Robust Up-Regulation of Nuclear Red Fluorescent-Tagged Fos Marks Neuronal Activation in Green Fluorescent Vasopressin Neurons after Osmotic Stimulation in a Double-Transgenic Rat
Robust Up-Regulation of Nuclear Red Fluorescent-Tagged Fos Marks Neuronal Activation in Green Fluorescent Vasopressin Neurons after Osmotic Stimulation in a Double-Transgenic Rat
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DOI：
10.1210/en.2009-0796
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发表时间：
2009-12-01
期刊：
影响因子：
4.8
通讯作者：
Murphy, David
中科院分区：
文献类型：
--
作者：
Fujihara, Hiroaki;Ueta, Yoichi;Murphy, David
C-fos基因编码的mRNA或蛋白表达上调被广泛用作各种刺激诱导神经元激活的标志。为了便于检测激活的神经元，我们制备了c-fos编码序列与c-fos基因调控序列控制下的mRFP1融合基因的转基因大鼠(c-fos-mRFP1大鼠)。C-fos-mRFP1转基因大鼠ip高渗盐水90min后，在视前正中核、终板血管器、视上核、室旁核和穹隆下器等渗透敏感脑区可观察到丰富的mRFP1核荧光。Fos蛋白免疫组织化学证实，非转基因大鼠的Fos样免疫反应与转基因大鼠ip高渗盐水后mRFP1的荧光分布相似。将表达精氨酸加压素增强绿色荧光蛋白融合基因的转基因大鼠(AVP-EGFP大鼠)与c-fos-mRFP1大鼠配对，获得多只双转基因大鼠。在这些双转基因大鼠中，给予高渗盐水90min后，几乎所有视上核和PVN内的EGFP神经元都表达了核mRFP1荧光。C-fos-mRFP1大鼠是一种强大的工具，可以方便地识别神经系统中激活的神经元。此外，当在细胞特异性调控序列的控制下与表达另一个荧光团的转基因结合时，可以很容易地检测到特定神经细胞类型对生理信号的激活。(内分泌学150：5633-5638,2009)
The up-regulation in the expression of mRNA or protein encoded by the c-fos gene is widely used as a marker of neuronal activation elicited by various stimuli. To facilitate the detection of activated neurons, we generated transgenic rats expressing a fusion gene consisting of c-fos coding sequences in frame with monomericred fluorescent protein 1 (mRFP1) under the control of c-fos gene regulatory sequences (c-fos-mRFP1 rats). In c-fos-mRFP1 transgenic rats, 90 min after hypertonic saline ip administration, nuclear mRFP1 fluorescence was observed abundantly in brain regions known to be osmosensitive, namely the median preoptic nucleus, organum vasculosum lamina terminalis, supraoptic nucleus, paraventricular nucleus, and subfornical organ. Immunohistochemistry for Fos protein confirmed that the distribution of Fos-like immunoreactivity in nontransgenic rats was similar to those of mRFP1 fluorescence after ip administration of hypertonic saline in the transgenic rats. Several double-transgenic rats were obtained from matings between transgenic rats expressing an arginine vasopressin-enhanced green fluorescent protein fusion gene (AVP-eGFP rats) and c-fos-mRFP1 rats. In these double-transgenic rats, almost all eGFP neurons in the supraoptic nucleus and PVN expressed nuclear mRFP1 fluorescence 90 min after hypertonic saline administration. The c-fos-mRFP1 rats are a powerful tool that enables the facile identification of activated neurons in the nervous system. Furthermore, when combined with transgenes expressing another fluorophore under the control of cell-specific regulatory sequences, activation of specific neuronal cell types in response to physiological cues can be readily detected. (Endocrinology 150: 5633-5638, 2009)