The Neurophysiological Basis of Ammonia Toxicity and Tolerance in Fishes

我的研究的首要目标是确定鱼类如何产生和排泄氨等含氮废物，以及它们如何科普这种废物的潜在毒性积累。氨通常由过量氨基酸的分解产生，但在高浓度下具有神经毒性作用。鱼类通常比哺乳动物对氨的耐受性更强，哺乳动物需要将氨解毒为尿素才能生存。然而，人们对这些差异的潜在机制知之甚少。我们最近的工作表明，金鱼（Carassius auratus）和鲫鱼（Carassius carassius）等耐缺氧鱼类已经进化出了耐受O2饥饿的神经生理机制，也具有耐氨性。事实上，表征脑缺氧/缺血的级联事件与氨中毒发生的事件相似。在哺乳动物中，过量的氨会导致大脑中谷氨酸受体的过度激活，导致兴奋性毒性，其特征是星形胶质细胞产生活性氧（ROS）和水积聚，最终导致潜在的致命性脑肿胀。然而，我们的研究结果表明，鱼很容易耐受氨引起的脑肿胀。我建议使用一个综合的方法来测试的工作假设，更大的氨耐受性的鱼类相比，哺乳动物有关的大脑抵抗氨的神经毒性作用的能力更强。同时，本研究也提出相关假说，即金鱼的耐缺氧生理适应，也可以解释其更强的耐氨性。在接下来的5年里，我的研究计划的目标将是：（一）表征急性氨毒性和耐氨金鱼和氨敏感鳟鱼中枢神经系统耐受性的潜在机制（虹鳟鱼）;（II）确定与鳟鱼相比，金鱼对活性氧的高耐受性是否会导致更大的氨耐受性;（III）确定导致鱼类脑肿胀的潜在机制，以应对氨和其他水生应激因素;（IV）确定长期亚致死氨暴露和喂养如何影响金鱼和鳟鱼的氨耐受性。我将在我的研究中使用的综合方法将包括整个动物模型，培养的脑切片模型，分子技术，免疫组织化学和电生理学方法。对鱼类氨毒性和耐受性的神经机制的进一步了解将提高我们对脊椎动物如何科普氨的基本理解，并阐明导致脊椎动物氨解毒和处理不同机制进化的选择压力。这项工作也将通过更好地解释鱼类如何应对和容忍水生生态系统中氨的积累而产生实际影响。

The overarching aim of my research is to determine how fishes produce and excrete nitrogenous wastes such as ammonia, and how they cope with potentially toxic build-ups of this waste product. Ammonia normally arises from the breakdown of excess amino acids, but has neurotoxic effects at high concentrations. Fish are generally much more tolerant to ammonia than mammals, which need to detoxify ammonia to urea to survive. Yet little is known about the underlying mechanisms for these differences. Our recent work suggests that anoxia tolerant fishes, such as goldfish (Carassius auratus) and crucian carp (Carassius carassius), that have evolved neurophysiological mechanisms to withstand O2 starvation, are also ammonia tolerant. Indeed, the cascade of events that characterize anoxia/ischemia in the brain are similar to those that occur with ammonia toxicity. In mammals, excess ammonia causes overactivation of glutamate receptors in the brain, leading to excitotoxicity characterized by the generation of reactive oxygen species (ROS) and water accumulation by astrocyte cells, culminating in potentially fatal brain swelling. Our findings suggest, however, that fish readily tolerate ammonia-induced brain swelling. I propose to use an integrated approach to test the working hypothesis that the greater ammonia tolerance of fishes compared to mammals is related to a greater ability of the brain to resist the neurotoxic effects of ammonia. I also propose to test the related hypothesis that the physiological adaptations that contribute to the anoxia tolerance of the goldfish, also explains their greater ammonia tolerance. Over the next 5 years, the objectives of my research program will be to: (I) Characterize the underlying mechanism(s) of acute ammonia toxicity and tolerance in the central nervous system of ammonia-tolerant goldfish and ammonia-sensitive trout (Oncorhynchus mykiss); (II) Determine if high tolerance to ROS results in greater ammonia tolerance in goldfish compared to trout; (III) Identify the underlying mechanisms that lead to brain swelling in fishes in response to ammonia and other aquatic stressors; (IV) Determine how prolonged sub-lethal ammonia exposure and feeding affect ammonia tolerance in goldfish and trout. The integrative approach I will use in my research will include whole animal models, cultured brain slice models, molecular techniques, immunohistochemistry, and electrophysiology methods. An improved understanding of the neural mechanism(s) of ammonia toxicity and tolerance in fishes will improve our fundamental understanding how vertebrates cope with ammonia, and shed light on the selective pressures that led to the evolution of different mechanisms of ammonia detoxification and handling in the vertebrates. This work will also have practical implications by better explaining how fishes respond to and tolerate build-ups of ammonia in aquatic ecosystems.