鼠视觉核团结构模式以及动物皮质差异机制的研究

Auditory and somatosensory nuclei could be divided into a core region and a diffuse shell region, according to their embryonic genesis, constitution organization or physiological functions. The core-to-shell organization is only evident in the amphibian mesencephalic auditory areas, but not present in amphibian diencephalic auditory areas or the mesencephalic and diencephalic auditory areas of fishes. Nevertheless, the areas that lack clear core-to-shell organization in amphibians and fishes exhibit features that are similar to those of the shell areas of amniotes. The cladistic analyses suggest that the shell region might be a more phylogenetically ancient configuration than the core region across the evolutionary course of vertebrates. The present study is designed to extend the study performed in the auditory and somatosensory nuclei to the mammalian visual nuclei. Combined the neural tract tracing with the immunohistochemistry for parvalbumin (PV) or calbindin (CB) which are generally distributed in the core or shell regions of auditory or somatosensory nuclei, respectively, we will determine the neural connections of PV or CB cells and their distributions in lateral posterior (Lp) in the tectofugal pathway in the mouse. Then, by using the double labeling for BrdU (to label the proliferating cells) and PV or CB, we will determine the embryonic genesis of PV and CB cells in the visual nuclei in the two visual pathways, and finally know whether the core-to-shell organization is present in the mammalian visual nuclei. . After a genomic comparative analysis of the genes critical for the development of the pallium (Pax6, Tbr1, and Emx1/2) and the subpallium (Dlx2 for the neostriatum and Nkx1/2 for the pallidum), it has been shown that there is an additional fragment of about 21 amino acids in Emx1 protein or poly-(Ala)6-8 in Emx2 protein in nearly all the examined mammals (Eutheria), but not in almost non-mammals. To explore whether such alterations in Emx1/2 result in the differences in the pallium between the mammals and nonmammals, following the injections of lentiviruses containing the mouse Emx1 and Emx2 into the lateral ventricle in E3 (embryonic day 3) chick embryos, we will examine the changes in the expression of some genes such as Reelin, Vimentin, and etc. which have been shown to be involved in the brain development, the neural connectivity between the two cerebral hemispheres, and the genesis pattern of embryonic cells on E12 and E16, or at post-hatch. We will also examine the changes in the ability of the memory of learning, and the moving or exploring ability in newborn chicks (within 5 days post-hatch) by performing one-trial passive avoidance learning task and the open field test. Our study will be helpful to further understand the origin and evolution of nuclei in the brain, and the mechanism underlying the large variations in the cerebral cortex between the mammals and non-mammals.

高等动物听觉和体感觉核团多由中央核心区和周围壳区组成，两者在胚胎发生以及结构和功能上的差异一定程度反映了核团的进化。为弄清核壳模式是否也存在于视觉核团，本项目通过神经示踪结合Parvalbumin（PV，主要分布核心区）或Calbindin（CB，主要分布壳区）免疫双标，确定PV和CB在离视盖通路间脑后外侧核的分布和神经联系，再通过BrdU和PV或CB免疫双标，确定两条视觉通路核团内的神经发生尤其是PV或CB细胞是否符合核壳模式。此外，为弄清哺乳类皮质发育控制基因Emx1/2是否由较非哺乳类多获取一段序列而导致皮质发育不同，将含鼠Emx1、2基因的慢病毒同时注入鸡胚（E3）端脑，检测鸡脑皮质发育相关的分子变化，如Reelin、Vimentin等以及细胞发育梯度的改变，并对出生后5日龄内小鸡，测定有关行为学变化。本项目可进一步揭示脑核团的起源和进化以及哺乳和非哺乳动物皮质发育差异的机制。

在进化的过程中，哺乳动物新皮质的出现使其更加适应复杂多变的环境，哺乳动物神经元“由内向外”迁移形成新皮质的特有的六层结构是进化过程中至关重要的一次创新，对于了解人类智商、情绪、认知和神经系统类疾病奠定了基础，因此，对于新皮质进化机制的研究就显得尤为重要。本论文首先对哺乳动物和非哺乳动物的皮质发育相关基因（Pax6、Tbr1、Emx1/2）及端脑皮质下区域（纹状体和苍白球）发育相关基因（Dlx2、Nkx2.1）进行比较分析，弄清是否由于这些基因的变异引起了哺乳动物新皮质的出现。本论文在基因组序列已公布的所有物种间发现以上基因均未发生重复、丢失，以及达尔文选择（Ka/Ks<1）等变化，但Emx1和Emx2基因编码的蛋白质序列发生了改变：哺乳动物的Emx1蛋白质比非哺乳动物多了一段包含21个氨基酸的序列；除了单孔类和有袋类Emx2蛋白质有2个多聚丙氨酸序列外，其它哺乳动物的Emx2蛋白质均有一段6-8个多聚丙氨酸的序列，而非哺乳动物基本没有。通过生物信息学分析发现：哺乳动物Emx2蛋白质的6-8个多聚丙氨酸能形成一个螺旋，这可能导致哺乳动物Emx2基因活性改变。为检验此推测，我们在体外培养鸡胚端脑神经元中转入含鸡Emx1/2基因、鼠Emx1/2基因以及变色蜥Emx2基因表达质粒，结果发现仅鼠Emx2基因能显著提高下游基因reelin的表达水平。随后，我们将鼠Emx1、2基因或其干扰RNA包装到慢病毒，注入E3（胚胎期第三天）鸡胚端脑中，使其在全脑均匀表达。转入慢病毒后的胚胎继续孵化。我们检测了胚胎发育期间以及孵化后第九天端脑内与脑发育相关的重要分子变化（reelin、GABA、Vimentin等），发现以上实验均可明显影响到脑组织结构变化。 我们还通过[3H]-thymidine放射自显影方法与Calbindin和Parvalbumin免疫组织化学染色方法相结合的双重标记法来确定小鼠胚胎期视觉脑区不同区域的神经发生起始时间和高峰时间显示，视觉核团的不同区域在神经发生时间上存在显著差异，揭示哺乳动物视觉脑区存在核心区-壳区模式结构。