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

在本研究中，作为了解孟加拉雀鸟学习歌曲的大脑机制的一步，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和直接测序的方法确定了Zenk基因的1549bp的序列。该基因与人、小鼠和斑马鱼的同源性分别为68.1%、67.1%和66.3%。将FITC标记的反义ODN包裹在阳离子脂质体中，然后注射入禽脑内，研究应用ODN的定位。结果表明，持续荧光定位于细胞核内至少1周，并且在距注射部位约100~200μm的范围内分布有FITC标记的细胞和ZENK免疫阴性细胞。Zenk免疫阳性细胞的分布太窄，不能判断是注射反义ODN还是手术损伤抑制了Zenk的表达。一些方法学试验，涉及长期将反义寡核苷酸注射到行为鸟的大脑中，并对其影响进行行为量化，未能获得定量数据来明确大脑内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