Journal: Neuron

Article Title: Loss of adaptive myelination contributes to methotrexate chemotherapy-related cognitive impairment

doi: 10.1016/j.neuron.2019.04.032

Figure Lengend Snippet: A) Representative image demonstrating RNAscope visualization of frontal cortex deep layer neurons (NeuN, white), astrocytes (Glast, green), and Bdnf mRNA (red). DAPI, blue. Scale bar = 20 μm

Article Snippet: RNAscope was performed according to Fluorescent Multiplex Reagent Kit protocol using RNAscope probes against BDNF (Advanced Cell Diagnostics, 424821), RbFox3 (NeuN; Advanced Cell Diagnostics 313311-C2) and Slc1a3 (Glast; Advanced Cell Diagnostics, 320851-C3).

Techniques:

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Behavioral screening of LS and HS subpopulations of mice exposed to 3-week CMS. ( B ) Quantitative PCR to examine mRNA expression of cytokines, chemokines, growth factors, and trophic factors in the hippocampus and prefrontal cortex of HS and LS mice. Data are standardized to control group ( n = 4 to 6, each sample in triplicate). ( C ) Enzyme-linked immunosorbent assay to assay the protein level of IL4 in prefrontal cortex, cerebellum, hippocampus, amygdala, and olfactory bulb of control, HS, and LS mice. Each circle represents one mouse ( n = 6, each sample in triplicate). ( D ) Western blotting shows the levels of IL4, IL4 receptor α chain (IL4Rα), STAT6, and pSTAT6 in the hippocampus of control, HS, and LS mice. IL4 and IL4Rα are normalized to β-actin, and the pSTAT6 is normalized to STAT6. ( n = 4 to 6, each sample in triplicate). ( E ) Correlation between sucrose preference or immobility duration in TST, hippocampal Arg1 levels, and hippocampal IL4 levels in CMS-exposed mice. Each circle represents one mouse ( n = 6). ( F ) Morphological characters of hippocampal microglia in control, HS, and LS mice. Scale bars, 50 μm (top) and 10 μm (bottom). The histogram represents the quantification of number and branch length of Iba1 + cells in hippocampus. Each dot in the bar graph represents the average of all micrograph (five to six micrographs for each mouse) in each mouse ( n = 5). ( G ) Immunofluorescence micrographs and quantification of Arg1 + microglia in hippocampus of control, LS, and HS mice ( n = 5). Scale bars, 50 μm. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, one-way ANOVA with Tukey test (B, C, D, F, and G). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Experimental timeline of IL4 overexpression in the hippocampus and CMS protocol. ( B ) Volcano maps indicate differentially expressed genes (DEGs) in the brains of CMS-exposed and/or AAV-IL4–treated mice. ( C ) Quantitative PCR examination of the expression of several DEGs in the hippocampus of mice ( n = 4 to 6). ( D ) Western blotting shows the levels of IL4, Arg1, and iNOS in the hippocampus of CMS-exposed and/or AAV-IL4–treated mice ( n = 5 to 6). ( E ) Morphological characters of Arg1 + microglia in hippocampus. Scale bar, 100 μm. The histogram represents the quantification of number and branch length of Arg1 + -Iba1 + cells in hippocampus. Each dot in the bar graph represents the average of five to six micrographs in each mouse ( n = 6). ( F ) Flow cytometry of single-cell suspension of the whole hippocampus for microglia (CD45 int -CD11b + cells), activated microglia (CD86 + cells), and anti-inflammatory microglia (CD86 + -CD206 + cells). The histogram represents the quantification of CD45 int -CD11b + cells, CD86 + cells, and CD86 + -CD206 + cells ( n = 5 to 6). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 versus AAV + Ctrl group, # P < 0.05, ## P < 0.01, ### P < 0.005 versus AAV + CMS group, two-way ANOVA with Tukey test (C); * P < 0.05, ** P < 0.01, *** P < 0.005, two-way ANOVA with Tukey test (D, E, and F). FITC A, fluorescein isothiocyanate area (FITC-A).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Flow Cytometry, Suspension

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Immunofluorescence micrographs and quantification show the hippocampal neurogenesis of LS and HS mice ( n = 5). ( B ) GO enrichment analysis for DEGs in the hippocampus of CMS-exposed mice. ER, endoplasmic reticulum; FDR, false discovery rate. ( C ) Effects of IL4 overexpression and/or microglial IL4Rα down-regulation on NSPC proliferation and differentiation in the hippocampus of CX 3 CR1 Cre/ERT2 mice. Representative fluorescence micrographs showing DCX expression and BrdU incorporation in the subgranular zone (SGZ). Scale bar, 100 μm. Quantification of total number of BrdU + cells, total number of DCX + -BrdU + cells, and percentage of DCX + -BrdU + cells out of all BrdU + cells in the neurogenic zones ( n = 5). ( D ) Effects of IL4 overexpression and/or microglial IL4Rα down-regulation in the hippocampus (before IL4 overexpression) on NSPC survival and maturation. Representative fluorescence micrographs illustrating NeuN expression and BrdU incorporation in the SGZ. Scale bar, 100 μm. Quantification of total number of BrdU + cells, BrdU + -NeuN + cells, and DCX + -NeuN + cells in DG ( n = 5). ( E ) Effects of conditioned medium from microglia isolated from hippocampus on NSPC proliferation. Proliferation was monitored by quantifying the percentage of BrdU + cells ( n = 6). Scale bar, 50 μm. Representative micrographs of NSPCs cultured for 7 days in conditioned culture medium from microglia isolated from hippocampus. Cells were immunolabeled with glial fibrillary acidic protein (GFAP) to identify astrocytes and MAP2 to identify neurons. Percentages of GFAP + cells and MAP2 + cells are quantified ( n = 6). Scale bar, 30 μm. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, one-way ANOVA with Tukey test (A) and two-way ANOVA with Tukey test (C, D, and E).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Immunofluorescence, Over Expression, Fluorescence, Expressing, BrdU Incorporation Assay, Isolation, Cell Culture, Immunolabeling

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Western blotting shows the levels of BDNF and TrkB in the hippocampus of LS and HS mice. BDNF and TrkB are normalized to β-actin ( n = 3). ( B ) Western blotting shows the levels of BDNF and TrkB in the hippocampus of CMS and/or AAV-IL4–treated mice. BDNF and TrkB are normalized to β-actin ( n = 4). ( C ) BDNF was detected in hippocampal microglia (Iba1 + cells) using Bdnf -RNAscope. ( D ) The mRNA expression of BDNF was observed in hippocampal microglia of AAV-IL4–treated mice ( n = 4). ( E ) Double immunohistochemical staining of BDNF and Arg1 in the hippocampus of AAV-IL4 mice. Scale bar, 20 μm. ( F ) Effects of CMS, knockdown of microglial IL4Rα, and overexpression of IL4 on BDNF protein levels in the hippocampus ( n = 5). ( G ) Effects of k252a treatment to block the BDNF/TrkB pathway on hippocampal neurogenesis in AAV-IL4 mice under CMS exposure. Representative fluorescence micrographs showing DCX expression and BrdU incorporation in the SGZ. Scale bar, 100 μm. Quantification of the number of DCX + cells and total number of DCX + -BrdU + cells in the neurogenic zones ( n = 5). DMSO, dimethyl sulfoxide. ( H ) The expression and secretion of BDNF in primary microglia after IL4 treatment in vitro ( n = 6). ( I ) Effects of anti-BDNF antibody or K252a on NSPC proliferation (BrdU + cells) in the presence of the conditioned culture medium from IL4-treated microglia ( n = 5). Scale bar, 20 μm. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, one-way ANOVA with Tukey test (A, G, and I), two-way ANOVA with Tukey test (B, D, and F), and two-tailed t test (H).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Western Blot, RNAscope, Expressing, Immunohistochemical staining, Staining, Knockdown, Over Expression, Blocking Assay, Fluorescence, BrdU Incorporation Assay, In Vitro, Two Tailed Test

Journal: Science Advances

Article Title: IL4-driven microglia modulate stress resilience through BDNF-dependent neurogenesis

doi: 10.1126/sciadv.abb9888

Figure Lengend Snippet: ( A ) Effects of overexpression of IL4 in the hippocampus on stress-induced depressive-like behaviors. Mice were injected stereotactically with AAV-IL4 or AAV, allowed to recover for 2 weeks, and then subjected to a CMS protocol consisting of exposure to three random stressors daily for 4 weeks. The mice were assessed by SPT and FST ( n = 8). ( B ) Correlations between sucrose preference and percentage of Arg1 + microglia in hippocampus, immobility time in FST and percentage of Arg1 + microglia in hippocampus, sucrose preference and concentration of BDNF in hippocampus, immobility time in FST and concentration of BDNF in hippocampus, sucrose preference and number of BrdU + -DCX + cells in DG, and immobility time in FST and number of BrdU + -DCX + cells in DG. Each circle represents one mouse ( n = 6). ( C ) Effects of microglial IL4Rα down-regulation (before IL4 overexpression) on stress-induced depressive-like behaviors, as assessed by SPT and FST ( n = 8 to 10). ( D ) Assessment of stress-induced depressive-like behaviors in WT mice when treated with K252a or TMZ with sCMS exposure ( n = 8). ( E ) Assessment of stress-induced depressive-like behaviors in AAV-IL4 mice when treated with k252a or TMZ ( n = 8 to 10). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, two-way ANOVA with Bonferroni test (A and C) and one-way ANOVA with Bonferroni test (D and E).

Article Snippet: In situ hybridization was performed according to the protocol of the Bdnf -RNAscope Multiplex Fluorescent Probe Reagent Kit v2 and Mm- Il4 -RNAscope 2.5 VS Fluorescent Probe Reagent Kit (Advanced Cell Diagnostics, California, USA).

Techniques: Over Expression, Injection, Concentration Assay

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Stable loss of major histocompatibility complex class I (MHC-I) gene DNA methylation (DNAm) in intestinal epithelial organoids (IEOs) derived from patients with Crohn’s disease (CD). (A) (i) Overview of experimental set-up and sample generation. (ii) Representative brightfield images of IEOs. Scale bars: 300 µm. (B) (i) Correlation heat map of comethylated CpG modules identified by weighted gene coexpression network analysis (WGCNA) in terminal ileum (TI) IEOs. Module 17 (ME17) demonstrates hypomethylation and the strongest association with CD diagnosis (R=−0.43, p value<0.001). (ii) Gene set enrichment analysis performed on module 17, showing a significant loss of DNAm in CD organoids compared with healthy controls and UC in TI. (C) DNAm (beta value) of four representative MHC-I related Differetial Methylated Positions (DMPs) showing CD-associated loss of DNAm in TI and sigmoid colon (SC) but not duodenum (DUO) organoids (DUO=54, TI=127 and SC=131). (D) Average DNAm (beta value) of all CpGs located in MHC-I related genes for IEOs split by diagnosis, gut segment and inflammatory status. (E) (i) Correlation of nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm between early and later passage IEOs from the same individuals including patients diagnosed with CD (blue), UC (yellow) and controls (grey, n=22 patients. (ii) DNAm (beta values) of CpGs located in NLRC5 and TAP1 at high passage (>7) IEOs (cohort 1, n=22). (F) Average MHC-I (i) and NLRC5 (ii) DNAm as well as NLRC5 gene expression (iii) in control patient-derived TI IEOs stimulated with proinflammatory cytokines interferon γ (IFNγ) and tumour necrosis factor α (TNFα) (n=5). (False Discovery Rate (FDR) * < 0.05, FDR **< 0.01, FDR***< 0.001, FDR**** < 0.0001, ns=not significant.)

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Derivative Assay, Biomarker Discovery, Methylation, Binding Assay, Gene Expression, Control

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Loss of major histocompatibility complex class I (MHC-I) DNA methylation (DNAm) correlates with increased gene expression in primary intestinal epithelium of patients with Crohn’s disease (CD). (A) Overview of patient cohort, sample preparation and data generation. (B, C) DNAm and gene expression in purified terminal ileum (TI) (B) and sigmoid colon (SC) (C) epithelium. (i) Average DNAm (beta value) of all and selected MHC-I pathway-related CpGs showing significant, CD-associated loss of DNAm. (ii) Correlation between beta values and corresponding gene expression (R=Spearman’s rank correlation). (D) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) promoter DNAm in the IE of healthy, patients with UC and CD at the point of diagnosis and during reassessment. (E) Correlation of NLRC5 promoter DNAm in intestinal epithelial organoids (IEOs) obtained from the same patient at diagnosis and reassessment (Spearman’s rank correlation). (F) NLRC5 promoter DNAm in TI IEOs derived from patients with CD, UC and control (n=3 IEO per condition, two-way analysis of variance (ANOVA) with Turkey’s test for multiple comparisons. ****p<0.0001). (G) NLRC5 mRNA expression in TI IEOs derived from patients with controls, UC and CD at baseline and on interferon γ (IFNγ) treatment (10 ng/mL for 6 hours). Data are normalised to the mean of control lines and shown as mean±SEM (two-way ANOVA with Turkey’s test for multiple comparisons. **P<0.01, *p<0.05, ns=not significant). n=3 IEO lines in each group for three independent experiments.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, DNA Methylation Assay, Gene Expression, Sample Prep, Purification, Binding Assay, Biomarker Discovery, Derivative Assay, Control, Expressing

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acts as transcriptional transactivator of intestinal epithelial cell (IEC) major histocompatibility complex class I (MHC-I) and potentiates the effect of interferon γ (IFNγ). (A) Overview of experimental set-up. (B) Heatmap showing gene expression (RNAseq) of MHC-I pathway genes in terminal ileum (TI) intestinal epithelial organoids (IEOs)± NLRC5 overexpression (dox), and ±exposure to IFNγ (n=4 independent replicates). (C) RNA transcription of HLA-A / -B / -C / -E / -F / -G in response to IFNγ and tumour necrosis factor α (TNFα) in wild type (WT) and NLRC5 OE TI IEOs. (D) Relative expression for MHC-I pathway genes in WT ( NLRC5 +/+ ) and corresponding NLRC5 deficient ( NLRC5 −/ − ) TI IEOs±IFNγ (n=3 replicates. Two-way analysis of variance (ANOVA) with Bonferroni’s test for multiple comparisons, **p<0.01, ***p<0.001, ****p<0.0001). Data are representative of two independent experiments. (E) Immunofluorescence spinning disc microscopy of organoids described in D, ±IFNγ (48 hours). (i) Representative images of untreated (BSA) and treated (IFNγ) WT ( NLRC5 +/+ ) and NLRC5 deficient ( NLRC5 −/ − ) TI IEOs taken by Opera Phoenix. Scale bar=2 mm. (ii) HLA-A,B,C mean intensity quantification of BSA and IFNγ NLRC5 +/+ and NLRC5 −/ − TI IEOs. (n=3 independent replicates. Two-way ANOVA with Bonferroni multiple comparisons test, **p<0.01, ***p<0.001, ****p<0.0001.) (F) Correlation between mRNA gene expression of NLRC5 and (i) HLA-B and (ii) HLA-E, in purified TI and sigmoid colon (SC) epithelium (cohort 2) (Spearman’s rank correlation).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Binding Assay, Immunopeptidomics, Gene Expression, Over Expression, Expressing, Immunofluorescence, Microscopy, Purification

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Crohn’s disease (CD)-associated increased intestinal epithelial major histocompatibility complex class I (MHC-I) expression affects the stem cell compartment and follows a crypt-villus gradient. (A) Summary of experimental set-up. (B) (i) Schematic representation of intestinal epithelial cell (IEC) subtypes and their location within the small bowel (terminal ileum (TI)) crypt-villus structure (TA—transiently amplifying cells). (ii) Uniform manifold approximation and projection (UMAP) plot demonstrating single IEC transcriptomes present in TI mucosal biopsies obtained from children newly diagnosed with CD and non-IBD controls. (C) Top panel: violin plots showing crypt-villus scores of cells within each identified cell subtype (top left) and total number of cells (top right). Bottom panel: correlation between MHC-I summary score and crypt-villus scores for all IEC transcriptomes. Best fitting correlation is displayed as individual lines for CD (blue), UC (yellow) and non-IBD control samples (grey) (bottom left). Bottom right: box plots of summary MHC-I single-cell transcriptional score split by diagnosis. (D) Summary/average MHC-I score in individual IEC subtypes comparing CD, UC and controls. (E) Nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) expression in TI IEC of patients with CD colocalises with CD8+ T cells. RNA scope of TI biopsies from healthy donors and patients with CD. EPCAM (cyan), NLRC5 (white), TAP1 (yellow), CD8A (red), IFNG (green) and nuclei (DAPI, blue). Proximity of CD8 + T-cells with NLRC5 + EPCAM + cells in the CD biopsy is shown with arrows. Representative images are shown. Scale bar=100 µm and zoom in scale bar=10 µm.

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, Expressing, Control, Biomarker Discovery, Binding Assay, RNAscope

Journal: Gut

Article Title: Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn’s Disease

doi: 10.1136/gutjnl-2024-332043

Figure Lengend Snippet: Intestinal epithelial cells (IECs) present antigen via major histocompatibility complex class I (MHC-I) and activate CD8 + T cells in vitro with nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 ( NLRC5 ) acting as key modulator of mucosal inflammation in vivo. (A) Overview of experimental set-up. (B) Quantification of H2K b -SIINFEKL and pan-H2K b flow cytometry on live EpCAM + cells in murine intestinal epithelial organoids (IEOs) stimulated with or without interferon γ (IFNγ) (48 hours) and pulsed with or without OVA257–264 peptide (SIINFEKL) peptide. Data are representative of two independent experiments run in triplicates. GMFI, geometric mean fluorescence intensity; AU, arbitrary units. P values were calculated by two-way analysis of variance (ANOVA) with Bonferroni test for multiple comparisons (**p<0.01, ****p<0.0001). (C) Overview of experimental design. (D) Quantitative PCR gene expression of Ifng for coculture experiment in murine IEOs±SIINFEKL peptide pulse and cocultured with SIINFEKL-activated OTI T-cells. Data are presented as fold change over unstimulated OTI cells minus murine IEOs, normalised to Cd8a . P values were calculated using two-way ANOVA with Bonferroni’s multiple comparisons test (***p<0.001, ns=not significant). (E) Body weight changes over time during and after a 6-day course of 2% dextran sulphate sodium (DSS) exposure. (n=8 and n=5 Nlrc5fl/fl and Nlrc5-/- mice, respectively. P values calculated by multiple t-tests with Holm-Šídák correction for multiple comparisons.) (F) Quantification of H2K b surface expression on EpCAM+ cell populations within the lamina propria extractions of DSS-treated mice. All panels: data are representative of two independent experiments (**p<0.01). (G) Colon weight per unit length and mesenteric lymph node (MLN) weight and spleen weight of Nlrc5 wild type and knockout mice, on day 14 after initiation of 6-day course of 2% DSS (**p<0.01).

Article Snippet: The human NLRC5 cDNA ( myc-NLRC5 ) was obtained from AddGene ( # 37509).

Techniques: Immunopeptidomics, In Vitro, Binding Assay, In Vivo, Flow Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Knock-Out

Journal: Nature Communications

Article Title: Subcortical serotonin 5HT 2c receptor-containing neurons sex-specifically regulate binge-like alcohol consumption, social, and arousal behaviors in mice

doi: 10.1038/s41467-023-36808-2

Figure Lengend Snippet: A Representative CtB 647 infection in BNST, females. Similar images were obtained in n = 3 mice B Representative CtB 555 infection, females. Similar images were obtained in n = 3 mice. C Representative CtB 647 infection in BNST, males. Similar images were obtained in n = 4 mice. D Representative CtB 555 infection in LHb, males. Similar images were obtained in n = 4 mice. E Representative CtB 647 labeling in DRN, females. Similar images were obtained in n = 3 mice. F Representative CtB 555 labeling in DRN, females. Similar images were obtained in n = 3 mice. G Representative CtB 647 infection in BNST, males. Similar images were obtained in n = 4 mice. H Representative CtB 555 infection, males. Similar images were obtained in n = 4 mice. I Representative 5HT labeling in DRN, females. Similar images were obtained in n = 3 mice. J Representative labeling in DRN for CtB 647, CtB 555, and 5HT, females. Similar images were obtained in n = 3 mice. K Representative 5HT labeling in DRN, males. Similar images were obtained in n = 4 mice. L Representative labeling in DRN for CtB 647, CtB 555, and 5HT, males. Similar images were obtained in n = 4 mice. M Percent of DRN-LHb neurons positive for 5HT N Percent of DRN-BNST neurons positive for 5HT. O Percent of CtB 555 neurons that co-express CtB 647 in DRN. P Percent of CtB 647 neurons that co-express CtB 555 in DRN. Q In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in BNST, females. Similar images were obtained in n = 3 mice. R In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in BNST, males. Similar images were obtained in n = 4 mice. S , In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in LHb, females. Similar images were obtained in n = 3 mice. T In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in LHb, males. Similar images were obtained in n = 4 mice. U Dorsal BNST 5HT2c neuron overlaps with vGAT or vGlut2. V Ventral BNST 5HT2c neuron overlaps with vGAT or vGlut2. W LHb 5HT2c neuron overlap with vGAT or vGlut2. n = 3 males, n = 4 females, 2 slices/mouse. No statistical comparisons were performed on this data as they are intended to be descriptive. All data are represented as mean ± SEM. Source data are provided as a file. Created with Biorender.com .

Article Snippet: ISH was performed to fluorescently label mRNA for mouse serotonin receptor 2c (Mm-Htr 2c , probe#: 401001), vesicular GABA transporter (Mm-Slc32a1, probe#:319191), and vesicular glutamate transporter 2 (Mm-Slc17a6, probe#: 319171) using the RNAscope Fluorescence Multiplex Assay Kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions.

Techniques: Infection, Labeling, In Situ Hybridization

Journal: The FASEB Journal

Article Title: Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion

doi: 10.1096/fj.201700197RR

Figure Lengend Snippet: Gβ5 knockout inhibits M3R-stimulated insulin secretion. Cholinergic stimulation of insulin secretion was tested in primary islets (A, B) or MIN6 cells lacking Gβ5 (E). A) Islets from Gnb5−/− (KO, red) and wild-type (WT, black) mice were perifused. After equilibration with media containing 3 mM glucose (3G), islets were challenged with 16.7 mM glucose (16G), with or without 10 μM Oxo-M. The data points are the mean ± sd of insulin concentrations measured in collected fractions from experiments on 4 independent animal cohorts (3–5 animals/genotype). ***P < 0.001 in the peak; for other data points, P < 0.05. B) Quantification of total secreted insulin (area under the curve shown in A). C) Immunoblot analysis of islets from Gnb5−/− mice (top) and CRISPR-Cas9-knockout MIN6 clones (bottom). Total cell lysates were probed for Gβ5, Gβ1, Gαq, and actin (representative of at least 2 experiments). D) In situ RNA hybridization of mouse pancreatic slices using RNAscope probes against Gnb5 (green) and Chrm3 (red) gene products was performed. Shown are phase-contrast (left) and fluorescence (right) images of a representative islet. The inset shows magnification of the fluorescent dots representing the single target mRNA molecules. E) Wild-type MIN6 cells, control CRISPR (LZ), and 2 stable clones after CRISPR-Cas9-mediated knockout of the Gnb5 gene (#1 and #2) were stimulated with 16.7 mM glucose or 16.7 mM glucose plus 100 μM Oxo-M. Data are means ± sd of insulin concentration in the supernatant from the cultured cells. *P < 0.05; **P < 0.01.

Article Snippet: Localization of mRNA in paraffin-embedded slices of the mouse pancreata and eyes was performed with custom fluorescence RNAscope probes (Advanced Cell Diagnostics, Newark, CA, USA), according to the manufacturer’s instructions, with minor modifications described earlier ( 27 ).

Techniques: Knock-Out, Western Blot, CRISPR, Clone Assay, In Situ, Hybridization, RNAscope, Fluorescence, Control, Concentration Assay, Cell Culture