轴突线粒体三维结构可反映果蝇的年龄

doi:10.3969/j.issn.1673-5374.2013.07.005

摘要/Abstract

摘要：

应用电子显微镜观察5d龄和20d龄果蝇背侧纵肌运动神经元的轴突，Fiji and Neurolucida软件重建两者轴突线粒体的三维结构图。结果显示20d与5d龄果蝇背侧纵肌运动神经元轴突线粒体总面积和总体积接近，但20d龄果蝇的线粒体总面积与轴突总面积的比值及线粒体密度低于5d龄果蝇，而体积小于1000000μm³和体积在1000000-10000000 μm³的线粒体的数量均比5d龄果蝇要多。2种日龄的果蝇轴突中体积在1000000-10000000 μm³的线粒体数量均比小于1000000 μm³或大于10000000 μm3的线粒体数量多；其中体积大于100000000 μm³的线粒体数量较小，但占到总体积的70%；体积在1000000-10000000 μm³的线粒体虽数量较大，但体积仅占总体积的不到30％。提示背侧纵肌运动神经元轴突线粒体的形态和数量在果蝇中存在日龄差异。

关键词: 神经再生, 神经退行性变, 线粒体, 果蝇, 轴突, 三维模型, 电子显微镜, 年龄相关的神经退行性疾病, 线粒体形态, 细胞显微结构, 图片文章

Abstract:

This study aimed to reconstruct a three-dimensional map of axonal mitochondria using Fiji and Neurolucida software, and to observe directly the morphology and distribution of mitochondria in axons of motor neurons in dorsal longitudinal flight muscles of drosophila aged 5 days and 20 days, using electron microscopy. Results indicated that there was no difference in the total area and volume of mitochondria between 5-day-old drosophila and 20-day-old drosophila in all sections, but the ratio of mitochondrial total areas to axon total areas, as well as mitochondrial density of 20-day-old drosophila, was lower than that of 5-day-old drosophila. The number of mitochondria, whose volume was less than 1 000 000 μm³, and between 1 000 000 μm³ and 10 000 000 μm³, was higher in 20-day-old drosophila than that in 5-day-old drosophila. The number of mitochondria with a volume between 1 000 000 μm³ and 100 000 000 μm³ was apparently higher than those with a volume less than 1 000 000 μm³or larger than 100 000 000 μm³. In addition, the number of mitochondria with a volume more than 100 000 000 μm³ was small; however, the volume was nearly 70% of the total volume in both 5-day-old and 20-day-old drosophila. In contrast, the number of mitochondria with a volume between 1 000 000 μm³ and 10 000 000 μm³ was large, but the volume was less than 30% of the total volume. These experimental findings suggest that changes in mitochondrial morphology and number in motor neurons from the dorsal longitudinal muscle of drosophila are present during different ages.

Key words: neural regeneration, neurodegenerative diseases, neurogenesis, mitochondrion, drosophila, axon, three-dimensional model, electron microscopy, age-related neurodegenerative diseases, mitochondrial morphology, cell microstructure, photographs-containing paper, neuroregeneration

. 轴突线粒体三维结构可反映果蝇的年龄[J]. 中国神经再生研究(英文版), 2013, 8(7): 616-621.

Honglian Zhu, Xiaojiang Sun. Three-dimensional structure of axonal mitochondria reflects the age of drosophila[J]. Neural Regeneration Research, 2013, 8(7): 616-621.

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引言

材料方法

Design
A randomized, controlled, animal experiment.

Time and setting
This study was performed at the Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China from October 2008 to March 2011.

Materials
A total of 10 drosophila were provided by the Drosophila Bloomington Stock Center, University of Indiana, USA. Drosophila stocks were cultured with standard medium at 24°C. After eclosion, the adult drosophila were cultured on standard medium and entrained into a 12-hour light/dark cycle at 28°C. W¹¹¹⁸ female flies were used. The giant fiber system of drosophila mediates escape behavior through a small number of neurons to innervate the dorsal longitudinal flight muscles^[12]. The P [Gal4]- A307 was used to drive the expression in the giant fiber system and other components of the nervous system. Drosophila were maintained in vials containing a mixture of agar, yeast, white cornmeal, molasses and water. Our experimental study used 5-day-old and 20-day-old drosophila to observe mitochondrial morphous and distribution.

Methods
Observations of axons in motor neurons using light and electron microscopy
Drosophila were anesthetized using carbon monoxide and were decapitated. The head, legs, wings and abdomen were removed^[22]. The thoracic segment was fixed with 4% (w/v) paraformaldehyde^[23] and 2.5% (v/v) glutaraldehyde for 48 hours at 4°C, and then washed in PBS for 10 minutes and transferred to 1% (w/v) osmium tetroxide for 2 hours^[24], followed by a series of graded alcohols and embedded in Epon812^[25]. Sections were polymerized for 48 hours at 60°C. The resin-embedded specimen was cut into 1-μm-thick sections^[26]. These sections were stained with toluidine blue[^27]. After staining, we investigated the structure of the axon of motor neurons innervating the dorsal longitudinal muscle^[28] under the light microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA).

Next, we used the ultramicrotomy to cut sections at 70 nm thickness^[29]. The distance between sections was 70 nm. Thus, we obtained 14-μm-long axon sections. Ultrathin sections were collected on formvar-coated copper grids, were stained with uranyl acetate and lead citrate^[30]and were viewed with the JEOL JEM-1230 transmission electron microscope (Leica Microsystems Inc.). After scanning of each slice, images at 5 000-fold magnification were saved.

Three-dimensional models and mitochondrial parameters
The software of Fiji and Neurolucida (MBF Bioscience, Williston, WT, USA) was used to reconstruct the three-dimensional coordinate models from serial sections^[31-32]. First, we imported images of 100 pieces of 70-nm-thick sequential sections into the software of Fuji. Second, we improved the local contrast of the pictures using CLAHE. Third, we aligned the 100 images and saved them as 1 stack of TIF images. We inputted the stack in the software of Neurolucida, and drew the contour of every mitochondrion and axon. Finally, the software of Neurolucida automatically formed the three-dimensional structure of axonal mitochondrion and simultaneously calculated the number-volume and surface areas of the mitochondria.

Statistical analysis
Data were analyzed with SPSS 16.0 software (SPSS, Chicago, IL, USA). The mean differences were tested using t-test and a P < 0.05 level was considered statistically significant.

Acknowledgments: We greatly thank Dr Qian Hu (Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China) who designed the method of using the software of Fiji and Neurolucida to reconstruct the three-dimensional structure of axonal mitochondria.
Author contributions: Honglian Zhu designed and performed this study, analyzed experimental data and wrote the manuscript. Xiaojiang Sun was the counselor. Both authors approved the final version of the manuscript.
Conflicts of interest: None declared.
Ethical approval: Experiments were approved by the Animal Experimental Ethical Committee of Shanghai Jiao Tong University School of Medicine, China.
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application/funding source disputations.
Supplementary information: Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org.