Kevin Yackle, M.D. Ph.D. - US grants

Project Summary There are two critical pacemakers for life: the cardiac pacemaker and the breathing pacemaker, the preBötzinger Complex (preBötC). The preBötC is a cluster of ~3000 neurons in the brainstem that are cyclically active, with each burst of activity initiating a breath. In contrast to the cardiac pacemaker, the molecular and cellular basis of breathing rhythm generation remains unknown, as do diseases associated with it, such as central sleep apnea and sudden infant death. The prevailing model of preBötC rhythm generation, called the `group-pacemaker' model, proposes that each breath is triggered by an emergent preBötC network phenomena. An important assumption of this model is that there are not dedicated breath-initiating neurons. However, based on the observed variety of preBötC neuron firing patterns, including ones that fire just before each breath (pre-inspiratory), and the unexpected molecular and functional diversity of the preBötC neurons I discovered during my Ph.D., I hypothesize that, as in the heart, there are specific neurons that initiate each breath, breathing pacemaker neurons, and propose to identify and characterize them in this research proposal. As a UCSF Sandler Fellow and recipient of the Early Independence Award, I plan to first comprehensively map preBötC cell types with single cell gene expression analysis and identify candidate breathing pacemaker neurons by their expression of the same ion channels important for cardiac pacemaking. Additionally, I plan identify candidate breath-initiating neurons by their anticipated activity during breathing (pre-inspiratory) and their autonomous, rhythmic activity in vitro (pacemaker activity). Lastly, I will identify candidate pacemakers by their proposed connectivity to ~175 preBötC neurons I identified in my Ph.D. that receive breathing pacemaker activity. I predict that these three independent approaches will converge on the same preBötC subtypes, the presumed breathing pacemaker neurons and I will then use intersectional genetic strategies to test if the identified neurons have breathing pacemaker properties: autonomous rhythmic activity, pre-inspiratory activity, ability to initiate a breath, and requirement for breathing. The molecular and functional identification of respiratory pacemaker neurons will be a transformative discovery, leading to the eventual resolution of how respiratory rhythms and arrhythmias, some of the most deadly diseases in infants, are generated. This mechanistic understanding of breathing rhythm generation will provide an avenue to develop pharmacological approaches to control ventilation, which would impact multiple medical fields, especially neonatology and critical care medicine. In my recent Ph.D. work, I have demonstrated extraordinary molecular diversity within the preBötC and demonstrated that small numbers of molecularly distinct preBötC cell types have highly specific functions in the breathing behavior. I am poised to continue this dissection with the objective of identifying the core neurons that initiate a breath and control the pace of breathing.

PROJECT SUMMARY This proposal seeks to leverage the emerging understanding of cellular and subcellular heterogeneity in opioid signaling for therapeutic discovery. The project develops new optogenetic and chemogenetic tools to achieve local control of opioid receptor activity and signaling in neural circuits and at a subcellular level of resolution. The proposal seeks to develop a versatile toolbox and provide proof-of-concept for in vivo precision pharmacology that have not yet been applied to the opioid system. The first phase (R61) is a high-risk technology enabling phase, based on combining our collaborative team's expertise in developing receptor-blocking intracellular nanobodies with established methods for conferring local control of nanobody concentration in the cytoplasm and specific membrane domains, using optogenetic and chemogenetic strategies that have already been validated but never before applied to opioid receptors. The R61 phase will develop these tools (Specific Aim 1) and validate them in neuronal culture and acute slice preparations (Specific Aim 2). These studies will provide not only proof-of-concept for in vivo precision pharmacology of opioid receptors, but also for G protein-coupled receptors as a class. We have set a specific series of milestones to evaluate progress, and to assess feasibility for advance to in vivo studies. If the milestones are successfully met, the second phase (R33) will apply the new tools to selectively target opioid signaling in neural circuits in vivo, focusing on analgesia, behavioral reinforcement and respiratory depression by clinically relevant opioid drugs. Mice are used as a well-established and relevant model (Specific Aim 3). These studies leverage the combined expertise of our collaborative team, and will establish powerful optogenetic and chemogenetic manipulations for the study of in vivo opioid function for the first time. Accordingly, the proposed studies to provide the first-in-class application of optogenetic and chemogenetic control to the opioid system in vivo, and they will explore a precision pharmacology of opioids based on location in neural circuits.