Ueta Y, Fujihara H, Dayanithi G, Kawata M, Murphy D
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
J Neuroendocrinol. 2008 Jun;20(6):660-4. doi: 10.1111/j.1365-2826.2008.01706.x.
The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca(2+) imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion.
抗利尿激素精氨酸加压素（AVP）在下丘脑室旁核（PVN）和视上核（SON）的大细胞神经分泌细胞（MNCs）中合成。利用识别AVP和神经垂体素II的特异性抗体通过免疫组织化学技术，并根据其电生理特性，可以鉴定出轴突终末投射到垂体后叶的含AVP的MNCs。最近，我们培育出了在含AVP的MNCs中表达AVP增强型绿色荧光蛋白（eGFP）融合基因的转基因大鼠。在这种转基因大鼠中，在PVN和SON中观察到了eGFP mRNA，在PVN、SON以及垂体后叶中都能看到eGFP荧光，这表明转基因蛋白沿着MNC轴突向下运输并储存于神经终末。脱水和慢性盐负荷后，PVN和SON中AVP-eGFP转基因的表达以及eGFP荧光显著增加。另一方面，参与下丘脑-垂体-肾上腺轴激活的PVN中含AVP的小细胞神经分泌细胞在双侧肾上腺切除和腹腔注射脂多糖后表现出强烈的AVP-eGFP荧光。在正中隆起，渗透压刺激和应激条件下，内层和外层分别对eGFP显示出强烈荧光，这再次表明转基因翻译产物的运输是合适的。脑片、急性解离的MNCs和轴突终末在荧光显微镜下观察时也表现出强烈荧光。因此，AVP-eGFP转基因动物是独特的，为在体内研究分泌AVP的细胞用于电生理学、细胞内Ca(2+)成像等成像分析、器官培养以及AVP分泌动态变化的体内监测提供了一个有用的工具。
