Itoi Keiichi, Talukder Ashraf Hossain, Fuse Toshimitsu, Kaneko Takuji, Ozawa Ryo, Sato Takayuki, Sugaya Takuma, Uchida Katsuya, Yamazaki Maya, Abe Manabu, Natsume Rie, Sakimura Kenji
Laboratory of Information Biology (K.I., A.H.T., T.F., T.K., R.O., T.Sa., T.Su., K.U.), Graduate School of Information Sciences, Tohoku University, Sendai 980-8579, Japan; Department of Neuroendocrinology (K.I.), Graduate School of Medicine, Tohoku University, Sendai 980-8579, Japan; and Department of Cellular Neurobiology (M.Y., M.A., R.N., K.S.), Brain Research Institute, Niigata University, Niigata 951-8585, Japan.
Endocrinology. 2014 Oct;155(10):4054-60. doi: 10.1210/en.2014-1182. Epub 2014 Jul 24.
Corticotropin-releasing factor (CRF) is the key regulator of the hypothalamic-pituitary-adrenal axis. CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), and yet its expression is driven by the CRF promoter and responds to changes in the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including the PVH. The majority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venus-expressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the Tg(CAG-CAT-EGFP/wt);CRF(iCre/wt) (EGFP/CRF-iCre) mouse, in which enhanced green fluorescent protein (EGFP) is driven by the CAG promoter. EGFP was expressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.
促肾上腺皮质激素释放因子（CRF）是下丘脑 - 垂体 - 肾上腺轴的关键调节因子。在未进行免疫染色的情况下，CRF神经元在下丘脑室旁核（PVH）中无法从形态上与其他神经内分泌神经元区分开来。因此，我们构建了一种基因敲入小鼠，其在CRF神经元中表达修饰的黄色荧光蛋白（Venus）（CRF-Venus），并且其表达由CRF启动子驱动并对内部环境的变化做出反应。在CRF-Venus小鼠中，表达Venus的神经元分布在含有CRF神经元的脑区，包括PVH。在PVH中，大多数表达Venus的神经元与表达CRF的神经元重叠，但在生理糖皮质激素条件下，许多神经元仅表达Venus或CRF。然而，在糖皮质激素剥夺后，Venus表达增强，并且大多数Venus神经元共表达CRF。相反，Venus表达被过量的糖皮质激素抑制。血管加压素基因内编码的一种肽—— copeptin的表达，在糖皮质激素剥夺时在PVH-Venus神经元中被诱导，而在给予糖皮质激素时被抑制。因此，Venus神经元重现了PVH-CRF神经元中糖皮质激素依赖性血管加压素的表达。去甲肾上腺素增加了在电压钳模式下从表达Venus的神经元记录到的谷氨酸依赖性兴奋性突触后电流的频率。此外，将CRF-iCre基因敲入小鼠与CAG-CAT-EGFP报告基因小鼠杂交，产生Tg（CAG-CAT-EGFP/wt）;CRF（iCre/wt）（EGFP/CRF-iCre）小鼠，其中增强型绿色荧光蛋白（EGFP）由CAG启动子驱动。EGFP在EGFP/CRF-iCre小鼠的PVH中表达更稳定。因此，CRF-Venus在监测不同糖皮质激素状态下CRF神经元和CRF网络的动态变化方面可能具有优势。
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