Rhythm control and regulation of Lymnaea respiratory central pattern generator neurons

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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.