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 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 a set of novel mass defect- based multiplex dimethyl pyrimidinyl ornithine (DiPyrO) tags for accurate and high throughput MS1-based relative quantification of in vivo expression changes of neuropeptides under different feeding conditions; (2) Developing a sub-ambient ionization MALDI-based mass spectral imaging (MSI) technique for mapping neuropeptides and amine neurotransmitters in identified neurons and feeding circuits, with enhanced spatial resolution and speed; (3) Developing a multi-target affinity-enhanced microdialysis in vivo sampling technique for MS detection and quantitation of circulating neuropeptides and biogenic amines in response to food intake, via a novel hybrid absolute and relative quantification (HARQ) strategy using isobaric dimethylated leucine (DiLeu) and isotopic DiLeu tags; and (4) Determining the functional consequences of neuropeptide isoforms and assaying functional activities of hormonal cocktails by combining mass spectrometric, electrophysiological, and behavioral studies. Novel neuropeptides will be evaluated for functional roles in feeding regulation. 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 tempo...