Rhythm control and regulation of Lymnaea respiratory central pattern generator neurons

呼吸对生命至关重要。其基本节律由中枢模式发生器（CPG）网络控制，该网络由能够产生内源性节律活动的相互作用神经元组成。CPG中的节律产生依赖于突触相互作用和固有膜特性。呼吸CPG （rCPG）受多种病理生理因素调控，包括低氧水平。慢性缺氧诱导适应性反应，促进呼吸运动活动，也可导致呼吸和神经功能障碍。由于哺乳动物呼吸神经网络的解剖学和功能复杂性，调控rCPG节律性活动的基本机制在很大程度上仍不清楚。**突触和内在膜特性的原理在物种间是保守的。因此，使用具有大神经元和良好映射的神经网络的无脊椎动物模型来研究CPG节律特性是有利的。池塘蜗牛（L. stagnation）是一种水生的呼吸空气的肺类动物。它的呼吸节律活动由一个简单的rCPG网络控制，该网络由三个可识别的大神经元组成，包括一个多巴胺能起搏器神经元RPeD1。这些神经元可以进行直接的电生理评估和分子操作，因此停滞乳杆菌是研究rCPG节律产生和调控基本机制的理想模式生物。**在2009-14年度NSERC Discovery Grant期间，我们以L. alis为模型研究了慢性缺氧诱导的神经可塑性调节呼吸节律，建立了本地和国际合作，培养了8名研究生和32名本科生。总的来说，我们(1)报道了应激蛋白在应对环境缺氧时调节神经行为和呼吸节律；(2)发现了在缺氧诱导的神经适应中调控的磷酸化蛋白；(3)发现u型通道是多巴胺能起搏器神经元的节律调节因子；(4)主持了第一个国际合作项目——停滞扁豆转录组测序。我们的初步数据表明，慢性缺氧启动呼吸促进，调节u型通道，突触前和突触后分子和应激诱导的调节蛋白，以及多巴胺能神经元的突触活动。这些新发现提出了缺氧诱导的呼吸可塑性是由rCPG神经元的u型通道和多巴胺依赖的突触传递介导的假设。为了验证这一假设，我们提出了未来5年的以下三个短期目标。* * 1。确定u型通道在慢性缺氧调节的rCPG神经元节律性放电模式中的作用。* 2。慢性缺氧对rCPG神经元多巴胺介导的突触活性的调节作用。确定呼吸可塑性所需的关键基因和蛋白质。**该研究项目的长期目标是：(1)确定多巴胺介导的突触可塑性和CPG神经元节律性活动的调节机制，(2)开发模拟或阻止这种调节的分子工具，最终用于潜在的治疗干预。这个项目的新颖之处在于我们能够使用先进的现代技术来揭示单个识别的rCPG神经元水平上的新机制，直到单个系统中的动物行为。**意义：本研究不仅为呼吸CPG活动的节律控制和调控提供了基础知识，而且为多巴胺依赖性突触调控提供了基础知识。它们将有助于确定新的靶标，用于开发在病理生理条件下操纵节律活动的试剂。

Respiration is vital to life. Its essential rhythm is controlled by central pattern generator (CPG) networks comprising interactive neurons capable of generating an endogenous rhythmic activity. Rhythm generation in CPG relies on synaptic interactions and intrinsic membrane properties. Respiratory CPG (rCPG) is regulated by multiple pathophysiological factors, including low oxygen levels. Chronic hypoxia induces adaptive responses to facilitate respiratory motor activities and can also result in respiratory and neural dysfunction. The fundamental mechanisms by which the rhythmic activities of rCPG are regulated remain largely unclear, primarily because of the anatomical and functional complexities of the respiratory neuronal network in mammals. **The principles of synaptic and intrinsic membrane properties are conserved across species. Thus, using invertebrate models that have large neurons and well-mapped neuronal networks to study the CPG rhythmic properties is advantageous. The pond snail, L. stagnalis, is an aquatic air-breathing pulmonate. Its respiratory rhythmic activity is controlled by a simple rCPG network consisting of three large and identifiable neurons, including a dopaminergic pacemaker neuron, RPeD1. These neurons allow for direct electrophysiological assessments and molecular manipulation, and thus L. stagnalis is an ideal model organism for studying fundamental mechanisms of rhythmic generation and regulation of rCPG. **During the last NSERC Discovery Grant (2009-14), we adopted L. stagnalis as a model to study chronic hypoxia-induced neural plasticity regulating respiratory rhythm, established local and international collaborations, and trained 8 graduate and 32 undergraduate students. Collectively, we (1) reported the stress proteins regulating neural behaviours and respiratory rhythm in response to environmental hypoxia; (2) discovered the phosphoproteins regulated in hypoxia-induced neural adaptation; (3) identified U-type channel as rhythmic regulator in the dopaminergic pacemaker neuron; and (4) led an international collaborative project of the first L. stagnalis transcriptome sequencing. Our preliminary data show that chronic hypoxia initiates facilitation of respiration, regulates U-type channels, pre- and postsynaptic molecules and stress-induced regulatory proteins, and synaptic activities of dopaminergic neurons. These new findings lead to the hypothesis that hypoxia-induced respiratory plasticity is mediated by U-type channels and dopamine-dependent synaptic transmission in rCPG neurons. To test this hypothesis, we propose the following three short-term objectives for the next 5 years. **1. Determine the role of the U-type channel in chronic hypoxia-regulated rhythmic firing pattern of rCPG neurons. *2. Determine the modulatory effect of chronic hypoxia on dopamine-mediated synaptic activity in rCPG neurons.*3. Identify critical gene and proteins required for respiratory plasticity.**The long-term goals of the research program are (1) to identify the regulatory mechanisms underlying dopamine-mediated synaptic plasticity and rhythmic activity of CPG neurons, and (2) to develop molecular tools mimicking or preventing such regulation, ultimately for potential therapeutic intervention. The novelty of this program lies in our ability to use advanced modern technologies to reveal the new mechanisms at the level of individually identified rCPG neurons up to animal behaviour in a single system.**Significance: The studies will provide fundamental knowledge of not only rhythm control and regulation of respiratory CPG activities, but also dopamine-dependent synaptic regulation. They will assist in identifying new targets for developing reagents that manipulate rhythmic activity in pathophysiological conditions.