Whole-Brain Mapping of Choline Acetyltransferase Neurons Expression in Tree Shrew Compared with Mice

The central cholinergic system regulates diverse neurological functions, including memory, learning, attention, arousal, sleep, emotion regulation and behavior control. As a close evolutionary relative of primates, tree shrew is a valuable comparative model for neurobiological investigation. However, the anatomical distribution of choline acetyltransferase-immunoreactive (ChAT-ir) neurons in its brain remains poorly characterized. Using ChAT immunofluorescence, we systematically mapped the whole-brain distribution and morphology of ChAT-ir neurons in tree shrew and compared them with mouse. In the neocortex, ChAT-ir neurons were absent in tree shrew, whereas mouse showed sparse cortical labeling. Semi-quantitative analyses of subcortical regions revealed the overall distribution was largely conserved, whereas tree shrew exhibited higher ChAT-ir neuronal density in the trochlear and prepositus nuclei compared to mice. ChAT-ir neurons were detected in the suprachiasmatic and supraoptic nuclei in the hypothalamus in tree shrew but not in mouse. Tree shrew ChAT-ir neurons showed greater dendritic complexity in caudate nucleus, medial septum, pedunculopontine/dorsal tegmental nuclei, and prepositus nucleus, while the horizontal limb of the diagonal band and oculomotor nuclei exhibited similar complexity across species. In the putamen, tree shrew has more complex distal dendrites but less complex proximal branches than mice. ChAT-ir neurons displayed a rostral-to-caudal density gradient in caudate, while mice showed largely uniform distributions of ChAT-, CB-, PV-, and CR-positive neurons across caudate putamen medial and lateral side. These findings provide a comprehensive mapping of ChAT-ir neurons in the tree shrew brain, highlighting significant interspecies differences and offering a structural framework for investigating cholinergic roles in diverse neural functions.