The extracellular space (ECS) is the microenvironment of the nerve cells and an important communication channel, allowing for long distance extrasynaptic communication between cells. Changes in ECS size, geometry, and composition have been reported in diverse (patho)physiological states, including aging. In the present study, real-time tetramethylammonium (TMA(+)) iontophoresis was used to quantify ECS diffusion parameters in different brain regions of adult and behaviorally characterized aged rats. Prior to ECS diffusion measurement, superior and inferior learners were selected from a large group of aged rats, according to their performance in the open-field water maze. The main finding was that the degree of impaired maze performance of old rats correlates, firstly, with decrease in ECS volume, loss of diffusion anisotropy in hippocampus, and degree of astrogliosis, and secondly, with disorganization of the astrocytic processes and reduction of hippocampal ECS matrix molecules. Importantly, no significant differences were found in the density of neurons in any region of the hippocampus or dentate gyrus. The alterations in hippocampal diffusion parameters evident in aged animals with severe learning deficits could account for the learning impairment, due to their effects on extrasynaptic volume transmission and/or on the "cross-talk" between synapses, which has been suggested to be involved in neural processes associated with learning and memory formation. (C) 2002 Wiley-Liss, Inc.