Oral Pontine Reticular Formation Research Articles

GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: Implications for REM sleep control

The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABAA receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABAA receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABAA receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABAA receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABAA receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABAA receptor subunit protein γ2. This finding implicates a specific subtype and location of GABAA receptors in PnO of rat in the control of REM sleep.

The medial paralemniscal nucleus and its afferent neuronal connections in rat.

Previously, we described a cell group expressing tuberoinfundibular peptide of 39 residues (TIP39) in the lateral pontomesencephalic tegmentum, and referred to it as the medial paralemniscal nucleus (MPL). To identify this nucleus further in rat, we have now characterized the MPL cytoarchitectonically on coronal, sagittal, and horizontal serial sections. Neurons in the MPL have a columnar arrangement distinct from adjacent areas. The MPL is bordered by the intermediate nucleus of the lateral lemniscus nucleus laterally, the oral pontine reticular formation medially, and the rubrospinal tract ventrally, whereas the A7 noradrenergic cell group is located immediately mediocaudal to the MPL. TIP39-immunoreactive neurons are distributed throughout the cytoarchitectonically defined MPL and constitute 75% of its neurons as assessed by double labeling of TIP39 with a fluorescent Nissl dye or NeuN. Furthermore, we investigated the neuronal inputs to the MPL by using the retrograde tracer cholera toxin B subunit. The MPL has afferent neuronal connections distinct from adjacent brain regions including major inputs from the auditory cortex, medial part of the medial geniculate body, superior colliculus, external and dorsal cortices of the inferior colliculus, periolivary area, lateral preoptic area, hypothalamic ventromedial nucleus, lateral and dorsal hypothalamic areas, subparafascicular and posterior intralaminar thalamic nuclei, periaqueductal gray, and cuneiform nucleus. In addition, injection of the anterograde tracer biotinylated dextran amine into the auditory cortex and the hypothalamic ventromedial nucleus confirmed projections from these areas to the distinct MPL. The afferent neuronal connections of the MPL suggest its involvement in auditory and reproductive functions.

Carbachol induction of REM sleep in the rat is more effective at lights-out than lights-on

Long-lasting increases in REM sleep are induced in the rat following injection of small amounts of muscarinic receptor agonists into the caudal oral pontine reticular formation. By injecting carbachol at the beginning of the light period or beginning of the dark period, we sought to determine whether the muscarinic, REM sleep induction is influenced by the time of day it is initiated. We found that carbachol is more effective at increasing REM sleep when administered at the beginning of the dark in 87% of the cases. Of these cases, 43% showed evidence of a decreased potency of carbachol by a shift in the dose–response curve to the right. The lack of agreement in efficacy and potency to increase REM sleep supports a conclusion that alterations in local muscarinic receptors are not mediating the effect of time of day. REM sleep control mechanisms down stream of the muscarinic receptors may be the responsible factors.

Adenosine A1 receptors mediate inhibition of cAMP formation in vitro in the pontine, REM sleep induction zone

Microinjection of adenosine A1 receptor agonist or an inhibitor of adenylyl cyclase into the caudal, oral pontine reticular formation (PnOc) of the rat induces a long-lasting increase in REM sleep. Here, we report significant inhibition of forskolin-stimulated cAMP in dissected pontine tissue slices containing the PnOc incubated with the A1 receptor agonist, cyclohexaladenosine (10 −8 M). These data are consistent with adenosine A1 receptor agonist actions on REM sleep mediated through inhibition of cAMP.

Paradoxical REM sleep promoting and permitting neuronal networks.

Since its electrophysiological identification in the 1950's, the state of REMS or PS has been shown through multiple lines of evidence to be generated by neurons in the oral pontine tegmentum. The perpetration of this paradoxical state that combines cortical activation with the most profound behavioral sleep occurs through interplay between PS-promoting (On) and PS-permitting (Off) cell groups in the pons. Cholinergic cells in the LDTg and PPTg promote PS by initiating processes of both forebrain activation and peripheral muscle atonia. Bearing alpha1-adrenergic receptors, cholinergic cells, which likely project to the forebrain, are excited by NA and active during both W and PS (W/PS-On), when they promote cortical activation. Bearing alpha2-adrenergic receptors, other cholinergic cells, which likely project to the brainstem, are inhibited by NA and thus active selectively during PS (PS-On), when they promote muscle atonia. Noradrenergic, together with serotonergic, neurons, as PS-Off neurons, thus permit PS in part by lifting their inhibition upon the cholinergic PS-On cells. The noradrenergic/serotonergic neurons are inhibited in turn by local GABAergic PS-promoting neurons that may be excited by ACh. Other similarly modulated GABAergic neurons located through the brainstem reticular formation become active to participate in the inhibition of reticulo-spinal and raphe-spinal neurons as well as in the direct inhibition of motor neurons. In contrast, a select group of GABAergic neurons located in the oral pontine reticular formation and possibly inhibited by ACh turn off during PS. These GABAergic PS-permitting neurons release from inhibition the neighboring large glutamatergic neurons of the oral pontine reticular formation, which are likely concomitantly excited by ACh. In tandem with the cholinergic neurons, these glutamatergic reticular neurons propagate the paradoxical forebrain activation and peripheral inactivation that characterize PS.

Enhancement of rapid eye movement sleep in the rat by actions at A1 and A2a adenosine receptor subtypes with a differential sensitivity to atropine

The adenosine agonist cyclohexaladenosine injected into the medial pontine reticular formation of the rat induces a long-lasting increase in rapid eye movement sleep. To investigate the adenosine receptor-subtype(s) mediating this effect, the dose-response relationships for increasing rapid eye movement sleep by two highly selective adenosine receptor agonists were compared. Rats were surgically prepared for chronic sleep recording and bilateral guide cannulae were aimed at medial sites in the caudal, oral pontine reticular formation. Injections were made unilaterally in 60 nl volumes within 1 h after lights-on. The adenosine agonists used were A1-selective cyclohexaladenosine (10 −6−10 −4 M) and A2a-selective CGS 21680 (10 −7−10 −3 M). Each animal also received a series of three, paired-consecutive injections of the muscarinic receptor antagonist atropine (4×10 −3 M) followed by the lowest effective dose of each agonist or saline as control. The A2a receptor agonist, CGS 21680, was one order of magnitude more potent than the A1 receptor agonist, cyclohexaladenosine, in inducing rapid eye movement sleep increases. Preinjection of atropine at a dose that did not itself affect rapid eye movement sleep resulted in antagonism of CGS 21680, but not cyclohexaladenosine-induced rapid eye movement sleep. The differential sensitivity of these ligands to antagonism by atropine supports the conclusion that both A1 and A2a adenosine receptor subtypes in the reticular formation subserve agonist-induced rapid eye movement sleep and that they do so by independent mechanisms. The A2a mechanism requires the cholinergic system and may act through the increased release of acetylcholine. The A1 mechanism operates at a different locus possibly through an inhibition of GABA neurotransmission.

Rapid Eye Movement Sleep induction by microinjection of the GABA-A antagonist bicuculline into the dorsal subcoeruleus area of the rat

In cats, rapid eye movement sleep (REMS) can be induced rapidly and reliably by injections of the cholinergic agonist carbachol into the anterodorsal pontine tegmentum, also recognized as the perilocus coeruleus alpha, and designated the REMS Induction Zone (RIZ). In rats, the RIZ has been ascribed to a much larger and more ventral region within the entire oral pontine reticular formation. However, carbachol injections throughout this area produce only small, unreliable, and long latency REMS enhancements. The present study investigated whether REMS induction in the rat is possible by microinjection into the dorsal subcoeruleus nucleus (SubCD), a region with similarities to the cat RIZ. In freely moving unanaesthetized rats, microinjection of the GABA-A antagonist bicuculline significantly increased the amount and reduced the latency to REMS during a 2-h recording in the mid-light period. However, at effective doses, bicuculline usually also produced intermittent ipsiversive circling behavior that disrupted REMS maintenance. Attempts at eliminating this side-effect by: (i) coinjection of bicuculline with the NMDA antagonist, APV, (ii) lower bicuculline doses, or (iii) injection of the GABA-B antagonist, phaclofen, were unsuccessful. Other drugs injected into this area did not induce REMS; these included carbachol, the acetylcholinesterase inhibitor neostigmine, the glutamate agonist kainate, and vasopressin. In the rat, the SubCD is a highly sensitive region for both REMS induction and locomotor effects.

Infusion of adenylyl cyclase inhibitor SQ22,536 into the medial pontine reticular formation of rats enhances rapid eye movement sleep

Microinjection of cholinergic and adenosinergic agonists into the medial pontine reticular formation of rats produces long lasting increases in the time spent in rapid eye movement sleep. Several G-protein-coupled muscarinic and adenosinergic receptors share a common action of inhibition of adenylyl cyclase and inhibition of cyclic adenosine monophosphate. Inhibition of cyclic adenosine monophosphate has been implicated in the mechanism of rapid eye movement sleep induction in the cat. We sought to determine whether a direct inhibitor of adenylyl cyclase microinjected into the rat reticular formation at sites where muscarinic and adenosinergic agonists are effective in producing long lasting elevations in rapid eye movement sleep also result in similar effects on the sleep/wake cycle. The caudal, oral pontine reticular formation was unilaterally infused with 60 nl volumes of carbachol (0.1–1.1 mM) and N 6-cyclohexyladenosine (0.1 mM) each within 1 h of lights on. Sites effective for significantly elevating rapid eye movement sleep for the 8 h following microinjection of both receptor agonists were additionally injected with the adenylyl cyclase inhibitor, SQ22,536 (0.1 M). Pontine injections of SQ22,536 resulted in significant mean increases in rapid eye movement sleep time and rapid eye movement sleep period frequency at all of these sites. As with the receptor agonists, SQ22,536 did not alter latency to rapid eye movement sleep onset. Rapid eye movement sleep amounts were observed to be significantly elevated by SQ22,536 at two days, but not at four days, following a single microinjection. These data implicate inhibition of cyclic adenosine monophosphate in the pons of the rat as a mechanism involved in the long-term modulation of rapid eye movement sleep. This mechanism may underlie the homeostatic regulation exhibited by this sleep-state.

Trigemino-reticulo-facial and trigemino-reticulo-hypoglossal pathways in the rat.

This study was undertaken to identify premotor neurons in the pontomedullary reticular formation serving as relay neurons between the sensory trigeminal complex and the motor nuclei of the VIIth and XIIth nerves. Trigeminoreticular projections were first investigated after injections of anterogradely transported tracers (biotinylated dextran amine, biocytin) into single subdivisions of the sensory trigeminal complex. The results show that the trigeminoreticular projections were abundant from the pars interpolaris (5i) and caudalis (5c) and moderate from pars oralis (5o) of the spinal trigeminal nucleus. Injections into the 5i and 5c produce dense anterograde labeling (1) in the dorsal medullary reticular field; (2) in the parvocellular reticular field, medially adjacent to the 5i; and (3) more rostral in the region dorsal and lateral to the superior olivary nucleus. Some labeled terminals were also found in the intermediate reticular field, whereas only light anterograde labeling was observed in the gigantocellular and oral pontine reticular formation. The 5o sends fibers and terminals throughout the whole reticular formation, with no clear preferential projections within a particular field. Only light projections originated from the principal nucleus (5P). In a second series of experiments, we examined whether premotor neurons in the reticular formation are afferented by trigeminal fibers. Double labeling was performed by injection of an anterograde tracer in the 5i and 5c and retrograde tracer (gold-horseradish peroxidase complex) into the VII or the XII motor nucleus on the same side. Retrogradely labeled neurons in contact with anterogradely labeled boutons were found throughout the reticular formation with predominance in the parvocellular and intermediate reticular fields. These experiments demonstrate the existence of trigeminal disynaptic influences, via reticular neurons of the pontomedullary reticular formation, in the control of orofacial motor behaviors.

Enhancement of rapid eye movement sleep in the rat by cholinergic and adenosinergic agonists infused into the pontine reticular formation

The cholinergic agonist carbachol (1.1 mM) and the adenosinergic agonist cyclohexyladenosine (0.1 mM) were microinjected (60 nl) into the region of the caudal, oral pontine reticular formation of the rat. Local intracerebral infusion of each receptor agonist resulted in significant, long-lasting (at least 8 h) elevations in rapid eye movement sleep without reduction in latency to onset. The effects of carbachol were reduced by the muscarinic receptor antagonist atropine, while those of cyclohexyladenosine were reduced by the adenosinergic receptor antagonist 8-cyclopentyltheophylline. Atropine failed to antagonize the long-term induction of rapid eye movement sleep following cyclohexyladenosine, but did appear to suppress increases in the first 2 h. Similarity of effects on sleep parameters and the lack of additivity when injected consecutively are consistent with these agonist ligands targeting the same cellular mechanisms through their respective receptors. These findings suggest that transitory increases in the pons of either acetylcholine or adenosine may underlie long-lasting elevations in the amount of rapid eye movement sleep. Adenosine may play a role in the increased rapid eye movement sleep following prolonged wakefulness, as well as following conditions of stress and learning.

Premotor neurons for trigeminal motor nucleus neurons innervating the jaw‐closing and jaw‐opening muscles: Differential differential in the lower brainstem of the rat

The distribution of premotor neurons for trigeminal motor nucleus neurons innervating the jaw-closing and jaw-opening muscles was examined in the lower brainstem of the rat by using retrograde and anterograde labeling techniques. First, Fluorogold, a fluorescent retrograde tracer, was injected into the dorsolateral or ventromedial division of the trigeminal motor nucleus, each of which contains motoneurons innervating the jaw-closing or jaw-opening muscles, respectively. Second, Phaseolus vulgaris-leucoagglutinin, an anterograde tracer, was injected into each of the lower brainstem sites, where clusters of retrogradely labeled premotor neurons had been seen in the first set of experiments. Third, after injection of the anterograde tracer into a lower brainstem site, followed by injection of the retrograde tracer cholera toxin B subunit into a masticatory muscle, termination of anterogradely labeled axons onto retrogradely labeled motoneurons was confirmed with the aid of a confocal laser-scanning microscope. It was found that the premotor neurons distributed in the mesencephalic trigeminal nucleus, medial part of the parabrachial region, supratrigeminal region, and dorsal parts of the principal sensory, oral spinal and interpolar spinal trigeminal nuclei project preferentially to the dorsolateral division of the trigeminal motor nucleus, whereas those in the lateral part of the parabrachial region, intermediate parts of the principal sensory, oral spinal and interpolar spinal trigeminal nuclei, and alpha part of the gigantocellular reticular nucleus project preferentially to the ventromedial division of the trigeminal motor nucleus. The dorsal and lateral parts of the medullary reticular formation and the medullary raphe nuclei contain premotor neurons of both types. Group k motoneurons, a cluster of trigeminal motoneurons that innervate the tensor tympani muscle, receive projection fibers predominantly from the dorsolateral part of the oral pontine reticular formation.

The efferent projections of the rat lateral habenular nucleus revealed by the PHA-L anterograde tracing method

The efferent connections of the rat lateral habenular nucleus (LHb) were demonstrated using anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). Following PHA-L injections into the LHb, neuronal somata located in the lateral two thirds of the LHb were labeled with PHA-L. Individual axonal fibers and terminal specializations were clearly visible. This permitted detailed mapping of both efferent fiber pathways and terminal distributions. Previous reports on fiber pathways were substantially confirmed and several new findings were revealed. (1) Major rostrally oriented fibers enter the medial forebrain bundle via 3 routes which initially branch from the fasciculus retroflexus: the mediodorsal thalamic nucleus and ventromedial thalamic nucleus; the zona incerta and fields of Forel; and the ventral tegmental area of Tsai. (2) A major decussation to the contralateral thalamic nuclei occurs in the central medial thalamic nucleus. (3) Caudally directed fibers follow two courses: one to the deep mesencephalic nucleus, central grey, dorsal raphe nucleus and the deep layers of the superior colliculus; and the other to the median raphe nucleus, oral pontine reticular formation and raphe pontis nucleus. The present results offer more detailed information concerning the dorsal diencephalic system.

Retrograde labeling of neurons in the brain stem following injections of [ 3H]choline into the rat spinal cord

In an attempt to identify cholinergic neurons in the brain stem which project to the spinal cord, [ 3H]choline (100,20, 10, 5 or 1 μ Ci) was injected into the upper cervical spinal cord in 55 rats. The animals were killed 20 h later and the brains processed for autoradiography of diffusible substances. At all doses of [ 3H]choline, cells were consistently, retrogradely labeled in the medial medullary reticular formation, the lateral vestibular nucleus, the dorsolateral pontine tegmentum and the red nucleus. The retrogradely labeled cells were found to be moderately to darkly stained for acetylcholinesterase. Injection of [ 3H]noradrenaline (50 μ Ci) into the upper cervical spinal cord resulted in retrograde labeling of cells in the locus coeruleus, subcoeruleus and the ventrolateral pontine tegmentum, that correspond in position to the neurons of the A6, A7 and A5 catecholamine cell groups, respectively. Injection of [ 3H]serotonin (20 μ Ci) into the upper cervical spinal cord was associated with retrograde labeling of cells in the raphe pallidus, obscurus and magnus nuclei that correspond in position to those of the B1, B2 and B3 serotonin cell groups, respectively. Injection of True Blue into the upper cervical spinal cord was followed by retrograde labeling of a large number of cells located in the areas where cells were retrogradely labeled by [ 3H]choline, [ 3H]noradrenaline and [ 3H]serotonin, and additionally, in the solitary tract nucleus, the lateral, parvicellular medullary reticular formation, the caudal and oral pontine reticular formation, the mesencephalic reticular formation and the superior colliculus. These results indicate that from the cervical spinal cord, [ 3H]choline selectively retrogradely labels a certain population of non-monaminergic, acetylcholinesterase-positive cells localized in the medial medullary, and secondarily the dorsolateral pontine, reticular formation, the lateral vestibular nucleus, and the red nucleus.