Transcriptional and functional profiles of muscarinic receptor-expressing neurons in primate lateral prefrontal and anterior cingulate cortices

Acetylcholine differentially modulates anterior cingulate (ACC) and lateral prefrontal (LPFC) cortices for cognitive-emotional integration, but cell-specific expression and function of muscarinic receptors (mAChR) and corresponding CHRM1-4 genes in these areas of the primate brain are largely unknown. Our single-nucleus RNA sequencing and mRNA-protein histology in macaques revealed CHRM3 as the most enriched mAChR gene in neurons, while m1 predominates at the protein level, likely due to nuclear retention of CHRM3 and cytoplasmic trafficking of CHRM1. CHRM3 and CHRM1 showed strong co-expression and functional overlap, and were transcriptomically-distinct from CHRM2, which was uniquely enriched in deep layer excitatory and PVALB+ inhibitory neurons. Between-region comparisons showed that CHRM3 is enriched in LPFC relative to ACC excitatory neurons. Further, CHRM1-3+ neurons showed region-specific transcriptomic signatures, with upregulation of synaptic plasticity genes in ACC relative to LPFC. Functional in vitro experiments confirmed a robust cholinergic-mediated decrease in excitatory and increase in inhibitory synaptic tone specific to ACC neurons, accompanied by changes in spine morphology. In contrast, cholinergic stimulation reduced inhibitory current amplitude in LPFC, shifting the microcircuit towards a stronger excitatory tone. These findings highlight region-specific acetylcholine signaling essential for flexible processing, learning and memory, which may underlie neurochemical circuit imbalance in neuropsychiatric disorders.