应用透明脑技术对斑马鱼Orexin能神经元三维动态可视化研究

Orexin is a vital neuropeptide involved arousal in central nervous system. The distribution of orexin-producing nuclei and projection of its fibers determine its physiological functions.We have previously mapped the locations of the subregion of orexinergic neurons in mice. In recent years, zebrafish (Danio rerio) have emerged as an excellent model for sleep research. Although orexinergic cell groups were densely found in the brain of zebrafish, the construction of the micro neuroanatomical network is lacked, the features of distribution and morphological structure of the orexinergic neurons in its growth and development process is unknown, and the projecting relations between the orexinergic neurons and cerebral cortex and subcenter in nervous system remain unclear.We then hypothesized that CLARITY technology which could make brain tissue transparent would offer a three-dimensional view of orexin neural networks in zebrafish. Based on the zebrafish model, combined with transgenosis, CLARITY technology, three-dimensional reconstruction and sleep-wake behaviors recording and analysis, the present study was established to (1) observe the development, distribution and projection of orexinergic neurons in different growth phase of zebrafish larvae; (2) construct the neuroanatomical networks of zebrafish orexinergic neurons in three-dimension; (3) identify the effects of sleep-wake circulation change on neuroanatomical networks of orexinergic neurons. These results will provide complete histology and morphology basis for further study of orexinergic system of zebrafish in sleep research field.

Orexin是一种重要的中枢觉醒肽。Orexin能神经元核团分布及神经系维投射决定其生理功能。我们前期也开展了Orexin分布亚区的形态学研究。近年来斑马鱼已成为睡眠研究的热点动物模型。Orexin能神经元虽密集分布于斑马鱼脑内，但缺乏完整的微观解剖网络构建，其核团分布及形态在生长发育过程中有何特点，与大脑皮质及下位中枢的投射关系如何，尚未明确。据此我们提出“利用透明脑技术可三维原位呈现斑马鱼Orexin能神经元网络结构”的工作假说。拟利用斑马鱼模型优势，综合运用转基因技术、透明脑技术、三维重建技术及睡眠行为记录分析解决以下问题：（1）动态观察斑马鱼幼体不同生长期Orexin能神经元发育、分布及投射；（2）实体原位呈现斑马鱼Orexin能神经元神经解剖网络；（3）睡眠-觉醒周期改变对Orexin能神经元网络影响。以期为斑马鱼Orexin系统在睡眠领域的进一步研究提供完整的组织形态学依据。

Orexin是一种重要的中枢觉醒肽。Orexin能神经元核团分布及神经系维投射决定其生理功能。本研究运用序列切片组织成像技术结合三维重建技术、透明脑技术结合免疫荧光技术等，实体原位重现斑马鱼脑内Orexin神经元的微观解剖学信息。重要成果和关键数据：(1)通过透明脑观察到成年野生型斑马鱼脑内完整的Orexin神经元系统；(2)通过序列组织成像观察鲤科和啮齿类模式生物斑马鱼、小鼠Orexin神经元的分布和趋势：在斑马鱼脑内，Orexin神经元主要位于间脑前部的视顶盖室周灰质（periventricular grey zone of optic tectum，PGZ）下方，后屈束（fasciculus retroflexus，FR）、水平连合（commissura horizontalis，Chor）与后连合（commissura posterior，Cpost）之间；向后则集中位于被盖吻核（rostral tegmental nucleus，RT）、内侧前球核（medial preglomerular nucleus，PGm）、丘脑后核（posterior thalamus nucleus，P）等结构，此外，亦出现在间脑室外侧隐窝（lateral recess of diencephalic ventricle，LR）周围；在小鼠脑内，从Bregma -1.94mm层面开始，Orexin 神经元密集表达于下丘脑穹窿周核（perifornical nucleus，PeF）和外侧区（lateral hypothalamic area，LH），另外，零星散在的对称分布于第三脑室（3rd ventricle，3V）的两侧，此特点持续至PeF和LH消失，呈现从前至后为稀疏-密集-稀疏的分布状态；(3)通过核团三维重建，可立体直观呈现Orexin神经元解剖范围。在斑马鱼占据厚约450μm的间脑组织；在小鼠占据厚约1mm厚度的下丘脑组织。本项目提供了完整的斑马鱼和小鼠Orexin能神经元神经网络3D图像，为Orexin系统功能的进一步研究提供组织形态学依据。