A novel role of pedunculopontine tegmental kainate receptors: a mechanism of rapid eye movement sleep generation in the rat.

Considerable evidence suggests that pedunculopontine tegmental cholinergic cells are critically involved in normal regulation of rapid eye movement sleep. The major excitatory input to the cholinergic cell compartment of the pedunculopontine tegmentum arises from glutamatergic neurons in the pontine reticular formation. Immunohistochemical studies reveal that both ionotropic and metabotropic receptors are expressed in pedunculopontine tegmental cells. This study aimed to identify the role of endogenous glutamate and its specific receptors in the pedunculopontine tegmentum in the regulation of physiological rapid eye movement sleep. To identify this physiological rapid eye movement sleep-inducing glutamate receptor(s) in the pedunculopontine tegmental cholinergic cell compartment, specific receptors were blocked differentially by local microinjection of selective glutamate receptor antagonists into the pedunculopontine tegmental cholinergic cell compartment while quantifying the effects on rapid eye movement sleep in freely moving chronically instrumented rats. By comparing the alterations in the patterns of rapid eye movement sleep following injections of control vehicle and selective glutamate receptor antagonists, contributions made by each receptor subtype in rapid eye movement sleep were evaluated. The results demonstrate that when kainate receptors were blocked by local microinjection of a kainate receptor selective antagonist, spontaneous rapid eye movement sleep was completely absent for the first 2 h, and for the next 2 h the total percentage of rapid eye movement sleep was significantly less compared to the control values. In contrast, when N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, groups I, II, and III metabotropic receptors were blocked, total percentages of rapid eye movement sleep did not change compared to the control values. These findings suggest, for the first time, that the activation of kainate receptors within the cholinergic cell compartment of the pedunculopontine tegmentum is a critical step for the regulation of normal rapid eye movement sleep in the freely moving rat. The results also suggest that the different types of glutamate receptors within a small part of the brainstem may be involved in different types of physiological functions.

Excitation of the pedunculopontine tegmental NMDA receptors induces wakefulness and cortical activation in the rat.

Microinjection of the excitatory amino acid, L-glutamate into the brainstem pedunculo pontine tegmentum (PPT) has been shown to induce wakefulness, however, it has been unclear that receptors mediate this effect. The aim of this study was to test the hypothesis that in the PPT, L-glutamate induces cortical activation and wakefulness via activation of NMDA receptors. To test this hypothesis, three sets of micro-injections into the PPT were carried out on two different groups of rats that were then allowed to move freely although chronic instrumentation recorded sleep/wake states. Three days after the initial control injections of saline, in a contra-lateral site, Group I was micro-injected with saline + glutamate (saline first, and glutamate 15 min later); after another 3 days, the same rats were micro-injected with the NMDA-receptor-specific antagonist, 2-amino-5-phosphonopentanoic acid, (AP5) + glutamate. Group II received the same initial control injections (saline only), then AP5 + glutamate and the saline + glutamate micro-injections last. In rats that were not pretreated with AP5, microinjection of a 90 ng dose of L-glutamate (0.48 nmol in a volume of 0.1 microl vehicle) kept animals awake for 2-3 hr by eliminating both slow-wave sleep (SWS) and rapid eye movement (REM) sleep. These behavioral state changes were accompanied by concomitant increase in the power of gamma (gamma) frequency (20-60 Hz) waves in the cortical EEG. Pretreatment of L-glutamate injection sites with 0.48 nmol of AP5 blocked L-glutamate-induced-wakefulness and preserved a normal amount of wakefulness and sleep. Pretreatment with AP5 decreased the power of gamma-wave activity below its control level. These results support the hypothesis that the glutamate-induced-wakefulness and cortical activation effects are mediated via the NMDA receptors.

Microinjection of glutamate into the pedunculopontine tegmentum induces REM sleep and wakefulness in the rat.

The aim of this study was to test the hypothesis that the cells in the brain stem pedunculopontine tegmentum (PPT) are critically involved in the normal regulation of wakefulness and rapid eye movement (REM) sleep. To test this hypothesis, one of four different doses of the excitatory amino acid L-glutamate (15, 30, 60, and 90 ng) or saline (control vehicle) was microinjected unilaterally into the PPT while the effects on wakefulness and sleep were quantified in freely moving chronically instrumented rats. All microinjections were made during wakefulness and were followed by 6 h of polygraphic recording. Microinjection of 15- ng (0.08 nmol) and 30-ng (0.16 nmol) doses of L-glutamate into the PPT increased the total amount of REM sleep. Both doses of L-glutamate increased REM sleep at the expense of slow-wave sleep (SWS) but not wakefulness. Interestingly, the 60-ng (0.32 nmol) dose of L-glutamate increased both REM sleep and wakefulness. The total increase in REM sleep after the 60-ng dose of L-glutamate was significantly less than the increase from the 30-ng dose. The 90-ng (0.48 nmol) dose of L-glutamate kept animals awake for 2-3 h by eliminating both SWS and REM sleep. These results show that the L-glutamate microinjection into the PPT can increase wakefulness and/or REM sleep depending on the dosage. These findings support the hypothesis that excitation of the PPT cells is causal to the generation of wakefulness and REM sleep in the rat. In addition, the results of this study led to the identification of the PPT dosage of L-glutamate that optimally induces wakefulness and REM sleep. The knowledge of this optimal dose will be useful in future studies investigating the second messenger systems involved in the regulation of wakefulness and REM sleep.

Prenatal protein malnourished rats show changes in sleep/wake behavior as adults.

Prenatal protein malnutrition significantly elevates brain levels of serotonin in rats, and these levels remain elevated throughout their lives. This biogenic amine is involved in the regulation of many physiological functions, including the normal sleep/wake cycle. The present study examined the effects of prenatal protein malnutrition on the sleep/wake cycle of freely moving adult rats. Six prenatally protein malnourished (6% casein) and 10 well-nourished (25% casein) male rats (90-120-day-old) were chronically implanted with a standard set of electrodes (to record cortical electroencephalogram, neck muscle electromyogram, electrooculogram, and hippocampal theta wave) to objectively measure states of sleep and wakefulness. Six-hour polygraphic recordings were made between 10.00 and 16.00 h; a time when the rats normally sleep. Prenatally malnourished rats spent 20% more time in slow wave sleep (SWS) compared to the well-nourished rats. The total percentage of time spent in rapid eye movement (REM) sleep was 61% less in prenatally malnourished rats compared to well-nourished control rats. These findings demonstrate the adverse consequences of prenatal protein malnutrition on the quality and quantity of adult sleep in rats. These sleep changes are potentially detrimental to normal social behavior and cognitive functions. Prenatally malnourished rats are an excellent animal model to study the role of endogenous serotonin in the regulation of the normal sleep/wake cycle.

Brainstem afferents of the cholinoceptive pontine wave generation sites in the rat.

The present study was designed to investigate the distribution of brainstem neurons projecting to the pontine wave (P-wave)-generating sites in the rat. In six rats, biotinylated dextran amine (BDA) was microinjected into the physiologically identified cholinoceptive P-wave generation site. In all cases, microinjections of BDA in the cholinoceptive P-wave generating site resulted in retrograde labeling of cell bodies in many parts of the brainstem. The majority of those retrogradely labeled cells were in the pedunculopontine tegmentum, pontine reticular nucleus oralis, parabrachial nucleus, vestibular nucleus, and gigantocellular reticular nucleus. The results presented in this study provide anatomical evidence that the cholinoceptive P-wave generation site in the rat receives anatomical projections from other parts of the brainstem known to be involved in the REM sleep-generation mechanism.

Localization of pontine PGO wave generation sites and their anatomical projections in the rat.

A number of experimental and theoretical reports have suggested that the ponto-geniculo-occipital (PGO) wave-generating cells are involved in the generation of rapid eye movement (REM) sleep and REM sleep dependent cognitive functions. No studies to date have examined anatomical projections from PGO-generating cells to those brain structures involved in REM sleep generation and cognitive functions. In the present study, pontine PGO wave-generating sites were mapped by microinjecting carbachol in 74 sites of the rat brainstem. Those microinjections elicited PGO waves only when made in the dorsal part of the nucleus subcoeruleus of the pons. In six rats, the anterograde tracer biotinylated dextran amine (BDA) was microinjected into the physiologically identified cholinoceptive pontine PGO-generating site to identify brain structures receiving efferent projections from those PGO-generating sites. In all cases, small volume injections of BDA in the cholinoceptive pontine PGO-generating sites resulted in anterograde labeling of fibers and terminals in many regions of the brain. The most important output structures of those PGO-generating cells were the occipital cortex, entorhinal cortex, piriform cortex, amygdala, hippocampus, and many other thalamic, hypothalamic, and brainstem nuclei that participate in the generation of REM sleep. These findings provide anatomical evidence for the hypothesis that the PGO-generating cells in the pons could be involved in the generation of REM sleep. Since PGO-generating cells project to the entorhinal cortex, piriform cortex, amygdala, and hippocampus, these PGO-generating cells could also be involved in the modulation of cognitive functions.

Endogenous and exogenous nitric oxide in the pedunculopontine tegmentum induces sleep.

Mesopontine cholinergic cells in the pedunculopontine tegmental (PPT) nuclei modulate the control of the wake-sleep cycle by releasing acetylcholine to their target structures. These cells also synthesize nitric oxide (NO) which diffuses into the extracellular space and acts as a neuronal messenger. The present study is based on the hypothesis that NO synthesis and its presence in the extracellular space in the PPT play a functional role in regulating the behavioral states of waking and sleep. This hypothesis was tested by microinjecting a control vehicle, NO donor, S-Nitroso-N-acetylpenicillamine (SNAP) and a competitive inhibitor of NO synthase enzyme (NOS), N(G)-Nitro-L-arginine methylester hydrochloride (L-NAME) into the PPT while quantifying the effects on wakefulness and sleep. Six cats were implanted with bilateral guide tubes for PPT microinjection and with standard electrodes to measure waking, slow-wave sleep (SWS), and rapid eye movement (REM) sleep. Five-hour free-moving polygraphic recordings were made following each microinjection (0.25 microl) of control saline, SNAP or L-NAME. Following microinjection of SNAP into the cholinergic cell compartments of the PPT, SWS and REM sleep were increased by 41.65% and 72.10% respectively, compared to the control microinjection. Microinjection of L-NAME reduced SWS and REM sleep by 40.33% and 62.05%, respectively, compared to controls. The present results demonstrate that endogenous NO synthesized within the PPT cholinergic cells functions as a paracrine signal in the control of waking and sleep by modulating local cholinergic cells.