3D ultrastructural comparison of synapses in thalamic and non-thalamic recipient layers of macaque V1

Abstract Understanding synaptic characteristics across cortical layers is essential for elucidating the functional architecture of brain regions. In this study, we compared the synaptic structure in the input layers of primate primary visual cortex (layer 4C) and the output layer (layer 3B) using focused ion beam scanning electron microscopy. We quantified synaptic density, classified boutons by synapse number and mitochondrial content, and measured morphometric parameters—including bouton volume, postsynaptic density area and morphology, mitochondrial volume, and postsynaptic targets—in each layer. Our results revealed that all layers show a higher proportion of single-synapse boutons without mitochondria. Multisynaptic boutons containing mitochondria, likely corresponding to thalamocortical terminals, were significantly more abundant in thalamocortical recipient layers 4Cα and 4Cβ. These multisynaptic boutons containing mitochondria were larger, more likely to contact dendritic spines, and contained more mitochondria than other boutons. In contrast, layer 3B displayed a lower prevalence of multisynaptic boutons containing mitochondria; these boutons were smaller than those in layer 4C and formed fewer synapses. These findings highlight laminar differences in bouton architecture and support the idea that thalamocortical synapses are structurally adapted for high synaptic efficacy, providing a quantitative framework for understanding synaptic organization in primate V1 and implications for sensory processing and cortical circuit function.