Liquid Chromatography-Mass Spectrometry Strategies for in vivo Neurochemical Monitoring with Microdialysis.

Liquid chromatography mass spectrometry (LC-MS) is a powerful analytical tool for multi-analyte quantification. This method can be combined with microdialysis sampling to study small molecules and neuropeptides within discrete brain regions. This thesis focuses on the development of targeted LC-MS assays to analyze dialysate samples collected from awake animals to correlate neurochemical dynamics with behavior. Previous LC-MS assays used benzoyl chloride derivatization to enable quantification of 17 neurotransmitters and metabolites in dialysate samples. In this work, derivatization conditions were modified to improve sensitivity up to 25-fold and reduce complexity of the procedure. The assay was also expanded to 70 compounds including amino acids, polyamines (e.g., putrescine, spermidine, spermine), compounds from catecholamine biosynthesis pathways (e.g., tyrosine and tryptophan metabolic pathways), and trace amines (e.g., tyramine, octopamine, synephrine). Besides measurements in dialysate, the method was able to analyze plasma and cerebrospinal fluid samples. This work improves the utility of benzoyl chloride, which labels multiple important functional groups, for widely targeted metabolomics methods. Neuropeptides constitute the largest group of neurotransmitters in the central nervous system. Neuropeptide signaling is involved in many physiological functions but detection in vivo is challenging due to low picomolar concentrations. Targeted capillary LC-MS methods were developed for neurotensin, oxytocin, dynorphin, and enkephalins. The assays utilize desalting and preconcentration on a single analytical column to achieve low picomolar limits of detection. Detection was improved by optimizing all facets of neuropeptide handling from sampling to detection with capillary LC-MS. These techniques were applied to examine several aspects of neuronal function. Specific neuronal circuits were defined, confirmed, and targeted by combining these analytical tools with pharmacogenetic and optogenetic methods. Novel pathophysiological changes to opioid neuropeptide (dynorphin and enkephalins) dynamics were elucidated in a rat model of Parkinson’s disease, and a potential neuropeptide-based treatment was established to reduce the abnormal dyskinetic movements associated with chronic dopamine replacement therapies in Parkinson’s Disease. These new multiplexed approaches will advance our understanding of the complex processes underlying neuronal function at the molecular and circuit levels, as well as provides an improved set of experimental tools to better understand lingering questions in the field of neuroscience.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135792/1/jmtwong_1.pd