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

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 an

Inferior Olive HCN1 Channels Coordinate Synaptic Integration and Complex Spike Timing

Acknowledgments This work was supported by the Medical Research Council (G0501216), the Wellcome Trust (093295/Z/10/Z and 086602/Z/08/Z), and the BBSRC (Bb/H020284/1). We thank Paolo Puggioni for help with motion analysis and the IMPACT facility at the University of Edinburgh for imaging resources.Peer reviewe

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

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

Inferior olive HCN1 channels coordinate synaptic integration and complex spike timing

Cerebellar climbing-fiber-mediated complex spikes originate from neurons in the inferior olive (IO), are critical for motor coordination, and are central to theories of cerebellar learning. Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels expressed by IO neurons have been considered as pacemaker currents important for oscillatory and resonant dynamics. Here, we demonstrate that in vitro, network actions of HCN1 channels enable bidirectional glutamatergic synaptic responses, while local actions of HCN1 channels determine the timing and waveform of synaptically driven action potentials. These roles are distinct from, and may complement, proposed pacemaker functions of HCN channels. We find that in behaving animals HCN1 channels reduce variability in the timing of cerebellar complex spikes, which serve as a readout of IO spiking. Our results suggest that spatially distributed actions of HCN1 channels enable the IO to implement network-wide rules for synaptic integration that modulate the timing of cerebellar climbing fiber signals

Extraction of synaptic input properties in vivo

Knowledge of synaptic input is crucial for understanding synaptic integration and ultimately neural function. However, in vivo, the rates at which synaptic inputs arrive are high, so that it is typically impossible to detect single events. We show here that it is nevertheless possible to extract the properties of the events and, in particular, to extract the event rate, the synaptic time constants, and the properties of the event size distribution from in vivo voltage-clamp recordings. Applied to cerebellar interneurons, our method reveals that the synaptic input rate increases from 600 Hz during rest to 1000 Hz during locomotion, while the amplitude and shape of the synaptic events are unaffected by this state change. This method thus complements existing methods to measure neural function in vivo

Differentiation of Human Embryonic Stem Cells to Regional Specific Neural Precursors in Chemically Defined Medium Conditions

Background: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury. Methodology and Principal Findings: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis. Conclusions/Significance: These findings suggest that our differentiation protocol has the capacity to generate regionspecific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-cultur