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The anterior cingulate cortex (ACC) is critical for pain perception, emotion and cognition. Previous studies showed that the ACC has a complex network architecture, which can receive some projection fibers from many brain regions, including the thalamus, the cerebral cortex and other brain regions. However, there was still a lack of whole-brain mapping of the ACC in adult mice. In the present study, we utilized a rabies virus-based retrograde trans-monosynaptic tracing system to map whole-brain afferents to the unilateral ACC in adult mice. We also combined with a new high-throughput, high-speed and high-resolution VISoR imaging technique to generate a three-dimensional whole-brain reconstruction. Our results showed that several principal groups of brain structures send direct monosynaptic inputs to the ACC, including the cerebral cortex, amygdala, striatum, the thalamus, and the brainstem. We also found that cortical neurons in the ACC mainly receive ipsilateral monosynaptic projections. Some cortical areas and forebrain regions also bilaterally projected to the ACC. These findings provide a complete analysis of the afferents to the ACC in adult mice, and whole-brain mapping of ACC afferents would provide important anatomic evidence for the study of pain, memory, and cognition. [ABSTRACT FROM AUTHOR]

The anterior cingulate cortex (ACC) is a key cortical region that plays an important role in pain perception and emotional functions. Previous studies of the ACC projections have been collected primarily from monkeys, rabbits and rats. Due to technological advances, such as gene manipulation, recent progress has been made in our understanding of the molecular and cellular mechanisms of the ACC-related chronic pain and emotion is mainly obtained from adult mice. Few anatomic studies have examined the whole-brain projections of the ACC in adult mice. In the present study, we examined the continuous axonal outputs of the ACC in the whole brain of adult male mice. We used the virus anterograde tracing technique and an ultrahigh-speed imaging method of Volumetric Imaging with Synchronized on-the-fly-scan and Readout (VISoR). We created a three-dimensional (3D) reconstruction of mouse brains. We found that the ACC projected ipsilaterally primarily to the caudate putamen (CPu), ventral thalamic nucleus, zona incerta (ZI), periaqueductal gray (PAG), superior colliculus (SC), interpolar spinal trigeminal nucleus (Sp5I), and dorsal medullary reticular nucleus (MdD). The ACC also projected to contralateral brain regions, including the ACC, reuniens thalamic nucleus (Re), PAG, Sp5I, and MdD. Our results provide a whole-brain mapping of efferent projections from the ACC in adult male mice, and these findings are critical for future studies of the molecular and synaptic mechanisms of the ACC and its related network in mouse models of brain diseases. [ABSTRACT FROM AUTHOR]