Novel tools to study circuit function, development, and periods of vulnerability

描述（由申请人提供）：神经回路功能和发育的研究将大大推进的工具，能够“拨号”或“关闭”从选择细胞在清醒的，自由行为的小鼠神经传递。特别强大的将是能够影响神经传递的诱导性和可逆性抑制的工具，并且可以以很大的细胞特异性来实现这一点-其中待沉默的细胞类型的选择基于基因表达的组合密码，因此对于特定的神经元亚型具有高度选择性。这样的工具将允许在高度选择的神经回路、基因表达（同时或先行）和动物行为之间定义因果关系。此外，诱导性和可逆性的特征将允许沉默在离散的发育期期间被触发，然后恢复;由此，例如，将有可能揭示回路发育期，在该发育期期间，选择神经元活动可能对以后的回路健康至关重要-没有该发育期，成熟的回路功能可能会受损或过早衰退。因此，可以确定遗传或环境损害的脆弱性的发育窗口。在这里，我们建议开发和测试四种遗传工具-四个独立的小鼠品系-每一种都旨在提供这些能力，但每一种都可能在神经元失活和恢复的动力学和效率方面有所不同，因此在最适合研究的回路和行为类型方面也有所不同。我们将建立在实验室已经建立的一套先决条件和令人兴奋的工具之上：我们将从一组等位基因中提取组成元件，我们最近已经证明这些等位基因能够以高度选择性的方式可视化和沉默广泛的神经元亚型，现在将沿着这些已被证明的元件以及其他应该允许诱导和可逆沉默的序列。通过将这些元素工程化为单个广泛适用的等位基因，我们简化了时间和费用方面的实验范式。这项工作的创新之处不在于单个的遗传元素，而在于这些元素如何一起使用-所产生的工具应该有可能通过许多研究人员使用它们来研究小鼠神经系统中几乎任何回路的功能和发育，从而大大改变神经科学研究。公共卫生相关性当代神经科学的一个基本挑战是确定给定脑回路活动与特定动物行为之间的因果关系，包括将这种关系联系在一起的发育和分子事件。为了应对这一挑战（由本RFA-MH-08-060定义），我们建议开发用于可视化和操纵清醒行为小鼠或在子宫内未受干扰的发育胚胎中离散神经回路（分子定义）的发育和活动的工具。如果成功，这些试剂将有可能从根本上推进基础和转化神经科学研究的许多领域;事实上，我们希望这些由单一实验室和R21资助机制产生的工具将被许多神经科学家利用，以推进对小鼠神经系统中几乎任何神经回路的研究。

DESCRIPTION (provided by applicant): Studies of neural circuit function and development would be advanced considerably by tools capable of "dialing down" or "turning-off" neurotransmission from select cells in the awake, freely behaving mouse. Especially powerful would be tools capable of effecting inducible and reversible suppression of neurotransmission, and which could do so with great cellular specificity - where the choice of cell type to be silenced is based on combinatorial codes of gene expression and thus is highly selective for a particular neuron subtype. Such a tool would allow causal relationships to be defined between a highly select neural circuit, gene expression (concurrent or antecedent) and animal behavior. Further, the features of inducibility and reversibility would allow silencing to be triggered during discrete developmental periods followed by recovery; from this it would become possible, for example, to reveal periods of circuit development during which select neuron activity may be critical for later circuit health - without which mature circuit function may be compromised or decline prematurely. Thus developmental windows of vulnerability for genetic or environmental insult could be identified. Here we propose developing and testing four genetic tools - four independent mouse strains - each designed to offer these capabilities, but each likely to differ in the kinetics and efficiency of neuron inactivation and recovery and thus in the types of circuits and behaviors most suitable for study. We will build upon a prerequisite and exciting set of tools already established in the lab: we will take component elements from a set of alleles, ones that we have recently shown able to visualize and silence a wide range of neuron subtypes in highly selective fashion, and now incorporate along with these proven elements other sequences that should allow for inducible and reversible silencing. By engineering these elements into single broadly applicable alleles, we simplify the experimental paradigm with respect to both time and expense. The innovation of the proposed work lies not in an individual genetic element but rather in how these elements will be used together - the resultant tools should have the potential to dramatically change neuroscience research through their use by many investigators to study the function and development of virtually any circuit in the mouse nervous system. PUBLIC HEALTH RELEVANCE A fundamental challenge of contemporary neuroscience is to define causal relationships between the activity of a given brain circuit and a particular animal behavior, including the developmental and molecular events that tie this relationship together. Towards meeting this challenge (one defined by this RFA-MH-08-060), we propose to develop tools for visualizing and manipulating the development and activity of discrete neural circuits (molecularly defined) in the awake, behaving mouse or in developing embryos otherwise undisturbed in utero. If successful, these reagents will have the potential to radically advance numerous areas of basic and translational neuroscience research; indeed, our hope is that these tools, generated by a single lab and R21 funding mechanism, will be leveraged, through their use by many neuroscientists, to advance studies of virtually any neural circuit in the mouse nervous system.