In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.

This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.

Astrocytes need to preserve constant volume in the face of osmolarity perturbations to function properly. To regain their original volume after hyposmotically induced swelling, they extrude intracellular electrolytes and organic osmolytes, such as inorganic ions, excitative amino acids or polyols, accompanied by osmotically driven water. This process is termed regulatory volume decrease and is ensured by various ion channels and transporters. Recently, much attention has been focused on the ubiquitous volume-regulated anion channels activated by cell swelling. VRACs are moderately outwardly rectifying with intermediary conductance, permeable to inorganic anions and organic osmolytes and sensitive to broad-spectrum anion channels blockers. Using patch-clamp technique we aimed to characterize VRACs in cultured cortical astrocytes isolated from neonatal Wistar rats and to elucidate the effect of intracellular Na+ on VRAC activity. In addition, we also intended to characterize these channels in situ in brain slices of 10 - 12 days old rats, focusing mainly on hippocampal astrocytes. To induce astrocytic swelling, we exposed astrocytes to hypotonic solution (250 mOsm). In agreement with previous findings, we showed that cultured cortical astrocytes activate VRAC currents upon exposure to hypotonic stress, which...

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....

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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The mammalian adrenal gland (or suprarenal gland) is a small organ located on the superior aspect of the kidney. The central region of the gland, the medulla, consists of chromaffin cells, which release catecholamines into the blood during periods of stress. This is best known as the 'fight or flight' response and is regulated, in the adult animal, by neuronal signals from the cholinergic sympathetic fibres of the splanchnic nerve. Interestingly, in some mammals, such as rat and human, sympathetic innervation is immature at birth, yet the chromaffin cells can still secrete catecholamines in response to physiological stessors, e.g. hypoxia. Increased plasma catecholamines is thought to provide a vital protective role for the neonatal animal during, and following birth. This is mediated in part by promoting lung fluid absorption, surfactant secretion, heart rate stabilization, and brown fat mobilization. The observation that, in the neonate, catecholamines are secreted in the absence of functional sympathetic innervation suggests that the chromaffin cells possess other mechanisms for directly 'sensing' a fall in blood O2 tension (hypoxia). The primary goal of this thesis was to uncover the mechanisms of oxygen-sensing in developing chromaffin cells from the rat and mouse, using primary short-term cell cultures of chromaffin cells. The experimental approaches relied on patch clamp techniques to record ionic currents and membrane potential, carbon fibre electrochemistry to record catecholamine secretion from cell clusters, and fluorescent indicators to measure reactive oxygen species generation. Hypoxic chemosensitivity was found in embryonic and neonatal, but not juvenile chromaffin cells from both the rat and mouse. Exposure to hypoxia or anoxia caused a reversible suppression of whole-cell current, which was comprised of the differential modulation of three K+ currents: (1) suppression of a large-conductance Ca2+-dependent K+ current; (2) suppression of a delayed rectifier K+ current; and (3) activation of an ATP-sensitive K+ current. Hypoxia also induced membrane depolarization that was not initiated by any of these three voltage-dependent K+ currents. Additionally, hypoxia broadened action potentials in chromaffin cells that showed spontaneous activity, and this was mediated by a prolongation of the time course of membrane repolarization. All of these factors likely contribute to catecholamine secretion by enhancing the influx of Ca2+ through depolarization-activated L-type Ca2+ channels. Two sets of experiments were designed to identify the oxygen sensor in neonatal chromaffin cells. First, cells from transgenic mice, deficient in the gp91^phox component of the putative O2-sensor protein, NADPH oxidase, responded to hypoxia in the same way as wild type cell, indicating that NADPH oxidase is not primarily responsible for oxygen sensitivity in these cells. Second, inhibitors of the proximal electron transport chain (e.g. rotenone and antimycin A) mimicked and attenuated the hypoxic response, while inhibitors of the distal electron transport chain (cyanide) and uncouplers of oxidative phosphorylation (2,4-dinitrophenol) had no effect. Furthermore, reactive oxygen species production, primarily H2O2, decreased during exposure to hypoxia or inhibitors of the proximal electron transport chain, revealing a potential mitochondrial mechanism for 'sensing' of the hypoxic stimulus. Reduced oxygen availability to the electron transport chain is proposed to cause a fall in cellular reactive oxygen species (ROS), principally H2O2. This fall in ROS signals closure of Ca2+-dependent and Ca2+-independent K+ channels, which causes broadening action potentials and increases Ca2+ influx. The latter is further enhanced by the hypoxia-induced membrane depolarization, which in turn increases the probability of cell firing. The rise in intracellular Ca2+ then acts as the signal for catecholamine release from the chromaffin cells. / Thesis / Doctor of Philosophy (PhD)

La prolongation de l'intervalle QT par les médicaments est un des effets indésirables les plus redoutés par l'industrie pharmaceutique. Ce retard de repolarisation ventriculaire cardiaque peut être précurseur d'arythmies ventriculaires potentiellement mortelles, particulièrement les torsades de pointes (TdP). Plusieurs médicaments de diverses classes pharmacologiques ont été impliqués dans le déclenchement d'arythmies ventriculaires malignes. Afin d'éviter ces effets indésirables des médicaments, il est essentiel de bien comprendre les mécanismes électrophysiologiques et pharmacologiques en cause. Les effets de la palipéridone, de la galantamine, de la tizanidine, de la rosuvastatine et du bupropion sur les courants potassiques rectifiants retardés (IKT et IKS) ainsi que sur la repolarisation ventriculaire cardiaque, ont été évalués à l'aide de trois modèles expérimentaux; la technique de patch-clamp en configuration cellule entière, la rétroperfusion de Langendorff et la télémétrie cardiaque sans fil. De plus, les effets du bupropion sur le courant sodique ont aussi été évalués à l'aide de la technique de patch-clamp en configuration cellule entière puis les effets sur les jonctions communicantes de cette même molécule ont été caractérisés par la technique de redistribution de fluorescence après photoblanchiment (FRAP). Cette étude démontre que l'inhibition du courant potassique rectifiant retardé par la palipéridone, la galantamine, la tizanidine et la rosuvastatine contribue à la prolongation de la repolarisation cardiaque. Elle met aussi en évidence que le bupropion retarde la conduction de l'impulsion d'une cellule cardiaque à l'autre en réduisant le couplage intercellulaire.

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