Circuits for spontaneous behavior and phototaxis in a simple model chordate

这项提议将在一种新兴的神经回路模型中研究驱动负趋光性的神经回路。 分析：原始脊索动物Ciona的幼虫。乔纳幼虫有许多特征使它们 非常适合这个项目。它们很小，而且是透明的，只有177个CNS神经元。此外， 已从Ciona连接体中鉴定出可能的趋光性回路。乔纳的负趋光性 幼虫由两个阶段组成。首先，幼虫进行短暂的定向游泳，试图辨别 通过移动他们的身体来改变环境照明的方向。第二，如果幼虫探测到光线的变化 当它们的光感受器离开光源时，持续的负趋光性游泳结果。 然而，许多定向游泳在没有持续游泳的情况下就结束了。这项提案将审查以下两个方面 趋光性：1)神经回路如何调节定向游泳的频率；以及2)如何定向和 持续的游泳是在赛道层面上联系在一起的。斑潜蝇幼虫在时间间隔内分布较广 在短距离定向游泳之间。然而，对游泳间隔时间的大型数据集的分析指向潜在的 支配自发游泳频率的振荡，主导周期为每两秒一次 (O.5赫兹)。在初步研究中，我们发现了一种VGAT阳性神经元，其振荡频率为 在乔纳中枢神经系统一个称为前脑泡的区域，频率为0.5赫兹。我们将这种振荡的神经元命名为 前脑小泡振荡器(ABVO)。此外，突变和药理学研究都指向aBVO 可能是自发游泳频率的调节器。在特定目标1中拟议的实验将针对 使用光遗传学工具和激光消融BVO神经元，然后评估行为结果。特定的 目标2将解决第二个问题：短时自发定向和短时定向之间的联系是什么 持续趋光性游泳？我们记录Vacht阳性神经元GCaMP活性的初步研究 建议一种合理且可测试的电路模型。我们观察到幼虫的短自发尾巴运动 在同一个Vacht阳性中间神经元中伴随着钙瞬变，而Vacht阳性中间神经元是 光感受器-一类被称为光感受器中继神经元(PrRns)的神经元。此外，连接体 使我们能够识别与aBVO对应的可能的候选神经元，基于它们的3D位置和 这些候选神经元的主要突触靶点也是prRN。因此，我们 假设短定向游泳与持续趋光性游泳是在相同的回路中开始的，并且 光感受器的输入的存在与否决定了是否开始持续的游泳。 特定目标2中的实验将通过靶向prRN来验证这一假设，以确定它们是否 对于开始定向游泳来说是必要的。我们还假设，在没有BVO抑制的情况下， PrRN会振荡。我们将通过检查一个缺乏ABV区域的突变株来检验这一假设 中枢神经系统。

This proposal will investigate neural circuits driving negative phototaxis in an emerging model for neural circuit analysis: larvae of the primitive chordate Ciona. Ciona larvae have a number of features that make them ideally suited for this project. They are small and transparent, and have only 177 CNS neurons. Moreover, putative circuits for phototaxis have been identified from the Ciona connectome. Negative phototaxis in Ciona larvae consists of two phases. First, the larvae perform short orienting swims in which they attempt to discern the direction of ambient lighting by moving their bodies. Second, if the larva detects a change in light falling on their photoreceptors as they turn away from the light source, a sustained negative phototactic swim results. However, many orienting swims terminate without a sustained swim. This proposal will examine two aspects of phototaxis: 1) how neural circuits regulate the frequency of orienting swims; and 2) how orienting and sustained swims are linked at the circuit level. Ciona larvae show a wide distribution in the time intervals between short orienting swims. However, analysis of a large dataset of swim interval times points to underlying oscillations governing spontaneous swims frequency, with the dominant period being once every two seconds (O.5 Hz). In preliminary studies we have identified a VGAT-positive neuron that oscillates with a frequency of 0.5 Hz in a region of Ciona CNS called the anterior brain vesicle. We have named the oscillating neuron the anterior brain vesicle oscillator (aBVO). Moreover, mutant and pharmacological studies both point to the aBVO as a likely regulator of spontaneous swim frequency. Proposed experiments in Specific Aim 1 will target the aBVO neuron using optogenetic tools and laser ablation, and then assess the behavioral outcomes. Specific Aim 2 will address the second question: what is the circuit link between short spontaneous orienting and sustained phototactic swims? Our preliminary studies recording GCaMP activity in VACHT-positive neurons suggest a plausible and testable circuit model. We observed that short spontaneous tail movements in larvae were accompanied by Ca2+ transients in the same VACHT-positive interneurons that are the primary targets of the photoreceptors - a neuron class called photoreceptor relay neurons (prRNs). Moreover, the connectome allowed us to identify likely candidate neurons corresponding to the aBVO, based on their 3D locations and connectivities, and the major synaptic targets of these candidate neurons are also the prRNs. Thus, we hypothesize that short orienting swims are initiated in the same circuit as the sustained phototactic swims, and it is the presence or absence of input from the photoreceptors that determines if a sustained swim is initiated. Experiments in Specific Aim 2 will test this hypothesis by targeting the prRNs to determine if they are necessary for the initiation of orienting swims. We also hypothesize that in the absence of aBVO inhibition the prRNs would oscillate. We will test this hypothesis by examining a mutant line that lacks the aBV region of the CNS.