Neuroethological analysis of song learning mechanism by using antisense DNA method in birds

在本研究中，我试图利用反义DNA的方法来阐明脑内zenk的功能，作为了解孟加拉雀鸣叫学习的大脑机制的一步。为了设计oligodeoxynucleotide (ODN)，我利用5'/3'RACE和直接测序技术确定了zenk cDNA中1549bp的序列。与人、鼠、斑马鱼的同源性分别为68.1%、67.1%和66.3%。为了研究应用ODN的定位，我们将fitc标记的反义ODN包裹在阳离子型hvj脂质体复合体中，然后注射到鸟的大脑中。结果显示，荧光在细胞核内持续定位至少1周，fitc标记细胞和zenk免疫阴性细胞分布在距注射部位约100-200μm的范围内。zenk免疫阴性细胞分布过窄，无法判断是否通过注射反义ODN或手术损伤抑制了zenk的表达。多次在行为鸟脑内长期注射反义ODN，并对其效果进行行为量化的方法学试验，均未能获得定量数据来明确脑内zenk表达与特定行为之间的因果关系

In the present study, as a step to understand the brain mechanisms of song learning in Bengalese finch, I attempted to elucidate the functions of intracerebral zenk using the antisense DNA method. For designing oligodeoxynucleotide (ODN), I decided 1549bp of sequence in zenk cDNA using 5'/3'RACE and direct sequencing. The sequence of Bengalese finch's zenk showed 68.1 %, 67.1 % and 66.3% homology with those of human, mouse and zebra fish, respectively. To investigate the localization of applied ODN, FITC-labeled antisense ODN was enwrapped in HVJ-liposome complex of cation type, and then injected into the brain of bird. The result exhibited that sustained fluorescence localized in the nucleus for at least 1 week, and that FITC-labeled cells as well as ZENK-immunonegative cells was distributed within a distance of about 100-200μm from the injection site. The distribution of ZENK-immunonegative cells was too narrow to judge wether the cells were suppressed zenk expression by injected antisense ODN or by surgical damage. Several methodological trials, concerning the chronic injection of antisense, ODN into the brain of behaving bird and the behavioral quantification of the effects, failed to obtain the quantitative data to clear the causal relationship between intracerebral zenk expression and specific behavior