Three-dimensional ultrastructural differences between thalamic and non-thalamic recipient layers in macaque V1

Understanding the synaptic characteristics of each cortical layer is essential for elucidating the functional architecture of each brain region. In the current study, we made a detailed quantitative comparison of the synaptic structure in the predominantly input layers of primate primary visual cortex (layer 4C) and in the predominant output layer (layer 3B) using focused ion beam scanning electron microscopy (FIB/SEM). We quantified the synaptic density in each layer, classified synaptic boutons according to their number of synapses and mitochondrial content, and quantified key morphometric parameters, including bouton volume, postsynaptic density (PSD) area and morphology, volume occupied by mitochondria, and postsynaptic targets. Our results revealed that for all the layers there is a higher proportion of single-synapse boutons without mitochondria. Multisynaptic boutons containing mitochondria (MSBm+)— which likely correspond to TC terminals —were significantly more abundant in the thalamocortical recipient layers 4Cα and 4Cβ. These MSBm+ boutons were also larger, more likely to contact dendritic spines, and contained more mitochondria than other bouton categories. In contrast, layer 3B, displayed a lower prevalence of MSBm+ boutons, these boutons were smaller than those in layer 4C and made fewer synapses. These findings highlight laminar differences in bouton architecture and support the idea that TC synapses are structurally adapted to support high synaptic efficacy. Together, our data provide a detailed quantitative framework for understanding the synaptic organization of primate V1, with implications for sensory processing and cortical circuit function.