Impaired GABA and glycine transmission triggers cardinal features of rapid eye movement sleep behavior disorder in mice

Abstract

Rapid eye movement (REM) sleep behavior disorder (RBD) is a neurological disease characterized by loss of normal REM motor inhibition and subsequent dream enactment. RBD is clinically relevant because it predicts neurodegenerative disease onset (e.g., Parkinson's disease) and is clinically problematic because it disrupts sleep and results in patient injuries and hospitalization. Even though the cause of RBD is unknown, multiple lines of evidence indicate that abnormal inhibitory transmission underlies the disorder. Here, we show that transgenic mice with deficient glycine and GABA transmission have a behavioral, motor, and sleep phenotype that recapitulates the cardinal features of RBD. Specifically, we show that mice with impaired glycine and GABA(A) receptor function exhibit REM motor behaviors, non-REM muscle twitches, sleep disruption, and EEG slowing--the defining disease features. Importantly, the RBD phenotype is rescued by drugs (e.g., clonazepam and melatonin) that are routinely used to treat human disease symptoms. Our findings are the first to identify a potential mechanism for RBD--we show that deficits in glycine- and GABA(A)-mediated inhibition trigger the full spectrum of RBD symptoms. We propose that these mice are a useful resource for investigating in vivo disease mechanisms and developing potential therapeutics for RBD.

Figure 1.

Transgenic mice exhibit an RBD phenotype. Typical raw EMG and EEG traces from a wild-type (top trace) and a transgenic (bottom trace) mouse during an episode of REM sleep. In contrast to wild-type mice, transgenics display elaborate motor behaviors that closely mimic RBD symptoms. While periods of motor quiescence still occur, transgenic mice exhibit overt motor behaviors throughout REM sleep. Group data for wild-type (Wt; white bars) and transgenic (Tg; black bars) mice demonstrating that transgenics have significantly higher muscle activity in the masseter (Wt: n = 16; Tg: n = 14), limb (Wt: n = 3; Tg: n = 4), and neck (Wt: n = 12; Tg: n = 8) during REM sleep. *p < 0.05; a.u., arbitrary units. All values are mean ± SEM.

Figure 2.

Exaggerated muscle twitches during REM sleep trigger RBD behaviors. A, EMG and EEG traces showing that phasic masseter muscle twitches are potently increased in transgenic versus wild-type mice. B, During REM sleep, transgenic mice (Tg; n = 16) have more frequent twitches (267% increase) that are longer-lasting (86% increase) and are of greater magnitude (65% increase) than wild types (Wt; n = 19). C, Transgenic and wild-type mice have similar levels of tonic muscle tone (i.e., atonia) during REM sleep. *p < 0.05; a.u., arbitrary units. All values are mean ± SEM.

Figure 3.

Verification that RBD behaviors occur during REM sleep. Because RBD behaviors are reminiscent of waking activity, we used post hoc analyses to confirm that such behaviors occur during REM sleep. A, In transgenic mice (Tg; n = 16), we show that there is a marked difference in the distribution of spectral power between periods of identified wakefulness and REM sleep. B, We also show that the length of identified REM sleep episodes is significantly shorter than periods of waking. C, Compared to wild-type mice (Wt; n = 19), transgenic mice spend the same amount of time in REM sleep. D, Also similar to wild-type mice, EEG spectral power is concentrated in the theta range during periods of identified REM sleep in transgenic mice. *p < 0.001. All values are mean ± SEM.

Figure 4.

Muscle tone is increased during waking in transgenic mice. EMG and EEG traces from a wild-type (top left trace) and a transgenic (top right trace) mouse during waking. Group data for wild-type (Wt; white bars) and transgenic (Tg; black bars) mice demonstrating that transgenics have significantly higher muscle activity during waking in the masseter (Wt: n = 16; Tg: n = 14) and limb (Wt: n = 3; Tg: n = 4), but not the neck (Wt: n = 12; Tg: n = 8). *p < 0.05; a.u., arbitrary units. All values are mean ± SEM.

Figure 5.

Transgenic mice exhibit RBD symptoms during NREM sleep. Typical raw EMG and EEG traces from a wild-type (top left trace) and a transgenic (top right trace) mouse during NREM sleep. Group data for wild-type (Wt; white bars) and transgenic (Tg; black bars) mice for the masseter (Wt: n = 16; Tg: n = 14), limb (Wt: n = 3; Tg: n = 4), and neck (Wt: n = 12; Tg: n = 8) during NREM sleep. While muscle tone is not elevated during this state, all transgenic mice experience brief, repetitive muscle twitches/jerks during NREM sleep (arrows). Such repetitive myoclonic twitching is not seen in wild-type mice (inset). a.u., Arbitrary units. All values are mean ± SEM.

Figure 6.

Sleep is disrupted in transgenic mice. A, Hypnograms showing that unlike wild-type mice, transgenics have markedly fragmented sleep. The white and black bars under each hypnogram indicate the light and dark periods, respectively. A zoomed-in half-hour portion of the hypnogram (shaded, outlined region) is shown for each mouse depicting the increased number of state transitions occurring in transgenic versus wild-type mice. B, Compared to wild-type mice (Wt; white bars, n = 19), transgenics (Tg; black bars, n = 16) have more arousals from NREM and REM sleep. W, Wake; N, NREM; R, REM. *p < 0.01. All values are mean ± SEM.

Figure 7.

Sleep is disrupted in transgenic mice. A, Sleep disruptions experienced by transgenic mice (Tg; black bars, n = 16) increase the amount of wakefulness and decrease NREM sleep in these mice compared to their wild-type littermates (Wt; white bars, n = 19). REM sleep amounts are unchanged. The increase in wakefulness is due to an increase in the amount of time spent in quiet wake (inset). B, The decrease in NREM sleep is due to a decrease in the length, not number, of NREM episodes. *p < 0.01. All values are mean ± SEM.

Figure 8.

EEG slowing in transgenic mice. EEG spectral profiles for wild-type (n = 15; dotted line) and transgenic (n = 11; solid line) mice for wake (A) and NREM (B) sleep. Transgenics have more power in the lower frequency ranges and less power in the higher frequency ranges, resulting in an overall EEG slowing in these states. *p < 0.01. All values are mean ± SEM.

Figure 9.

Clonazepam and melatonin rescue the RBD phenotype. A, In transgenic mice, a single dosage of clonazepam (3 mg/kg, i.p.) reduced masseter muscle tone by 26% in REM sleep. B, Long-term oral melatonin treatment (2 mg/kg/d for 2–4 weeks) also suppressed muscle tone during REM sleep, reducing it by 43% in transgenics. Clonazepam, but not melatonin, reduced NREM muscle twitches in transgenics (insets). *p < 0.05; a.u., arbitrary units. All values are mean ± SEM.

Figure 10.

Melatonin, but not clonazepam, improves sleep and alleviates sleep fragmentation in transgenic mice. A, Clonazepam treatment (3 mg/kg, i.p.) does not affect amounts of wake, NREM, or REM sleep in treated versus untreated (control) transgenic mice (n = 7). Clonazepam treatment does not reduce the number of arousals out of NREM or REM sleep, i.e., sleep fragmentation (insets). B, Melatonin treatment (2 mg/kg/d for 2–4 weeks) reduced wakefulness and increased NREM sleep in treated (n = 7) versus untreated (control; n = 14) transgenic mice. Treatment had no effect on REM sleep amounts. Melatonin treatment relieved the sleep fragmentation associated with NREM sleep, but it did not reduce REM sleep disruption (insets). *p < 0.05. All values are mean ± SEM.