Browsing by Subject "Nervenzelle"

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Der Beitrag von Neurofascin zur Entstehung und Lokalisation von Gephyrinclustern während der inhibitorischen Synaptogenese im ZNS
(2008) Burkarth, Nadine; Volkmer, Hansjürgen
Membrane-bound cell adhesion molecules (CAMs) were discovered to be key players in initiating excitatory synapse formation in the CNS. However, so far little is known about the role of CAMs in inhibitory synapse development. In particular, a functional link between CAMs and the clustering of postsynaptic scaffold component gephyrin, which is a critical determinant of y-aminobutyric acid A (GABAA) clustering, still needs to be elaborated. Neurofascin belongs to the L1-subgroup of the immunoglobuline like CAMs (IgCAMs). In vivo Neurofascin has been shown to direct the formation and localization of GABAergic Input on cerebellar Purkinje neurons. Thus it serves as a candidate molecule recruiting gephyrin to inhibitory postsynaptic sites. At early stages of inhibitory synaptogenesis, formation of gephyrin clusters and their translocation to the axon hillock correlated with a somatic expression of neurofascin in rat hippocampal neurons. Furthermore, a neurofascin splice variant lacking the extracellular fifth fibronectine-III like domain (NF-5te FN-III) was predominantely expressed at early stages of synapse formation. In contrast expression of NF+5te FN-III harbouring the fifth fibronectine-III like domain was prominent only in adult brain. Transfection of expression vectors for different splice variants and deletion mutants of neurofascin revealed that the embryonic neurofascin isoform NF-5te FN-III is required for the formation of gephyrin clusters. This process is presumably dependent on extracellular interactions with molecules on pre- and postsynaptic terminals, respectively. However, possible interaction partners for neurofascin are unknown. Point mutations in the cytoplasmatic domain of neurofascin inhibited the formation of gephyrin clusters suggesting intracellular signal transduction pathways triggered by neurofascin. Furthermore, expression of neurofascin is necessary for the translocation of gephyrin clusters to the axon hillock of hippocampal neurons as shown by shRNA-mediated knockdown. In addition, overexpression of an embryonic neurofascin isoform was sufficient for functional rescue after knockdown of endogenous neurofascin. Expression of NF-5te FN-III resulted in the accumulation of exogenous GFP-Gephyrin at the axon initial segment AIS suggesting a functional link between neurofascin and gephyrin. However, colocalization studies in HEK293 and PC12E2 cells, respectively, did not provide an indication of direct neurofascin gephyrin interactions leading to the observed effects.
Patch clamp experiments with human neuron-like cells under different gravity conditions
(2010) Kohn, Florian Peter Michael; Hanke, Wolfgang
Gravitation influences many physical, chemical and biological processes. Cells and their behaviour are no exception. The gravitational impact on the activity of neuronal cells is very important with the perspective of manned space missions. Previous experiments with vertebrates showed that the velocity of neuronal impulses (action potentials) in nerve fibres is decreasing under zero gravity and is increasing at high gravity. There are many theories about the changed properties of the nervous systems under zero gravity, but the molecular principles are mostly unexplored. For this dissertation hardware for patch clamp experiments under microgravity was developed. The patch clamp technique is a common used tool in investigating the electrophysiological properties of cells and single ion channels. Since the conditions during parabolic flights render classic patch clamp nearly impossible, advanced chip-based planar patch-clamp hardware, the Port-a-Patch from Nanion Technologies was integrated into a setup. This setup had to comply with the mandatory design and safety regulations to be used in parabolic flights. Two parabolic flight campaigns were used to validate the hardware, adapt the patch clamp procedures to the special conditions during a parabolic flight (as time pressure and vibrations) and to choose a suitable cell line from the available cell lines. As the laboratory conditions on ground were insufficient at the beginning of the project, the main focus had to lie on robust cells. The SH-SY5Y cell line was chosen for their robustness (they already have been used successfully by other teams) and their origin from the human brain (glioblastoma). During two subsequent parabolic flight campaigns patch clamp experiments were performed and whole cell currents of SH-SY5Y cells were recorded with increasing success rate. Pulse protocols were used to create current-voltage (I-V) characteristics. To obtain 20 seconds of microgravity, two phases of hypergravity, each lasting 20 seconds, had to be endured by the passengers. This fact allowed the subsequent recording of whole cell currents of the same cell during normal Gravity (1g), hypergravity (1.8g) and microgravity (approx. 10-3g) to compare the I-V characteristics of the different gravity conditions. For SH-SY5Y cells it was shown that a gravity dependence of the whole cell currents exists. The micro- and hypergravity whole cell currents were changed compared to 1g flight controls. At -20 and -10mV, the hypergravity current was significantly decreased compared to 1g, by 13.5% at -20mV and by 7.4% at -10mV. A significant 1.8g current < 0g current relation could be observed at potentials between -20 and +10mV. At -20mV the microgravity whole cell currents were increased by 13.5%, at -10mV by 7.4%, at 0mV by 6.2% and at +10mV by 3.9%. The duration and complexity of the used pulse protocols were limited by the time of each gravity phase (20-22 seconds). In a last parabolic flight campaign, therefore the passive electrophysiological properties of the cell membrane were investigated. As the laboratory conditions at the site were greatly improved in the meantime, especially for cell culture, new cell lines could be tested for their usability. SNB19 cells, also originating from the human brain (astrocytoma) were chosen due to their good sealing quality and stability compared to SH-SY5Y cells and constant pulse protocols near the estimated resting potential (-80 and -60mV) were performed. At constant -80mV and -60mV, it was shown that the whole cell currents during the variable gravity conditions are significantly increased compared to the 1g in-flight controls. The hypergravity current of SNB19 was increased by 2.2% at -80V and by 8.2% at -60mV. The microgravity current was increased by 4.1% at -80mV and 3.7% at -60mV. The acquired I-V characteristic of SNB19 differs from the I-V characteristic of SH-SY5Y. These findings show that the planar patch clamp technique can be used in parabolic flights to investigate the electrophysiological properties of single. Furthermore the findings suggest that the electrophysiological properties of single cells originating from the human brain exist are gravity dependent.