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Identification of changes in membrane properties of astrocytes in a mouse model of amyotrophic lateral sclerosis
Vaňátko, Ondřej ; Turečková, Jana (advisor) ; Vlachová, Viktorie (referee)
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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The interaction of kainate subtypes of glutamate receptors with steroid compounds.
Fraňková, Denisa ; Krůšek, Jan (advisor) ; Adámek, Pavel (referee)
Kainate receptors belong to the family of glutamate receptors, which include NMDA, AMPA and δ receptors. Glutamate receptors are widely found in the brain and therefore they are very dynamically investigated, especially from view of pharmacology, because there is great potential for finding new and more specific modulators which could be used in the treatment of neurodegenerative diseases. The aim of this work was to extend the knowledge about the influence of neurosteroids on homomeric kainate receptors (GluK1, GluK2, GluK3) in which is the study of modulation by neurosteroids still at the beginning. We have investigated interactions of homomeric kainate receptors with selected neurosteroids (pregnenolone sulfate, pregnanolone sulfate, dehydroepiandrosterone, dehydroepiandrosterone sulfate) by using patch clamp method in the configuration of whole-cell recording and also by using microfluorometry. We have found out that the biggest modulating effect on homomeric kainate receptors is caused by pregnenolone sulfate, which inhibits glutamate responses of these receptors. Keywords kainate receptor, glutamate, neurosteroids, steroids, patch-clamp technique
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Study of neurosteroid effect on the NMDA subtype of glutamate receptor.
Krausová, Barbora ; Vyklický, Ladislav (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are glutamatergic ionotropic receptors involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They are heteromeric complexes of GluN1 combined with GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Many allosteric modulators can influence the activity of these receptors including neurosteroids. Pregnanolone sulfáte (3α5βS) is an endogenous neurosteroid that inhibits NMDA receptors in a use-dependent manner and has neuroprotective effect. Binding site for 3α5βS on the NMDA receptor molecule is still not indentified. The aim of my work was to contribute to the identification of the biding site by kinetic analysis of rate of response return from 3α5βS inhibition. Using the point mutation we also attempted to identify the amino acids residues that could be involved in the neurosteroid binding. In order to study the effect of 3α5βS on NMDA receptors the electropfysiological recordings on human embryonic kidney 293T cells expressing recombinant GluN1/GluN2B receptors was performed. We confirm that the effect of 3α5βS on GluN1/GluN2B receptors is voltage-independent. The results of my work indicate that steroids can reach the binding site on the NMDA receptors through the membrane rather than directly from the aqueous...
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Volume-regulated anion channels in astrocytes- in vitro and in situ analysis
Harantová, Lenka ; Anděrová, Miroslava (advisor) ; Vargová, Lýdia (referee)
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...
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Preparation of neuroactive steroids for study of NMDA receptors
Vidrna, Lukáš ; Hodek, Petr (advisor) ; Pouzar, Vladimir (referee)
Neurosteroids are an important group of substances that affect communication between neurons. They act as allosteric modulators of membrane receptors for neurotransmitters. One of the most important systems influenced by neurosteroids are NMDA receptors; however, a binding site(s) for their inhibition by steroids have not been found yet. This work is focused on the synthesis of fluorescently labeled photoaffinity probe, which may help explain the structure and location of binding site(s) and simplify the development of new neuroprotectives. A structural analogue of the endogenous neurosteroid, (20S)-20-Azido-5β-pregnan- 3α-yl N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-L-glutamyl 1-ester (8), was prepared. The structure of compound 8 includes photolabile azido group, as well as covalently bounded fluorescent NBD group. In addition, a photoaffinity probe with a modified steroid skeleton - pyridinium 17aα-azido-17α-methyl-17a-homo-5β-androstan-3α-yl 3-sulfate (29) - was synthesized. The ability of compound 8 and 29 to inhibit activated NMDA receptor has been verified for recombinant NR1-1a/NR2B receptors expressed in HEK293 cells using a patch-clamp technique. Additionally, the IC50 values of compounds 8 and 29 have been calculated. (In Czech) Key words: neuroactive steroid, NMDA receptor, photoffinity...
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