Activity Dependent Control of Visual System Development

描述（由申请人提供）：gaba能抑制是电路发育和功能所必需的。在发育中的视觉系统中，gaba能抑制决定了眼优势可塑性关键期的开始和结束。此外，gaba能突触传递的发展受视觉经验的调控。这些研究表明，感觉输入活动调节gabaergy神经元及其突触连接的发育，反过来，抑制性gabaergy突触传递调节视觉回路的发育和可塑性。尽管这一假设在神经回路发育中起着关键的抑制作用，但无法在完整的发育回路中可视化和干扰gaba能抑制神经元的连接，阻碍了对这一假设的批判性评估。我们已经开发了工具来解决这两个限制，现在提出实验来确定控制gaba能神经元的发育机制及其在完整的爪蟾蝌蚪视觉顶盖中的突触连接，并确定gaba能输入如何控制视觉感受野和视觉引导行为的发展。为了在完整动物中可视化gabaergy神经元，我们将使用VGAT启动子来驱动荧光蛋白（FPs）和其他感兴趣的基因在gabaergy神经元中的表达。我们将通过vgat驱动的肽表达来减少突触传递到gaba能神经元上，我们已经证明这些肽以细胞自主的方式特异性地抑制gaba能或谷氨酸能突触传递。在Aim 1中，我们将收集表达FP和FP标记的突触蛋白的单个gabaergy顶端神经元的延时图像，以确定控制gabaergy神经元发育的机制。我们将通过表达多肽来阻断突触传递到成像细胞，来测试谷氨酸能和gaba能突触传递在gaba能神经元发育中的作用。在Aim 2中，我们将结合体内延时成像来识别稳定和动态树突，并结合回顾性连续切片电子显微镜（EM）来确定gaba能神经元发育过程中发生的突触重排。目的1和目的2的数据将展示gaba能神经元体内控制形态发育和突触连接的活动依赖机制。在目标3和4中，我们将测试阻断抑制性和兴奋性传递对顶叶视觉感受野特性的影响，使用细胞连接和完整记录，以及视觉回避试验，这将扩展我们的研究到行为领域。抑制回路的异常发育和功能被认为是多种发育性神经系统疾病的基础，包括自闭症、精神分裂症和抑郁症，然而控制抑制回路发展的基本机制相对未知。本文提出的实验应阐明gaba能回路在体内发育的活动依赖机制。

DESCRIPTION (provided by applicant): GABAergic inhibition is essential for circuit development and function. In the developing visual system, GABAergic inhibition determines the onset and close of the critical period for ocular dominance plasticity. Furthermore, the development of GABAergic synaptic transmission is regulated by visual experience. These studies suggest that sensory input activity regulates the development of GABAergic neurons and their synaptic connections, and that, in turn, inhibitory GABAergic synaptic transmission regulates the development and plasticity of visual circuits. Despite this postulated pivotal role for inhibition in circuit development, the inability to visualize and perturb connectivity of GABAergic inhibitory neurons in an intact developing circuit has prevented a critical evaluation of this hypothesis. We have developed tools to address both of these limitations and now propose experiments to determine mechanisms controlling the development of GABAergic neurons and their synaptic connections in the intact Xenopus tadpole optic tectum and to determine how GABAergic inputs control the development of visual receptive fields and visually guided behavior. To visualize GABAergic neurons in the intact animal, we will use the VGAT promoter to drive expression of fluorescent proteins (FPs) and other genes of interest specifically in GABAergic neurons. We will decrease synaptic transmission onto GABAergic neurons by VGAT-driven expression of peptides which we have shown specifically inhibit GABAergic or glutamatergic synaptic transmission in a cell autonomous manner. In Aim 1, we will collect time-lapse images of individual GABAergic tectal neurons expressing FP and FP-tagged synaptic proteins to identify mechanisms that control GABAergic neuronal development. We will test the role of glutamatergic and GABAergic synaptic transmission in GABAergic neuron development by expressing peptides to block synaptic transmission onto the imaged cell. In Aim 2, we will combine in vivo time-lapse imaging, to identify stable and dynamic dendrites, and retrospective serial section electron microscopy (EM) to determine the synaptic rearrangements that occur during GABAergic neuronal development. Data from Aims 1 and 2 will demonstrate the activity-dependent mechanisms that control the morphological development and synaptic connectivity of GABAergic neurons in vivo. In Aims 3 and 4 we will test the effect of blocking inhibitory and excitatory transmission on tectal visual receptive field properties, using cell-attached and whole recordings, and a visual avoidance assay, that will extend our studies into the behavioral arena. The aberrant development and function of inhibitory circuits is thought to underlie a variety of developmental neurological disorders, including autism, schizophrenia and depression, however the basic mechanisms governing the development of inhibitory circuits are relatively unknown. The experiments proposed here should illuminate activity-dependent mechanisms of GABAergic circuit development in vivo. PUBLIC HEALTH RELEVANCE: This project will determine the mechanisms governing the development of GABAergic inhibitory neurons and their synaptic connections in the intact developing brain. We will determine the role of inhibitory neurons in perceiving visual information and in visually guided behavior. These studies will help us understanding developmental errors in brain circuit formation.