GABAB agonism promotes sleep and reduces cataplexy in murine narcolepsy

Abstract

γ-Hydroxybutyrate (GHB) is an approved therapeutic for the excessive sleepiness and sudden loss of muscle tone (cataplexy) characteristic of narcolepsy. The mechanism of action for these therapeutic effects is hypothesized to be GABAB receptor dependent. We evaluated the effects of chronic administration of GHB and the GABAB agonist R-baclofen (R-BAC) on arousal state and cataplexy in two models of narcolepsy: orexin/ataxin-3 (Atax) and orexin/tTA; TetO diphtheria toxin mice (DTA). Mice were implanted for EEG/EMG monitoring and dosed with GHB (150 mg/kg), R-BAC (2.8 mg/kg), or vehicle (VEH) bid for 15 d-a treatment paradigm designed to model the twice nightly GHB dosing regimen used by human narcoleptics. In both models, R-BAC increased NREM sleep time, intensity, and consolidation during the light period; wake bout duration increased and cataplexy decreased during the subsequent dark period. GHB did not increase NREM sleep consolidation or duration, although NREM delta power increased in the first hour after dosing. Cataplexy decreased from baseline in 57 and 86% of mice after GHB and R-BAC, respectively, whereas cataplexy increased in 79% of the mice after VEH. At the doses tested, R-BAC suppressed cataplexy to a greater extent than GHB. These results suggest utility of R-BAC-based therapeutics for narcolepsy.

Keywords: GABA; cataplexy; hypocretin; narcolepsy; orexin; therapeutics.

Figure 1.

GHB and R-BAC occasionally induced hypersynchronous slow waves and/or a spike and wave discharge pattern that was distinct from NREM sleep. A, Representative NREM EEG and EMG traces in Atax and DTA transgenic mice ∼1 h after dosing with VEH, GHB, or R-BAC (each trace from a different mouse). B, NREM EEG power density for VEH, GHB, or R-BAC corresponding to A. C, Representative EEG and EMG traces from a single Atax mouse during the drug-induced state within 1 h after dosing with GHB and R-BAC. Calibration: vertical bar, 200 μV; horizontal bar, 10 s.

Figure 2.

Cumulative minutes of wakefulness (A), NREM sleep (B), REM sleep (C), drug-induced state (D), and accumulated NREM delta power (E) after GHB, R-BAC, or VEH administered 2 h after light onset (ZT2) and ZT6 (triangles) in Atax mice (n = 10). Latency to NREM sleep (F) after dosing at ZT2. Data are mean ± SEM; *p < 0.05 VEH versus R-BAC (light blue = 2.8 mg/kg; medium blue = 5.6 mg/kg; dark blue = 11.2 mg/kg; black = all doses); ⁺p < 0.05 VEH versus GHB (medium green = 100 mg/kg; dark green = 150 mg/kg; black = all doses).

Figure 3.

Percentage time spent awake (A, B), in NREM sleep (C, D), in REM sleep (E, F), and in cataplexy (G, H) on day 15 of chronic dosing with GHB (150 mg/kg), R-BAC (2.8 mg/kg), or VEH versus baseline in Atax and DTA mice (n = 6–8/drug group per model). GHB, R-BAC, or VEH were administered 2 h after light onset (ZT2) and ZT6 (triangles). Data are the mean ± SEM. Statistical comparisons of these data are shown as the cumulative change from baseline in Figures 4 and 7.

Figure 4.

R-BAC promoted NREM sleep during the light period with sustained suppression of REM sleep. Minutes of wakefulness (A, D), NREM sleep (B, E), and REM sleep (C, F) on day 15 of chronic dosing with GHB (150 mg/kg), R-BAC (2.8 mg/kg), or VEH plotted as the cumulative change from baseline in Atax and DTA mice (n = 6–8/drug group per model). Positive slopes indicate increased time in state relative to baseline. GHB, R-BAC, or VEH were administered at ZT2 and ZT6 (triangles). Data are mean ± SEM; *p < 0.05 R-BAC versus VEH; ⁺p < 0.05 GHB versus VEH. For data from which these plots were derived, see Figure 3.

Figure 5.

NREM EEG spectral power in Atax (A) and DTA (B) mice during the second half of the light period after GHB (150 mg/kg), R-BAC (2.8 mg/kg), or VEH. Insets, NREM EEG power spectra from 0.49–10 Hz. Data are the mean ± SEM (n = 6–7/drug group per model).

Figure 6.

R-BAC increased NREM sleep intensity and consolidation during the light period and subsequently lengthened wake bouts during the dark period. NREM delta power (A, D) and distribution of NREM sleep bout durations by proportion of NREM sleep time (B, E) during the last 6 h of the light period on day 15 of chronic dosing with GHB (150 mg/kg), R-BAC (2.8 mg/kg), or VEH versus baseline (BL) in Atax and DTA mice (n = 6–8/drug group per model). Distribution of wake bout durations by proportion of time spent awake (C, F) during the first 6 h of the dark period on dosing day 15. Data are mean ± SEM. p < 0.05: black asterisk = R-BAC day 15 versus VEH day 15; blue asterisk = R-BAC day 15 versus R-BAC BL; black + = GHB day 15 versus VEH day 15; green + = GHB day 15 versus GHB BL.

Figure 7.

R-BAC reduced time in cataplexy and cataplexy density. A, Cataplexy time during 24 h baseline in Atax (n = 19) and DTA (n = 23) mice. B, Cataplexy time during the dark period on day 15 of chronic dosing with GHB (150 mg/kg), R-BAC (2.8 mg/kg), or VEH in Atax and DTA mice (n = 6–8/drug group per model); *p < 0.05 versus VEH. For data from which these plots were derived, see Figure 3. C, Cataplexy density (number of cataplexy bouts per hour of wakefulness) during the dark period on baseline versus dosing day 15. Data pooled between mouse models (n = 14 per drug group). Linear regression: *p < 0.05; ANCOVA equality of intercepts: ⁺p < 0.05 versus GHB. D, Percentage of mice from both models that showed an increase or decrease in cataplexy density on dosing day 15 versus baseline; χ², p < 0.001.

Figure 8.

Chronic dosing protocol altered parameters related to metabolism in Atax and DTA mice administered GHB (150 mg/kg, green), R-BAC (2.8 mg/kg, blue), or VEH (white). A, Body weight change during Hcrt neurodegeneration (Dox(−)) and chronic dosing in DTA mice. 18 ± 3.3 d elapsed between Dox(−) day 28 and dosing day 1. B, Mean core body temperature (T_b) during the light (top) and dark (bottom) periods in Atax and DTA mice. C, Time spent wheel running and (D) gross motor activity during the first half of the dark period in Atax and DTA mice on dosing day 15 versus baseline. Data are mean ± SEM (n = 6–8/drug group per model); p < 0.05: black asterisk = main effect, day, + = main effect, model; green asterisk = interaction effect, GHB dosing day 15 versus baseline.