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The study of functional and pharmacological properties of glutamate receptors
Kysilov, Bohdan ; Vyklický, Ladislav (advisor) ; Valeš, Karel (referee) ; Novotný, Jiří (referee)
N-methyl-D-Aspartate receptors (NMDAR) are ionotropic glutamate receptors that are involved in the regulation of nearly every process in the brain. Therefore, even a subtle disturbance in NMDAR function may result in severe pathological consequences. Loss-of- function mutations in the NMDAR-encoding genes have been implicated in numerous neuropsychiatric disorders, including intellectual disability, developmental delay, schizophrenia, autism spectrum disorders, epilepsy, and movement disorders. Insufficient NMDAR function can be rectified by positive allosteric modulators, including neurosteroids; however, the mechanism underlying the potentiating effect of steroids is not well understood. By employing patch-clamp electrophysiology we assessed the effect of newly synthesized neurosteroid-like pregnane analogues on recombinant GluN1/GluN2B receptors. We demonstrated that compounds with short C3 residues, such as pregnanolone acetate (PA- Ace) and pregnanolone carboxylate (PA-Car), are negative modulators of NMDAR, whereas compounds with longer C3 residues, such as pregnanolone butyrate (PA-But) and epipregnanolone butyrate (EPA-But), are positive modulators of NMDARs. Furthermore, we revealed that EPA-But has a disuse-dependent positive allosteric effect, being similar in that regard to endogenous...
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Mouse models for Angelman syndrome: generation and characterization
Syding, Linn Amanda ; Sedláček, Radislav (advisor) ; Vyklický, Ladislav (referee) ; Valeš, Karel (referee)
Angelman syndrome (AS) is a neurodevelopmental disease found in 1 to 10,000 to 40,000 births, exhibiting an equal gender ratio. Key characteristics of the disease include an ataxic gait with tremor, severe mental retardation, profound speech impairment and seizures. Behavioral deficits such as increased anxiety and autism spectrum disorder features is found in affected individuals as well. The disease stems from the imprinted region 15q11.2-13q where genes are either maternally or paternally expressed as a result of parent-of-origin specific expression of the alleles. There are four main genetic etiologies causing AS namely, i) a large deletion ranging from 4-6 Mb on the maternally inherited allele including imprinted and bi-allelically expressed genes, ii) maternal deletion of the Ubitiquin ligase E3 (UBE3A) gene, iii) paternal uniparental disomy and iv) imprinting defect leading to inappropriate methylation of the locus. So far, there is no cure for AS rather the symptoms are ameliorated using a multidisciplinary approach. The goal of the doctoral study was to further decipher the role of Ube3a and Gabra5 using two mouse models to gain more knowledge about the involvement of these two genes for future therapeutic interventions in for Angelman syndrome. One model generated was a full gene deletion...
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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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Neurosteroid modulation of ligand-gated ion-channel activity
Krausová, Barbora ; Vyklický, Ladislav (advisor) ; Moravec, Jan (referee)
The term neurosteroids refers to steroids that are synthetized in the nervous tissue from cholesterol or steroidal precursors from peripheral sources. These compounds affect the neuronal excitability by modulating the function of some ligand-gated ion channels. NMDA (N methyl D aspartate) receptors are glutamate gated ion channels involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. GABAA ( aminobutyric acid type A) receptors mediate most of the inhibitory synaptic transmission in the mammalian brain and are targeted by many clinically important drugs. Function of NMDA and GABAA receptors can by affected by neurosteroids, both positively and negatively. The aim of this work is to summarize the current knowledge about the neurosteroid effects on the function of GABAA a NMDA receptors and suggest the physiological role and the potential therapeutic use of the neurosteroids as a regulator of some functions of the central nervous system.
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Function of the GABA and glycine receptors in the mammalian MNTB
Hrušková, Bohdana ; Tureček, Rostislav (advisor) ; Vyklický, Ladislav (referee) ; Horák, Martin (referee)
GABA and glycine are major inhibitory neurotransmitters in the central nervous system. They act on ionotropic and metabotropic receptors that form oligomeric complexes in plasma membrane of neuronal cells. Pharmacological properties, subcellular distribution and function of GABA and glycine receptors depend on their subunit composition. The thesis was aimed to find subunit composition and physiological role of ionotropic glycine and metabotropic GABAB receptors in neurons of medial nucleus of trapezoid body, MNTB. The nucleus resides in the mammalian auditory brainstem and it is characterized by well defined excitatory and inhibitory inputs. Excitatory fibers form giant glutamatergic nerve terminals, calyces of Held, whereas inhibitory fibers form small GABA/glycinergic boutons. Both types of afferents innervate somatodenritic parts of MNTB principal neurons. The nucleus thus represents a suitable model for studying molecular and cellular mechanisms of interactions between excitation and inhibition. Our experiments were performed using electrophysiology and immunohistochemistry methods. Patch clamp technique was used to record membrane currents and voltages from neurons in live MNTB slices isolated from rats or mice. Spontaneous and sound-evoked activity of murine MNTB neurons was recorded by in...
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Věkové a pohlavní rozdíly v GABAergní transmisi v substantia nigra pars reticulata u krys.
Chudomel, Ondřej ; Bojar, Martin (advisor) ; Vyklický, Ladislav (referee) ; Mareš, Pavel (referee)
Many experimental as well as clinical studies have shown that subcortical neuronal circuitries including the anterior part of the substantia nigra pars reticulata (SNR) are closely involved in the control of seizures propagation and termination. In vivo studies in rats demonstrated that inhibition of the GABAergic SNRA electrical output increases the seizure threshold in the flurothyl model of generalized clonic seizures. The anticonvulsant properties of the SNRA are largely age- and sex-depended. In the current experiments were used 3 age groups of Sprague Dawley rats (PN5-9, PN11-16 and PN25-32 days, PN = postnatal) to study GABAergic inhibition. Two types of GABAergic inhibition were studied: a) a synaptic inhibition, which is generated by α1 and α3-containing GABAARs and b) a tonic inhibition mediated by extrasynaptic δ subunit-containing GABAARs. Immunohistochemistry showed that the α1 subunit expression was generally more abundant in adult rats and females while the α3 subunit dominated in the early development and gradually decreased by the age of PN30. The more α1 subunit was expressed the faster were the kinetics, higher the mean amplitudes and frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs). The α subunit subtype underlies sensitivity to zolpidem, which preferentially...
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Membrane properties of neural stem/progenitor cells during in vitro differentiation and after transplantation into the rat brain
Prajerová, Iva ; Chvátal, Alexandr (advisor) ; Vyklický, Ladislav (referee) ; Mazurová, Yvona (referee)
AOSTRACT The aim oflhis .....orl:. was to elucidate the dilTercntiation mechanisms of neural stcmlpmgenitor cells in vitro and their potentiallO survive and ditfcn:ntiate in \'il'o, al\er trarlSplnntatioo into thc injured mt conex. IImnunohistochcmistI)' "'lIS used for cell idcntiticatioos. and lhe p'0pCl ties ofK' aod Na' voltagc-gated ion ehnnnels were studiC<! using the pat,h-(:Iamp teclmique, We Ilave demonstrnted lhal inunortalised green lluort:SCCnll)r()lein (GFP)INE-4C neural stem cells derivtXI fmm tbc neurocpithelium of p5l'<lctieicm mouse cmbryos iII cmbryonic day (E)9 are ablc to dilferentiate i"to !leUrQns in \1"0, After tmnsplantalioo illto the site of a photlxhcmical lC'iion of aduh rol5, GFP!NE4C cdls slIn'ive and give Tise to neurons. astrocytt5 and oHgodmdrocytcs. Prima!)' embryonic ncura.l Slem cells were isoLaled frum O6IGFP ntice, in which GFP is expressed un(\cr tbc IXlfItrol of06. 11 promoter of thc mDachl gene. which is in\'Olved in tbe development of Ihc concx. At E1 2. D6 is specifically exprcssed in loc neurd.! stem cclLs of the (\(nal tclcnocephalon. from whieh cortical neurons arise, We have shown lila! D61GFP neurJI stem odLs isolated fmm 1312 embfyos are able to give rlse to neuroos and glial fibrillary acidic protcin (GFAP)-posilivc cel1s ill \'i"o nnd that after...
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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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