Fig 1: The simultaneous intervention of KORs and the BLA to vHip projections prevented the reinstatement of morphine CPP and anxiety-like behaviors in morphine-withdrawn mice. (A) Timeline of experiment. (B) The inhibition of the BLA to vHip projections combined with nor-BNI administration increased the open-arm time in and entries into the EPM by the Mor-A mice. (C) The inhibition of the BLA to vHip projections combined with nor-BNI administration increased the time spent and distances traveled in the central area of the OFT by the Mor-A mice. In (B,C), the blue histogram represents the hM4Di (CNO) + BNI group, in which the mice were first injected with the chemogenetic virus (hM4Di). Then, the mice morphine withdrawal anxiety model was constructed, and BNI was injected 2 weeks after withdrawal. The next day, the mice were injected with CNO and tested for anxiety-like behavior 30 min later. CNO and BNI were dissolved in 0.5% DMSO. The white histogram represents the hM4Di (DMSO) + DMSO group. (D) The inhibition of the BLA to vHip projections combined with nor-BNI administration prevented the reinstatement of morphine CPP. nor-BNI: a long-lasting KOR antagonist. Data are expressed as the mean ± SEM. In the CPP results, *: comparison between the extinction group and the post-test group. #: comparison between the 2 indicated groups. n = 7–10/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ###P < 0.001, ####P < 0.0001.
Fig 2: Mu receptor protein levels are decreased in the dorsal striatum of LgA rats after a month of forced abstinence. a–c Quantification of protein expression and representative images of Western blots showing levels of mu (OPRM1), delta (OPRD1), and kappa (OPRK1) proteins in rat striata. a LgA-L and LgA-H show decreased striatal OPRM1 protein levels. b Striatal OPRD1 protein levels show significant increases in the ShA rats. c Striatal OPRK1 protein levels were not significantly affected in any of the groups. d OPRM1 protein expression shows negative correlation to individual oxycodone intake. e OPRD1 and f OPRK1 protein expression show no significant relationship to drug intake. For quantitative Western blot analysis, the bands were normalized to cyclophilin B (CYPB) or a-tubulin (a-Tub). The values in the bar graphs represent means ± SEM (n = 5–6 rats per group). Key to statistics: *, **, p < 0.05, 0.01, respectively, in comparison to saline rats; ##p < 0.05 in comparison to ShA rats
Fig 3: Mu and kappa protein levels are increased in the hippocampus of LgA-H rats after 1 month of forced abstinence and incubated behaviors. a, b, c Quantitative measures and representative images showing Western blot analyses for mu (OPRM1), delta (OPRD1), and kappa (OPRK1) receptor proteins, respectively, in the rat hippocampus. LgA-H rats show increased OPRM1 (a) and OPRK1 (c) receptor protein expression after a month of forced abstinence. b There were no significant changes in OPRD1 receptor protein levels. Individual oxycodone intake showed positive correlation to d OPRM1 and f OPRK1 protein levels in the hippocampus. e There was no significant relationship between the OPRD1 and oxycodone intake. For quantification, Western blotting for OPRM1, OPRD1, and OPRK1 proteins were normalized to cyclophilin B (CYPB) and then analyzed. The values represent means ± SEM (n = 5–6 rats per group). Note the differences in scales on the Y-axis. Key to statistics: *p < 0.05 in comparison to saline rats, #p < 0.05 in comparison to ShA rats; $p < 0.05 in comparison to LgA-L
Fig 4: Differential changes in striatal and hippocampal mRNA expression after a month of forced abstinence from oxycodone SA. a Increased Oprm1 receptor mRNA levels in the striatum of LgA-H rats. b Striatal Oprd1 mRNA levels are increased in ShA rats. c Striatal Oprk1 expression showed no significant changes. d Decreased hippocampal Oprm1 receptor mRNA levels in LgA rats. e Hippocampal Oprd1 mRNA levels show no significant changes. f Hippocampal Oprk1 mRNA levels are decreased in all oxycodone groups. The values in the bar graphs represent means ± SEM (n = 5–12 animals per group). Note the differences in scales on the Y-axis. Key to statistics: *, **, *** = p < 0.05, 0.01, 0.001, respectively, in comparison to saline rats; ##p < 0.01 in comparison to ShA rats; $p < 0.05 in comparison to LgA-L
Fig 5: Specific knockdown of the kappa opioid receptor in the BLA to vHip projections ameliorate anxiety-like behaviors in morphine-withdrawn mice. (A) Immunoblots and quantification analysis of KOR levels in the BLA. KOR protein expression levels increased in the BLA of the Mor-A mice. (B) (Left) Representative images of KOR mRNA (red) in the BLA of the Mor-A mice. The nuclei were stained with DAPI (blue). Scale bar: 200 µm. BLA regions enclosed by a green box are shown at higher magnification in images to the right. Scale bars: 50 µm. (Right) KOR+ cell numbers per mm2 in the BLA of the Mor-A mice were increased. (C) Co-labeled neurons for KOR mRNA and FG in the BLA. Retrograde FG was injected into the vHip. (Left) Representative images of KOR mRNA (green) and fluorogold (FG; red) immunostaining in the BLA. Scale bar: 200 µm. BLA regions enclosed by a red box are shown at higher magnification in the images to the right. Arrows indicate neurons co-labeled with FG and KOR mRNA. Scale bars: 50 µm. (Right) The percentage of neurons co-labeled with KOR mRNA and FG relative to FG in the BLA. (D) (Left) Schematic of the KOR knockdown from the BLA to vHip projections in the KORloxp/loxp mice. (Right) Representative coronal images of virus injection in the BLA and vHip. Scale bar: 100 µm (E) KOR mRNA and (F) KOR protein expression levels in the BLA of the KORloxp/loxp mice with Cre-recombinase expression were decreased. (G) The Mor-A mice with KOR knockdown exhibited increases in open-arm time and entries in the EPM. (H) The Mor-A mice with KOR knockdown exhibited increases in central time and central distances in the OFT. (I) Knockdown of KOR in the BLA to vHip projections did not affect the morphine CPP. Data are expressed as the mean ± SEM. The KOR knockdown experiment compared the Mor-A mice expressing Cre-recombinase with Mor-A mice expressing GFP. In the CPP results, *: comparison between the extinction group and the post-test group. #: comparison between the 2 indicated groups. n = 6–8/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ##P < 0.01, ####P < 0.0001.
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