Sleep and GABA levels in the oral part of rat pontine reticular formation are decreased by local and systemic administration of morphine.

Morphine, a mu-opioid receptor agonist, is a commonly prescribed treatment for pain. Although highly efficacious, morphine has many unwanted side effects including disruption of sleep and obtundation of wakefulness. One mechanism by which morphine alters sleep and wakefulness may be by modulating GABAergic signaling in brain regions regulating arousal, including the pontine reticular nucleus, oral part (PnO). This study used in vivo microdialysis in unanesthetized Sprague-Dawley rat to test the hypothesis that mu-opioid receptors modulate PnO GABA levels. Validation of the high performance liquid chromatographic technique used to quantify GABA was obtained by dialyzing the PnO (n=4 rats) with the GABA reuptake inhibitor nipecotic acid (500 microM). Nipecotic acid caused a 185+/-20% increase in PnO GABA levels, confirming chromatographic detection of GABA and demonstrating the existence of functional GABA transporters in rat PnO. Morphine caused a concentration-dependent decrease in PnO GABA levels (n=25 rats). Coadministration of morphine (100 microM) with naloxone (1 microM), a mu-opioid receptor antagonist, blocked the morphine-induced decrease in PnO GABA levels (n=5 rats). These results show for the first time that mu-opioid receptors in rat PnO modulate GABA levels. A second group of rats (n=6) was used to test the hypothesis that systemically administered morphine also decreases PnO GABA levels. I.v. morphine caused a significant (P<0.05) decrease (19%) in PnO GABA levels relative to control i.v. infusions of saline. Finally, microinjections followed by 2 h recordings of electroencephalogram and electromyogram tested the hypothesis that PnO morphine administration disrupts sleep (n=8 rats). Morphine significantly (P<0.05) increased the percent of time spent in wakefulness (65%) and significantly (P<0.05) decreased the percent of rapid eye movement (REM) sleep (-53%) and non-REM sleep (-69%). The neurochemical and behavioral data suggest that morphine may disrupt sleep, at least in part, by decreasing GABAergic transmission in the PnO.

Three-dimensional structure of the mammalian suprachiasmatic nuclei: a comparative study of five species.

The suprachiasmatic nuclei from five mammalian species (rat, hamster, cat, rhesus, and squirrel monkey) were reconstructed in three-dimensions by use of computer graphics and conventional histological techniques. The gross nuclear structures of the suprachiasmatic nuclei have complex three-dimensional geometries in every plane of orientation, and between the five species there are marked differences in the three-dimensional morphology of the suprachiasmatic nuclei. These dimensionally accurate reconstructions are discussed in relation to previous data suggesting morphological and/or functional specialization within specific regions of the suprachiasmatic nuclei.

Regional brain glucose metabolism is altered during rapid eye movement sleep in the cat: a preliminary study.

Glucose utilization was measured in 74 brain regions of the cat during states of wakefulness or rapid eye movement (REM) sleep. These data were obtained from intact, unanesthetized animals which were instrumented for objectively measuring states of consciousness. Through a chronically implanted intravenous catheter, the cats received 250 microCi of magnitude of 6-14C glucose during REM sleep (N = 3) or during wakefulness (N = 3). After spending approximately 8 min in REM sleep or in quiet wakefulness, the cats were administered a lethal dose of barbiturate and the brains were removed and processed for autoradiography. The results revealed site-specific changes in glucose metabolism during REM sleep. Significant alterations in glucose use occurred in the thalamus, the limbic system, and specific regions of the pontine reticular formation. These data demonstrate for the first time that during states comprised entirely of REM sleep there are anatomically specific changes in cerebral glucose metabolism. The majority of brain regions exhibiting REM sleep-dependent changes in glucose metabolism either overlapped with regions known to contain cholinergic cell bodies, or with areas that receive prominent cholinergic input.

The carbachol-induced enhancement of desynchronized sleep signs is dose dependent and antagonized by centrally administered atropine.

Considerable data show that microinjection of carbachol into the pontine reticular formation produces a desynchronized (D) sleep-like state. The present study examined the hypothesis that this carbachol-induced enhancement of D sleep signs is mediated by muscarinic, cholinergic receptors. This hypothesis was tested by quantifying the dose-dependent effects of centrally administered carbachol on the D sleep-like state and by pretreating the animals with centrally administered atropine. Six dosages of carbachol were microinjected into the pontine reticular formation of conscious cats and polygraphic measures of behavioral state were recorded. The percentage, latency, duration, frequency, and time course of the carbachol-induced D sleep-like state were dose dependent. Centrally administered atropine competitively antagonized the ability of carbachol to induce the D sleep-like state, whereas pontine administration of L-glutamate did not significantly alter D sleep. These data demonstrate that muscarinic, cholinergic receptors within the pontine reticular formation mediate the phenomenon of cholinoceptive D sleep sign enhancement.

Carbachol in the pontine reticular formation of C57BL/6J mouse decreases acetylcholine release in prefrontal cortex.

The prefrontal cortex and brainstem modulate autonomic and arousal state control but the neurotransmitter mechanisms underlying communication between prefrontal cortex and brainstem remain poorly understood. This study examined the hypothesis that microdialysis delivery of carbachol to the pontine reticular formation (PRF) of anesthetized C57BL/6J (B6) mouse modulates acetylcholine (ACh) release in the frontal association cortex. Microdialysis delivery of carbachol (8.8 mM) to the PRF caused a significant (P<0.01) decrease (-28%) in ACh release in the frontal association cortex, a significant (P<0.01) decrease (-23%) in respiratory rate, and a significant (P<0.01) increase (223%) in time to righting after anesthesia. Additional in vitro studies used the [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) assay to test the hypothesis that muscarinic cholinergic receptors activate guanine nucleotide binding proteins (G proteins) in the frontal association cortex and basal forebrain. In vitro treatment with carbachol (1 mM) caused a significant (P<0.01) increase in [(35)S]GTPgammaS binding in the frontal association cortex (62%) and basal forebrain nuclei including medial septum (227%), vertical (210%) and horizontal (165%) limbs of the diagonal band of Broca, and substantia innominata (127%). G protein activation by carbachol was concentration-dependent and blocked by atropine, indicating that the carbachol-stimulated [(35)S]GTPgammaS binding was mediated by muscarinic cholinergic receptors. Together, the in vitro and in vivo data show for the first time in B6 mouse that cholinergic neurotransmission in the PRF can significantly alter ACh release in frontal association cortex, arousal from anesthesia, and respiratory rate.

M2 muscarinic receptors in pontine reticular formation of C57BL/6J mouse contribute to rapid eye movement sleep generation.

Microinjecting the acetylcholinesterase inhibitor neostigmine into the pontine reticular formation of C57BL/6J (B6) mouse causes a rapid eye movement (REM) sleep-like state. This finding is consistent with similar studies in cat and both sets of data indicate that the REM sleep-like state is caused by increasing levels of endogenous acetylcholine (ACh). Muscarinic cholinergic receptors have been localized to the pontine reticular formation of B6 mouse but no previous studies have examined which of the five muscarinic receptor subtypes participate in cholinergic REM sleep enhancement. This study examined the hypothesis that M2 receptors in pontine reticular formation of B6 mouse contribute to the REM sleep-like state caused by pontine reticular formation administration of neostigmine. B6 mice (n=13) were implanted with electrodes for recording states of sleep and wakefulness and with microinjection cannulae aimed for the pontine reticular formation. States of sleep and wakefulness were recorded for 4 h following pontine reticular formation injection of saline (control) or neostigmine. Experiments designed to gain insight into the muscarinic receptor subtypes mediating REM sleep enhancement involved pontine reticular formation administration of neostigmine after pertussis toxin, neostigmine after methoctramine, and neostigmine after pirenzepine. Pertussis toxin was used to block effects mediated by M2 and M4 receptors. Methoctramine was used to block M2 and M4 receptors, and pirenzepine was used to block M1 and M4 receptors. Pertussis toxin and methoctramine significantly decreased the neostigmine-induced REM sleep-like state. In contrast, pretreatment with pirenzepine did not significantly decrease the REM sleep-like state caused by neostigmine. These results support the interpretation that M2 receptors in the pontine reticular formation of B6 mouse contribute to the generation of REM sleep.

Acetylcholine release in the pontine reticular formation of C57BL/6J mouse is modulated by non-M1 muscarinic receptors.

Pontine acetylcholine (ACh) contributes to the regulation of electroencephalographic and behavioral arousal in all mammals so far investigated. The mouse is recognized as a powerful model for pharmacogenomics but the synaptic mechanisms regulating ACh release in mouse pontine reticular formation have not been characterized. Drug delivery by microdialysis was used in isoflurane-anesthetized C57BL/6J (B6) mice (n=33) to test the hypothesis that muscarinic autoreceptors modulate ACh release in the pontine reticular nucleus, oral part (PnO). Dialysis delivery of tetrodotoxin to the PnO significantly decreased ACh by 58% below control levels, confirming that measured ACh reflected neurotransmitter release. The muscarinic antagonist scopolamine increased ACh release in the PnO by 21% (3 nM), 48% (10 nM), 56% (30 nM), and 104% (100 nM). The muscarinic agonist bethanechol dialyzed into the PnO significantly decreased ACh release by 60% compared with control. Dialysis delivery of relatively subtype selective muscarinic antagonists to the PnO revealed the following order of potency for increasing ACh release: scopolamine (3 nM)>AF-DX 116 (100 nM)=pirenzepine (100 nM). These data support the conclusion that ACh release in PnO of B6 mouse is modulated by non-M1 muscarinic receptors.

Upper airway function during sleep and wakefulness: experimental studies on normal and anesthetized cats.

Normal (N = 6) and anesthetized (N = 70) cats were used to study the laryngeal abductors, the posterior cricoarytenoid (PCA) muscles, during sleep and wakefulness and to investigate sites within the brainstem that influenced PCA and diaphragmatic activity. The findings were as follows: 1. During wakefulness, PCA activity occurred throughout the respiratory cycle but was most intense during inspiration. Both expiratory and inspiratory PCA activity declined during sleep--the former more so than the latter. The decline in abductor activity was maximal in REM sleep. 2. Barbiturate anesthesia, according to the dosage, produced PCA activity patterns characteristic of either wakefulness or sleep. 3. The brainstem between A4 and P14 was mapped with stimulating electrodes. Rostral brainstem sites showed predominantly facilitatory effects of PCA activity; caudal sites produced predominantly blocking effects. 4. PCA facilitation consisted of (a) an increase in the duration of the PCA burst, (b) and increase in the discharge frequency of the PCA motor units, and (c) a recruitment of larger motor units. PCA blocking effects were the opposite, i.e., burst durations were shortened and motor units were decruited. 5. Facilitatory sites produced clear change in intensity and duration of PCA activity at stimulation intensities below those necessary to obtain changes in the intensity of diaphragmatic activity. 6. Stimulation of facilitatory sites during expiration caused phase switching to inspiration. In some cases, stimulation during inspiration caused phase switching to expiration. The results are discussed in terms of their implications for the obstructive apneas of sleep and in terms of the neural control of breathing.

M2 muscarinic receptor subtype in the feline medial pontine reticular formation modulates the amount of rapid eye movement sleep.

Rapid eye movement (REM) sleep is generated, in part, by activating muscarinic cholinergic receptors (mAChRs) in the medial pontine reticular formation (mPRF). Molecular cloning has identified five mAChR subtypes, and this study tested the hypothesis that the M2 subtype in the mPRF modulates the amount of REM sleep. This hypothesis cannot be tested directly, due to lack of subtype selective muscarinic agonists. However, the amount of REM sleep can be enhanced by mPRF microinjection of a muscarinic agonist, and the relative potencies of muscarinic antagonists to block the REM sleep enhancement can be determined. Two muscarinic antagonists, methoctramine and 4-DAMP, were studied. Six concentrations of each antagonist were microinjected into the mPRF of conscious cat 15 min prior to the agonist bethanechol. Nonlinear regression analysis was used to calculate the dose of antagonist that caused a 50% inhibition (ID50) of bethanechol-induced REM sleep. Bethanechol significantly increased (442%) the amount of time spent in REM sleep. Both methoctramine and 4-DAMP significantly blocked the bethanechol-induced REM sleep increase, with an ID50 of 1.8 microM and 0.6 microM, respectively. The ID50 ratio for methoctramine-to-4-DAMP (3.0) was similar to the affinity ratio of methoctramine-to-4-DAMP only at the M2 subtype (3.5), suggesting that the M2 subtype in the mPRF modulates the amount of REM sleep. This study also tested the null hypothesis that sleep-dependent respiratory depression evoked by mPRF cholinomimetics would not be antagonized by pretreatment of the mPRF with muscarinic antagonists. Neither methoctramine nor 4-DAMP antagonized the bethanechol-induced decrease in respiratory rate.

Cholinomimetics, but not morphine, increase antinociceptive behavior from pontine reticular regions regulating rapid-eye-movement sleep.

Sleep disruption is a significant problem associated with the subjective experience of pain. Both rapid-eye-movement (REM) sleep and nociception are modulated by cholinergic neurotransmission, and this study tested the hypothesis that antinociceptive behavior can be evoked cholinergically from medial pontine reticular formation (mPRF) regions known to regulate REM sleep. The foregoing hypothesis was investigated by quantifying the effect of mPRF drug administration on tail flick latency (TFL) of cat during polygraphically defined sleep/wake states. The mPRF was microinjected with 0.25 ml saline, carbachol (4.0 microg), neostigmine (6.7 microg), or morphine sulfate (14.7 microg), and TFL measures were obtained in response to radiant heat. During wakefulness TFL (% increase) was not increased by morphine or saline, but was significantly increased by mPRF administration of carbachol (42.4%) and neostigmine (35.2%). Cortical somatosensory potentials (SSEPs) were reliably evoked by tail stimulation before and after mPRF microinjections of carbachol. The results show for the first time that mPRF administration of cholinomimetics significantly increased TFL. During NREM sleep and REM sleep, TFL was significantly increased compared to waking TFL (110% and 321%, respectively). The finding of sleep-dependent alterations in TFL demonstrates that mPRF regions known to regulate REM sleep can modulate supraspinal cholinergic antinociceptive behavior.

G protein activation in rat ponto-mesencephalic nuclei is enhanced by combined treatment with a mu opioid and an adenosine A1 receptor agonist.

STUDY OBJECTIVES: Opioids delivered to the pons inhibit REM sleep, whereas pontine administration of adenosine enhances REM sleep. In other brain areas opioids and adenosine interact to produce antinociception. Adenosine A1 receptors and mu opioid receptors each activate Gi/Go proteins. This study tested the hypothesis that combined treatment with the adenosine A1 receptor agonist SPA and the mu opioid agonist DAMGO would enhance G protein activation to a greater level than produced by either agonist alone. G protein activation was quantified in seven brainstem regions regulating sleep and nociception. This study also tested the hypothesis that G protein activation caused by SPA would be concentration dependent and blocked by the adenosine A1 receptor antagonist DPCPX. DESIGN: Activation of G proteins was assessed autoradiographically by agonist stimulation of [35S]GTPgammaS binding in slide-mounted sections of rat brainstem. G protein activation was quantified in nCi/g tissue for pontine reticular formation, dorsal raphe, ventrolateral and dorsomedial periaqueductal gray, and laterodorsal and pedunculopontine tegmental nuclei. SETTING: N/A. PATIENTS OR PARTICIPANTS: N/A. MEASUREMENTS AND RESULTS: Combined treatment with SPA and DAMGO caused a partially additive increase in G protein activation that was significantly (p<0.01) greater than G protein activation caused by either agonist alone. Treatment with SPA alone caused a concentration dependent (p<0.001) increase in [35S]GTPgammaS binding that was blocked by DPCPX. CONCLUSION: Agonist activation of adenosine A1 receptors stimulates G proteins in brainstem nuclei regulating sleep and nociception. In these same nuclei, G protein activation by combined treatment with DAMGO and SPA was partially additive, suggesting that mu opioid and adenosine A1 receptors activate some common G protein pools.

Suprachiasmatic region of the human hypothalamus: homolog to the primate circadian pacemaker?

The suprachiasmatic nuclei (SCN) of the hypothalamus in mammals, including nonhuman primates, contain a key pacemaker of the circadian timing system. Examination of the histology of the anterior hypothalamus in human fetal, child, and adult brains indicates that there is a cluster of neurons which may be homologous to SCN. These neurons are more diffusely organized and laterally placed in human brains than is the SCN of nonhuman primates.

The effects of ethanol upon threshold and response rate for self-stimulation.

The hypothesis that ethanol would reduce the threshold for self-stimulation of the medial forebrain bundle was not supported. Ten rats, implanted with electrodes in the lateral hypothalamus, were shaped to bar press for electrical brain stimulation. The effects of 0.6, 0.9, and 1.2 g/kg ethanol injections upon threshold and response rate for self-stimulation were measured. The lowest dose had no effect upon self-stimulation threshold while 0.9 and 1.2 g/kg ethanol raised thresholds. Rate of bar pressing was increased by 0.6 g/kg ethanol but was not affected by higher doses. Results were discussed in terms of a postulated dual effect of ethanol upon a brain arousal system and upon a reward system.

Muscarinic cholinoceptive reticular mechanisms and parabrachial neuron discharge: a novel experimental approach.

This study examined the hypothesis that cholinergic receptor mechanisms within the gigantocellular tegmental field (FTG) of the medial pontine reticular formation can cause state-dependent changes in the firing rates of parabrachial nuclear complex (PBNC) neurons. Using intact, unanesthetized cats, long-term recordings were obtained from single PBNC neurons following FTG administration of cholinergic agonists and antagonists. We show that the state-dependent decrease in PBNC cell discharge reported in this study can be attributed to muscarinic mechanisms within the FTG. Our pharmacological blocking results further suggest that diminished PBNC discharge was mediated by non-M1 muscarinic receptors in the FTG. This study demonstrates the usefulness of combining long-term neuronal recordings in the PBNC with multiple pharmacological manipulations within the FTG.

Nitric oxide synthase inhibition decreases pontine acetylcholine release.

This study tested the hypothesis that inhibition of nitric oxide synthase (NOS) in the medial pontine reticular formation (mPRF) would cause decreased acetylcholine (ACh) release. Microdialysis of cat mPRF permitted measurement of ACh during states of wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep. ACh release during microdialysis with Ringers (control) was compared to ACh release during microdialysis with 10 mM NG-nitro-L-arginine (NLA). The NOS inhibitor NLA caused a significant reduction in ACh released from the mPRF during wakefulness, NREM sleep, and REM sleep. This reduction in mPRF ACh release elicited by NLA suggests that nitric oxide (NO) contributes to cholinergic neurotransmission in the pontine reticular formation.

Pontomedullary neurons transsynaptically labeled by laryngeal pseudorabies virus.

The activity of the laryngeal abductor, the posterior cricoarytenoid (PCA) muscles, is diminished with the loss of wakefulness. During sleep, PCA muscles become hypotonic, suggesting that neural mechanisms which control states of consciousness also contribute to state-dependent changes in upper airway patency. The medial pontine reticular formation (mPRF) has long been known to play a key role in sleep control. Microinjection of cholinergic agonists into the mPRF produces a rapid eye movement (REM) sleep-like state and PCA muscle hypotonia, but the neuroanatomical pathways mediating this hypotonia are not clear. Therefore, the present study used retrograde transsynaptic viral labeling to define multisynaptic pathways from the PCA muscles to the mPRF. Pseudorabies virus was injected into the laryngeal PCA muscles of anesthetized rats. The distribution of retrogradely labeled neurons was studied in the brain stem 4 days post-inoculation. The results show that brain stem areas known to be involved in sleep cycle control are part of a multisynaptic pathway providing input to the laryngeal PCA muscles.

Opioids activate G proteins in REM sleep-related brain stem nuclei of rat.

Mu opioid receptors within the pontine reticular formation contribute to opioid-induced rapid eye movement (REM) sleep inhibition. Mu receptors are coupled to guanine nucleotide binding (G) proteins and this study tested the hypothesis that the micro opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) would activate G proteins in rat brain stem nuclei known to regulate REM sleep. In vitro autoradiography of DAMGO-stimulated [35S]GTPgammaS binding showed that, compared with basal [35S]GTPgammaS binding, DAMGO significantly increased G protein activation in the nucleus pontis oralis (56.2%), nucleus pontis caudalis (57.3%), laterodorsal tegmental nucleus (75.8%), pedunculopontine tegmental nucleus (72.4%), nucleus locus coeruleus (77.2%) and dorsal raphe nucleus (73.4%). DAMGO stimulation of [35S]GTPgammaS binding in nuclei regulating REM sleep suggests that opioid-induced REM sleep inhibition involves activation of G proteins.

Vesicular acetylcholine transport inhibitor suppresses REM sleep.

The vesamicol-like compound (+/-)-4-aminobenzovesamicol (ABV) non-competitively inhibits vesicular packaging of acetylcholine (ACh) in presynaptic terminals. This study tested the hypothesis that microinjection of ABV into the medial pontine reticular formation (mPRF) of intact, unanesthetized cats would inhibit rapid eye movement (REM) sleep. Microinjection of ABV alone or before administration of the acetylcholinesterase inhibitor neostigmine was used to evaluate the effects of ABV on natural REM sleep and on the neostigmine-induced REM sleep-like state. ABV decreased (24.8%) REM sleep and significantly reduced (33.6%) the neostigmine-induced REM sleep-like state. The results show for the first time that REM sleep generation can be disrupted by blocking a synaptic vesicle protein that modulates ACh transport in localized regions of the mPRF.

Halothane decreases pontine acetylcholine release and increases EEG spindles.

This study tested the hypothesis that halothane anesthesia would cause decreased acetylcholine (ACh) release within the medial pontine reticular formation (mPRF). ACh was collected by microdialysis and measured by high pressure liquid chromatography during wakefulness and during halothane-induced anesthesia. The electroencephalogram (EEG) showed that spindles were a reliable indicator of anesthetic depth. There was a statistically significant disease in ACh release during halothane anesthesia compared with ACh release during wakefulness. Spindles always disappeared during noxious stimulation and during emergence from anesthesia when pontine ACh levels began to increase. These results are consistent with previous data concerning brain stem cholinergic influences on thalamocortical spindle generation, and suggest that similar mechanisms generate cortical spindles during natural sleep and halothane anesthesia.

Pontine acetylcholine release is regulated by muscarinic autoreceptors.

Acetylcholine (ACh) in the medial pontine reticular formation (mPRF) originates from the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei and contributes to generating rapid eye movement (REM) sleep. The mechanisms controlling mPRF ACh levels are incompletely understood. This study tested the hypothesis that mPRF ACh release is regulated, in part, by muscarinic autoreceptors. The mPRF of intact, halothane-anesthetized cats was dialyzed with Ringer's solution (control) or Ringer's containing the muscarinic antagonist scopolamine, Scopolamine caused a dose-dependent increase in mPRF ACh release and a concomitant decrease in the number of halothane-induced cortical EEG spindles. These data suggest that presynaptic muscarinic receptors, presumed to reside on cholinergic LDT/PPT terminals in the mPRF, play a role in regulating mPRF ACh release, REM sleep and EEG spindles.