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, we test this assumption by analyzing approximately 117 million synaptic clefts from two independent 1 mm³ electron microscopy datasets: the human H01 middle temporal gyrus and the mouse MICrONS primary visual cortex, using three independent cleft-extraction methods. Across both volumes, we observe that 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, a difference that may reflect the expanded dendritic arbors and greater integrative demands of human pyramidal neurons. We propose that cleft orientation bias is a geometric consequence of the axonal and dendritic architecture that shapes synapse formation, representing a new candidate organizational feature of cortical microarchitecture with potential implications for circuit computation and neuromodulation. These findings motivate targeted physiological studies to determine whether synaptic orientation contributes causally to cortical function.