Role of an arousal circuit in respiration and opioid-induced respiratory depression

项目总结 这个K99/R00独立之路奖旨在让候选人实现她的长期 目标是建立一个独立的研究生涯，专注于大脑中的唤醒回路如何控制 呼吸，以及阿片类药物如何影响这些神经机制。这项建议是专门为 对候选人的神经科学和呼吸神经生理学背景进行补充 有研究体内和体外呼吸控制回路的知识和技能，并将使她成为理想的 适合在她的职业道路上取得成功。阿片类药物过量致死的主要原因是呼吸系统 抑郁症。阿片类药物的镇静作用通过在很大程度上未被探索的方式进一步损害呼吸动力 机械装置。尽管过量的治疗通常使用阿片类拮抗剂纳洛酮、纳洛酮 逆转所有阿片类药物的作用，导致疼痛和戒断。我们目前对阿片类药物影响的理解 呼吸回路尚未显示出理想的治疗目标，以最大限度地减少呼吸抑制 严重的副作用。因此，至关重要的是确定新的策略来缓解呼吸抑制和 刺激呼吸。这一提议试图揭示重要的新见解，即蓝斑(LC)， 一种广为人知的有助于唤醒的大脑结构，影响着关键的Kӧlliker-fuse(KF) 呼吸区，对阿片类药物高度敏感，对阿片类药物引起的呼吸抑制至关重要。 跨细胞类型特定神经示踪、体外脑片记录和体内的初步数据 光遗传学和纤维光度学支持LC在呼吸中对KF的输入作用，这一作用尚未得到 之前很欣赏。根据初步数据，总体假设是LCKF电路是一个关键 呼吸功能的调节剂，特别是在阿片类药物引起的呼吸抑制期间。候选人的 之前接受过脑片记录、清醒动物体内电生理学和交叉病毒的培训- 遗传方法为最先进的体内外光遗传学和纤维提供了坚实的基础 建议书中的测光技术。这种多层次的方法将允许应试者检验假设 KF神经元从LC神经元接受阿片敏感的单突触兴奋性输入(目标1)，即KF 投射LC神经元的活动与体内的呼吸作用是耦合的(目标2)，LCKF电路起关键作用 在呼吸和阿片类药物引起的呼吸抑制中的作用(目标3)。总之，这项提案的目标将是 产生关于大脑控制呼吸的新信息，并将提供强大的概念和 方法培训，使开发一个有影响力和成功的独立研究计划成为可能。 由Erica Levitt博士和David Fuller博士以及由以下人员组成的指导委员会提供强大的指导 资深教授，他们是拟议技术方面的专家，并得到了广泛的指导 经验，将帮助应聘者实现本提案的研究和职业发展目标。

PROJECT SUMMARY This K99/R00 Pathway to Independence Award is designed to allow the candidate to achieve her long-term goal to establish an independent research career focused on how arousal circuits in the brain control breathing, and how these neural mechanisms are influenced by opioids. This proposal has been tailored to supplement the candidate’s background in neuroscience and respiratory neurophysiology with additional knowledge and technical skills to study respiratory control circuits in vivo and ex vivo, and will make her ideally suited to succeed on her career path. The primary cause of death from an opioid overdose is respiratory depression. The sedative effect of opioids further impairs respiratory drive through largely unexplored mechanisms. Although overdoses are routinely treated with the opioid antagonist, naloxone, naloxone reverses all opioid effects, causing pain and withdrawal. Our current understanding of opioid effects on the respiratory circuitry has not revealed an ideal therapeutic target to minimize respiratory depression without serious side effects. Therefore, it is essential to identify new strategies to alleviate respiratory depression and stimulate breathing. This proposal seeks to uncover important new insights whereby the locus coeruleus (LC), a brain structure widely known for its contributions to arousal, influences the Kӧlliker-Fuse (KF), a key respiratory region, which is highly sensitive to opioids and critical for opioid-induced respiratory depression. Preliminary data spanning cell-type specific neural tracing, ex vivo brain slice recordings, and in vivo optogenetics and fiber photometry, support a role for LC input to the KF in respiration, which has not been previously appreciated. Based on preliminary data, the overall hypothesis is that the LCKF circuit is a critical modulator of respiratory function, particularly during opioid-induced respiratory depression. The candidate’s prior training in brain slice recordings, in vivo electrophysiology in awake animals, and intersectional viral- genetic methods provides a solid foundation for the state-of-the-art ex vivo and in vivo optogenetics and fiber photometry techniques in the proposal. This multi-level approach will allow the candidate to test the hypothesis that KF neurons receive opioid-sensitive, monosynaptic excitatory input from LC neurons (Aim 1), that KF projecting LC neuron activity is coupled with respiration in vivo (Aim 2), and the LCKF circuit plays a key role in respiration and opioid-induced respiratory depression (Aim 3). Together, the aims of this proposal will yield novel information regarding the brain’s control of respiration and will also provide strong conceptual and methodological training, enabling development of an impactful and successful independent research program. Strong mentorship by Drs. Erica Levitt and David Fuller, as well as a Mentoring Committee comprised of established professors, who are experts in the proposed techniques and have extensive mentoring experience, will help the candidate achieve the research and career development goals of this proposal.