This proposal brings together two laboratories with complementary experimental and computational expertise to determine the complexity of the extracellular space (ECS) in the brain at nanoscopic resolution in live tissue and its effects on neuronal function. The Spanish laboratory has spearheaded the development of super-resolution shadow imaging (SUSHI). The US team will use computer models to study the consequences of using competing hypotheses of how the ECS complexity affects the diffusion of neurotransmitters, which they will test experimentally. Neuronal structural complexity can cause a breakdown of classical diffusion, resulting in anomalous diffusion. A molecule can experience anomalous and normal diffusion at different temporal scales. Depending on which diffusional process dominates will affect the spread and relative concentration of ECS diffusing signals, such as neurotransmitters. SUSHI experiments will measure the structure of the ECS in live hippocampal organotypic and acute slices focusing on perisynaptic excitatory and inhibitory areas of CA1 pyramidal neurons. The values of the ECS volume fraction and the pore-size of the interstitial fluid (ISF) will be determined as a function of age, which is known to affect the ECS structure. SUSHI will be used to measure diffusional properties of the ECS at nanoscopic resolution over microscopic regions. These measurements will determine whether diffusion is anomalous or normal and over which periods of time. Monte Carlo and differential equation models and theory will examine the anatomical properties that cause anomalous or normal diffusion. The models will be extended to predict the effects of ECS structure on biochemical signaling at different spatio-temporal scales. The team will test the hypothesis that modifying the ECS volume directly affects the relative strength of neurotransmitter signals. Experiments will use a combination of electrophysiology with pharmacological and optogenetic manipulations to determine the effects of changes in ECS volume on the relative strength of excitatory and inhibitory (tonic and phasic) synapses. The experimental manipulations will mimic pathological conditions such as epilepsy and gliosis. The experiments will be the basis to build biophysical detailed models of pyramidal hippocampal cells to determine how the excitability of these cell changes as a function of ECS structure.