Idegi ős/progenitor sejtek fejlődésének fiziológiai jellemzése = Physiological characterisation of developing neural stem/progenitor cells

1) Az MTA KOKI Idegi Sejt és Fejlődésbiológia Laboratóriumában felszereltem és beállítottam az elekrofiziológiai mérésekhez szükséges munkaállomást. A rendszer segítségével whole cell patch-clamp méréseket végeztem fejlődő idegi stem/progenitor sejteken. 2) Az NE-4C idegi őssejtek mellett jellemeztem a primer agyszövetből izolált radiális gliasejtek elektrofiziológiai sajátságait. Megállapítottuk, hogy az idegi ős-progenitorsejtekre általánosan jellemző a többszörös GJ kapcsoltság és ennek függvényében a passzív konduktancia. A sejt „ingerlékenységét”, külső stimulusokra adott válaszoló képességét feltehetően a passzívan mozgó kálium-és kloridionok eloszlásának finom összjátéka szabályozza. A differenciálódás során a GJ kapcsolatok megszűnnek és feszültségfüggő ioncsatornák szabályozzák a sejten belüli ionkoncentrációt. Az idegsejt irányú elköteleződés legelső fiziológia jelei ezek a változások. Eredményeink alapján, a nagyon korán megjelenő KDR áram fejlődési állapot specifikus alegység összetétellel bír, ami további indikátora lehet az idegi differenciálódás megfelelő stádiumainak. 3) Elsőként kimutattuk, hogy az idegi ős-progenitorsejtekben expresszálódik és működik az eddig főleg kifejlett idegrendszerben ismert EAAT4 glutamát transzporter. A transzporter jelenlétét in vivo is igazoltuk a fejlődő agyhólyag neurogén területein. A transzporter korai jelenléte a sejtek kloridion háztartása szemponjából lehet fontos. Az EAAT4 jelentős kloridion konduktanciával rendelkezik, a kloridion áramot a sejtek nyugalmi potenciálja és a sejten kivüli glutamát koncentráció szabályozza. | 1) The electrophysiological workstation had been set up and whole cell patch-clamp assays have been running. 2) Besides the NE-4C neuroectodermal stem cells, we characterized the electrophysiological properties of radial glial cells isolated from mouse brain tissue. The main bioelectric characteristics of stem-progenitors cells is the multiple ionic coupling and as a consequence, the high passive conductance. While these features may contribute to the maintenance of the non-committed stem-like phenotype, the passive movement of potassium and chloride ions may sensibly tune the excitability and responsiveness of the coupled group of progenitor cells. With the appearance of neuronal morphology, neighboring cells cease GJ communication and voltage dependent ionic channels take the role in setting the IC ion concentration. The changes of these bioelectrical properties are the first physiological signs of neuronal commitment. Delayed rectifier potassium currents (KDR) are present already in stem cell stage but the current is hindered by multiple GJ coupling. The pharmacology of KDR seems to be characteristic to defined developmental stages. 3) We have shown that the EAAT4 glutamate transporter is present and has function in neural stem cells. The in vivo expression of the protein was verified at the neurogenic regions of developing forebrain. The early presence of the transporter may play important roles in regulating the chloride homeostasis. EAAT4 has significant chloride conductance regulated by the resting membrane potential and by the EC glutamate concentration

Aktivitás- és információfüggő szinaptikus plaszticitás in vitro neuronhálózatokban = Activity- and Information-Dependent Synaptic Plasticity in Neural Networks in vitro

Kompakt neuronhálózatok szinaptikus szerveződését és dinamikai viselkedését tanulmányoztuk a preszinaptikus aktivitás függvényében a nagy mocsári csiga (Lymnaea stagnalis) idegrendszerében. Hagyományos és modern elektrofiziológiai mószerek alkalmazásával kerestünk olyan újszerű hálózatszerveződési elveket, amelyek a neuronok közötti szinaptikus kapcsolatok hosszútávú, finom változásait eredményezhetik. Vizsgálataink első részében részletesen jellemeztük a kardiorespiratorikus neuronok spontán működését és válaszaikat szimulált szinaptikus bemenetekre. Fő kísérleti eszközként a mintázat clamp technikát alkalmaztuk, amellyel kiválasztott preszinaptikus neuronok aktivitását tudtuk tetszőlegesen vezérelni és azok hatását kvantitatíve jellemezni a neuronhálózatban. Oszcilláló tipusú preszinaptikus tüzelési mintázatok alkalmazásakor új, frekvenciaszelektív dinamikai viselkedést mutattunk ki. Ez a rezonáns viselkedés a neuronok belső biofizikai tulajdonságainak és a szinaptikus bemenetek időviszonyainak kölcsönhatásából alakul ki. Megállapítottuk, hogy a felnőtt állatokban a neuronhálózatok szinaptikus összeköttetéseit kevésbé befolyásolják plasztikus változások, mint a neurohumorális hatások. A sejtszintű memória folyamatokban lényeges szerepet játszó neuromodulátorokkal (5-HT, DA) végzett kísérleteink a hálózat dinamikus újraszerveződését tárták fel. Eszerint a molluskákban a már kialakult szinaptikus kapcsolatok is figyelemreméltó változatosságot produkálnak, mert a neurokémiai környezet változásai folyamatosan újrakonfigurálják azokat. | In the current project we studied how presynaptic activity shapes the dynamics and synaptic organisation of compact neuronal networks in the pond snail Lymnaea stagnalis. We used both conventional and novel methods of electrophysiology seeking novel principles of plasticity, which might result in long-term, fine changes in the synaptic connectivity. In the first part of our investigation we performed a detailed characterization of the spontaneous firing patterns of cardiorespiratory neurons as well their responses to simulated synaptic inputs. We used the pattern clamp technique, which allowed us to control the activity of selected presynaptic neurons in a flexible manner. When using oscillatory presynaptic firing patterns as input we detected a novel, frequency selective dynamical behavior in several neurons of the cardiorespiratory circuit. This resonant behavior is an outcome of the interplay between the intrinsic biophysical properties of the neurons and the temporal structure of the synaptic inputs they receive. We found that in adult animals the synaptic interconnections are affected mainly by neurohormonal factors rather than by activity-dependent plastic changes. We observed dynamic reconfiguration of the neural connections in experiments with neuromodulators such as 5-HT and dopamine known to play important role in memory formation at the cellular level. Therefore, in molluscs, even mature synaptic connections display a remarkable degree of interanimal variability, reflecting the differential actions of neurochemical factors

Activity and Coupling to Hippocampal Oscillations of Median Raphe GABAergic Cells in Awake Mice.

Ascending serotonergic/glutamatergic projection from the median raphe region (MRR) to the hippocampal formation regulates both encoding and consolidation of memory and the oscillations associated with them. The firing of various types of MRR neurons exhibits rhythmic modulation coupled to hippocampal oscillatory activity. A possible intermediary between rhythm-generating forebrain regions and entrained ascending modulation may be the GABAergic circuit in the MRR, known to be targeted by a diverse array of top-down inputs. However, the activity of inhibitory MRR neurons in an awake animal is still largely unexplored. In this study, we utilized whole cell patch-clamp, single cell, and multichannel extracellular recordings of GABAergic and non-GABAergic MRR neurons in awake, head-fixed mice. First, we have demonstrated that glutamatergic and serotonergic neurons receive both transient, phasic, and sustained tonic inhibition. Then, we observed substantial heterogeneity of GABAergic firing patterns but a marked modulation of activity by brain states and fine timescale coupling of spiking to theta and ripple oscillations. We also uncovered a correlation between the preferred theta phase and the direction of activity change during ripples, suggesting the segregation of inhibitory neurons into functional groups. Finally, we could detect complementary alteration of non-GABAergic neurons’ ripple-coupled activity. Our findings support the assumption that the local inhibitory circuit in the MRR may synchronize ascending serotonergic/glutamatergic modulation with hippocampal activity on a subsecond timescale

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

Isolation of Radial Glia-Like Neural Stem Cells from Fetal and Adult Mouse Forebrain via Selective Adhesion to a Novel Adhesive Peptide-Conjugate

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes

Regulated Appearance of NMDA Receptor Subunits and Channel Functions during In Vitro Neuronal Differentiation

The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes.

Different neuronal phenotypes developed from radial glia-like cells.

<p>Neurons with different neurotransmitter-phenotypes developed from cloned populations of RGl-cells. Genes indicating noradrenergic (<i>Dbh</i>), serotonergic (<i>Tph2</i>) and cholinergic (<i>Chat</i>) neurotransmitter phenotypes were not expressed in neuron-rich cultures of any RGl-cells regardless of fetal or adult origin (<b>a</b>). GABA- and VGAT-immunopositive GABAergic (<b>b</b>, <b>c</b> respectively) and VGlut2-immunopositive glutamatergic (<b>d</b>) neurons developed in embryo-derived clones upon EGF-withdrawal (cells from clone C4 are shown). All adult-derived clones generated GABAergic neurons. Hippocampus- derived HC_A cells produced <i>VGlut1</i>-expressing (<b>a</b>) neurons, and all SVZ-clones gave rise to tyrosine-hydroxylase (TH)-positive (<b>e, e’</b>) but <i>dbh</i>-negative (<b>a</b>), neurons (<b>e</b>, <b>e’</b>: neurons from SVZ_M clone are shown). The pictures were taken on the 11^th day after EGF-withdrawal.</p

Characteristics of radial glia-like cell clones derived from adult mouse brain.

<p>Adult brain-derived radial glia-like cells (after the first passage) showed elongated cell shape (<b>a</b>), nestin- (<b>b</b>) RC2- (<b>d</b>) and Pax6- (<b>c</b>) immunoreactivity, like those derived from fetal forebrains, but in contrast to embryonic clones, they displayed GFAP-immunoreactivity (<b>c</b>). Cells of adult-derived RGl clones expressed genes characteristic to radial glial cells and many of the investigated positional genes (<b>e</b>).</p

PCR primer sequences.

<p>PCR primer sequences.</p

Radial glia-like cell clones.

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