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通过能够“降低”或“关闭”来自清醒，自由行为的小鼠中选定细胞的神经传递的工具大大推进。尤其强大的工具将能够影响诱导和可逆的神经传递抑制，并且可以具有很大的细胞特异性-其中沉默细胞类型的选择是基于基因表达的组合密码，因此对特定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（并发或先行）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在离散的发育期间触发沉默，随后是恢复；例如，从这一点来看，揭示神经回路发育的某些时期可能成为可能，在这一时期，选定的神经元活动可能对后来的神经回路健康至关重要——没有这一时期，成熟的神经回路功能可能会受到损害或过早衰退。因此，遗传或环境侮辱的脆弱性发育窗口可以确定。在这里，我们建议开发和测试四种遗传工具——四种独立的小鼠品系——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上有所不同，因此在环路和行为的类型上最适合研究。我们将以实验室中已经建立的一套先决条件和令人兴奋的工具为基础：我们将从一组等位基因中提取组成元素，这些等位基因最近被证明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，现在与这些已证实的元素一起纳入其他序列，这些序列应该允许诱导和可逆的沉默。通过将这些元素设计成单个广泛适用的等位基因，我们在时间和费用方面简化了实验范式。这项工作的创新之处不在于单个遗传因素，而在于如何将这些因素结合在一起——通过许多研究人员使用这些工具来研究小鼠神经系统中几乎任何回路的功能和发育，由此产生的工具应该有可能极大地改变神经科学研究。当代神经科学的一个基本挑战是确定特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RFA-MH-08-060定义），我们建议开发工具来可视化和操纵在清醒的、行为正常的小鼠或发育中的胚胎中离散神经回路（分子定义）的发育和活动。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，我们的希望是，这些由一个实验室和R21资助机制产生的工具，将通过许多神经科学家的使用，来推动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.