Novel tool for sampling in vivo neurochemicals from the central nervous system

Understanding the role of neurochemicals in the central nervous system (CNS) remains one of the most daunting tasks in neuroscience today. Chemical analysis of the extracellular space of CNS will provide functional information regarding the roles of these signals during normal and disease conditions. The goal of this thesis is to develop a novel in vivo sampling method to collect diverse chemicals from specific regions of the CNS. The push-pull perfusion (PPP) technique was among the earliest scientific approaches to collecting extracellular fluid from the brain. Although it was largely successful for nearly any signaling molecule, it suffered relative to other sampling techniques due to the significant amount of tissue damage that occurred with sample collection. I developed a low-flow, in vivo collection method based on the classical PPP concept that provides reduced tissue damage. It is capable of collecting extracellular fluid from different regions of the CNS with minor tissue damage, high collection efficiency and high special resolution. In vitro studies demonstrate that the recovery of designed push-pull probe was 70--80% over the 10--50 nL/min flow rates.; Primarily, this technique is applied for in vivo studies of the rat striatum. Collected samples are assayed for amino acids by capillary electrophoresis with laser-induced fluorescence. The basal glutamate level at the striatum was determined to be 1.97 +/- 0.70 muM and is similar to other reports. During sample collection, injection of a glutamate reuptake blocker, L-trans-pyrrolidine-2-4-dicarboxylic acid (PDC) leads to a 4000% average increase of basal glutamate level. This confirms that the source of glutamate is neuronal, a requisite consideration for studying the glutamate neurotransmitter system. The push-pull probe was miniaturized for in vivo collection from the vitreous and vitreo-retinal interface (VRI) of the rat eye. Sampling from these regions demonstrates that there is no significant difference between amino acid basal levels at vitreous and VRI. The possibility of studying specific regions of the retina was tested. PDC infusion at the optic nerve head (ONH) shows an average increase of glutamate that is significantly lower than infusion at peripheral sites. This confirms the high spatial resolution of this technique and opens the door to further studies of disease models where different regions of the retina are effected. Glaucoma is one such disease and experiments with rat glaucoma models demonstrate that there is a higher glutamate level with induced glaucoma compared to controls.; Finally, this sampling method is broadly applicable to most of the known neurochemical messengers, with an ability to collect the wide diversity of molecular signals with high regional specificity. It will be possible to provide new insights into functional and disease states of the CNS.