几种类型鸟鸣啭学习行为差异与鸣唱语句产生和维持的生理学机制

Song learning is a complicated and learned behavior, birdsong relies on auditory feedback during song learning process. Studies on song learning and its neural basis have important significance to reveal mechanisms about human language learning and mammalian sex differences. Previous studies have shown auditory feedback is necessary to maintain the stability of birdsongs in zebra finches and Bengalese finches. Removal of auditory feedback by deafening can make the singing behavior diminished dramatically. This project plans to use the neural tracing techniques, electrophysiological methods, fluorescence double-labeling, in vitro tissue culture，gene knockdown and overexpression as well as many other methods to study the relationship between song learning behavior and related brain structure in different kinds of songbirds, compare birds song learning behavior with the neural basis of auditory vocal center development and its regulatory mechanism, also we will examine male and female differences in the regulation of brain development. Our work will be divided into following parts: 1) explore the relationship between song learning and vocal control nuclei in different kinds of songbirds; examine the difference in nuclei connections and whether the abundance of nuclei connections can contribute to the song complexity; 2) study the correlation between song complexity and newborn neurons by examining the generation, migration, differentiation and apoptosis of newborn neurons at different stages of song learning in songbirds; check whether newborn neurons added to the vocal center is required for maintaining a continuous learning statement at adult; 3) use gene microarray screening to identify differentially expressed genes between male and female, then we can study the functions of these male-specific genes during song learning process; 4) Examine the changes in brain ultrastructures(the class and number of the synapses, dentritic spines, synaptic vesicles and the length of postsynaptic density) and the expressions of related proteins in songbirds with surgery damage induced deafness to reveal the relationship between auditory feedback and the song learning process. This study will greatly expand our knowledge in the neural mechanisms of male and female behavior differences, provide some theoretical basis of birds song learning behavior and song control nuclei development as well as neural plasticity, and will also provide important implications for the mammalian brain differences between male and female.

鸣禽的鸣唱学习及其脑内结构的研究对于揭示人类语言学习及哺乳动物性别差异等方面都有重要的意义。本项目拟通过示踪神经技术、电生理、荧光双标、体外组织块培养以及基因抑制、过表达等多种方法研究不同鸣禽发声行为与脑内结构之间的关系，比较鸟类发声学习行为与听觉发声中枢的发育及其调控机制，以及脑发育调控的雌雄差异的神经机理。拟开展如下工作：1）比较不同种类鸣禽鸣唱学习与发声控制核团之间的关系；2）通过鸣唱学习不同阶段的鸣禽脑内新生神经元的发生，研究鸣唱复杂性与新生神经元发生的关系；3）通过基因差筛得到具有雌雄表达差异的基因，研究具体基因对鸣唱学习的影响；4）通过损毁致聋技术研究听觉损伤后的脑内超微结构变化及相关分子蛋白水平的变化差异。本研究为揭示脑发育调控的雌雄差异的神经机制，鸟类鸣啭学习行为与其发声控制中枢的发育及神经可塑性机理提供一定的理论依据，也将为理解哺乳类脑的雌雄差异现象提供重要的参考资料。

用电生理、荧光双标、体外组织块培养、基因抑制和过表达研究了：1）白腰文鸟、斑胸草雀和百灵鸟鸣唱学习复杂性与发声核团大小等成正相关；2）鸣唱学习中脑内新生神经元的发生与鸣唱的复杂性成正相关。体外培养脑片，雄鸟VZ 区细胞线密度高于雌鸟。加了雌激素后，雌鸟VZ 区细胞的线密度有显著提高；3）基因差筛得到雌雄表达差异的基因，Notch基因显著影响了VZ及HVC区的发育，雌激素对Notch1 mRNA 水平表达有显著的影响，转染Notch1干扰慢病毒后雄性斑胸草雀HVC核团内BrdU、NeuN双标数、HVC、RA体积增加，鸣曲音节重复数增多。基因沉默HVC的ERBIN和ERBB2表达后，改变了鸣唱语句和音节，HVC的体积变小，抑制了HVC背侧VZ区的细胞增殖和分化。4）双侧损毁NIf，非典型鸣曲缺失或缩短，降低了鸣曲的复杂性；Uva损毁后HVC中间神经元的放电率显著上升，Uva抑制了HVC中间神经元的活性。5）分别损毁幼鸟的发声中枢和致聋后，NM 核团的体积下降，RA突触数量增加，突触后致密带（PSD）的长度降低、轴-树突触的PSD厚度升、凸型突触降低、平型突触增加、穿孔突触减少； RA神经元的放电率降低，峰峰间隔增加, 致聋鸟比隔离鸟更为严重，均干扰了HVC对RA间投射的建立；6）耳蜗摘除导致听觉剥夺，脑内听觉及发声通路中抑制性神经递质r-氨基丁酸(GABA) 及其受体的表达，听觉通路内GABA和GABAA-R的阳性标记细胞增加, 发声运动通路中GABA和GABA-R的阳性标记细胞数均显著减少；致聋后HVC、RA内表达Calbindin D-28K、 Calretinin的标记细胞减少, X区内表达P物质的阳性纤维末梢减少，表明Calbindin D-28K及Calretinin两种钙结合蛋白、P物质参与了鸣唱与听觉的整合。本研究为揭示脑发育调控的雌雄差异的神经机制，鸟类鸣啭学习行为与其发声控制中枢的发育及神经可塑性机理提供重要的参考资料。