c57bl 6j strain background  (Jackson Laboratory)

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Ileums from 3-week-old Tac2 knockout (KO) and wild-type (WT) littermates immunostained as in . Scale bar, 50 μm. (B) Number of S100B + glia in each spatial-morphological class of the ileum in Tac2 KO (orange) and WT (gray) mice ( n = 4–5 mice/genotype). (C) Number of ANNA-1 + neurons in each enteric plexus in Tac2 KO and WT mice. (D) Whole-mount preparation from adult Tac2 Cre/+ Rosa26 tdTomato/+ mouse ileum immunolabeled with S100B (green). Scale bar, 100 μm. Graph shows percentage of tdTomato + neurons in the myenteric plexus. (E) Whole-mount preparation of Tac2 Cre/+ Rosa26 tdTomato/+ ileum immunostained for CALB2 (green) and shown at the same scale as (A). (F) Association of TAC3 SNPs with hard stool (top) and human esophagus muscularis eQTLs (bottom) from UK Biobank and GTEx Portal data, respectively. Genomic region analyzed is shown and arrow depicts the direction of TAC3 transcription. For (B) and (C), two-way ANOVA with Šidák’s multiple comparisons test was used (* p < 0.05 and **** p < 0.0001). See also .

Article Snippet: Mouse: Tac2 Cre/+ Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:018938.

Techniques: Knock-Out, Immunolabeling

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Schematic of experimental design. GFP + (glia) and GFP − (non-glia) cells were isolated from the mucosa and muscularis compartments of small intestines from Plp1 eGFP mice. (B) Volcano plots of top 5 significantly up- and downregulated genes in GFP + compared with GFP − cells in total tissue (pooling compartments), muscularis, and mucosal compartments. Dashed line indicates p adj. < 0.05. (C) Volcano plot of macrophage ( Cd300a , Csf1r , Mpeg1 , Cx3cr1 , and Adgre1 ) and glial markers ( S100b , Plp1 , and Gfap ) enriched in mucosal GFP + cells. (D) Csf1r transcripts (yellow) labeled by in situ hybridization in the small intestinal mucosa of a Plp1 CreERT2 Rosa26 tdTomato/+ Cx3cr1 GFP/+ reporter mouse showing glial (tdTomato + ; arrowhead) and macrophage (GFP + ) expression. Scale bar, 10 μm. (E) Proportions of Plp1 tdTomato+ glia (pink) and Cx3cr1 GFP+ macrophages (green) that were labeled by in situ hybridization for putative macrophage transcripts (807 cells, 4 mice). (F) Biological pathways upregulated in mucosal and muscularis glia. (G) Uniform manifold approximation and projection (UMAP) plots of re-analyzed single-cell glial transcriptomes from the CNS and PNS. Heatmap of normalized gene set enrichment scores (NESs) of mucosal and muscularis glia. See also and .

Article Snippet: Mouse: Rosa26 DTA/DTA Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:009669.

Techniques: Isolation, Labeling, In Situ Hybridization, Expressing

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Volcano plots of most significantly upregulated genes in GFP + cells in the muscularis compartment and the subset enriched in muscularis glia. (B) Whole-mount preparations of ileum and distal colon from adult Plp1 eGFP mice with immunohistochemical labeling of TACR3 (magenta) imaged at the myenteric plexus. Insets show magnified view of boxed regions. Arrowheads mark TACR3-immunoreactive GFP + glia surrounding larger GFP − soma (asterisk), presumably neuronal. (C) Schematic illustrating spatial-morphological classification of glia in the small intestine. (D) Schematic and image of the distal colon from a 3-week-old Tacr3 IRES-Cre/+ Rosa26 tdtomato/+ mouse showing tdTomato expression throughout the myenteric plexus (scale bar, 1 mm). (E) Whole-mount preparations of duodenum, ileum, and distal colon from 3-week-old Tacr3 IRES-Cre/+ Rosa26 tdtomato/+ mice imaged at the myenteric plexus. TdTomato expression colocalizes with S100B (cyan) in myenteric glia (arrowheads and arrows mark example intraganglionic and connective glia, respectively) but not extranganglionic glia (asterisks). (F) Tacr3 reporter expression in ANNA-1 + neurons (cyan) in the myenteric (MP) and submucosal (SMP) plexuses. For (E) and (F), n = 5 mice; scale bar, 20 μm; see also . For (E), one-way ANOVA with Tukey’s multiple comparisons test was used (* p < 0.05, ** p < 0.005, **** p < 0.0001, and ns, not significant). For (F), unpaired t test was used.

Article Snippet: Mouse: Rosa26 DTA/DTA Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:009669.

Techniques: Immunohistochemical staining, Labeling, Expressing

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Ileums from 3-week-old Tac2 knockout (KO) and wild-type (WT) littermates immunostained as in . Scale bar, 50 μm. (B) Number of S100B + glia in each spatial-morphological class of the ileum in Tac2 KO (orange) and WT (gray) mice ( n = 4–5 mice/genotype). (C) Number of ANNA-1 + neurons in each enteric plexus in Tac2 KO and WT mice. (D) Whole-mount preparation from adult Tac2 Cre/+ Rosa26 tdTomato/+ mouse ileum immunolabeled with S100B (green). Scale bar, 100 μm. Graph shows percentage of tdTomato + neurons in the myenteric plexus. (E) Whole-mount preparation of Tac2 Cre/+ Rosa26 tdTomato/+ ileum immunostained for CALB2 (green) and shown at the same scale as (A). (F) Association of TAC3 SNPs with hard stool (top) and human esophagus muscularis eQTLs (bottom) from UK Biobank and GTEx Portal data, respectively. Genomic region analyzed is shown and arrow depicts the direction of TAC3 transcription. For (B) and (C), two-way ANOVA with Šidák’s multiple comparisons test was used (* p < 0.05 and **** p < 0.0001). See also .

Article Snippet: Mouse: Rosa26 DTA/DTA Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:009669.

Techniques: Knock-Out, Immunolabeling

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Schematic of experimental design. GFP + (glia) and GFP − (non-glia) cells were isolated from the mucosa and muscularis compartments of small intestines from Plp1 eGFP mice. (B) Volcano plots of top 5 significantly up- and downregulated genes in GFP + compared with GFP − cells in total tissue (pooling compartments), muscularis, and mucosal compartments. Dashed line indicates p adj. < 0.05. (C) Volcano plot of macrophage ( Cd300a , Csf1r , Mpeg1 , Cx3cr1 , and Adgre1 ) and glial markers ( S100b , Plp1 , and Gfap ) enriched in mucosal GFP + cells. (D) Csf1r transcripts (yellow) labeled by in situ hybridization in the small intestinal mucosa of a Plp1 CreERT2 Rosa26 tdTomato/+ Cx3cr1 GFP/+ reporter mouse showing glial (tdTomato + ; arrowhead) and macrophage (GFP + ) expression. Scale bar, 10 μm. (E) Proportions of Plp1 tdTomato+ glia (pink) and Cx3cr1 GFP+ macrophages (green) that were labeled by in situ hybridization for putative macrophage transcripts (807 cells, 4 mice). (F) Biological pathways upregulated in mucosal and muscularis glia. (G) Uniform manifold approximation and projection (UMAP) plots of re-analyzed single-cell glial transcriptomes from the CNS and PNS. Heatmap of normalized gene set enrichment scores (NESs) of mucosal and muscularis glia. See also and .

Article Snippet: Mouse: Rosa26 tdTomato/tdTomato Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:007909.

Techniques: Isolation, Labeling, In Situ Hybridization, Expressing

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Volcano plots of most significantly upregulated genes in GFP + cells in the muscularis compartment and the subset enriched in muscularis glia. (B) Whole-mount preparations of ileum and distal colon from adult Plp1 eGFP mice with immunohistochemical labeling of TACR3 (magenta) imaged at the myenteric plexus. Insets show magnified view of boxed regions. Arrowheads mark TACR3-immunoreactive GFP + glia surrounding larger GFP − soma (asterisk), presumably neuronal. (C) Schematic illustrating spatial-morphological classification of glia in the small intestine. (D) Schematic and image of the distal colon from a 3-week-old Tacr3 IRES-Cre/+ Rosa26 tdtomato/+ mouse showing tdTomato expression throughout the myenteric plexus (scale bar, 1 mm). (E) Whole-mount preparations of duodenum, ileum, and distal colon from 3-week-old Tacr3 IRES-Cre/+ Rosa26 tdtomato/+ mice imaged at the myenteric plexus. TdTomato expression colocalizes with S100B (cyan) in myenteric glia (arrowheads and arrows mark example intraganglionic and connective glia, respectively) but not extranganglionic glia (asterisks). (F) Tacr3 reporter expression in ANNA-1 + neurons (cyan) in the myenteric (MP) and submucosal (SMP) plexuses. For (E) and (F), n = 5 mice; scale bar, 20 μm; see also . For (E), one-way ANOVA with Tukey’s multiple comparisons test was used (* p < 0.05, ** p < 0.005, **** p < 0.0001, and ns, not significant). For (F), unpaired t test was used.

Article Snippet: Mouse: Rosa26 tdTomato/tdTomato Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:007909.

Techniques: Immunohistochemical staining, Labeling, Expressing

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Ileums from 3-week-old Tac2 knockout (KO) and wild-type (WT) littermates immunostained as in . Scale bar, 50 μm. (B) Number of S100B + glia in each spatial-morphological class of the ileum in Tac2 KO (orange) and WT (gray) mice ( n = 4–5 mice/genotype). (C) Number of ANNA-1 + neurons in each enteric plexus in Tac2 KO and WT mice. (D) Whole-mount preparation from adult Tac2 Cre/+ Rosa26 tdTomato/+ mouse ileum immunolabeled with S100B (green). Scale bar, 100 μm. Graph shows percentage of tdTomato + neurons in the myenteric plexus. (E) Whole-mount preparation of Tac2 Cre/+ Rosa26 tdTomato/+ ileum immunostained for CALB2 (green) and shown at the same scale as (A). (F) Association of TAC3 SNPs with hard stool (top) and human esophagus muscularis eQTLs (bottom) from UK Biobank and GTEx Portal data, respectively. Genomic region analyzed is shown and arrow depicts the direction of TAC3 transcription. For (B) and (C), two-way ANOVA with Šidák’s multiple comparisons test was used (* p < 0.05 and **** p < 0.0001). See also .

Article Snippet: Mouse: Rosa26 tdTomato/tdTomato Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:007909.

Techniques: Knock-Out, Immunolabeling

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Schematic of experimental design. GFP + (glia) and GFP − (non-glia) cells were isolated from the mucosa and muscularis compartments of small intestines from Plp1 eGFP mice. (B) Volcano plots of top 5 significantly up- and downregulated genes in GFP + compared with GFP − cells in total tissue (pooling compartments), muscularis, and mucosal compartments. Dashed line indicates p adj. < 0.05. (C) Volcano plot of macrophage ( Cd300a , Csf1r , Mpeg1 , Cx3cr1 , and Adgre1 ) and glial markers ( S100b , Plp1 , and Gfap ) enriched in mucosal GFP + cells. (D) Csf1r transcripts (yellow) labeled by in situ hybridization in the small intestinal mucosa of a Plp1 CreERT2 Rosa26 tdTomato/+ Cx3cr1 GFP/+ reporter mouse showing glial (tdTomato + ; arrowhead) and macrophage (GFP + ) expression. Scale bar, 10 μm. (E) Proportions of Plp1 tdTomato+ glia (pink) and Cx3cr1 GFP+ macrophages (green) that were labeled by in situ hybridization for putative macrophage transcripts (807 cells, 4 mice). (F) Biological pathways upregulated in mucosal and muscularis glia. (G) Uniform manifold approximation and projection (UMAP) plots of re-analyzed single-cell glial transcriptomes from the CNS and PNS. Heatmap of normalized gene set enrichment scores (NESs) of mucosal and muscularis glia. See also and .

Article Snippet: Mouse: Rosa26 GCaMP6f/+ Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:028865.

Techniques: Isolation, Labeling, In Situ Hybridization, Expressing

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Volcano plots of most significantly upregulated genes in GFP + cells in the muscularis compartment and the subset enriched in muscularis glia. (B) Whole-mount preparations of ileum and distal colon from adult Plp1 eGFP mice with immunohistochemical labeling of TACR3 (magenta) imaged at the myenteric plexus. Insets show magnified view of boxed regions. Arrowheads mark TACR3-immunoreactive GFP + glia surrounding larger GFP − soma (asterisk), presumably neuronal. (C) Schematic illustrating spatial-morphological classification of glia in the small intestine. (D) Schematic and image of the distal colon from a 3-week-old Tacr3 IRES-Cre/+ Rosa26 tdtomato/+ mouse showing tdTomato expression throughout the myenteric plexus (scale bar, 1 mm). (E) Whole-mount preparations of duodenum, ileum, and distal colon from 3-week-old Tacr3 IRES-Cre/+ Rosa26 tdtomato/+ mice imaged at the myenteric plexus. TdTomato expression colocalizes with S100B (cyan) in myenteric glia (arrowheads and arrows mark example intraganglionic and connective glia, respectively) but not extranganglionic glia (asterisks). (F) Tacr3 reporter expression in ANNA-1 + neurons (cyan) in the myenteric (MP) and submucosal (SMP) plexuses. For (E) and (F), n = 5 mice; scale bar, 20 μm; see also . For (E), one-way ANOVA with Tukey’s multiple comparisons test was used (* p < 0.05, ** p < 0.005, **** p < 0.0001, and ns, not significant). For (F), unpaired t test was used.

Article Snippet: Mouse: Rosa26 GCaMP6f/+ Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:028865.

Techniques: Immunohistochemical staining, Labeling, Expressing

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Representative fluorescence images (left) or heatmaps of fluorescence intensity standard deviation (right) demonstrating glial Ca 2+ responses in the mid-colons of adult Kir4.1-GCaMP6f mice induced by brush stimulation of the distal mucosa. Responses to evoked contractions were measured at baseline and after blockade of TACR3 signaling with 1 μM fezolinetant. Examples of intraganglionic (asterisks), connective (arrowhead), and intramuscular (arrow) glia are marked. (B) Example Ca 2+ responses in individual glia (colored traces) following an evoked neurogenic contraction (black trace) at baseline and after fezolinetant. Traces are shown for cells marked by colored boxes in (A): intraganglionic (red and green), connective (magenta), and intramuscular (orange) glia. (C) Quantification of features of glial Ca 2+ responses to neurogenic contractions at baseline (gray) and with fezolinetant (blue; n = 8 mice). Unpaired t tests were used to compare baseline and fezolinetant conditions in each group of cells (** p < 0.01, *** p < 0.001, and **** p < 0.0001). See . (D) Gastrointestinal transit time (GITT) in adult males ( n = 10/group) and females ( n = 18/group) following administration of fezolinetant (10 mg/kg) or vehicle. (E) GITT in adult males ( n = 8–11/group) and females ( n = 18/group) following administration of TACR3 agonist senktide (5 mg/kg) or vehicle. (F) Representative heatmaps of motor activity in colons from wild-type adult male mice imaged ex vivo with 1 μM fezolinetant or vehicle. (G) Quantification of colonic migrating motor complex (CMMC) frequency, velocity, duration, and length in vehicle and fezolinetant conditions ( n = 8 mice/condition). For (D) and (E), unpaired t tests were used for males and paired t tests were used for females, which were studied in a crossover design (* p < 0.05, ** p < 0.005, *** p < 0.0005, and **** p < 0.0001). Unpaired t tests were used for (G). For (D)–(G), see also .

Article Snippet: Mouse: Rosa26 GCaMP6f/+ Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:028865.

Techniques: Fluorescence, Standard Deviation, Activity Assay, Ex Vivo

Journal: Neuron

Article Title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system

doi: 10.1016/j.neuron.2025.11.030

Figure Lengend Snippet: (A) Ileums from 3-week-old Tac2 knockout (KO) and wild-type (WT) littermates immunostained as in . Scale bar, 50 μm. (B) Number of S100B + glia in each spatial-morphological class of the ileum in Tac2 KO (orange) and WT (gray) mice ( n = 4–5 mice/genotype). (C) Number of ANNA-1 + neurons in each enteric plexus in Tac2 KO and WT mice. (D) Whole-mount preparation from adult Tac2 Cre/+ Rosa26 tdTomato/+ mouse ileum immunolabeled with S100B (green). Scale bar, 100 μm. Graph shows percentage of tdTomato + neurons in the myenteric plexus. (E) Whole-mount preparation of Tac2 Cre/+ Rosa26 tdTomato/+ ileum immunostained for CALB2 (green) and shown at the same scale as (A). (F) Association of TAC3 SNPs with hard stool (top) and human esophagus muscularis eQTLs (bottom) from UK Biobank and GTEx Portal data, respectively. Genomic region analyzed is shown and arrow depicts the direction of TAC3 transcription. For (B) and (C), two-way ANOVA with Šidák’s multiple comparisons test was used (* p < 0.05 and **** p < 0.0001). See also .

Article Snippet: Mouse: Rosa26 GCaMP6f/+ Strain Background: C57BL/6J , The Jackson Laboratory , RRID: IMSR_JAX:028865.

Techniques: Knock-Out, Immunolabeling