论文总字数：27811字

摘 要

T-bar结构是果蝇突触前致密区一个特化的结构，它是果蝇和昆虫所特有，因其在电镜下像一个“T”形，故称为 T-bar。T-bar可为突触囊泡提供附着位点以及介导囊泡的释放中。BRP是果蝇T-bar的一个组成部分，在构建T-bar中发挥着重要的作用。研究表明，果蝇的神经突触发育呈现一定的规律性，受多种基因的调控。果蝇是一种极为重要的模式动物，但是目前关于果蝇神经突触在发育中的精细结构还未能很好的阐明，其精细发育模式，尤其是在电镜下的发育模式人们还知之甚微。本课题选取模式生物果蝇，通过对不同时期果蝇幼虫的腹神经索进行观察，探索果蝇突触中T-bar结构的发育模式，并力图通过免疫标记BRP，研究BRP在突触活跃区参与T-bar等结构组装中的作用，从而构建初步的动物神经突触的发育模式，以为神经信号的传递等分子机制研究提供形态学基础。

目的:探索果蝇突触T-bar的发生规律，描绘出果蝇突触T-bar结构发育的模式图。利用BRP抗体免疫标记不同发育阶段的果蝇，探讨BRP在T-bar结构组装中的作用。

方法：利用常规电镜包埋技术对不同时期的野生型（CS）果蝇进行包埋处理，并置于常规电镜下观察。对不同时期果蝇幼虫的突触进行形态学观察，统计突触前T-bar结构的“棒”状结构和“帽”状结构，二者均为T-bar结构的前体，再统计成熟T-bar结构、多T-bar结构、总T-bar结构的数据分布规律。对三龄的CS和Brp-RNAi突变体果蝇进行免疫电镜技术包埋以及免疫荧光处理，在电镜下进行观察。

结果：随着果蝇的发育，T-bar的棒状结构、帽状结构、多T-bar和总T-bar的数量先增长，然后下降，最后趋于稳定；成熟T-bar的数量持续增长，最后保持在稳定的数目。这种数量的变化反应了果蝇神经突触发育的动态变化，也能够为T-bar结构的发育模式提供统计学上的支持。

关键词：黑腹果蝇；腹神经索；突触；突触活跃区；T-bar；BRP；囊泡；

The assembly pattern of synaptic structure in Drosophila

No：41112119 Student:Chao Li

Supervisor:Guangming Gan

Abstract

The T-bar is a specialized structure of presynaptic dense area in Drosophila, it is unique to insects like Drosophila, and because it is like a "T" shape in the electron microscope, it is known as the T-bar. T-bar can provides the attachment sites for synaptic vesicles and plays an important role in mediating vesicle release. BRP is a component of the T-bar in Drosophila, which plays an important role in the formation of T-bar. The research shows that the nerve synapse development of Drosophila has a certain regularity, which is regulated by many genes. Drosophila is a very important model animal, but at present, the developing of synaptic fine structure of Drosophila also still failed be clarified well, its fine development mode, especially the development pattern under the electron microscope, people still know little. This topic selected model organism-- Drosophila melanogaster to explore developmental pattern of synaptic T-bar structure in the Drosophila through observing different stages of ventral nerve cord of larval Drosophila. We try to immunolabelling BRP to study what the role BRP plays in assembling T-bar structure at synaptic active region, so as to construct the developmental pattern of preliminary animal synapses to provide morphological basis for transmission mechanism of neural signal molecular .

Objective: Exploring the occurrence regularity of synaptic T-bar in Drosophila, depicting the development pattern of Drosophila synaptic T-bar structure. Studying The effect of BRP on the assembly of T-bar structure in Drosophila by using BRP antibody immunolabelling different developmental stages of Drosophila.

Methods: the wild type(CS) Drosophila in different periods were embedded by using conventional electron microscope embedding technology, and observed by conventional electron microscope to observed the morphology of Drosophila larval synapses in different periods and statistical distribution of presynaptic” stick” T-bar” and” cap” T-bar, which are both precursors of T-bar structure. Then counting the statistical distribution of mature T-bar structure, multi T-bar structure and total T-bar. The three instar CS and Brp-RNAi mutants of Drosophila were embedded by immuno electron microscopy technology and stained by immunofluorescence, then observed under the electron microscope.

Results: with the development of the Drosophila, the number of” stick” T-bar, ” cap” T-bar, multi T-bar and total T-bar growth firstly, then decreased, finally tends to be stable; the number of mature T-bar continued growth in the first time and finally keeping in the stable level. The changes of number reflect the dynamic change of synaptic development in Drosophila nerve, which also can provide statistical support for the development model of the T-bar structure.

Key words：Drosophila; Ventral nerve cord; Synapse; synaptic active region; T-bar; BRP; Vesicle;

目录

第一章 绪论 5

1.1神经系统与神经组织 5

1.1.1神经系统 5

1.1.2神经组织 5

1.2突触的结构及组分 6

1.2.1突触的结构 6

1.2.2突触前膜 6

1.2.3突触间隙 6

1.2.4突触后膜 6

1.3突触的功能 6

1.4.1BRP的结构 7

1.4.2BRP的功能 7

1.4.2.1BRP是T-bar形成的必要组件 7

1.4.2.2BRP对 Ca2 通道集群的影响 8

1.4.2.3BRP影响突触囊泡的释放 8

1.5问题及展望 9

第二章材料和方法 10

2.1实验材料 10

2.1.1果蝇 10

2.1.1.1果蝇品系 10

2.1.1.3培养基制备程序 10

2.1.1.4果蝇的饲养 10

2.1.2实验试剂及溶液 11

2.1.3实验仪器及设配 12

2.2实验方法 13

2.2.1果蝇腹神经索常规电镜样品制备 13

2.2.1.1 样品收集 13

2.2.1.2果蝇腹神经索常规电镜样品处理 13

2.2.1.2.1漂洗与固定 13

2.2.1.2.2脱水和渗透 13

2.2.1.2.3样品包埋 13

2.2.1.2.4切片、染色、观察 13

2.2.2果蝇腹神经索免疫电镜样品制备 14

2.2.2.1样品收集 14

2.2.2.2果蝇腹神经索免疫电镜样品处理 14

2.2.2.2.1漂洗、脱水与渗透 14

2.2.2.2.2样品包埋 14

2.3实验数据整理 14

第三章实验结果与分析 15

3.1果蝇发育过程中突触前膜出现各种形态的T-bar结构 15

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