Pozícionális gének aktivitásának szerepe az idegsejt-fenotípus meghatározásában = Role of positional genes in the determination of the neuronal phenotype

A jellegzetes mintázatban aktiválódó pozícionális gének agyfejlődésben játszott szerepét vizsgálva az alábbi eredményeket nyertük: 1. A korai NE-4C embrionális idegi őssejtek indukálatlan állapotban regionálisan nem elkötelezettek; a neuron-képzés időszakában a regionális gének széles skálája aktiválódik; a sejtekből GABAerg, glutamaterg és szerotonin termelő neuronok is fejlődnek. 2. Az NE-4C sejtvonal transzfekciójával 11 idegi őssejtklónt alapítottunk; ezek a fejlődés minden szakaszában expresszálták az emx2 (antero-dorzális telencephalon) regionális gént; az NE-4Cemx2+ sejtek adhéziós sajátságai megváltoztak és katekolamin termelő neuronokat is képeztek. 3. Idegi őssejtvonalakat izoláltunk embrionális és kifejlett egéragyból új, szintetikus adhezív peptidkonjugátumok alkalmazásával; 4. A különböző agyi régiókból származó idegi őssejtvonalak és a belőlük in vitro fejlődő idegszöveti sejttípusok sok eredetre jellemző sajátságot megőriztek, de a hagyományos regionális gén-mintázat ezt az „emlékezetet” nem tükrözte; 5. Az egyes őssejt-klónok retinoid-érzékenysége és inherens retinsav metabolizmusa eltérő. Igazoltuk, hogy felnőtt agy neurogén régiói magas retinsav-tartalommal bírnak. 6. A kifejlett agyi parenchyma nem nyújt befogadó környeztet az őssejtek számára; a sérült előagyban az ős/progenitor sejtek szaporodnak, de nem differenciálódnak; túlnyomásos O2-kezelés hatására sporadikus idegsejt irányú fejlődés indítható. | Studies on developmental roles of „positional genes” in the formation of regional brain features led to the following results: 1. Early embryonic neural stem cells (NE-4C) are regionally not determined; in neuron-fromation phase, however, many regional genes got activated; GABAergic and glutamatergic neurons developed from the clon. 2. Inserting the Emx2 (antero-dorsal telencephalic) positional gene into NE-4C cells, 11 sub-clones were established, all expressing Emx2 throughout the entire differentiation period. The NE-4Cemx2+ cells displayed altered adhesive characteristics, and could generate catecholamine producing neurons. 3. By using novel synthetic adhesive peptide-conjugates, neural stem/progenitor clones had been established from different ages and regions of the mouse brain. 4. Neural stem/progenitor clones preserved several features characteristic to their origin, while the expression profile of traditional „regional genes” failed to reflect the regional „memory”. 5. The stem/progenitor clones displayed important differences in retinoid sentitivity and metabolism. In the neurogenic zones of the adult brain, enhanced retinoic acid contaent was demonstrated. 6. The adult brain parenhcyma is not permissive for the implanted stem/progenitor cells, regardless of their origin. In cortical lesion sites, the stem/progenitor cells proliferate, but do not differentiate. Hyperbaric O2-treatment was shown to allow sporadic neuronal differentiation

Retinoid Machinery in Distinct Neural Stem Cell Populations with Different Retinoid Responsiveness

Retinoic acid (RA) is present at sites of neurogenesis in both the embryonic and adult brain. While it is widely accepted that RA signaling is involved in the regulation of neural stem cell differentiation, little is known about vitamin A utilization and biosynthesis of active retinoids in the neurogenic niches, or about the details of retinoid metabolism in neural stem cells and differentiating progenies. Here we provide data on retinoid responsiveness and RA production of distinct neural stem cell/neural progenitor populations. In addition, we demonstrate differentiation-related changes in the expression of genes encoding proteins of the retinoid machinery, including components responsible for uptake (Stra6) and storage (Lrat) of vitamin A, transport of retinoids (Rbp4, CrbpI, CrabpI-II), synthesis (Rdh10, Raldh1-4), degradation of RA (Cyp26a1-c1) and RA signaling (Raralpha,beta,gamma, Rxralpha,beta,gamma). We show that both early embryonic neuroectodermal (NE-4C) stem cells and late embryonic or adult derived radial glia like progenitors (RGl cells) are capable to produce bioactive retinoids but respond differently to retinoid signals. However, while neuronal differentiation of RGl cells can not be induced by RA, neuron formation by NE-4C cells is initiated by both RA and RA-precursors (retinol or retinyl acetate). The data indicate that endogenous RA production, at least in some neural stem cell populations, may result in autocrine regulation of neuronal differentiation

Egy új adamantán-származék (p-bróm-benzoil-metil-adamantilamin) K+ csatorna blokkoló hatásának vizsgálata emlős és békaszíven

Megvizsgáltuk egy új adamantán-származék , a p-brom-benzoil-metil-adamantilamin (BMA) hatását emlős szívizom preparátumok transzmembrán potenciáljaira és kontraktilitására, valamint békaszív pitvari rostok transzmembrán ionáramaira. Megállapítottuk, hogy a BMA Ю- 5—10~ 4 M koncentráció tartományban szelektív К csatorna blokkoló hatással rendelkezik. Ezt támasztják alá a következő kísérleti adatok: 1. a vegyület extrém módon késlelteti az akciós potenciál repolarizációját, 2. pacemaker aktivitást indukál kamrai munkaizomzatban, mely alapmechanizmusa bizonyítottan a K-permeabilitás csökkenése, 3. lassú akciós potenciálokat indukál K-depolarizált rostokon, ami inkább tulajdonítható a depolarizáció okozta shunt-konduktancia csökkentésének, mint a lassú Ca csatorna foszforiláció-függő aktiválásának, 4. voltage clamp kísérletekben csökkenti a kifelé irányuló К ionáram nagyságát, de nem hat sem a gyors Na, sem a lassú Ca ionáramokra

A novel approach in the mineralogy of Carpathian mahogany obsidian using complementary methods

Carpathian obsidians can have various macroscopic features. They are typically black or grey and their transparency ranges from clear to opaque. The Tolcsva source, very rarely, can yield brown or red (‘mahogany’ type) obsidian. Archaeological, as well as geological pieces of mahogany obsidian were previously identified and characterised using Prompt Gamma Activation Analysis (PGAA). In 2007, the exact location of the red variant's outcrop was identified on the Szokolya hill (Tolcsva). The aim of this study was to better understand the possible reasons for the colouring of red obsidian. A novel approach was applied, using multiple methods for the analysis of the samples. For comparison, other Carpathian € y (Anatolia) were also studied. type, namely black obsidian from Tolcsva, and red obsidian from Bogazk o € ssbauer spectroscopy and Besides the PGAA measurements of the bulk elemental composition, M o transmission electron microscopy (TEM) were used to study the samples in order to identify the presence of ferrous or ferric iron. With the help of Small Angle Neutron Scattering (SANS), the bulk nanostructures of the samples have been investigated and their surface or volume fractal dimensions have been determined. Black obsidians showed isotropy, while mahogany samples displayed a considerable anisotropy in the bulk pore orientation. According to our results, a large amount of the iron is dominantly located in different phases in the case of mahogany and black obsidians. Based on the summarised re- sults, the differences between the red and black variants can be also explained by the different oxidation states of the Fe-ions, which may explain the colour difference

Radial glia-like cell clones.

<p>Radial glia-like cell clones.</p

Differentiation-Dependent Motility-Responses of Developing Neural Progenitors to Optogenetic Stimulation

During neural tissue genesis, neural stem/progenitor cells are exposed to bioelectric stimuli well before synaptogenesis and neural circuit formation. Fluctuations in the electrochemical potential in the vicinity of developing cells influence the genesis, migration and maturation of neuronal precursors. The complexity of the in vivo environment and the coexistence of various progenitor populations hinder the understanding of the significance of ionic/bioelectric stimuli in the early phases of neuronal differentiation. Using optogenetic stimulation, we investigated the in vitro motility responses of radial glia-like neural stem/progenitor populations to ionic stimuli. Radial glia-like neural stem cells were isolated from CAGloxpStoploxpChR2(H134)-eYFP transgenic mouse embryos. After transfection with Cre-recombinase, ChR2(channelrhodopsin-2)-expressing and non-expressing cells were separated by eYFP fluorescence. Expression of light-gated ion channels were checked by patch clamp and fluorescence intensity assays. Neurogenesis by ChR2-expressing and non-expressing cells was induced by withdrawal of EGF from the medium. Cells in different (stem cell, migrating progenitor and maturing precursor) stages of development were illuminated with laser light (λ = 488 nm; 1.3 mW/mm2; 300 ms) in every 5 min for 12 h. The displacement of the cells was analyzed on images taken at the end of each light pulse. Results demonstrated that the migratory activity decreased with the advancement of neuronal differentiation regardless of stimulation. Light-sensitive cells, however, responded on a differentiation-dependent way. In non-differentiated ChR2-expressing stem cell populations, the motility did not change significantly in response to light-stimulation. The displacement activity of migrating progenitors was enhanced, while the motility of differentiating neuronal precursors was markedly reduced by illumination

Rate of neuron- and oligodendrocyte production.

<p>The rate of neuron and oligodendrocyte production by adult-derived clones showed marked differences. After six days of EGF-withdrawal, adult cortex-derived RGl cells (CTX) gave rise to significantly less neurons than any other clones (<b>a</b>). RGl cells derived from the fetal ventral forebrain (clone A2) produced almost twofold more oligodendrocytes than those of dorsal origin (clone C4) (<b>b</b>). Adult RGl cells with hippocampal (HC; clone HC_A) and cortical (CTX; clone CTX_H) origin generated significant amount of oligodendrocytes (O4-immunopositive cells; <b>c</b>), while those of SVZ- (clone: SVZ_M) and midbrain (MID; clone MES_D)-origin produced significantly less O4-positive cells (∼1% of total cells).</p

Electrophysiological characteristics of radial glia-like cells.

<p>Electrophysiological characteristics of cloned RGl-1 cells (<b>a</b>, <b>b</b>) and RGl-1 derived neurons (<b>c</b>, <b>d</b>) were detected by whole-cell patch-clamp recording. Large passive conductance together with K_DR current (<b>a</b>) and current/voltage (I-V) relationship (<b>b</b>) are shown from a representative RGl-1 cell. Voltage-dependent inward Na-currents with small amplitude (<b>c</b>) were detected from primitive, differentiating RGl-derived neurons (n = 8). A representative current-profile and its current/voltage (I-V) relationship (<b>d</b>) are shown. The current traces were obtained by clamping the cell membrane from a -70 mV holding potential to values ranging from -160 mV to +20 mV, at 10 mV intervals.</p