Developmental gene networks of 5HT neurons in addiction, aggression, and anxiety

描述（由申请人提供）：本研究将尖端的遗传工具用于理解攻击和动机行为背后的神经回路。我们最近的进展表明，小鼠的这些行为是由两个分子上不同的脑5-羟色胺能（5-HT）神经元的小亚群调节的：一个亚群，约3000个神经元，通过D1多巴胺受体（Drd1a基因）的表达在5-HT神经元中独特地定义，另一个亚群，约1000个神经元，通过D2多巴胺受体（Drd2基因）的表达。这些结果与基因工具相结合，就像一把锋利的楔子，现在可以用来“打开”并进入这些结果行为背后的电路、细胞特性和分子途径。在这里，我们建议以四个目标的形式应用这个“楔子”来回答：这些行为关键的5-HT神经元独有的细胞和分子特性是什么？正如受体表达所暗示的那样，这些5-HT神经元是否对DA（一种通常与大脑奖励系统相关的神经化学物质）有反应？这些亚型通过什么前脑回路调节攻击性和动机？这些参数是否会随着人的一生而改变，也许与人类年龄相关的冲动、攻击性和药物滥用倾向有关？在Aim 1中，我们将通过转录谱分析（RNA-seq）和电生理记录来鉴定Drd1a和Drd2 5-HT神经元亚型的功能特性。这项工作是通过神经元亚型特异性标记的新型遗传工具实现的，适用于神经元亚型分类和分子分析，以及全细胞记录。这些相同的遗传工具不仅可以进入5-HT神经元亚型的体细胞，还可以进入轴突和终末，从而可以精确识别神经元亚型控制下的目标大脑区域-这是Aim 2的目标。功能性突触后连接也将被探索，我们的交叉遗传标记工具赋予前所未有的分辨率，经典的束追踪技术以及涉及跨突触示踪剂的尖端病毒方法。因此，Aim 2将定义、标记并允许这些回路下游“攻击相关”突触后神经元的分子表征。在Aim 3中，我们将更深入地探讨这两种5-HT神经元亚型所调节的行为方面，以及它们在不同生命周期中的贡献是否不同。类似的亚型特异性沉默方法将采用基础攻击研究，但现在将询问额外的社会行为和神经功能。在aims 4中，我们将使用药物遗传学（DREADDs）在“童年”期间暂时沉默每个Drd 5-HT神经元亚型，询问是否发生了持久的变化，使成年后易患过度攻击和改变社会动机，正如人类研究预测的那样，通过5-HT系统，童年压力和成年病理性攻击将遗传易感性联系起来。我们的方法在技术上和概念上都是创新的，是发现新的、潜在的行为选择性的、适合年龄的治疗方法的基础。结果迫使重新定义5-HT系统组织。

DESCRIPTION (provided by applicant): The proposed research brings cutting-edge genetic tools to bear on understanding the neural circuitry under- lying aggression and motivated behavior. Our recent progress reveals that these behaviors in mice are regulated by two molecularly distinct, small subsets of brain serotonergic (5-HT) neurons: one subset, ~3,000 neurons, uniquely defined among 5-HT neurons by expression of the D1 dopamine (DA) receptor (Drd1a gene), and the other, ~1,000 neurons, by the D2 DA receptor (Drd2 gene). These results combined with the enabling genetic tools, like a powerfully sharpened wedge, can now be used to "break open" and access the circuitry, cellular properties, and molecular pathways underlying these consequential behaviors. Here we propose applying this "wedge" in the form of four aims to answer: What cellular and molecular properties are unique to these behavior-critical 5-HT neurons? As suggested by receptor expression, are these 5-HT neurons responsive to DA - a neurochemical commonly associated with the reward system of the brain? Through what forebrain circuitry do these subtypes modulate aggression and motivation? Do these parameters change across the life span, perhaps bearing on human age-related propensity for impulsivity, aggression, and substance abuse? In Aim 1, we will identify functional properties of the Drd1a and Drd2 5-HT neuron subtypes by transcriptional profiling (RNA-seq) and electrophysiological recording. This work is enabled through novel genetic tools for neuron subtype-specific marking, suitable for neuron subtype sorting and molecular profiling, and for whole-cell recording. These same genetic tools not only offer access to the soma of a 5-HT neuron subtype, but also to axons and terminals, thus allowing precise identification of target brain regions under neuron-subtype control - the goal of Aim 2. Functional postsynaptic connections will also be explored, with our intersectional genetic marking tools conferring unprecedented resolution to classic tract- tracing techniques as well as to cutting-edge viral approaches that involve trans-synaptic tracers. Thus, Aim 2 will define, label, and allow for molecular characterization of "aggression-relevant" postsynaptic neurons downstream in these circuits. In Aim 3, we will explore more deeply the behavioral facets modulated by these two 5-HT neuron subtypes and if their contributions vary across life span. Similar subtype-specific silencing methods will be employed as in the foundational aggression studies, but now additional social behaviors and neurological functions will be queried. In Aim 4, we will use pharmacogenetics (DREADDs) to transiently silence each Drd 5-HT neuron subtype during "childhood," asking if lasting changes occur that predispose to hyperaggression and altered social motivation in adulthood, as predicted by human studies that associate genetic predisposition via the 5-HT system, childhood stress, and adult pathological aggression. Our approaches are technically and conceptually innovative, and are foundational for discovering new, potentially behavior-selective, age-suitable therapeutics. Results compel a redefinition of 5-HT system organization.