Establishment of the cell type-specific gene expression technique to study the regulatory mechanism of oxytocin release
Date Issued
2016
Date
2016
Author(s)
Liou, Shih-Yuan
Abstract
Oxytocin (OT) regulates various physiological functions and behaviors. OT is mainly secreted from the magnocellular neurons (MCNs) of supraoptic nuclei (SON) and paraventricular nuclei in the hypothalamic-neurohypophysial system (HNS). The MCNs are responsible for producing OT and thus named as oxytocinergic neurons (OT neurons). In HNS, OT neurons project their axon terminals to posterior pituitary. OT is then released into cerebrospinal fluid (CSF) from the somatodendrites or axon collaterals. In addition, OT can be secreted to posterior pituitary and further into peripheral plasma from the main axon terminals. In HNS, there two distinct kinds of vesicles, large-dense core vesicles (LDCVs) and microvesicles (MVs), and the release from both types of vesicles are mediated by Ca2+-regulated exocytosis. In the previous study, we found that Synapsin Ia, a SVs/MVs-specific protein, in the SON can increase LDCV-packaged OT secretion by phosphorylation of the amino acid (Ser-62). In this study, to further clarify the effects of Syn Ia on OT secretion can occur in OT neurons exclusively, we established a cell type-specific gene expression technique in OT neurons. First, we used the 563 bp-promoter and the enhancer (IGR 182 bp) to build up a OT neuron-specific exocytosis reporter, VAMP2-pHVenus. In addition, we transfected the constructs into hypothalamic oxytocinergic GT1-7 cells and performed immunostaining to confirm the successful expression. By live-imaging the exocytosis reporter, we found that Syn Ia increased the releasing probability, but this effect was weakened by the MAPK site-phosphodeficient mutant (Syn Ia-S62A). Furthermore, we verified that Syn Ia did not change the vesicle amounts or Ca2+ entry to affect the vesicle release. To further transfect the OT neuron-specific DNA into the OT neurons of SON, we performed in vivo electroporation in adult male rat brain. By conducting immunostaining, we observed that the successful expression of these constructs in the OT neurons. Moreover, the vesicles expressing the exocytosis reporter can be found in the somadendrites and the axons of OT neurons. In conclusion, we set up a OT neuron-specific gene expression technique. In the future, we can detect how Syn Ia may affect OT secretion from the somadendrites and axons of the OT neurons.
Subjects
oxytocin
hypothalamic-neurohypophysial system
exocytosis reporter
calcium imaging
in vivo electroporation
Type
thesis
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