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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.

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.

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.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

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.

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.

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)

The noradrenergic neurones of the locus coeruleus (LC) discharge tonically during wakefulness, decrease their activity during slow wave sleep and are virtually quiescent during paradoxical sleep. We recently demonstrated an inhibitory glycinergic input to the locus coeruleus and proposed that this could be responsible for inhibition of the LC during paradoxical sleep. To test this proposal, we developed a method combining polygraphic recordings, iontophoresis and single-unit extracellular recordings in the unanaesthetized head-restrained rat. Iontophoretically applied strychnine, a specific glycine antagonist, induced strong excitation of LC neurones during paradoxical sleep, but also during slow wave sleep and wakefulness. These results suggest that glycine tonically inhibits noradrenergic LC neurones throughout the entire sleep-waking cycle and not only during paradoxical sleep.

To localize glycinergic cell bodies and fibers in the rat brain, we developed a sensitive immunohistochemical method combining the use of specific glycine antibodies (Campistron G. et al. (1986) Brain Res. 376, 400-405; Wenthold R. J. et al. (1987) Neuroscience 22, 897-912) with the streptavidin-horseradish peroxidase technique and 3,3'-diaminobenzidine.4HCl-nickel intensification. We confirmed the presence of numerous glycine-immunoreactive cell bodies and fibers in the cochlear nuclei, superior olivary complex, nucleus of the trapezoid body, cerebellar cortex, deep cerebellar nuclei and area postrema. For the first time in rats, we described a large to very large number of cell bodies in the medial vestibular ventral part, prepositus hypoglossal, gracile, raphe magnus and sensory trigeminal nuclei. A large number of cells was also observed in the oral and caudal pontine, parvocellular, parvocellular pars alpha, gigantocellular and gigantocellular pars alpha reticular nuclei. In addition, glycine-immunoreactive cells were seen in the ambiguous and subtrigeminal nuclei, the lateral habenula and the subfornical organ. We also provide the first evidence in rats for a very large number of fibers in the trigeminal, facial, ambiguous and hypoglossal motor nuclei, all nuclei of the medullary and pontine reticular formation, and the raphe and trigeminal sensory nuclei. We further revealed the presence of a substantial number of fibers in regions where glycine was not considered as a main inhibitory neurotransmitter, such as the pontine nuclei, the periaqueductal gray, the mesencephalic reticular formation, the anterior pretectal nucleus, the intralaminar thalamic nuclei, the zona incerta, the fields of Forel, the parvocellular parts of the paraventricular nucleus, the posterior hypothalamic areas, the anterior hypothalamic area, and the lateral and medial preoptic areas. These results indicate that, in contrast to previous statements, glycine may be an essential inhibitory neurotransmitter not only in the lower brainstem and spinal cord, but also in the upper brainstem and the forebrain.

In order to determine the localization of the glycinergic neurones responsible for the hyperpolarization of the rat trigeminal motoneurones during paradoxical sleep, we developed a new double immunohistochemical method combining the b subunit of the cholera toxin (CTb), a very sensitive retrograde tracer, with glycine immunohistochemistry. After iontophoretic injections of CTb into the trigeminal motor nucleus (Mo5), a large number of double-labelled cells was observed bilaterally in the parvocellular reticular nucleus alpha, dorsolateral to the descending branch of the facial nerve. A moderate number of double-labelled neurones was found in the ipsilateral parvocellular reticular nucleus at the level of the facial nucleus, and bilaterally in the raphe magnus and the gigantocellular reticular alpha nuclei. These results suggest that the glycinergic neurones hyperpolarizing the trigeminal motoneurons during paradoxical sleep might be localized in the parvocellular reticular nucleus alpha.

A large body of data suggests that the activation of alpha 1 receptors by a tonic noradrenergic input might be responsible for the tonic discharge of the serotonergic neurons of the dorsal raphe nucleus (DRN). To test this hypothesis, it was necessary to determine the origin of the noradrenergic and adrenergic innervation of these neurons. For this purpose, we combined small iontophoretic injections of the sensitive retrograde tracer cholera toxin b subunit (CTb) in the different subdivisions of the DRN with tyrosine hydroxylase immunohistochemistry. After CTb injections in the ventral or dorsal parts of the central DRN, a small number of double-labeled cells was observed in the locus coeruleus (A6 noradrenergic cell group), the A5 noradrenergic group, the dorsomedial medulla (C3 adrenergic cell group), and the lateral paragigantocellular nucleus (C1 adrenergic cell group). After CTb injections in the lateral wings or the dorsal part of the rostral DRN, a similar number of double-labeled cells was seen in C3. Slightly more double-labeled cells were seen in A6 and A5. In addition, a substantial to large number of double-labeled cells appeared in C1, the commissural part of the nucleus of the solitary tract (A2 noradrenergic cell group) and the caudoventrolateral medulla (A1 noradrenergic cell group). These results indicate that the noradrenergic and adrenergic inputs to the DRN arise from all the catecholaminergic cell groups of the lower brainstem except the A7 noradrenergic group. They further reveal the existence of a topographical organization of these afferents to the different subdivisions of the DRN.

The aim of the present study was to describe the distribution of dopamine (DA) fibres in the dorsal raphe nucleus (DRN) and to determine their neurones of origin. Using an anti-DA antibody, we observed a moderate density of DA varicose fibres over the DRN and a dense plexus of DA fibres in the ventrolateral central grey. With a sensitive retrograde tracing technique combining the use of cholera toxin subunit b with tyrosine hydroxylase immunohistochemistry, after tracer injections in the DRN, a few double-labelled cells were observed in the ventral tegmental area and the A10 dorsocaudal DA cell group, as already described. In addition, a moderate number of double-labelled cells was seen in the A11 hypothalamic DA cell group.

The microinjection of carbachol into the nucleus pontis oralis produces a state which is polygraphically and behaviorally similar to active sleep (rapid eye movement sleep). In the present study, using double-labeling techniques for serotonin and the protein product of c-fos (Fos), we sought to examine whether immunocytochemically identified serotonergic neurons of the raphe nuclei of the cat were activated, as indicated by their expression of c-fos, during this pharmacologically-induced behavioral state (active sleep-carbachol). Compared with control cats, which were injected with saline, active sleep-carbachol cats exhibited a significantly greater number of c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus. Whereas most of the c-fos-expressing neurons in the raphe dorsalis were small, those in the raphe magnus were medium-sized and in the raphe pallidus they were small and medium-sized. The mean number of serotonergic neurons that expressed c-fos (i.e. double-labeled cells) was similar in control and active sleep-carbachol cats. These data indicate that there is an increased number of non-serotonergic, c-fos-expressing neurons in the raphe dorsalis, magnus and pallidus during the carbachol-induced state.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to examine anatomical evidence in cats of whether the nucleus reticularis parvicellularis (Pc) is part of the circuit responsible for the inhibition of brainstem motoneurons during paradoxical sleep. For this purpose, we made iontophoretic injections of the retrograde and anterograde tracer cholera toxin B subunit (CTb) in the Pc. After CTb injections in the Pc, a large number of retrogradely labeled neurons were seen in the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the posterior hypothalamic areas, the mesencephalic reticular formation, the nucleus locus subcoeruleus, the nucleus pontis caudalis, other portions of the Pc, the nucleus reticularis dorsalis, the trigeminal sensory complex, and the nucleus of the solitary tract. We further found that the Pc receives 1) serotoninergic afferents from the raphe dorsalis, magnus, and obscurus nuclei; 2) noradrenergic inputs from the dorsolateral pontine tegmentum; 3) cholinergic afferents from the lateral medullary reticular formation; 4) substance P-like afferents from the central nucleus of the amygdala, bed nucleus of the stria terminalis, periaqueductal gray, and nucleus of the solitary tract; and 5) methionine-enkephalin-like projections from the periaqueductal gray, the nucleus of the solitary tract, the lateral pontine and medullary reticular formation, and the spinal trigeminal nucleus. We further found that the Pc do not receive afferents from brainstem structures responsible for muscle atonia, such as the ventromedial medulla and the dorsomedial pontine tegmentum, and therefore may not be part of the circuit inhibiting the brainstem motoneurons during paradoxical sleep.

Using a specific glycine antiserum, we determined the localization of glycinergic neurones and fibres in the cat brain stem reticular formation. We visualized a large number of glycine-immunoreactive cell bodies and fibres in the medullary reticularis gigantocellularis, magnocellularis, paragigantocellularis lateralis and parvocellularis nuclei. The pontis oralis and caudalis and the raphe magnus nuclei also contained a large number of glycine-immunoreactive fibres but fewer neurones. Using a double staining method, we further observed glycine-immunoreactive boutons over (1) noradrenergic neurones in the locus coeruleus complex and the ventrolateral and dorsomedial medulla, (2) serotoninergic neurones in and outside the raphe nuclei and (3) cholinergic neurones in the pedunculopontine and laterodorsal tegmental nuclei. These results suggest that glycinergic neurones in the reticular formation may be involved in aspects of paradoxical sleep, including the general muscle atonia seen during this sleep state.

The distribution of enkephalin (ENK)-like immunoreactivity (LI) in spinal cord and medulla oblongata of cat and gray monkey (Macaca fascicularis) was studied by use of immunofluorescence and peroxidase antiperoxidase (PAP) techniques. Possible coexistence between ENK- and 5-hydroxytryptamine (5-HT)-LI was also analyzed with double labeling immunofluorescence. Furthermore, in situ hybridization was used to demonstrate cell bodies in the brain stem expressing mRNA encoding for ENK. ENK-immunoreactive (IR) axonal varicosities and fibers were demonstrated throughout the spinal cord gray matter, with the highest density in the superficial dorsal horn, the area around the central canal, the intermediolateral cell column, the sacral parasympathetic nucleus, and in Onuf's nucleus. In the monkey ventral horn, ENK-IR varicose fibers could in some cases be demonstrated in very close apposition to cell bodies. A low degree of co-localization between ENK- and 5-HT-LI was seen in the spinal cord of both species. Still, fibers containing both compounds could as a rule be demonstrated in every section studied. The highest degree of coexistence was encountered in the motor nucleus of the ventral horn. Six weeks after a low thoracic spinal cord transection a decreased staining for ENK-LI was demonstrated in the ventral horn motor nucleus, whereas other parts of the spinal cord appeared unaffected. In the brain stem of cats after colchicine treatment, ENK-LI was found in a majority of the 5-HT-IR cell bodies in the raphe nuclei (nucleus raphe magnus, pallidus and obscurus) and in the lateral reticular nucleus (rostroventrolateral reticular nucleus). In cat not pretreated with colchicine, a few weakly stained ENK-IR cell bodies could be found in the midline raphe nuclei and in the lateral reticular nucleus with the PAP technique. In the monkey brain stem without colchicine treatment, using the PAP technique, heavily stained ENK-IR cell bodies could be seen in the lateral reticular nucleus whereas, as in the cat, only a few, weakly stained ENK-IR cell bodies could be seen in the midline raphe nuclei. Using in situ hybridization technique, ENK mRNA expressing cells were demonstrated in the lateral reticular nucleus while no convincing mRNA signal could be found over cell bodies in the raphe nuclei. It is concluded that part of the ENKergic innervation of the cord in both species derives from supraspinal or suprasegmental levels.(ABSTRACT TRUNCATED AT 400 WORDS)

In this report, we demonstrate that cholera-toxin B subunit (CTb) is a very sensitive retrograde tracer in the central nervous system when recognized by streptavidin-peroxidase immunohistochemistry. We further show that: (1) injection of a small volume of CTb gives rise to small sharply defined injection sites limited to the cell group of interest associated with the labeling of all the known afferent projections, (2) CTb is taken up, and anterogradely as well as retrogradely transported in damaged but not intact fibers of passage, (3) CTb can be applied iontophoretically, allowing us to study the afferents to small cell groups without any evidence of tissue necrosis in the sites and therefore without artefactual labeling due to uptake by damaged fibers of passage, (4) the use of 4% paraformaldehyde fixative ideally suited for the preservation of most neural antigens, the addition of a 48 h colchicine treatment and the development of a double immunohistochemical method allow the biochemical characterization of the cell of origin of particular pathways in the CNS, (5) CTb is also anterogradely transported with an extensive filling of axons and axon terminals and thereby opens up the possibility of identifying simultaneously the afferents as well as the efferents of the group of cells studied and finally (6) the very long conservation of the preparation, the possibility of counterstaining it and of making camera lucida drawings allow easy and precise localization of the retrogradely labeled cells.

The aim of the present study was to determine the brainstem afferents and the location of neurons giving rise to monoaminergic, cholinergic, and peptidergic inputs to the cat trigeminal motor nucleus (TMN). This was done in colchicine treated animals by using a very sensitive double immunostaining technique with unconjugated cholera-toxin B subunit (CT) as a retrograde tracer. After CT injections in the TMN, retrogradely labeled neurons were most frequently seen bilaterally in the nuclei reticularis parvicellularis and dorsalis of the medulla oblongata, the alaminar spinal trigeminal nucleus (magnocellular division), and the adjacent pontine juxtatrigeminal region and in the ipsilateral mesencephalic trigeminal nucleus. We further observed that inputs to the TMN arise from the medial medullary reticular formation (the nuclei retricularis magnocellularis and gigantocellularis), the principal bilateral sensory trigeminal nucleus, and the dorsolateral pontine tegmentum. In addition, the present study demonstrated that the TMN received 1) serotonergic afferents, mainly from the nuclei raphe obscurus, pallidus, and dorsalis; 2) catecholaminergic afferent projections originating exclusively in the dorsolateral pontine tegmentum, including the Kölliker-Fuse, parabrachialis lateralis, and locus subcoeruleus nuclei; further, that 3) methionin-enkephalin-like inputs were located principally in the medial medullary reticular formation (nuclei reticularis magnocellularis and gigantocellularis and nucleus paragigantocellularis lateralis), in the caudal raphe nuclei (Rpa and Rob) and the dorsolateral pontine tegmentum; 4) substance P-like immunoreactive neurons projecting to the TMN were present in the caudal raphe and Edinger-Westphal nuclei; and 5) cholinergic afferents originated in the whole extent of the nuclei reticularis parvicellularis and dorsalis including an area located ventral to the nucleus of the solitary tract at the level of the obex. In the light of these anatomical data, the present report discusses the possible physiological involvement of TMN inputs in the generation of the trigeminal jaw-closer muscular atonia occurring during the periods of paradoxical sleep in the cat.

Using a double-immunostaining technique with cholera toxin (CT) as a retrograde tracer, the authors examined the cells of origin and the histochemical nature of lower brainstem afferents to the cat posterior hypothalamus. The posterior hypothalamus, in particular the lateral hypothalamic area, receives substantial afferent projections from: substantia nigra, peripeduncular nucleus, ventral tegmental area, periaqueductal grey, mesencephalic reticular formation, peribrachial region including the locus coeruleus complex, rostral raphe nuclei and the rostral part of the nucleus magnus. In addition, a moderate number of retrogradely labeled neurons was found in: Edinger-Westphal nucleus, nucleus reticularis pontis oralis, nucleus reticularis magnocellularis, caudal lateral bulbar reticular formation around the nucleus ambiguus and lateral reticular nucleus and the nucleus of the solitary tract. The posterior hypothalamus receives: 1) dopaminergic inputs from A8, A9 and A10 cell groups; 2) noradrenergic inputs from A6 and A7 pontine, as well as A1 and A2 bulbar cell groups; 3) adrenergic inputs from C1 cell group in the caudal medulla; 4) serotoninergic inputs from the rostral raphe nuclei (B6, B7 and B8 cell groups); 5) cholinergic inputs from the peribrachial region of the dorsal pontine tegmentum as well as from the nucleus reticularis magnocellularis of the medulla; 6) peptidergic inputs such as methionine-enkephalin, substance P, corticotropin-releasing factor and galanin that originate mainly in the mesencephalic periaqueductal grey, the dorsal raphe nucleus and the peribrachial region of the dorsal pontine tegmentum.

Using a highly specific antiserum to Glycine and a very sensitive immunohistochemical technique with streptavidin-HRP, we visualized for the first time a considerable number of glycine immunoreactive cell bodies and fibers in the cat medulla oblongata. These results suggest that glycine may play an essential role in nearly all the physiological functions involving the medulla oblongata, including the muscular atonia occurring during paradoxical sleep.

The localization of dopaminergic and non-dopaminergic neuronal perikarya sending axons to the median eminence was investigated in the cat by using two colour double-immunostaining techniques. Unconjugated cholera toxin and wheat germ agglutinin were used as retrograde tracers and injected respectively into the median eminence and the neuro-intermediate pituitary of the same animal. As controls, cholera toxin was also injected into the arcuate (infundibular) nucleus or third ventricle. The retrograde labelling of one of the tracers was combined with tyrosine hydroxylase immunohistochemistry as a marker for dopaminergic neurons. The retrograde labelling studies of cholera toxin alone and the double-immunostaining of cholera toxin and wheat germ agglutinin on the same sections revealed that the cat median eminence receives major afferent projections originating in midline hypothalamic nuclear groups such as the anterior periventricular nucleus, the periventricular part of the paraventricular nucleus and the arcuate nucleus; minor afferent projections arise from the anterior hypothalamic area, the rostral part of the medial preoptic area around the organum vasculosum of the lamina terminalis and to a lesser extent from the posterior hypothalamic region. We further determine that the rostral part of the parvocellular arcuate neurons constitutes the main source of dopaminergic afferents to the median eminence in the cat brain.

Using a sensitive double immunostaining technique with unconjugated cholera-toxin B subunit as a retrograde tracer, the authors determined the nuclei of origin of monoaminergic, peptidergic, and cholinergic afferent projections to the cat facial nucleus (FN). The FN as a whole receives substantial afferent projections, with relative subnuclear differences, from the following areas: 1) the perioculomotor areas, the contralateral paralemniscal region, and the mesencephalic reticular formation dorsal to the red nucleus; 2) the ipsilateral parabrachial region and the nucleus reticularis pontis, pars ventralis; and 3) the nuclei reticularis parvicellularis, magnocellularis, ventralis, and dorsalis of the medulla. In addition, the present study demonstrated that the lateral portion of the FN receives specific projections from the contralateral medial and olivary pretectal nuclei and the ipsilateral reticular formation of the pons. It was also found that the FN receives: 1) serotoninergic inputs mainly from the nuclei raphe obscurus, pallidus, magnus, and the caudal ventrolateral bulbar reticular formation; 2) catecholaminergic afferent projections from the A7 noradrenaline cell group located in the Kölliker-Fuse, parabrachialis lateralis, and locus subcoeruleus nuclei; 3) methionin-enkephalin-like inputs originating in the pretectal complex, the nucleus paragigantocellularis lateralis and the caudal raphe nuclei; 4) substance P-like afferent projections mainly from the Edinger-Westphal complex and the caudal raphe nuclei; and 5) cholinergic afferents from an area located ventral to the nucleus of the solitary tract at the level of the obex. In the light of these anatomical data, the present report discusses the physiological significance of FN inputs relevant to tonic and phasic events occurring at the level of the facial musculature during the period of paradoxical sleep in the cat.

Using immunohistochemical techniques, we demonstrated oxytocin (OT) and vasopressin (AVP) neurons in the cat hypothalamus. The OT immunoreactive neurons were found mainly in the paraventricular nucleus, supraoptic nucleus and dorsal accessory group located lateral to the fornix. In addition to these hypothalamic structures, the AVP immunoreactive neurons were observed in the suprachiasmatic nucleus, ventral accessory group located in the retrochiasmatic area and lateral accessory group, dorsal to the supraoptic nucleus caudally, and ventral to the medial part of the internal capsule rostrally. We further demonstrated a different localization of the OT and AVP immunoreactive neurons in the paraventricular and supraoptic nuclei.

The localization of cholinergic neurons in the cat lower brain stem was determined immunocytochemically with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. ChAT-positive neurons were observed in four major cell groups: cranial nerve motor and special visceromotor neurons: parasympathetic preganglionic visceromotor neurons; neurons located in the ponto-mesencephalic tegmentum including area X (or pedunculopontine tegmental nucleus), nucleus laterodorsalis tegmenti (Ldt) of Castaldi, and peri-locus coeruleus alpha (peri-alpha); and neurons located in nucleus reticularis magnocellularis (Mc) and adjacent nucleus reticularis gigantocellularis (Gc) of the medulla.

The topographic distribution of neurophysin-immunoreactive (NP-IR) cells projecting to the posterior pituitary gland has been studied in the cat using a double staining method: immunohistochemistry of neurophysin in conjunction with horseradish peroxidase (HRP) retrograde tracer technique. We found that almost all the hypothalamic NP-IR cells project directly to the neurohypophysis except those localized in the suprachiasmatic nucleus and in the caudal part of the paraventricular nucleus.

By means of a double immunostaining technique using unconjugated cholera-toxin B subunit (CTb) as a retrograde tracer combined with phenylethanolamine-N-methyltransferase (PNMT) immunohistochemistry, we demonstrated that the nucleus raphe pallidus of the cat receives a major projection from the ventrolateral part of the rostral medulla corresponding to the nucleus paragigantocellularis lateralis and the ventrolateral medullary reticular formation just caudal to it. We further showed that nearly 60% of the total CTb-labeled cells in this region are immunoreactive to PNMT. These double-labeled cells constitute one-third of the total PNMT-immunoreactive cells.

Using a double immunostaining technique with cholera toxin (CT) as a retrograde tracer, we examined the cells of origin and the histochemical nature of afferents to the cat posterior hypothalamus. After injection in the tuberomamillary nucleus, a number of CT-labeled cells were observed in: medial preoptic area, nuclei of the septum and the stria terminalis, amygdaloid complex, anterior hypothalamic, ventromedial hypothalamic and premamillary nuclei. CT injections in the lateral hypothalamic area gave an additional heavy labeling of neurons in: lateral preoptic area, nuclei of the diagonal band of Broca, substantia innominata, and nucleus accumbens. The posterior hypothalamus receives: 1) cholinergic inputs from the septum, the lateral preoptic area and the nuclei of the diagonal band of Broca; 2) dopaminergic afferents from A11, A13, and A14 groups; 3) histaminergic afferents from the posterior hypothalamus; and 4) peptidergic afferents such as methionin-enkephalin, galanin and neurotensin, substance P and corticotropin-releasing factor from the medial preoptic area, the nucleus of the stria terminalis and/or the posterior hypothalamic structures.

Using a sensitive double-immunostaining technique with nonconjugated cholera toxin B subunit (CT) as a retrograde tracer, we examined the cells of origin and the histochemical nature of afferents to the cat nucleus reticularis magnocellularis (Mc) of the medulla oblongata. After injections of CT confined to the Mc, we found that the major afferents to the Mc arise from: (1) the lateral part of the bed nucleus of the stria terminalis, the nucleus of the anterior commissure, the preoptic area, the central nucleus of the amygdala, the posterior hypothalamus, and the nucleus of the fields of Forel; (2) the Edinger-Westphal nucleus, the mesencephalic reticular formation, and the ventrolateral part of the periaqueductal grey; (3) the nuclei locus coeruleus alpha (LC alpha), peri-LC alpha, locus subcoeruleus, and reticularis pontis oralis and caudalis; (4) the caudal raphe nuclei; and (5) the nucleus reticularis ventralis of the medulla.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticotropin releasing factor (CRF)-immunoreactive (IR) perikarya, visualized by the indirect immunoperoxidase method in colchicine-pretreated cats, were localized in many discrete regions of the medulla oblongata. They were found mainly in the dorsal aspect and midline of the medulla oblongata, and more rostrally in the ventrolateral portion. Our results also demonstrated CRF-IR neurons in the rostrocaudal extent of the inferior olive, probably projecting to the cerebellar cortex via thick axons visualized along the lateral edge of the medulla. CRF-IR olivary cells were also found in the pontine cat from which the forebrain was removed, but neither in hypophysectomized nor adrenalectomized cats.

In order to study putative hypothalamic mechanisms of sleep waking cycle regulation we destroyed, by electrolytic coagulation, a large part of the medial hypothalamus overlapping the paraventricular nucleus in 6 adult cats. We never observed any modification of light slow wave sleep. Three of the six cats presented no paradoxical sleep (PS) impairment, despite an almost total destruction of neurophysin-immunoreactive cells of PVN in two cats and marked signs of diabetes insipidus in the third. Further, in the other three animals a statistically significant decrease of daily quantities of PS and deep slow wave sleep (SWS2) were related to an extensive destruction of the anterior hypothalamic area. These results suggest lack of influence of the PVN in sleep regulation and an involvement of the anterior hypothalamus in the onset of SWS2 and PS.

The present study examines the distribution and morphological characteristics of neurons containing immunoreactivity of tyrosine hydroxylase in the cat hypothalamus. We used the indirect immunoperoxidase technique on vibratome sections. Tyrosine hydroxylase-immunoreactive cell bodies were widely distributed in discrete regions of the cat hypothalamus. Several principal cell groups were identified. They were seen in the posterior and dorsal hypothalamic areas, zona incerta, dorsomedial and lateral hypothalamic areas, arcuate nucleus, periventricular nucleus, paraventricular nucleus, and an area of the tuber cinereum and preoptic area. These cells presented two different morphological characteristics; small with two to three short processes and medium to large, multipolar with three to five long dendritic trees. The atlas is presented in twelve cross-sectional drawings of the cat hypothalamus from the level A8.5 to A15 of the Horsley-Clarke stereotaxic planes. We also examined the distribution of hypothalamic catecholamine fluorescent neurons by using the aqueous aldehyde method in combination with glyoxylic acid applied to vibratome sectioned tissues, which improves sensitivity. Comments are made on the relative localizations of the tyrosine hydroxylase-immunoreactive and aldehyde-induced histofluorescent cells, as well as on species differences between the cat, rat, and mouse.

The localization of histaminergic neurons in the cat brain was determined immunohistochemically with an antibody against histamine. We found that histamine-immunoreactive neurons are observed exclusively in the posterior hypothalamus of colchicine treated cats. The larger group of neurons was found in the ventrolateral part of the posterior hypothalamus, including the tuberomammillary nucleus. Histamine-positive neurons were also observed in the supramammillary area and adjacent posterior hypothalamic area, as well as in the peri- and premammillary regions. In addition, numerous histamine immunoreactive fibers were detected, not only in the posterior hypothalamus, but also in other brain areas, such as the preoptic area of the anterior hypothalamus.

Using a double immunostaining technique with unconjugated cholera toxin (CT) as a retrograde tracer, we have demonstrated in the cat that the nucleus raphe pallidus receives two major afferent projections from the hypothalamus: the preoptic periventricular nucleus; and the peri- and paraventricular zones of the posterior hypothalamic area. Some CT-labeled neurons in the preoptic periventricular nucleus showed Met-Enk-like immunoreactivity, while many CT-labeled neurons in the posterior hypothalamic area presented either corticotropin-releasing-factor-like or Met-Enk-like immunoreactivity.