Dendrite Growth and Synaptogenesis in Zebrafish CNS

描述(申请人提供)：体内双光子成像方法将被用来探索斑马鱼视网膜顶盖通路中树突生长和突触发生的机制。该项目将特别关注视觉刺激活动对树突生长和突触形成的影响。斑马鱼的顶盖非常适合双光子荧光成像，而视网膜顶盖投射的发育取决于活动。新的遗传编码荧光蛋白(FP)标记和自动双光子(2P)显微镜将被用来有效地研究长时间段高时间分辨率的树枝生长和突触发生的动态。这项研究涉及四个具体目标：(1)测定基线(暗，未刺激)条件下树突状细胞生长和突触发生的动力学参数；(2)测量特定视觉刺激对树突生长和突触发生动力学参数的影响；(3)检测选择性谷氨酸受体拮抗剂对基线和视觉刺激条件下树突状细胞生长和突触发生参数的影响；(4)测量和分析图形视觉刺激诱发的树突状细胞钙瞬变。这些研究将是第一批将树枝生长的动态成像与突触发生过程的可视化相结合的研究之一。基于这一新能力的实验将测试各种当前的假说，这些假说将树突生长和突触发生的动力学相互联系起来，并与功能神经回路的活动依赖的发展有关。 要了解正常或病理性的脑发育，显然有必要了解树突的生长和突触发生。现在看来，树突生长和突触发生过程也可能在成熟的大脑中持续存在，因此我们对斑马鱼发育中这些过程的研究可能会促进对成熟神经系统的可塑性和病理学的理解。此外，获得的发展洞察力可能指向意想不到的治疗机会，用于治疗视力障碍，以及修复神经创伤、神经退行性变和药物成瘾的效果。

DESCRIPTION (provided by applicant): In vivo two-photon imaging methods will be used to explore the mechanisms of dendrite growth and synaptogenesis in the zebrafish retinotectal pathway. The project will focus especially on effects of visually stimulated activity on dendrite growth and synaptogenesis. The zebrafish tectum is very well suited to two-photon fluorescence imaging, and development of the retinotectal projection is known to depend on activity. Novel genetically encoded fluorescent protein (FP) markers and automated two-photon (2P) microscopy will be used to efficiently study dynamics of dendritic arbor growth and synaptogenesis at high time resolution over long time periods. The research involves four specific aims: (1) determine kinetic parameters of dendrite growth and synaptogenesis under baseline (dark, unstimulated) conditions, (2) measure effects of defined visual stimuli on kinetic parameters of dendrite growth and synaptogenesis, (3) test selective glutamate receptor antagonists for effects on growth and synaptogenesis parameters under baseline and visual stimulation conditions, and (4) measure and analyze dendritic Ca transients evoked by patterned visual stimulation. These studies will be among the first to integrate dynamic imaging of dendritic arbor growth with visualization of synaptogenesis processes. Experiments based on this new capability will test various current hypotheses relating the dynamics of dendrite growth and synaptogenesis to each other and to the activity-dependent development of functional neural circuitry. To understand normal or pathological brain development, it is obviously necessary to understand dendrite growth and synaptogenesis. It now appears that dendrite growth and synaptogenesis processes may persist in mature brains, as well, so our studies of these processes in developing zebrafish may advance understanding of the plasticity and pathologies of mature nervous systems. In addition, the developmental insights obtained may point to unexpected therapeutic opportunities for treating disorders of vision, and repairing effects of neurological trauma, neurodegeneration, and drug addiction.