Glutamatergic and cholinergic projections to the pontine inhibitory area identified with horseradish peroxidase retrograde transport and immunohistochemistry

Previous studies in our laboratory have shown that microinjection of acetylcholine and non-N-methyl-D-aspartate (NMDA) glutamate agonists into the pontine inhibitory area (PIA) induce muscle atonia. The present experiment was designed to identify the PIA afferents that could be responsible for these effects, by use of retrograde transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP), glutamate immunohistochemistry and NADPH-diaphorase staining techniques. Experiments were performed in both decerebrate and intact cats. Dense retrograde WGA-HRP labelling was found in neurons in the periaqueductal gray (PAG) and mesencephalic reticular formation (MRF) at the red nucleus (RN) level, ventral portion of paralemniscal tegmental field (_VFTP), retrorubral nucleus (RRN), contralateral side of PIA (_CPIA), pontis reticularis centralis caudalis (PoC), and most rostral portion of the nucleus parvicellularis (NPV) and nucleus praepositus hypoglossi (PH) at the level of the pontomedullary junction; moderate labelling was seen in pedunculopontine nucleus, pars compacta (PPNc), laterodorsal tegmental nucleus (LDT), superior colliculus (SC), MRF and PAG at the level caudal to RN, medial and superior vestibular nuclei, and principle sensory trigeminal nucleus (5P); and light labelling was seen in dorsal raphe (DR) and locus coeruleus complex (LCC). The projection neurons were predominantly ipsilateral to the injection site, except for both vFTP and RRN, which had more projection cells on the contralateral side. Double labelled WGA-HRP/NADPH-d neurons could be found in PPNc and LDT. Double labelled WGA-HRP/glutamatergic neurons could be seen at high densities in MRF, RRN, vFTP, and cPIA, moderate densities in SC, LDT, PPNc, PoC, and NPV, and low densities in PH, 5P, DR, LCC, and PAG. No cells in LDT and PPNc were triple labelled with NADPH-d, glutamate antibody and WGA-HRP. The mesopontine efferents identified here may mediate the suppression of muscle tone in REM sleep and coordinate muscle tone during head and neck movements. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Corticotropin-releasing factor mediated muscle atonia in pons and medulla

The dorsolateral pontine inhibitory area (PIA) and medial medullary reticular formation (MMRF) have been found to mediate the muscle atonia of REM sleep. Our previous studies have shown that acetylcholine (ACh) microinjection in the PIA and in the nucleus paramedianus of the medial medulla produces muscle atonia. Glutamate microinjection in both PIA and nucleus magnocellularis (NMC) of the medial medulla also produces muscle atonia. Since immunohistochemical studies have identified corticotropin-releasing factor (CRF) as a potential dorsolateral pontine and NMC transmitter, the present study was undertaken to determine whether this transmitter could produce suppression of muscle tone. Experiments were performed on unanesthetized, decerebrated cats. CRF was microinjected into points in the PIA and NMC at which electrical stimulation produced bilateral inhibition of muscle tone. We found that CRF produced a dose-dependent muscle tone suppression. At 10 nM concentration, the latency and duration of muscle inhibition produced by CRF injection were comparable with those ofl-glutamate, at 18.8 s and 4.1 min, respectively. This CRF-induced muscle inhibition was blocked by the CRF antagonist, α-helical [Glu²⁷]corticotropin-releasing factor 9–41 (CRF 9–41). Microinjection of CRF and non-NMDA agonists, kainate and quisqualate, into the same sites in PIA and NMC produced muscle atonia. Pontine sites at which CRF injection induces atonia are identical to those at which acetylcholine microinjection produces atonia. These results indicate that CRF may interact with glutamate and acetylcholine in the generation of muscle atonia.     

A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice

During rapid eye movement (REM) sleep, anti-gravity muscle tone and bodily movements are mostly absent, because somatic motoneurons are inhibited by descending inhibitory pathways. Recent studies showed that glycine/GABA neurons in the ventromedial medulla (VMM; Gly^VMM neurons) play an important role in generating muscle atonia during REM sleep (REM-atonia). However, how these REM-atonia-inducing neurons interconnect with other neuronal populations has been unknown. In the present study, we first identified a specific subpopulation of Gly^VMM neurons that play an important role in induction of REM-atonia by virus vector-mediated tracing in male mice in which glycinergic neurons expressed Cre recombinase. We found these neurons receive direct synaptic input from neurons in several brain stem regions, including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD; Glu^SLD neurons). Silencing this circuit by specifically expressing tetanus toxin light chain (TeTNLC) resulted in REM sleep without atonia. This manipulation also caused a marked decrease in time spent in cataplexy-like episodes (CLEs) when applied to narcoleptic orexin-ataxin-3 mice. We also showed that Gly^VMM neurons play an important role in maintenance of sleep. This present study identified a population of glycinergic neurons in the VMM that are commonly involved in REM-atonia and cataplexy.SIGNIFICANCE STATEMENT We identified a population of glycinergic neurons in the ventral medulla that plays an important role in inducing muscle atonia during rapid eye movement (REM) sleep. It sends axonal projections almost exclusively to motoneurons in the spinal cord and brain stem except to those that innervate extraocular muscles, while other glycinergic neurons in the same region also send projections to other regions including monoaminergic nuclei. Furthermore, these neurons receive direct inputs from several brainstem regions including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD). Genetic silencing of this pathway resulted in REM sleep without atonia and a decrease of cataplexy when applied to narcoleptic mice. This work identified a neural population involved in generating muscle atonia during REM sleep and cataplexy.     

Enhanced glutamate release during REM sleep in the rostromedial medulla as measured by in vivo microdialysis

Anatomical studies and stimulation studies in the decerebrate animal have suggested that the muscle atonia of rapid eye movement (REM) sleep is mediated by a projection from cholinoceptive glutamatergic neurons in the pons to the nucleus magnocellularis (NMC) of medulla. This model suggests that glutamate release in NMC should be enhanced in REM sleep. In the present study, glutamate release across the sleep–wake cycle in NMC was measured by in vivo microdialysis. We found that glutamate release in NMC was significantly higher (p=0.0252) during REM sleep than during wakefulness (W). Glutamate release during REM sleep was not elevated either in nucleus paramedianus (NPM) or in the pontine inhibitory area (PIA) regions where cholinergic stimulation suppresses muscle tone. Acetylcholine (ACh) microinjection into PIA enhanced glutamate release in NMC. These results support the hypothesis that a glutamatergic pathway from PIA to NMC is responsible for the suppression of muscle tone in REM sleep.     

Pontine regulation of REM sleep components in cats: integrity of the pedunculopontine tegmentum (PPT) is important for phasic events but unnecessary for atonia during REM sleep.

Transection, lesion and unit recording studies have localized rapid eye movement (REM) sleep mechanisms to the pons. Recent work has emphasized the role of pontine cholinergic cells, especially those of the pedunculopontine tegmentum (PPT). The present study differentiated REM sleep deficits associated with lesions of the PPT from other pontine regions implicated in REM sleep generation, including those with predominantly cholinergic vs non-cholinergic cells. Twelve hour polygraphic recordings were obtained in 18 cats before and 1-2 weeks after bilateral electrolytic or radio frequency lesions of either: (1) PPT, which contains the dorsolateral pontine cholinergic cell column; (2) laterodorsal tegmental nucleus (LDT), which contains the dorsomedial pontine cholinergic cell column; (3) locus ceruleus (LC), which contains mostly noradrenergic cells; or (4) subceruleus (LC alpha, peri-LC alpha and the lateral tegmental field), which also contains predominantly noncholinergic cells. There were three main findings: (i) Only lesions of PPT and subceruleus significantly affected REM sleep time. These lesions produced comparable reductions in REM sleep time but influenced REM sleep components quite differently: (ii) PPT lesions, estimated to damage 90 +/- 4% of cholinergic cells, reduced the number of REM sleep entrances and phasic events, including ponto-geniculooccipital (PGO) spikes and rapid eye movements (REMs), but did not prevent complete atonia during REM sleep: (iii) Subceruleus lesions eliminated atonia during REM sleep. Mobility appeared to arouse the cat prematurely from REM sleep and may explain the brief duration of REM sleep epochs seen exclusively in this group. Despite the reduced amount of REM sleep, the total number of PGO spikes and REM sleep entrances increased over baseline values. Collectively, the results distinguish pontine loci regulating phasic events vs atonia. PPT lesions reduced phasic events, whereas subceruleus lesions created REM sleep without atonia. Severe REM sleep deficits after large pontine lesions, including PPT and subceruleus, might be explained by simultaneous production of both REM sleep syndromes. However, extensive loss of ACh neurons in the PPT does not disrupt REM sleep atonia.     

The neuronal network responsible for paradoxical sleep and its dysfunctions causing narcolepsy and rapid eye movement (REM) behavior disorder

Rapid eye movement (REM) sleep behavior disorder (RBD) is a parasomnia characterized by the loss of muscle atonia during paradoxical (REM) sleep (PS). Conversely, cataplexy, one of the key symptoms of narcolepsy, is a striking sudden episode of muscle weakness triggered by emotions during wakefulness, and comparable to REM sleep atonia. The neuronal dysfunctions responsible for RBD and cataplexy are not known. In the present review, we present the most recent results on the neuronal network responsible for PS. Based on these results, we propose an updated integrated model of the mechanisms responsible for PS and explore different hypotheses explaining RBD and cataplexy. We propose that RBD is due to a specific degeneration of a sub-population of PS-on glutamatergic neurons specifically responsible of muscle atonia, localized in the caudal pontine sublaterodorsal tegmental nucleus (SLD). Another possibility is the occurrence in RBD patients of a specific lesion of the glycinergic/GABAergic pre-motoneurons localized in the medullary ventral gigantocellular reticular nucleus. Conversely, cataplexy in narcoleptics would be due to the activation during waking of the caudal PS-on SLD neurons responsible for muscle atonia. A phasic glutamatergic excitatory pathway from the central amygdala to the SLD PS-on neurons activated during emotion would induce such activation. In normal conditions, the glutamate excitation would be blocked by the simultaneous excitation by the hypocretins of the PS-off GABAergic neurons localized in the ventrolateral periaqueductal gray and the adjacent deep mesencephalic reticular nucleus, gating the activation of the PS-on SLD neurons.     

Olivopontocerebellar degeneration, abnormal sleep, and REM sleep without atonia

Polygraphic studies during sleep performed in two patients with olivopontocerebellar degeneration (OPCD) revealed an abnormal control of muscle tone. It was demonstrated by bursts of EMG activity during sleep and progressive disappearance of muscle atonia during sleep. Muscle atonia disappeared during rapid eye movement (REM) sleep, permitting movements and expression of feelings probably associated with REM sleep-related oneiric activity. Patients, unaware of their nocturnal sleep disturbance, complained only of the resulting daytime tiredness and sleepiness.     

REM sleep behavioral disorders

REM sleep behaviors were recently described as wild, dream-enacting behaviors during REM sleep with loss of usual atonia on submental muscles. We examined 6 patients (5 M, 1F) with characteristic episodes of behavioral manifestations during REM sleep. Polysomnographic data indicate a decrease in first REM latency, an absence of stage 4 NREM, altered phasic motor activity and behavioral episodes during REM sleep even with normal chin muscle atonia. Three patients had Shy-Drager syndrome, 1 olivopontocerebellar atrophy and 2 patients had no neurological disease. The crucial importance of a disinhibited locomotor system during sleep appears to be responsible for this REM parasomnia.     

New aspects in the pathophysiology of rapid eye movement sleep behavior disorder: the potential role of glutamate,gamma-aminobutyric acid,and glycine

Rapid eye movement sleep behavior disorder (RBD) is a parasomnia characterized by the occurrence of intense movements during rapid eye movement (REM) sleep, also named paradoxical sleep. The neuronal dysfunctions at the origin of the loss of atonia in RBD patients are not known. One possibility is that RBD is due to the degeneration of neurons inducing the muscle atonia of REM sleep. Therefore, in our paper we review data on the populations of neurons responsible for the atonia of REM sleep before discussing their potential role in RBD. We first review evidence that motoneurons are tonically hyperpolarized by gamma-aminobutyric acid (GABA) and glycine and phasically excited by glutamate during REM sleep. Then, we review data indicating that the atonia of REM sleep is induced by glycinergic/GABAergic REM-on premotoneurons contained within the raphe magnus and the ventral and alpha gigantocellular reticular nuclei localized in the ventral medullary reticular formation. These neurons are excited during REM sleep by a direct projection from glutamatergic REM-on neurons localized in the pontine sublaterodorsal tegmental nucleus (SLD).From these results, we discuss the possibility that RBD is due to a specific degeneration of descending REM-on glutamatergic neurons localized in the caudal SLD or that of the REM-on GABA/glycinergic premotoneurons localized in the ventral medullary reticular formation. We then propose that movements of RBD are induced by descending projections of cortical motor neurons before discussing possible modes of action of clonazepam and melatonin.     

Juvenile Parkinson's disease with REM sleep behavior disorder, sleepiness, and daytime REM onset

We describe an unmedicated patient with juvenile PD with difficulties maintaining wakefulness and the atonia of REM sleep. Laboratory testing showed enhanced muscle activity in REM sleep consistent with a history of dream enactment behavior (i.e., REM sleep behavior disorder) and daytime sleepiness, and REM-sleep onsets on multiple sleep latency testing. The results emphasize the potential role of dopamine and basal ganglia circuits in the modulation of activated behavioral states (e.g., wakefulness and REM sleep).     

REM sleep behavior disorder and REM sleep without atonia in Parkinson's disease

OBJECTIVE: To determine the frequency of REM sleep behavior disorder (RBD) among patients with PD using both history and polysomnography (PSG) recordings and to further study REM sleep muscle atonia in PD. BACKGROUND: The reported occurrence of RBD in PD varies from 15 to 47%. However, no study has estimated the frequency of RBD using PSG recordings or analyzed in detail the characteristics of REM sleep muscle atonia in a large group of unselected patients with PD. METHODS: Consecutive patients with PD (n = 33) and healthy control subjects (n = 16) were studied. Each subject underwent a structured clinical interview and PSG recording. REM sleep was scored using a method that allows the scoring of REM sleep without atonia. RESULTS: One third of patients with PD met the diagnostic criteria of RBD based on PSG recordings. Only one half of these cases would have been detected by history. Nineteen (58%) of 33 patients with PD but only 1 of 16 control subjects had REM sleep without atonia. Of these 19 patients with PD, 8 (42%) did not present with behavioral manifestations of RBD, and their cases may represent preclinical forms of RBD associated with PD. Moreover, the percentage of time spent with muscle atonia during REM sleep was lower among patients with PD than among healthy control subjects (60.1% vs 93.2%; p = 0.003). CONCLUSIONS: RBD and REM sleep without atonia are frequent in PD as shown by PSG recordings.     

Motor dyscontrol in narcolepsy: rapid-eye-movement (REM) sleep without atonia and REM sleep behavior disorder.

Narcolepsy involves abnormalities of rapid-eye-movement (REM) sleep, including a short latency to the onset of REM sleep, hypnagogic hallucinations, and sleep paralysis. In addition, persistence of muscle tone by electromyographic criteria or excessive muscle twitching during REM sleep or both have been reported in treated and untreated narcoleptic patients. We report that another previously described abnormality of REM sleep, REM sleep behavior disorder, may also be a symptom of narcolepsy. This disorder was found in 10 narcoleptic patients during routine clinical evaluations involving polysomnography and multiple sleep latency tests. During REM sleep, 7 additional narcoleptic patients displayed persistent muscle tone and/or excessive twitching, which we believe to be subclinical components of REM sleep behavior disorder. These 17 patients, diagnosed by established criteria for narcolepsy and for REM sleep behavior disorder, ranged in age from 8 to 74 years. Seventy-one percent were male. Narcolepsy and REM sleep behavior disorder most commonly emerged in tandem. In 3 patients, treatment of narcolepsy-cataplexy with stimulants and tricyclics either induced or exacerbated REM sleep behavior disorder.     

Raphe unit activity during REM sleep in normal cats and in pontine lesioned cats displaying REM sleep without atonia

Previous studies have shown that the activity of serotonin-containing raphe neurons in cats is almost completely suppressed during rapid eye movement (REM) sleep. However, since raphe unit activity is known to be grossly correlated with the level of behavioral arousal or tonic motor activity, this decrease in activity during REM sleep may be simply due to the fact that tonic EMG activity or motoric output is at a minimum. On the other hand, raphe unit activity may be related to the state (i.e. REM sleep) of the organism. To test these competing hypotheses, in the present study we compared raphe unit activity in normal cats with that in cats that display REM sleep without atonia (produced by bilateral lesions of the pontine tegmentum). These lesioned cats manifest episodes which, by all criteria, appear to be REM sleep except that they display overt behavior, presumably because the mechanism normally responsible for producing atonia has been disrupted. Although the activity of raphe neurons in lesioned cats during REM sleep without atonia was significantly below that seen in these cats during waking, the level of activity was often impressive. This is especially true when those animals that displayed the greatest degree of tonic motor activity during REM sleep (group IV animals) are considered separately. In these cats, the depression was only 40.5% below their quiet waking level, whereas in lesioned cats displaying less tonic motor activity (Group II animals), raphe discharge rate was 65.6% below their quiet waking level. The discharge rate of raphe neurons during REM sleep in lesioned cats was more than 6-fold greater than that seen in normal animals. These data, in conjunction with other recent results from our laboratory, suggest that the decrease in raphe unit activity during REM sleep is largely a concomitant of the atonia which characterizes that state. These data are discussed within the general context of the relationship between raphe unit discharge and the activity of central motor systems.     

An episode of geniospasm in sleep: Toward new insights into pathophysiology?

Polysomnography and needle electromyography were performed on three members of a family with hereditary geniospasm. Electromyography showed simultaneous bilateral discharges exclusively in the mentalis muscle. In one subject we documented a paroxysm of geniospasm during sleep phase 2. This activity ceased with the onset of REM sleep. In view of the mechanism of REM atonia and the bilateral chin EMG discharges, our findings support a supranuclear origin of the peculiar mentalis muscle paroxysms. © 2007 Movement Disorder Society     

REM sleep without atonia is associated with increased rigidity in patients with mild to moderate Parkinson’s disease

ObjectiveIncreased muscle activity during rapid eye movement (REM) sleep (i.e. REM sleep without atonia) is common in people with Parkinson’s disease (PD). This study tested the hypotheses that people with PD and REM sleep without atonia (RSWA) would present with more severe and symmetric rigidity compared to individuals with PD without RSWA and age-matched controls.MethodsSixty-one individuals participated in this study (41 PD, 20 controls). An overnight sleep study was used to classify participants with PD as having either elevated (PD-RSWA+) or normal muscle activity (PD-RSWA−) during REM sleep. Quantitative measures of rigidity were obtained using a robotic manipulandum that passively pronated and supinated the forearm.ResultsQuantitative measures of forearm rigidity were significantly higher in the PD-RSWA+ group compared to the control group. Rigidity was significantly more asymmetric between limbs in the PD-RSWA− group compared with controls, while there was no significant difference in symmetry between the control and PD-RSWA+ groups.ConclusionIn people with mild to moderate PD, RSWA is associated with an increased and more symmetric presentation of upper limb rigidity.SignificanceDysfunction of brainstem systems that control muscle tone during REM sleep may contribute to increased rigidity during wakefulness in people with PD.     

Medullary regions mediating atonia

Electrical stimulation studies have implicated the medial medulla in the inhibition of muscle tone. In the present report we present evidence for suppression of muscle tone by chemical activation of the medial medulla. We find 2 distinct zones within the classically defined medial medullary inhibitory area. A rostral region corresponding to the nucleus magnocellularis (NMC) is sensitive to glutamate. Atonia produced by activation of this region is mediated by non-NMDA receptors. A caudal region, corresponding to the nucleus paramedianus (NPM) is sensitive to ACh. Atonia produced by activation of this region is mediated by muscarinic receptors. Activation of these regions both in acute decerebrate and intact cats suppresses muscle tone. We find that the cholinoceptive dorsolateral pontine region, previously implicated in atonia control, can be activated by glutamate-sensitive non-NMDA receptors. Microinjection of atropine into the NPM or of glutamylglycine into the NMC blocks atonia elicited by pontine carbachol injection. The medullary regions identified here are hypothesized to mediate the suppression of muscle tone that occurs in rapid eye movement sleep and in cataplexy and may have a role in postural control in waking.     

Descending projections from the dorsolateral pontine tegmentum to the paramedian reticular nucleus of the caudal medulla in the cat.

We examined whether the dorsolateral pontine cholinergic cells project to the paramedian reticular nucleus (PRN) of the caudal medulla. In 3 cats, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the PRN and we noted cells in the dorsolateral pons that contained the HRP reaction product, cells that were immunolabeled for choline acetyltransferase (ChAT), and cells that contained the HRP reaction product and were ChAT positive. We found cholinergic projections from the pedunculopontine tegmental and laterodorsal tegmental nuclei to the PRN. This finding is consistent with studies indicating a cholinoceptive region in the medial medulla mediating suppression of muscle tone. Our results demonstrate that this medullary region has monosynaptic input from pontine neurons implicated in generating the atonia of rapid eye movement sleep.     

Pedunculopontine tegmental nucleus-induced inhibition of muscle activity in the rat

The connections of the pedunculopontine tegmental nucleus (PPN) have led us to propose that this structure mediates striatally induced inhibition of muscle activity by directing basal ganglia output to an inhibitory reticulospinal system (nucleus reticularis gigantocellularis and ventralis, nrGi-V). We conducted experiments in order to examine the effects of electrical stimulation of the PPN on the activity of selected neck and shoulder muscles. PPN stimulation at low rates (0.1 Hz) elicited bilateral muscle excitation. As the rate of stimulation was increased (e.g. to 10 Hz), less excitation was observed. Anodal DC current inactivation of the nrGi-V during concurrent 10 Hz PPN stimulation resulted in an augmentation of muscle activity above the levels observed during 10 Hz PPN stimulation alone. PPN stimulation (10 Hz) also profoundly inhibited cortically-induced muscle activity. Further support for our proposal stems from increased baseline activity (0.1 Hz PPN-induced excitation) in animals with ibotenic acid lesions of the PPN as compared to normal animals. Apparently, destruction of the PPN releases the musculature from tonic and/or phasic inhibition. A model is discussed which attempts to account for both the rate-dependent changes in excitation and the inhibition of cortically induced muscle activity.     

Pontomedullary glutamate receptors mediating locomotion and muscle tone suppression.

Microinjection of NMDA and non-NMDA agonists into the same sites in pontomedullary motor "inhibitory" areas of decerebrate animals produced opposite effects on muscle tone. Microinjection of non-NMDA agonists into peri-locus coeruleus alpha (peri-LC alpha) and nucleus magnocellularis (NMC) suppressed muscle tone, while injection of NMDA agonists at the same sites increased muscle tone and produced locomotion. The latency, duration, and magnitude of muscle tone change after both NMDA and non-NMDA agonist injections were dose dependent. Increased muscle tone and locomotor effects were blocked by NMDA antagonists, and muscle tone suppression effects were blocked by non-NMDA antagonists. We conclude that pontomedullary non-NMDA receptors mediate muscle tone suppression, and that NMDA receptors mediate locomotion and muscle tone facilitation. Activation of both NMDA and non-NMDA pontomedullary receptors by glutamate release in REM sleep can explain the combination of motor activation and loss of muscle tone that characterizes this state. In the waking animal, the co-localization of these mechanisms may facilitate the coordination of locomotion with postural adjustments.     

Polysomnographic features of REM sleep behavior disorder: development of a scoring method.

REM sleep behavior disorder (RBD) is characterized by the intermittent absence of REM sleep EMG atonia and the appearance of elaborate motor activity associated with dream mentation. There are no specific diagnostic criteria for RBD based upon polysomnographic findings. We describe a new scoring method and show its sensitivity to treatment with clonazepam. An increased phasic submental EMG density occurs in RBD patients, but REM density is similar to that of controls. Clonazepam selectively decreases REM sleep phasic activity but exerts no effect on REM sleep atonia. Periodic limb movements in sleep (PLMS) occur equally in both REM and NREM sleep in RBD patients, suggesting that normal suppression of PLMS in REM sleep is due to motor inhibition.