Dendrite Growth and Synaptogenesis in Zebrafish CNS

描述（由申请人提供）：体内双光子成像方法将用于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发生的影响。斑马鱼顶盖非常适合于双光子荧光成像，并且已知视网膜顶盖投射的发展取决于活动。新的基因编码的荧光蛋白（FP）标记物和自动双光子（2P）显微镜将被用来有效地研究动态的树突状乔木生长和突触在很长一段时间内在高时间分辨率。该研究涉及四个具体目标：（1）确定基线下树突生长和突触发生的动力学参数（黑暗，未刺激）条件下，（2）测量确定的视觉刺激对树突生长和突触发生的动力学参数的影响，（3）测试选择性谷氨酸受体拮抗剂在基线和视觉刺激条件下对生长和突触发生参数的影响，以及（4）测量和分析由图案化视觉刺激诱发的树突状细胞Ca瞬变。这些研究将是第一个将树突状乔木生长的动态成像与突触发生过程的可视化相结合的研究。基于这一新能力的实验将测试目前各种假设有关的树突生长和突触相互之间的动态和功能性神经回路的活动依赖性发展。 要了解正常或病理的大脑发育，显然有必要了解树突生长和突触发生。现在看来，树突生长和突触发生过程也可能持续存在于成熟的大脑中，因此我们对发育中的斑马鱼的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，所获得的发展见解可能指向意想不到的治疗机会，用于治疗视力障碍，修复神经创伤，神经变性和药物成瘾的影响。

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.