Zelluläre Kontrolle adulter Neurogenese - Expression und Funktion des VEGF-Rezeptor-1 (Flt-1) im adulten Säugerhirn

Neurale Stammzellen sind im adulten Säugerhirn in der Subventrikulären Zone (SVZ) der Lateralventrikel und dem Hippokampus lokalisiert. In der SVZ entstandene neurale Zellen migrieren entlang eines von Astrozyten umgebenen Pfades, dem Rostralmigratorischen Strom (RMS), zum Olfaktorischen Bulbus (OB), wo sie zu olfaktorischen Interneuronen differenzieren. Vaskuläre Wachstumsfaktoren, wie VEGF-A beeinflussen die adulte Neurogenese. Die vorliegende Arbeit beschreibt erstmalig detailliert die spezifische Expression des VEGF-Rezeptor-1 (VEGFR-1) in den Regionen olfaktorischer und hippokampaler Neurogenese des adulten ZNS. Die Ergebnisse zeigen, dass VEGFR-1 im adulten Hirn hauptsächlich in GFAP-positiven Zellen in der SVZ, dem RMS, dem OB, dem Corpus callosum und dem Hippokampus exprimiert ist. In vivo-Analysen transgener Mäuse (Flt-1TK-/-), denen die Signaltransduktionsdomäne des VEGFR-1 fehlt, demonstrieren hier erstmals eine Rolle des VEGFR-1 in adulter Neurogenese. Flt-1TK-/- weisen eine erhöhte Proliferation neuronaler Vorläuferzellen der SVZ auf. Im RMS ist jedoch 6 Tage nach BrdU-Administration die Anzahl markierter Zellen im Vergleich zum Wildtyp (wt) um 47,97% reduziert, ohne dass es zu einer Akkumulation in der SVZ kommt. Zusammen mit der in Kulturversuchen stark erhöhten Migrationsgeschwindigkeit von Neuroblasten der Flt-1TK-/- und einer verminderten Abwanderung von Zellen aus dem RMS ins Corpus callosum der Flt-1Tk-/-, weist dies auf eine gesteigerte Migration zum OB hin. Tatsächlich war der OB der Flt-1TK-/-, vor allem die Plexiform- und Periglomerulärzellschicht (PGL), signifikant vergrößert. Im OB der transgenen Tiere migrieren zudem signifikant mehr BrdU-markierte Zellen in die PGL. Dort differenzieren signifikant mehr Neurone als im wt. Subtypisierungen zeigen, zudem eine erhöhte Differenzierung in dopaminerge Interneurone in der PGL der Flt-1TK-/-. Im Gehirn Flt-1TK-/- war die Konzentration von VEGF-A erhöht. Intrazerebroventrikuläre Infusion von VEGF-A in wt-Tiere erbrachte den eindeutigen Nachweis, dass die Erhöhung der VEGF-A-Konzentration im Gehirn der Flt-1TK-/- ursächlich für die in diesen Tieren beobachtete Reduktion der BrdU-positiven Zellen im RMS ist. Dies ist gleichzeitig der erste Nachweis einer Wirkung von VEGF-A auf Neuroblasten im RMS in vivo unter physiologischen Bedingungen. Die erhöhte VEGF-A-Konzentration könnte auch den anderen hier dargelegten Effekten zugrunde liegen. VEGFR-1 ist somit ein regulatorischer Faktor für die adulte olfaktorische Neurogenese und spielt eine potentielle Rolle in der Differenzierung dopaminerger Interneurone.

Membrane properties and synaptic integration of identified neuronal populations in the developing rodent neocortex

In my PhD work I concentrated on three elementary questions that are essential to understand the interactions between the different neuronal cell populations in the developing neocortex. The questions regarded the identity of Cajal-Retzius (CR) cells, the ubiquitous expression of glycine receptors in all major cell populations of the immature neocortex, and the role of taurine in the modulation of immature neocortical network activity.rnTo unravel whether CR cells of different ontogenetic origin have divergent functions I investigated the electrophysiological properties of YFP+ (derived from the septum and borders of the pallium) and YFP− CR cells (derived from other neocortical origins). This study demonstrated that the passive and active electrophysiological properties as well as features of GABAergic PSCs and glutamatergic currents are similar between both CR cell populations. These findings suggest that CR cells of different origins most probably support similar functions within the neuronal networks of the early postnatal cerebral cortex.rnTo elucidate whether glycine receptors are expressed in all major cell populations of the developing neocortex I analyzed the functional expression of glycine receptors on subplate (SP) cells. Activation of glycine receptors by glycine, -alanine and taurine elicited membrane responses that could be blocked by the selective glycinergic antagonist strychnine. Pharmacological experiments suggest that SP cells express functional heteromeric glycine receptors that do not contain 1 subunits. The activation of glycine receptors by glycine and taurine induced a membrane depolarization, which mediated excitatory effects. Considering the key role of SP cells in immature cortical networks and the development of thalamocortical connections, this glycinergic excitation may influence the properties of early cortical networks and the formation of cortical circuits.rnIn the third part of my project I demonstrated that tonic taurine application induced a massive increase in the frequency of PSCs. Based on their reversal potential and their pharmacological properties these taurine-induced PSCs are exclusively transmitted via GABAA receptors to the pyramidal neurons, while both GABAA and glycine receptors were implicated in the generation of the presynaptic activity. Accordingly, whole-cell and cell-attached recordings from genetically labeled interneurons revealed the expression of glycine and GABAA receptors, which mediated an excitatory action on these cells. These findings suggest that low taurine concentrations can tonically activate exclusively GABAergic networks. The activity level maintained by this GABAergic activity in the immature nervous system may contribute to network properties and can facilitate the activity dependent formation of adequate synaptic projections.rnIn summary, the results of my studies complemented the knowledge about neuronal interactions in the immature neocortex and improve our understanding of cellular processes that guide neuronal development and thus shape the brain.rn

Resonance properties of different neuronal populations in the immature mouse neocortex

Subthreshold resonance is a characteristic membrane property of different neuronal classes, is critically involved in the generation of network oscillations, and tunes the integration of synaptic inputs to particular frequency ranges. In order to investigate whether resonance properties of distinct neuronal populations in the immature neocortex contribute to these network oscillations, I performed whole-cell patch-clamp recordings from visually identified neurons in tangential and coronal neocortical slices from postnatal day (P) P0-P7 C57Bl/6 and P6-P13 GAD67-GFP knock-in mice. Subthreshold resonance was analyzed by sinusoidal current injection of varying frequency. All Cajal-Retzius cells showed subthreshold resonance with an average frequency of 2.6 ± 0.1 Hz (n=60), which was massively reduced by ZD7288, a blocker of hyperpolarization-activated cation currents. About 65.6% (n=61) of the supragranular pyramidal neurons showed subthreshold resonance with an average frequency of 1.4 ± 0.1 Hz (n=40). Application of 1 mM Ni2+ suppressed subthreshold resonance, suggesting that low-threshold Ca2+ currents contribute to resonance in these neurons. About 63.6% (n=77) of the layer V pyramidal neurons showed subthreshold resonance with an average frequency of 1.4 ± 0.2 Hz (n=49), which was abolished by ZD7288. Only 44.1% (n=59) of the subplate neurons showed subthreshold resonance with an average frequency of 1.3 ± 0.2 Hz (n=26) and a small resonance strength. Finally, 50% of the investigated GABAergic interneurons showed subthreshold resonance with an average frequency of 2.0 ± 0.2 Hz (n=42). Membrane hyperpolarization to –86 mV attenuated the frequency and strength of subthreshold resonance. Subthreshold resonance was virtually abolished in the presence of 1 mM Ni2+, suggesting that t-type Ca2+ currents are critically involved in the generation of resonance, while ZD7288 had no effect. Application of 0.4 µM TTX suppressed subthreshold resonance at depolarized, but not hyperpolarized membrane potential, suggesting that persistent Na+ current contribute to the amplification of membrane resonance. rnIn summary, these results demonstrate that all investigated neuronal subpopulations reveal resonance behavior, with either hyperpolarization-activated cation or low-threshold Ca2+ currents contributing to the subthreshold resonance. GABAergic interneurons also express subthreshold resonance at low frequencies, with t-type Ca2+ and persistent Na+ currents underlying the generation of membrane resonance. The membrane resonance of immature neurons may contribute to the generation of slow oscillatory activity pattern in the immature neocortex and enhance the temporal precision of synaptic integration in developing cortical neurons.rn