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

在本研究中，作为了解孟加拉雀鸣唱学习的脑机制的一个步骤，我试图用反义DNA方法阐明大脑内zenk的功能。为了设计寡脱氧核苷酸（ODN），我用5 '/3' RACE和直接测序法确定了zenk cDNA中1549 bp的序列。孟加拉雀的zenk基因与人、小鼠和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，将FITC标记的反义ODN包埋在HVJ-阳离子型脂质体复合物中，然后注射到鸟脑内。结果显示，荧光持续定位于细胞核中至少1周，并且FITC标记的细胞以及ZENK免疫阴性细胞分布在距注射部位约100-200μm的距离内。ZENK免疫阴性细胞的分布太窄，无法判断是否注射反义ODN或手术损伤抑制了ZENK表达。几个方法学试验，关于慢性注射反义核酸，ODN到行为鸟的脑内和行为量化的影响，未能获得定量数据来明确脑内zenk表达和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