GABAergic structures in the ventral part of the oral pontine reticular nucleus: An ultrastructural immunogold analysis

GABA mediates inhibitory effects in neurons of the ventral part of the oral pontine reticular nucleus (vRPO). Evidence increasingly suggests that GABA plays an important role in the modulation of rapid eye movement (REM) sleep generation in the cat vRPO. Here, we investigate the anatomical substrate of this modulation using GABA immunocytochemistry. Immunoperoxidase labeling revealed a few small GABA-immunoreactive cell bodies scattered throughout the vRPO. The numerical densities of all vRPO synapses and the GABA-immunoreactive synapses were estimated, at the electron microscopical level, by using a combination of the physical disector and the post-embedding immunogold techniques. We estimated that 30% of all vRPO synaptic terminals were immunoreactive to GABA. Our findings support the hypothesis that vRPO neuron activity is significantly controlled by inhibitory GABAergic terminals that directly target somata and the different parts of the dendritic tree, including distal regions. GABAergic input could inhibit vRPO REM sleep-inducing neurons during other states of the sleep-wakefulness cycle such as wakefulness or non-REM sleep.     

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase–wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus and peri-, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.     

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.     

Role of the thalamic reticular nucleus in relations between the mesencephalic reticular formation and lateral geniculate body

Brain Research Institute, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. N. Smol'yannikov.) Translated from Byulleten Éksperimental'noi Biologii i Meditsiny, Vol. 106, No. 12, pp. 643–645, December, 1988.     

GABAergic and pallidal terminals in the thalamic reticular nucleus of squirrel monkeys

The ultrastructure of synaptic terminals from the external segment of the globus pallidus and of other synaptic terminals positive for gamma-aminobutyric acid (GABA) was examined in the thalamic reticular nucleus (TRN) of squirrel monkeys. Two GABA-positive terminals types were commonly encountered within the TRN neuropil. The most common type of GABAergic terminals (F terminals) are filled with dispersed pleomorphic synaptic vesicles and clusters of mitochondria. These terminals establish multiple symmetric synapses upon the somata and dendrites of TRN neurons. The external pallidal terminals, labeled with WGA-HRP, arise from thinly myelinated axons and correspond to the medium to large F terminals. A less prevalent population of smaller GABAergic synaptic profiles was also identified. The synaptic profiles in this second group contain considerably fewer pleomorphic synaptic vesicles in small irregular clusters and fewer mitochondria, establish symmetric synapses, are postsynaptic to other axonal terminals, are presynaptic to dendrites and soma, and are unlabeled following pallidal injections of WGA-HRP.     

Glycine inhibits startle-mediating neurons in the caudal pontine reticular formation but is not involved in synaptic depression underlying short-term habituation of startle

The mammalian startle response is controlled by glycine inhibition in the spinal cord. Evidence for additional glycine inhibition on the level of the brainstem, namely in the caudal pontine reticular nucleus (PnC), is controversial. Startle mediating PnC neurons receive fast input from sensory pathways and project to cranial and spinal motoneurons. Synaptic depression in the sensory synapses in the PnC has been indicated as underlying mechanism of short-term habituation of startle. We here performed patch-clamp recordings of PnC giant neurons in rat brain slices to test the hypothesis that the activation of glycine receptors inhibits PnC neurons and that this inhibition is involved in synaptic depression in the PnC.Glycine strongly inhibited PnC neuron activity and synaptic signalling, indicating that functional glycine receptors mediate a powerful inhibition of PnC neurons over a wide range of glycine concentrations. Strychnine reversed all glycine effects, but had no effect on PnC neurons itself. Thus, we found no evidence for a tonic glycine inhibition or for glycine activation within the primary startle pathway indicating that baseline startle reactions are unlikely to be controlled by glycine in the PnC. Most importantly, synaptic depression underlying short-term habituation was not affected by glycine or strychnine.     

Ultrastructural study of vestibular and reticular projections to the abducens nucleus

Summary The origin of the synaptic boutons in the abducens nucleus was studied following lesions of the contralateral medial vestibular nucleus, the ipsilateral paramedian pontine reticular formation and the contralateral dorsomedial part of the reticular formation caudal to the abducens nucleus.Lesions in the rostral part of the contralateral medial vestibular nucleus resulted in degeneration of boutons located mainly on dendritic processes. On the other hand, lesions in both ipsilateral and contralateral reticular formations provoked degenerating terminals on the somata of the abducens neurones and on proximal dendrites in the abducens nucleus beneath the genu of the facial nerve.This work was supported by INSERM (U6), CNRS (GR 45), and grant DGRST 78.7.3017     

Supramedullary projections to the dorsal and ventral divisions of the paramedian reticular nucleus in the cat

Summary Injections of combined lectin-conjugated and unconjugated horseradish peroxidase were made in the dorsal (d) and ventral (v) divisions of the paramedian reticular nucleus (PRN), a precerebellar relay nucleus, of the cat. The origins of supramedullary afferent projections to the PRN were identified in the pons, midbrain and cerebral cortex using the transverse plane of section. The data indicate a segregation of input from a number of sites to the dPRN and vPRN. The interstitial nucleus of Cajal projects bilaterally to the dPRN and predominantly to the ipsilateral side. The vPRN receives only a unilateral projection from the ipsilateral nucleus of Cajal. Major afferent projections to the vPRN arise from the ipsilateral nucleus of Darkschewitsch and the intermediate layer of the contralateral superior colliculus. Neither of these sites projected to the dPRN. The raphe nuclei and medial reticular formation of the pons and midbrain contribute a moderate input to both divisions of the PRN. A moderate bilateral cerebral cortical projection arises from the first somatomotor area (SMI). The ventral coronal and anterior sigmoid gyri project mainly to the dPRN and vPRN respectively. Smaller afferent projections arise from the posterior sigmoid gyri and area 6 of Hassler and Mühs-Clement (1964) in the medial wall of the anterior sigmoid gyrus. Inputs from the accessory oculomotor nuclei, tectal regions and the first somatomotor cortex suggest a role in postural control for the PRN which may underlie its involvement in mediating orthostatic reflexes.Abbreviations 3N oculomotor nerve - 5ME mesencephalic nucleus (trigeminal) - 5MN motor nucleus (trigeminal) - 5PN sensory nucleus, parvocellular division (trigeminal) - 5SM sensory nucleus, magnocellular division (trigeminal) - 12M hypoglossal nucleus - 12N hypoglossal nerve - AQ aqueduct - BC brachium conjunctivum - BP brachium pontis - CAE nucleus caeruleus - Cl inferior central nucleus (raphe) - CM centromedian nucleus - CNF cuneiform nucleus - CS superior central nucleus (raphe) - D nucleus of Darkschewitsch - DRM dorsal nucleus of the raphe (median division) - EW Edinger-Westphal nucleus - FTC central tegmental field - FTG gigantocellular tegmental field - FTP paralemniscal tegmental field - ICA interstitial nucleus of Cajal - ICC inferior colliculus (central nucleus) - INC nucleus incertus - INT nucleus intercalatus - ION inferior olivary nucleus - LLV ventral nucleus of lateral lemniscus - LP lateral posterior complex of thalamus - MGN medial geniculate nucleus - MLF medial longitudinal fasciculus - TN nucleus of optic tract - P pyramidal tract - PCN nucleus of posterior commissure - PF parafascicular nucleus - PH nucleus praepositus hypogloss - PRN paramedian reticular nucleus (a — accessory division; d — dorsal division; v — ventral division) - PUL pulvinar - SCD superior colliculus (deep layer) - SNC substantia nigra (compact division) - SON superior olivary nucleus - RM red nucleus (magnocellular) - RR retrorubral nucleus - TB trapezoid body - TDP dorsal tegmental nucleus (pericentral division) - TRC tegmental reticular nucleus (central division) - TV ventral tegmental nucleus - V3 third ventricle - V4 fourth ventricle - VB ventrobasal complex of thalamus - VIN inferior vestibular nucleus - VSN superior vestibular nucleus - ZI zona incerta Supported by the Medical Research Council of Canada     

Investigation of the connection between the substantia nigra and the medullary reticular formation in the rat

Unfixed, slide-mounted tissue sections from the rat forebrain have been incubated in the presence of 20 and 100 nM [3H]kainic acid ([3H]KA). For the last 2 min of incubation, 10 micrometers unlabelled KA was added to displace [3H]KA from binding sites with high on-off rate. Washed and dried slices were exposed on [3H]Ultrofilm for 178 days. Our results confirm the high density of KA receptors in the terminal field of the hippocampal mossy fibre system which is shown to be due to receptors with slow dissociation rate. Furthermore, the concentration dependency of the specific labelling, as quantified by microdensitometry, allows some suggestions concerning the local binding affinities involved.     

Enhancement of the acoustic startle response by stimulation of an excitatory pathway from the central amygdala/basal nucleus of Meynert to the pontine reticular formation

The acoustic startle response (ASR) is a simple motor reaction to intense and sudden acoustic stimuli. The neural pathway underlying the ASR in rats is already fairly well understood. As the ASR is subject to a variety of modulations, this reaction can serve as a model for vertebrate neuroethologists to investigate the neural mechanisms mediating sensorimotor transfer and their extrinsic modulation. We report here on experiments in rats which were undertaken in order to investigate the neural mechanisms underlying the enhancement of the ASR. An increased amplitude of the ASR can be observed during states of conditioned and unconditioned fear. By employing neuroanatomical tract tracing methods, we describe a pathway from neurons of the medial division of the central amygdaloid nucleus (cA) and the basal nucleus of Meynert (B) to the caudal pontine reticular nucleus (PnC), an important relay station in the acoustic startle pathway. Extracellular recordings from acoustically responsive neurons in the PnC showed that electrical stimulation of the cA/B facilitates the tone evoked response of these neurons. Behavioural tests following chemical stimulation of the cA/B with NMDA (N-methyl-d-aspartate) in awake rats indicated that activation of this pathway increases the ASR. The lack of sufficient spatial resolution of our stimulation techniques did not allow us to differentiate the relative contributions of the cA and the B to this effect. As the amygdaloid complex has been implicated in emotional behaviour, particularly in the mediation of fear, these findings substantiate the concept that the amygdaloid complex plays a key role for the enhancement of the ASR by conditioned and unconditioned fear.     

Effects of bilateral microinjections of ibotenic acid in the thalamic reticular nucleus on delta oscillations and sleep in freely-moving rats

The thalamic reticular nucleus (NRT) consists of a large pool of GABAergic neurons located on each side on the anterior, lateral, and ventral surfaces of the dorsal thalamus. The NRT is divided up into sectors. The aim of this study was to investigate the effects of bilateral lesions of the NRT on sleep and sleep oscillations. Only the results concerning delta oscillations will be reported here. As a first step we produced stereotaxically placed electrolytic lesions. The rats presented continuous circling behavior with electroencephalographic (EEG) theta and delta activity and subsequent sudden death. To avoid disruption of the bundles of fibers that pass through the NRT to and from the cerebral cortex, we used the excitotoxic ibotenic acid. Given its high toxicity, we concentrated on the rostral pole of the NRT, which is believed to have powerful effects on the synchronization of oscillatory activity during sleep. Immediately after surgery, the rats fell into a deep sleep during which there was an increase in EEG slow-wave activity and no spindles. On postoperative day 2, corresponding to the destruction period, the sleep/wake cycle partially recovered, but NREM sleep was quantitatively diminished and showed abnormalities (increased latency to sleep onset, sleep fragmentation, gradual elimination of the delta rhythm). It is concluded that the rostral pole of the NRT contributes to normal and pathological EEG synchronization and the organization of sleep in rats.     

Microinjection of 70-kDal heat shock protein into the oral reticular nucleus of the pons suppresses rapid eye movement sleep in pigeons

Previous studies have demonstrated that increases in the duration of slow-wave sleep and decreases in somatovisceral measures in response to microinjections of 70-kDal heat shock protein (Hsp70) into the third ventricle in pigeons may be due to activation of GABA_A receptors in the preoptic area of the hypothalamus. With the aim of identifying the transmitter mechanisms whose activation is temporally (2–3 h) linked with suppression of rapid eye movement sleep, the present studies were based on injection of Hsp70 into the oral reticular pontine nucleus (nucleus reticularis pontis oralis, NRPO), whose cholinergic neurons are critical for generating rapid eye movement sleep. Hsp70 was found to induce earlier (within the first 2 h) decreases in the number of episodes and the total duration of rapid eye movement sleep, with decreases in electroencephalogram (EEG) spectral power in the range 9–14 Hz, the level of muscle contractile activity, and brain temperature. It is hypothesized that the effects of Hsp70 are mediated by activation of GABA_A receptors in the NRPO, evoking suppression of the cholinergic mechanisms initiating rapid eye movement sleep. The increase in the total duration of slow-wave sleep occurring with a long latent period (8–12 h after injection of Hsp70 into the NRPO) may be due to the influence of Hsp70 on the population of neurons responsible for maintaining slow-wave sleep outside the NRPO. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 3, pp. 301–311, March, 2008.     

Postnatal development of spontaneous neuronal discharges in the pontine reticular formation of free-moving rats during sleep and wakefulness

Summary A developmental study has been made of spontaneous neuronal activity within the pontine reticular formation (giant cell field: FTG) of the rat between one week and one month after birth. Through day 14, the recorded FTG neurons discharged more frequently during quiet sleep (QS) than was generally true in older animals. In addition, they were active to the same extent during active-sleep (AS) as during waking-with-movements (AW). In contrast, most of the cells recorded from day 15 on were considerably more active during AS and AW, relative to the QS level, than had hitherto been the case. This new class of neurons, in turn, fell into two sub-groups, one of which was most active during AW while the other was more active during AS. Clomipramine selectively suppressed AS along with the neuronal activity patterns associated with it, and in many cases the QS firing level was even lower than it had been prior to the injection. It is concluded that FTG unit activity is an excellent monitor for controlling the effectiveness of experimental manipulations of AS but is probably not involved in its generation.     

Increased extracellular 5-HT but no change in sleep after perfusion of a 5-HT1A antagonist into the dorsal raphe nucleus of rats

Aim: The 5‐HT_1A receptor antagonist 4‐Iodo‐N‐[2‐[4‐(methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐2‐pyridinyl‐benzamide hydrochloride (p‐MPPI) (10 μm ) was perfused into the dorsal raphe nucleus (DRN) to study simultaneously the effects of the drug on the DRN and frontal cortex extracellular serotonin (5‐hydroxytryptamine, 5‐HT) levels and concurring behavioural states. Methods: Waking, slow wave sleep and rapid eye movement sleep were determined by polygraphic recordings during microdialysis perfusion and extracellular sample collection. The samples were analysed by microbore high‐performance liquid chromatography coupled with electrochemical detection for analysis of 5‐HT. Results: p‐MPPI perfusion into the DRN (n = 6) produced a sixfold 5‐HT increase in the DRN during all behavioural states. The increased 5‐HT level was most likely related to the blockage of 5‐HT_1A receptors in the DRN by p‐MPPI. No significant effect was seen on sleep. Conclusion: Despite the dramatic increase in DRN extracellular 5‐HT produced by p‐MPPI, only a transient and nonsignificant effect on sleep was recorded. It is suggested that the usual coupling between 5‐HT level and behavioural state may be lost when an excessive serotonergic output is pharmacologically achieved.     

Ultrastructural relationships between serotonin and dopamine neurons in the rat arcuate nucleus and medial zona incerta: a combined radioautographic and immunocytochemical study

Combined radioautographic and immunocytochemical detection of [3H]serotonin-labeled axon terminals and tyrosine hydroxylase-immunoreactive processes in the same thin sections allowed for electron microscopic demonstration of direct appositions between serotoninergic axonal varicosities and dopaminergic nerve cell bodies and/or dendrites in the anterior part of the arcuate nucleus and in the medial zona incerta. Although no junctional specializations were apparent at the sites of contacts, it is proposed that the observed appositions may represent a serotonin input onto tubero-infundibular and incerto-hypothalamic dopaminergic neurons. This innervation could account for some of the central neuroendocrine effects of serotonin, particularly its regulatory role on prolactin and gonadotropin secretion.     

Cytoarchitecture and cytology of the lateral reticular nucleus in the rat

Summary The cytoarchitecture and cytology of the rat lateral reticular nucleus (LRN) was studied in serial sections of paraffin embedded tissue stained with cresyl violet. Cell outlines and nuclear outlines were drawn in the transverse plane and the nucleus was serially reconstructed. The LRN in the rat begins at a point just caudal to the caudal limit of the inferior olivary nucleus and extends to the mid-olivary level. The nucleus can be subdivided into a predominantly small-celled parvocellular division ventrally, a predominantly large-celled magnocellular division dorsomedially, and a subtrigeminal division dorsolaterally containing predominantly medium-sized cells. At rostral levels the nucleus comprises two parts, a medial principal portion and a lateral subtrigeminal division. Measurements of neuronal diameters yielded size distributions which confirmed the predominance of large cells in the magnocellular division, small cells in the parvocellular division and medium-sized cells in the subtrigeminal division. The neurons display multipolar, triangular, piriform and fusiform somata. All types show a range in size from small to large. The larger cells have abundant Nissl bodies which are coarse in nature and voluminous cytoplasm. The smaller cells have poorly developed Nissl bodies and scant amounts of cytoplasm.     

The lateral reticular nucleus in the cat—II. Effects of lateral reticular lesions on posture and reflex movements

Unilateral lesion of the lateral reticular nucleus produced a postural asymmetry characterized by ipsilateral hypertonia and contralateral hypotonia of the limb extensor muscles. Soon after the operation the cat was unable to stand or walk, and it laid on one side. Within 3 days, it began to walk, but it often deviated to its contralateral side, 'frequently falling in this direction. Some compensation for both postural and motor deficits occurred in chronic preparations maintained up to 154 days after the lesion. The postural asymmetry was reversed by the following operations: (1) section of the ipsilateral VIIIth nerve, (2) electrolytic lesion of the ipsilateral Deiters' nucleus, or (3) ablation of the contralateral vermal cortex of the cerebellar anterior lobe.The lateral reticular nucleus lesion also produced, in the ipsilateral limbs, a transient loss of the proprioceptive placing reaction and a persistent deficit of the tactile placing reflex. These effects on the ipsilateral side were not reversed by the procedures described above.All of these behaviors depended on selective destruction of the lateral reticular nucleus and were not due to damage to the nearby main reticular formation or ascending and descending pathways. Moreover, the postural changes did not involve mesencephalic or higher mechanisms, since they were still observed after decerebration. The forelimbs were affected primarily by lesions involving the dorsomedial, magnocellular part of the lateral reticular nucleus, whereas the hindlimbs also were affected by lesions including the ventrolateral, parvicellular part of the lateral reticular nucleus.The postural asymmetry is attributed to interruption of the crossed spinoreticulocerebellar pathway, acting on the vermal cortex of the anterior lobe and the fastigial nucleus, while the ipsilateral loss of the placing reaction is attributed to interruption of the uncrossed spinoreticulocerebellar pathway acting on the intermediate cortex of the anterior lobe and the interpositus nucleus. The lateral reticular nucleus appears, therefore, to be composed of two, more or less independent, parts, a crossed one being related to major changes in postural tone, equilibrium and locomotion of the entire body, and an uncrossed one restricted to discrete movements of the ipsilateral limbs.     

Visual signals in the dorsolateral pontine nucleus of the alert monkey: Their relationship to smooth-pursuit eye movements

Summary The visual properties of 77 dorsolateral pontine nucleus (DLPN) cells were studied in two alert monkeys. In 41 cells, presentation of a moving random dot background pattern, while the monkeys fixated a stationary spot, elicited modulations in discharge rate that were related either to (i) the velocity of background motion in a specific direction or to (ii) only the direction of background movement. Thirty-six DLPN cells exhibited responses to small, 0.6–1.7 deg, visual stimuli. Nine such cells exhibited non-direction selective receptive fields that were eccentric from the fovea. During fixation of a stationary bluish spot, the visual responses of 27 DLPN cells to movement of a small, white test spot were characterized by two components: (1) as the test spot crossed the fovea in a specific direction, transient velocity-related increases in discharge rate occurred and (2) a maintained, smaller increase in activity was observed for the duration of test spot movement in the preferred direction. This DLPN activity associated with small visual stimuli was also observed during smooth-pursuit eye movements when, due to imperfect tracking, retinal image motion of the target produced slip in the same direction. These preliminary results suggest that the DLPN could supply the smooth-pursuit system with signals concerning the direction and velocity of target image motion on the retina.This study was supported by NSF Grant BNS-8107111, NIH Grant R01 EY04552-01, and the Smith-Kettlewell Eye Research Foundation Dedication. This paper is dedicated to Dr. Kitsuya Iwama, Emeritus Professor of Osaka University Medical School, on his retirement. The first author is grateful for the inspiration and guidance that Dr. Iwama provided during the early part of the author's education in neurophysiology.