Collaborative Research: Structural and Functional Connectivity of Squid Chromatophores

鱿鱼和它们的亲戚（其他头足类动物，如章鱼）有能力改变皮肤颜色的色素细胞，显微镜下的肌肉器官是在神经系统的控制下。所有关于鱿鱼色素细胞控制的细胞机制的工作都集中在三个相关的物种上，这些物种栖息在相对较浅的沿海地区，这些地区具有海底海藻，岩石和珊瑚等突出特征。这些物种的肤色变化与伪装有关，是同一物种个体之间的信号，也是对其他物种的威胁。更深的开阔海洋呈现出一个完全不同的环境，也有许多鱿鱼居住，主要是不同的分类家庭从一个通常居住在沿海沃茨。一个重要的公海家庭包括洪堡鱿鱼（Dosidicus gigas）。在这些乌贼居住的海洋深处，白天几乎没有光线，珊瑚和岩石等视觉特征也不存在。Dosidicus的新颜色变化行为包括重复的全身“闪烁”，用于该物种个体之间的信号传递，以及混乱的“闪烁”，这可能是开放海洋中伪装的基础。虽然这些动态行为与沿海物种典型的静态模式形成对比，但这两个家族的鱿鱼在不同程度上采用了时间和空间模式。因此，控制色素细胞网络的基本机制在大多数（如果不是全部）鱿鱼中可能是相同的。从大脑到色素细胞肌肉的“垂直”控制在沿海鱿鱼中是已知的，并且可以解释这些物种中大多数基于色素细胞的行为，但是像深水物种中的闪烁行为可能更多地受到皮肤本身的过程的影响，这些过程允许色素细胞的变化从一个传播到另一个，而不直接涉及神经系统。这种假设的途径将定义一个“水平”或分布式控制系统的外围，将允许自主行为内的色素细胞网络。这个问题是本项目的首要意义。了解色素细胞水平控制的基本原理有可能改变该领域，因为目前的范式是所有控制都直接由大脑施加。水平控制与脊椎动物的循环系统、肠道功能和神经系统微回路向局部组织输送血液有关。因此，该项目的结果也将更广泛地影响对局部控制的理解。从更广泛的角度来看，该项目的结果将提供深入了解分布式（水平）和自上而下（垂直）控制机制的相互作用，这是一个与复杂系统产生不可预测的紧急现象的一般能力相关的主题。这一概念是一个广泛的社会部门的根本利益，从工程到经济学到politics.A综合的方法将允许测试的假设，即鱿鱼的色素细胞网络控制涉及外周机制，是从神经元马达控制通路，从大脑下降不同。自发的色素细胞的活动，是独立的典型的神经控制将被隔离的实验操作在沿海loliginid鱿鱼（Doryteuthis opalescens），包括慢性去神经和河豚毒素的神经元活动的药理学阻断。此外，比较的方法将利用海洋ommastrephid物种，Dosidicus gigas，其中自发的，河豚毒素抗性色素活性是非常突出的。相关方法包括分子转录组学、细胞电生理学、免疫组织化学共聚焦显微镜和高分辨率电子显微镜。具体目标是：1）识别控制操作单个色素细胞器官的桡肌纤维中的兴奋性的相关离子通道和受体的分子和生理特性; 2）定义相邻色素细胞的肌纤维之间的耦合机制的结构、分子和生理特征，所述耦合机制定义皮肤内的兴奋性传递途径;（3）阐明5-羟色胺对色素细胞自发活动的抑制作用; 4）在Dosidicus中进行平行实验，生态学上重要的乌贼家族的一员，其色素细胞的研究从未被进行过。该项目将支持本科生和研究生的培训，并包括从STEM中代表性不足的群体中吸收学生的重大努力。

Squid and their relatives (other cephalopods such as octopuses) have the ability to change skin color with chromatophores, microscopic muscular organs that are under control of the nervous system. All work on the cellular mechanisms of chromatophore control in squid has focused on three related species that inhabit relatively shallow coastal areas that have prominent features like seaweed, rocks and coral on the ocean floor. Skin-color changes in these species are associated with camouflage, signaling between individuals of the same species and threat displays with other species. The deeper open ocean presents a radically different environment that is also inhabited by many squids, primarily of different taxonomic families from the one commonly inhabiting coastal waters. An important open-ocean family includes the Humboldt squid (Dosidicus gigas). There is little light in the ocean at depths inhabited by these squid during daytime, and visual features such as coral and rocks are non-existent. Novel color-change behaviors in Dosidicus include repetitive whole-body "flashing," used for signaling between individuals of this species, and chaotic "flickering" that may underlie camouflage in the open ocean. Although these dynamic behaviors contrast with the more static patterns typical of coastal species, squids of both families employ temporal and spatial patterning to varying degrees. It is therefore likely that basic mechanisms for controlling the chromatophore network are the same in most, if not all, squids. "Vertical" control from the brain to the chromatophore muscles is known in the coastal squids, and may account for most chromatophore-based behaviors in those species, but behaviors like flickering in deeper-water species may be more influenced by processes within the skin itself that permit changes in chromatophores to spread from one to another without directly involving the nervous system. This hypothetical pathway would define a "horizontal" or distributed control system in the periphery that would permit autonomous behavior within the chromatophore network. This issue is the primary significance of the project. Understanding the fundamentals of horizontal control of chromatophores has the potential of being transformative to the field, because the current paradigm is that all control is directly exerted by the brain. Horizontal control is relevant to blood delivery to local tissues by circulatory systems, gut function and nervous system micro-circuits in vertebrates. Therefore, results from this project would also influence understanding of local control more broadly. From a wider perspective, results of this project will provide insight into the interactions of distributed (horizontal) and top-down (vertical) control mechanisms, a subject relevant to the general ability of complex systems to generate non-predictable, emergent phenomena. This concept is of fundamental interest to a broad sector of society, ranging from engineering to economics to politics.An integrated approach will permit testing the hypothesis that control of the chromatophore network in squid involves peripheral mechanisms that are distinct from the neuronal motor-control pathway that descends from the brain. Spontaneous chromatophore activity that is independent of canonical neural control will be isolated by experimental manipulations in coastal loliginid squid (Doryteuthis opalescens), including chronic denervation and pharmacological block of neuronal activity with tetrodotoxin. In addition, a comparative approach will take advantage of an oceanic ommastrephid species, Dosidicus gigas, in which spontaneous, tetrodotoxin-resistant chromatophore activity is extremely prominent. Relevant methods involve molecular transcriptomics, cellular electrophysiology, immunohistochemistry with confocal microscopy and high-resolution electron microscopy. Specific aims are: 1) identify molecular and physiological properties of relevant ion channels and receptors that control excitability in the radial muscle fibers that operate individual chromatophore organs; 2) define structural, molecular and physiological features of coupling mechanisms between muscle fibers of neighboring chromatophores that define an excitatory transmission pathway within the skin; 3) elucidate the inhibitory role in controlling spontaneous chromatophore activity played by serotonin; 4) carry out parallel experiments in Dosidicus, a member of a family of ecologically important squid in which cellular studies of chromatophores have never been carried out. This project will support undergraduate and graduate student training, and includes significant efforts to involve students from groups underrepresented in STEM.