Whole-brain mesoscale mapping of primates has been hindered by large brain size and the relatively low throughput of available microscopy methods. Here, we present an integrative approach that combines primate-optimized tissue sectioning and clearing with ultrahigh-speed, large-scale, volumetric fluorescence microscopy, capable of completing whole-brain imaging of a rhesus monkey at 1 µm × 1 µm × 2.5 µm voxel resolution within 100 hours. A progressive strategy is developed for high-efficiency, long-range tracing of individual axonal fibers through the dataset of hundreds of terabytes, establishing a "Serial sectioning and clearing, 3-dimensional Microscopy, with semi-Automated Reconstruction and Tracing" (SMART) pipeline. This system supports effective connectome-scale mapping of large primates that reveals distinct features of thalamocortical projections of the rhesus monkey brain at the level of individual axonal fibers.

The anterior cingulate cortex (ACC) is located in the frontal part of the cingulate cortex, and plays important roles on pain perception and emotion. The thalamocortical pathway is the major sensory input to the ACC. Previous studies show that several different thalamic nuclei receive projection fibers from spinothalamic tract, that in turn send efferents to the ACC by using neural tracers and optical imaging methods. Most of these studies were performed in monkeys, cats and rats, few studies were reported systematically in adult mice. Adult mice, especially genetically modified mice, have provided molecular and synaptic mechanisms for cortical plasticity and modulation in the ACC. In the present study, we utilized rabies virus-based retrograde tracing system to map thalamic-anterior cingulate monosynaptic inputs in adult mice. We also combined with a new high-throughput VISoR imaging technique to generate a three-dimensional whole-brain reconstruction, especially the thalamus. We found that cortical neurons in the ACC received direct projections from different subnuclei in the thalamus, including the anterior, ventral, medial, lateral, midline and intralaminar thalamic nuclei. These findings provide key anatomic evidences for the connection between the thalamus and ACC.

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