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Mechanisms of dendritic peptide releaseMonteiro, Olivia F. de S. January 2010 (has links)
Magnocellular neurones (MCNs) are capable of secreting vasopressin and oxytocin from the somato-dendritic compartment, which can occur independently to secretion from nerve terminals. One hypothesis of the mechanism that regulates this differential release is that dendrites utilise different vesicle pools compared to those found in terminals. Little is known for the function of neuronal dendrites, especially the mechanism for peptide release. One theory is that vesicles stored in dendrites are non-released vesicles ready for recycling or degradation. Immunofluorescent labelling was performed on hypothalamic slices of the transgenic rat where enhanced green fluorescent protein (eGFP) was tagged to vasopressin. Lysosomes were detected by the lysosome-associated membrane protein LAMP1. Correlation analysis of LAMP1 labelling and VP-eGFP had shown that localisation of lysosomes in dendrites is positively correlated to loci of high vasopressin expression. This suggests active degradation of vesicles in dendrites. It is not known whether preferential release of peptides occurs along the profile of dendrites. Experiments were carried out using a temperature block to block exit of vesicles from the Golgi apparatus. Release of the temperature block triggered release of a wave of newly synthesised vesicles from the Golgi apparatus. Measurement of the fluorescent intensity of VP-eGFP showed that preferential release of peptides does not occur along the profile of dendrites. I have also utilised confocal live cell imaging to study the dynamics of dendritic vasopressin release using VP-eGFP slice explants. Experiments using high potassium stimulation showed significant increase in the release of vasopressin after priming with thapsigargin (intracellular calcium mobiliser), in accordance to in vitro release and microdialysis studies. These results demonstrate that live cell imaging can be achieved in magnocellular neurons, providing a robust model system in the study of dendritic peptide release. Large dense core vesicles (LDCVs) in other cell types such as bovine adrenal chromaffin cells were shown to segregate according to vesicle age, suggesting that vesicle age is an important factor in the regulation of peptide release. Whether vesicles of different age groups exist in magnocellular dendrites is not known. Thus, biolistic transfection with exogenous fluorescent proteins for expression under temporal control was carried out. However, low transfection rate in magnocellular neurones and the high background fluorescence caused by scattered gold particles used as bullets for transfection deemed this method inappropriate for the purpose of imaging vesicles. Hence, development of an adenoviral transduction system was employed. By using an inducible adenovirus gene construct coupled with a fluorescent reporter gene, it is possible to visualise vesicle pool segregation under different experimental conditions. Subcloning of a red fluorescent construct tagged to ppANF was tested on PC12 cells to show targeting of fluorescence expression to LDCVs. Successful production of an inducible adenoviral DNA with the red fluorescent construct insert was confirmed by PCR and DNA sequencing. Whilst the generation of viral particles is still to be achieved, successful production of the virus will be an invaluable system for inducible gene expression in neurones.
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Emergence and Homeostasis of Functional Maps in Hippocampal NeuronsRathour, Rahul Kumar January 2014 (has links) (PDF)
Systematic investigations through several experimental techniques have revealed that hippocampal pyramidal neurons express voltage gated ion channels (VGICs) with well-defined gradients along their dendritic arbor. These actively maintained gradients in various dendritic VGICs effectuate several stereotypic, topographically continuous functional gradients along the topograph of the dendritic arbor, and have been referred to as intraneuronal functional maps. The prime goal of my thesis was to understand the emergence and homeostasis of the several coexistent functional maps that express within hippocampal pyramidal neurons.
In the first part of the thesis, we focus only on spatial interactions between ion channels and analyzed the role of such interactions in the emergence of functional maps. We developed a generalized quantitative framework, the influence field, to analyze the extent of influence of a spatially localized VGIC cluster. Employing this framework, we showed that a localized VGIC cluster could have spatially widespread influence, and was heavily reliant on the specific physiological property and background conductances. Using the influence field model, we reconstructed functional gradients from VGIC conductance gradients, and demonstrated that the cumulative contribution of VGIC conductances in adjacent compartments plays a critical role in determining physiological properties at a given location. These results suggested that spatial interactions among spatially segregated VGIC clusters are necessary for the emergence of the functional maps.
In the second part of the thesis, we assessed the specific roles of only kinetic interactions between ion channels in determining physiological properties by employing a single-compartmental model. In doing this, we analyzed the roles of interactions among several VGICs in regulating intrinsic response dynamics. Using global sensitivity analysis, we showed that functionally similar models could be achieved even when underlying parameters displayed tremendous variability and exhibited weak pair-wise correlations. These results suggested that that response homeostasis could be achieved through several non-unique channel combinations, as an emergent consequence of kinetic interactions among these channel conductances.
In the final part of the thesis, we analyzed the combined impact of both spatial and kinetic interactions among ion channel conductances on the emergence and homeostasis of functional maps in a neuronal model endowed with extensive dendritic arborization. To do this, we performed global sensitivity analysis on morphologically realistic conductance-based models of hippocampal pyramidal neurons that coexpressed six functional maps. We found topographically continuous functional maps to emerge from disparate model parameters with weak pair-wise correlations between parameters. These results implied that individual channel properties need not be set at constant values in achieving overall homeostasis of several coexistent functional maps. We suggest collective channelostasis, where several channels regulate their properties and expression profiles in an uncorrelated manner, as an alternative for accomplishing functional map homeostasis. Finally, we developed a methodology to assess the contribution of individual channel conductances to the various functional measurements employing virtual knockout simulations. We found that the deletion of individual channels resulted in variable, measurement-and location-specific impacts across the model population.
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