建立細胞專一性的基因表現技術探討催產素釋放的調控機制

催產素(OT)是一種神經傳導物質，調控著許多生理及行為反應。催產素主要是由下視丘-腦下垂體系統中視上核和室旁核的magnocellular neruons (MCNs)所分泌，而負責製造催產素的MCNs，被稱為催產素神經元。在下視丘-腦下垂體系統中，催產素神經元會將軸突末端投射投射到腦下垂體後葉。催產素會進而經由神經元的樹突和軸突分支釋放到腦脊髓液中，或是沿著軸突主幹分泌到腦下垂體後葉並且進入周邊血液系統中。在下視丘-腦下垂體中，神經傳導物質主要是被包覆在兩種囊泡中，分別是large-dense core vesicles (LDCVs)和microvesicles (MVs)，兩者皆受到鈣離子調控的胞吐作用所調節。在先前的研究中，我們發現一種synaptic vesicles (SVs)/MVs的蛋白-Synpsin Ia (Syn Ia)會表現在視上核時，藉由其第62個胺基酸位置的磷酸化會增加被包覆在LDCVs中催產素的釋放。因此，為了更進一步釐清Syn Ia在催產素神經元中對於催產素釋放的影響，我們建立了一套能夠在催產素神經元專一表現基因的技術。首先，我們採用催產素啟動子(563bp)和增強子(IGR 182bp)來建構一個能夠同時專一在催產素神經元表現胞吐作用報導蛋白VAMP2-pHVenus以及Syn Ia的DNA。接著，我們將這些DNA轉染到會分泌催產素的下視丘細胞株-GT1-7 cells中，並且利用免疫螢光染色確認DNA能夠成功表現。利用胞吐作用報導蛋白，發現Syn Ia能夠增加囊泡釋放的機率，但此效應會被無法被MAPK磷酸化的Syn Ia (Syn Ia-S62A)所弱化。此外，我們藉由胞吐作用報導蛋白和鈣離子顯像技術確認Syn Ia不是透過囊泡數目的改變或鈣離子流入量去影響到囊泡的釋放。為了更進一步的將此專一表達在催產素神經元的DNA表現於活體中，我們利用活體電穿孔方式將DNA送入視上核的催產素神經元中。之後再利用免疫螢光染色，發現DNA可成功地被表現在催產素神經元中。這些表現胞吐作用報導蛋白的囊泡會經由催產素神經元的軸突被傳送至腦下腺後葉。綜合以上所述，我們建立了一套能夠專一表現在催產素神經元的技術，並且未來可能透過這個技術來了解在催產素神經元中，Syn Ia對於催產素在樹突和軸突釋放的影響。

關鍵字

催產素；下視丘-腦下垂體系統； synapsin Ia ；胞吐作用報導蛋白；鈣離子顯像技術；活體電穿孔； GT1-7 cells

並列摘要

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.

並列關鍵字

oxytocin ； hypothalamic-neurohypophysial system ； synapsin Ia ； exocytosis reporter ； calcium imaging ； in vivo electroporation ； GT1-7 cells

參考文獻

Adan, R. A., Van Leeuwen, F. W., Sonnemans, M. A., Brouns, M., Hoffman, G., Verbalis, J. G., & Burbach, J. P. (1995). Rat oxytocin receptor in brain, pituitary, mammary gland, and uterus: partial sequence and immunocytochemical localization. Endocrinology, 136(9), 4022-4028.

Aihara, H., & Miyazaki, J. (1998). Gene transfer into muscle by electroporation in vivo. Nat Biotechnol, 16(9), 867-870.

Akaneya, Y., Jiang, B., & Tsumoto, T. (2005). RNAi-induced gene silencing by local electroporation in targeting brain region. J Neurophysiol, 93(1), 594-602.

Archer, B. T., 3rd, Ozcelik, T., Jahn, R., Francke, U., & Sudhof, T. C. (1990). Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2. J Biol Chem, 265(28), 17267-17273.

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建立細胞專一性的基因表現技術探討催產素釋放的調控機制

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