Mechanisms of dendritic peptide release

Abstract

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.