Cellular and molecular dissection of the breathing pacemaker [electronic resource]

There are two critical pacemakers for life: the cardiac pacemaker and the breathing rhythm generator. The cardiac pacemaker in the sinoatrial node of the heart is composed of several thousand cardiomyocyte pacemaker cells that use at least thirteen ion channels to autonomously and periodically generate action potentials that trigger cardiac contraction. Additionally, this in depth cellular and molecular understanding of cardiac pacemaking has provided a framework to explain cardiac arrhythmias and sudden cardiac death and enabled the development of drugs used to treat and prevent them. The other critical pacemaker for life, the breathing rhythm generator, called the preBötzinger Complex (preBötC, breathing pacemaker), is a cluster of ~4000 neurons in the medulla of the murine brainstem that are required for breathing and 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 largely unknown, as does the origin of diseases associated with it, such as central sleep apnea and sudden infant death. The leading model of preBötC rhythm generation, called the 'group-pacemaker' model, proposes that a fraction of preBötC neurons become active late in expiration and this activity percolates among interconnected neurons, eventually building up enough potential to trigger activity throughout the preBötC, leading to inspiration. An important assumption of this model is that there are not dedicated breath-initiating neurons and that all preBötC neurons function redundantly to trigger a breath, but this has never been definitively demonstrated. In this dissertation, I call into question this core assumption with my discovery of molecularly distinct groups of preBötC neurons have dedicated and specific functions in breathing. I created a molecular map of preBötC cell types by screening > 19,000 mRNA expression patterns that identified genes specific to the preBötC, and subsequently characterized the cellular colocalization of 21 genes in detail. This revealed over 70 molecularly distinct preBötC cell types. In studies to define the function of the first five subtypes, I have shown that this extraordinary molecular diversity has revealed preBötC cell types that have exquisitely specific, distinct, interesting, and novel functions. For example ~200 preBötC neurons composing three cell types, are sufficient to induce and selectively required for the control of sighing, a separate cell type of ~50 neurons extends the length of expiration for calm breaths, and yet another cell type, ~175 preBötC neurons, projects to, synapses with, and activates the locus coeruleus, which is a higher order brain center that promotes arousal. This work definitively demonstrates that the preBötC is composed of an extraordinary number of neuronal subtypes, each with novel, distinct and important functions in breathing. In addition to identifying new roles of the preBötC in breathing, the functional specificity of neurons reshapes the understanding of the preBötC and how it could generate a breath, contradicting the central assumption of the group-pacemaker hypothesis that preBötC neurons functional redundancy. These studies imply that one or more of the remaining > 65 subtypes I identified will be a key cell type that triggers a breath or controls the pace of breathing, the breathing pacemakers.

Keywords: Biochemistry.
Advisor: Krasnow Mark
Brown Patrick
Chen Xiaoke advisor. Thesis advisor
Clandinin Thomas R. (Thomas Robert)
Harbury Pehr advisor. Thesis advisor