REGULATING THE FATE OF SEROTONERGIC NEURONS

调节血清素能神经元的命运

• 批准号: 6319169
• 负责人: BARRY G CONDRON
• 金额: $ 4.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6319169
• 关键词: Insecta; animal genetic material tag; antisense nucleic acid; developmental genetics; developmental neurobiology; fluorescence microscopy; gene induction /repression; homeobox genes; invertebrate embryology; neurogenesis; neurogenetics; neurons; nucleic acid sequence; oligonucleotides; protein structure function; serotonin; stem cells; transcription factor

DESCRIPTION: This application proposes experiments that approach the problem of neural fate determination. The model system is a pair of neurons (s1 and s2) in the grasshopper CNS which express the neurotransmitter serotonin. s1 and s2 are sibling neurons born from the first ganglion mother cell (GMC1) of neuroblast Nb7-3. At an early stage, the entire lineage of Nb7-3 expresses the homeobox gene engrailed (en); en-expression then fades and is turned up to a high level exclusively in the serotonergic neurons at a time when these neurons have started to differentiate, more specifically, when the growth cones of these neurons approach the midline. There is good preliminary evidence showing that en-expression is causally related to later serotonin expression. The PI wants to address when en is required to induce serotonin expression, and how the surge in en-expression in s1 and s2 is regulated. The hypothesis to be tested in regard to the second aim is that environmental cues associated with the midline act upon the growth cone, which in turn signals back to the nucleus to turn up en-expression. Specific Aim 1 is technical, optimizing the technique used to block en-expression. This technique is to inject en-antisense oligonucleotides into different cells of the Nb7-3 lineage. It will be tested whether reduction in en-expression and serotonin expression can be adequately quantified, using a fluorescence microscopy/CCD camera setup. In addition, the effectiveness of en-suppression using oligos of different length, different concentration and complementarity will be established. Specific Aim 2 is to inject en-oligos into cells of the Nb7-3 lineage at different stages (neuroblast itself, GMC1, s1/s2 at different stages) and assay for expression of serotonin and other biochemical differentiation markers, as well as morphological differentiation (i.e., midline-crossing and contralateral arborization of s1/s2 axons). The hypothesis is that only late en-expression in the s1/s2 neurons themselves is essential for expressing the proper phenotype. Specific Aim 3 tests the possibility that the cues resulting in upregulation of en are picked up by the growth cone at different points along its trajectory. Favored by the P.I. is the possibility that these cues are associated with the midline cells, which are known to relay a wealth of guidance cues to axons in other systems. It is proposed to surgically transsect s1/s2 axons which had been previously filled with a fluorescent dye at different stages of their pathway, and to monitor the resulting effect on en-expression.

描述：此应用程序提出了实验 神经命运的问题。 模型系统是一对神经元 （S1和S2）在表达神经递质的蚱hopper中枢神经系统中 5-羟色胺。 S1和S2是从第一个神经节出生的兄弟神经元 神经细胞NB7-3的母细胞（GMC1）。 在早期，整个阶段 NB7-3的谱系表达植入的同源基因（EN）；表达 然后逐渐消失，并仅在血清素能中升至高水平 这些神经元开始分化的神经元，更多 具体而言，当这些神经元的生长锥接近中线时。 有良好的初步证据表明，表达是有因果的 与后来的5-羟色胺表达有关。 PI想解决en时 需要诱导5-羟色胺表达，以及如何表达激增 在S1和S2中受到调节。 关于 第二个目的是与中线行为相关的环境提示 生长锥，进而信号回到细胞核以升高 表达。 特定目标1是技术，优化用于阻止的技术 表达。 该技术是注入启动的寡核苷酸 进入NB7-3谱系的不同细胞。 它将测试是否 可以充分地降低表达和5-羟色胺的表达 使用荧光显微镜/CCD摄像头设置进行了量化。 此外， 使用不同长度的寡聚物的抑制的有效性， 将建立不同的集中度和互补性。 具体目标2是将en-Oligos注入NB7-3谱系的细胞中 不同的阶段（神经细胞本身，GMC1，S1/S2在不同阶段）和 表达5-羟色胺和其他生化分化的测定 标记物以及形态学分化（即中线交叉 S1/S2轴突的对侧树博化）。 假设是只有 S1/S2神经元本身中的晚期表达对于 表达适当的表型。 特定的目标3测试了导致上调的提示的可能性 EN的生长锥沿其不同点拾取了 弹道。 受到P.I.的青睐这些提示是 与中线细胞相关联，众所周知会中线的财富 在其他系统中轴突的指导线索。 它是通过外科手术提出的 以前填充荧光的Transect S1/S2轴突 染料在其路径的不同阶段，并监视结果 对表达的影响。