The overall goal is to characterize how substances diffuse in the extracellular space of the brain. This will clarify the structure of extracellular space, demonstrate the potential for extracellular chemical communication. The study will help improve therapeutic intervention and aid targeting of drugs and bioactive substances in brain tissue. Appropriate solutions to the diffusion equation will be used to interpret experimental results. All experiments will be made on slices from various brain regions of Sprague-Dawley rats. The three Specific Aims are to: 1) improve optical methods for measuring diffusion 2) quantify the diffusion and uptake of dopamine 3) describe diffusion in the developing brain.
Specific Aim 1 is further development of quantitative optical imaging and related theory for analyzing diffusion. New experimental and theoretical methods will use high-resolution imaging of molecules tagged with fluorescent dyes. The technique will be extended to fluorescent dextrans above 70 kDa in molecular weight and proteins. It will measure extracellular volume fraction as well as apparent tortuosity in homogeneous and heterogeneous regions of extracellular space and account for loss of the diffusing substance to the intracellular compartment and at the surfaces of slices. These methods will have wide applicability beyond this proposal.
Specific Aim 2 is to study the relative roles of Michaelis-Menten kinetics and diffusion in the migration of dopamine in the extracellular space. Fast-scan cyclic voltametry and numerical solutions to the diffusion equation will describe the behavior of dopamine release by pressure ejection from a micropipette into the neostriatum, the nucleus accumbens and the substantia nigra. This work will help to decide the potential of dopamine as an agent for extra-synaptic volume transmission and will provide data for the modeling of dopamine replacement strategies to ameliorate Parkinson's Disease.
Specific Aim 3 is to characterize the diffusion properties of the extracellular space in the normal developing brain and under hypoxia, ischemic and osmotic stress. Fluorescent dextrans and proteins will be used with quantitative optical imaging to determine how the changing structures of the extracellular space affect diffusion in slices of the neocortex, from post- natal day 1 to day 21 and in the adult brain. Slices will also be subjected to hypoxia with and without glucose (to simulate ischemia) and to hypo- and hyperosmotic stress to see how the developing brain structure reacts to these insults. A Monte Carlo simulation will be programmed to model data that cannot be described by analytical or numerical solutions to the diffusion equation. These experiments will aid discussions of the role of diffusion in the developmental process and how susceptible is the structure of extracellular space to various insults at different ages.
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