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，以及它们如何应对这种废物的潜在有毒堆积。氨通常产生于过量氨基酸的分解，但在高浓度时会产生神经毒性作用。鱼类通常比哺乳动物对氨的耐受性要强得多，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，耐缺氧的鱼类，如金鱼(Carassius Auratus)和鲫鱼(Carassius Carassius)，已经进化出抵抗O2饥饿的神经生理机制，也耐氨性。事实上，表征大脑缺氧/缺血的一连串事件与氨中毒所发生的事件相似。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物种(ROS)和水分积累，最终导致潜在的致命脑肿胀。然而，我们的发现表明，鱼很容易耐受氨引起的脑肿胀。我建议使用一种综合的方法来检验一个有效的假设，即鱼类比哺乳动物对氨的耐受性更强，与大脑抵抗氨的神经毒性影响的能力更强有关。我还建议检验相关假设，即有助于金鱼耐缺氧的生理适应也解释了它们更强的氨耐受性。在接下来的5年里，我的研究计划的目标将是：(I)描述耐氨性金鱼和氨敏性鲑鱼(Oncorhynchus MykISS)中枢神经系统急性氨毒性和耐受性的潜在机制(S)；(Ii)确定金鱼是否对ROS的高耐受性导致金鱼比鲑鱼对氨的耐受性更高；(Iii)确定鱼类对氨和其他水生应激源反应导致脑肿胀的潜在机制；(Iv)确定长期亚致死性氨暴露和摄食如何影响金鱼和鲑鱼的氨耐力。我将在我的研究中使用的综合方法将包括完整的动物模型、培养的脑片模型、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(S)的深入理解将提高我们对脊椎动物如何应对氨的基本理解，并有助于揭示导致脊椎动物氨解毒和处理的不同机制进化的选择性压力。这项工作还将通过更好地解释鱼类如何响应和容忍水生生态系统中氨的积累而具有实际意义。

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.