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Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons

Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons

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SCHIMMELMANN, Melanie Joan von, 2009. Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons

@phdthesis{Schimmelmann2009Chann-8656, title={Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons}, year={2009}, author={Schimmelmann, Melanie Joan von}, address={Konstanz}, school={Universität Konstanz} }

Schimmelmann, Melanie Joan von 2009 Schimmelmann, Melanie Joan von 2011-03-24T17:45:27Z 2011-03-24T17:45:27Z application/pdf Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons Regulierung von Ionenkanälen in der Wegfindung von Wachstumskegeln infolge von Semaphorin 3A in Spinalneuronen von Xenopus laevis deposit-license Growth cone intrinsic properties, such as intracellular cyclic nucleotide signalling pathways and ion channels in the growth cone plasma membrane, are important determinants of whether a guidance molecule functions as either an attractant or a repellent. Binding of guidance molecules to their receptor or receptor complexes triggers intracellular second messenger signalling cascades, e.g., via cAMP, cGMP and Ca2+, which ultimately leads to the regulation of appropriate cytoskeleton proteins in order to impose directional guidance on growth cones of either axons or dendrites.<br />Based on this background, this dissertation examined the mechanisms of second messenger signalling in response to diffusible guidance proteins, in particular the repellent Sema3A. Various techniques were utilized, including observations of single cell behaviour by growth cone turning assays combined with electrophysiology, immunocytochemistry, and Ca2+ imaging of cultured, developing Xenopus laevis spinal neurons. This work resulted in three specific conclusions: First, diffusible guidance molecules cause membrane potential shifts of about 15 mV; attractants cause depolarization and repellents cause hyperpolarization. Sema3A-induced cGMP production causes membrane hyperpolarization by the activation of Cl channels. In addition, pharmacological increase of cGMP levels leads to PKG-dependent activation of saxitoxin-sensitive channels, which converts Sema3A-induced repulsion to attraction. Thus, bi-directional turning responses are likely mediated by shifts of the membrane potential and the Sema3A-induced growth cone turning direction is imposed by the level of intracellular cGMP demonstrating a molecular switching mechanism of cGMP-dependent repulsion/hyperpolarization to PKG-dependent attraction/depolarization.<br />Second, Ca2+ influx through CNGCs is required and sufficient for Sema3A-induced growth cone repulsion. Electrophysiological studies indicated that CNG leak currents are enhanced in response to Sema3A. Xenopus spinal neuron growth cones express the CNGA1 subunit of rod-type CNGCs, which is co-expressed with the Sema3A receptor Npn-1. Loss-of-function studies, in which either antisense morpholino oligonucleotides against the Xenopus CNGA1 subunit were injected to eliminate the CNGA1 protein or a CNGA1 subunit mutant that has a deletion at the cGMP binding domain was overexpressed, abolish Sema3A-induced leak currents and convert the repulsion to attraction. Moreover, this conversion into attraction correlates with the conversion of membrane hyperpolarization to depolarization. Taken together, these observations indicate that Sema3A-induced bi-directional growth cone turning is caused by cGMP-dependent activation of CNGCs and likely VDCCs, depending on the level of intracellular cGMP. CNGCs or VDCCs in turn conduct Ca2+ in which the magnitude of Ca2+ entry determines the direction of growth cone turning.<br />Third, establishment and characterization of a culture system of dissociated primary mature Xenopus laevis spinal neurons allow the study of cellular and molecular mechanisms of nerve regeneration. This culture system reveals that older neurons display distinctive morphologies as well as different abilities to grow and survive than do their embryonic counterparts. In addition, consistent with the development in vivo, the polarity of their neurites in culture progressively switches from axons to dendrites as they are derived from increasingly mature developmental-stage animals. Importantly, unlike neurons in most other culture systems, these cultured neurons maintain their mature intrinsic properties. Interestingly, the culture conditions seem to favour the survival of commissural interneurons. Therefore, this culture system is a resource in which to study the mechanisms of nervous system function in mature animals and to examine the developmental changes that occur in the intrinsic properties of growth cones that may be responsible for the failure of adult nerve regeneration.<br />In summary, the three conclusions of this study contribute to our better understanding of the intracellular signalling mechanisms that regulate bidirectional growth cone turning in response to a diffusible guidance protein Sema3A, which functions not only during nervous system development, but also in various other important cellular processes, i.e., nerve regeneration, tissue differentiation and apoptosis. Finally, this study provides a pivotal research resource in form of a primary culture system for mature Xenopus spinal neurons that favours the survival of a distinct class of spinal neurons for the study of the cellular and molecular mechanisms of nerve regeneration. eng

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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