PROJECT SUMMARY/ABSTRACT Feeding behavior is critical for animal survival, and is also a fundamental aspect of energy homeostasis. This process is regulated by highly complex neurochemical pathways involving a multitude of neuropeptides and biogenic amines. Despite decades of work on individual neurochemical systems, the general organizational principles underlying neuromodulation are still poorly understood. This is mainly due to the fact that modulation of neural circuit has so far been studied primarily one modulator at a time without the knowledge about co-modulation of networks. The latter information would require the development of sensitive and selective analytical tools to precisely identify these low abundance endogenous signaling molecules and accurately measure their behaviorally-relevant concentrations in a complex microenvironment. Our proposed research aims to address this critical knowledge and technological gap by developing new bioanalytical methods to elucidate the complex identities and functional roles of neuropeptides in food intake via combined mass spectrometric and physiological approaches. We employ the crustacean stomatogastric nervous system, cardiovascular system, and its associated neuroendocrine organs as a test-bed for technology development and validation due to the unique advantages and biological significance of this model system. In parallel, we aim to translate our technology development for neuropeptide discovery and analysis to the mammalian central nervous system. To this end, we propose to focus on key brain regions in a rat model at progressively more complex levels of feeding-related information processing. The specific aims include: (1) Developing and applying mass defect-based, amine reactive chemical tags coupled with data-independent acquisition (DIA) mass spectrometry (MS)-based strategy for multiplexed quantitation of neuropeptide changes under different feeding conditions; (2) Developing a nanosecond photochemical reaction (nsPCR)-assisted MALDI-based mass spectral imaging (MSI) technique for mapping co-localization patterns of individual isoforms of extended peptide families and amine neurotransmitters in identified neurons and the feeding circuits, with enhanced sensitivity and chemical information; and (3) Assessing functional roles of neuropeptides in feeding and cardiac regulation using a multi-pronged approach integrating in vivo microdialysis and ex vivo physiological and behavioral measurements. Novel neuropeptides will be evaluated for functional roles in feeding regulation at the neuronal network and system levels. The outcome of the proposed research will be a suite of new analytical tools enabling quantitative assessment of the interplay of neuropeptides and biogenic amines with high spatial, chemical and temporal information. The parallel application of these new methods to both crustacean and mammalian nervous systems in feeding will accelerate our pace towards the development of new...