Transcriptional and functional profiles of muscarinic cholinergic receptor-expressing neurons in the primate prefrontal cortex

Acetylcholine modulates the anterior cingulate (ACC) and lateral prefrontal (LPFC) cortices via specific m1-m4 muscarinic receptor subtypes (mAChR) encoded by CHRM1-4 genes. 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, differing from CHRM2 mainly in calcium signaling-related genes. CHRM2 is uniquely enriched in deep layer excitatory and PVALB+ inhibitory neurons. Although CHRM+ cell distributions are similar between areas, CHRM1–3+ excitatory neurons in ACC exhibited upregulation of synaptic plasticity genes. Functional in vitro experiments confirm a more robust cholinergic-mediated decrease in excitatory:inhibitory synaptic ratio in ACC than in LPFC neurons, accompanied by compensatory changes in spine morphology. These findings highlight region-specific acetylcholine signaling essential for flexible processing, learning and memory. Overall design: snRNA-seq of ACC and LPFC tissue from 4 adult male rhesus monkeys (Macaca mulatta; ages= 8.5, 8.9, 20.8, 24.8 y.o.)