Advances in in Vivo Neurochemical Monitoring Using Microdialysis with Liquid Chromatography-Mass Spectrometry

Abstract

Improving our understanding of the central nervous system necessitates developing techniques capable of making neurochemical measurements in the complex extracellular environment of the brain within living animal models. Microdialysis is a method in which a semipermeable membrane is implanted into the brain and snapshots of the chemical content surrounding the probe are collected and analyzed offline using a variety of analytical assays. Liquid chromatography-mass spectrometry is a powerful method for separation and detection of biomolecules in dialysate. The aim of this thesis is to improve microdialysis collection and analysis using liquid chromatography-mass spectrometry by improving quantification, analysis speed, and simplicity of operation. A method was developed for the analysis of 26 neurochemicals in dialysate with comparable figures of merit at 30% of the analysis time of previous reports. The methods are applied to the study of addiction and regulation of amino acid metabolism in the brain. Determining absolute in vivo concentrations from analysis of dialysate is challenging because the mass transport processes in the extracellular space have unknown effects on probe recovery. Several methods for calibrating microdialysis in vivo have been implemented, though all require assumptions regarding calibrant mass transport or sacrifice of temporal information. Retrodialysis of stable-isotope-labeled forms of endogenous compounds can be used for accurate recovery calibration without losing temporal resolution. We applied this method to the study of dopamine in rat models of cocaine addiction and as a result were able to differentiate between groups with different self-administration experience and correlate this to behavioral indicators of addiction. Microdialysis calibration was also utilized for accurate basal measurement of serine, glycine, threonine, alanine, glutamate, and γ-aminobutyric acid in mice. A comparative study was performed which demonstrated a 25% decrease in extracellular D-Ser levels in ASCT1-KO animals. Benzoyl chloride derivatization of neurochemicals prior to LC-MS/MS analysis improves the assay; however, derivatizing large sample sets is labor intensive and prone to error. Moreover, collecting small fractions (≤ 1 min) presents challenges to benzoylation accuracy and precision. The development of a novel microfluidic device for automated, on-chip benzoylation is described. This method yielded similar limits of detection, repeatability, and linearity to offline derivatization. The platform was successfully implemented for quantitation of basal and potassium/bicuculline-stimulated neurochemicals in awake rats. In vivo neuropeptide monitoring presents challenges due to low extracellular concentrations and poor microdialysis recovery. A capillary liquid chromatography-mass spectrometry assay for the quantitation of sub-pM levels of three neuropeptides believed to be released by proopiomelanocortin neurons: α-melanocyte-stimulating hormone (1-13), α-melanocyte-stimulating hormone (1-12), and [des-acetyl] α-melanocyte-stimulating hormone is described. An in vivo study of mice showed that extracellular levels of the three isoforms were below our limits of detection. Our methods will be adapted for future use in measuring other neuropeptides in dialysate.