Neurophysiological and Metabolic Adaptations to Increased Ammonia and Oxygen Starvation in Fishes.

我的研究的首要目标是确定水生脊椎动物如何应对环境压力，如体内氨的积累和氧气饥饿。氨是一种代谢废物，由过量的氨基酸分解而产生，但在浓度升高时会产生神经毒性。为此，需要将氨排出或转化为毒性较低的含氮废物(N废物)，如尿素或尿酸，这些产品是由陆生脊椎动物产生的。由于其高度的水溶性，氨是鱼类的主要氮废物，因为它可以相对容易地通过鳃排泄到水中。然而，在摄食、运动或暴露在高外氨环境中后，鱼类经常不得不应对体内氨的波动。在哺乳动物中，将氨解毒为尿素的能力受损(例如由于肝病)会导致体内氨的高度升高。这会导致“兴奋性毒性”，其特征是中枢神经系统(CNS)过度激活，产生活性氧物种，并增加大脑水分含量，导致潜在的致命脑肿胀。相反，我们证明了当暴露在氨和低环境氧气中时，鲫鱼和金鱼很容易耐受脑肿胀。这一观察结果表明，这些鱼类具有保护中枢神经系统免受氨伤害的新的生理机制。我的NSERC发现工作的另一个值得注意的发现是，对高氨的耐受性与长期氧气饥饿的生存能力有关。在接下来的5年里，我的学生们将检验这一假设，即这种缺氧-氨交叉耐受是由于这些鱼类防止兴奋性毒性的能力、对氧化应激的高耐受性以及对脑肿胀的弹性。我的具体研究目标将是：(I)表征保护金鱼中枢神经系统免受缺氧或缺氧时兴奋性毒性细胞死亡的神经生理适应；(Ii)比较氨敏性虹鱼和耐氨性金鱼中枢氨毒性和耐受性的机制(S)；(Iii)确定氧化应激与缺氧、缺氧或氨暴露下金鱼脑肿胀发展之间的联系(S)；(4)确定升高的氨和摄食量如何影响太平洋七鳃鳗和七鳃鳗这两种能够承受氨和低氧气的无颌鱼的抗氧化能力和神经生理过程。我将使用一项综合性研究来解决这些目标，包括完整的动物模型、分离的线粒体、培养的脑片模型、分子技术、免疫组织化学和电生理学。这项工作最终将加深我们对鱼类应对体内氨和氧气饥饿增加所使用的生理适应的理解，并确定缺氧-氨交叉耐受的潜在机制。

The overarching goal of my research is to determine how aquatic vertebrates cope with environmental stressors such as build-ups of internal ammonia and O2 starvation. Ammonia is a metabolic waste product that arises from the breakdown of excess amino acids, but at elevated concentrations it is neurotoxic. For this reason ammonia needs to be excreted or converted to less toxic nitrogenous waste (N-waste) products such as urea or uric acid, which are produced by terrestrial vertebrates. Due to its high water solubility, ammonia is the primary N-waste product of fishes because it can be excreted with relative ease across the gills to the water. However, fishes frequently have to cope with fluctuations in internal ammonia following feeding, exercise or exposure to high external ammonia. In mammals, an impaired ability to detoxify ammonia to urea (e.g. due to liver disease) leads to highly elevated ammonia in the body. This then results in “excitotoxicity” characterized by hyper-activation of the central nervous system (CNS), reactive oxygen species generation and increased brain water content leading to potentially fatal brain swelling. In contrast, we demonstrated that that brain swelling is readily tolerated by crucian carp and goldfish when exposed to ammonia and low environmental O2. This observation suggests that these fishes possess novel physiological mechanisms to protect the CNS from ammonia. Another notable finding of my NSERC Discovery work was that tolerance to elevated ammonia was linked to an ability to survive prolonged O2 starvation. Over the next 5 years, my students will test the hypothesis that this anoxia-ammonia cross-tolerance is due to the ability of these fishes to prevent excitotoxicity, high tolerance to oxidative stress, and resilience to brain swelling. My specific research objectives will be to: (I) Characterize the neurophysiological adaptations that protect the goldfish CNS from excitotoxic cell death during exposure to hypoxia or anoxia; (II) Contrast the mechanism(s) of ammonia toxicity and tolerance in the CNS of ammonia sensitive rainbow trout (Oncorhynchus mykiss) to those of the ammonia-tolerant goldfish; (III) Identify the link(s) between oxidative stress and the development of brain swelling in goldfish subjected to anoxia, hypoxia or ammonia exposure; (IV) Determine how elevated ammonia and feeding affect antioxidant capacity and neurophysiological processes in the Pacific hagfish and sea lamprey, two jawless fish species able to withstand ammonia and low O2. I will use an integrative research to address these objectives including whole animal models, isolated mitochondria, cultured brain slice models, molecular techniques, immunohistochemistry, and electrophysiology. This work will ultimately improve our understanding of the physiological adaptations used by fishes to cope with increased internal ammonia and O2 starvation, and identify the underlying mechanisms of anoxia-ammonia cross tolerance.