Development of Tools for Chemical Sampling and Analysis of Neuronal Tissue Ex Vivo

Characterizing the role of neurotransmitters in the central nervous system (CNS) is an important task in neuroscience. Chemical analysis of the extracellular fluid from the CNS will provide functional information regarding signaling processes during normal or disease conditions. In vivo sampling methods such as microdialysis, low flow push pull perfusion (LFPS) and direct sampling have helped in monitoring neurotransmitters in the CNS. While in vivo models are useful and important, excised CNS tissue is one of the most used models because it maintains a functional circuitry once removed from the organism. The aim of this thesis is to develop a new approach for excised neuronal tissue, for collect its extracellular fluid and analyze its chemical content. In the first series of experiments I present the feasibility of using in vivo sampling methods to collect extracellular fluid from excised rat retinas. Microdialysis, LFPS and direct sampling approaches were used to collect and monitor endogenous amino acid levels of excised retinas. These methods were used to monitor fluctuations in amino acid concentrations when excised retinas were exposed to dark and light environments. Results suggest that LFPS is the best approach to monitor the chemical content in excised tissue, due to the ability to directly access the target sampling area while causing minor tissue damage. A set of experiments with LFPS was used to characterize a new nano-liter ejection device that can manipulate the microenvironment of excised tissue with a degree of focal delivery. These experiments have shown the capabilities of the LPFS sampling method to characterize such device along with microscopy data to form a clear picture of the ejection process. This made possible the calculation of recovered concentration that lead to the discovery of the major role of dead volume and chromatographic broadening in the sampling process. The use of LFPS was further expanded when applied to collect samples from thin brain slices of mice. The amino acid concentrations were monitored at the hippocampus of control and mutant mice that do not express the cysteine glutamate transporter protein (xCT). A 61% difference in the observed extracellular glutamate was found between control and xCT-/- mice, as seen by others. Collecting samples at a higher sampling frequency following a significant temperature change in tissue bath does not appear to influence observed glutamate levels. Moreover probe placement in acutely sliced tissue does not appear to show a contribution due to the likely presence of blood that affects perfusate glutamate levels. Finally LFPS findings were used to complement electrophysiology data. In the future the combination of electrophysiological data collection with LFPS should provide a more complete picture on the state of the tissue in brain slice experiments.