Abstract
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.
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Data availability
Raw and processed snRNA-seq data generated in this study have been deposited in the NCBI Gene Expression Omnibus database (GEO accession # GSE296153). Source data are provided in the Source Data files in Supplementary Materials.
Code availability
The R scripts used for data analysis are available on GitHub [https://github.com/campbio-manuscripts/Muscarinic_snRNAseq_ePhys].
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Acknowledgements
We thank Drs. Douglas Rosene and Tara Moore for access to resources and providing tissue for the experiments; Bethany Bowley, Samantha Calderazzo, Alexander Hsu, Eli Shobin, Penny Shultz, Ajay Uprety, Katelyn Batterman, Karen Bottenfield, Karen Slater, and Veronica Go for technical assistance during perfusion and brain cutting. We also thank Dr. Yuriy Alekseyev and the members of the Boston University Chobanian & Avedisian School of Medicine Microarray and Sequencing Resource Core and Flow Cytometry Core for their help and guidance with the sequencing experiments. We are grateful to NIH agencies for their support: NIH/NIMH R21MH126250 (MPI: M. Medalla and E. Zeldich), NIH/NIA R21AG072069 (MPIs: J.I. Luebke and E. Zeldich), NIH/NIMH R01 MH116008 (PI: M. Medalla); NIH/NIA: R01-AG059028 (MPIs: J.I. Luebke and P. Hof), R01LM013154 (PI: J.D.C.), R01/RF1AG068168 (PI: T.L. Moore), and RF1-AG043640 (PI: D.L. Rosene).
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A.T., C.A.M., and M.M. designed and performed aspects of all the experiments and aided in the bioinformatics analysis, interpreted the results, and wrote the first drafts of the paper. M.M., E.Z., and J.L. conceived and oversaw the project, designed and performed aspects of all experiments and analyses, provided guidance, interpreted the results, and wrote the paper. S.D.K., R.Y., S.A., and J.D.C. designed and performed bioinformatics and statistical analyses for the snRNA-seq dataset and edited the paper. B.J.S., W.C., T.G.V., J.G., A.C., Y.Z., I.L.T., and J.M. helped perform experiments, gather and analyze data, and edit the manuscript.
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Tsolias, A., Mojica, C.A., Yamani, R. et al. Transcriptional and functional profiles of muscarinic receptor-expressing neurons in primate lateral prefrontal and anterior cingulate cortices. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09866-7
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DOI: https://doi.org/10.1038/s42003-026-09866-7
