Simultaneously cholinergic projection in Ascending and Descending Circuits from Midbrain

The midbrain participates in complex neural information processing in the ascending and descending circuits, but their organization remains unclear due to the lack of comprehensive dissection of the characterization of individual neurons. Combining fluorescent micro-optical sectional tomography with sparse labeling, we acquired the whole-brain dataset with high resolution and reconstructed the detailed morphology of the pontine-tegmental cholinergic neurons (PTCNs). As the main cholinergic system of the midbrain, the individual PTCNs own abundant axons with length up to 60 cm and 5000 terminal branches and innervate multiple brain regions from the spinal cord to cortex in both hemispheres. According to various targeting regions in the ascending and descending circuits, individual PTCNs could be grouped into four types and the axonal fibers of cholinergic neurons in the pedunculopontine nucleus present more divergent while neurons in the laterodorsal tegmental nucleus contain richer axonal fibers and dendrites. In the axonal targeting nuclei, such as in the thalamus or cortex, the individual neurons innervate multiple sub-regions with separate pathways. These results provide the detailed organization characterization of the cholinergic neurons to understand the connection logic of the midbrain.