Collaborative Research: Structural and Functional Connectivity of Squid Chromatophores

合作研究：鱿鱼色素细胞的结构和功能连接

• 批准号: 1557754
• 负责人: William Gilly
• 金额: $ 61.46万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1557754&HistoricalAwards=false
• 关键词: Collaborative; Research; Structural; Functional; Connectivity

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.

鱿鱼及其亲戚（其他头足动物，例如章鱼）具有通过染色体，微观肌肉器官控制的能力，这些器官正在控制神经系统。鱿鱼中色谱控制的细胞机制的所有工作都集中在三种相关物种上，这些物种居住在相对较浅的沿海地区，这些物种具有突出特征，例如海藻，岩石和珊瑚在海底。这些物种的皮肤变化与伪装有关，同一物种的个体之间的信号以及与其他物种的威胁显示。较深的开放海洋呈现出一个根本不同的环境，许多鱿鱼也居住，主要是通常居住在沿海水域的不同分类家庭。一个重要的开放海洋家庭包括洪堡鱿鱼（Dosidicus gigas）。在白天，这些鱿鱼居住的深处，海洋几乎没有光，珊瑚和岩石等视觉特征不存在。剂型中的新型色彩变化行为包括重复的全身“闪烁”，用于该物种的个体之间的信号，以及混乱的“闪烁”，这可能是在开阔的海洋中伪装的基础。尽管这些动态行为与沿海物种典型的更静态模式形成鲜明对比，但两个家庭的鱿鱼在不同程度上采用时间和空间图案。因此，控制色谱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。从大脑到色谱肌肉的“垂直”控制在沿海鱿鱼中是已知的，可能是这些物种中的大多数基于色谱的行为，但是诸如在较深的水物种中闪烁的行为可能会受到皮肤内部过程的影响更大，从而使皮肤中的过程更具影响，从而使染色体的变化允许染色体变化无需直接涉及神经系统。该假设的途径将在外围定义一个“水平”或分布式控制系统，该系统将允许在色谱网络中自主行为。这个问题是该项目的主要意义。了解染色体水平控制的基本原理具有转化为田间的潜力，因为当前的范式是所有控制均由大脑直接施加。水平控制与循环系统，肠道功能和脊椎动物中神经系统微电路的血液传递有关。因此，该项目的结果还将更广泛地影响对当地控制的理解。从更广泛的角度来看，该项目的结果将洞悉分布式（水平）和自上而下的（垂直）控制机制的相互作用，这是与复杂系统产生不可预测的，新兴现象的一般能力有关的主题。这个概念对社会的广泛部门具有根本的兴趣，从工程到经济学再到政治。一种综合方法将允许检验以下假设：squid中对色谱网络的控制涉及与大脑下降的神经元运动控制途径不同的外围机制。独立于规范神经控制的自发染色体活性将通过沿海loliginid鱿鱼（Doryteuthis opalescens）中的实验操作分离，包括慢性神经神经毒素和四二毒素神经元活性的药理阻滞。此外，一种比较方法将利用海洋性粘剂物种，即dosidicus gigas，其中自发，耐二毒素耐药的色子能活性极为突出。相关方法涉及分子转录组学，细胞电生理学，具有共聚焦显微镜和高分辨率电子显微镜的免疫组织化学。具体目的是：1）确定相关离子通道和受体的分子和生理特性，这些通道和受体控制着操作单个色素器官的径向肌肉纤维中的兴奋性； 2）定义邻近色谱的肌肉纤维之间耦合机制的结构，分子和生理特征，该肌肉纤维定义了皮肤内的兴奋性传播途径； 3）阐明抑制性作用在控制5-羟色胺的自发色谱活性中； 4）在剂型中进行平行实验，这是生态上重要的鱿鱼家族的成员，其中从未进行过染色体的细胞研究。该项目将支持本科生和研究生培训，并包括让STEM中代表性不足的团体的学生参与的重大努力。