Effects of progesterone on T-type-Ca2+-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-Ca2+-channels Cav3.1, Cav3.2, and Cav3.3 as they helped determine neuronal plasticity by regulating Ca2+-influx in neuronal cells. The results showed direct effects of progesterone on the mRNA expression of T-type-Ca2+-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-Ca2+-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-Ca2+-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 Cav3.1 and Cav3.3 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+ 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.

Disabling VEGF-Response of Purkinje Cells by Downregulation of 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.

Cultivation of Purified Primary Purkinje Cells from Rat Cerebella.

Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.