Rhythm control and regulation of Lymnaea respiratory central pattern generator neurons

呼吸对生命至关重要。它的基本节律由中枢模式产生器(CPG)网络控制，该网络由能够产生内源性节律活动的交互神经元组成。CPG节律的产生依赖于突触的相互作用和固有的膜特性。呼吸性CPG(RCPG)受多种病理生理因素的调节，包括低氧水平。慢性低氧可诱导适应性反应，促进呼吸运动活动，也可导致呼吸和神经功能障碍。调节rCPG节律活动的基本机制仍然很大程度上尚不清楚，主要是因为哺乳动物呼吸神经元网络的解剖和功能复杂。**突触和固有膜特性的原理在物种之间是保守的。因此，使用具有大型神经元和映射良好的神经元网络的无脊椎动物模型来研究CPG的节律特性是有利的。池塘蜗牛，L.stagnalis，是一种水生呼吸空气的肺动物。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型神经元组成，其中包括一个多巴胺能起搏神经元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，Stagnalis是研究rCPG节律产生和调节的基本机制的理想模式生物。**在上一次NSERC发现资助期间(2009-14)，我们采用STESTNALIS作为研究慢性低氧诱导的调节呼吸节律的神经可塑性的模型，建立了本地和国际合作，培养了8名研究生和32名本科生。总之，我们(1)报道了调节神经行为和呼吸节律以响应环境低氧的应激蛋白；(2)发现了在低氧诱导的神经适应中调节的磷蛋白；(3)确定了U型通道是多巴胺能起搏神经元中的节律调节器；(4)领导了一个国际合作项目，首次对L.stagnalis转录组进行了测序。我们的初步数据表明，慢性缺氧启动了呼吸的易化，调节了U型通道、突触前和突触后的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。这些新的发现导致了一种假设，即低氧诱导的呼吸可塑性是通过U型通道和多巴胺依赖的突触传递在rCPG神经元上介导的。为了验证这一假设，我们提出了以下三个未来5年的短期目标。**1.确定U型通道在慢性低氧调节的rCPG神经元节律性放电模式中的作用。*2.确定慢性低氧对rCPG神经元中多巴胺介导的突触活动的调节作用。*3.确定呼吸可塑性所需的关键基因和蛋白质。**该研究计划的长期目标是(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，以及(2)开发模拟或防止这种调节的分子工具，最终用于潜在的治疗干预。这个项目的新颖性在于，我们能够利用先进的现代技术，在单个系统中揭示单个rCPG神经元水平上的新机制，直到动物的行为。**意义：这些研究不仅将提供呼吸CPG活动的节律控制和调节的基础知识，还将提供多巴胺依赖的突触调节的基础知识。他们将帮助确定新的靶点，以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.