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 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.

Brain extracellular glucose assessed by voltammetry throughout the rat sleep-wake cycle.

In the present study, cortical extracellular levels of glucose were monitored for the first time throughout the sleep-wake states of the freely moving rat. For this purpose, polygraphic recordings (electroencephalogram of the fronto-occipital cortices and electromyogram of the neck muscles) were achieved in combination with differential normal pulse voltammetry (DNPV) using a specific glucose sensor. Data obtained reveal that the basal extracellular glucose concentration in the conscious rat is 0.59 +/- 0.3 m M while under chloral hydrate anaesthesia (0.4 g/kg, i.p.) it increases up to 180% of its basal concentration. Regarding the sleep-wake cycle, the existence of spontaneous significant variations in the mean glucose level during slow-wave sleep (SWS = +13%) and paradoxical sleep (PS = -11%) compared with the waking state (100%) is also reported. It is to be noticed that during long periods of active waking, glucose level tends towards a decrease that becomes significant after 15 min (active waking = -32%). On the contrary, during long episodes of slow-wave sleep, it tends towards an increase which becomes significant after 12 min (SWS = +28%). It is suggested that voltammetric techniques using enzymatic biosensors are useful tools allowing direct glucose measurements in the freely moving animal. On the whole, paradoxical sleep is pointed out as a state highly dependent on the availability of energy and slow-wave sleep as a period of energy saving.

Effect of decerebration on blood pressure during paradoxical sleep in cats.

We investigated the effects of decerebration on long-term variations in arterial blood pressure during paradoxical sleep (PS) in cats. In normal cats, the blood pressure decreased during the transition from slow wave sleep to PS and maintained its lower level throughout PS for several days after surgery. After this early postoperative stage, however, the arterial hypotension was replaced by tonic and phasic rises in blood pressure during PS. Such long-term changes in blood pressure were completely abolished when the brain stem was transected at the ponto-mesencephalic junction, and the cats consistently exhibited a sustained fall in blood pressure throughout the survival periods of 1 month or more.

Long-term variations of arterial blood pressure during sleep in freely moving cats.

Using a new telemetric system for arterial blood pressure recordings, we have investigated long-term postoperative changes in blood pressure during sleep in freely moving cats. Particular attention was paid to the transitional periods at the beginning and end of paradoxical sleep (PS), as well as to the relationship between the blood pressure and ponto-geniculo-occipital (PGO) waves. In the initial postoperative stage lasting 2 to 5 days, the blood pressure decreased during the transition from slow wave sleep (SWS) to PS and maintained its lower level until the end of PS. In contrast, in the later chronic stage, the blood pressure increased tonically during the transition from SWS to PS and maintained its higher level throughout PS on which several phasic rises in blood pressure were superimposed. A significant increase in arterial pressure during the transitional period began shortly after the first appearance of PGO waves. On the other hand, significant phasic rises in arterial pressure during PS shortly preceded the onset of PGO wave bursts.

Power spectral analysis of blood pressure fluctuations during sleep in normal and decerebrate cats.

Arterial blood pressure fluctuations during sleep were investigated with power analysis technique in both normal and decerebrate cats. In the initial postoperative stage lasting about 3 to 4 days, intact cats displayed, during paradoxical sleep, phasic increases in arterial blood pressure which were superimposed on a tonic hypotension. In the later chronic stage, however, the animals showed the phasic hypertension being superimposed on the background of a tonic hypertension. Regardless of these stages, the blood pressure during paradoxical sleep exhibited a 1/f-like spectrum, expressed by the power spectral density which is inversely proportional to the Fourier frequency f. On the other hand, a power spectral profile of the blood pressure during slow wave sleep presented a white noise-like pattern within the same frequency range of 0.1-0.01 Hz. After brainstem transections at the pontomesencephalic border, the cats exhibited consistently a sustained fall in blood pressure during paradoxical sleep and the power spectral density of the blood pressure displayed a white noise-like pattern throughout the survival periods of one month or more. These observations indicate that the blood pressure fluctuations in the 1/f spectrum during paradoxical sleep originate in rostral brain structures.

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.

Lower brainstem afferents to the cat posterior hypothalamus: a double-labeling study.

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.

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.

Cells of origin of histaminergic afferents to the cat median eminence.

The exact origin of histaminergic neuronal perikarya sending axons to the median eminence and posterior pituitary was investigated in the cat by using two colour double-immunostaining methods: unconjugated wheat germ agglutinin or cholera toxin as retrograde tracers combined with histamine (HA) immunohistochemistry. HA immunohistochemistry revealed the presence of HA-immunoreactive terminal-like fibers both in the external layer of the median eminence and neural lobe of the pituitary. The double-labeling studies further demonstrated the histaminergic innervation of the median eminence and neural lobe by a few HA-immunoreactive neuronal perikarya located in the posterior hypothalamus.

Forebrain afferents to the cat posterior hypothalamus: a double labeling study.

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.

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.

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)

Peptidergic hypothalamic afferents to the cat nucleus raphe pallidus as revealed by a double immunostaining technique using unconjugated cholera toxin as a retrograde tracer.

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.

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 cholinergic neurons in the cat lower brain stem]. / Localisation des neurones cholinergiques dans le tronc cérébral inférieur chez le chat.

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.

[Histamine-immunoreactive neurons in the hypothalamus of cats]. / Neurones immunoréactifs à l'histamine dans l'hypothalamus chez le chat.

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.

Possible role of serotonin in the secretory activity of the subcommissural organ of the cat.

The possible role of serotonin (5-hydroxytryptamine, 5-HT) in the secretory activity of the subcommissural organ (SCO) was investigated in the cat by using 5-HT immunohistochemistry and Gomori's secretory staining technique. Administration of para-chlorophenylalanine, a potent inhibitor of 5-HT synthesis, was found to produce a marked decrease in both 5-HT-immunoreactive materials and secretory granules in the SCO. In contrast, administration of 5-hydroxytryptophan, an immediate precursor of 5-HT, to animals pretreated with para-chlorophenylalanine resulted in an increase in the amount of both secretory granules and 5-HT immunoreactivities in this circumventricular organ. These results strongly suggest that the secretory activity of the SCO of cats is regulated by 5-HT.