Article Figures & Data
Figures
- Figure 1.
Fate-mapping of VGLUT2 expression in VGLUT2-Cre x zsGreen mice indicates that the majority of mesopontine cholinergic neurons are positive for the fluorescent reporter zsGreen. A, Schematic for VGLUT2-Cre dependent expression of zsGreen. B, Percentage of ChAT-positive neurons expressing zsGreen; n = 3–4 animals/group. C, Tiled midbrain section from a VGLUT2-Cre x zsGreen mouse showing endogenous zsGreen fluorescence (green) and immunostaining for ChAT (red) as well as counterstaining with DAPI (blue). Cholinergic nuclei PBG, PPTg and 3N are denoted. Scale bar, 500 μm. D, Higher-power images of PBG, PPTg, 3N, and LDTg. Note that images are not from section illustrated in panel C. Scale bar, 100 μm.
- Figure 2.
VGAT-Cre x zsGreen mice reveal that mesopontine cholinergic neurons are rarely coexpressing the VGAT fluorescent reporter zsGreen. A, Schematic for VGAT-Cre dependent expression of zsGreen. B, Percentage of ChAT-positive neurons expressing zsGreen; n = 3–4 animals/group. C, Tiled midbrain section from VGAT-Cre x zsGreen mouse showing endogenous zsGreen fluorescence (green) and immunostaining for ChAT (red) and nuclear labeling with DAPI (blue). Cholinergic nuclei PBG, PPTg, and 3N are denoted. Scale bar, 500 μm. D, Higher-power images of PBG, PPTg, 3N, and LDTg. Note that images are not from section illustrated in panel C. Scale bar, 100 μm.
- Figure 3.
Multiplex fluorescent in situ hybridization reveals that most PBG but not PPTg or 3N cholinergic neurons express VGLUT2 mRNA in the adult. A, Schematic of fluorescent multiplex in situ hybridization (RNAscope) approach using antisense probes directed against ChAT (red), VGLUT2 (green), and VGAT (blue) mRNAs. B, Quantification of ChAT+/VGLUT2+ and ChAT+/VGAT+ neurons in the PBG, PPTg, and 3N; n = 3 animals/group. C, C57BL/6 mice (10–15 weeks) were stained for ChAT (red), VGLUT2 (green), and VGAT (blue) mRNAs and counterstained with DAPI. Scale bar, 50 μm. D, Magnified images of PBG, PPTg, and 3N labeled for ChAT (red), VGLUT2 (green), and VGAT (blue) mRNAs. Scale bar, 25 μm. White arrows indicate colocalization of ChAT and VGLUT2; yellow arrow indicates colocalization of ChAT and VGAT.
- Figure 4.
Viral tracing in PBG neurons of ChAT-Cre and VGLUT2-Cre mice. A, Schematic of AAV5-DIO-ChR2:eYFP injection into the PBG of Cre lines. B, Quantification of ChR2+ cells co-positive for ChAT counted in the PBG of VGLUT2-Cre mice; n = 4 animals. C, D, Tiled midbrain images of ChR2:eYFP (green) expression in the PBG of ChAT-Cre (C) and VGLUT2-Cre (D) mice stained for ChAT (red) and counterstained with DAPI (blue). E, F, Higher-power images showing colocalization of ChR2:eYFP (green) with ChAT (red) in the PBG of ChAT-Cre (E, top left) and VGLUT2-Cre (F, top right) mice. Bottom, ChR2:eYFP-positive terminals (green) in the main PBG projection targets SC, LGN, Opt, and LA of ChAT-Cre (E) and VGLUT2-Cre (F). Scale bar, 100 μm.
- Figure 5.
Partial colocalization of VGLUT2 and VAChT in PBG terminals in SC. ChAT-Cre mice injected with AAV5-DIO-ChR2:eYFP into the PBG were immunostained for ChR2 (blue), VGLUT2 (red), and VAChT (green). A, Tiled SC image and overview of VGLUT2, VAChT, and ChR2 expression in SC sublayers; white squares labeled B and C indicate higher-magnification images shown in B and C. PAG, Periaqueductal gray. Scale bar, 200 μm. B, C, High-power images demonstrating ChR2-labeled processes primarily in the SL (B) and sparser expression in the IL (C); white squares labeled D and E indicate zoomed-in images shown in D and E. Scale bar, 20 μm. D, E, High-magnification images in SL (D) and IL (E) demonstrate partial colocalization of ChR2, VAChT, and VGLUT2 (indicated by white arrows) and partial colocalization of VAChT and VGLUT2 in ChR2-negative terminals (indicated by black/white arrows). Scale bar, 5 μm.
- Figure 6.
Viral tracing in PPTg neurons of ChAT-Cre and VGLUT2-Cre mice. A, Schematic of AAV-DIO-ChR2 injection into the PPTg. B, Quantification of ChR2+ cells co-positive for ChAT counted in the PPTg of VGLUT2-Cre mice; n = 6 animals. C, D, Tiled midbrain images of ChR2 (green) expression in the PPTg of ChAT-Cre (C) and VGLUT2-Cre (D) mice stained for ChAT (red) and counterstained with DAPI (blue). E, F, Higher-power images showing colocalization of ChR2 (green) with ChAT (red) in the PPTg of ChAT-Cre (E, top left) and VGLUT2-Cre (F, top right ) mice. ChR2-positive terminals (green) in the selected major PPTg projection targets VTA, SNc, GPi, LH, STN, and CeA/BLA of ChAT-Cre (E, bottom left ) and VGLUT2-Cre (F, bottom right ). Scale bar, 100 μm.
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