Abstract
BACKGROUND: The brain extracellular space (ECS) is a convoluted compartment of nano- to microscale interconnected ducts filled with interstitial fluid and lined by neural cell membranes. A key step in signaling between neural cells is diffusion through the ECS of transmitter molecules released from point sources distributed throughout the parenchyma. Yet, signaling is generally considered solely from the stance of cellular properties, while disregarding ECS sojourn time and putative signal modulation at this phase. Where ECS diffusion is considered, it is commonly done based on volume-averaging techniques blind to individual signaling events or actual ECS structural geometry. This has precluded knowledge on how specific ECS geometries may impact diffusion and modulate signaling arising from individual transmitter release events. We hypothesized that ECS geometry can shape local diffusion gradients resulting from individual point source release events and thereby tune signaling, and we further propose that this modulation can impart non-random functionality. METHODS: To access the scale of individual transmitter release events and true ECS geometries, we used super-resolution STED microscopy to image the ECS in the hippocampal CA1 stratum radiatum of live mouse brain slices. We then developed a computational diffusion model, DifFlux, based on super-resolved images of hippocampal ECS and applied this along single molecule Monte Carlo diffusion simulations. Our approach allows us to simulate diffusion of molecules of our choosing in actual live ECS geometries. RESULTS: We observed local anomalous and anisotropic diffusion imposed by ECS geometry, whereby diffusion along larger structures was more directional than in denser neuropil of finer cellular structures. Further, we identified that the perisynaptic ECS geometries around respective glutamatergic and GABAergic synapses imposed distinct functional advantages, shedding light on the longstanding conundrum of why glutamatergic and GABAergic synapses are so conspicuously morphologically different. CONCLUSIONS: Our modelling broadly identifies ECS structure as a direct modulator of extrasynaptic signaling that can operate in parallel to conventional regulation mechanisms. This ultimately provides a metabolic and computational advantage to the brain. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-026-00797-3.