Are Synaptic Clefts Directionally Oriented?

Synapses are fundamental building blocks of cortical circuits, yet their geometry is typically regarded as a local property, independent of mesoscale architecture. The prevailing assumption is that synaptic clefts are isotropically oriented in space. Here, this assumption was tested by analyzing approximately 117 million synaptic clefts from two independent 1 mm ³ electron microscopy datasets: the human H01 middle temporal gyrus and the mouse MICrONS primary visual cortex. Across both volumes, synaptic cleft orientations are not randomly distributed, but instead show statistically significant and spatially coherent directional biases across cortical layers. This mesoscale anisotropy is conserved across species, yet is stronger and more consistent in human association cortex than in mouse sensory cortex. These findings reveal an unrecognized dimension of cortical microarchitecture and suggest that synaptic geometry contributes to circuit organization, mesoscale connectivity, and interactions with endogenous or externally applied electric fields.