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