Fig 1.
[Ala11, D-Leu15]-orexin-B (AL-OXB) selectively activates OX2R in vitro and in vivo.
(A) Dose-response curves of intracellular Ca2+ transients induced by OXA and AL-OXB in CHO/OX1R cells (left) and CHO/OX2R cells (right). Data represent the mean ± SEM from two independent assays. (B) Representative images of c-fos immunoreactivity (green) after ICV administration of OXA or AL-OXB in tyrosine hydroxylase-positive (red) noradrenalin neurons of the LC and histidine decarboxylase-positive (red) histamine neurons of the TMN. 4V: 4th ventricle. Regions marked by white rectangles are magnified in small panels. Scale bars: small panels, 25 μm; large panels, 100 μm. (C) Quantification of c-fos-positive cells in LC-noradrenergic neurons (left) and TMN-histaminergic neurons (right). Data represent the means ± SEM from 4–5 mice. Statistical analysis: one-way ANOVA followed by Bonferroni’s multiple comparisons test.
Fig 2.
ICV AL-OXB prevents both cataplexy-like episodes and fragmentation of wakefulness in OXKO mice.
(A) Experimental schedule (left) and surgery design (right). (B) Representative hypnograms showing the effect of ICV OXA or AL-OXB on cataplexy-like episodes in OXKO mice. R, REM state; NR, non-REM state; W, wake state. Red arrows: cataplexy-like states. (C) The number of cataplexy-like states during 3h (left) and latency to first cataplexy-like state (right) after ICV administration. Data represent the means ± SEM from 6 mice. Statistical analysis: one-way ANOVA followed by Bonferroni’s multiple comparisons test. (D) Experimental schedule for evaluation of wakefulness fragmentation. (E) Representative hypnograms showing the effect of ICV OXA or AL-OXB on wakefulness fragmentation in OXKO mice. (F) Hourly plots of wake time (left) and wake episode duration (right) during 3 h after ICV administration. Data represent the means ± SEM from 6 mice. *p < 0.05, **p < 0.01, ****p < 0.0001 for OXA vs. vehicle; #p < 0.05, ##p < 0.01, ####p < 0.0001 for AL-OXB vs. vehicle; two-way repeated-measures ANOVA followed by Bonferroni’s multiple comparisons test.
Fig 3.
ICV AL-OXB prevents sleep/wake fragmentation in OXKO mice.
The number of transitions between sleep/wake stages over 3 h after ICV administration of vehicle (left), 3 nmol OXA (middle), and 3 nmol AL-OXB (right) at ZT11-12. Data represent the means ± SEM from 6 mice. *p < 0.05, **p < 0.01, ****p < 0.0001 for OXA vs. vehicle; #p < 0.05, ##p < 0.01, ####p < 0.0001 for AL-OXB vs. vehicle; one-way ANOVA followed by Bonferroni’s multiple comparisons test.
Fig 4.
Effect of continuous ICV administration of OXA and AL-OXB during dark phase on sleep-wake cycle in OXKO mice.
(A) Experimental schedule of ICV infusion and EEG/EMG recording. (B) Total wake time duration during 12-h ICV administration. Data represent the means ± SEM from 3 mice. Statistical analysis: one-way ANOVA followed by Bonferroni’s multiple comparisons test. (C and D) Hourly plots of wake time (C) and wake episode duration (D) before, during and after continuous ICV administration. Data represent the means ± SEM from 3 mice. Statistical analysis: two-way repeated-measures ANOVA.
Fig 5.
OXA but not AL-OXB induces conditioned place preference.
(A) Experimental schedule of CPP test. (B and C) Effects of different doses of OXA (B) and AL-OXB (C) on CPP score. Same CPP data for vehicle injections are displayed twice in (B) and (C). Data represent the means ± SEM from 5 mice for 1 and 10 nmol OXA, 6 mice for 1 and 10 nmol AL-OXB, 9 mice for 3 nmol OXA or AL-OXB and 8 mice for vehicle. Statistical analysis: one-way ANOVA followed by Bonferroni’s multiple comparisons test.