Collaborative Research: Structural and Functional Connectivity of Squid Chromatophores

鱿鱼及其近亲（其他头足类动物，如章鱼）有能力通过色素体改变皮肤颜色，色素体是神经系统控制下的微观肌肉器官。所有关于鱿鱼色素体控制的细胞机制的研究都集中在三种相关的物种上，它们生活在相对较浅的沿海地区，这些地区有明显的特征，如海底的海藻、岩石和珊瑚。这些物种的皮肤颜色变化与伪装有关，是同一物种个体之间的信号，也是对其他物种的威胁。更深的开放海洋呈现出一个完全不同的环境，也栖息着许多鱿鱼，主要是不同的分类科，通常居住在沿海水域。一个重要的远洋家族包括洪堡乌贼（Dosidicus gigas）。白天，海洋深处几乎没有光线，这些鱿鱼居住在那里，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，以及可能在开放海洋中伪装的混乱“闪烁”。尽管这些动态行为与沿海物种典型的静态模式形成鲜明对比，但这两个科的鱿鱼都在不同程度上采用了时间和空间模式。因此，控制染色质网络的基本机制可能在大多数（如果不是全部的话）乌贼中是相同的。从大脑到色素体肌肉的“垂直”控制在沿海鱿鱼中是已知的，这可能解释了这些物种中大多数基于色素体的行为，但深水物种的行为，如闪烁，可能更多地受到皮肤内部过程的影响，这些过程允许色素体的变化在不直接涉及神经系统的情况下从一个传播到另一个。这种假设的途径将在外围定义一个“水平”或分布式控制系统，允许染色质网络内的自主行为。这个问题是这个项目的首要意义。理解染色质水平控制的基本原理有可能改变这个领域，因为目前的范式是所有的控制都是由大脑直接施加的。水平控制与脊椎动物循环系统、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地方控制的理解。从更广泛的角度来看，这个项目的结果将提供对分布式（水平）和自上而下（垂直）控制机制的相互作用的洞察，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。从工程学到经济学再到政治学，这一概念与社会广泛领域的根本利益相关。一种综合的方法将允许测试这样的假设，即鱿鱼中染色质网络的控制涉及不同于来自大脑的神经元运动控制途径的外周机制。独立于典型神经控制的自发染色质活性将通过实验操作在沿海低脂鱿鱼（Doryteuthis opalescens）中分离出来，包括慢性去神经支配和河豚毒素对神经元活性的药物阻断。此外，一种比较方法将利用一种海洋巨蝮物种Dosidicus gigas，其中自发的、抗河豚毒素的染色质活性非常突出。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。具体目的是：1)识别控制单个染色质器官的径向肌纤维兴奋性的相关离子通道和受体的分子和生理特性；2)定义定义皮肤内兴奋传递通路的相邻染色质肌纤维之间耦合机制的结构、分子和生理特征；3)阐明血清素在控制自发染色质活性中的抑制作用；4)在Dosidicus中进行平行实验，Dosidicus是生态学上重要的鱿鱼科成员，其色素体的细胞研究从未进行过。该项目将支持本科生和研究生的培训，并努力让STEM中代表性不足的群体的学生参与进来。

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.