Long-range neuronal circuits play an important role in motor and sensory information processing. Determining direct synaptic inputs of excited and inhibited neurons is important for understanding the circuit mechanisms involved in regulating movement. Here, we used the monosynaptic rabies tracing technique, combined with fluorescent micro-optical sectional tomography, to characterize the brain-wide input to the motor cortex (MC). The whole brain dataset showed that the main excited and inhibited neurons in the MC received inputs from similar brain regions with a quantitative difference. With 3D reconstruction we found that the distribution of input neurons, that target the primary and secondary MC, had different patterns. In the cortex, the neurons projecting to the primary MC mainly distributed in the lateral and anterior portion, while those to the secondary MC distributed in the medial and posterior portion. The input neurons in the subcortical areas also showed the topographic shift model, as in the thalamus, the neurons distributed as outer and inner shells while the neurons in the claustrum and amygdala were in the ventral and dorsal part, respectively. These results lay the anatomical foundation to understanding the organized pattern of motor circuits and the functional differences between the primary and secondary MC.

Resin embedding has been widely used for precise imaging of fluorescently labeled biological samples with optical and electron microscopy. The low preservation rate of fluorescence, especially for red fluorescent proteins, has limited the application of resin embedding in multifluorescent protein-labeled samples. Here, we optimized the embedding method to retain the intensity of multiple fluorescent proteins during resin embedding. By reducing the polymerization temperature from 50 to 35°C and adding a fluorescent protein protection reagent during the embedding process, we successfully increased the fluorescence preservation rate by nearly twofold for red fluorescent proteins, including tdTomato, mCherry, and DsRed. Meanwhile, the background fluorescence decreased significantly in the optimized embedding method. This method is suitable not only for red fluorescent protein-labeled samples but also for blue (BFP) and green fluorescent protein (GFP)-labeled samples. We embedded brains labeled with BFP, DsRed, and GFP via AAV and rabies virus and acquired the distribution of input neurons to different cortical areas. With GFP/tdTomato double-labeled samples in resin, we obtained the cholinergic projectome of the pedunculopontine tegmental nucleus (PPTg) and the distribution of cholinergic neurons at single-neuron resolution in the whole brain simultaneously. Input cholinergic terminals from the PPTg were found to innervate the cholinergic soma and fiber in the neocortex, basal forebrain and brainstem, indicating that local cholinergic neurons received long-range cholinergic modulation from the midbrain. Our optimized method is useful for embedding multicolor fluorescent protein-labeled samples to acquire multidimensional structural information on neural circuits at single-neuron resolution in the whole brain.

Dissection of the neural circuits of the cerebral cortex is essential for studying mechanisms underlying brain function. Herein, combining a retrograde rabies tracing system with fluorescent micro-optical sectional tomography, we investigated long-range input neurons of corticotropin-releasing hormone containing neurons in the six main cortical areas, including the prefrontal, somatosensory, motor, auditory, and visual cortices. The whole brain distribution of input neurons showed similar patterns to input neurons distributed mainly in the adjacent cortical areas, thalamus, and basal forebrain. Reconstruction of continuous three-dimensional datasets showed the anterior and middle thalamus projected mainly to the rostral cortex whereas the posterior and lateral projected to the caudal cortex. In the basal forebrain, immunohistochemical staining showed these cortical areas received afferent information from cholinergic neurons in the substantia innominata and lateral globus pallidus, whereas cholinergic neurons in the diagonal band nucleus projected strongly to the prefrontal and visual cortex. Additionally, dense neurons in the zona incerta and ventral hippocampus were found to project to the prefrontal cortex. These results showed general patterns of cortical input circuits and unique connection patterns of each individual area, allowing for valuable comparisons among the organisation of different cortical areas and new insight into cortical functions.