Almorexant promotes sleep and exacerbates cataplexy in a murine model of narcolepsy

Abstract

Study objectives: Humans with narcolepsy and orexin/ataxin-3 transgenic (TG) mice exhibit extensive, but incomplete, degeneration of hypo-cretin (Hcrt) neurons. Partial Hcrt cell loss also occurs in Parkinson disease and other neurologic conditions. Whether Hcrt antagonists such as almorexant (ALM) can exert an effect on the Hcrt that remains after Hcrt neurodegeneration has not yet been determined. The current study was designed to evaluate the hypnotic and cataplexy-inducing efficacy of a Hcrt antagonist in an animal model with low Hcrt tone and compare the ALM efficacy profile in the disease model to that produced in wild-type (WT) control animals.

Design: Counterbalanced crossover study.

Setting: Home cage.

Patients or participants: Nine TG mice and 10 WT mice.

Interventions: ALM (30, 100, 300 mg/kg), vehicle and positive control injections, dark/active phase onset.

Measurements and results: During the 12-h dark period after dosing, ALM exacerbated cataplexy in TG mice and increased nonrapid eye movement sleep with heightened sleep/wake fragmentation in both genotypes. ALM showed greater hypnotic potency in WT mice than in TG mice. The 100 mg/kg dose conferred maximal promotion of cataplexy in TG mice and maximal promotion of REM sleep in WT mice. In TG mice, ALM (30 mg/ kg) paradoxically induced a transient increase in active wakefulness. Core body temperature (Tb) decreased after acute Hcrt receptor blockade, but the reduction in Tb that normally accompanies the wake-to-sleep transition was blunted in TG mice.

Conclusions: These complex dose- and genotype-dependent interactions underscore the importance of effector mechanisms downstream from Hcrt receptors that regulate arousal state. Cataplexy promotion by ALM warrants cautious use of Hcrt antagonists in patient populations with Hcrt neurodegeneration, but may also facilitate the discovery of anticataplectic medications.

Citation: Black SW; Morairty SR; Fisher SP; Chen TM; Warrier DR; Kilduff TS. Almorexant promotes sleep and exacerbates cataplexy in a murine model of narcolepsy. SLEEP 2013;36(3):325-336.

Keywords: Cataplexy; hypocretin; mouse; narcolepsy; orexin.

Figure 1

Selective ablation of hypocretin (Hcrt) neurons in orexin/ataxin-3 transgenic(TG) mice. Matched brain sections from representative wild-type (WT, left panels) and TG (right panels) mice stained with anti-Hcrt2 (A, B) and anti-MCH (C, D) antisera at the tuberal region of the hypothalamus. Whereas Hcrt-IR fibers are readily evident in the locus coeruleus region of WT mice (E, G) few if any fibers are present in the same region in TG mice (F, H). G, H are enlargements of the boxed areas in E, F. f, fornix; ic, internal capsule; mt, mammillothalamic tract; scp, superior cerebellar peduncle; 4V, fourth ventricle. Bars = 100 μm.

Figure 2

Orexin/ataxin-3 transgenic (TG) mice exhibited episodes of cataplexy distinct from wake, nonrapid eye movement (NREM) and rapid eye movement (REM) sleep. Representative electroencephalograph (EEG) and electromyography (EMG) traces from a single TG mouse during wake, NREM sleep, REM sleep, and cataplexy (bracket). Immobility during cataplexy was confirmed by video (see Video 1 in supplemental material). Four cataplexy-like events were recorded in wild-type (WT) mice after almorexant (ALM); see Results for details (also see Video 2 in supplemental material). Videos show five-times real-time speed, vertical stationary line demarcates synchronous time point of electrophysiologic signals and videographed behavior, vertical moving lines separate 10-sec epochs. Traces on videos (in order from top to bottom): locomotor activity counts, EEG, EMG, and EEG periodogram (0-25 Hz per 10-sec epoch).

Figure 3

Almorexant (ALM) increased cataplexy in orexin/ataxin-3 transgenic (TG) mice. Time spent in cataplexy (A), mean cataplexy bout duration (B), and number of cataplexy bouts (C) per 12-h dark period after dosing with ALM (30, 100, or 300 mg/kg), quinpirole (QNP, 0.5 mg/kg) or vehicle (VEH). One-way repeated-measures analysis of variance and post hoc Bonferroni t tests: *P < 0.05. (D) Time course of cataplexy bouts over the 12-h dark period. Two-way repeated-measures analysis of variance and post hoc Bonferroni t tests: P < 0.05 ⁺ALM-100 versus vehicle (VEH), ^#ALM-300 versus VEH. Data represent the mean ± standard error of the mean; n = 9. ZT, Zeitgeber time.

Figure 4

Almorexant (ALM) dose-dependently promoted nonrapid eye movement (NREM) sleep and was more potent in wild-type (WT) than orexin/ataxin-3 transgenic (TG) mice. Almorexant enhanced REM sleep in a dose × time-dependent manner. Time spent awake (A-C), in NREM (D-F), and REM sleep (G-I) over the 12-h dark period in WT (n = 10) and TG (n = 9) mice after treatment with ALM (30, 100, or 300 mg/kg), quinpirole (QNP, 0.5 mg/kg), or vehicle (VEH). For 12-h binned data (A, D, and G), two-way mixed-model analysis of variance and post hoc Bonferroni t tests: on between-subjects factor “genotype,” *P < 0.05; on within-subjects factor “drug condition,” P < 0.05 ⁺versus VEH. For cumulative time series data (B, C, E, F, H, I), two-way repeated-measures analysis of variance and post hoc Bonferroni t tests (P < 0.05 versus VEH) on factor “drug condition” at each h: *ALM-100 and ALM-300, ⁺ALM-100, #ALM-300. Data represent the mean ± SEM. ZT, Zeitgeber time.

Figure 5

Almorexant (ALM) at a low dose (30 mg/kg) induced paradoxical behavioral activation in orexin/ataxin-3 transgenic (TG) mice. Latency to nonrapid eye movement (NREM) sleep (A), number of wheel-running bouts (B) and locomotor activity (C) during the fi rst h after dosing with ALM (30, 100, or 300 mg/kg), quinpirole (QNP, 0.5 mg/kg), or vehicle (VEH) in wild type (WT, n = 10) and TG (n = 9) mice. Two-way mixed-model analysis of variance and post hoc Bonferroni t tests: on between-subjects factor “genotype,” *P < 0.05; on within-subjects factor “drug condition,” P < 0.05 ⁺versus VEH (main effect in number of wheel-running bouts for ALM-300 and QNP). Data represent the mean ± standard error of the mean.

Figure 6

Almorexant (ALM, 100 mg/kg) promoted rapid eye movement (REM) sleep in wild-type (WT) mice. Latency to REM sleep after dosing (A), time interval between REM sleep bouts (B), and ratio of REM to nonrapid eye movement sleep (C) in WT (n = 10) and TG (orexin/ataxin-3 transgenic, n = 9) mice per 12-h period after dosing with ALM (30, 100, or 300 mg/kg), quinpirole (QNP, 0.5 mg/kg), or vehicle (VEH). Two-way mixed-model analysis of variance and post hoc Bonferroni t tests: on between-subjects factor “genotype,” *P < 0.05; on within-subjects factor “drug condition,” P < 0.05 ⁺versus VEH (main effect in latency to REM for ALM-100). Data represent the mean ± standard error of the mean.

Figure 7

Almorexant (ALM, 300 mg/kg) reduced nonrapid eye movement (NREM) delta power similarly in both wild-type (WT) and orexin/ataxin-3 transgenic (TG) mice. Overall, TG mice had higher NREM delta power than WT mice, regardless of drug condition. Normalized NREM delta power in WT and TG mice per 12-h period after dosing with ALM (30, 100, 300 mg/kg), quinpirole (QNP, 0.5 mg/kg) or vehicle (VEH). Two-way mixed-model analysis of variance and post hoc Bonferroni t tests: main effect on between-subjects factor “genotype,” *P < 0.05; main effect on within-subjects factor “drug condition,” P < 0.05 ⁺versus VEH. Data represent the mean ± standard error of the mean.

Figure 8

Hypocretin (Hcrt) cell loss diminished thermolysis during sleep. Almorexant (ALM, 300 mg/kg) reduced core body temperature (T_b) in wild-type (WT) mice. T_b (mean ± standard error of the mean) during wakefulness (A-C), nonrapid eye movement (NREM) sleep (D-F) and rapid eye movement (REM) sleep (G-I) in WT (n = 10) and orexin/ataxin-3 transgenic (TG) mice (n = 9) per 12-h period post dosing with ALM (30, 100, or 300 mg/kg), quinpirole (QNP, 0.5 mg/kg), or vehicle (VEH). For 12-h binned data (A, D, and G), two-way mixed-model analysis of variance and post hoc Bonferroni t tests: on between-subjects factor “genotype,” *P < 0.05 (main effect in REM T_b); on within-subjects factor “drug condition,” P < 0.05 ⁺versus VEH. For time series data (B, C, E, F, H, I), two-way repeated-measures analysis of variance and post hoc Bonferroni t tests (P < 0.05 versus VEH) on factor “drug condition” at each h: *ALM-300, +ALM-100, ^#ALM-30. Contrasts for QNP not shown for simplicity. (J) Correlation between number of Hcrt cells in TG mice and T_b change from wakefulness (mean per 12-h dark period) during NREM (closed circles) and REM sleep (open circles). ZT, Zeitgeber time.