Expression Pattern of T-Type Ca2+ Channels in Cerebellar Purkinje Cells after VEGF Treatment

T-type Ca2+ channels, generating low threshold calcium influx in neurons, play a crucial role in the function of neuronal networks and their plasticity. To further investigate their role in the complex field of research in plasticity of neurons on a molecular level, this study aimed to analyse the impact of the vascular endothelial growth factor (VEGF) on these channels. VEGF, known as a player in vasculogenesis, also shows potent influence in the central nervous system, where it elicits neuronal growth. To investigate the influence of VEGF on the three T-type Ca2+ channel isoforms, Cav3.1 (encoded by Cacna1g), Cav3.2 (encoded by Cacna1h), and Cav3.3 (encoded by Cacna1i), lasermicrodissection of in vivo-grown Purkinje cells (PCs) was performed, gene expression was analysed via qPCR and compared to in vitro-grown PCs. We investigated the VEGF receptor composition of in vivo- and in vitro-grown PCs and underlined the importance of VEGF receptor 2 for PCs. Furthermore, we performed immunostaining of T-type Ca2+ channels with in vivo- and in vitro-grown PCs and showed the distribution of T-type Ca2+ channel expression during PC development. Overall, our findings provide the first evidence that the mRNA expression of Cav3.1, Cav3.2, and Cav3.3 increases due to VEGF stimulation, which indicates an impact of VEGF on neuronal plasticity

Der Einfluss von VEGF auf die synaptische Plastizität von Purkinjezellen

Ziel dieser Arbeit war es, die Effekte des vaskulären endothelialen Wachstumsfaktors (VEGF) auf die funktionellen Aspekte der synaptischen Plastizität zu erforschen. Hierzu wurde die mRNA-Expression von NMDA-Rezeptoren, AMPA-Rezeptoren und T-Typ-Calciumkanälen in einer aufgereinigten Purkinjezellkultur, in in vivo gewachsenen Purkinjezellen und in Abhängigkeit von VEGF bestimmt. Außerdem wurden mögliche Veränderung der Funktion von T-Typ-Calciumkanäle, hervorgerufen durch VEGF, elektrophysiologisch untersucht. Hierzu wurden ein spezielles Zellkulturaufreinigungsprotokoll, Lasermikrodissektion, Immunhistochemie, quantitative Polymerasenkettenreaktion und Zwei-Elektroden-Spannungsklemmtechnik genutzt. Erstmalig konnte eine Veränderung der mRNA-Expression der NMDA-Rezeptoren, AMPA-Rezeptoren und T-Typ-Calciumkanälen, ausgelöst von VEGF, festgestellt werden. Eine fünftägige Stimulation mit VEGF führte bei den T-Typ-Calciumkanälen zu einem frühzeitigen erhöhten Calciumstrom

Disabling VEGF-Response of Purkinje Cells by Downregulation of <em>KDR</em> via miRNA-204-5p

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC

Effects of progesterone on T-type-Ca2+^{2+}-channel expression in Purkinje cells

Plasticity of cerebellar Purkinje cells (PC) is influenced by progesterone via the classical progesterone receptors PR-A and PR-B by stimulating dendritogenesis, spinogenesis, and synaptogenesis in these cells. Dissociated PC cultures were used to analyze progesterone effects at a molecular level on the voltage-gated T-type-Ca$^{2+}$-channels Ca$_v$3.1, Ca$_v$3.2, and Ca$_v$3.3 as they helped determine neuronal plasticity by regulating Ca$^{2+}$-influx in neuronal cells. The results showed direct effects of progesterone on the mRNA expression of T-type-Ca$^{2+}$-channels, as well as on the protein kinases A and C being involved in downstream signaling pathways that play an important role in neuronal plasticity. For the mRNA expression studies of T-type-Ca$^{2+}$-channels and protein kinases of the signaling cascade, laser microdissection and purified PC cultures of different maturation stages were used. Immunohistochemical staining was also performed to characterize the localization of T-type-Ca$^{2+}$-channels in PC. Experimental progesterone treatment was performed on the purified PC culture for 24 and 48 hours. Our results show that progesterone increases the expression of Ca$_v$3.1 and Ca$_v$3.3 and associated protein kinases A and C in PC at the mRNA level within 48 hours after treatment at latest. These effects extend the current knowledge of the function of progesterone in the central nervous system and provide an explanatory approach for its influence on neuronal plasticity

Expression pattern of T-type Ca2+Ca^{2+} channels in cerebellar Purkinje cells after VEGF treatment

T-type $Ca^{2+}$ channels, generating low threshold calcium influx in neurons, play a crucial role in the function of neuronal networks and their plasticity. To further investigate their role in the complex field of research in plasticity of neurons on a molecular level, this study aimed to analyse the impact of the vascular endothelial growth factor (VEGF) on these channels. VEGF, known as a player in vasculogenesis, also shows potent influence in the central nervous system, where it elicits neuronal growth. To investigate the influence of VEGF on the three T-type $Ca^{2+}$ channel isoforms, Cav3.1 (encoded by $\it Cacna1g$), Cav3.2 (encoded by $\it Cacna1h$), and Cav3.3 (encoded by $\it Cacna1i$), lasermicrodissection of in vivo-grown Purkinje cells (PCs) was performed, gene expression was analysed via qPCR and compared to in vitro-grown PCs. We investigated the VEGF receptor composition of in vivo- and in vitro-grown PCs and underlined the importance of VEGF receptor 2 for PCs. Furthermore, we performed immunostaining of T-type $Ca^{2+}$ channels with in vivo- and in vitro-grown PCs and showed the distribution of T-type $Ca^{2+}$ channel expression during PC development. Overall, our findings provide the first evidence that the mRNA expression of Cav3.1, Cav3.2, and Cav3.3 increases due to VEGF stimulation, which indicates an impact of VEGF on neuronal plasticity

Disabling VEGF-response of Purkinje Cells by downregulation of KDR\it KDR via miRNA-204-5p

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC

Herausforderung Wissenstransfer in Clustern : neues Wissen vom Biotechnologiestandort Martinsried

Wissen stellt in forschungsintensiven Industrien eine Schlüsselressource dar. Vor dem Hintergrund der noch weitgehend ungeklärten Frage nach den Konkurrenzbeziehungen und Kausalitäten in Wissenstransferprozessen ergründet der vorliegende Beitrag die verschiedenen Koordinationsweisen von Wissenstransfer am Beispiel des Biotechnologiestandorts Martinsried. Die empirisch auf qualitative Experteninterviews und eine Online-Befragung gestützte Untersuchung gibt Aufschluss über die Potentiale und Probleme von Wissensvernetzung, Wissensteilung und Wissensabfluss als zentrale Mechanismen des interorganisationalen Wissenstransfers. Die Ergebnisse der Fallstudie liefern sowohl konzeptionelle Einsichten in die raumzeitlichen Dynamiken von Wissenstransfer als auch praxisorientierte Hinweise bezüglich der Herausforderungen der Wissensintermediation im Biotech-Cluster Martinsried