This project will characterize and quantify the diffusion of molecules in brain extracellular microenvironment (BEM). The results will also reveal the geometry of the extracellular space and how substances are removed by uptake processes. The work will use a micropipette as a point-source to release molecules into the brain by iontophoresis, a pressure pulse or a controlled infusion. The subsequent distribution of the molecules will be measured using ion-selective microelectrodes (for tetramethylammonium and potassium), fast scan cyclic voltammetry (for dopamine) and integrative optical imaging (for fluorescent dextrans and albumins). Experiments will be made on the rat cortical slice, the isolated turtle cerebellum and the anesthetized frog. There are 3 specific aims: 1) To analyze the structural properties of the BEM. This will seek to discover if some regions are inaccessible to large molecules but not to small ones and whether hyaluronate or similar molecules impede diffusion. Other experiments will explore the transition from diffusion to bulk flow when molecules are infused into the brain at increasing rates and investigate whether there is evidence for endogenous bulk flow in the brain. 2) To compare regional uptake kinetics and diffusion of dopamine. Using an extension of the diffusion equation with Michaelis Menten uptake kinetics to interpret the data, dopamine diffusion in the striatum, nucleus accumbens, substantia nigra and ventral tegmental area will be studied. Uptake blockers will be used to further characterize the kinetic properties. 3) To quantify the contribution of spatial buffering to potassium homeostasis. The diffusion equation will be extended to accommodate spatial buffering according to the methods of Gardner-Medwin and the point source paradigm will be used to determine the contribution of the spatial buffer to removal of potassium from the BEM under different conditions. Blockers of the inward rectifier channels responsible for the spatial buffer and the role of gap junctions will be tested. Further experiments will investigate other mechanisms for potassium homeostasis such as KCl uptake. This study is relevant to nonsynaptic communication between cells by means of diffusing chemical messengers, the characterization of the role of diffusible factors in development, dopamine replacement therapies to alleviate Parkinson's disease, prevention of potassium-induced cell damage and the formulation of strategies for drug delivery to the brain.
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