactory attributes of
an odour, whereas SP cells are more heavily implicated in the
associative aspect of olfactory processing.
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Shute, Lauren. "The effects of neuropeptide Y on dissociated subfornical organ neurons." 2017. http://hdl.handle.net/1993/32071.
Full textAbstract:
The subfornical organ (SFO) is a sensory circumventricular organ, lacking a proper blood-brain barrier. Neurons of the SFO are exposed directly to the ionic environment and circulating signaling molecules in the plasma, providing a unique window for communication of physiological status from the periphery to the central nervous system (CNS). The SFO is recognized as a key site for hydromineral balance, cardiovascular regulation and energy homeostasis. Neuropeptide Y (NPY) is a potent stimulator of food intake when released centrally, and has well-documented pressor effects when released peripherally. It has been demonstrated that the SFO expresses NPY receptors, however the effects of NPY on SFO neurons has never been investigated. The aim of this study was to determine the effects of NPY on the electrophysiological properties of SFO neurons dissociated from Sprague Dawley rats. Using whole cell patch clamp techniques in the current-clamp configuration, we report that 300 nM NPY caused 16% of SFO neurons to depolarize and 26% to hyperpolarize. The remaining neurons were insensitive to NPY. These effects were dose-dependent with a combined EC50 of 3.7 nM. Specific NPY receptor antagonists were applied, suggesting that the Y5 receptor predominately elicited a hyperpolarizing effect, while the Y1 receptor had a mixed response that was predominately hyperpolarizing, and the Y2 receptor had a mixed response that was predominately depolarizing. Using the voltage-clamp configuration, it was also observed that NPY caused an increase in the voltage-gated K+ current density as well as a shift in membrane activation of the persistent Na+ current, mediating the hyperpolarizing and depolarizing effects, respectively. These findings indicate that NPY elicits electrophysiological changes on SFO neurons, suggesting that the SFO is a key site of action for NPY in mediating energy regulation and/or cardiovascular output.February 2017
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HAYTON, SCOTT JOSEPH. "LEARNING IMPULSE CONTROL IN A NOVEL ANIMAL MODEL: SYNAPTIC, CELLULAR, AND PHARMACOLOGICAL SUBSTRATES." Thesis, 2011. http://hdl.handle.net/1974/6600.
Full textAbstract:
Impulse control, an executive process that restrains inappropriate actions, is impaired in numerous psychiatric conditions. This thesis reports three experiments that utilized a novel animal model of impulse control, the response inhibition (RI) task, to examine the substrates that underlie learning this task.
In the first experiment, rats were trained to withhold responding on the RI task, and then euthanized for electrophysiological testing. Training in the RI task increased the AMPA/NMDA ratio at the synapses of pyramidal neurons in the prelimbic, but not infralimbic, region of the medial prefrontal cortex. This enhancement paralleled performance as subjects underwent acquisition and extinction of the inhibitory response.
AMPA/NMDA was elevated only in neurons that project to the ventral striatum. Thus, this experiment identified a synaptic correlate of impulse control.
In the second experiment, a separate group of rats were trained in the RI task prior to electrophysiological testing. Training in the RI task produced a decrease in membrane excitability in prelimbic, but not infralimbic, neurons as measured by maximal spiking evoked in response to increasing current injection. Importantly, this decrease was strongly correlated with successful inhibition in the task. Fortuitously, subjects trained in an operant control condition showed elevated infralimbic, but not prelimbic, excitability, which was produced by learning an anticipatory signal that predicted imminent reward availability. These experiments revealed two cellular correlates of performance, corresponding to learning two different associations under distinct task conditions.
In the final experiment, rats were trained on the RI task under three conditions: Short (4-s), long (60-s), or unpredictable (1-s to 60-s) premature phases. These conditions produced distinct errors on the RI task. Interestingly, amphetamine increased premature responding in the short and long conditions, but decreased premature responding in the unpredictable condition. This dissociation may arise from interactions
between amphetamine and underlying cognitive processes, such as attention, timing, and conditioned avoidance.
In summary, this thesis showed that learning to inhibit a response produces distinct synaptic, cellular, and pharmacological changes. It is hoped that these advances will provide a starting point for future therapeutic interventions of disorders of impulse control.Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2011-07-11 09:44:54.815
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Mihic, Anton. "Interaction of hERG Channels and Syntaxin 1A." Thesis, 2009. http://hdl.handle.net/1807/17438.
Full textAbstract:
The human ether-à-go-go related gene (hERG) encodes the pore-forming voltage-gated K+ channel that is essential for cardiac repolarization. Dr. Tsushima’s laboratory has previously characterized the endogenous expression of SNARE proteins in the mammalian heart, and the interaction of the SNARE protein syntaxin 1A (STX1A) with several cardiac ion channels. Here, we utilize a multi-disciplinary approach to describe the inhibitory effect of STX1A on hERG channel function. STX1A impairs hERG channel maturation and trafficking to the plasma membrane and induces a hyperpolarizing shift in the voltage-sensitivity of steady-state inactivation. We identify the residues involved in this protein-protein interaction through the use of hERG truncation mutations. We also describe the pharmacological and temperature-mediated rescue of hERG channel trafficking in the presence of STX1A. The regulation of cardiac ion channels by SNARE proteins represents a novel biological mechanism that may have universally intrinsic implications for normal and diseased heart function.
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(6803957), Qiuyu Wu. "MAPPING BRAIN CIRCUITS IN HEALTH AND DISEASE." Thesis, 2019.
Find full textAbstract:
Intricate neural circuits underlie all brain functions. However, these neural circuits are highly dynamic. The ability to change, or the plasticity, of the brain has long been demonstrated at the level of isolated single synapses under artificial conditions. Circuit organization and brain function has been extensively studied by correlating neuronal activity with information input. The primary visual cortex has become an important model brain region for the study of sensory processing, in large part due to the ease of manipulating visual stimuli. Much has been learned from studies of visual cortex focused on understanding the signal-processing of visual inputs within neural circuits. Many of these findings are generalizable to other sensory systems and other regions of cortex. However, few studies have directly demonstrated the orchestrated neural-circuit plasticity occurring during behavioral experience.
It is vital to measure the precise circuit connectivity and to quantitatively characterize experience-dependent circuit plasticity to understand the processes of learning and memory formation. Moreover, it is important to study how circuit connectivity and plasticity in neurological and psychiatric disease states deviates from that in healthy brains. By understanding the impact of disease on circuit plasticity, it may be possible to develop therapeutic interventions to alleviate significant neurological and psychiatric morbidity. In the case of neural trauma or ischemic injury, where neurons and their connections are lost, functional recovery relies on neural-circuit repair. Evaluating whether neurons are reconnected into the local circuitry to re-establish the lost connectivity is crucial for guiding therapeutic development.
There are several major technical hurdles for studies aiming to quantify circuit connectivity. First, the lack of high-specificity circuit stimulation methods and second, the low throughput of the gold-standard patch-clamp technique for measuring synaptic events have limited progress in this area. To address these problems, we first engineered the patch-clamp experimental system to automate the patching process, increasing the throughput and consistency of patch-clamp electrophysiology while retaining compatibility of the system for experiments in ex vivo brain slices. We also took advantage of optogenetics, the technology that enables control of neural activity with light through ectopic expression of genetically encoded photo-sensitive channels in targeted neuronal populations. Combining optogenetic stimulation of pre-synaptic axonal terminals and whole-cell patch-clamp recording of post-synaptic currents, we mapped the distribution and strength of synaptic connections from a specific group of neurons onto a single cell. With the improved patch-clamp efficiency using our automated system, we efficiently mapped a significant number of neurons in different experimental conditions/treatments. This approach yielded large datasets, with sufficient power to make meaningful comparisons between groups.
Using this method, we first studied visual experience-dependent circuit plasticity in the primary visual cortex. We measured the connectivity of local feedback and recurrent neural projections in a Fragile X syndrome mouse model and their healthy counterparts, with or without a specific visual experience. We found that repeated visual experience led to increased excitatory drive onto inhibitory interneurons and intrinsically bursting neurons in healthy animals. Potentiation at these synapses was absent or abnormal in Fragile X animals. Furthermore, recurrent excitatory input onto regular spiking neurons within the same layer remained stable in healthy animals but was depressed in Fragile X animals following repeated visual experience. These results support the hypothesis that visual experience leads to selective circuit plasticity which may underlie the mechanism of visual learning. This circuit plasticity process is impaired in a mouse model of Fragile X syndrome.
In a separate study, in collaboration with the laboratory of Dr. Gong Chen, we applied the circuit-mapping method to measure the effect of a novel brain-repair therapy on functional circuit recovery following ischemic injury, which locally kills neurons and creates a glial scar. By directly reprogramming astrocytes into neurons within the region of the glial scar, this gene-therapy technology aims to restore the local circuit and thereby dramatically improve behavioral function after devastating neurological injury. We found that direct reprogramming converted astrocytes into neurons, and importantly, we found that these newly reprogrammed neurons integrated appropriately into the local circuit. The reprogramming also improved connections between surviving endogenous neurons at the injury site toward normal healthy levels of connectivity. Connections formed onto the newly reprogrammed neurons spontaneously remodeled, the process of which resembled neural development. By directly demonstrating functional connectivity of newly reprogrammed neurons, our results suggest that this direct reprogramming gene-therapy technology holds significant promise for future clinical application to restore circuit connectivity and neurological function following brain injury.
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Vaňátko, Ondřej. "Identifikace změn membránových vlastností astrocytů u myšího modelu amyotrofické laterální sklerózy." Master's thesis, 2020. http://www.nusl.cz/ntk/nusl-435853.
Full textAbstract:
Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder of the central nervous system characterized by loss of motor neurons and voluntary muscle degeneration. Astrocytes play a major role in regulation of the disease onset and progression due to their intimate association with neurons. Regulation of ionic homeostasis is one of their key functions and its failure has been linked to several neurological diseases. The aim of this thesis was to explore differences in membrane properties of astrocytes in ALS. To fulfill this aim, a double transgenic mouse strain with ALS-like phenotype and a specific expression of enhanced green fluorescent protein in astrocytes was generated. To phenotype this strain, two sensorimotor tests, wire grid hang test and rotarod test, were conducted. Immunohistochemistry was used to characterize the strain on a cellular level and to explore changes of specific ion channels. Functional properties of astrocytes were explored using the patch clamp technique. The double transgenic strain has the characteristic ALS-like phenotype and is comparable to the original strain with differences in symptom onset and progression between models and sexes. On the cellular level, there are characteristic ALS features, specifically loss of motor neurons and astrogliosis....
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Lin, Kun-Han. "Functional properties and Ca2+-dependent feedback modulation of voltage-gated Ca2+ channels in glutamatergic nerve terminals of the mammalian auditory brainstem." Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-0006-AE4C-9.
Full text
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Wadel, Kristian. "The mechanism mediating fast neurotransmitter release at the calyx of Held synapse." Doctoral thesis, 2008. http://hdl.handle.net/11858/00-1735-0000-0006-B4F6-F.
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Caro, Lydia. "CONCEPTION ET CARACTERISATION DE BIOCAPTEURS BASES SUR L'ASSOCIATION DE RECEPTEURS ET CANAUX IONIQUES." Phd thesis, 2010. http://tel.archives-ouvertes.fr/tel-00544513.
Full textAbstract:
Les canaux sensibles à l'ATP (KATP) résultent de l'association unique d'une protéine ABC (le récepteur des sulfonylurées, SUR) et d'un canal potassique rectifiant entrant (Kir6.x). Suite à la liaison de divers effecteurs (nucléotides, molécules pharmacologiques), SUR module l'activité de Kir6.x. Dans l'organisme, les canaux KATP lient le niveau énergétique de la cellule au potentiel membranaire. De ce fait, leur implication dans diverses fonctions physiologiques telles que la sécrétion pancréatique d'insuline ou le contrôle du tonus vasculaire en fait des cibles thérapeutiques. Ainsi, dans le cadre d'une collaboration, nous avons testé des composés (dérivés benzothiazine) sur le canal KATP. Quatre d'entre eux se sont avérés être des ouvreurs des canaux KATP. Nous avons également exploré l'effet de deux insecticides, l'amitraz et le diflubenzuron sur le canal KATP. En outre, dans une optique de généralisation du concept d'Ion Channel-Coupled Receptor (ICCR) mis au point par l'équipe, nous avons élaboré des biocapteurs reposant sur l'assemblage de récepteurs couplés aux protéines G (GPCR) et Kir6.2. Cette association, inspirée par le canal KATP, est réalisée de telle manière que la fixation d'un ligand sur le GPCR entraîne la modulation de l'activité de Kir6.2. L'ingénierie moléculaire nous a permis de fusionner Kir6.2 aux récepteurs β2 adrénergique, dopaminergique D3, cannabinoïde 1 (CB1), des CC-chimiokines 2 et à l'opsine. La méthode de double-microélectrodes nous a permis d'identifier trois ICCR fonctionnels basés sur les récepteurs β2, D3 et CB1. Ces biocapteurs présentent des applications dans le cadre du criblage haut débit, la caractérisation fonctionnelle des GPCR ou la compréhension de la régulation de l'activité de Kir6.2.
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Barrantes-Freer, Alonso. "Functional properties of the plasma membrane of human glioma initiating cells." Doctoral thesis, 2012. http://hdl.handle.net/11858/00-1735-0000-000D-F0B9-D.
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