Project summary/abstract Heart failure (HF) is major cause of morbidity and mortality and its incidence is expected to increase over the next decade. Characteristics of HF include an imbalance in the autonomic nervous system, with increased sympathetic tone and parasympathetic withdrawal, as well as chronic inflammation. Factors that contribute to increased sympathetic tone include increased release of the primary neurotransmitter norepinephrine (NE) and co-transmitter, neuropeptide Y (NPY). Although b-blocker therapy is a well-established approach for modulating the effects of NE during heart failure, even high levels of these agents do not fully prevent the effects of sympathetic activation. Recent work has demonstrated that increased plasma levels of NPY are correlated with worse outcomes in HF, including increased sudden cardiac deaths, suggesting that the co-release of NPY contributes to disease pathology and may be arrhythmogenic. Indeed, addition of an NPY blocker together with b-blocker therapy has shown promise in preventing the effects of increased sympathetic activation. Despite evidence that overactivity of sympathetic neurons contributes to HF, the mechanisms underlying the enhanced release of NPY have yet to be investigated. Importantly, direct observations of NPY transport in sympathetic neurons and knowledge of the molecular pathways involved are lacking. We hypothesize that inflammatory signals that are elevated during cardiovascular disease potentiate NPY trafficking and release, contributing to disease progression. Although treatments can help stabilize or slow disease progression for patients with heart failure, prognosis remains poor with a 5-year survival rate of approximately 50%. Thus, understanding the molecular changes that underly the dynamic regulation of sympathetic neurons will enable the development of novel therapeutic interventions. We recently developed a novel imaging technique, optical pulse-chase axonal long-distance (OPAL) imaging, that enables the visualization of axonal trafficking of proteins with single-molecule resolution. Using this and other imaging techniques, we propose to investigate the trafficking of NPY-containing vesicles in cardiac sympathetic neurons from neonatal mice cultured in compartmentalized microfluidic chambers. We will investigate the molecular motors and trafficking machinery involved in the long-distance axonal transport of NPY, including Rab-GTPases and kinesin motors. Elucidation of this pathway will provide targets of opportunity for therapeutic interventions for conditions such as HF. Additionally, we propose to investigate the dynamic regulation of NPY trafficking and release in response to inflammatory cytokines found in HF. Together, these will provide the first report of dynamic regulation of vesicular trafficking and neuropeptide release in sympathetic neurons, and could transform how we detect and treat some cardiovascular diseases.