The long-term goal is to understand the biophysics of substance diffusion in brain extracellular space (ECS). Biomedically, this proposal will enhance understanding of impediments to drug delivery in brain and factors that affect chemical signaling in the ECS. The ECS comprises the ensemble of narrow interstitial spaces between brain cells and harbors an extracellular matrix (ECM) of long chain glycosylated molecules. Hindrance to diffusion in the ECS is characterized by a composite parameter, the tortuosity, that includes a) increase in geometric path-length, b) dead-space microdomains, c) obstruction and interstitial viscosity, d) specific binding to ECM and e) nonspecific interaction with fixed negative charges on the ECM. These factors will be explored through the diffusion of substances using integrative optical imaging of fluorescent macromolecules combined with realtime iontophoresis of tetramethylammonium and other ions both in vivo and in brain slices. Aim 1: What is the largest molecule or particle that can traverse the ECS? Preliminary experiments show that macromolecules with 40 nm diameter can diffuse in vivo. A further range of molecular sizes will be explored in normal and osmotically modified ECS. Aim 2: How much does specific ECM binding hinder diffusion of biologically important molecules? Diffusion of the naturally occurring model protein, lactoferrin, will assay specific binding with heparan sulfate proteoglycans of the ECM. Aim 3: Is there charge discrimination in the ECS? This aim will study the non-specific interaction of diffusing mono- and multivalent ions and biogenic amines with the fixed negative charges of the ECM. Aim 4: Model dead-space microdomains, binding and charge interaction and unify software. Models and algorithms for data analysis will be developed and the custom software unified to facilitate outside users