Article Figures & Data
Figures
- Figure 1.
The involvement of β-EP and MORs on opioid-dependent stress-induced analgesia. A–C, Left, Tail-flick latency under transitory swim (A), moderate swim (B), or prolonged swim (C) exposure in mice pretreated with saline or β-FNA; n = 12 for each group, **p < 0.01. Right, Equated MPE percentage from left groups, with data shown as the mean ± SEM. **p < 0.01 versus saline. D, β-EP levels in serum analysis using ELISA. n = 6 for each group, data are shown as mean ± SEM. *p < 0.05; **p < 0.01 versus control.
- Figure 2.
Generation of conditional knock-out mice specifically lacking MORs on glutamatergic and GABAergic neurons. A, Schematic of in situ hybridization for Oprm1 mRNA and GAD2 mRNA in the PAG areas in MORGABA+/+ and MORGABA−/− mice. The nucleus is stained in blue (DAPI), GAD2 mRNA is stained in green, and Oprm1 mRNA is stained in red. Scale bar, 100 μm. B, Higher-magnification images of the fields in the PAG areas in MORGABA+/+ and MORGABA−/− mice. The white arrowhead indicates a double-labeled cell with Oprm1 mRNA and GAD2 mRNA; the purple arrowheads represent Oprm1 mRNA localization in GAD2-negative cells; and the yellow arrowheads represent GAD2-positive cells without Oprm1 mRNA. Scale bar, 20 μm. C, Schematic of in situ hybridization for Oprm1 mRNA and vGlut1 mRNA in the ACC areas in MORGlut+/+ and MORGlut−/− mice. The nucleus is stained in blue (DAPI), vGlut1 mRNA is stained in green, and Oprm1 mRNA is stained in red. Scale bar, 200 μm. D, Higher-magnification images of the fields in the ACC areas in MORGlut+/+ and MORGlut−/− mice. The white arrowhead indicates a double-labeled cell with Oprm1 mRNA and vGlut1 mRNA; the purple arrowheads represent Oprm1 mRNA localization in vGlut1-negative cells; and the yellow arrowheads represent vGlut1-positive cells without Oprm1 mRNA. Scale bar, 20 μm. E, Quantitative analysis of the percentage of double-positive neurons (Oprm1 and GAD2) against the GAD2-positive neurons. *p < 0.05 versus MORGABA+/+, unpaired t test (n = 3 in each group). F, Quantitative analysis of the percentage of double-positive neurons (Oprm1 and vGlut1) against the vGlut1-positive neurons. **p < 0.01 versus MORGlut+/+, unpaired t test (n = 3 in each group).
- Figure 3.
A–D, The contributions of MORs expressed in glutamatergic (B, D) and GABAergic (A, C) neurons to stress-induced analgesia under transitory (A, B) or moderate (C, D) swim exposure. Left, Tail-flick latency. Right, Equated MPE percentage; n = 12 for each group; data are shown as the mean ± SEM. **p < 0.01.
- Figure 4.
The responses of PAG-RVM neurons to thermal stimulus. A, Schematic representation of the virus injection sites and optic fiber insertion sites, and corresponding schematic of GCaMP expression on RVM and PAG. Scale bar, 100 μm. B, Typically representative photometry traces from GCaMP (top) or EYFP (bottom) mouse relative to the onset in response to 55°C (left) and 25°C (right) water tail immersion. Each red arrow represents an event (tail immersion stimulus). C, Time course of the averaged fluorescence signal change in response to tail immersion. Light-colored shadow indicates the SEM; n = 6 for each group. D, Comparison of the averaged fluorescence signal change between EYFP and GCaMP groups during the onset period (0–5 s) for each stimulation. **p<0.01.
- Figure 5.
Changes in fluorescence intensities of PAG-RVM neurons induced by thermal stimulus under transitory swim exposure. A–C, Top, Heat map of averaged fluorescence dynamics of GCaMP relative to the onset prestress and poststress in response to 55°C water tail immersion on wild-type, MORGABA−/−, or MORGlut−/− mice; n = 6 for each group. Bottom, Time course of the averaged fluorescence intensities prestress and poststress on wild-type, MORGABA−/−, or MORGlut−/− groups. D–F, Comparison of the averaged fluorescence intensities between prestress and poststress during the onset period (0–5 s) for each stimulation on wild-type, MORGABA−/−, and MORGlut−/− mice. *p < 0.05.
- Figure 6.
Changes in the fluorescence intensities of PAG-RVM neurons induced by thermal stimulus under moderate swim exposure. A–C, Top, Heat map of averaged fluorescence dynamics of GCaMP relative to the onset prestress and poststress in response to 55°C water tail immersion on wild-type, MORGABA−/−, or MORGlut−/− mice; n = 6 for each group. Bottom, Time course of the averaged fluorescence intensities prestress and poststress on wild-type, MORGABA−/−, or MORGlut−/− groups. D–F, Comparison of the averaged fluorescence intensities between prestress and poststress during the onset period (0–5 s) for each stimulation on wild-type, MORGABA−/−, and MORGlut−/− mice. *p < 0.05, **p < 0.01.
- Figure 7.
Schematic illustration showing the proposed mechanism of the contributions of MORGABA and MORGlut to analgesia induced by different intensities of stress. Under transitory swim stress, the activation of MORGlut and MORGABA by the released β-endorphin equally inhibits glutamatergic and GABAergic inputs on PAG-RVM neurons, respectively. These opposite effects on PAG-RVM neurons should neutralize each other and keeps the activity of PAG-projecting neurons almost unchanged. In contrast, under moderate swim stress, MORGABA are further activated by the more elevated β-endorphin to cause imbalance of excitatory–inhibitory synaptic inputs on PAG neurons, and therefore leads to disinhibition of PAG-RVM neurons and induces analgesia.
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