Developmental shifts in gene expression in the auditory forebrain during the sensitive period for song learning

A male zebra finch begins to learn to sing by memorizing a tutor’s song during a sensitive period in juvenile development. Tutor song memorization requires molecular signaling within the auditory forebrain. Using microarray and in situ hybridizations, we tested whether the auditory forebrain at an age just before tutoring expresses a different set of genes compared with later life after song learning has ceased. Microarray analysis revealed differences in expression of thousands of genes in the male auditory forebrain at posthatch day 20 (P20) compared with adulthood. Furthermore, song playbacks had essentially no impact on gene expression in P20 auditory forebrain, but altered expression of hundreds of genes in adults. Most genes that were song-responsive in adults were expressed at constitutively high levels at P20. Using in situ hybridization with a representative sample of 44 probes, we confirmed these effects and found that birds at P20 and P45 were similar in their gene expression patterns. Additionally, eight of the probes showed male–female differences in expression. We conclude that the developing auditory forebrain is in a very different molecular state from the adult, despite its relatively mature gross morphology and electrophysiological responsiveness to song stimuli. Developmental gene expression changes may contribute to fine-tuning of cellular and molecular properties necessary for song learning. Post-hatch day 20 male zebra finches that had been raised in acoustic isolation with a foster female or adult male zebra finches were placed in a song playback chamber. The next day, birds heard either silence (control) or 30 minutes of novel song. All samples were hybridized against the universal SoNG RNA reference pool, 6 biological replicates per group in each of 4 groups.

雄性斑胸草雀在幼鸟发育的敏感期内，通过记忆导师的鸣曲启动歌唱学习行为。对导师鸣曲的记忆需要听觉前脑（auditory forebrain）内的分子信号传导。本研究借助基因芯片（microarray）与原位杂交（in situ hybridization）技术，探究了刚进入鸣曲学习前的幼鸟，其听觉前脑的基因表达谱与鸣曲学习完成后的成年个体是否存在差异。基因芯片分析结果显示，孵化后第20日龄（posthatch day 20，P20）的雄性斑胸草雀听觉前脑，与成年个体相比，数千个基因的表达水平存在显著差异。进一步研究发现，鸣曲回放对P20个体听觉前脑的基因表达几乎无影响，但可改变成年个体数百个基因的表达水平。绝大多数在成年个体中对鸣曲产生响应的基因，在P20个体中呈组成型高表达状态。本研究采用44个探针的代表性样本进行原位杂交验证，证实了上述结果，同时发现P20与孵化后第45日龄（posthatch day 45，P45）的幼鸟，其基因表达模式较为相似。此外，8个探针对应的基因存在雌雄表达差异。我们得出结论：尽管发育中的听觉前脑在大体形态与对鸣曲刺激的电生理响应上已相对成熟，但其分子状态与成年个体截然不同。发育过程中的基因表达变化，可能参与微调鸣曲学习所需的细胞与分子特性。

本研究将饲养于听觉隔离环境中、仅与寄养雌鸟或成年雄性斑胸草雀一同成长的P20日龄雄性斑胸草雀，放置于鸣曲回放舱内。次日，各组个体分别聆听静音（对照组）或30分钟的新奇鸣曲。所有样本均与通用SoNG RNA参考池进行杂交，4个实验组每组设置6个生物学重复。