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一种提高海兔神经节成像清晰度的清晰化技术

陈婷婷, 郁炜, 刘丹丹, 余可, 陈宋安, 王晔, 杨少忠, 贾若楠, 郑予桐, 黄振, Ferdinand S Vilim, 董磊, Elizabeth C Cropper, Klaudiusz R Weiss, 景键

南京大学生命科学学院医药生物技术国家重点实验室，生命科学高等研究院，南京 210046；西奈山伊坎医学院神经科学系和弗里德曼脑研究所，纽约 NY10029，美国

摘要

无论在脊椎动物还是无脊椎动物中，提高神经环路的成像效果对于神经环路的功能研究都是必不可少的。因此，一种新的基于鼠脑的成像技术——清晰化技术(CLARITY)，正吸引越来越多的研究者将它运用到其它物种中。这里我们将改进过的清晰化技术应用于无脊椎动物海兔的神经节的清晰化成像中。改进之处包括为海兔神经节修改了水凝胶溶液配方并设计了一个专用的容器。我们使用预先经过免疫组化处理的神经节，再使用改进的清晰化技术。我们采用荧光和共聚焦成像检查这些技术的相容性以及成像效果的改善程度。结果显示，这种改进过的清晰化技术确实可以使样本更清晰，并且进一步提高了海兔神经节中神经肽CP2免疫阳性神经元的清晰度。例如，这种方法提高了神经节深处的一些弱免疫阳性神经元的可视化效果。这种改良方法使得清晰化技术不仅更适合海兔整体神经节的结构成像，也会对其它的无脊椎动物神经节的成像起到帮助作用。

关键词： 清晰化技术; 成像改进 ; 神经结构 ; 海兔 ; 免疫组化

分类号：Q-334；Q-336；Q421

A clarifying method that improves imaging of Aplysia ganglia

CHEN Ting-Ting, YU Wei, LIU Dan-Dan, YU Ke, CHEN Song-An, WANG Ye, YANG Shao-Zhong, JIA Ruo-Nan, ZHENG Yu-Tong, HUANG Zhen, Ferdinand S Vilim, DONG Lei, Elizabeth C Cropper, Klaudiusz R Weiss, JING Jian

1State Key Laboratory of Pharmaceutical Biotechnology, Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University, Nanjing 210046, China； 2Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Abstract

Improvements in the imaging of neural circuits are essential for studies of network function in both invertebrates and vertebrates. Therefore, CLARITY, a new imaging enhancement technique developed for mouse brains has attracted broad interest from researchers working on other species. We studied the potential of a modified version of CLARITY to enhance the imaging of ganglia in an invertebrate Aplysia. For example, we have modified the hydrogel solution and designed a small container for the Aplysia ganglia. The ganglia were first processed for immunohistochemistry, and then for CLARITY. We examined the compatibility of these techniques and the extent to which the imaging of fluorescence improved using confocal microscopy. We found that CLARITY did indeed enhance the imaging of CP2 immunopositive neurons in Aplysia ganglia. For example, it improved visualization of small, weak immunoreactive neurons deep in the ganglia. Our modifications of CLARITY make this new method suitable for future use in Aplysia experiments. Furthermore, our techniques are likely to facilitate imaging in other invertebrate ganglia.

Key words: CLARITY; imaging enhancement ; neuronal structure ; Aplysia ; immunohistochemistry

收稿日期：2017-01-22　　录用日期：2017-02-22

通讯作者：景键　　E-mail: jingj01@nju.edu.cn

引用本文：

陈婷婷, 郁炜, 刘丹丹, 余可, 陈宋安, 王晔, 杨少忠, 贾若楠, 郑予桐, 黄振, Ferdinand S Vilim, 董磊, Elizabeth C Cropper, Klaudiusz R Weiss, 景键. 一种提高海兔神经节成像清晰度的清晰化技术[J]. 生理学报 2017; 69 (4): 461-466.

CHEN Ting-Ting, YU Wei, LIU Dan-Dan, YU Ke, CHEN Song-An, WANG Ye, YANG Shao-Zhong, JIA Ruo-Nan, ZHENG Yu-Tong, HUANG Zhen, Ferdinand S Vilim, DONG Lei, Elizabeth C Cropper, Klaudiusz R Weiss, JING Jian. A clarifying method that improves imaging of Aplysia ganglia. Acta Physiol Sin 2017; 69 (4): 461-466