Journal: bioRxiv

Article Title: The lateral hypothalamus and orexinergic transmission in the paraventricular thalamus promote the attribution of incentive salience to reward-associated cues

doi: 10.1101/2020.03.01.972174

Figure Lengend Snippet: Experiment 1: a) Schematic of the excitotoxic lesion of the LH and photomicrograph of a representative lesion, and b) experimental timeline. Rats received 0.6 □l of NMDA in the right and left LHA. Sham rats underwent the same surgery but no injection was performed. After recovery from surgery, rats were trained in a Pavlovian Conditioned Approach (PavCA) paradigm for 7 consecutive sessions. Experiment 2: c) Schematic of the cannulation surgery to target the aPVT and pPVT and representative images of aPVT and pPVT cannulation sites, and d) experimental timeline. After recovery from surgery, rats were trained in a Pavlovian Conditioned Approach (PavCA) paradigm for 5 consecutive sessions (Acquisition) and phenotyped as sign-(STs) or goal-trackers (GTs). On the following day rats went through a PavCA test session (session 6) in order to assess the effects of OX1 or OX2 receptor antagonism in the PVT on the expression of Pavlovian conditioned approach behavior. The next day, all rats went through an additional PavCA session (session 7) with no drug or vehicle infusions to assess any lasting effects of drug infusion on PavCA behavior. Following the completion of PavCA training, all subjects were tested in a conditioned reinforcement (CRT) paradigm, in order to assess the effects of OX1 or OX2 receptor antagonism in the PVT on the conditioned reinforcing properties of the lever-CS.

Article Snippet: To block orexin 2 receptors, the selective OX2r antagonist TCS-OX2-29 (TCS; Lots 2A/179223, 2B191601, Tocris Bioscience) was dissolved in 0.9% sterile saline at a concentration of 15 μg per 300 nl.

Techniques: Injection, Expressing

Journal: bioRxiv

Article Title: The lateral hypothalamus and orexinergic transmission in the paraventricular thalamus promote the attribution of incentive salience to reward-associated cues

doi: 10.1101/2020.03.01.972174

Figure Lengend Snippet: Left panel, mean ± SEM for a ) number of lever contacts in sign-trackers during Session 5, 6 and 7 of PavCA training. There was a significant Treatment x Session interaction (p = 0.004). Post-hoc analyses revealed that, compared to control rats (SAL), rats that received a single administration of the OX2r antagonist TCS-OX2-29 (TCS) before Session 6 showed a decrease in lever contacts (*p = 0.039 vs. SAL). c) Number of magazine entries in sign-trackers during Session 5, 6 and 7 of PavCA training. No effects were found for magazine entries in sign-trackers. b) number of lever contacts and d) magazine entries in goal-trackers during Session 5, 6 and 7 of PavCA training. There were no significant effects in goal trackers. N = 7 STs-SAL, 16 STs-TCS, 4 GTs-SAL, 7 GTs-TCS. Right panel, mean ± SEM for e) nosepokes, g) lever contacts, and i) incentive value index in STs. A significant reduction in lever contacts and incentive index was apparent in STs who received infusion of the OX2r antagonist TCS OX2 29 into the PVT (vs. controls). For GTs, there was no significant effect of drug administration for f) nosepokes, h) lever contacts, or the j) incentive value index. N = 7 STs-SAL, 16 STs-TCS, 4 GTs-SAL, 7 GTs-TCS.

Article Snippet: To block orexin 2 receptors, the selective OX2r antagonist TCS-OX2-29 (TCS; Lots 2A/179223, 2B191601, Tocris Bioscience) was dissolved in 0.9% sterile saline at a concentration of 15 μg per 300 nl.

Techniques: Control

Journal: Neuroscience

Article Title: Galanin and the orexin 2 receptor as possible regulators of enkephalin in the PVN: Relation to dietary fat

doi: 10.1016/j.neuroscience.2011.07.057

Figure Lengend Snippet: In Experiment 4, the density of orexin 2 receptor (OX 2 R) and enkephalin (ENK) single-labeled as well as double-labeled neurons was increased in the parvocellular region of the posterior paraventricular nucleus after high-fat diet intake.

Article Snippet: The primary antibodies used were: guinea-pig anti-GAL 1:500 (Peninsula Laboratories, Torrance, CA); monoclonal mouse anti-ENK 1:200 (Millipore, Temecula, CA), polyclonal rabbit anti-GalR1 1:100 (Novus Biologicals, St. Charles, MO), rabbit anti-OX 1 R 1:500 (Imgenex, San Diego, CA) and rabbit anti-OX 2 R 1:200 (Enzo Life Sciences, Plymouth Meeting, PA).

Techniques:

Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: (A-D) Phosphorylation of pErk (n=16, from 8 experiments), p-Akt473 (n=14, from 7 experiments), p-Akt308 (n=14, from 7 experiments) and p-rpS6 (n=7, from 7 experiments) in U937-CD200R-GFP (Blue) or CD200RΔ-GFP (Gray) cells after 1h stimulation with anti-CD200R (filled histogram) or isotype control (open histogram) as determined by intracellular flow cytometry. Significance was determined by a t test with Welch correction. The data are normalized to isotype stimulated cells within experiments. See supplemental figure 1A for expression of CD200R and CD200RΔ and gating strategy. ( E-H ) The effect of removal of Dok2 or RasGAP, using two different guide RNA per target, on the inhibitory capacity of CD200R-GFP on ( E ) p-Erk (n=8, from 4 experiments), ( F ) IL-8 secretion (n=4, from 4 experiments), ( G ) p-Akt473 (n=13, from 7 experiments) and ( H ) p-Akt308 (n=13, from 7 experiments). CD200R was ligated with an agonistic antibody or isotype control on U937-CD200R-GFP polyclonal lines deficient for Dok2 or RasGAP. Within experiments, data were normalized to the parental cell line and tested for significance by one-way ANOVA with Holm-Sidak’s correction. Data are obtained in at least 4 independent experiments. Boxplots: median with 25 and 75 percentiles and bars represent minimum and maximum values. ( I ) A model of CD200R signaling. CD200R-signaling bifurcates at the level of Dok2. Dok2 is required for inhibition of Akt and recruitment of RasGAP. Subsequently, RasGAP inhibits Ras/Erk signaling.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: ( A ) Phosphorylation of p-Akt308, p-Akt473, p-Erk, and p-S6 after 1h stimulation of CD200R with agonistic-antibodies or isotype control in CD200R-U937-cells that over-express full-length RasGAP/p120 or RasGAP 1-455 . Data were normalized to isotype-stimulated cells. Significance was determined by a t test with Welch correction (n=6). ( B ) Caspase activity in fresh and IFNα stimulated PBMC as measured by CaspGlo 3/7 assay. Significance was determined by a t test (n=4). ( C, D ) Western blot analysis of RasGAP and Dok-2 protein expression and cleavage in fresh (Fr.) or 16h IFNα stimulated cells. Blots are lysates of (C) CD14 + monocytes and (D) PBMC cultures. >: full length RasGAP/p120; <: published RasGAP cleavage fragment . H3: Histone 3. HSP: heat shock protein 90. ( E ) Quantification of . Data represent the amount full length RasGAP/p120 or the amount of cleaved RasGAP as percentage of signal in the whole lane. Significance was determined by paired-t test.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Activity Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: ( A ) Cytokine secretion by moDC stimulated with R848 for 6h in presence of control, Akt-inhibitor AktVIII or Mek-inhibitor U0126. IL-1β: n=5, IL-8: n=8. Significance was determined by one-sample t test. ( B, C ) Phosphorylation of p-Akt473 and p-rpS6 in HC moDC. Fresh replated (n=8, 3 samples without IFN-counterpart) or IFNα-stimulated (16h, n=5, paired with fresh samples) moDCs were seeded on plates coated with control (recombinant human CD200R1, rhCD200R1) or recombinant human CD200 (rhCD200). After 2h pre-incubation, the moDC were stimulated with 1µg/ml R848 for 30 minutes. Phosphorylation was assessed by flow cytometry. Depicted is the ratio of phosphorylation in CD200-stimulated conditions over control stimulated conditions. Significance between the effect of CD200R on fresh and IFNα stimulated cells was determined by t test. Significance of the effect of CD200R in medium or IFNα stimulated cells was determined by one-sample t test. ( D, E ) mRNA (n=11) and protein (n=6) data on R848-induced IFNG or IFNγ production in PBMC. Donors with blue lines are matched between mRNA and protein data. Significance was determined by Wilcoxon test. ND = not detectable. ( F ) The ratio of R848 induced IFNG mRNA production of PBMC stimulated with anti-CD200R over PBMC stimulated with isotype. IFNG mRNA production was measured in healthy control PBMC that were used fresh after isolation (Fr., n=14), or have been cultured for 16h in medium (Med, n=20), 10 (n=8), 100 (n=8) or 1000 (n=20) U/ml of IFNα prior to ligation of CD200R with an agonistic antibody and stimulation of TLR7/8 with R848 for 4h. Significance was determined by a paired t test with Welch correction.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Recombinant, Incubation, Flow Cytometry, Isolation, Cell Culture, Ligation

Journal: bioRxiv

Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

doi: 10.1101/2020.02.06.933739

Figure Lengend Snippet: ( A, B ) IFNG mRNA production relative to GAPDH (A) or B2M (B) by healthy control and SLE patients’ PBMC in two cohorts. PBMC were seeded on an isotype control (open circles) or an agonistic anti-CD200R antibody (filled circles) prior to stimulation with R848 for 4h. Each connected dot represents one patient/control. The color of the connecting arrow indicates: inhibition by CD200R ligation (blue, >15% inhibition compared to isotype control); potentiation by CD200R ligation (red, >15% potentiation compared to isotype control); or no effect of CD200R ligation (grey, <15 % inhibition or <15% potentiation compared to isotype control). ( C ) Ratio of R848-induced IFNG mRNA production from data in and , grouped to HC, SLE patients without lupus nephritis (LN-) and patients with (a history of) lupus nephritis (LN+). Significance was determined with a one-way ANOVA.

Article Snippet: For CD200R-ligation experiments, 10μg/ml culture grade anti-CD200R or isotype control; or 3μg/ml CD200his and CD200Rhis (both Sino Biologicals) were coated onto MaxiSORB (NUNC) plates at 4°C overnight in PBS.

Techniques: Inhibition, Ligation

Journal: Frontiers in Cellular Neuroscience

Article Title: Orexin-A Excites Airway Vagal Preganglionic Neurons via Activation of Orexin Receptor Type 1 and Type 2 in Rats

doi: 10.3389/fncel.2019.00478

Figure Lengend Snippet: Retrogradely labeled airway vagal preganglionic neurons (AVPNs) in the external portion of the nucleus ambiguus (eNA) were positively immunoreactive to anti-orexin receptor type 1 (OX 1 R) and anti-OX 2 R antibodies. (A1,B1) Retrogradely labeled AVPNs (green) in the ventrolateral medulla. (A2,B2) Neurons in the ventrolateral medulla that were positively stained (red) by anti-OX 1 R (A2) and anti-OX 2 R antibody (B2) . (A3,B3) Merged images showing the positive immunoreactivity of retrogradely labeled neurons in the eNA to anti-OX 1 R ( A3 , marked by *) and anti-OX 2 R antibody ( B3 , marked by *). Note that retrogradely labeled AVPNs in the cNA were also positively immunoreactive to anti-OX 1 R antibody, but not anti-OX 2 R antibody. Dashed circles represent the range of cNA. Dorsal (d) and lateral (l) directions are indicated.

Article Snippet: Sections from three rats were incubated at 4°C overnight in a PBS-Triton solution containing rabbit anti-OX 2 R antibody (no. AOR002, lot AN-01, 1:200; Alomone, Jerusalem, Israel).

Techniques: Labeling, Staining

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Effects of Sleep Deprivation (SD) on Rats via ERK1/2 Signaling Pathway

doi: 10.12659/MSM.913839

Figure Lengend Snippet: Effects of sleep deprivation (SD) on the expression of Orexin-A, OX1R, OX2R, PARP-1, ERK1/2, and p-ERK1/2 in rat hippocampal tissue. Total protein and total RNA were collected from hippocampal tissue using tissue extraction reagent. Western blot analysis was used to assess total protein, separation of protein, protein transference, incubation of primary antibody and secondary antibody, and coloration. We used qRT-PCR for cDNA synthesis and amplification. ( A ) Relative mRNA level was measured using qRT-PCR. ( B, C ) Relative protein level was evaluated using Western blot analysis. ANOVA with t tests was used to analyze data (* vs. control group; * p<0.05, ** p<0.01).

Article Snippet: TCSOX229, the OX2 receptor (OX2R) antagonist [ , ], and orexin-A were purchased from MCE (NJ, USA).

Techniques: Expressing, Extraction, Western Blot, Incubation, Quantitative RT-PCR, Amplification, Control

Journal: Scientific Reports

Article Title: Influence of light/dark cycle and orexins on breathing control in green iguanas ( Iguana iguana )

doi: 10.1038/s41598-020-79107-2

Figure Lengend Snippet: Effect of SB-334867 and Almorexant on basal ventilation. Effect of the i.c.v. injection of SB-334867 and its vehicle on fR, V T and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{\text{V}}}_{{\text{I}}}$$\end{document} V ˙ I in normocapnic normoxia in green iguanas during light or dark phases ( A ). Effect of the i.c.v. injection of Almorexant and its vehicle on fR, V T and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{\text{V}}}_{{\text{I}}}$$\end{document} V ˙ I in normocapnic normoxia in green iguanas during light or dark phases ( B ). * Means different from light phase at P < 0.05.

Article Snippet: For the Almorexant (OX 1 R and OX 2 R antagonist, Cayman Chemical Company, USA), the same procedure was made, however, saline was used instead of aCSF.

Techniques: Injection

Journal: Scientific Reports

Article Title: Influence of light/dark cycle and orexins on breathing control in green iguanas ( Iguana iguana )

doi: 10.1038/s41598-020-79107-2

Figure Lengend Snippet: Breathing pattern.

Article Snippet: For the Almorexant (OX 1 R and OX 2 R antagonist, Cayman Chemical Company, USA), the same procedure was made, however, saline was used instead of aCSF.

Techniques: Injection

Journal: Scientific Reports

Article Title: Influence of light/dark cycle and orexins on breathing control in green iguanas ( Iguana iguana )

doi: 10.1038/s41598-020-79107-2

Figure Lengend Snippet: Ventilatory parameters.

Article Snippet: For the Almorexant (OX 1 R and OX 2 R antagonist, Cayman Chemical Company, USA), the same procedure was made, however, saline was used instead of aCSF.

Techniques: Injection

Journal: Scientific Reports

Article Title: Influence of light/dark cycle and orexins on breathing control in green iguanas ( Iguana iguana )

doi: 10.1038/s41598-020-79107-2

Figure Lengend Snippet: Effect of Almorexant on hypoxic chemoreflex. Effect of the i.c.v. microinjection (MI) of Almorexant and its vehicle on f R , V T and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{\text{V}}}_{{\text{I}}}$$\end{document} V ˙ I in green iguanas exposed to acute hypoxia (5% O 2 ) during light or dark phases. Data reported as means ± s.e.m. The effect of hypoxia (vehicle group, no drug) is indicated by * compared to control, while + represent the statistical differences caused by the effect of the drug.

Article Snippet: For the Almorexant (OX 1 R and OX 2 R antagonist, Cayman Chemical Company, USA), the same procedure was made, however, saline was used instead of aCSF.

Techniques: Microinjection, Control

Journal: Scientific Reports

Article Title: Influence of light/dark cycle and orexins on breathing control in green iguanas ( Iguana iguana )

doi: 10.1038/s41598-020-79107-2

Figure Lengend Snippet: Effect of Almorexant on CO 2 -chemoreflex. Effect of the i.c.v. microinjection (MI) of Almorexant and its vehicle on f R , V T and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{\text{V}}}_{{\text{I}}}$$\end{document} V ˙ I in green iguanas exposed to acute hypercarbia (5% CO 2 ) during light or dark phases. Data reported as means ± s.e.m. * means different from vehicle. The effect of hypercarbia (vehicle group, no drug) is indicated by * compared to control, while + represent the statistical differences caused by the effect of the drug.

Article Snippet: For the Almorexant (OX 1 R and OX 2 R antagonist, Cayman Chemical Company, USA), the same procedure was made, however, saline was used instead of aCSF.

Techniques: Microinjection, Control