Journal: Cell reports

Article Title: Septo-hypothalamic regulation of binge-like alcohol consumption by the nociceptin system

doi: 10.1016/j.celrep.2025.115482

Figure Lengend Snippet: (A) Atlas image of regions of interest for the dLS, iLS, and vLS for analysis of Pnoc and Oprl1 expression across the A-P gradient. (B) Representative image of the LS visualizing probes for VGAT (GABA), Pnoc (nociceptin), and Oprl1 (NOP) mRNA in DAPI + nuclei. Scale bar, 100 μM. (C) Representative images of VGAT , Pnoc , and Oprl1 overlay with DAPI. Scale bar, 50 μM. (D–F) Quantification of mRNA within the dLS, iLS, and vLS. Values are collapsed across sex given the lack of main effect and factor interaction. (D) Pnoc and Oprl1 are expressed to a similar extent in VGAT + cells throughout the A-P gradient of the dLS, with the exception of 0.86 mm where Oprl1 expression is less than Pnoc (* p < 0.05). Cells expressing both Pnoc and Oprl1 are expressed to a lesser extent than Pnoc ($ p < 0.01) and Oprl1 (∧ p < 0.001). (E) Oprl1 expression is greater than Pnoc (* p < 0.001) and Pnoc and Oprl1 colocalization ($ p < 0.001) across the A-P gradient in the iLS. Pnoc expression was also greater than Pnoc and Oprl1 colocalization ($ p < 0.001). (F) Oprl1 expression is greater than Pnoc (* p < 0.001) and Pnoc and Oprl1 colocalization ($ p < 0.001) across the A-P gradient in the vLS. Pnoc expression was also greater than Pnoc and Oprl1 colocalization across the A-P gradient ($ p < 0.001). (G) Pnoc expression is most abundant in the dLS, being greater than iLS and vLS in all but the posterior-most LS ( ** p < 0.01, *** p < 0.005, **** p < 0.001). (H) Oprl1 mRNA is fairly evenly represented across the LS. (I) Colocalized Pnoc and Oprl1 mRNA was most abundant in the dLS, exceeding the iLS and vLS (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001). Data are presented as mean ± SEM. Triangles, females; Circles, males.

Article Snippet: Nociceptin , Tocris , Cat# 0910.

Techniques: Expressing

Journal: Cell reports

Article Title: Septo-hypothalamic regulation of binge-like alcohol consumption by the nociceptin system

doi: 10.1016/j.celrep.2025.115482

Figure Lengend Snippet: (A) Depiction of systemic NOP antagonist (SB-612111; 10 mg/kg) treatment prior to alcohol drinking. (B) Systemic NOP antagonist treatment decreased alcohol intake (drug [ F (1, 30) = 5.55, p = 0.025]). While females generally consumed more alcohol than males (sex [ F (1, 30) = 6.69, p = 0.015]), a post hoc analysis indicated that the ability of a NOP antagonist to reduce alcohol drinking was primarily driven by males ( p = 0.02). (C) Depiction of site-specific NOP antagonist (SB-612111; 100 μM) microinjection into the LS prior to alcohol drinking. (D) Intra-LS injection of SB-612111 decreased alcohol intake (drug [ F (1, 21) = 11.22, p = 0.003]) and post hoc indicated a reduction of alcohol drinking in males ( p = 0.015). (E) Depiction of patch-clamp electrophysiology assessing effect of nociceptin and SB-612111 on membrane potential in the LS. (F) Bath application of SB-612111 did not affect membrane potential in male and female mice. However, membrane potential was more hyperpolarized in females compared with males (sex [ F (1, 15) = 9.51, p = 0.008]). (G) Representative traces depicting hyperpolarizing effect of nociceptin in the LS and blockade with SB-612111. (H) Nociceptin application drove a hyperpolarizing shift in membrane potential in LS cells (agonist [ F (1, 15) = 17.13, p < 0.001]) in males ( p = 0.012) and females ( p = 0.030). Pre-application with SB-612111 blocked the effect of nociceptin. See also . Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

Article Snippet: Nociceptin , Tocris , Cat# 0910.

Techniques: Microinjection, Injection, Patch Clamp, Membrane

Journal: Communications Biology

Article Title: Dynamics of agonist-evoked opioid receptor activation revealed by FRET- and BRET-based opioid receptor conformation sensors

doi: 10.1038/s42003-025-07630-x

Figure Lengend Snippet: A , B Averaged FRET-based measurement of HEK293 cells stably expressing the KOR conformation sensor measured in a 96-well plate format at a plate reader (schematic image of a 96-well plate created with BioRender, licence INC-12457). Increasing concentrations of dynorphine A were applied followed by a saturating concentration of dynorphine A and the antagonist naloxone. The agonist-induced activation was normalized to the maximal activation (mean ± SEM, n = 5 of independent transfections measured in triplets) and plotted as concentration-response curve for dynorphine A in ( B ) (EC 50 : 219 nM). C Inhibition curve for the FRET KOR sensor activated with 500 nM dynorphine A and inhibited with increasing concentrations of the antagonist naloxone (IC 50 : 716 nM; pKi: 314 nM). D , E Averaged FRET-based measurement of HEK293 cells stably expressing the NOP conformation sensor measured in a 96-well plate format at a plate reader (schematic image of a 96-well plate created with BioRender, licence INC-12457). Increasing concentrations of nociceptin were applied followed by a saturating concentration of nociceptin. The agonist-induced activation was normalized to the maximal activation (mean ± SEM, n = 3 of independent transfections measured in triplets) and plotted as concentration-response curve for nociceptin in E (EC 50 : 63 nM). F Inhibition curve for the FRET NOP sensor activated with 100 nM nociceptin and inhibited with increasing concentrations of the antagonist JTC-801 (IC 50 : 473 nM; pKi: 298 nM). G Averaged BRET-based measurement of HEK293 cells stably expressing the KOR BRET-sensor. Increasing concentrations of dynorphine A were applied followed by a saturating concentration. The agonist-induced activation was normalized to the maximal activation and plotted as a concentration–response curve ( H ) (mean ± SEM, n = 3 of independent measurements performed in triplets). H Concentration–response curve for dynorphine A at the KOR BRET (blue) and KOR FRET (magenta) receptor conformation sensor. I Calculated EC 50 values for dynorphine A (H) were comparable between BRET (209 nM) and FRET-based sensor (219 nM) ( P = 0.7294, unpaired t test with Welch’s correction).

Article Snippet: Morphine hydrochloride was purchased from Merck (Darmstadt, Germany), naloxone-HCl and dynorphin A TFA salt were purchased from Cayman Chemical (Ann Arbor, Michigan, USA), l -methadone-HCl was purchased from Hoechst AG (Frankfurt, Germany), nociceptin (1–13) amide TFA was purchased from TargetMol (Boston, MA, USA) and JTC-801 was purchased from Tocris Bioscience (Bristol, United Kingdom).

Techniques: Stable Transfection, Expressing, Concentration Assay, Activation Assay, Transfection, Inhibition

Journal: Cells

Article Title: Nuclear Factor-κB Signaling Regulates the Nociceptin Receptor but Not Nociceptin Itself

doi: 10.3390/cells13242111

Figure Lengend Snippet: Effects of PMA on NOP ( A ) and nociceptin ( B ) mRNA and protein levels in THP-1 cells. Cells were treated with PMA 5 ng/mL or without PMA (controls) for 24 h. Median with interquartile range and 10–90 percentiles; mean “+”; mRNA and protein data are representative of six and twelve independent experiments, respectively. Wilcoxon tests * p < 0.05; ** p < 0.005; *** p < 0.001, compared to the respective controls.

Article Snippet: To stain intracellular nociceptin, cells were fixed and permeabilized (BD Cytofix/CytopermTM Kit, BD Biosciences, Allschwil, Switzerland) and incubated with rabbit anti-human nociceptin antibody (Phoenix Pharmaceuticals, Karlsruhe, Germany) or isotype-control antibody (Abcam, Cambridge, UK) at a final concentration of 5 μg/mL for one hour at room temperature (RT).

Techniques: