Iosif_2026_J.Neurosci__

The cerebellum plays a key role in coordinating balance and movement control. Most studies of cerebellar function focus on the role of cerebellar glutamatergic inputs, thus the roles of neuromodulatory inputs remain unexplored. We sought to determine whether the cholinergic projections from the pedunculopontine nuclei (PPN) to the interpositus nuclei of the cerebellum are involved in modulating performance of a beam traversal task in rats. We manipulated cholinergic signalling in the cerebellum using chemogenetic and pharmacological methods. Experiments were conducted in male rats, except for studies specifically targeting PPN cholinergic neurons, which included rats of both sexes. Chemogenetic inhibition of either a mixed population of projections from the PPN to interpositus nuclei, or specific inhibition of cholinergic PPN projections, improved balance and foot placement on the beam traversal task. This effect is likely mediated by nicotinic receptors, as infusion of the nicotinic receptor antagonist mecamylamine improved balance and foot placement accuracy. In contrast, enhancing cholinergic signalling using the cholinesterase inhibitor physostigmine reduced accuracy of foot placement. Surprisingly, infusion of muscarinic receptor antagonists mimicked the effect of cholinesterase inhibition leading to impaired motor performance. We investigated the cellular effects of cholinergic receptor activation using adult rat cerebellar slices. Interpositus nuclear neurons exhibited decreased intrinsic excitability and reduced responsivity to synaptic inputs in presence of a cholinergic agonist. Together, our findings indicate that low levels of acetylcholine in the cerebellar interpositus are optimal for performance of the beam traversal task, while enhancing cholinergic signalling decreases interpositus excitability and impairs task performance.Significance statement Pontine cholinergic centres are impacted in various disease states, with changes in cholinergic signalling linked to severe motor deficits such as in Parkinson's disease. The cerebellum is central to the regulation of motor control, but the roles of acetylcholine in cerebellar motor control are unclear. We set out to identify the function of the cholinergic pedunculopontine nuclei to cerebellar nuclei pathway in the beam traversal task in rats and found that inhibiting this pathway improved motor performance. Our results suggest that low levels of acetylcholine in the cerebellum are optimal for motor performance. The identified role of this pathway in normal motor behaviour will reveal how this pathway may be targeted to improve motor symptoms in disease states.