Analyse Neurone-generierender Zellteilungen im Neuroepithel der Maus

In Vertebraten wird Dauer der Proliferationsphase neuronaler Vorläuferzellen im Neuroepithel (Neuroepithelzellen) und damit die Neuronenzahl des Gehirns vermutlich durch den Übergang von im cell fate symmetrischen, proliferativen (Generierung zweier Neuroepithelzellen) zu asymmetrischen differenzierenden Zellteilungen (Generierung einer Neuroepithelzelle und eines Neurons) kontrolliert. Direkte Beweise für die Existenz dieser asymmetrischen Teilungen stehen noch aus, genauso wie zellbiologische Mechanismen und genetische Kontrolle des „Umschaltens“ unbekannt sind. Durch die Expression von GFP unter Kontrolle regulatorischer Sequenzen des TIS21-Gens wurden spezifisch Neurone-generierende Neuroepithelzellen markiert, um (i) die Symmetrie der Neurone-generierenden Zellteilung videomikroskopisch zu untersuchen und (ii) eine differenzielle Analyse der Zellbiologie (z.B. Verteilung apikaler Membran, Zellzyklusdauer) und Genexpression von proliferierenden vs. Neurone-generierenden Neuroepithelzellen zu ermöglichen. Es wurden drei transgene und eine TIS21-GFP-knock in-Mauslinie etabliert. Im TIS21-GFP-knock in ersetzt eine GFP-Expressionskassette das offene Leseraster von TIS21 in Exon 1. Der TIS21-GFP-knock in exprimiert entlang des ganzen Neuralrohrs GFP in Neurone-generierenden Neuroepithelzellen, die transgenen Linien jeweils nur in bestimmten Abschnitten. Die Expression von TIS21 im Neuroepithel wird offensichtlich von verschiedenen Elementen gesteuert, die jeweils bestimmte Regionen im Neuralrohr abdecken. In Schnittkulturen von Neuralrohr-Explantaten aus TIS21-GFP-knock in-Embryonen wurden mit Multiphoton-Videomikroskopie zu Beginn der Neurogenese (E12 Telencephalon) neben sich apikal teilenden Zellen auch überraschend Zellen entdeckt, die sich auf der basalen Seite des Neuroepithels teilen. Apikalen Teilungen bilden wahrscheinlich ein Neuron und eine Neuroepithelzelle, basale bilden zwei Neurone. Demnach existieren in Vertebraten zwei Typen Neurone-generierender Teilungen: (i) im cell fate asymmetrische, apikale Teilungen typischer Neuroepithelzellen und (ii) symmetrische, basale Teilungen einer hier erstmals beschriebenen Zellpopulation. Die Verteilung apikaler Membran und die Zellzykluslänge wurden in proliferierenden vs. Neurone-generierenden Neuroepithelzellen als mögliche Steuerungsmechanismen des Übergangs von Proliferation zu Neurogenese untersucht. Die Orientierung der Teilungsebene (abgeleitet aus der Chromatinfärbung in der Anaphase) relativ zur apikalen Membran identifiziert als Lücke in der basolateralen, Cadherin-positiven Plasmamembran in frühen Stadien der Neurogenese (E10..5-E11.5) im TIS21-GFP-knock in zeigt, dass der Schritt von Proliferation zu Neurogenese durch einen Wechsel in der Verteilung apikaler Membrankomponenten kontrolliert werden könnte: Apikale Teilungen mit symmetrischer Verteilung der apikalen Membran sind größtenteils proliferierend, Teilungen mit asymmetrischer Verteilung der apikalen Membran Neurone-generierend. Zudem korreliert die Differenzierung mit einer Verlängerung des Zellzyklus: Durch eine kumulative BrdU-Markierung konnte gezeigt werden, dass sich zu Beginn der Neurogenese (E10.5 Telencephalon) der Zellzyklus der Neurone-generierenden Neuroepithelzellen um 40% auf 13 h, gegenüber 9 h in proliferierenden Neuroepithelzellen, verlangsamt. Eine ursächliche Beteiligung von TIS21, das spezifisch in Neurone-generierenden also sich langsamen teilenden Neuroepithelzellen exprimiert ist und bekanntermaßen Zellzyklus-verlangsamend wirkt, an diesem Phänomen ist noch nicht geklärt. Homozygote TIS21-GFP-knock in-Tiere, als TIS21-knock out-Modell, zeigen zumindest keinen offensichtlichen qualitativ veränderten Ablauf der Neurogenese. Dagegen verschiebt sich im Blutbild das Gleichgewicht von Lymphozyten zu Granulocyten. In Verbindung mit dem in dieser Arbeit beschriebenen embryonalen und adulten Aktivitätsmuster - TIS21 ist in differenzierenden Zellpopulationen verschiedener Gewebe exprimiert - impliziert dies eine allgemeine physiologische Funktion von TIS21 bei Proliferations- und Differenzierungsprozessen. In einer unabhängigen Serie von Experimenten wurde die Möglichkeit untersucht, durch RNA-interference (RNAi) spezifisch die Expression von Genen in postimplantorischen Mausembryonen zu unterdrücken. Konkret wurde esi-(endoribonuclease III-prepared short interfering)-RNA (esiRNA) in E10 Mäuseembryonen intraventrikulär injiziert, durch Elektroporation in Neuroepithelzellen transfiziert und der Effekt von RNAi nach 24 h in vitro-Kultur der Embryonen analysiert. esiRNA gegen b-Galaktosidase, koelektroporiert mit einem b-Galaktosidase- und einem GFP-Expressionsvektor, inhibierte spezifisch die b-Galaktosidase- nicht aber die GFP-Expression. In einem analogen Experiment in E10 TIS21-GFP-knock in Embryonen verhinderte die Elektroporation von esiRNA gerichtet gegen GFP die GFP-Expression zu Beginn der Neurogenese – d.h. die Expression eines endogen exprimierten Gens

Central amygdala PKC-δ^+ neurons mediate the influence of multiple anorexigenic signals

Feeding can be inhibited by multiple cues, including those associated with satiety, sickness or unpalatable food. How such anorexigenic signals inhibit feeding at the neural circuit level is not completely understood. Although some inhibitory circuits have been identified, it is not yet clear whether distinct anorexigenic influences are processed in a convergent or parallel manner. The amygdala central nucleus (CEA) has been implicated in feeding control, but its role is controversial. The lateral subdivision of CEA (CEl) contains a subpopulation of GABAergic neurons that are marked by protein kinase C-δ (PKC-δ). We found that CEl PKC-δ^+ neurons in mice were activated by diverse anorexigenic signals in vivo, were required for the inhibition of feeding by such signals and strongly suppressed food intake when activated. They received presynaptic inputs from anatomically distributed neurons activated by different anorexigenic agents. Our data suggest that CEl PKC-δ^+ neurons constitute an important node that mediates the influence of multiple anorexigenic signals

Tis21 Expression Marks Not Only Populations of Neurogenic Precursor Cells but Also New Postmitotic Neurons in Adult Hippocampal Neurogenesis

During embryonic cortical development, expression of Tis21 is associated with cell cycle lengthening and neurogenic divisions of progenitor cells. We here investigated if the expression pattern of Tis21 also correlates with the generation of new neurons in the adult hippocampus. We used Tis21 knock-in mice expressing green fluorescent protein (GFP) and studied Tis21-GFP expression together with markers of adult hippocampal neurogenesis in newly generated cells. We found that Tis21-GFP 1) was absent from the radial glia–like putative stem cells (type-1 cells), 2) first appeared in transient amplifying progenitor cells (type-2 and 3 cells), 3) did not colocalize with markers of early postmitotic maturation stage, 4) was expressed again in maturing neurons, and 5) finally decreased in mature granule cells. Our data show that, in the course of adult neurogenesis, Tis21 is expressed in a phase additional to the one of the embryonic neurogenesis. This additional phase of expression might be associated with a new and different function of Tis21 than during embryonic brain development, where no Tis21 is expressed in mature neurons. We hypothesize that this function is related to the final functional integration of the newborn neurons. Tis21 can thus serve as new marker for key stages of adult neurogenesis

Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin–driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors

Genetic dissection of an amygdala microcircuit that gates conditioned fear

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ^+ neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ^− neurons in CEl. Electrical silencing of PKC-δ^+ neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called Cel_(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing

Ultrathin Coatings from Isocyanate Terminated Star PEG Prepolymers: Patterning of Proteins on the Layers

This study presents the easy and fast patterning of low molecular weight molecules that act as binding partners for proteins on Star PEG coatings. These coatings are prepared from isocyanate terminated star shaped prepolymers and form a highly cross-linked network on the substrate in which the stars are connected via urea groups and free amino groups are present. Streptavidin has been patterned on these layers by microcontact printing (μCP) of an amino reactive biotin derivative and consecutive binding of streptavidin to the biotin. Patterns of Ni^(2+)-nitriltriacetic acid (NTA) receptors have been prepared by printing amino functional NTA molecules in freshly prepared Star PEG layers that still contain amino reactive isocyanate groups. Complexation of the NTA groups with Ni(II) ions enabled the binding of His-tag enhanced green fluorescent protein (EGFP) in the desired pattern on the substrates. Since the unmodified Star PEG layers prevent unspecific protein adsorption, His-EGFP could selectively be bound to the sample by immersion into crude, nonpurified His-tag EGFP containing cell lysate