Collaborative Research: Nervous System Adaptations in a Highly Neurotoxic Organism

粗糙皮肤的蝾螈（Taricha granulosa）是已知毒性最强的动物之一：有些个体拥有大量的河豚毒素（TTX），这会阻止脑细胞（神经元）相互传递信号。科学家们对TTX通常如何阻止神经活动了解很多，但对拥有TTX的动物如何能够抵抗其影响却知之甚少。蝾螈抵抗TTX的能力特别令人困惑，因为它们的几个基因必须一致突变，因为只有一个受影响基因的突变会使蝾螈容易受到TTX的影响，并可能死亡。奇怪的是，蝾螈不仅对TTX的影响有抵抗力，而且实际上被TTX的气味所吸引。在拟议的工作中，该团队将使用成熟的方法来量化蝾螈体内TTX的分布，识别可能参与TTX抗性的突变，检查TTX对神经元的影响，并发现参与嗅觉的神经元如何被TTX激活。这项基础研究将帮助科学家了解神经元如何在分子水平上工作，这将增加或了解健康大脑功能的“螺母和螺栓”，以及动物如何适应毒素的存在。来自代表性不足群体的本科生，包括来自俄勒冈州美洲原住民的学生，将被招募参加与这项工作相关的暑期研究项目。这些经验将帮助学生为未来在STEM相关领域的职业生涯做好准备。技术段落TTX是有毒的，因为它阻断电压门控钠通道（NaVs），这对动作电位的产生和传播至关重要。拟议的工作将确定不同形式的NaV在蝾螈的神经元和肌肉中表达的位置，确定这些通道中的特定突变，使它们能够抵抗TTX的毒性作用，并确定这些结构变化如何改变神经元功能。使用分析化学，组织化学和分子生物学，TTX在不同组织中的水平以及六种不同NaV的位置和结构将在高毒性和无毒蝾螈中进行检查，以量化TTX抗性水平并识别赋予抗性的突变。将表征异源表达系统中的NaVs和蝾螈脑中的神经元的电生理学特性，以确定TTX抗性是否以及如何改变通道和神经元功能。此外，使用技术的组合，在嗅觉上皮TTX检测的适应将被确定，以了解这种不寻常的能力是如何演变的。拟议的工作将有助于理解生理上至关重要的一类离子通道，以及分子水平上的进化如何塑造神经系统功能和动物行为，从而加深我们对神经元如何在进化过程中发挥作用和变化的理解。此外，拟议的工作将沉浸在科学的性质和实践的本科生，特别是通过在密歇根州州立大学的REU计划的威廉斯大学的学生参与。

Non-Technical ParagraphRough-skinned newts (Taricha granulosa) are among the most toxic animals known: some individuals possess enormous quantities of tetrodotoxin (TTX), which prevents brain cells (neurons) from signaling to each other. Scientists know a great deal about how TTX normally blocks neural activity, but not much about how animals that possess TTX are able to resist its effects. The ability of newts to resist TTX is particularly puzzling because several of their genes must be mutated in concert, as a mutation in only one affected gene would leave the newt vulnerable to TTX's affects, and likely dead. Strangely, newts are not simply resistant to TTX's effects, but are actually attracted to the smell of TTX. In the proposed work, the team will use well-established methods to quantify the distribution of TTX inside newts' bodies, identify mutations that are likely involved in TTX resistance, examine the effects of TTX on their neurons, and discover how the neurons involved in smelling are activated by TTX. This basic research will help scientists understand how neurons work at a molecular level, which will increase or understanding of the "nuts and bolts" of healthy brain function, as well as how animals adapt to the presence of toxins. Undergraduate students from under-represented groups, including students from Native American populations in Oregon, will be recruited to participate in summer research programs associated with the work. These experiences will help prepare a diverse population of students for future careers in STEM related fields.Technical ParagraphTTX is toxic because it blocks voltage-gated sodium channels (NaVs), essential for the generation and propagation of action potentials. The proposed work will identify where the different forms of NaVs are expressed in neurons and muscles in newts, identify specific mutations in these channels that allow them to resist the toxic effects of TTX, and determine how these structural changes alter neuron function. Using analytical chemistry, histochemistry, and molecular biology, levels of TTX in different tissues as well as the location and structure of the six different NaVs will be examined in both highly toxic and non-toxic newts to quantify levels of TTX resistance and identify mutations that confer resistance. The electrophysiological properties of NaVs in a heterologous expression system and of neurons in the brain of newts will be characterized to determine whether and how TTX resistance alters channel and neuron function. In addition, using a combination of techniques, the adaptations underlying TTX detection in the olfactory epithelium will be identified to understand how this unusual ability evolved. The proposed work will contribute to understanding a physiologically vital class of ion channels, as well as the ways in which evolution at the molecular level shapes nervous system function and animal behavior, thereby deepening our understanding of how neurons function and change over evolutionary time. In addition, the proposed work will immerse undergraduate students in the nature and practice of science, particularly through the involvement of Williamette University students in an REU program at Michigan State University.