Whole-brain mapping of choline acetyltransferase neurons expression in tree shrew compared with mice

The central cholinergic system regulates diverse neurological functions, learning, attention, arousal, sleep, emotion regulation and behavior control. As a close evolutionary relative of primates, the 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 the tree shrew and compared them with those in the mouse. In the neocortex, ChAT-ir neurons were absent in the tree shrew, whereas the mouse showed sparse cortical labeling. Semi-quantitative analyses of subcortical regions revealed the overall distribution was largely conserved, whereas the 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 the tree shrew but not in the mouse. Tree shrew ChAT-ir neurons showed greater dendritic complexity in the 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, the 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 the caudate, while mice showed largely uniform distributions of ChAT-, CB-, PV-, and CR-positive neurons across the medial and lateral sides of the caudate putamen. 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.