non peptide orphanin receptor antagonist j 113397  (Tocris)

Journal: Nature Communications

Article Title: Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release

doi: 10.1038/s41467-024-49712-0

Figure Lengend Snippet: a Structural model of NOPLight and screening summary. n ≥ 3 cells for each variant. b Representative images of HEK293T cells (top; scale bars, 10 µm) and neurons (bottom; scale bars, 20 µm) expressing NOPLight before and after application of 1 µM N/OFQ and ΔF heat map. c Responses of NOPLight or NOPLight-ctr in HEK293T cell and neurons to 1 µM N/OFQ, followed by competition with 10 µM J-113397. d Quantification of maximal responses from ( c ), n = 38, 30, 22, 27 cells from three independent experiments, respectively. Data shown as mean ± SD. (One-sided Mann–Whitney U -test, **** P < 0.0001, P = 1.003 × 10 −12 , and 1.262 × 10 −9 for NOPLight versus NOPLight-ctr in HEK293T and neurons). e Normalized response of HEK293T cells and neurons expressing NOPLight to N/OFQ titrations (mean ± SD) fitted with three-parameter Hill equation. n = 3 independent experiments per concentration. f Line-scan time plot of NOPLight ΔF/F 0 (gray) from membrane-pixels shown in bottom. Right, quantification of time constant (τ) from four independent experiments. Data represented as mean ± SD. g Left: experimental schematics and images (scale bars, 20 µm), right: time plot of normalized ΔF/F 0 from a representative experiment (gray) with quantified time constant (τ off ). Data represented as mean ± SD. h Maximal NOPLight response to: NCS, Nocistatin; J11, J-113397; UFP, UFP-101; RO, Ro 64-6198; MCO, MCOPPB; OFQ1-11, Orphanin-FQ (1–11)). * P < 0.05, ** P < 0.01, P = 0.024, Nocistatin; 0.0012, J-113397; 0.0012, UFP-101. n = 9 cells except MCO ( n = 8) from three independent experiments. i Maximal NOPLight response to endogenous opioid ligands (1 µM). ** P < 0.01, P = 0.003, Dynorphin A; P = 0.003, Dynorphin-B; P = 0.003, Leu-Enkephalin; P = 0.003, Met-Enkephalin; P = 0.003, β-endorphin. n = 9 from three independent experiments. Statistical comparisons in ( h , i ) by one-sided pairwise Mann–Whitney rank test with post hoc Bonferroni correction, data represented as mean ± SEM. j NOPLight response to neurotransmitters (DA dopamine, ACh acetylcholine, GABA gamma-Aminobutyric, all 1 mM). n = 29 (DA) or 30 (ACh, GABA) cells from three independent experiments (two-sided paired t -test, **** P < 0.0001, P = 2.45 × 10 −22 , 7.46 × 10 −19 , and 7.82 × 10 −21 , respectively).

Article Snippet: For pharmacological characterizations, the following compounds were used: Nocistatin (Abbiotec); J-113397 (Sigma-Aldrich); UFP-101 (Sigma-Aldrich); Ro 64-6198 (Sigma-Aldrich); MCOPPB (Cayman); Orphanin-FQ (1–11) (Tocris); Leu-Enkephalin (Cayman); Met-Enkephalin (Cayman); Dynorphin A (Cayman); Dynorphin-B (Cayman); β-Endorphin (Sigma-Aldrich); γ-Aminobutyric acid (Sigma-Aldrich); Dopamine hydrochloride (Sigma-Aldrich), Acetylcholine bromide (Sigma-Aldrich), Glutamate (Sigma-Aldrich).

Techniques: Variant Assay, Expressing, MANN-WHITNEY, Concentration Assay, Membrane

Journal: Nature Communications

Article Title: Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release

doi: 10.1038/s41467-024-49712-0

Figure Lengend Snippet: a Fiber photometry schematic. Cartoon of NOPLight viral injection and fiber implant in the VTA. b Representative image showing DAPI (blue) and NOPLight (green) expression with fiber placement. c Left: Averaged traces of NOPLight fluorescence after systemic (i.p.) injection of vehicle (veh, black; n = 11 mice) or 1, 5, or 10 mg/kg of selective NOPR agonist Ro 64-6198 (RO, green; n = 5, 12, or 16 mice/group, respectively). Right: Mean NOPLight fluorescence 20–25 min after RO injection increases dose-dependently (two-tailed Mann–Whitney test, ** p = 0.0012, **** p < 0.0001). Data represented as mean ± SEM. d Left: Averaged traces of NOPLight fluorescence after 10 mg/kg RO (pink), 10 mg/kg selective NOPR antagonist LY2940094 (LY, o.g., blue), or both (purple) ( n = 4 mice). Right: Increase in signal 20–25 min after RO is blocked by LY pretreatment (two-tailed Mann–Whitney test, * p = 0.0286, # p = 0.0286, n = 4 mice). Data represented as mean ± SEM. e Same as ( d ) for NOPR antagonist J-113397 (J11, i.p.) (two-tailed Mann–Whitney test, ## p = 0.0028, #### p < 0.0001, **** p < 0.0001, n = 9 mice, RO; 4 mice, J11; 9 mice, J11 + RO). Data represented as mean ± SEM. f Top: Cartoon of FLEX-NOPLight or NOPLight-ctr viral injection and fiber implant in the VTA. Bottom: Representative image showing expression of DAPI (blue) and FLEX-NOPLight (left, green) or NOPLight-ctr (right, green), with fiber placement in VTA. g Averaged traces of NOPLight (green), FLEX-NOPLight (blue), or NOPLight-ctr (gray) fluorescence ( n = 16, 3, 6 mice, respectively) after systemic (i.p.) injection of 10 mg/kg RO. Data represented as mean ± SEM. h Area under the curve (AUC) of each NOPLight variant after RO injection (two-tailed Mann–Whitney test, * p = 0.0275, *** p = 0.0001, **** p < 0.0001, ns not significant ( p = 0.9577), # p = 0.0238, #### p < 0.0001. Group sizes left to right: n = 11, 5, 12, 16, 3, and 6 mice). Data represented as mean ± SEM. PN paranigral VTA, veh vehicle, BL baseline.

Article Snippet: For pharmacological characterizations, the following compounds were used: Nocistatin (Abbiotec); J-113397 (Sigma-Aldrich); UFP-101 (Sigma-Aldrich); Ro 64-6198 (Sigma-Aldrich); MCOPPB (Cayman); Orphanin-FQ (1–11) (Tocris); Leu-Enkephalin (Cayman); Met-Enkephalin (Cayman); Dynorphin A (Cayman); Dynorphin-B (Cayman); β-Endorphin (Sigma-Aldrich); γ-Aminobutyric acid (Sigma-Aldrich); Dopamine hydrochloride (Sigma-Aldrich), Acetylcholine bromide (Sigma-Aldrich), Glutamate (Sigma-Aldrich).

Techniques: Injection, Expressing, Fluorescence, Two Tailed Test, MANN-WHITNEY, Variant Assay

Journal: Nature Communications

Article Title: Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release

doi: 10.1038/s41467-024-49712-0

Figure Lengend Snippet: a Fiber photometry schematic. Cartoon of viral injection of NOPLight and fiber implant in VTA of wild-type mice ( n = 7 mice). b Cartoon depicting head-fixed cued-sucrose setup and trial structure. c Averaged traces of NOPLight fluorescence following pretreatment with vehicle (veh, green) or 20 mg/kg NOPR antagonist J-113397 (J11, blue), aligned to tone onset (magenta, shaded). Data represented as mean ± SEM. d Average licks made during vehicle or J-113397 sessions (two-tailed Wilcoxon test, p = 0.1649, ns not significant, n = 7 mice). Data represented as mean ± SEM. e Heat maps of NOPLight fluorescence, rows correspond to trials averaged in ( c ) for vehicle (left) and J11 (right) sessions. f Area under the curve (AUC) for averaged traces from ( c ), calculated over 5-s intervals surrounding cued-sucrose events. Decrease in NOPLight fluorescence during 10% sucrose access (two-tailed Wilcoxon test, ** p = 0.0034, n = 7 mice) is blocked by J11 pretreatment (two-tailed Mann–Whitney test, ## p = 0.0022, n = 7 mice). Data represented as mean ± SEM. g Top: Fiber photometry schematic. Middle: Cartoon depicting fiber implant and viral injection of DIO-GCaMP6m, FLEX-NOPLight, or NOPLight-ctr into the VTA of PNOC-Cre, OPRL1-Cre, or WT mice, respectively. Bottom: Session trial structure. h Left: Averaged trace of pnVTA PNOC GCaMP6m activity during tail lift. Right: AUC for photometry trace calculated over 5-s intervals surrounding tail lift (two-tailed Wilcoxon test, *** p = 0.0002 (5–10 s) or 0.0003 (10–15 s); **** p < 0.0001, n = 4 mice). Data represented as mean ± SEM. i Left: Averaged traces of FLEX-NOPLight (green) and NOPLight-ctr (gray) fluorescence during the tail lift. Right: AUC for photometry traces calculated over 5-s intervals surrounding tail lift. FLEX-NOPLight fluorescence increases during tail lift (two-tailed Wilcoxon test, ** p = 0.0034 (0–5 s) or 0.0093 (5–10 s); n = 3 mice), NOPLight-ctr fluorescence remains unchanged (two-tailed Mann–Whitney test, # p = 0.0264 (0–5 s) or 0.0326 (5–10 s); n = 8 mice, NOPLight-ctr; 3 mice, FLEX-NOPLight). Data represented as mean ± SEM. VITI variable inter-trial interval.

Article Snippet: For pharmacological characterizations, the following compounds were used: Nocistatin (Abbiotec); J-113397 (Sigma-Aldrich); UFP-101 (Sigma-Aldrich); Ro 64-6198 (Sigma-Aldrich); MCOPPB (Cayman); Orphanin-FQ (1–11) (Tocris); Leu-Enkephalin (Cayman); Met-Enkephalin (Cayman); Dynorphin A (Cayman); Dynorphin-B (Cayman); β-Endorphin (Sigma-Aldrich); γ-Aminobutyric acid (Sigma-Aldrich); Dopamine hydrochloride (Sigma-Aldrich), Acetylcholine bromide (Sigma-Aldrich), Glutamate (Sigma-Aldrich).

Techniques: Injection, Fluorescence, Two Tailed Test, MANN-WHITNEY, Activity Assay