The role of genetic and activity dependent cues in the generation of 5HT3aR interneuron diversity

项目概要（项目1） ! 项目一将探索内在遗传程序和活动对人类基因组的互补影响。 5 HT 3aR中间神经元的发育。目的1将研究Prox 1的转录和表观遗传功能， 5HT3aR cINs。通过检测5 HT 3aR细胞中Prox 1的靶点，我们将提供对5 HT 3aR细胞中Prox 1基因的遗传学研究。 产生这些群体的机制，以及Prox 1指导的基础 突触发生、形态学和电路连接（Aim 2）。将研究活动如何改变基因表达 与5 HT 3aR cINs的关系以及L型钙通道在活动依赖性发育和可塑性中的作用 5 HT 3aR cIN。这一目标将分两个子部分进行：（子目标2a）在这里，我们将使用交叉遗传学， 靶向5种L1-3 5 HT 3aR中间神经元亚型的方法，通过细胞自主抑制（Kir2.1）或 增加（NachBac）它们的活性，并使用RNA-seq来确定它如何改变特定细胞内的基因表达。 亚群以及（子目标2b）其中我们将使用条件遗传方法来去除L-型Ca 2 + 5 HT 3aR cIN的功能（Cav1.2，Cav1.3）。这一目标将产生对细胞自主要求的见解 5 HT 3aR cIN群体中的L型通道。（目标3）。将考察建立的发展性 5 HT 3aR cIN的连接性。这一目标还将从两个子部分着手：（子目标3a）将审查 浅层皮质内中间神经元/锥体细胞群的发育。这将使用一个组合 单突触狂犬病追踪，遗传编码钙传感器（gCaMP/rCaMP）和光遗传学， 检查L1中特定5 HT 3aR cIN亚型的传入和传出连接。特别感兴趣的是， 将这种回路与成熟的听觉和躯体感觉皮层的回路进行比较 和（subaim 2b）扩展了我们对Prox 1和L-型Ca 2+通道的研究，它们影响了 在开发过程中5 HT 3aR cIN的连接性。具体而言，审查如何改变 内在遗传程序（Aim 1）或活动介导的成熟（Aim 2）表现在回路的变化中 5 Ht 3aR中间神经元的功能（如Rudy博士在项目2中所探索的）和胆碱能传入的能力 和L1中间神经元影响可塑性和注意力在一个去-不去任务，其中小鼠被检查为他们的 对突出线索和非突出线索的反应（Froemke在项目3中）。项目1将深入了解 L1-3电路的组装如何严重依赖于5 Ht 3aR中间神经元影响成人连接， 可塑性和功能。

Project Summary (Project 1) ! Project One will explore the complementary influences of intrinsic genetic programs and activity to the development of 5HT3aR interneurons. Aim 1 will examine the transcriptional and epigenetic function of Prox1 in 5HT3aR cINs. By examining the targets of Prox1 in 5HT3aR cells we will provide insight in the genetic mechanisms that generate these populations, as well as the basis by which Prox1 directs synaptogenesis,morphology and circuit connectivity (Aim 2). Will examine how activity alters gene expression with 5HT3aR cINs and the role of L-type Ca2+ channels in the activity-dependent development and plasticity of 5HT3aR cINs. This aim will proceed in two subparts: (subaim 2a) Where we will use intersectional genetic methods to target the five L1-3 5HT3aR interneuronal subtypes, by cell autonomously suppressing (Kir2.1) or increasing (NachBac) their activity and use RNA-seq to determine how it alters gene expression within specific subpopulations. And (subaim 2b) Where we will use conditional genetic methods to remove L-Type Ca2+ function (Cav1.2, Cav1.3) from 5HT3aR cINs. This aim will yield insights into the cell autonomous requirement for L-type channels in 5HT3aR cIN populations. , (Aim 3). Will examine the establishment the developmental connectivity of 5HT3aR cINs. This aim will also proceed with two subparts: (subaim 3a) which will examine the development of interneuron/pyramidal cells ensembles within the superficial cortex. This will use a combination of mono-synaptic rabies tracing, genetic encoded Calcium-sensors (gCaMP/rCaMP) and optogenetics to examine the afferent and efferent connectivity of specific 5HT3aR cIN subtypes in L1. Of particular interest will be to compare how this circuitry compares with that seen in the mature auditory and somatosensory cortices and (subaim 2b) which extend our examination of Prox1 and L-Type Ca2+ channels affect the formation of the connectivity of the 5HT3aR cINs during development . Specifically, examination as to how changes in the intrinsic genetic program (Aim1) or activity-mediated maturation (Aim 2) manifest in changes in the circuit function of 5Ht3aR interneurons (as explored by Dr. Rudy in Project 2) and the ability of cholinergic afferents and L1 interneurons to influence plasticity and attention in a Go-No go task where mice are examined for their responses to salient versus non-salient cues (Froemke in Project 3). Together project 1 will provide insight into how assembly of L1-3 circuits that are critically dependent upon 5Ht3aR interneurons impact adult connectivity, plasticity and function.!