Journal: Nature Communications

Article Title: Orexin modulates behavioral fear expression through the locus coeruleus

doi: 10.1038/s41467-017-01782-z

Figure Lengend Snippet: Role of OX1R in NA LC neurons in cued fear memory retrieval. a Schematic drawing of fear conditioning paradigm used in this study. b Cell-type-selective deletion of OX1R in NA LC neurons. LoxP sites are introduced in the OX1R allele in OX1R f/f mice to delete exons 5 and 6 by Cre-mediated recombination. c , d After cued fear conditioning, OX1R f/f ;NAT-Cre mice reduced freezing responses in test session. e Pharmacological blockade of OX1R by OX1R antagonist (SB334867) in cued fear conditioning paradigm. f , g After cued fear conditioning, i.p. administration of SB334867 1 h before the test session significantly attenuated cued fear memory retrieval. ** p < 0.01, *** p < 0.001. Values are mean ± SEM

Article Snippet: The orexin receptor-1 antagonist, SB334867 (Tocris) (5 mg/kg), was dissolved in 1% dimethyl sulfoxide in distilled water.

Techniques:

Journal: Nature Communications

Article Title: Orexin modulates behavioral fear expression through the locus coeruleus

doi: 10.1038/s41467-017-01782-z

Figure Lengend Snippet: Optogenetic excitation of orexin LH→LC -induced freezing behavior in Context A′. a Experimental procedure. b Schematic representation showing sites of AAV 10 -FLEX-ChR2-EYFP delivery and optic fiber implantation in Orexin-Cre mice. c Focal expression of Cre-dependent ChR2 in orexin LH neurons (as shown with white arrow heads) and their axon terminals in the LC. Scale bars: upper panel, 100 μm; lower panel, 50 μm; right panel, 100 μm. d , e After cued fear conditioning, optogenetic stimulation of orexin LH to LC was applied in context A′ for 150 s. f Control experiments indicating that laser stimulation did not show any effect on freezing without fear conditioning. g Optogenetic stimulation of orexin fibers induced robust Fos expression in NA LC neurons (white arrow heads). Scale bars: right panel, 100 μm. h Fos + /TH+cells were markedly increased by optogenetic manipulation as compared to the control group ( Orexin-Cre-;ChR2) and the WT (CS+) groups. i , j After cued fear conditioning, SB334867 administration 1 h before test session blocked optogenetically induced increase of freezing. k Laser stimulation itself did not show any effect on freezing without prior fear learning. * p < 0.05, ** p < 0.01, *** p < 0.001. Values are presented as mean ± SEM

Article Snippet: The orexin receptor-1 antagonist, SB334867 (Tocris) (5 mg/kg), was dissolved in 1% dimethyl sulfoxide in distilled water.

Techniques: Expressing, Control

Journal: Nature Communications

Article Title: Orexin modulates behavioral fear expression through the locus coeruleus

doi: 10.1038/s41467-017-01782-z

Figure Lengend Snippet: Physiological activation of orexin LH to NA LC pathway increased freezing behavior. a Experimental procedure. b Mouse’s body weight was significantly decreased after fasting. c Representative image showing the bilateral placement of stainless cannula above the LC. d Gradual increase of freezing was observed during conditioning. e Six hours after cued fear conditioning following fasting for 16 h, mice showed increased freezing response in context A′ compared to the fed groups, and this response was attenuated by injection of SB334867 in the LC 1 h before the test session. f There were negative correlation between freezing time and weight change of Fed + Veh and Fasted + Veh groups. g There were no correlation between freezing time and weight change of Fed + Veh and Fasted + SB groups. h Orexin neurons, NA LC neurons, and LA neurons were highly activated by fasting condition, and focal administration of SB334867 into the LC inhibited activation of NA LC and downstream LA neurons. Images show the Fos + /Orexin + cells in orexin neurons (left column, scale bar: 50 μm), Fos + /TH + cells in NA LC neurons (center column, scale bar: 50 μm), Fos + cells in the LA region (mm 2 ) (right column, scale bar: 100 μm) in each group (Fed + Veh, upper; Fasted + Veh,middle; Fasted + SB, bottom, Fig. 7h). Bottom figures showed the percentage of these cells in different regions regarding Fed + Veh, Fasted + Veh, Fasted + SB groups (Fig. 7h). * p < 0.05, ** p < 0.01, *** p < 0.001. Values are mean ± SEM

Article Snippet: The orexin receptor-1 antagonist, SB334867 (Tocris) (5 mg/kg), was dissolved in 1% dimethyl sulfoxide in distilled water.

Techniques: Activation Assay, Injection

Journal:

Article Title: SB-334867-A: the first selective orexin-1 receptor antagonist

doi: 10.1038/sj.bjp.0703953

Figure Lengend Snippet: SB-334867-A inhibits human OX1 receptor-mediated calcium responses in a concentration-dependent manner. CHO-OX1 cells were preincubated with SB-334867-A (10 pM – 10 μM) for 30 min and then [Ca2+]i was monitored using Fluo-3AM before and after addition of orexin-A (10 nM), orexin-B (100 nM) or UTP (3 μM). Responses were measured as peak increase in fluorescence minus basal and are given as mean±s.e.mean, where n=8.

Article Snippet: Materials Orexin-A and orexin-B were synthesized for SmithKline Beecham at California Peptides, CA, U.S.A. All other peptides were supplied by Bachem, U.K. SB-334867-A and N 6,10 -RG-orexin-A were manufactured at SmithKline Beecham.

Techniques: Concentration Assay, Fluorescence