what neurotransmitter should i take to stop sleep paralysis

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Identification of the Transmitter and Receptor Mechanisms Responsible for REM Sleep Paralysis

Periodical of Neuroscience eighteen July 2012, 32 (29) 9785-9795; DOI: https://doi.org/10.1523/JNEUROSCI.0482-12.2012

Abstract

During REM sleep the CNS is intensely active, simply the skeletal motor arrangement is paradoxically forced into a country of muscle paralysis. The mechanisms that trigger REM sleep paralysis are a affair of intense debate. Two competing theories argue that it is caused past either agile inhibition or reduced excitation of somatic motoneuron activeness. Hither, we identify the transmitter and receptor mechanisms that function to silence skeletal muscles during REM sleep. We used behavioral, electrophysiological, receptor pharmacology and neuroanatomical approaches to determine how trigeminal motoneurons and masseter muscles are switched off during REM sleep in rats. We evidence that a powerful GABA and glycine drive triggers REM paralysis by switching off motoneuron action. This drive inhibits motoneurons by targeting both metabotropic GABA_B and ionotropic GABA_A/glycine receptors. REM paralysis is only reversed when motoneurons are cut off from GABA_B, GABA_A and glycine receptor-mediated inhibition. Neither metabotropic nor ionotropic receptor mechanisms lone are sufficient for generating REM paralysis. These results demonstrate that multiple receptor mechanisms trigger REM slumber paralysis. Breakup in normal REM inhibition may underlie mutual sleep motor pathologies such as REM sleep behavior disorder.

Introduction

Skeletal muscle paralysis (motor atonia) is a defining characteristic of normal REM sleep (Jouvet, 1967; Brooks and Peever, 2008a). It may function to prevent the dreaming brain from triggering unwanted and potentially dangerous sleep movements (Mahowald and Schenck, 2005; Brooks and Peever, 2011). Breakdown in REM slumber mechanisms is linked to common slumber disorders such equally narcolepsy/cataplexy and REM sleep behavior disorder (RBD) (Mahowald and Schenck, 2005; Lu et al., 2006; Siegel, 2006; Burgess et al., 2010). The mechanisms responsible for REM sleep paralysis remain a matter of considerable debate (Brooks and Peever, 2008a; Chase, 2008).

Classic studies showed that skeletal motoneurons are hyperpolarized by glycine-mediated IPSPs during REM sleep (Nakamura et al., 1978; Soja et al., 1991), which led to the prevailing hypothesis that REM paralysis is unmarried-handedly triggered by glycinergic inhibition of motoneurons (Chase et al., 1989; Hunt and Morales, 2005). However, contempo studies found that REM paralysis remained even after glycine receptors were blocked on motoneurons (Kubin et al., 1993; Morrison et al., 2003; Brooks and Peever, 2008b). Later on, it was hypothesized that REM paralysis is caused past loss of serotonergic and noradrenergic excitation of motoneurons (Fenik et al., 2005), but motor paralysis could non be overridden by direct chemic stimulation (e.g., glutamate, noradrenaline, serotonin) of motoneurons (Jelev et al., 2001; Chan et al., 2006; Burgess et al., 2008). These findings betoken that REM paralysis is triggered by a powerful, yet unidentified, inhibitory mechanism.

Several lines of evidence point that both metabotropic GABA_B and ionotropic GABA_A/glycine receptor-mediated inhibition of skeletal motoneurons underlies REM sleep atonia. Start, brainstem circuits that control REM slumber incorporate GABA and glycine neurons that project to and synapse on motoneurons (Holstege, 1996; Rampon et al., 1996; Morales et al., 2006). Second, somatic motoneurons themselves express GABA_B, GABA_A and glycine receptors, which when activated trigger cellular hyperpolarization (Lalley, 1986; Araki et al., 1988; Persohn et al., 1992; Okabe et al., 1994; Margeta-Mitrovic et al., 1999; O'Brien and Berger, 1999; Charles et al., 2003; O'Brien et al., 2004). Third, motoneurons are hyperpolarized by large-amplitude IPSPs during REM sleep (Nakamura et al., 1978). Yet, it is unknown whether REM slumber atonia is triggered by activation of both metabotropic GABA_B and ionotropic GABA_A/glycine receptors.

Here, we aimed to identify the transmitter and receptor mechanisms responsible for REM sleep paralysis. We studied trigeminal motoneurons and the masseter muscles they innervate considering this motor system experiences typical REM paralysis and contributes to sleep pathologies such as RBD (Schenck and Mahowald, 2002; Burgess et al., 2008; Brooks and Peever, 2011). We found that inactivation of both metabotropic GABA_B and ionotropicGABA_A/glycine receptors prevented and indeed reversed REM paralysis. However, neither metabotropic nor ionotropic pathways lone are sufficient for inducing REM inhibition. REM paralysis is only reversed when motoneurons are cutoff from both metabotropic and ionotropic receptor-mediated inhibition. These results reshape our understanding of the transmitter and receptor mechanisms underlying REM sleep paralysis.

Materials and Methods

Animals

All procedures and experiments were approved by the University of Toronto'south Animate being Care Committee and were in accord with the Canadian Council on Animate being Intendance. Rats were housed individually and maintained on a 12:12 light/dark cycle (lights on at 0700 h) and both food and water were available ad libitum. Procedures and experimental protocols are similar in nature to our previously published work (Brooks and Peever, 2008b; Burgess et al., 2008).

Surgical preparation for sleep and microdialysis studies

Studies were performed using male Sprague Dawley rats (boilerplate weight: 391 ± vi g). To implant electroencephalographic (EEG) and electromyographic (EMG) electrodes and a microdialysis probe, sterile surgery was performed nether anesthesia induced with intraperitoneal ketamine (85 mg/kg) and xylazine (15 mg/kg). Isoflurane (0.5–ii%) was also used to maintain depth of anesthesia, which was determined by absenteeism of the pedal withdrawal and blink reflexes. Body temperature was monitored with a rectal probe (CWE) and maintained at 37 ± 1°C.

Three insulated, multistranded stainless steel wire EMG electrodes (Cooner Wire) were implanted into the left and right masseter muscles. The wires were tunneled subcutaneously to an incision along the dorsal surface of the cranium. Three EMG electrode wires were also inserted into the nuchal musculus. 4 stainless steel screws (JI Morris) fastened to insulated 34 gauge wire (Cooner Wire) were implanted in the skull for recording cortical EEG (coordinates: two mm rostral and 2 mm to the left and right of bregma, and 3 mm caudal and two mm to the left and correct of bregma).

To implant a microdialysis probe into the left trigeminal motor puddle, a ∼2 mm burr hole was fabricated at nine.iv mm caudal and 1.8 mm lateral to bregma (Paxinos and Watson, 1998). A microdialysis guide cannula (CMA) was then lowered eight.2 mm below the skull surface by stereotaxic manipulation. Dental cement (1234, Lang Dental) secured the cannula in identify and later on the cement was dry, EEG and EMG electrodes were continued to pins (Centrolineal Electronics) and inserted into a custom-made head-plug (Allied Electronics) that was affixed to the skull with dental cement.

Afterwards surgery rats were given a subcutaneous injection of ketoprofen (v mg/kg) and 5% dextrose in 0.9% saline and kept warm past a heating pad. They were also given a dietary supplement (Nutri-Cal) and soft nutrient for the 2 d post-obit surgery. Rats recovered for at least 7–10 d before experiments began.

Experimental procedures for sleep and microdialysis studies

Recording surroundings.

During experiments, animals were housed in a Raturn system (BAS), which is a motion-responsive caging organization eliminating the need for a commutator or liquid hinge. This caging system was housed within a sound-attenuated, ventilated, and illuminated (lights on: 110 lux) sleeping accommodation.

Electrophysiological recordings.

EEG and EMG activities were recorded by attaching a lightweight cable to the plug on the rat's caput, which was connected to a Super-Z head-stage amplifier and BMA-400 AC/DC Bioamplifier (CWE). EEG signals were amplified 1000 times and bandpass filtered between 1 and 100 Hz. EMG signals were amplified between 500 and 1000 times and bandpass filtered between 30 Hz and thirty kHz. All electrophysiological signals were digitized at 500 Hz (Spike 2 Software, 1401 Interface, CED) and monitored and stored on a estimator.

Microdialysis probes.

A microdialysis probe was used to perfuse candidate drugs into the trigeminal motor pool. The microdialysis probe (6K Da cutoff; membrane length and diameter: 1 mm by 250 μm, CMA) was placed into the left trigeminal nucleus. The microdialysis probe was connected to Teflon tubing (inner diameter = 0.1 mm; Eicom), which was continued to a ane ml gastight syringe via a liquid switch (BAS). The probe was continuously perfused with filtered (0.2 μm PVDF, Fisher Scientific) artificial CSF (aCSF: 125 thoum NaCl, 5 m1000 KCl, one.25 mm KH_twoPO₄, 24 kg NaHCO₃, 2.five mg CaCl_ii, 1.25 thousandg MgSO₂, 20 mthou d-glucose) at a flow rate of 2 μl/min using a syringe pump (BAS).

Drug preparation.

All drugs were made immediately before each experiment and dissolved in aCSF. The following drugs were used to manipulate GABA and glycine receptors: CGP52432 (GABA_B antagonist; FW: 420.27; Tocris Bioscience), baclofen (GABA_B agonist; FW: 213.66; Tocris Bioscience), bicuculline (GABA_A antagonist; FW: 435.87; Tocris Bioscience) and strychnine (glycine antagonist; FW: 370.9; Sigma-Aldrich). The AMPA receptor agonist (α-amino-three-hydroxy-5-methylisoxazole-iv-propionic acid, FW: 186.17; Tocris Bioscience) was prepared in accelerate and stored in stock solutions at −twenty°C. All drugs were vortexed and filtered (0.22 μm PVDF, Fisher Scientific) before use.

Experimental protocols

Each experiment took ii d to consummate. On the showtime mean solar day at 0800–1000 h, animals were placed into a recording sleeping room and given at least one h to habituate before being connected to the recording tether. They were then given a minimum of 3 h to habituate to this before recordings began. Baseline recordings (without the microdialysis probe in identify) were established on Day 1 of experiments, between 1300 and 1600 h. The microdialysis probe was inserted at 1700 h and aCSF perfused throughout the night. Probes were inserted the night before experiments began considering previous studies demonstrate that probe insertion induces spontaneous neurotransmitter release and local neuronal activation (Di Chiara, 1990; Kodama et al., 1998). On the second twenty-four hour period of experimentation, candidate drug treatments (meet below, Studies 1 and two) were perfused in random order betwixt 0800–1800 h. Because baseline (i.east., 1300–1600 h) and drug treatment (i.e., 0800–1800 h) times were overlapping, potential furnishings of candidate drugs on REM sleep could exist compared and adamant. Each drug was practical onto the trigeminal nucleus for ii–4 h, which typically allowed sufficient time for rats to transition through iii complete sleep cycles (i.eastward., wake to NREM to REM slumber). An aCSF washout period of at least two h followed every drug treatment.

Report i: Does GABA_B receptor activation at the trigeminal motor pool underlie REM slumber paralysis?

We addressed this question in two ways. Commencement, we activated GABA_B receptors past perfusing baclofen (GABA_B receptor agonist) into the left trigeminal motor pool while monitoring left masseter muscle EMG activity. We did this to determine whether receptor activation could trigger motor paralysis. We used 0.5 one thousandm baclofen because previous studies showed that this concentration can activate GABA_B receptors both in vitro and in vivo (Okabe et al., 1994; Ouyang et al., 2007; Matsuki et al., 2009).

Next, we wanted to determine whether there is an endogenous GABA_B-mediated drive onto trigeminal motoneurons during either sleep or waking and whether removal of this drive in REM sleep could forbid REM paralysis of masseter muscle. We antagonized GABA_B receptors in a dose-dependent manner by applying increasing concentrations of CGP52432 (0.01 chiliadm, 0.05 km, 0.1 mthou, and 0.2 mm) at the trigeminal motor pool.

Written report 2: Is REM sleep paralysis triggered by activation of metabotropic GABA_B and ionotropic GABA_A/glycine receptors?

Nosotros hypothesize that REM paralysis is caused past activation of both metabotropic GABA_B and ionotropic GABA_A/glycine receptors considering (1) GABA and glycine are released onto motoneurons during REM sleep (Chase et al., 1989; Morrison et al., 2003; Brooks and Peever, 2008b), and (2) motoneurons limited all three receptor types (Araki et al., 1988; Persohn et al., 1992; Margeta-Mitrovic et al., 1999). To test this hypothesis, we simultaneously antagonized GABA_B, GABA_A and glycine receptors by perfusing 0.ii gm CGP52432, 0.1 mthousand bicuculline and 0.i one thousandm strychnine onto trigeminal motoneurons during sleep and waking. We used this concentration of CGP52432 because results from Study ane showed it triggers potent increases in masseter tone when applied to motoneurons. This is supported by in vitro and in vivo studies showing GABA_B receptors are finer antagonized 0.2 km CGP52432 (Westerink et al., 1996; Fedele et al., 1997; Chéry and De Koninck, 2000). We applied 0.1 mm bicuculline/strychnine considering we previously showed that such concentrations antagonize GABA_A and glycine receptor-mediated neurotransmission at the trigeminal motor puddle (Morrison et al., 2003; Brooks and Peever, 2008b).

Considering we had concerns that drug perfusion (via microdialysis) for extended periods of time (i.eastward., 2–4 h) might spread to REM slumber circuits near the trigeminal motor pool or that inadequate receptor antagonism may non cake GABA and glycine inhibition on motoneurons, nosotros directly microinjected high concentrations of receptor antagonists (0.3 mthou strychnine/bicuculline and 0.6 mm CGP52432) at the trigeminal motor puddle but during REM sleep (north = 31 rats).

Microinjections were performed by placing injection probes (i.e., CMA/11 microdialysis probes with dialysis membranes removed) into a guide cannula situated in the left trigeminal motor puddle. A 1 μl Hamilton syringe was then attached to each probe by a 30 cm length of tubing and 0.2 μl of a candidate drug was applied over a 15 s period. All injections were confined to private REM slumber episodes with each injection starting time at the transitioned from NREM into REM sleep. Drug effects on masseter atonia were nerveless and analyzed only for the REM period in which receptor antagonists were applied onto trigeminal motoneurons. This arroyo enabled united states to determine how rapid and focal animosity of GABA_B and GABA_A/glycine receptors at the trigeminal motor pool influences REM masseter atonia during a detached REM episode.

Verification of microdialysis probe location

Two procedures were used to demonstrate that microdialysis probes were both functional and located in the left trigeminal motor pool. At the end of each experiment, 0.1 1000m AMPA was perfused into the trigeminal nucleus. If the probe is functional and at the motor pool then glutamatergic activation of motoneurons should increase left masseter musculus tone. Nosotros also used postmortem histological assay to demonstrate that microdialysis probe lesion sites were physically located in the left trigeminal nucleus.

Histology

Nether deep anesthesia (ketamine: 85 mg/kg and xylazine: 15 mg/kg, i.p.) rats were decapitated, brains removed and placed in chilled four% paraformaldehyde (in 0.one m PBS) for 24 h. Brains were cryoprotected in 30% sucrose (in 0.1 m PBS) for 48 h; they were then frozen in dry out water ice and transversely sectioned in 40 μm slices using a microtome (Leica). Brain sections were mounted, dried and stained with Neutral Red. Tissue sections were viewed using a light microscope (Olympus) and the location of probe lesion tracts were plotted on standardized brain maps (Paxinos and Watson, 1998).

Data assay

Behavioral land.

We classified iii behavioral states. Waking (W) was characterized by high-frequency, low-voltage EEG signals coupled with high levels of EMG activity. NREM sleep was characterized by high-amplitude, low-frequency EEG signals and minimal EMG action. REM sleep was characterized by depression-aamplitude, high-frequency theta-like EEG activity and REM atonia interspersed by periodic muscle twitches. Slumber states were visually identified and analyzed in 5 s epochs using the Sleepscore v1.01 script (CED).

EMG assay.

Raw EMG signals were full-wave rectified, integrated and quantified in arbitrary units (a.u.). Boilerplate EMG activity for left and right masseter and neck muscle activity was quantified in five due south epochs for each behavioral state. EMG activity was non analyzed during the first 30 min of drug perfusion because of the commitment latency from the syringe pump to the microdialysis probe. At least three episodes of each behavioral country (i.e., Westward, NREM, and REM) were analyzed for each experimental status. In each rat, the average EMG activity was calculated for each behavioral state for each drug perfused into the trigeminal motor pool.

EMG assay in REM sleep.

REM slumber consists of both tonic and phasic motor events. The stereotypical periods of motor atonia occur during tonic REM sleep and the periodic musculus twitches that punctuate REM atonia occur during phasic REM slumber (i.e., during rapid heart-movements). Because the goal of this study was to decide the role for GABA_B inhibition in REM motor control, nosotros used a previously established method for identifying and quantifying the phasic (i.e., musculus twitches) and tonic (i.e., REM atonia) periods of REM sleep. In each rat, REM atonia and musculus twitches were quantified for each REM episode during the baseline condition and for each drug perfused into the trigeminal motor pool (Brooks and Peever, 2008b; Burgess et al., 2008).

EEG spectral analysis.

EEG spectral analysis was calculated using fast Fourier transformation of each v s epoch, yielding a power spectra profile inside four frequency bands. The band limits used were delta (δ): 0.48–4 Hz; theta (θ): 4.25–8 Hz; alpha (α): viii.25–15 Hz; beta (β): xv.25–35 Hz. A hateful EEG spectrum contour was obtained for each epoch and then, to minimize nonspecific differences in absolute power between individuals, EEG ability in each frequency bin was expressed every bit a percentage of the total EEG power in the epoch. The spectral profiles of each behavioral state were and so compared between treatments.

Statistical analyses

All statistical analyses were performed using Sigmastat (SPSS Inc.) and applied a disquisitional two-tailed α value of p < 0.05. All comparisons made between baseline and drug treatments were determined using ANOVA with repeated measures (RM-ANOVA) and postal service hoc comparisons were performed using a Student–Newman–Keuls (SNK) examination. Comparisons for the microinjection experiments (i.e., drug treatments versus aCSF) were fabricated using t tests. All data are expressed as mean ± SEM.

Results

Drug manipulations affect trigeminal motoneuron beliefs

Our start aim was to show that receptor manipulations target motoneurons in the trigeminal nucleus. First, we showed that all probes were located in the left trigeminal motor pool (Fig. 1a,b) and then we showed that drug manipulations but influenced the activity of the muscle (i.e., left masseter) innervated past these cells. Drug manipulations at the left trigeminal motor pool never influenced the activity of right masseter or neck muscles (Fig. 1c,d). Probe insertion into the left motor pool caused an immediate, merely transient (<1 min), activation of only left masseter muscle tone (paired t test, t _(four) = three.022, p = 0.039; Fig. ic). Neither correct masseter (paired t test, t ₍₄₎ = 0.152, p = 0.887) nor neck musculus activity (paired t examination, t ₍₄₎ = ane.265, p = 0.275; data non shown) were affected past this intervention, suggesting that these interventions selectively targeted trigeminal motoneurons in the left motor pool.

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Effigy 1.

Drug interventions preferentially target trigeminal motoneurons. a , A histological example showing the tip of a probe tract (red circle) in the trigeminal motor pool (blueish circumvolve). Scale bar, 500 μm. b, Locations of the 31 probe tracts in the trigeminal motor pool plotted on standardized brain maps. Red dots represent probe tip locations in the trigeminal motor pool. Although probes were just placed in the left motor pool, we plotted probe locations in both left and right motor pools in this figure for the sake of visual clarity. c, EEG and EMG traces (acme) and grouping information (bottom) showing that inserting a probe into the left trigeminal motor puddle only increases left masseter muscle activeness (left EMG), right masseter (correct EMG) activeness is unaffected. d, EEG and EMG traces (top) and group data (lesser) showing that AMPA perfusion at the left motor pool only increases left masseter muscle activity. *p < 0.05. All values are mean ± SEM.

To confirm that practical drugs bear upon motoneurons in the targeted motor puddle, nosotros perfused AMPA at the end of each experiment. AMPA triggered a robust motoneuron excitation that resulted in rapid and forceful activation of only left masseter EMG tone (paired t test, t _{(half dozen)} = 3.231, p = 0.018), neither right masseter (paired t test, t _{(half dozen)} = 1.082, p = 0.321) nor neck muscle activity (paired t examination, t ₍₆₎ = 0.042, p = 0.968; data not shown) were affected (Fig. 1d). This finding shows that applied drugs targeted motoneurons in merely the left motor pool, which themselves remained viable throughout experiments. It besides confirms that probes remained functional during the class of experimental interventions.

Drug manipulations have negligible effects on REM-generating circuits

Although drug application targets motoneurons, we wanted to verify that cells bordering the trigeminal motor pool remained unaffected. The sublaterodorsal nucleus (SLD) sits beside the trigeminal motor pool (0.1–0.2 mm dorsomedial) and controls REM slumber (Boissard et al., 2002; Lu et al., 2006). Previous studies evidence that REM sleep is influenced by GABA_A receptor antagonism at the SLD (Boissard et al., 2002; Pollock and Mistlberger, 2003; Sanford et al., 2003). Importantly, we constitute that antagonism of GABA_A, GABA_B and glycine receptors at the trigeminal motor puddle reversed REM masseter muscle paralysis (Fig. 2a), only it had no effect on REM sleep expression (REM slumber corporeality: baseline vs drug, SNK, q = 0.215, p = 0.879; Fig. 2b). Blockade of only GABA_A and glycine receptors also had no effect on REM sleep expression (REM amount: baseline vs drug, SNK, q = 0.760, p = 0.853; Fig. 2b). EEG spectral power during REM slumber was too unaffected by pharmacological interventions (RM ANOVA, F _(3,9) = 1.336 p = 0.232; Fig. iic). These findings bear witness that changes in REM sleep muscle tone are not caused by indirect modulation of REM-generating SLD circuits, rather they upshot from direct manipulation of motoneurons themselves. Our results therefore document the transmitter and receptor mechanisms responsible for controlling trigeminal motoneurons and masseter muscles in natural REM sleep.

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Figure 2.

Drug manipulations exercise non affect REM-generating circuits. a , REM sleep paralysis is abolished by preventing GABA and glycine receptor-mediated inhibition of trigeminal motoneurons. EEG and EMG traces (i.e., masseter musculus) showing that blockade of GABA_B/GABA_A/glycine receptors on trigeminal motoneurons reversed and prevented masseter REM slumber paralysis. Neither REM sleep amounts ( b ) nor REM sleep EEG spectral power ( c ) were afflicted when ionotropic GABA_A/glycine and metabotropic GABA_B receptors were antagonized at the trigeminal motor puddle. Perfusion of either 0.one mm bicuculline/strychnine or 0.two mgrand CGP52432 and 0.1 thousandchiliad strychnine/bicuculline at the trigeminal nucleus had no affects on REM slumber amounts or EEG power, indicating that applied drugs did non spread to and influence REM-regulating circuits in the nearby sublaterodorsal nucleus. However, metabotropic GABA_B and ionotropic GABA_A/glycine receptor antagonism on trigeminal motoneurons had profound affects on masseter tone during REM slumber ( a ). All values are mean ± SEM.

Neither metabotropic GABA_B nor ionotropic GABA_A/glycine receptor-mediated inhibition themselves can trigger REM paralysis

Information technology is unknown whether metabotropic GABA_B receptors modulate motoneuron physiology during natural motor behaviors. We establish that activating GABA_B receptors (by baclofen) on trigeminal motoneurons reduced waking masseter muscle tone by 78 ± 5% (baseline vs baclofen; paired t test, t _(iv) = 3.960, p = 0.017; Fig. iiia,b), indicating that receptor activation influences motoneuron behavior. Yet, GABA_B receptor agonism did not trigger consummate muscle paralysis since waking masseter tone remained twofold in a higher place normal REM slumber levels (baclofen during waking: 1.i ± 0.ane a.u. vs baseline REM: 0.5 ± 0.05 a.u.; t exam, t ₍₁₈₎ = four.431, p < 0.001; Fig. iiib). This finding suggests that GABA_B receptor activation solitary is incapable of inducing REM slumber musculus paralysis.

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Figure three.

GABA_B receptor activation on trigeminal motoneurons reduces masseter tone, merely does not trigger muscle paralysis. a , EEG and EMG traces showing that GABA_B receptor agonism by baclofen (0.5 1000yard) perfusion at the left trigeminal motor pool markedly reduces (compared with baseline) left masseter tone (left EMG) during waking. Right masseter musculus tone is unaffected. b , Group information (n = 5) showing that compared with baseline baclofen-induced activation of GABA_B receptors on trigeminal motoneurons reduces waking masseter tone. However, this intervention does not reduce waking masseter tone to normal REM sleep levels. All values are mean ± SEM.

It is unknown whether metabotropic GABA_B receptor-mediated inhibition underlies the motoneuron hyperpolarization that causes REM paralysis (Okabe et al., 1994; Brooks and Peever, 2008b). Therefore, we determined whether REM motor paralysis could be prevented by antagonizing GABA_B receptors (using CGP52432) on motoneurons. CGP52432 awarding heightened masseter tone during both waking (RM ANOVA, F _(5,4) = 3.147, p = 0.037) and NREM slumber (RM ANOVA, F _(v,four) = 3.052, p = 0.041; Fig. iv), suggesting that an endogenous GABA drive functions to inhibit motoneurons during these states by a GABA_B receptor mechanism. But surprisingly, GABA_B receptor antagonism had no effect on masseter muscle tone during REM slumber (RM ANOVA, F _(v,iv) = ane.521, p = 0.234). Specifically, information technology had no effect on either masseter paralysis (SNK, q = 0.901, p = 0.528) or REM muscle twitch activeness (baseline vs CGP52432; duration: SNK, q = 0.722, p = 0.613; frequency: SNK, q = 1.551, p = 0.280; aamplitude: SNK, q = 1.852, p = 0.198; Fig. 5), suggesting that GABA_B receptor-mediated inhibition alone plays a trivial function in REM motor control. This finding likewise suggests that a remainder inhibitory drive must continue to hyperpolarize motoneurons and cause REM paralysis.

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Figure 4.

GABA_B receptor animosity on trigeminal motoneurons does not prevent REM paralysis. a , EEG and EMG traces showing that GABA_B receptor blockade by CGP52432 perfusion (0.ii one thousandm) at the trigeminal motor pool causes robust increases in masseter action during both waking and NREM slumber, but it does not affect levels of masseter tone during REM slumber. b , Group data (north = 7) showing that CGP52432 perfusion (0.01–0.2 mm) heightens masseter EMG activity during waking and NREM slumber, only it does not prevent REM atonia. *p < 0.004. All values are mean ± SEM.

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Figure 5.

Ionotropic GABA_A/glycine receptor-mediated inhibition functions to suppress REM muscle twitches. a , EMG and EEG traces illustrating how masseter muscle twitches during REM sleep are affected past animosity of GABA_B (0.two mg CGP52432), GABA_A/glycine (0.ane thouthousand bicuculline/strychnine) and GABA_B/GABA_A/glycine (0.two 1000grand CGP52432 and 0.1 mchiliad bicuculline/strychnine) receptors at the trigeminal motor pool. b–d , Grouping data (n = 14) demonstrating how metabotropic, ionotropic, and combined metabotropic/ionotropic receptor blockade on trigeminal motoneurons affects the duration ( b ), frequency ( c ) and amplitude ( d ) of REM muscle twitches. *p < 0.05. All values are mean ± SEM.

All the same, we show that this residue inhibition is not mediated by ionotropic GABA_A and glycine receptors considering antagonizing them has no outcome on REM atonia. We found that ionotropic receptor antagonism increased masseter tone during both waking (SNK, q = 10.258, p < 0.001) and NREM sleep (SNK, q = 7.751, p < 0.05), suggesting that motoneurons are inhibited during these states (Fig. 6). Receptor antagonism besides triggered marked increases in the size (SNK, q = 4.576, p = 0.007) and frequency (SNK, q = 5.483, p = 0.001) of muscle twitches during REM sleep (Fig. 5), demonstrating that an inhibitory bulldoze is present during REM slumber and that it functions to suppress musculus twitches. This finding is consistent with intracellular recordings, which show that motoneurons are maximally inhibited when REM muscle twitches occur (Chase and Morales, 1983; Brooks and Peever, 2008b). However, we prove that GABA_A and glycine receptor antagonism on trigeminal motoneurons had no effect on REM masseter paralysis (SNK, q = 2.147, p = 0.294; Figs. half-dozen, 7). In fact, removal of merely GABA_A and glycine receptor-mediated inhibition still immune the normal drop in basal muscle tone from NREM to REM sleep (Figs. half dozena, 7d). These observations signal that an additional, simply unidentified machinery, continues to inhibit motoneurons during REM sleep. To place this mechanism we simultaneously antagonized both metabotropic GABA_B and ionotropic GABA_A/glycine receptors at the trigeminal motor pool during REM sleep.

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Figure 6.

GABA_A and glycine receptor antagonism increases masseter tone during waking and NREM sleep, but it does not foreclose REM atonia. a , An EMG and EEG trace showing the abrupt loss of masseter tone on entrance into REM despite continued antagonism of GABA_A/glycine receptors. b , Group data (n = xiv) showing that bicuculline and strychnine perfusion (0.1 mm for each) onto trigeminal motoneurons increases masseter EMG activity during both waking and NREM sleep. Even so, this same intervention has no bear on on basal levels of muscle tone during REM slumber. *p < 0.001. All values are mean ± SEM.

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Figure 7.

Activation of both metabotropic GABA_B and ionotropic GABA_A/glycine receptors is required for REM sleep paralysis. a , EMG and EEG traces illustrating that masseter REM atonia remains intact when just GABA_A/glycine receptors are antagonized, but prevented and overridden when both GABA_B and GABA_A/glycine receptors are simultaneously antagonized on trigeminal motoneurons. b, Group data showing that bicuculline and strychnine (0.1 mm) applied onto trigeminal motoneurons during REM slumber cannot prevent REM inhibition (n = 12). However, occludent of both metabotropic GABA_B and ionotropic GABA_A/glycine receptors by perfusion of CGP52432 (0.two chiliadk), bicuculline and strychnine (0.i thoug) abolishes masseter REM atonia (n = 13). c, Antagonism of GABA_B, GABA_A and glycine receptors at the trigeminal motor pool elevates REM masseter tone to baseline NREM sleep levels. However, despite continued receptor animosity REM masseter tone remains beneath normal (i.e., baseline) waking levels that occur immediately after REM episodes. This finding indicates that loss of motoneuron excitation helps to reinforce REM muscle paralysis. d, REM paralysis is still triggered even after antagonism of both GABA_A/glycine receptors. This graph shows the normal drib in masseter tone when NREM slumber is exited and REM slumber is entered despite connected ionotropic receptor blockade. *p < 0.004. All values are hateful ± SEM.

Blockade of GABA_B, GABA_A, and glycine receptors prevents REM motor paralysis

Metabotropic and ionotropic receptor-mediated mechanisms can deed synergistically to bear upon neuron function (Liu et al., 2000; Lee et al., 2002; Balasubramanian et al., 2004). Therefore, we hypothesized that REM motor inhibition may be driven by mechanisms that crave both metabotropic GABA_B and ionotropic GABA_A/glycine receptors. To test this hypothesis nosotros simultaneously antagonized GABA_B, GABA_A and glycine receptors to determine whether this intervention could prevent REM paralysis. We found that receptor animosity on trigeminal motoneurons not only increased masseter tone during waking and NREM sleep (waking: SNK, q = eight.616, p < 0.001; NREM: SNK, q = 7.246, p < 0.05; Fig. eight), it also triggered a potent activation of masseter tone during REM slumber (Figs. vii, 8). Specifically, we plant that perfusion of CGP52432 (0.2 mm) and bicuculline/strychnine (0.one mm for each) onto trigeminal motoneurons reversed motor paralysis past triggering a 105 ± 30% increase in basal levels of masseter tone during REM sleep (SNK, q = 5.237, p = 0.004; Figs. 7, 8). In fact, when all 3 receptors were antagonized masseter muscle tone increased to levels observed during normal NREM slumber (NREM baseline vs REM drug; paired t test, t ₍₁₃₎ = 1.929, p = 0.076; Fig. sevenc). Despite the reversal of REM atonia brief periods of depression masseter tone intermittently punctuated REM periods (Fig. 7a). This effect is in sharp contrast to occludent of only GABA_A and glycine receptors, which had no influence on REM atonia (Figs. 6, vii, viii). Together, these findings advise that REM paralysis is triggered when motoneurons are inhibited by concomitant activation of both metabotropic GABA_B and ionotropic GABA_A/glycine receptors.

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Figure 8.

Antagonism of GABA_B, GABA_A and glycine increases basal masseter tone during waking, NREM and REM sleep. a , An EMG and EEG trace showing that REM masseter atonia is not triggered on entrance into REM despite when both metabotropic and ionotropic receptors are antagonized. This is in marked dissimilarity to the consummate loss of masseter tone that occurs on entrance into REM sleep when merely GABA_A/glycine receptors are blocked. b , Group information (n = 13) showing that CGP52432 (0.2 yardm) and bicuculline/strychnine perfusion (0.1 chiliadm for each) at the trigeminal motor pool significantly increases basal levels of masseter EMG activity not but during waking and NREM slumber, only also during REM sleep. *p < 0.001. All values are mean ± SEM.

Even so, our results suggest that reduced motoneuron excitation also contributes to REM paralysis. Although trigeminal motoneurons were cutoff from GABA and glycine receptor-mediated inhibition, REM musculus tone nevertheless remained below normal waking levels (mail-REM waking during baseline vs REM drug; paired t test, t ₍₁₃₎ = 3542, p = 0.0036; Fig. viic ). This observation infers that loss of wake-active excitatory drives—probable stemming from glutamate, noradrenaline, hypocretin/orexin and dopamine sources (Peever et al., 2003; Fenik et al., 2005; Burgess et al., 2008; Schwarz et al., 2008; Schwarz and Peever, 2011)—as well functions to reduce motoneuron and musculus action during REM sleep.

Activation of both metabotropic GABA_B and iontotropic GABA_A/glycine receptors on motoneurons is required for triggering REM sleep paralysis

In the preceding experiments nosotros perfused (via reverse-microdialysis) GABA and glycine receptor antagonists onto trigeminal motoneurons for 2–4 h, which could cause receptor desensitization (Jones and Westbrook, 1995; Pitt et al., 2008; Bright et al., 2011) and thus insufficient receptor antagonism. To accost this concern we increased adversary concentrations (from 0.1 to 0.iii mone thousand) and applied them in a unmarried bolus (via microinjection, 0.2 μl) simply during REM slumber. Despite this additional precaution, we plant that REM masseter paralysis remained completely intact when GABA_A and glycine receptors on motoneurons were antagonized (aCSF_{( n = half dozen)} vs drug_{( n = 12)}; t test, t _(sixteen) = ane.593, p = 0.131; Fig. ix). However, this intervention triggered potent increases in the frequency, duration and amplitude of REM sleep muscle twitches (p < 0.05 for each variable; data not shown). Because intracellular studies evidence that motoneurons are maximally hyperpolarized during REM musculus twitches (Chase and Morales, 1983) and because GABA_A and glycine receptor animosity markedly increase twitch action (Brooks and Peever, 2008b), nosotros contend that these receptors were fully antagonized. Nosotros conclude that neither GABA_A nor glycine receptor-mediated inhibition is sufficient for generating REM paralysis.

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Figure ix.

REM atonia is triggered by activation of both metabotropic GABA_B and iontotropic GABA_A/glycine receptors on trigeminal motoneurons. a , EMG and EEG traces showing that REM atonia is reversed when GABA_B, GABA_A and glycine receptors are antagonized at the trigeminal motor puddle; however, atonia remains intact when only GABA_A and glycine receptors are blocked. b , Grouping data showing that even high concentrations of bicuculline and strychnine (0.three mm) practical onto trigeminal motoneurons during REM sleep cannot prevent REM inhibition (n = 12). Yet, blockade of both metabotropic GABA_B and ionotropic GABA_A/glycine receptors past microinjection of CGP52432 (0.6 1000m), bicuculline and strychnine (0.ii 1000m) abolishes masseter REM atonia (due north = xiii). *p < 0.003. All values are mean ± SEM.

Finally, we confirmed that motoneurons are just released from REM inhibition when GABA_B, GABA_A and glycine receptors are simultaneously antagonized. We did this by microinjecting CGP52432 (0.6 1000m) and bicuculline/strychnine (0.3 mm of each) at the trigeminal motor pool only during private REM sleep episodes. We establish that REM paralysis was rapidly overridden when both metabotropic GABA_B and ionotropic GABA_A/glycine receptors were blocked on trigeminal motoneurons (aCSF_{( due north = 6)} vs drug_{( n = 12)}; t test, t ₍₁₇₎ = 3.400, p = 0.003; Fig. nine). This result is consistent with microdialysis experiments (Figs. 7, 8) and confirms our findings that REM paralysis is merely reversed when motoneurons are cutoff from both metabotropic GABA_B and ionotropic GABA_A/glycine inhibition.

Discussion

Our results identify the transmitter and receptor mechanisms responsible for REM sleep paralysis. Nosotros show GABA and glycine inhibition causes motor paralysis by switching-off motoneurons during REM sleep. This bulldoze inhibits motoneurons by activating both metabotropic GABA_B and ionotropic GABA_A/glycine receptors. REM motor inhibition is but prevented when motoneurons are cutoff from all sources of GABA and glycine transmission. No single course of receptor-mediated inhibition is capable of triggering REM paralysis. Current results therefore advance our agreement of the synaptic mechanisms underlying REM sleep paralysis.

Technical considerations

Somatic motor pools contain both motoneurons and interneurons (Moriyama, 1987; Nozaki et al., 1993). Therefore, a potential technical caveat is that our drug manipulations at the trigeminal motor pool influenced REM atonia by affecting interneuron role. Withal, current and previous results (Brooks and Peever, 2008b; Burgess et al., 2008; Schwarz et al., 2008) advise that interneuron activity was negligibly affected past experimental manipulations. For example, we showed that drug interventions at the left motor pool merely influenced left masseter musculus tone, they never affected right masseter tone (Figs. 1, three). If changes in REM sleep atonia were in fact mediated by interneurons then both left and correct masseter musculus tone would be affected because interneurons synaptically control motoneurons in both left and right motor pools (Ter Horst et al., 1990; McDavid et al., 2006). Accordingly, we conclude that drug interventions at the trigeminal motor puddle predominantly affect motoneurons and therefore suggest that GABA and glycine receptor manipulations touch on REM slumber paralysis by directly impacting trigeminal motoneuron function.

Multiple mechanisms mediate REM sleep paralysis

Our findings are important because they refute the long-standing hypothesis that a one-transmitter, one-receptor miracle is responsible for REM paralysis (Chase, 2008; Soja, 2008). In fact, we discover no testify to support the theory that glycine inhibition is single-handedly responsible for REM sleep paralysis. Our results clearly show that both GABA and glycine transmission are disquisitional for normal motor control in REM slumber. Specifically, nosotros show that metabotropic GABA_B and ionotropic GABA_A/glycine receptor-mediated inhibition are required for generating REM motor paralysis.

Yet, our results back up the original concept that REM motor paralysis is caused by hyperpolarization of motoneurons (Nakamura et al., 1978). In fact, we show that an extraordinarily powerful GABA and glycine bulldoze onto motoneurons is switched-on specifically during REM sleep. This inhibitory drive is far more pervasive than the relatively weak bulldoze present during NREM sleep, which is easily blocked by inactivating either GABA or glycine receptors (Morrison et al., 2003; Brooks and Peever, 2008b, 2011). Past comparison, REM motor inhibition is only rendered ineffective when motoneurons are completely deprived of both metabotropic GABA_B and GABA_A/glycine receptor-mediated inhibition.

Metabotropic and ionotropic receptor activation could cause motoneuron inhibition and REM paralysis in a numbers of ways. Straightforward summation of long-lasting GABA_B and short-lasting GABA_A/glycine receptor-driven IPSPs could generate motor atonia past hyperpolarizing motoneurons. However, motoneuron inhibition could as well outcome from dynamic interactions betwixt metabotropic and ionotropic receptors (Obrietan and van den Political leader, 1998; O'Brien et al., 2004). Synergy betwixt metabotropic and ionotropic receptor function is a well documented phenomenon in neuroscience, particularly for glutamatergic receptors. But dynamic interaction between GABA_B, GABA_A and glycine receptors is also evident. For example, coactivation of GABA_B and GABA_A receptors produces more than hyperpolarization than predicted by summation lonely (Li et al., 2010) and cantankerous talk between GABA_B and ionotropic GABA_A/glycine receptors affects inhibition by second-messenger and receptor phosphorylation mechanisms (Kardos and Kovacs, 1991; Kardos et al., 1994; Barilà et al., 1999). It remains to exist determined how GABA and glycine inhibition functions to hyperpolarize motoneurons during REM sleep.

Additional synaptic mechanisms could as well influence motoneuron physiology and muscle tone during REM sleep. For example, activation of chloride channels of the cystic fibrosis transmembrane regulator (CFTR) results in motoneuron hyperpolarization in vitro (Morales et al., 2011). Modulation of the neuron-specific potassium chloride cotransporter-ii (KCC2), which enables chloride-mediated inhibition, also influences motoneuron inhibition and motor function (Hübner et al., 2001). In improver, activation of muscarinic receptors on motoneurons functions to suppress muscle tone (Liu et al., 2005). Because brainstem cholinergic neurons also regulate REM slumber (Kodama et al., 2003; McCarley, 2004; Jones, 2008) and innervate somatic motor pools then such mechanisms also could mediate REM atonia (Lydic et al., 1989). Whether any or all of these mechanisms control motoneuron behavior and REM paralysis remains to be adamant.

REM paralysis requires both GABA and glycine inhibition

Our results reconcile a longstanding argue in biology. Two competing theories take argued that REM paralysis results from either increased glycinergic inhibition or decreased monoaminergic excitation of motoneurons. Early experiments showed that some motoneurons are hyperpolarized by glycine-sensitive IPSPs during REM slumber (Chase et al., 1989; Soja et al., 1991). Yet, subsequent pharmacological studies plant that REM muscle paralysis was unaffected by direct animosity of glycine receptors on motoneurons (Kubin et al., 1993; Morrison et al., 2003; Brooks and Peever, 2008b). More recently we constitute that REM atonia was unperturbed by the loss of function of glycine receptors in transgenic mice. Specifically, nosotros showed that impaired glycine receptor function triggered robust REM sleep behaviors in mutant mice; however, these behaviors did not result from loss of REM sleep paralysis, but instead were caused by excessive muscle twitch activity during REM sleep (Brooks and Peever, 2011). Together, these observations refuted the merits that REM paralysis is caused by a glycine-dependent mechanism, simply supported the hypothesis that it is acquired by loss of motoneuron excitation. Even so, artificially restoring excitatory drives onto motoneurons by exogenous neurotransmitter application (e.one thousand., glutamate, noradrenaline, serotonin) failed to contrary REM paralysis (Jelev et al., 2001; Chan et al., 2006; Brooks and Peever, 2008b; Burgess et al., 2008). This finding provided strong back up for the concept that a powerful inhibitory mechanism acts to switch-off motoneurons during REM sleep.

Our current results evidence that motoneuron inhibition is indeed the driving force behind REM paralysis. However, nosotros also testify that reduced motoneuron excitation acts to reinforce muscle paralysis during REM sleep. Although preventing GABA_B and GABA_A/glycine receptor function reversed REM paralysis, it did non restore it to waking levels. This finding suggests that motoneuron inhibition forces motoneurons and muscles to remain silent during REM sleep, merely that reduced motoneuron excitation also contributes to REM atonia.

Current and previous work clearly indicate that REM paralysis results from a residue betwixt increased motoneuron inhibition and reduced motoneuron excitation. Biochemical studies show that noradrenaline/serotonin release decreases, whereas, GABA/glycine release increases within spinal/cranial motor pools during drug-induced REM slumber (Kubin et al., 1994; Lai et al., 2001; Kodama et al., 2003). Reduced excitatory bulldoze arising from glutamate, noradrenaline, dopamine and hypocretin neurons functions to weaken motoneuron activity and reinforce REM atonia (Lai et al., 2001; Kodama et al., 2003; Peever et al., 2003; Chan et al., 2006; Burgess et al., 2008; Schwarz and Peever, 2010, 2011). It is unknown how the inhibitory and excitatory neuro-circuits establish the balance between motoneuron inhibition and disfacilitation during REM sleep. But, breakdown of either side of this system could tip the normal balance of control and consequence in REM motor disruption such every bit in REM sleep behavior disorder.

Neuro-circuits underlying REM paralysis

Potential neuro-circuits responsible for REM paralysis take been identified and mapped-out. Cells in the ventromedial medulla (VMM) and the SLD region function to promote REM paralysis (Holmes and Jones, 1994; Boissard et al., 2002, 2003; Lu et al., 2006). Immunohistochemical studies show that cells in these regions comprise GABA and glycine (Holstege, 1996; Li et al., 1996). Electrophysiological and lesion studies show these cells are REM-agile and destroying them disturbs motor function during REM slumber (Schenkel and Siegel, 1989; Siegel et al., 1991; Maloney et al., 1999; Boissard et al., 2002, 2003; Lu et al., 2006; Vetrivelan et al., 2009). In addition, chemic and electrical stimulation of these REM regulating regions as well triggers muscle paralysis in anesthetized animals (Lai and Siegel, 1988, 1991). Although the VMM and SLD both promote REM motor inhibition, information technology is unclear how they communicate with each another to initiate and maintain REM paralysis.

Our information reshape our agreement of REM motor control and nosotros suggest a new model that incorporates both past and present data. We suggest that REM-on GABA and glycine neurons in VMM regions trigger REM paralysis by directly inhibiting motoneurons during REM sleep (Lai and Siegel, 1988; Lu et al., 2006; Vetrivelan et al., 2009). REM paralysis can besides exist initiated past REM-on glutamate neurons in the SLD region (Lu et al., 2006; Clément et al., 2011; Luppi et al., 2011). These cells indirectly trigger motor atonia past activating GABA/glycine interneurons, which in turn inhibit motoneurons. However, REM sleep paralysis is ultimately triggered when GABA and glycine co-release hyperpolarizes motoneurons past simultaneously activating both metabotropic GABA_B and ionotropic GABA_A/glycine receptors.

Understanding the mechanisms mediating REM slumber paralysis is clinically of import considering they could explain the nature of REM sleep disorders such equally RBD, sleep paralysis and cataplexy/narcolepsy. RBD results from loss of typical REM atonia, which allows pathological motor activation and dream enactment, which often lead to serious injuries (Mahowald and Schenck, 2005; Peever, 2011). Conversely, sleep paralysis and cataplexy result when REM atonia intrudes into wakefulness thus preventing normal behavior and movement (Siegel, 2006; Peever, 2011). Determining the mechanistic nature of REM slumber paralysis will better our agreement and treatment of such disorders.

Footnotes

• This study was funded by the Canadian Institutes of Wellness Research and the National Scientific discipline and Engineering Research Council of Canada. We give thanks members of our laboratory for reading this manuscript and providing helpful feedback. We also thank Clarissa Muere for her technical assistance with histology.

• Correspondence should be addressed to Dr. John Peever, Systems Neurobiology Laboratory, Section of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, M5S 3G5, Canada. John.Peever{at}utoronto.ca

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Source: https://www.jneurosci.org/content/32/29/9785

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