rnascope® fluorescent multiplex kit version 2  (Advanced Cell Diagnostics Inc)

Journal: Molecular Metabolism

Article Title: Modulation of stress-related behaviour by preproglucagon neurons and hypothalamic projections to the nucleus of the solitary tract

doi: 10.1016/j.molmet.2024.102076

Figure Lengend Snippet: Selective and efficient chemogenetic activation of cNTS PPG neurons in vivo. A) Schematic of injection protocol. B) Representative images of RNAscope in situ hybridisation for Ppg mRNA (yellow) and immunolabelling for mCherry (to detect hM3Dq:mCherry, red) in tissue from one Glu-Cre/tdRFP mice injected with AAV8-DIO-hM3Dq:mCherry. Scale bars: 100 μm. C) Percent of Ppg- expressing neurons in the cNTS also expressing hM3Dq:mCherry (efficiency), as well as percent of mCherry-expressing cNTS neurons also expressing Ppg (selectivity). Results from control mice (expressing mCherry only, n = 2) are indicated with black circles, while results from hM3Dq-expressing mice (n = 2) are indicated with green triangles. D) Representative images of immunohistochemical labelling for cFOS (black nuclear label) and dsRed (brown cytoplasmic label, detecting mCherry and tdRFP) in mice expressing mCherry only (control, top panels) or hM3Dq:mCherry (hM3Dq, bottom panels) injected with saline (2 ml/kg, left panels) or CNO (2 mg/kg, 2 ml/kg; right panels). Scale bar: 100 μm. E) Percent of mCherry-expressing cNTS neurons also labelled for cFOS in control (grey/black) and hM3Dq-expressing (green) mice injected with saline (2 ml/kg, pattern) or CNO (2 mg/kg, filled). Data from females are indicated by circles; males are indicated by triangles. Also shown is Gardner-Altman estimation plot showing the mean difference in activated neurons between saline and CNO-injected hM3Dq-expressing mice. Two-way drug × virus interaction: F(1,32) = 138.9, p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Tissue sections were processed using fluorescence in situ hybridisation (FISH, RNAscope Multiplex Fluorescent Reagent Kit version 2, Advanced Cell Diagnostics [ACD], #323100) to label preproglucagon ( Ppg ), Crhr1 and Crhr2 mRNA transcripts in the cNTS and Crh mRNA transcripts in the PVN.

Techniques: Activation Assay, In Vivo, Injection, RNAscope, In Situ, Hybridization, Expressing, Control, Immunohistochemical staining, Saline, Virus

Journal: Molecular Metabolism

Article Title: Modulation of stress-related behaviour by preproglucagon neurons and hypothalamic projections to the nucleus of the solitary tract

doi: 10.1016/j.molmet.2024.102076

Figure Lengend Snippet: Ch emogenetic inhibition of NTS-projecting PVN neurons. A) Diagram of the experimental paradigm for chemogenetic inhibition of NTS-projecting PVN neurons. B) cFOS immunoreactivity (magenta nuclei, pseudocolour) and EGFP or mCherry immunofluorescence (greyscale) labelling in the PVN of a representative control mouse (left, EGFP) and a representative hM4Di-expressing mouse (right, hM4Di) after i.p. injection of CNO (2 mg/kg, 2 ml/kg) followed by 30 min restraint stress. 3V: third ventricle. Scale bar: 100 μm. C) Calculated percentage of EGFP- vs. hM4Di:mCherry-expressing (fluorescent protein, FP+) cells that were also cFOS-positive in mice after i.p. injection of CNO (2 mg/kg, 2 ml/kg) followed by exposure to an acute stressor (novel environment or restraint stress). Unpaired T-test: t = 2.498, df = 6. D) Representative images of cFOS-immunoreactivity in the cNTS of mice expressing EGFP (top) or hM4Di (bottom) in NTS-projecting PVN neurons. Counted cFOS-immunoreactive cells indicated with rainbow-coloured mask to the right. All mice were injected with CNO (2 mg/kg, 2 ml/kg) and then exposed to novel environment or restraint stress. Scale bar: 100 μm. E) Counts of cFOS-immunoreactive nuclei in the cNTS in mice expressing EGFP or hM4Di in NTS-projecting PVN neurons and injected with CNO (2 mg/kg, 2 ml/kg) prior to stress exposure. Unpaired T-test: t = 3.603, df = 11. F) cFOS- and GLP-1-immunoreactivity in the cNTS of mice expressing EGFP (top) or hM4Di (bottom) in NTS-projecting PVN neurons and injected with CNO (2 mg/kg, 2 ml/kg) prior to stress exposure. Scale bar: 100 μm. G) Calculated percentage of GLP-1-immunoreactive cells in the cNTS that were also cFOS-IR. Unpaired T-test: t = 2.766, df = 6. H) Cumulative chow intake over 4 h after dark onset in mice expressing EGFP or hM4Di in NTS-projecting PVN neurons after injection with saline (2 ml/kg; dashed lines) or CNO (2 mg/kg, 2 ml/kg; solid lines). I) Chow intake over the first 2 h of dark phase in mice expressing EGFP or hM4Di in NTS-projecting PVN neurons after injection with saline (2 ml/kg) or CNO (2 mg/kg, 2 ml/kg). J) Chow intake during the first 2 h of dark onset of mice expressing EGFP or hM4Di in NTS-projecting PVN neurons and injected with CNO (2 mg/kg, 2 ml/kg) prior to 30 min restraint stress. Virus × stress interaction: F(1, 6) = 7.805, p = 0.0314. K) The relationship between activation of NTS-projecting PVN neurons and stress-induced hypophagia. The effect of stress on 2 h food intake in mice expressing EGFP (grey squares) or hM4Di (magenta circles) in NTS-projecting PVN neurons is plotted against the percentage of cFOS-positive NTS-projecting PVN neurons. L) Representative images of RNAscope in situ hybridisation for Crh (magenta in merged image) combined with immunolabelling for GFP (green in merged image) and oxytocin (cyan in merged image) in the PVN of mice expressing GFP in NTS-projecting PVN neurons. Scale bar: 100 μm. Yellow arrowheads indicate Crh- positive EGFP-labelled neurons; red arrows indicate oxytocin-positive EGFP-labelled neurons; yellow arrows indicate EGFP-labelled neurons positive for both Crh and oxytocin. M) Percentage of NTS-projecting PVN neurons expressing Crh and/or oxytocin (OT+). N = 3 mice, 2 sections containing the PVN from each mouse. N) Diagram illustrating RNAscope in situ labelling for Crh in cells with direct projections to NTS PPG neurons (RABV-GFP labelled). O) RNAscope in situ hybridisation for Crh in PPG-projecting PVN neurons (RABV-GFP labelled). Scale bar top panel: 100 μm. White arrowheads indicate Crh -positive RABV-GFP labelled neurons. Bottom panel: higher magnification image of inset in top panel. Scale bar: 20 μm. Gardner-Altman estimation plots in panels C, E, G, I, and J display estimated effect sizes for each assessed parameter. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Tissue sections were processed using fluorescence in situ hybridisation (FISH, RNAscope Multiplex Fluorescent Reagent Kit version 2, Advanced Cell Diagnostics [ACD], #323100) to label preproglucagon ( Ppg ), Crhr1 and Crhr2 mRNA transcripts in the cNTS and Crh mRNA transcripts in the PVN.

Techniques: Inhibition, Immunofluorescence, Control, Expressing, Injection, Saline, Virus, Activation Assay, RNAscope, In Situ, Hybridization