Collaborative Research: Nervous System Adaptations in a Highly Neurotoxic Organism

摘要：透皮蝾螈（Taricha granulosa）是已知毒性最强的动物之一：一些个体拥有大量的河豚毒素（TTX），这种毒素可以阻止脑细胞（神经元）相互传递信号。科学家们对TTX通常如何阻断神经活动了解甚多，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的能力尤其令人费解，因为它们的几个基因必须协同突变——只有一个受影响基因的突变会使蝾螈容易受到TTX的影响，甚至可能死亡。奇怪的是，蝾螈不仅对TTX的影响有抵抗力，而且实际上还被TTX的气味所吸引。在提议的工作中，科学家将使用成熟的方法来量化TTX在蝾螈体内的分布，识别可能与TTX抗性有关的突变，检查TTX对神经元的影响，并发现与嗅觉有关的神经元是如何被TTX激活的。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，以及动物如何适应毒素的存在。通过与这项工作相关的教学和培训活动，拟议的项目还将有助于培养具有科学素养的美国劳动力。技术摘要tttx是有毒的，因为它阻断了电压门控钠通道（NaVs），这是动作电位产生和传播所必需的。拟议的工作将确定不同形式的nav在蝾螈神经元和肌肉中的表达位置，这些通道中的特定突变使它们能够抵抗TTX的毒性作用，以及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的位置和结构将在剧毒和无毒蝾螈中进行检测，以量化TTX抗性水平并确定赋予抗性的突变。我们将对异源表达系统和蝾螈脑内神经元中nav的电生理特性进行表征，以确定TTX抗性是否以及如何改变通道和神经元功能。此外，嗅觉上皮中TTX检测的适应性将被确定，以了解这种不寻常的能力是如何进化的。这项提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，这两个都是神经科学的基本问题。此外，拟议的工作将使本科生沉浸在科学的本质和实践中，特别是通过威廉特大学的学生参与密歇根州立大学的REU项目。

General AbstractRough-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 - 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, scientists 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, as well as how animals adapt to the presence of toxins. Through teaching and training activities associated with this work, the proposed project will also contribute to the development of a science-literate American workforce.Technical AbstractTTX 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, specific mutations in these channels that allow them to resist the toxic effects of TTX, and 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, 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, both fundamental questions in neuroscience. 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.