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.

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.

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.

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.

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.

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.

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.

Afferents to the nucleus reticularis parvicellularis of the cat medulla oblongata: a tract-tracing study with cholera toxin B subunit.

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.

The nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat nucleus reticularis magnocellularis: a double-labeling study with cholera toxin as a retrograde tracer.

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)

Periventricular dopaminergic neurons terminating in the neuro-intermediate lobe of the cat hypophysis.

The aim of the present study was to determine the exact origins of the dopaminergic hypothalamohypophyseal projections in the cat brain. For this purpose, we used a retrograde tracer technique with horseradish peroxidase (HRP) in conjunction with tyrosine hydroxylase (TH) immunohistochemistry as a marker for the dopaminergic neurons. After injections of the tracer into the neuro-intermediate lobe, a substantial number of HRP-labeled neurons was observed in the supraoptic and paraventricular neurosecretory nuclei. Furthermore, a cluster of HRP-positive neurons was found in the tuberal component of the periventricular nucleus where few, if any, neurosecretory magnocellular cells are identified. TH immunohistochemistry on the same sections further revealed that virtually all these HRP-containing neurons showed TH immunoreactivity. These double-labeled neurons were medium in size and fusiform or ovoid and appeared to belong to the A14 dopamine cell group. In addition to these medium-sized double-labeled neurons, a magnocellular type of double-labeled cell body was identified just adjacent to the organum vasculosum of the lamina terminalis and in and around the supraoptic and paraventricular nuclei. These double-labeled cells appeared to be members of the A14 and A15 dopamine cell groups. In conclusion, the present study indicated that the dopaminergic projections to the cat neurointermediate lobe might originate mainly in the medium-sized cells located in the tuberal periventricular nucleus and partly in the large-sized cells located in and around the supraoptic and paraventricular neurosecretory nuclei.

Localization of tyrosine hydroxylase-immunoreactive neurons in the cat hypothalamus, with special reference to fluorescence histochemistry.

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.

Monoaminergic, peptidergic, and cholinergic afferents to the cat facial nucleus as evidenced by a double immunostaining method with unconjugated cholera toxin as a retrograde tracer.

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.

Lower brainstem catecholamine afferents to the rat dorsal raphe nucleus.

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.

Nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat trigeminal motor nucleus: a double-labeling study with cholera-toxin as a retrograde tracer.

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.

Distribution of glycine-immunoreactive cell bodies and fibers in the rat brain.

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.

Afferent projections to the rat locus coeruleus demonstrated by retrograde and anterograde tracing with cholera-toxin B subunit and Phaseolus vulgaris leucoagglutinin.

The aim of this study was to examine the afferents to the rat locus coeruleus by means of retrograde and anterograde tracing experiments using cholera-toxin B subunit and phaseolus leucoagglutinin. To obtain reliable injections of cholera-toxin B in the locus coeruleus, electrophysiological recordings were made through glass micropipettes containing the tracer and the noradrenergic neurons of the locus coeruleus were identified by their characteristic discharge properties. After iontophoretic injections of cholera-toxin B into the nuclear core of the locus coeruleus, we observed a substantial number of retrogradely labeled cells in the lateral paragigantocellular nucleus and the dorsomedial rostral medulla (ventromedial prepositus hypoglossi and dorsal paragigantocellular nuclei) as previously described. We also saw a substantial number of retrogradely labeled neurons in (1) the preoptic area dorsal to the supraoptic nucleus, (2) areas of the posterior hypothalamus, (3) the Kölliker-Fuse nucleus, (4) mesencephalic reticular formation. Fewer labeled cells were also observed in other regions including the hypothalamic paraventricular nucleus, dorsal raphe nucleus, median raphe nucleus, dorsal part of the periaqueductal gray, the area of the noradrenergic A5 group, the lateral parabrachial nucleus and the caudoventrolateral reticular nucleus. No or only occasional cells were found in the cortex, the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the vestibular nuclei, the nucleus of the solitary tract or the spinal cord, structures which were previously reported as inputs to the locus coeruleus. Control injections of cholera-toxin B were made in areas surrounding the locus coeruleus, including (1) Barrington's nucleus, (2) the mesencephalic trigeminal nucleus, (3) a previously undefined area immediately rostral to the locus coeruleus and medial to the mesencephalic trigeminal nucleus that we named the peri-mesencephalic trigeminal nucleus, and (4) the medial vestibular nucleus lateral to the caudal tip of the locus coeruleus. These injections yielded patterns of retrograde labeling that differed from one another and also from that obtained with cholera-toxin B injection sites in the locus coeruleus. These results indicate that the area surrounding the locus coeruleus is divided into individual nuclei with distinct afferents. These results were confirmed and extended with anterograde transport of cholera-toxin B or phaseolus leucoagglutinin. Injections of these tracers in the lateral paragigantocellular nucleus, preoptic area dorsal to the supraoptic nucleus, the ventrolateral part of the periaqueductal gray, the Kölliker-Fuse nucleus yielded a substantial to large number of labeled fibers in the nuclear core of the locus coeruleus.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

Catecholaminergic afferents to the cat median eminence as determined by double-labelling methods.

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.

Fos and serotonin immunoreactivity in the raphe nuclei of the cat during carbachol-induced active sleep: a double-labeling study.

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)