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GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUSStephens, Michelle Lee 01 January 2009 (has links)
A positive correlation exists between increasing age and the incidence of hippocampal-associated dysfunction and disease. Normal L-glutamate neurotransmission is absolutely critical for hippocampal function, while abnormal glutamate neurotransmission has been implicated in many neurodegenerative diseases. Previous studies, overwhelmingly utilizing ex vivo methods, have filled the literature with contradicting reports about hippocampal glutamate regulation during aging. For our studies, enzyme-based ceramic microelectrode arrays (MEA) were used for rapid (2 Hz) measurements of extracellular glutamate in the hippocampal trisynaptic pathway of young (3-6 months), late-middle aged (18 mo.) and aged (24 mo.) urethane-anesthetized Fischer 344 rats. Compared to young animals, glutamate terminals in cornu ammonis 3 (CA3) showed diminished potassium-evoked glutamate release in aged rats. In late-middle aged animals, terminals in the dentate gyrus (DG) showed increased evoked release compared to young rats. The aged DG demonstrated an increased glutamate clearance capacity, indicating a possible age-related compensation to deal with the increased glutamate release that occurred in late-middle age.
To investigate the impact of changes in glutamate regulation on the expression of a disease process, we modified the MEA technology to allow recordings in unanesthetized rats. These studies permitted us to measure glutamate regulation in the hippocampal formation without anesthetic effects, which showed a significant increase in basal glutamatergic tone during aging. Status epilepticus was induced by local application of 4-aminopyridine. Realtime glutamate measurements allowed us to capture never-before-seen spontaneous and highly rhythmic glutamate release events during status epilepticus. A significant correlation between pre-status tonic glutamate and the quantity of status epilepticus-associated convulsions and glutamate release events was determined. Taken together, this body of work identifies the DG and CA3 as the loci of age-associated glutamate dysregulation in the hippocampus, and establishes elevated levels of glutamate as a key factor controlling severity of status epilepticus in aged animals.
Based upon the potential ability to discriminate brain areas experiencing seizure (i.e. synchronized spontaneous glutamate release) from areas not, we have initiated the development of a MEA for human use during temporal lobe resection surgery. The final studies presented here document the development and testing of a human microelectrode array prototype in non-human primates.
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CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUSTalauliker, Pooja Mahendra 01 January 2010 (has links)
An overarching goal of the Gerhardt laboratory is the development of an implantable neural device that allows for long-term glutamate recordings in the hippocampus. Proper L-glutamate regulation is essential for hippocampal function, while glutamate dysregulation is implicated in many neurodegenerative diseases. Direct evidence for subregional glutamate regulation is lacking in previous in vivo studies because of limitations in the spatio-temporal resolution of conventional experimental techniques. We used novel enzyme-coated microelectrode arrays (MEAs) for rapid measurements (2Hz) of extracellular glutamate in urethane-anesthetized rats. Potassium-evoked glutamate release was highest in the cornu ammonis 1 (CA1) subregion and lowest in the cornu ammonis 3 (CA3). In the dentate gyrus (DG), evoked-glutamate release was diminished at a higher potassium concentration but demonstrated faster release kinetics. These studies are the first to show subregion specific regulation of glutamate release in the hippocampus.
To allow for in vivo glutamate measurements in awake rats, we have adapted our MEAs for chronic use. Resting glutamate measurements were obtained up to six days post-implantation but recordings were unreliable at later time points. To determine the cause(s) for recording failure, a detailed investigation of MEA surface characteristics was conducted. Scanning electron microscopy and atomic force microscopy showed that PT sites have unique surface chemistry, a microwell geometry and nanometer-sized features, all of which appear to be favorable for high sensitivity recordings. Accordingly, studies were initiated to improve enzyme coatings using a computer-controlled microprinting system (Microfab Technologies, Plano, TX). Preliminary testing showed that microprinting allowed greater control over the coating process and produced MEAs that met our performance criteria.
Our final studies investigated the effects of chronic MEA implantation. Immunohistochemical analysis showed that the MEA produced minimal damage in the hippocampus at all time points from 1 day to 6 months. Additionally, tissue attachment to the MEA surface was minimal. Taken together with previous electrophysiology data supporting that MEAs are functional up to six months, these studies established that our chronic MEAs technology is capable of maintaining a brain-device interface that is both functional and biocompatible for extended periods of time.
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Studies of emotionality in genetic mouse models of altered glutamate or 5-HT functionBarkus, Christopher January 2010 (has links)
No description available.
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Stříbrné amalgamové elektrody v elektroanalýze vybraných agrochemikálií / Silver Amalgam Electrodes in Electroanalysis of Selected AgrochemicalsDaňhel, Aleš January 2012 (has links)
4 ABSTRACT Development and testing of novel non-toxic electrode materials, detection arrangements and analytical methods applicable in determination of selected agrochemicals is the main aim of this Ph.D. Thesis. New working electrodes based on silver solid amalgam paste (AgSA-PE) with organic pasting liquid and other based on crystallic silver amalgam (CAgAE) were developed, their electrochemical behaviour investigated and further used in voltammetric determination of widespread and toxic environmental pollutant 4-nitrophenol (4-NP). This analyte could be determined by DPV at AgSA-PE with limit of detection (LD) 1×10í6 mol lí1 and using CAgAE with LD 4×10í7 mol lí1 , both in 0.2 mol lí1 acetate buffer pH 4.8. Attempts to decrease LDs by utilization of adsorptive stripping voltammetry were not successful in either case. Crystallic silver amalgam was also successfully used for construction of microcylindric flow-through cell and tested for amperometric determination of nitrophenol mixture in HPLC-ED system. Both novel electrodes were found to be suitable alternatives to toxic mercury electrodes and the CAgAE seems to be promising working electrode for flowing systems. Method for sample preparation and voltammetric determination of broad-spectrum herbicide Glyphosate in contaminated soil samples was also...
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Construction of an enzyme-free electrochemical sensor based on Ag-Fe2O3/POM/RGO novel nanocomposite for hydrogen peroxide detectionNqakala, Noniko Civilized January 2018 (has links)
>Magister Scientiae - MSc / The motivation to determine H2O2 lies in the fact that this chemical species plays a crucial role in diverse fields of practise such as cosmetic, food, diagnostic, pharmaceutical, clinical and environmental protection industries. Several methods such as chromatography, colorimetry, titrimetry and spectrophotometry have been developed for its detection. However, these methods are known to manifest underlying disadvantages such as high cost, time consuming, instability and complicated immobilization procedures. In this present study an enzyme-less electrochemical sensor based on Ag-Fe2O3/POM/RGO nanocomposite (POM stands for polyoxometalate and RGO stands for reduced graphene oxide) was successfully synthesised via a hydrothermal method and a photochemical reduction method for the detection of hydrogen peroxide (H2O2).
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Determinação biamperométrica de íons iodato e iodeto em água do mar, iodato em sal de cozinha e íons bromato em farinha / Biamperometry determination iodide and iodate ions in sea water, iodate in kitchen salt and bromate ions in flourDimas Augusto Morozin Zaia 28 February 1985 (has links)
Pelo uso da técnica biamperométrica modificada, pode-se determinar 35 ppb de íons iodato e 70 ppb de íons iodeto em água do mar; iodeto é determinado após sua oxidação a iodato com água de bromo no intervalo de pH entre 2,5 e 3,5 (o excesso de bromo é eliminado pelo aquecimento). Um estudo sistemático de pH foi efetuado para a elaboração do estudo de oxidação de íons iodeto a iodato com água de bromo, assim, como a oxidação de íons iodeto com água de bromo em várias temperaturas. Pode ser determinado 35 ppb de iodato em água do mar com %S de 3,9 e 70 ppb de íons iodeto em água do mar com %S de 5,8. O método proposto pode ser utililizado na determinação de íons iodato em sal de cozinha obtendo-se %S de 1,5. Por esta técnica também pode-se determinar íons bromato em farinha com melhores resultados em relação a volumetria. A análise de íons bromato, via biameprometria, pode ser efetuada por duas maneiras: diretamente na suspensão de farinha e por extração com Zn++/OH-, sendo que no primeiro caso pode-se determinar 15,2 ppm de íons bromato com %S de 7,00 e no segundo caso há necessidade de se utilizar um fator de correção, podendo-se chegar a concentrações de ppb. / Using the modified biamperometria technique, one can determine 35 ppb IO3- and 70 ppb I- is determined after its oxidation to IO3- with bromine in a range of pH between 2,5 - 3,5 (the excess of bromine is eliminated at 50º C). A sistematic study of pH for the oxidation of I- to IO3- was done as well for the oxidation of I- with bromine in a range of temperature. 35 ppb IO3- in sea water can be determined with accuracy of 3,9%; 70 ppb I- in sea water can be determined with accuracy of 5,8%. This method can be used for the determination of IO3- in kitchen salt in with accuracy of 1,5%. BrO3- in flours can be determined by the some technique with better results in comparison with the volumetric classical methods. The analysis of BrO3- in flours by the modified biamperometry can be made through two ways: directly in the suspension of the flours in water and by extraction with Zn++/OH-. By the first one can determine 15,2 ppm BrO3- with 7,00% of accuracy; by the second way one need to use a factor for correction, for determination of ppb level.
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Determinação biamperométrica de íons iodato e iodeto em água do mar, iodato em sal de cozinha e íons bromato em farinha / Biamperometry determination iodide and iodate ions in sea water, iodate in kitchen salt and bromate ions in flourZaia, Dimas Augusto Morozin 28 February 1985 (has links)
Pelo uso da técnica biamperométrica modificada, pode-se determinar 35 ppb de íons iodato e 70 ppb de íons iodeto em água do mar; iodeto é determinado após sua oxidação a iodato com água de bromo no intervalo de pH entre 2,5 e 3,5 (o excesso de bromo é eliminado pelo aquecimento). Um estudo sistemático de pH foi efetuado para a elaboração do estudo de oxidação de íons iodeto a iodato com água de bromo, assim, como a oxidação de íons iodeto com água de bromo em várias temperaturas. Pode ser determinado 35 ppb de iodato em água do mar com %S de 3,9 e 70 ppb de íons iodeto em água do mar com %S de 5,8. O método proposto pode ser utililizado na determinação de íons iodato em sal de cozinha obtendo-se %S de 1,5. Por esta técnica também pode-se determinar íons bromato em farinha com melhores resultados em relação a volumetria. A análise de íons bromato, via biameprometria, pode ser efetuada por duas maneiras: diretamente na suspensão de farinha e por extração com Zn++/OH-, sendo que no primeiro caso pode-se determinar 15,2 ppm de íons bromato com %S de 7,00 e no segundo caso há necessidade de se utilizar um fator de correção, podendo-se chegar a concentrações de ppb. / Using the modified biamperometria technique, one can determine 35 ppb IO3- and 70 ppb I- is determined after its oxidation to IO3- with bromine in a range of pH between 2,5 - 3,5 (the excess of bromine is eliminated at 50º C). A sistematic study of pH for the oxidation of I- to IO3- was done as well for the oxidation of I- with bromine in a range of temperature. 35 ppb IO3- in sea water can be determined with accuracy of 3,9%; 70 ppb I- in sea water can be determined with accuracy of 5,8%. This method can be used for the determination of IO3- in kitchen salt in with accuracy of 1,5%. BrO3- in flours can be determined by the some technique with better results in comparison with the volumetric classical methods. The analysis of BrO3- in flours by the modified biamperometry can be made through two ways: directly in the suspension of the flours in water and by extraction with Zn++/OH-. By the first one can determine 15,2 ppm BrO3- with 7,00% of accuracy; by the second way one need to use a factor for correction, for determination of ppb level.
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More transparency in bioanalysis of exocytosis : application of fluorescent false neurotransmitters in coupling methodology of electrochemistry with fluorescence microscopy at ITO microelectrodes / Bioanalyse microélectrochimique de l'exocytose vésiculaire : utilisation de faux neurotransmetteurs fluorescents dans la méthodologie de couplage de l'électrochimie avec la microscopie de fluorescence sur microélectrodes d'ITOLiu, Xiaoqing 26 September 2016 (has links)
L’exocytose vésiculaire est une voie physiologique majeure de la communication intercellulaire. Dans ce contexte, le TIRFM (microscopie de fluorescence par réflexion totale interne) et l’ampérométrie sont aujourd'hui les deux méthodes analytiques les plus fréquemment utilisées dans l’étude de l’exocytose. En raison de la complémentarité de ces deux techniques d’analyse pour le suivi de la sécrétion exocytotique, leur combinaison pour suivre la sécrétion exocytotique a d'abord été réalisée par notre groupe en 2011. Ce couplage a permis un enregistrement simultané des signaux fluorescents et ampérométriques avec une bonne résolution spatiale et temporelle. L'inconvénient majeur de ce travail reste le chargement indépendant des sondes optique et électrochimique dans les vésicules de sécrétion, ce qui entraîne la détection d’évènements « orphelins » ampérométriques ou optiques ainsi que la faible efficacité de détection des évènements couplés. Par conséquent, dans cette thèse, nous avons tenté de mettre à profit une sonde unique à la fois fluorescente et électroactive pour suivre l’exocytose par la méthodologie couplée TIRFM/ampérométrie. Ainsi, un analogue de neurotransmetteurs monoamine primaire, la 4-(2-amino-éthyl)-6-chloro-7-hydroxy-2H-1-benzopyran-2-one (nommé 1 dans ce travail), a été synthétisé.1 présente une fluorescence forte, stable et pH-dépendante. Lorsque cette entité est excitée à 405 nm, son intensité de fluorescence est presque doublée de pH 5 (valeur intra-vésiculaire) à 7 (valeur milieu extracellulaire). De plus, des études en voltammétrie ont pu mettre en évidence que 1 est oxydable sur électrode de carbone vitreux, microélectrode à fibre de carbone et ITO (oxyde d’indium dopé à l’étain), montrant ainsi une bonne électroactivité. La pénétration cellulaire dans les vésicules de cellules BON N13 a également été démontrée, prouvant la spécificité de l’interaction entre 1 et ces vésicules équipées d’un transporteur de monoamines primaires (VMAT). L’utilisation de 1 comme sonde unique optique et électrochimique pour le suivi de l'exocytose a ensuite été validée séparément dans des cellules BON N13 par TIRFM et ampérométrie. L’enregistrement simultané par fluorescence et électrochimie en utilisant 1 comme sonde double a ensuite été réalisé dans un microdispositif constitué d’électrodes ITO conductrice et transparente. Nos résultats basés sur la sonde unique 1 montrent qu’elle semble plus adaptée que toutes les stratégies antérieures impliquant deux sondes indépendantes. Les résolutions spatiale et temporelle de cette méthode combinée ont permis d'analyser des sécrétions d’exocytose de cellules marquées par 1. Une analyse ultérieure de ces signaux couplés optique et électrochimique sera à même d’étudier la corrélation entre le comportement du pore de fusion (dynamique d'ouverture/de fermeture, stabilité..) détecté par ampérométrie et le mouvement d'une vésicule en trois dimensions (ancrage, amarrage, fusion puis retrait dans le cytoplasme) détecté par TIRFM. / Vesicular exocytosis is a ubiquitous way for intercellular communications. TIRFM (total internal reflection fluorescence microscopy) and amperometry are nowadays the two most frequently used analytical methods with complementary features for its investigation. The combination of these two analytical techniques to track exocytotic secretions was firstly achieved by our group in 2011 and this new technique was demonstrated to show both high temporal and spatial resolutions by simultaneously recording the fluorescent and amperometric signals. The major disadvantage of this former work was the independent loading of optical and electrochemical probes to the secretory vesicles, which resulted in 'sightless' amperometric or optical signals as well as low coupling efficiency. Therefore, in this thesis, we attempted to develop a unique probe with dual fluorescent/electrochemical characteristics to track exocytotic process by TIRFM/amperometry coupling technique. This is why an analog of biogenic monoamine neurotransmitters, 4-(2-aminoethyl)-6-chloro-7-hydroxy-2H-1-benzopyran-2-one hydrochloride (named as 1 in this work) was synthesized. 1 exhibited bright, stable, pH-dependent fluorescence. When excited at 405 nm, its fluorescence intensity was almost doubled with the increase of pH values from 5 (similar to that in the vesicular lumen) to 7 (similar to the extracellular medium). Furthermore, in voltammetry, 1 was demonstrated to be easily electrooxidized on GCE (glassy carbon electrode), CFE (carbon fiber electrode) and ITO (indium tin oxide) electrodes surfaces, showing good electroactivity. 1 was also shown to penetrate easily into the vesicles of BON N13 cells within 1 hour incubation, testifying its specific affinity with these VMAT-equipped (vesicular monoamine transporter) vesicles. The applications of 1 as optical and electrochemical probes for exocytosis monitoring were then separately validated in BON N13 cells by TIRFM and amperometry measurements, respectively. Simultaneous recording of fluorescent and amperometric information by using 1 dual probes loaded cells was subsequently acquired in a microfabricated device constituted by conductive and transparent ITO electrodes. Our results based on the unique probe 1 for electrochemical and fluorescent detection of exocytotic release seemed more adapted than all the previous works involving independent probes. The high spatial and temporal resolutions of this combined method also allowed analyzing consecutive exocytotic secretions as well as overlapped events in 1-stained cells. Further analysis of these two signals with complementary information will shed more light on the correlation of the fusion pore behavior (opening/closure dynamics, stability…) measured by amperometry and the motion of a secretory vesicle in three dimensions (tethering, docking, fusion and retrieval) detected by TIRFM.
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Forebrain Acetylcholine in Action: Dynamic Activities and Modulation on Target AreasZhang, Hao January 2009 (has links)
<p>Forebrain cholinergic projection systems innervate the entire cortex and hippocampus. These cholinergic systems are involved in a wide range of cognitive and behavioral functions, including learning and memory, attention, and sleep-waking modulation. However, the <italic>in vivo</italic> physiological mechanisms of cholinergic functions, particularly their fast dynamics and the consequent modulation on the hippocampus and cortex, are not well understood. In this dissertation, I investigated these issues using a number of convergent approaches.</p><p> First, to study fast acetylcholine (ACh) dynamics and its interaction with field potential theta oscillations, I developed a novel technique to acquire second-by-second electrophysiological and neurochemical information simultaneously with amperometry. Using this technique on anesthetized rats, I discovered for the first time the tight <italic>in vivo</italic> coupling between phasic ACh release and theta oscillations on fine spatiotemporal scales. In addition, with electrophysiological recording, putative cholinergic neurons in medial setpal area (MS) were found with firing rate dynamics matching the phasic ACh release. </p><p> Second, to further elucidate the dynamic activities and physiological functions of cholinergic neurons, putative cholinergic MS neurons were identified in behaving rats. These neurons had much higher firing rates during rapid-eye-movement (REM) sleep, and brief responses to auditory stimuli. Interestingly, their firing promoted theta/gamma oscillations, or small-amplitude irregular activities (SIA) in a state-dependent manner. These results suggest that putative MS cholinergic neurons may be a generalized hippocampal activation/arousal network. </p><p> Third, I investigated the hypothesis that ACh enhances cortical and hippocampal immediate-early gene (IEG) expression induced by novel sensory experience. Cholinergic transmission was manipulated with pharmacology or lesion. The resultant cholinergic impairment suppressed the induction of <italic>arc</italic>, a representative IEG, suggesting that ACh promotes IEG induction. </p><p> In conclusion, my results have revealed that the firing of putative cholinergic neurons promotes hippocampal activation, and the consequent phasic ACh release is tightly coupled to theta oscillations. These fast cholinergic activities may provide exceptional opportunities to dynamically modulate neural activity and plasticity on much finer temporal scales than traditionally assumed. By the subsequent promotion of IEG induction, ACh may further substantiate its function in neural plasticity and memory consolidation.</p> / Dissertation
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Development of original strategies for the electrochemical detection of cell-penetrating peptides and for the electrochemical bleaching of fluorescent probes : an entry to the monitoring of translocation in phospholipid membranes / Développement de stratégies originales pour la détection électrochimique de peptides pénétrants et le blanchiment électrochimique de sondes fluorescentes : une contribution à l'étude de la translocation dans les membranes phospholipidiquesPerez Jimenez, Ana Isabel 19 September 2016 (has links)
Ce travail de thèse s’intéresse à l’introduction de méthodologies électrochimiques dans la problématique de la caractérisation du transport de peptides pénétrants (CPPs) à travers des membranes phospholipidiques. Malgré leur charge électrique globalement positive, ces peptides sont en effet capables de traverser les bicouches lipidiques de cellules réelles ou artificielles (liposomes) et il n’existe pas à ce jour de mécanisme d’internalisation universellement admis. Dans ce contexte, nous avons dans un premier temps développé des méthodologies visant à détecter des peptides pénétrants marqués par des sondes rédox en optimisant les conditions de volume et de confinement à l’électrode. Parallèlement, nous avons tenté d’observer le passage transmembranaire de ces peptides en utilisant un dispositif dérivé du patch-clamp, dans lequel un morceau (patch)de membrane lipidique est excisé d’une vésicule géante et le suivi du passage assuré par une détection ampérométrique sur ultramicro-électrode à proximité de la membrane. La sensibilité de la technique ampérométrique et le flux de CPP s’avérant faibles, nous nous sommes tournés vers une stratégie associant fluorescence (pour la sensibilité) et commande électrochimique (pour l’extinction de la fluorescence). Dans la mesure où les phospholipides constituent la barrière dynamique à travers laquelle doivent passer les CPPs, nous avons d’abord étudié l’extinction électrochimique de phospholipides marqués par une sonde à la fois rédox et fluorescente, le NBD. Observée sur des vésicules géantes au microscope confocale, la réduction électrochimique a permis l’extinction sélective des phospholipides situés sur le feuillet externe de la vésicule, un résultat que ne permettent ni la réduction par des agents chimiques (dithionite), ni les techniques de « photobleaching ». Cette propriété a été confirmée cette fois-ci pour des CPPs marqués par le NBD qui ont été mis à incuber en présence de vésicules géantes et pour lesquels un essai préliminaire semble confirmer une extinction de fluorescence essentiellement pour les peptides associés au feuillet externe de la vésicule. / This PhD work was aimed at introducing electrochemical strategies in the general topic devoted to the characterization of the passage of cell penetrating peptides (CPPs) across phospholipidic membranes. Although positively charged, CPPs are prone to cross lipidic bilayers of real and artificial cells (liposomes) and there is no commonly admitted internalization mechanism so far. Therefore, we first developed electrochemical setups aimed at improving the amperometric detection of redox-taggedCPPs, through optimization of volume and confinement. Additionally, we have made attempts to use patch-clamp inspired setups to monitor the passage of CPPs across a membrane patched from a giant vesicle using ultramicro-electrodes in the close vicinity of the patched membrane. Since the amperometric technique displayed poor sensibility and the flux of CPP was too narrow, we changed our strategy for a methodology coupling fluorescence (for the sensitivity) and an electrochemical command (to achieve fluorescence extinction). Considering that phospholipids are forefront actors of CPP internalization, we first focused on the electrochemical quenching of phospholipids tagged with a probe displaying both redox and fluorescent properties (NBD). Observed on giant unilamellar vesicles (GUVs) with confocal microscopy, the electrochemical reduction of the NBD probe led to the selective extinction of the phospholipids located on the outer leaflet of the vesicle, a selectivity which is not observed using chemical quenchers such as dithionite or photobleaching methods. That property was extended to NBD-tagged CPPs, previously incubated with unlabeled Guvs and that preliminary experiment confirmed that the electrochemical extinction mostly concerned peptides associated to the outer leaflet of the liposome.
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