A Species‐Dependent Spatial Patterning of FOXP1 and nNOS Protein Expression in the Adult Mammalian Subthalamic Nucleus

The subthalamic nucleus (STN) is a key component of basal ganglia circuitry involved in motor, cognitive, and limbic functions. While the transcription factor FOXP1 has been implicated in neurodevelopmental disorders and basal ganglia function, its expression in the adult STN remains insufficiently characterized. Our study investigated the expression and distribution of FOXP1 protein and its colocalization with neuronal nitric oxide synthase (nNOS) and a pan-neuronal marker (HuC/HuD) in the adult STN across three mammalian species: mouse, rat, and human. Using immunofluorescent double-labeling and quantitative analysis, we examined their spatial organization and relative proportions within the neuronal population. FOXP1 was identified as a substantial neuronal subpopulation, comprising approximately 40%-45% of STN neurons across all species. In contrast, nNOS-positive neurons showed marked interspecies variation, increasing from ∼32% in mice to ∼68% in humans. Notably, the spatial distribution of these markers differed significantly between species. In rodents, FOXP1+ and nNOS+ neurons were largely segregated along the dorsoventral axis, forming visibly distinct subdomains. Conversely, in the human STN, these populations were diffusely intermixed with a higher degree of colocalization. Additionally, both markers exhibited species-specific antero-posterior gradients. These findings provide the first evidence of FOXP1 protein expression in the adult rat and human STN and highlight fundamental interspecies differences in neuronal organization. The expanded nNOS+ population and increased FOXP1/nNOS colocalization in humans suggest evolutionary divergence in the mammalian STN cytoarchitecture. Overall, the results challenge simplified models of STN subdivision in mammals, primates especially, and underscore the need for caution when extrapolating rodent data to human neurophysiology.