Fig 2: Cholinergic agonist promoted motile post-synaptic spine morphologies in ACC and LPFC pyramidal neurons.a Box-and-whisker and vertical scatter plots of spine density (total and by subtype) on apical and basal dendrites of individual ACC and LPFC L3 pyramidal neurons in slices treated with control Ringer’s (n = 15 ACC, 9 LPFC cells from 7 cases), 10 min of 10 µM CCH (n = 5 ACC from 2 cases, 4 LPFC from 3 cases), and 10 min of 10 µM CCH followed by 15 min of washout in Ringer’s (n = 3 ACC, 6 LPFC from 2 cases). b Representative z-maximum projections of confocal images of mid-apical dendrites of control and CCH-treated ACC and LPFC L3 pyramidal neurons. The neurons were filled with biocytin and labeled with Streptavidin-Alexa 488. c Average proportion of spines per subtype on apical and basal dendrites of control (CTR), CCH, and CCH + Washout (W) treated L3 pyramidal neurons in ACC and LPFC, as represented by stacked bar graphs (Fisher’s exact test, p < 0.0001). d Box and whisker and vertical scatter plots of the average major diameter of spine subtypes on control, CCH, and CCH + Washout-treated pyramidal neurons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (see Supplementary Data 6). e Summary schematic of findings showing cell-specific CHRM1/3+ vs. CHRM2+ ExNs and InNs enrichment patterns, associated potential differences in mRNA–protein dynamics and trafficking of m1 vs. m3 (1), and regional differences in downstream functional effects of ACh on synaptic plasticity and function, within the context of known laminar and regional differences in excitatory and inhibitory connectivity, cellular composition (2) and mAChR subcellular localization (3, 4), based on current data and previous studies40,41.

Fig 3: CHRM enrichment defines cell-specific transcriptomic signatures aligning with mAChR functional classes.a Bar graph of the number of DEGs in total, ExN or InN enriched in CHRM1 relative to CHRM2 (light blue); enriched in CHRM3 relative to CHRM2 (dark blue); and enriched in CHRM1 relative to CHRM3 (purple). b Top significantly enriched KEGG pathway (top bars) and GO biological processes (bottom bars) terms generated based on DEGs between CHRM1+ relative to CHRM2+ ExNs (light blue) and c CHRM3+ relative to CHRM2+ ExNs (dark blue). The data was generated using EnrichR55 and significance enrichment was defined as p-value < 0.05, Benjamini. Fold-change of top CHRM1 vs. CHRM2 and CHRM3 vs. CHRM2 DEGs within ACC and LPFC ExNs, related to: d KEGG term “Glutamatergic Synapse” and GO term “Chemical Synaptic Transmission” (teal text in panels b and c); e KEGG term “Calcium Signaling”, and GO term “Calcium transmembrane import into the cytosol” (purple text in panels b and c)’ and f KEGG term “Axon Guidance”, and GO term “Nervous System Development” (pink text in panels b and c). g KEGG pathway and GO terms from DEGs between CHRM3+ relative to CHRM2+ InNs (dark blue), CHRM1+ relative to CHRM2+ InNs (light blue), and CHRM1+ relative to CHRM3+ InNs (purple). Fold-change of top CHRM1 vs. CHRM2, CHRM3 vs. CHRM2, and CHRM1 vs. CHRM3 DEGs within ACC and LPFC InNs, related to: h KEGG/GO term “Ca Signaling” and “Ca Ion Transport”, and KEGG term “Cholinergic Synapse” (purple text in panel g); i Plasticity related terms—GO terms “Nervous system development” and “Regulation of Growth Factor Stimulation” (pink text in panel g); j GO term “Chemical Synaptic Transmission” (teal text in panel g). See Supplementary Data 2 and 3 for a full list of DEGs and enriched terms, and Supplementary Fig. 3 for volcano plots.

Fig 4: Predominant neuronal expression and nuclear retention of CHRM3 in ACC and LPFC.a Photographs of the rhesus monkey brain showing locations of ACC (area 24) and LPFC (area 46) regions of interest, and a schematic of the experimental workflow. Brain images and FACS plot are from original data; other schematic images were created in BioRender (https://BioRender.com/uixnhif). b UMAP plot based on snRNA-seq showing transcriptomically distinct clusters corresponding to major cell types in ACC and LPFC (see Supplementary Fig. 1). c Violin plots showing the expression level of CHRM1, CHRM2, CHRM3, and CHRM4 within each major cell type in ACC vs. LPFC. d UMAP feature plots showing the expression pattern of the CHRM1–4 in ACC and LPFC. e Stacked horizontal bar graph showing percentage of total nuclei in ACC and LPFC expressing CHRM1, CHRM2, CHRM3, and CHRM4. f Proportion of nuclei expressing CHRM1–4 out of total nuclei by cell type for each area. g Representative z-maximum projection confocal images (five optical stacks) from ACC and LPFC L2–3 showing the cellular/subcellular distribution of m1 protein (green), m1 mRNA (magenta), m3 protein (cyan), and m3 mRNA (red). Note the predominantly cytoplasmic localization of CHRM1, versus the diffused distribution of CHRM3 within nuclear and cytoplasmic compartments. h Density of total cells immuno-labeled for m1+ (green) and m3+ (cyan) proteins in ACC and LPFC (n = 4 monkeys). In L2–3 of ACC, there was a significantly greater density of m1+ protein neurons in L2–3 compared to m3+ protein neurons (Welch t-test. **p < 0.001 in ACC; and a trend #p = 0.07 in LPFC). i Violin plots of mean intensity within individual cells expressing m1 and m3 proteins (left) and mRNA (right). Intensity of m1+ protein label is significantly greater than m3+ protein label in both areas (***p < 0.001; ACC: n = 3013 cells, and LPFC: n = 3344 cells, from 4 monkeys). j Proportion of total m1+ and total m3+ mRNA and protein-expressing cells in ACC and LPFC. Open bars show the proportion of cells that are mRNA negative but protein positive (mRNA−/PROT+), and filled bars show the proportion positive for mRNA with (mRNA+/PROT+, dark gray) or without (mRNA+/PROT−, cyan) protein. Note the presence of m3+ cells expressing mRNA but no protein (mRNA+/PROT−, cyan). k Scatter plot of mRNA/protein label intensity within individual cells and linear regression analyses in ACC and LPFC, showing correlations of m1 and m3 mRNA vs. protein. l Proportion of cells showing mRNA label localized only in the cytoplasm, or within nuclei and cytoplasm. Nuclear vs. cytoplastic localization was based on the central vs. peripheral localization of label and its overlap with MAP2 cytoplasmic labeling.

Fig 5: CHRM1–3 show distinct distribution across layer-specific ExNs and neurochemical InN subclasses in ACC and LPFC.a Dot plot showing the expression level of the top region-specific enriched genes in ACC vs. LPFC ExNs. b Top enriched Gene-Ontology (GO) Biological Processes terms associated with the region-specific genes generated using EnrichR55. c Pie chart showing the overall proportion of ExNs expressing/co-expressing specific CHRMs in ACC and LPFC. Inset shows UMAP highlighting the population of ExNs (green) together with other cell types. d UMAP plot (left) after re-clustering of ExNs annotated based on layer-specific subpopulations identified via expression of layer-specific genes in each cluster shown in dot plot (right; see Supplementary Fig. 2 and Supplementary Table 2). e Pie chart showing the overall proportion of InN expressing/co-expressing CHRMs in ACC and LPFC. Inset shows UMAP highlighting the population of InNs (blue) together with other cell types. f UMAP plot (left) after re-clustering of InNs showing 7 distinct clusters/subpopulations annotated based on expression of neurochemical markers. Dot plot shows the expression pattern of a subset of markers for distinct neurochemical subclasses of InNs based on the literature (see Supplementary Fig. 2). g Feature UMAP plots showing the expression pattern of CHRM1–4 following the re-clustering of ExNs and h InNs. i Within ExN laminar subpopulations (left) and InN subtypes (right): proportion of cells with single, co-expression (>1) or no expression of CHRM1–4 are presented as a stacked bar plot, UL upper layers, DL deep layers. j Within ExNs laminar subpopulations (left) and InN subtypes (right) with CHRM1–4 co-expression: proportion of cells with specific combinations of CHRM1–4 coexpression are presented as a stacked bar plot.