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Physiological and pathological potential of astroglial NMDA receptors
Džamba, Dávid ; Anděrová, Miroslava (advisor) ; Vyklický, Ladislav (referee) ; Paleček, Jiří (referee)
Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice during ageing as well as before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in cortical GFAP/EGFP- positive (GFAP/EGFP+ ) glial cells. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP+ glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP+ glial cells and that...
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Multiple regulatory roles of the transmembrane adaptor protein NTAL in gene transcription and mast cell physiology
Polakovičová, Iva ; Dráber, Petr (advisor) ; Vyklický, Ladislav (referee) ; Hašek, Jiří (referee)
(EN) This thesis focuses mainly on understanding of the regulatory roles of the transmembrane adaptor proteins, non-T cell activation linker (NTAL) and phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), in murine mast cell signaling. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) activation pathways in mast cells. Studies carried out on mast cells prepared from NTAL knock-out mice have indicated that NTAL is a negative regulator of FcεRI signaling, whereas experiments performed on human mast cells and rat basophilic leukemia cells with silenced NTAL expression have suggested its positive regulatory role. To thoroughly examine the involvement of NTAL in FcεRI-mediated signaling events in mouse mast cells and to determine whether different methodologies of NTAL ablation have different physiological consequences, we utilized a broad range of assays. Using bone marrow-derived mast cells (BMMCs) as a model, we obtained cells from NTAL wild type and knock-out cells and using lentiviral delivery approach we transduced part of the wild type cells, with vector bearing NTAL shRNA or empty vector to generate NTAL knock-down cells and control cells, respectively. Comparison of all four groups of generated cells in our assays...
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mmunohistochemical analysis of the inhibitory glycine and GABAB receptors in the MNTB
Trojanová, Johana ; Tureček, Rostislav (advisor) ; Druga, Rastislav (referee) ; Vyklický, Ladislav (referee)
Properties of glycine (GlyR) and GABAB (GABABR) receptors were studied in the adult rat medial nucleus of the trapezoid body (MNTB). MNTB belongs among brainstem auditory nuclei. Prevailing cell type in the MNTB is the principal cell (PC). Each PC receives two types of synaptic inputs. Excitatory input originating at contralateral anteroventral cochlear nucleus (AVCN), forms a giant glutamatergic nerve terminal, calyx of Held (CH). Inhibitory inputs are brought by glycinergic and GABAergic fibers of unclear origin. Synaptically released GABA and glycine modulate excitatory synaptic transmission in MNTB via receptors expressed by presynaptic and postsynaptic neurons. The goal of our work was to elucidate both the subcellular distribution and subunit composition of the receptors, thereby extend our knowledge of the function of the receptors in the MNTB. We used immunohistochemical staining methods. The receptors were labelled by specific antibodies localized in the nervous tissue by means of fluorescence or electron microscopy. The experimental work consisted of three phases. During the first phase we revealed that GlyRs in the MNTB form two distinct populations. Postsynaptic receptors form α1β heteromeric clusters on somatodendritic parts of PCs. These clusters colocalize with glycinergic endings...
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Neurogeneze a gliogeneze v dospělém mozku po ischemickém poškození
Honsa, Pavel ; Anděrová, Miroslava (advisor) ; Vyklický, Ladislav (referee) ; Mazurová, Yvona (referee)
Ischemic brain injury belongs to the most common cause of death in the developed countries. High complexity of this disorder significantly slows and limits the possible treatment. Currently, there is only one treatment available - the application of the thrombolytic, tissue plasminogen activator, while thousands of other drugs failed during clinical testing. Great expectations were seen in the stroke treatment employing neural stem cells obtained from several external sources; nevertheless, low survival rate, limited favorable outcome and enormous ethical issues complicate the application of such therapy. On the other hand, in the adult mammalian brain exist two endogenous processes - neurogenesis and gliogenesis. These processes need to be fully described and understood, in order to employ them as a source of new cells after injury. Therefore, this thesis focuses on the processes of adult neurogenesis and gliogenesis predominantly after ischemia. Adult neurogenesis and gliogenesis are processes, by which neurons or glial cells are generated from stem/progenitor cells. Both these processes are strongly influenced by brain injury; nevertheless, their contribution to regeneration after ischemia in the human brain is negligible. Here, we aimed to describe the role of polydendrocytes in the...
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Neuropharmacology of spatial navigation, cognitive coordination and flexibility tests in animal models
Prokopová, Iva ; Stuchlík, Aleš (advisor) ; Vyklický, Ladislav (referee) ; Blahoš, Jaroslav (referee)
Spatial navigation, cognitive coordination and behavioral flexibility belong amongst cognitive functions, which play a role in many neuropsychiatric disorders. Behavioral tasks have proved to be useful paradigms to test these functions in pharmacological or genetic animal models. First aim was to determine a potential interaction between β-adrenergic and α1-adrenergic or D2-dopaminergic systems. Spatial navigation and coordination were impaired in both studies during co-aplication of subthreshold doses of drugs. Used substances belong to group of widely prescribed drugs, thus our results could be implicated in clinical practice. Another study examined an acute effect of MK-801 (animal model of schizophrenia) on behavioral flexibility in Carousel maze and the Morris water maze (MWM). Carousel maze showed higher sensitivity with impairments from 0.08 mg.kg-1 compared to 0.10 mg.kg- 1 in MWM. The final experiment aimed at testing the effect of reduced expression of Nogo-A protein on spatial navigation and behavioral flexibility of rats. A battery of tests in the Carousel maze revealed impairment in cognitive functions, MWM showed unaffected working memory of rats. Our results support the hypothesis linking Nogo-A knock-down rats with neuropsychiatric symptoms and cognitive disorders. Key words:...
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Characterization of the effect of pregnanolone sulfate and its derivatives on NMDA receptors.
Švehla, Pavel ; Vyklický, Ladislav (advisor) ; Blahoš, Jaroslav (referee)
N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...
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Affecting the function of the central part of the auditory system of the adult rat by a brief noise exposure during development
Bartošová, Jolana ; Popelář, Jiří (advisor) ; Vyklický, Ladislav (referee) ; Maršálek, Petr (referee)
ln my the:!i" I aimed to dwal:teriu t.hc dumges in neuronil mvi'Y in tbe tenUlll nucleus of !hl' inferior coIlleulus (CIC) in ..:Iult fllI who ..ffertd fium a lack ohuditory stimulatiOll dl.lling their postnatal dcvdopment ""'..... by the Illmponry i~ase of auditory bearing thresholds after bOef noise e:o:poawe Oll postnItaJ day 14 (broad-bllrKl noise, US dB SPL., 11 min). AI the age of 3-6 IJlOI\tbs, lhe rtSpon5eII ofindividual neurons in tbc CIC 10 :taJnd wtre rcoordod in tb_ animall .nd eompared witb thoseoh~a\l;hcd eontrols. We foood lhal tbe neuronal response tbresholds were sinůlll" in tbe exposed and eontrol animals. However, tbe responJe propenies of high-frequtnCy neuronJ in the exposed rau had characteristic.s typical of the immature auditory system, Le. significantly widtr excitatory response II"talI (which indicatel impairod frcquoncy selectivity), a loogcr fil"Jl-spikc latcfJ(:y, a u.rrower dynamie range, lower maximum rcspon.<;c magniludes, a SUleper ,Iope nf tbc rare-inlensily flmClionllnd a lignifiClnLly lo"",r percelllage of monotonie neuron!. On the basis of thelle !inding" we investignled wllelher the reponed alterallon! nf neuronal relponlivenesl are llso Rccompanied by abnonnal behavioral responses 10 toUnd. The resoll howed a reduced strengtl1 nf the acousbc litanie reflex...
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Structure, function and pharmacology of NMDA receptors
Švehla, Pavel ; Vyklický, Ladislav (advisor) ; Bendová, Zdeňka (referee)
Glutamate is a major excitatory neurotransmitter between neurons in the central nervous system. The effect of glutamate is caused by the activation of distinct ionotropic glutamate channels: AMPA, kainate and NMDA receptors. NMDA receptors play a critical role in the synaptic plasticity and excitotoxicity. Despite the crucial role of these receptors in the right function of the brain, their overexcitation under pathological conditions may result in such neurological disorders as Alzheimers, Parkinsons and Huntingtons diseases. The aim of this work is to review available data concerning the molecular structure of NMDA subtype of ionotropic glutamate receptors, their pharmacology and therapeutic potential.
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Regulatory functions of the transmembrane adaptor protein NTAL in activation of mast cells
Tůmová, Magda ; Dráber, Petr (advisor) ; Vyklický, Ladislav (referee) ; Hašek, Jiří (referee)
Mast cells originate from hematopoietic pluripotent stem cells present in bone marrow. They do not circulate, but are spread throughout a body and reside in all vascularized tissues. Mast cells as a part of innate immune system are also able to influence adaptive immune system and are effectors in IgE-mediated allergic and inflammatory diseases. Mast cells possess high affinity receptor for IgE (FcεRI). Aggregation of the receptor triggers signaling cascades which lead to the release of preformed and de-novo synthesized effector molecules. Early phases of mast cell activation include tyrosine phosphorylation of numerous substrates and formation of signaling assemblies (also called signalosomes). Important structural components of the signalosomes are transmembrane adaptor proteins. One of them is the transmembrane adaptor protein NTAL (Non-T cell Activation Linker), which becomes rapidly tyrosine- phosphorylated upon FcεRI triggering and serves as a positive or a negative regulator of FcεRI signaling. To be able to study fine topography of NTAL and other plasma membrane components in mouse bone marrow-derived mast cells (BMMCs), we first developed a new method of plasma membrane sheets isolation from non-adherent cells. The method is based on adsorption of the cells to ultraclean glass cover-slips,...
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Functional role of purinergic P2X receptors in the supraoptic nuclei of the rat and structure-function relations of recombinant P2X receptors.
Vávra, Vojtěch ; Zemková, Hana (advisor) ; Novák, František (referee) ; Vyklický, Ladislav (referee)
Purinergic P2X receptors are non-selective cationic channels gated by extracellular ATP. Up to now, seven mammalian subunits, termed P2X1-X7, have been cloned and characterized. These receptors comprise a new membrane channel family with distinct structural and functional features. P2X receptors take part in a signalling network called "purinergic signalling" which is widely exploited in both somatic and neuronal tissues. In the central nervous system, they are highly expressed in the hypothalamus and hypophysis, where they participate in the regulation of homeostatic and reproductional functions. The main focus of my Thesis is on the expression and functional role of P2X receptors in supraoptic nuclei of the rat hypothalamus. These nuclei contain two populations of magnocellular neurons which produce either oxytocin or arginine-vasopressin. Delivery of the hormones into the systemic blood relies on the electrical activity of supraoptic neurons, which is in turn governed by the incomming synaptic inputs. It has been recently shown, that the process of hormone release from supraoptic neurons is regulated by extracellular ATP. However, purinergic signals that regulate hormone secretion are not well understood. The aim of my study was to identify subtypes of P2X receptors expressed in the supraoptic...
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