Sensory control of oxygen-dependent taxis behavior

描述（由申请人提供）：地球上高级生命形式的进化和生存依赖于富氧大气来驱动有效的能量代谢。生理和行为机制已经进化到维持足够的呼吸氧水平，同时限制有毒活性氧的形成。所有生物，无论是简单的还是复杂的，监测外部和内部氧气水平的能力是在正常环境条件下生存的关键。在临床情况下，患者可能面临病理状况，如心血管疾病、癌症或呼吸系统疾病，这些机制可能被破坏或被推向极限。基本的氧感应机制是多种多样的，但迄今知之甚少。在模型遗传系统（如秀丽隐杆线虫和果蝇）中使用广泛的实验工具可以显著促进我们对氧气感知机制的理解。我们利用遗传、分子和生理学的方法，将果蝇幼虫的基本飞行定向行为与外围感觉神经元子集的激活联系起来。初步研究表明，幼虫利用一种氧气感知机制来协调在化蛹和最终生存之前食物退出所必需的行为。这些幼虫的飞行偏好需要一小部分外周感觉神经元特异性表达DEG/ENaC离子通道亚基Pickpocket1 （PPK1）的功能。表达ppk1的神经元的转基因超敏化导致幼虫对增加的氧水平的敏感性急剧增加。使用破伤风毒素对表达ppk1的神经元进行转基因失活，导致对增加的氧气水平失去厌恶感。我们提出了一个模型，其中表达ppk1的神经元可能在幼虫阶段作为环境氧传感器起作用。在像果蝇这样的节肢动物中，氧气基本上是通过复杂的气管小管系统向每个细胞和组织扩散的。在幼虫觅食阶段，当幼虫对增加的氧气水平表现出最高的厌恶时，幼虫气管系统的外部开口，即后气门，是幼虫唯一凸出食物表面以允许进入大气的部分。初步结果表明，一个表达ppk1的神经元支配着每个后气门。这一建议的重点将是这些后气门神经元和他们的潜在作用，在氧气传感和空中战术行为。我将利用Ca2+敏感的G-CaMP成像技术来检查支配后气门的表达ppk1的神经元的直接氧依赖性激活。特异性Aim II将通过使用MARCM技术生成单细胞超敏神经元克隆，寻求将表达ppk1的后气孔神经元的功能与幼虫的空中定向行为联系起来。这些目标的成功完成将为未来环境氧传感机制的遗传和分子研究建立一个通用的和富有成效的遗传模型。公共卫生相关性：所有生物，无论是简单的还是复杂的，监测外部和内部氧气水平的能力是在正常环境条件下生存的关键。在临床情况下，患者可能面临病理状况，如心血管疾病、癌症或呼吸系统疾病，这些机制可能被破坏或被推向极限。尽管氧气感知在这个星球上的生命生存中起着核心作用，但我们对用于平衡氧气供应和代谢需求的各种机制知之甚少。

DESCRIPTION (provided by applicant): The evolution and survival of higher life forms on Earth has relied upon an oxygen-enriched atmosphere to drive efficient energy metabolism. Physiological and behavioral mechanisms have evolved to maintain adequate levels of respiratory oxygen while at the same time limiting toxic reactive oxygen species formation. The ability of all organisms, whether simple or complex, to monitor external and internal oxygen levels is key to survival under normal environmental conditions. In clinical situations where patients may be confronted with pathological conditions such as cardiovascular disease, cancer or respiratory illness, these mechanisms can be disrupted or pushed to their limits. Essential oxygen-sensing mechanisms are diverse and as yet poorly understood. Use of the broad range of experimental tools available in model genetic systems such as C. elegans and Drosophila can contribute significantly to advancing our understanding of mechanisms involved in oxygen-sensing. We have utilized genetic, molecular and physiological approaches to relate a fundamental aerotactic behavior in Drosophila larvae to the activation of a subset of peripheral sensory neurons. Preliminary studies suggest that larvae utilize an oxygen-sensing mechanism to coordinate behaviors necessary for food exit prior to pupariation and ultimate survival. These larval aerotactic preferences require function of a small subset of peripheral sensory neurons specifically expressing the DEG/ENaC ion channel subunit, Pickpocket1 (PPK1). Transgenic hypersensitization of ppk1-expressing neurons causes a dramatic increase in larval sensitivity to increased oxygen levels. Transgenic inactivation of ppk1-expressing neurons using tetanus toxin causes a loss of aversion to increased oxygen levels. We propose a model in which PPK1-expressing neurons may function as sensors of environmental oxygen during larval stages. In arthropods such as Drosophila, oxygen is supplied to essentially every cell and tissue by diffusion of air through a complex tracheal tubule system. During larval foraging stages, when larvae display the highest aversion to increased oxygen levels, the external openings of the larval tracheal system, the posterior spiracles, are the only part of the larva projected above the surface of the food to allow access to the atmosphere. Preliminary results show that a single ppk1-expressing neuron innervates each of the posterior spiracles. The focus of this proposal will be on those posterior spiracle neurons and their potential role in oxygen-sensing and aerotactic behavior. Specific Aim I will utilize Ca2+sensitive G-CaMP imaging techniques to examine direct oxygen-dependent activation of the PPK1-expressing neurons innervating the posterior spiracles. Specific Aim II will seek to correlate the function of the same PPK1-expressing posterior spiracle neurons with larval aerotactic behavior by generating single cell hypersensitized neuronal clones using the MARCM technique. Successful completion of these aims should establish a versatile and productive genetic model for use in future genetic and molecular studies of environmental oxygen sensing mechanisms. PUBLIC HEALTH RELEVANCE: The ability of all organisms, whether simple or complex, to monitor external and internal oxygen levels is key to survival under normal environmental conditions. In clinical situations where patients may be confronted with pathological conditions such as cardiovascular disease, cancer or respiratory illness, these mechanisms can be disrupted or pushed to their limits. Despite the central role of oxygen sensing in the survival of life on this planet, we know relatively little concerning the diverse mechanisms that are utilized to balance oxygen supply with metabolic demand.