Neurology and psychiatry: waking up to opportunities of sleep. : State of the art and clinical/research priorities for the next decade.

In recent years, evidence has emerged for a bidirectional relationship between sleep and neurological and psychiatric disorders. First, sleep-wake disorders (SWDs) are very common and may be the first/main manifestation of underlying neurological and psychiatric disorders. Secondly, SWDs may represent an independent risk factor for neuropsychiatric morbidities. Thirdly, sleep-wake function (SWF) may influence the course and outcome of neurological and psychiatric disorders. This review summarizes the most important research and clinical findings in the fields of neuropsychiatric sleep and circadian research and medicine, and discusses the promise they bear for the next decade. The findings herein summarize discussions conducted in a workshop with 26 European experts in these fields, and formulate specific future priorities for clinical practice and translational research. More generally, the conclusion emerging from this workshop is the recognition of a tremendous opportunity offered by our knowledge of SWF and SWDs that has unfortunately not yet entered as an important key factor in clinical practice, particularly in Europe. Strengthening pre-graduate and postgraduate teaching, creating academic multidisciplinary sleep-wake centres and simplifying diagnostic approaches of SWDs coupled with targeted treatment strategies yield enormous clinical benefits for these diseases.

Animal models of REM dysfunctions: what they tell us about the cause of narcolepsy and RBD?

Rapid eye movement 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 subpopulation 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 premotor-neurons 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 direct or indirect pathway activated during positive emotion from the central amygdala to the SLD PS-on neurons would induce such activation. In normal conditions, the activation of SLD neurons 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.

Rapid eye movement sleep behavior disorder: devising controlled active treatment studies for symptomatic and neuroprotective therapy--a consensus statement from the International Rapid Eye Movement Sleep Behavior Disorder Study Group.

OBJECTIVES: We aimed to provide a consensus statement by the International Rapid Eye Movement Sleep Behavior Disorder Study Group (IRBD-SG) on devising controlled active treatment studies in rapid eye movement sleep behavior disorder (RBD) and devising studies of neuroprotection against Parkinson disease (PD) and related neurodegeneration in RBD. METHODS: The consensus statement was generated during the fourth IRBD-SG symposium in Marburg, Germany in 2011. The IRBD-SG identified essential methodologic components for a randomized trial in RBD, including potential screening and diagnostic criteria, inclusion and exclusion criteria, primary and secondary outcomes for symptomatic therapy trials (particularly for melatonin and clonazepam), and potential primary and secondary outcomes for eventual trials with disease-modifying and neuroprotective agents. The latter trials are considered urgent, given the high conversion rate from idiopathic RBD (iRBD) to Parkinsonian disorders (i.e., PD, dementia with Lewy bodies [DLB], multiple system atrophy [MSA]). RESULTS: Six inclusion criteria were identified for symptomatic therapy and neuroprotective trials: (1) diagnosis of RBD needs to satisfy the International Classification of Sleep Disorders, second edition, (ICSD-2) criteria; (2) minimum frequency of RBD episodes should preferably be ⩾2 times weekly to allow for assessment of change; (3) if the PD-RBD target population is included, it should be in the early stages of PD defined as Hoehn and Yahr stages 1-3 in Off (untreated); (4) iRBD patients with soft neurologic dysfunction and with operational criteria established by the consensus of study investigators; (5) patients with mild cognitive impairment (MCI); and (6) optimally treated comorbid OSA. Twenty-four exclusion criteria were identified. The primary outcome measure for RBD treatment trials was determined to be the Clinical Global Impression (CGI) efficacy index, consisting of a four-point scale with a four-point side-effect scale. Assessment of video-polysomnographic (vPSG) changes holds promise but is costly and needs further elaboration. Secondary outcome measures include sleep diaries; sleepiness scales; PD sleep scale 2 (PDSS-2); serial motor examinations; cognitive indices; mood and anxiety indices; assessment of frequency of falls, gait impairment, and apathy; fatigue severity scale; and actigraphy and customized bed alarm systems. Consensus also was established for evaluating the clinical and vPSG aspects of RBD. End points for neuroprotective trials in RBD, taking lessons from research in PD, should be focused on the ultimate goal of determining the performance of disease-modifying agents. To date no compound with convincing evidence of disease-modifying or neuroprotective efficacy has been identified in PD. Nevertheless, iRBD patients are considered ideal candidates for neuroprotective studies. CONCLUSIONS: The IRBD-SG provides an important platform for developing multinational collaborative studies on RBD such as on environmental risk factors for iRBD, as recently reported in a peer-reviewed journal article, and on controlled active treatment studies for symptomatic and neuroprotective therapy that emerged during the 2011 consensus conference in Marburg, Germany, as described in our report.

Alternating vigilance states: new insights regarding neuronal networks and mechanisms.

Since the discovery of rapid eye movement (REM) sleep (also known as paradoxical sleep; PS), it is accepted that sleep is an active process. PS is characterized by EEG rhythmic activity resembling that of waking with a disappearance of muscle tone and the occurrence of REMs, in contrast to slow-wave sleep (SWS, also known as non-REM sleep) identified by the presence of delta waves. Here, we review the most recent data on the mechanisms responsible for the genesis of SWS and PS. Based on these data, we propose an updated integrated model of the mechanisms responsible for the sleep-wake cycle. This model introduces for the first time the notion that the entrance and exit of PS are induced by different mechanisms. We hypothesize that the entrance from SWS to PS is due to the intrinsic activation of PS-active GABAergic neurons localized in the posterior hypothalamus (co-containing melanin-concentrating hormone), ventrolateral periaqueductal gray and the dorsal paragigantocellular reticular nucleus. In contrast, the exit from PS is induced by the inhibition of these neurons by a PS-gating system composed of GABAergic neurons localized in the ventrolateral periaqueductal gray and just ventral to it, and waking systems such as the pontine and medullary noradrenergic neurons and the hypothalamic hypocretin neurons. Finally, we review human neurological disorders of the network responsible for sleep and propose hypotheses on the mechanisms responsible for REM behavior disorder and narcolepsy.

The satiety molecule nesfatin-1 is co-expressed with melanin concentrating hormone in tuberal hypothalamic neurons of the rat.

Overlapped in the tuberal hypothalamic area (THA), melanin-concentrating hormone (MCH) and hypocretin (Hcrt) neurons contribute to the integrated regulation of food intake, energy regulation and sleep. Recently, physiological role in appetite suppression has been defined for a novel hypothalamic molecule, nesfatin-1. Acute i.c.v. infusion of nesfatin-1 (nesf-1) promotes anorexia whereas chronic treatment reduces body weight in rats. This satiety molecule is expressed in neurons from areas prominently involved in appetite regulation including THA. We therefore sought functionally relevant to determine whether nesf-1 might be a reliable signaling marker for a new cell contingent within THA, in addition to MCH and Hcrt neurons. Thus, we completed a detailed topographical mapping of neurons immunostained for nesf-1 (nesf-1+) together with cell quantification in each discrete nucleus from THA in the rat. We further combined the immunodetection of nesf-1 with that of MCH or Hcrt to assess possible co-expression. More than three quarters of the nesf-1+ neurons were encountered in nuclei from the lateral half of THA. By double immunofluorescent staining, we showed that all neurons immunoreactive for melanin concentrating hormone (MCH+) neurons depicted nesf-1 immunoreactivity and approximately 80% of the nesf-1+ neurons were labeled for MCH. Maximal co-expression rates were observed in the lateral THA containing approximately 86% of the double-labeled neurons plotted in THA. The present data suggest that nesf-1 co-expressed in MCH neurons may play a complex role not only in food intake regulation but also in other essential integrative brain functions involving MCH signaling, ranging from autonomic regulation, stress, mood, cognition to sleep.

Role of the dorsal paragigantocellular reticular nucleus in paradoxical (rapid eye movement) sleep generation: a combined electrophysiological and anatomical study in the rat.

It is well known that noradrenergic locus coeruleus neurons decrease their activity during slow wave sleep and are quiescent during paradoxical sleep. It was recently proposed that their inactivation during paradoxical sleep is due to a tonic GABAergic inhibition arising from neurons located into the dorsal paragigantocellular reticular nucleus (DPGi). However, the discharge profile of DPGi neurons across the sleep-waking cycle as well as their connections with brain areas involved in paradoxical sleep regulation remain to be described. Here we show, for the first time in the unanesthetized rat that the DPGi contained a subtype of neurons with a tonic and sustained firing activation specifically during paradoxical sleep (PS-on neurons). Noteworthy, their firing rate increase anticipated for few seconds the beginning of the paradoxical sleep bout. By using anterograde tract-tracing, we further showed that the DPGi, in addition to locus coeruleus, directly projected to other areas containing wake-promoting neurons such as the serotonergic neurons of the dorsal raphe nucleus and hypocretinergic neurons of the posterior hypothalamus. Finally, the DPGi sent efferents to the ventrolateral part of the periaqueductal gray matter known to contain paradoxical sleep-suppressing neurons. Taken together, our original results suggest that the PS-on neurons of the DPGi may have their major role in simultaneous inhibitory control over the wake-promoting neurons and the permissive ventrolateral part of the periaqueductal gray matter as a means of influencing vigilance states and especially PS generation.

Brainstem glycinergic neurons and their activation during active (rapid eye movement) sleep in the cat.

It is well established that, during rapid eye movement (REM) sleep, somatic motoneurons are subjected to a barrage of inhibitory synaptic potentials that are mediated by glycine. However, the source of this inhibition, which is crucial for the maintenance and preservation of REM sleep, has not been identified. Consequently, the present study was undertaken to determine in cats the location of the glycinergic neurons, that are activated during active sleep, and are responsible for the postsynaptic inhibition of motoneurons that occurs during this state. For this purpose, a pharmacologically-induced state of active sleep (AS-carbachol) was employed. Antibodies against glycine-conjugated proteins were used to identify glycinergic neurons and immunocytochemical techniques to label the Fos protein were employed to identify activated neurons. Two distinct populations of glycinergic neurons that expressed c-fos were distinguished. One population was situated within the nucleus reticularis gigantocellularis (NRGc) and nucleus magnocellularis (Mc) in the rostro-ventral medulla; this group of neurons extended caudally to the ventral portion of the nucleus paramedianus reticularis (nPR). Forty percent of the glycinergic neurons in the NRGc and Mc and 25% in the nPR expressed c-fos during AS-carbachol. A second population was located in the caudal medulla adjacent to the nucleus ambiguus (nAmb), wherein 40% of the glycinergic cells expressed c-fos during AS-carbachol. Neither population of glycinergic cells expressed c-fos during quiet wakefulness or quiet (non-rapid eye movement) sleep. We suggest that the population of glycinergic neurons in the NRGc, Mc, and nPR participates in the inhibition of somatic brainstem motoneurons during active sleep. These neurons may also be responsible for the inhibition of sensory and other processes during this state. It is likely that the group of glycinergic neurons adjacent to the nucleus ambiguus (nAmb) is responsible for the active sleep-selective inhibition of motoneurons that innervate the muscles of the larynx and pharynx.

The endogenous somnogen adenosine excites a subset of sleep-promoting neurons via A2A receptors in the ventrolateral preoptic nucleus.

Recent research has shown that neurons in the ventrolateral preoptic nucleus are crucial for sleep by inhibiting wake-promoting systems, but the process that triggers their activation at sleep onset remains to be established. Since evidence indicates that sleep induced by adenosine, an endogenous sleep-promoting substance, requires activation of brain A(2A) receptors, we examined the hypothesis that adenosine could activate ventrolateral preoptic nucleus sleep neurons via A(2A) adenosine receptors in rat brain slices. Following on from our initial in vitro identification of these neurons as uniformly inhibited by noradrenaline and acetylcholine arousal transmitters, we established that the ventrolateral preoptic nucleus comprises two intermingled subtypes of sleep neurons, differing in their firing responses to serotonin, inducing either an inhibition (Type-1 cells) or an excitation (Type-2 cells). Since both cell types contained galanin and expressed glutamic acid decarboxylase-65/67 mRNAs, they potentially correspond to the sleep promoting neurons inhibiting arousal systems. Our pharmacological investigations using A(1) and A(2A) adenosine receptors agonists and antagonists further revealed that only Type-2 neurons were excited by adenosine via a postsynaptic activation of A(2A) adenosine receptors. Hence, the present study is the first demonstration of a direct activation of the sleep neurons by adenosine. Our results further support the cellular and functional heterogeneity of the sleep neurons, which could enable their differential contribution to the regulation of sleep. Adenosine and serotonin progressively accumulate during arousal. We propose that Type-2 neurons, which respond to these homeostatic signals by increasing their firing are involved in sleep induction. In contrast, Type-1 neurons would likely play a role in the consolidation of sleep.

Brainstem structures responsible for paradoxical sleep onset and maintenance.

This paper is dedicated to our mentor, Michel Jouvet who inspired our career and transmitted to us his passion for the study of the mechanisms responsible for paradoxical sleep genesis and also that of its still mysterious functions. We expose in the following the progresses in the knowledge in this field brought during 40 years by Michel Jouvet and his team and more recently by the members of a new CNRS laboratory in which we aim to pursue in the path opened by Michel Jouvet.

Effect of chronic treatment with milnacipran on sleep architecture in rats compared with paroxetine and imipramine.

A number of studies in humans and various other species have shown that chronic treatment with antidepressants, such as tricyclics or selective serotonin reuptake inhibitors (SSRIs), induces a decrease or suppression of rapid eye movement (REM) sleep. The effect of a new selective serotonin and noradrenaline reuptake inhibiting (SNRI) antidepressant, milnacipran, on REM sleep has been investigated and compared with that of the SSRI, paroxetine, and the tricyclic, imipramine. Rats injected with vehicle or milnacipran twice a day showed, over 24 h, a similar amount of REM sleep, number and duration of REM sleep episodes to control rats. In contrast, rats treated acutely with imipramine or paroxetine showed a statistically significant decrease in the total quantity of REM sleep. The number of REM sleep episodes was decreased while their duration was increased. A more detailed analysis showed further that the quantity of REM sleep was decreased for the first 4 h following the 9 a.m. injection but not the 7 p.m. injection for milnacipran, during the first 6 h for paroxetine and for the entire light-dark period for imipramine. For all drugs, the quantities of slow-wave sleep and waking over 24 h were not significantly different from control conditions and no rebound of REM sleep occurred during the day following withdrawal. Power spectrum analysis revealed no global changes in the different electroencephalogram (EEG) waves (delta, theta, gamma) between the control condition and the different treatments during waking, slow-wave sleep or REM sleep. Taken together our results indicate that the SNRI, milnacipran, at therapeutic doses, induces only minor disturbances of REM sleep compared with a SSRI and tricyclic antidepressant used. Possible mechanisms responsible for the difference of action on REM sleep of milnacipran are discussed.

Unrelated course of subthalamic nucleus and globus pallidus neuronal activities across vigilance states in the rat.

The pallido-subthalamic pathway powerfully controls the output of the basal ganglia circuitry and has been implicated in movement disorders observed in Parkinson's disease (PD). To investigate the normal functioning of this pathway across the sleep-wake cycle, single-unit activities of subthalamic nucleus (STN) and globus pallidus (GP) neurons were examined, together with cortical electroencephalogram and nuchal muscular activity, in non-anaesthetized head-restrained rats. STN neurons shifted from a random discharge in wakefulness (W) to a bursting pattern in slow wave sleep (SWS), without any change in their mean firing rate. This burst discharge occurred in the 1-2 Hz range, but was not correlated with cortical slow wave activity. In contrast, GP neurons, with a mean firing rate higher in W than in SWS, exhibited a relatively regular discharge whatever the state of vigilance. During paradoxical sleep, both STN and GP neurons increased markedly their mean firing rate relative to W and SWS. Our results are not in agreement with the classical 'direct/indirect' model of the basal ganglia organization, as an inverse relationship between STN and GP activities is not observed under normal physiological conditions. Actually, because the STN discharge pattern appears dependent on coincident cortical activity, this nucleus can hardly be viewed as a relay along the indirect pathway, but might rather be considered as an input stage conveying corticothalamic information to the basal ganglia.

Single-unit and polygraphic recordings associated with systemic or local pharmacology: a multi-purpose stereotaxic approach for the awake, anaesthetic-free, and head-restrained rat.

In order to avoid any artifactual pharmacological interferences with anaesthetic agents, a procedure has been developed for working on the awake, anaesthetic-free rat in a head-restrained condition. It allows, on the same animal and over several consecutive days, single-unit recordings in combination with systemic or local pharmacology (microiontophoresis or micropressure ejections), as well as monitoring vigilance states via the electroencephalogram and the electromyogram. After the cementing of a special "U"-shaped device on its skull under general anaesthesia, the animal is progressively habituated to stay daily, for several hours, under a painless corresponding stereotaxic restraint. This system can be easily adapted to different stereotaxic frames and, because of its spatial flexibility for targetting the desired rostrocaudal or lateral positions, allows access to a large number of cerebral structures. Experiments performed on Globus Pallidus, Substantia Nigra, and Locus Coeruleus neurons, combining the different possibilities of this system, are reported. They demonstrate, on the awake anaesthetic-free head-restrained rat, and under suitable ethical conditions, the feasibility of single-unit recordings of identified neurons associated with the study of their pharmacological reactivity after systemic or local drug administrations without any other drug interferences, and in physiologically relevant conditions such as the spontaneous alternance of vigilance states.

Identification of sleep-promoting neurons in vitro.

The neurons responsible for the onset of sleep are thought to be located in the preoptic area and more specifically, in the ventrolateral preoptic nucleus (VLPO). Here we identify sleep-promoting neurons in vitro and show that they represent an homogeneous population of cells that must be inhibited by systems of arousal during the waking state. We find that two-thirds of the VLPO neurons are multipolar triangular cells that show a low-threshold spike. This proportion matches that of cells active during sleep in the same region. We then show, using single-cell reverse transcriptase followed by polymerase chain reaction, that these neurons probably contain gamma-aminobutyric acid (GABA). We also show that these neurons are inhibited by noradrenaline and acetylcholine, both of which are transmitters of wakefulness. As most of these cells are also inhibited by serotonin but unaffected by histamine, their overall inhibition by transmitters of wakefulness is in agreement with their relative inactivity during waking with respect to sleep. We propose that the reciprocal inhibitory interaction of such VLPO neurons with the noradrenergic, serotoninergic and cholinergic waking systems to which they project is a key factor for promoting sleep.

Role and origin of the GABAergic innervation of dorsal raphe serotonergic neurons.

Extracellular electrophysiological recordings in freely moving cats have shown that serotonergic neurons from the dorsal raphe nucleus (DRN) fire tonically during wakefulness, decrease their activity during slow wave sleep (SWS), and are nearly quiescent during paradoxical sleep (PS). The mechanisms at the origin of the modulation of activity of these neurons are still unknown. Here, we show in the unanesthetized rat that the iontophoretic application of the GABA(A) antagonist bicuculline on dorsal raphe serotonergic neurons induces a tonic discharge during SWS and PS and an increase of discharge rate during quiet waking. These data strongly suggest that an increase of a GABAergic inhibitory tone present during wakefulness is responsible for the decrease of activity of the dorsal raphe serotonergic cells during slow wave and paradoxical sleep. In addition, by combining retrograde tracing with cholera toxin B subunit and glutamic acid decarboxylase immunohistochemistry, we demonstrate that the GABAergic innervation of the dorsal raphe nucleus arises from multiple distant sources and not only from interneurons as classically accepted. Among these afferents, GABAergic neurons located in the lateral preoptic area and the pontine ventral periaqueductal gray including the DRN itself could be responsible for the reduction of activity of the serotonergic neurons of the dorsal raphe nucleus during slow wave and paradoxical sleep, respectively.

Origins of the glycinergic inputs to the rat locus coeruleus and dorsal raphe nuclei: a study combining retrograde tracing with glycine immunohistochemistry.

The amino acid glycine is a major inhibitory neurotransmitter in the brainstem and is likely involved in the tonic inhibition of the monoaminergic neurons during all sleep-waking stages. In order to determine the neurons at the origin of the glycinergic innervation of the two principal monoaminergic nuclei, the locus coeruleus and the dorsal raphe of the rat, we applied a double-labelling technique, combining retrograde transport of cholera-toxin B subunit with glycine immunohistochemistry. Using this technique, we found that the locus coeruleus and dorsal raphe nuclei receive a common glycinergic innervation from the ventral and ventrolateral periaqueductal grey, including the adjacent deep mesencephalic reticular nucleus. Small additional glycinergic inputs to these nuclei originated from the lateral paragigantocellular nucleus and the rostral ventromedial medullary reticular formation. The potential role of these glycinergic inputs in the control of the excitability of the monoaminergic neurons of the locus coeruleus and dorsal raphe nuclei is discussed.

Electrophysiological evidence that noradrenergic neurons of the rat locus coeruleus are tonically inhibited by GABA during sleep.

It is well known that noradrenergic locus coeruleus (LC) neurons decrease their activity during slow wave sleep (SWS) and are virtually quiescent during paradoxical sleep (PS). It has been proposed that a GABAergic input could be directly responsible for this sleep-dependent neuronal inactivation. To test this hypothesis, we used a new method combining polygraphic recordings, microiontophoresis and single-unit extracellular recordings in unanaesthetized head-restrained rats. We found that iontophoretic application of bicuculline, a specific GABA(A)-receptor antagonist, during PS and SWS restore a tonic firing in the LC noradrenergic neurons. We further observed that the application of bicuculline during wakefulness (W) induced an increase of the discharge rate. Of particular importance for the interpretation of these results, using the microdialysis technique, Nitz and Siegel (Neuroscience, 1997; 78: 795) recently found an increase of the GABA release in the cat LC during SWS and PS as compared with waking values. Based on these and our results, we therefore propose that during W, the LC cells are under a GABAergic inhibitory tone which progressively increases at the entrance and during SWS and PS and is responsible for the inactivation of these neurons during these states.

Forebrain afferents to the rat dorsal raphe nucleus demonstrated by retrograde and anterograde tracing methods.

The dorsal raphe nucleus through its extensive efferents has been implicated in a great variety of physiological and behavioural functions. However, little is know about its afferents. Therefore, to identify the systems likely to influence the activity of serotonergic neurons of the dorsal raphe nucleus, we re-examined the forebrain afferents to the dorsal raphe nucleus using cholera toxin b subunit and Phaseolus vulgaris-leucoagglutinin as retrograde or anterograde tracers. With small cholera toxin b subunit injection sites, we further determined the specific afferents to the ventral and dorsal parts of the central dorsal raphe nucleus, the rostral dorsal raphe nucleus and the lateral wings. In agreement with previous studies, we observed a large number of retrogradely-labelled cells in the lateral habenula following injections in all subdivisions of the dorsal raphe nucleus. In addition, depending on the subdivision of the dorsal raphe nucleus injected, we observed a small to large number of retrogradely-labelled cells in the orbital, cingulate, infralimbic, dorsal peduncular, and insular cortice, a moderate or substantial number in the ventral pallidum and a small to substantial number in the claustrum. In addition, we observed a substantial to large number of cells in the medial and lateral preoptic areas and the medial preoptic nucleus after cholera toxin b subunit injections in the dorsal raphe nucleus excepting for those located in the ventral part of the central dorsal raphe nucleus, after which we found a moderate number of retrogradely-labelled cells. Following cholera toxin b subunit injections in the dorsal part of the central dorsal raphe nucleus, a large number of retrogradely-labelled cells was seen in the lateral, ventral and medial parts of the bed nucleus of the stria terminalis whereas only a small to moderate number was visualized after injections in the other dorsal raphe nucleus subdivisions. In addition, respectively, a substantial and a moderate number of retrogradely-labelled cells was distributed in the zona incerta and the subincertal nucleus following all tracer injections in the dorsal raphe nucleus. A large number of retrogradely-labelled cells was also visualized in the lateral, dorsal and posterior hypothalamic areas and the perifornical nucleus after cholera toxin b subunit injections in the dorsal part of the central raphe nucleus and to a lesser extent following injections in the other subdivisions. We further observed a substantial to large number of retrogradely-labelled cells in the tuber cinereum and the medial tuberal nucleus following cholera toxin b subunit injections in the dorsal part of the central dorsal raphe nucleus or the lateral wings and a small to moderate number after injections in the two other dorsal raphe nucleus subdivisions. A moderate or substantial number of labelled cells was also seen in the ventromedial hypothalamic area and the arcuate nucleus following cholera toxin injections in the dorsal part of the central dorsal raphe nucleus and the lateral wings and an occasional or small number with injection sites located in the other subdivisions. Finally, we observed, respectively, a moderate and a substantial number of retrogradely-labelled cells in the central nucleus of the amygdala following tracer injections in the ventral or dorsal parts of the central dorsal raphe nucleus and a small number after injections in the other subnuclei. In agreement with these retrograde data, we visualized anterogradely-labelled fibres heterogeneously distributed in the dorsal raphe nucleus following Phaseolus vulgaris-leucoagglutinin injections in the lateral orbital or infralimbic cortice, the lateral preoptic area, the perifornical nucleus, the lateral or posterior hypothalamic areas, the zona incerta, the subincertal nucleus or the medial tuberal nucleus. (ABSTRACT TRUNCATED)

Anatomical demonstration of a medullary enkephalinergic pathway potentially implicated in the oro-facial muscle atonia of paradoxical sleep in the cat.

The present study was aimed to compare in detail the distribution within the rostral ventromedial medulla of Methionin-Enkephalin-immunoreactive neurons with efferent projections to the facial or trigeminal motor nuclei, using a double immunostaining technique in colchicine-treated cats. Following cholera toxin B subunit injections in the facial or trigeminal motor nuclei, we found that respectively 55% and 65% of the medium to large-sized retrogradely labeled cells in the lateral part of the nucleus reticularis magnocellularis were Methionin-Enkephalin-positive. For both motor nuclei, the double-labeled neurons had similar morphology and size and were located exactly in the same area. They could therefore belong to the same population of reticular enkephalinergic neurons. Based on these and previous anatomical and electrophysiological data, we propose that these enkephalin-containing neurons could participate in the hyperpolarization of brainstem and spinal somatic motoneurons during paradoxical sleep.

Afferent projections to the rat nuclei raphe magnus, raphe pallidus and reticularis gigantocellularis pars alpha demonstrated by iontophoretic application of choleratoxin (subunit b).

The aim of the present study was to identify the specific afferent projections to the rostral and caudal nucleus raphe magnus, the gigantocellular reticular nucleus pars alpha and the rostral nucleus raphe pallidus. For this purpose, small iontophoretic injections of the sensitive retrograde tracer choleratoxin (subunit b) were made in each of these structures. In agreement with previous retrograde studies, after all injection sites, a substantial to large number of labeled neurons were observed in the dorsal hypothalamic area and dorsolateral and ventrolateral parts of the periaqueductal gray, and a small to moderate number were found in the lateral preoptic area, bed nucleus of the stria terminalis, paraventricular hypothalamic nucleus, central nucleus of the amygdala, lateral hypothalamic area, parafascicular area, parabrachial nuclei, subcoeruleus area and parvocellular reticular nucleus. In addition, depending on the nucleus injected, we observed a variable number of retrogradely labeled cells in other regions. After injections in the rostral nucleus raphe magnus, a large number of labeled cells were seen in the prelimbic, infralimbic, medial and lateral precentral cortices and the dorsal part of the periaqueductal gray. In contrast, after injections in the other nuclei, fewer cells were localized in these structures. Following raphe pallidus injections, a substantial to large number of labeled cells were observed in the medial preoptic area, median preoptic nucleus, ventromedial part of the periaqueductal gray, Kölliker-Fuse and lateral paragigantocellular reticular nuclei. Following injections in the other areas, a small to moderate number of cells appeared. After gigantocellular reticular pars alpha injections, a very large and substantial number of labeled neurons were found in the deep mesencephalic reticular formation and oral pontine reticular nucleus, respectively. After the other injections, fewer cells were seen. Following rostral raphe magnus or raphe pallidus injections, a substantial number of labeled cells were observed in the insular and perirhinal cortices. Following caudal raphe magnus or gigantocellular reticular pars alpha injections, fewer cells were found. After raphe magnus or gigantocellular reticular pars alpha injections, a moderate to substantial number of cells were localized in the fields of Forel, lateral habenular nucleus and ventral caudal pontine reticular nucleus. Following raphe pallidus injections, only a small number of cells were seen. Our data indicate that the rostral and caudal parts of the nucleus raphe magnus, the gigantocellular reticular nucleus pars alpha and the nucleus raphe pallidus receive afferents of comparable strength from a large number of structures. In addition, a number of other afferents give rise to stronger inputs to one or two of the four nuclei studied. Such differential inputs might be directed to populations of neurons with different physiological roles previously recorded specifically in these nuclei.