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. 2021 Jan 11;11(5):1548–1592.e1. doi: 10.1016/j.jcmgh.2020.12.014

Figure 2.

Figure 2

Expression patterns of neurotransmitters, neurotransmitter receptors, and common immunohistochemistry (IHC) markers, and validation of ENK and SP coexpression in adult mouse colon. (A) Average expression for neurotransmitters and IHC markers that were differentially expressed between distinct neuron and glial subclasses. (B) Proportion of cells per cluster with expression values >0 for differentially expressed neurotransmitters and IHC markers. (C) Average expression for neurotransmitter receptors and subunits that were differentially expressed between distinct neuron and glial subclasses. (D) Proportion of cells per cluster with expression values >0 for differentially expressed neurotransmitter receptors and subunits. (A, C) Color key represents loge(normalized average gene expression within each cluster). (E–G) ENK (green) colocalizes with SP (red) in myenteric neuron cell bodies in (E) proximal, (F) mid, and (H) distal colon. (H–J) Most myenteric intraganglionic neuron varicosities in (H) proximal, (I) mid, and (J) distal colon express both ENK (green) and SP (red). (K, L) Most enkephalin+ (green) neuron varicosities in circular smooth muscle in (K) mid colon and (L) distal colon also express SP (red), but only a subset of substance P-expressing neuron varicosities in circular smooth muscle express ENK. (E–L) Images representative of n = 3 preparations per colon region and n = 3 images per preparation. (M) Quantification of colocalization of ENK and SP in varicosities within mid colon myenteric ganglia and within circular smooth muscle. (N) EGFP (green) fluorescence signal colocalizes with TDTOMATO+ (red) neurons (blue) in Vglut2-IRES-Cre; R26R-TdTomato; ChAT-EGFP-L10A mice. (O) In a small subset of HuC/D+ neurons (blue), TDTOMATO+ (red) does not colocalize with EGFP fluorescence signal in Vglut2-IRES-Cre; R26R-TdTomato; ChAT-EGFP-L10A mice. (P) Quantification of the colocalization of EGFP fluorescence with TDTOMATO fluorescence in Vglut2-IRES-Cre; R26R-TdTomato mice. (E–G) Scale bar = 10 μm. (H–L) Scale bar = 5 μm. (E–L) White arrows point toward cells and varicosities that express both ENK and SP. (H–L) White arrowheads point toward varicosities that express enkephalin only. Empty arrowheads point toward varicosities that express SP only. (M, P) Mean ± SD.