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Role of NG2 expressing cells in murine terminal phalanx regenerationJanuary 2013 (has links)
Research using the adult mammalian model shows that regeneration in the limb is limited to the distal most portion of the terminal phalanx. Recent studies suggest that the cellular contributions made to the regenerating system are lineage restricted and that the niche bone marrow hematopoietic stem cell population’s contributions are minimal. These studies however, do not address other residing populations within the bone marrow, specifically the mesenchymal and endothelial stem cell populations. One of the residing populations, the reputed pericyte or perivascular cells, possesses the ability to differentiate into multiple other cell types. To assess the potential contribution of perivascular cells to the regeneration competency of the terminal phalanges, we began by identifying perivascular cells within the terminal phalanx by using two accepted pericyte markers: nerve-glial antigen 2 (NG2) and endosialin (TEM1). Using NG2 and TEM1 in conjunction with vascular marker Tie2 in the Tie2-EGFP murine reporter line, we confirm a large number of perivascular cells in the bone marrow’s unusually well-developed and organized vasculature and a lower density within the connective tissue microvasculature; implicating a great potential contribution from the bone marrow. Post-amputation, we observe a large population of NG2+ and TEM1+ cells within the regenerating blastema region. Co-immunohistochemical studies reveal the blastema have cells that co-express osteogenic and pericyte markers; strongly suggestive of a transdifferentiation event. We attempt to confirm our hypotheses made in our initial assessment by utilizing two independent cell tracing studies: a DiI labeling of the bone marrow of the terminal phalanx to identify a marrow derived cellular contribution to the regenerate and a genetic fate tracing study using transgenic NG2CreERTamR26REYFP mice to confirm a transdifferentiation event. Using a novel in vivo method , we DiI-label the bone marrow content before amputation and trace DiI labeled bone marrow derived cellular contributions to the regenerate. DiI labeled cells were observed within the blastema expressing either endothelial, perivascular, or osteogenic markers, confirming the bone marrow contributes multiple cell types during the regeneration process. Using a similar experimental design, we genetically label the terminal phalanx NG2 expressing cells using systemic tamoxifen induction of NG2CreERTamR26REYFP mice. We fate trace the initially labeled population during blastema formation and re-differentiation and observed transdifferentiation events of the perivascular cells into two distinctive lineages, endothelial and osteoprogenitor cells. Establishing a direct correlation between peri-vasculature and re-differentiation, we address NG2/perivascular necessity with a series of temporal loss of function studies using a blocking antibody (iNG2). We implant iNG2 soaked microcarrier beads into various regions of the terminal phalanx and during different stages of the regeneration process. The experiments confirm the necessity of NG2 expression for distal bone elongation, as well as ascertain the temporal nature of the NG2 expression in different microenvironments. These results establish the importance of NG2+ cells in the bone marrow during early stages of regeneration, with early iNG2 bone marrow implantation resulting in a complete failure of the regeneration process. In an attempt to rescue this iNG2 failed regeneration we employ an established position-specific fibroblast cell line that displays a surprising plasticity as a cell-based therapeutic. Through a series of RNAi lentiviral transfection of inhibitors of the TGFβ-BMP pathway we induce osteogenic plasticity in the line. These results reveal regeneration competency associated with the mammalian terminal phalanx is in part due to the ability to recruit local perivascular multipotent populations, which has great translational relevancy. / acase@tulane.edu
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Communication synaptique et non-synaptique entre neurones et cellules précurseurs d’oligodendrocytes dans le cortex somatosensoriel / Synaptic and non-synaptic communication between neurons and oligodendrocyte precursor cells in the somatosensory cortexMaldonado Rojas, Paloma P. 09 December 2013 (has links)
Les cellules précurseur d'oligodendrocytes (CPOs) représentent la majeure source d'oligodendrocytesmyélinisants durant le développement post-natal. Ces progéniteurs, identifiés par l'expression du protéoglycane NG2, sont non seulement extrêmement abondants avant la myélinisation, mais ils persistent aussi dans le cerveau mature. À l'instar d'autres cellules non-neuronales, elles expriment un large panel de canaux ioniques et de récepteurs pour des neurotransmetteurs. Cependant, ils sont uniques de part leur capacité à recevoir de véritables contacts synaptiques neuronaux glutamatergiqueset GABAergiques. Durant cette thèse, nous avons caractérisé les propriétés électrophysiologiques des CPOs durant le développement post-natal du cortex en champ de tonneaux de la souris (premier mois post-natal). En effectuant des enregistrements de patch-clamp, des analyses par RT-PCR sur cellule unique et des analyses pharmacologiques, nous avons observé que la courbe I-V à rectification sortante devient linéaire durant le développement, résultant d'une régulation positive de l'expression des canaux potassiques de type Kir4.1. Dotés de ces canaux, les CPOs adultes sont capables de détecter les augmentations locales de potassium extracellulaire générées par l'activité neuronale. Cette régulation positive développementale des canaux Kir4.1 dans les CPOs révèle que ces cellules ont un gain de fonction durant le développement, leur conférant la capacité de communiquer avec les neurones via un mécanisme non-synaptique lié au potassium. Ce changement développemental soutient aussi l'idée que les CPOs sont probablement plus que des progéniteurs. Dans la deuxième partie de cette thèse, nous nous sommes intéressés à l'étude des patrons de connectivité du réseau GABAergique interneurones-CPOs dans le cortex en champ de tonneaux jeune (deuxième semaine post-natale). Dans un premier temps, nous avons tiré avantage de la haute précision latérale et axiale de la photolyse holographique en mono-photon pour stimuler les interneurones GABAergiques avec une résolution à l'échelle de la cellule, de manière à évoquer un potentiel d'action. Nous avons ensuite utilisé cette technique pour cartographier la connectivité entre interneurones et CPOs. Nous avons trouvé que la probabilité de connexion des CPOs est près de moitié moins que celle des cellules pyramidales, et implique plutôt une microcircuitrie locale. De plus, en effectuant des enregistrements pairés, nous avons observé que les CPOs sont contactés transitoirement par des interneurones à décharge rapide et à décharge régulière. Ces connections se caractérisents pour la présence d'un ou deux sites de libération uniquement. Étonnamment, les sites post-synaptiques contenant des récepteurs GABAA avec la sous-unité γ2 sont principalement connectés par les interneurones à décharge rapide, indiquant que ces cellules constituent une afférence spécifique auprès des CPOs. Ici nous décrivons pour la première fois l'émergence de réseaux corticaux spécifiques entre neurones et cellules non-neuronales. / Oligodendrocyte precursor cells (OPCs) are the main source of myelinating oligodendrocytes during postnatal development. These progenitors, identified by the expression of the proteoglycan NG2, are extremely abundant before myelination, but also persist in the mature brain. Similarly to other non-neuronal cells they express a wide range of ionic and ligand-gated ion channels. However, they are unique by their ability to receive truly glutamatergic and GABAergic synaptic contacts from neurons. During this thesis, we characterized the electrophysiological properties of OPCs during the postnatal development of the mouse somatosensory cortex (post postnatal month). By performing patch-clamp recordings, single-cell RT-PCR analyses and pharmacological approaches, we found that outwardly rectifying I-V curves become linear during development, as the result of an upregulation of Kir4.1 potassium channels. Endowed with these channels, adult OPCs are able to sense local extracellular potassium increases generated by neuronal activity. This developmental upregulation of Kir4.1 channels in OPCs revealed that these cells gain physiological properties during development, conferring them the capacity to communicate with neurons, via a non-synaptic potassium-mediated mechanism. This developmental change also supports the view that OPCs are probably more than simple progenitors. In the second part of this thesis, we were interested in study the connectivity patterns underlying the GABAergic interneuron-OPC network in the young somatosensory cortex (second postnatal week). First, we took advantage of the high lateral and axial precision of one-photon holographic photolysis to stimulate GABAergic interneurons at a single cell resolution in order to evoke an action potential. We then used this technique to map the connectivity between interneurons and OPCs. We found that the connectivity probability of OPCs was around half less than that of pyramidal cells and involved more local microcircuits. In addition, by performing paired-recordings, OPCs showed to be transiently contacted by fast-spiking (FSI) and non-fast-spiking (NFSI) interneurons, through single or double release sites. Interestingly, postsynaptic sites containing GABAA receptors with the γ2 subunit were predominantly connected by FSI, indicating that these cells provide a specific input to OPCs. Here we described for the first time the emergence of specific cortical network between neurons and non-neuronal cells. In conclusion, this thesis contributed to get a better understanding of the different modes of communication between neurons and OPCs and the establishment of new signaling mechanisms used by neurons to control the activity of these precursors.
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