Activity Dependent Control of Visual System Development

描述(由申请人提供)：GABA能抑制对电路的发育和功能是必不可少的。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能突触传递的发育受视觉经验的调节。这些研究表明，感觉输入活动调节GABA能神经元及其突触连接的发育，而抑制性GABA能突触传递又调节视觉回路的发育和可塑性。尽管抑制在电路发育中起着关键作用，但由于无法显示和干扰完整发育电路中GABA能抑制神经元的连接，因此无法对这一假说进行关键评估。我们已经开发了工具来解决这两个限制，现在提出了实验来确定控制完整非洲爪哇蝌蚪顶盖中GABA能神经元及其突触连接的发育的机制，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。为了在完整的动物中显示GABA能神经元，我们将使用VGAT启动子来驱动荧光蛋白(FP)和其他感兴趣的基因在GABA能神经元中的表达。我们将通过VGAT驱动的肽的表达来减少对GABA能神经元的突触传递，我们已经证明这些肽以细胞自主的方式特异性地抑制GABA能或谷氨酸能突触的传递。在目标1中，我们将收集单个GABA能顶盖神经元表达FP和FP标记突触蛋白的时间推移图像，以确定控制GABA能神经元发育的机制。我们将通过表达多肽来阻断突触传递到成像细胞，来测试谷氨酸和GABA能突触传递在GABA能神经元发育中的作用。在目标2中，我们将结合体内时间推移成像，以确定稳定和动态的树突，并回顾连续切片电子显微镜(EM)，以确定发生在GABA能神经元发育过程中的突触重排。AIMS 1和AIMS 2的数据将展示体内控制GABA能神经元形态发育和突触连接的活动依赖机制。在目标3和4中，我们将使用细胞附着和整体记录以及视觉回避分析来测试阻断抑制性和兴奋性传递对顶盖视觉感受野特性的影响，这将把我们的研究扩展到行为领域。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育的基本机制相对未知。这里提出的实验应该能阐明活体内GABA能神经元发育的活性依赖机制。 公共卫生相关性：该项目将确定在完整发育的大脑中控制GABA能抑制神经元及其突触连接的发育机制。我们将确定抑制神经元在感知视觉信息和视觉引导行为中的作用。这些研究将帮助我们理解大脑回路形成中的发育错误。

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.