Sleep architecture and homeostasis in mice with partial ablation of melanin-concentrating hormone neurons.

Recent reports support a key role of tuberal hypothalamic neurons secreting melanin concentrating-hormone (MCH) in the promotion of Paradoxical Sleep (PS). Controversies remain concerning their concomitant involvement in Slow-Wave Sleep (SWS). We studied the effects of their selective loss achieved by an Ataxin 3-mediated ablation strategy to decipher the contribution of MCH neurons to SWS and/or PS. Polysomnographic recordings were performed on male adult transgenic mice expressing Ataxin-3 transgene within MCH neurons (MCH(Atax)) and their wild-type littermates (MCH(WT)) bred on two genetic backgrounds (FVB/N and C57BL/6). Compared to MCH(WT) mice, MCH(Atax) mice were characterized by a significant drop in MCH mRNAs (-70%), a partial loss of MCH-immunoreactive neurons (-30%) and a marked reduction in brain density of MCH-immunoreactive fibers. Under basal condition, such MCH(Atax) mice exhibited higher PS amounts during the light period and a pronounced SWS fragmentation without any modification of SWS quantities. Moreover, SWS and PS rebounds following 4-h total sleep deprivation were quantitatively similar in MCH(Atax)vs. MCH(WT) mice. Additionally, MCH(Atax) mice were unable to consolidate SWS and increase slow-wave activity (SWA) in response to this homeostatic challenge as observed in MCH(WT) littermates. Here, we show that the partial loss of MCH neurons is sufficient to disturb the fine-tuning of sleep. Our data provided new insights into their contribution to subtle process managing SWS quality and its efficiency rather than SWS quantities, as evidenced by the deleterious impact on two powerful markers of sleep depth, i.e., SWS consolidation/fragmentation and SWA intensity under basal condition and under high sleep pressure.

The supramammillary nucleus and the claustrum activate the cortex during REM sleep.

Evidence in humans suggests that limbic cortices are more active during rapid eye movement (REM or paradoxical) sleep than during waking, a phenomenon fitting with the presence of vivid dreaming during this state. In that context, it seemed essential to determine which populations of cortical neurons are activated during REM sleep. Our aim in the present study is to fill this gap by combining gene expression analysis, functional neuroanatomy, and neurochemical lesions in rats. We find in rats that, during REM sleep hypersomnia compared to control and REM sleep deprivation, the dentate gyrus, claustrum, cortical amygdaloid nucleus, and medial entorhinal and retrosplenial cortices are the only cortical structures containing neurons with an increased expression of Bdnf, FOS, and ARC, known markers of activation and/or synaptic plasticity. Further, the dentate gyrus is the only cortical structure containing more FOS-labeled neurons during REM sleep hypersomnia than during waking. Combining FOS staining, retrograde labeling, and neurochemical lesion, we then provide evidence that FOS overexpression occurring in the cortex during REM sleep hypersomnia is due to projections from the supramammillary nucleus and the claustrum. Our results strongly suggest that only a subset of cortical and hippocampal neurons are activated and display plasticity during REM sleep by means of ascending projections from the claustrum and the supramammillary nucleus. Our results pave the way for future studies to identify the function of REM sleep with regard to dreaming and emotional memory processing.

Genetic deletion of melanin-concentrating hormone neurons impairs hippocampal short-term synaptic plasticity and hippocampal-dependent forms of short-term memory.

The cognitive role of melanin-concentrating hormone (MCH) neurons, a neuronal population located in the mammalian postero-lateral hypothalamus sending projections to all cortical areas, remains poorly understood. Mainly activated during paradoxical sleep (PS), MCH neurons have been implicated in sleep regulation. The genetic deletion of the only known MCH receptor in rodent leads to an impairment of hippocampal dependent forms of memory and to an alteration of hippocampal long-term synaptic plasticity. By using MCH/ataxin3 mice, a genetic model characterized by a selective deletion of MCH neurons in the adult, we investigated the role of MCH neurons in hippocampal synaptic plasticity and hippocampal-dependent forms of memory. MCH/ataxin3 mice exhibited a deficit in the early part of both long-term potentiation and depression in the CA1 area of the hippocampus. Post-tetanic potentiation (PTP) was diminished while synaptic depression induced by repetitive stimulation was enhanced suggesting an alteration of pre-synaptic forms of short-term plasticity in these mice. Behaviorally, MCH/ataxin3 mice spent more time and showed a higher level of hesitation as compared to their controls in performing a short-term memory T-maze task, displayed retardation in acquiring a reference memory task in a Morris water maze, and showed a habituation deficit in an open field task. Deletion of MCH neurons could thus alter spatial short-term memory by impairing short-term plasticity in the hippocampus. Altogether, these findings could provide a cellular mechanism by which PS may facilitate memory encoding. Via MCH neuron activation, PS could prepare the day's learning by increasing and modulating short-term synaptic plasticity in the hippocampus.

Role of the lateral paragigantocellular nucleus in the network of paradoxical (REM) sleep: an electrophysiological and anatomical study in the rat.

The lateral paragigantocellular nucleus (LPGi) is located in the ventrolateral medulla and is known as a sympathoexcitatory area involved in the control of blood pressure. In recent experiments, we showed that the LPGi contains a large number of neurons activated during PS hypersomnia following a selective deprivation. Among these neurons, more than two-thirds are GABAergic and more than one fourth send efferent fibers to the wake-active locus coeruleus nucleus. To get more insight into the role of the LPGi in PS regulation, we combined an electrophysiological and anatomical approach in the rat, using extracellular recordings in the head-restrained model and injections of tracers followed by the immunohistochemical detection of Fos in control, PS-deprived and PS-recovery animals. With the head-restrained preparation, we showed that the LPGi contains neurons specifically active during PS (PS-On neurons), neurons inactive during PS (PS-Off neurons) and neurons indifferent to the sleep-waking cycle. After injection of CTb in the facial nucleus, the neurons of which are hyperpolarized during PS, the largest population of Fos/CTb neurons visualized in the medulla in the PS-recovery condition was observed in the LPGi. After injection of CTb in the LPGi itself and PS-recovery, the nucleus containing the highest number of Fos/CTb neurons, moreover bilaterally, was the sublaterodorsal nucleus (SLD). The SLD is known as the pontine executive PS area and triggers PS through glutamatergic neurons. We propose that, during PS, the LPGi is strongly excited by the SLD and hyperpolarizes the motoneurons of the facial nucleus in addition to local and locus coeruleus PS-Off neurons, and by this means contributes to PS genesis.

Brainstem mechanisms of paradoxical (REM) sleep generation.

Paradoxical sleep (PS) is characterized by EEG activation with a disappearance of muscle tone and the occurrence of rapid eye movements (REM) in contrast to slow-wave sleep (SWS, also known as non-REM sleep) identified by the presence of delta waves. Soon after the discovery of PS, it was demonstrated that the structures necessary and sufficient for its genesis are restricted to the brainstem. We review here recent results indicating that brainstem glutamatergic and GABAergic, rather than cholinergic and monoaminergic, neurons play a key role in the genesis of PS. We hypothesize that the entrance to PS from SWS is due to the activation of PS-on glutamatergic neurons localized in the pontine sublaterodorsal tegmental nucleus. The activation of these neurons would be due to a permanent glutamatergic input arising from the lateral and ventrolateral periaqueductal gray (vlPAG) and the removal at the onset of PS of a GABAergic inhibition present during W and SWS. Such inhibition would be coming from PS-off GABAergic neurons localized in the vlPAG and the adjacent deep mesencephalic reticular nucleus. The cessation of activity of these PS-off GABAergic neurons at the onset and during PS would be due to direct projections from intermingled GABAergic PS-on neurons. Activation of PS would depend on the reciprocal interactions between the GABAergic PS-on and PS-off neurons, intrinsic cellular and molecular events, and integration of multiple physiological parameters.

The neuronal network responsible for paradoxical sleep and its dysfunctions causing narcolepsy and rapid eye movement (REM) behavior disorder.

Rapid eye movement (REM) 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 sub-population 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 pre-motoneurons 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 phasic glutamatergic excitatory pathway from the central amygdala to the SLD PS-on neurons activated during emotion would induce such activation. In normal conditions, the glutamate excitation 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.

A very large number of GABAergic neurons are activated in the tuberal hypothalamus during paradoxical (REM) sleep hypersomnia.

We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD(67) mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD(67)in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD(+), Fos-ir/MCH(+), and GAD(+)/MCH(+) double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD(+) neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis.

Dopaminergic neurons expressing Fos during waking and paradoxical sleep in the rat.

Formerly believed to contribute to behavioural waking (W) alone, dopaminergic (DA) neurons are now also known to participate in the regulation of paradoxical sleep (PS or REM) in mammals. Indeed, stimulation of postsynaptic DA1 receptors with agonists induces a reduction in the daily amount of PS. DA neurons in the ventral tegmental area were recently shown to fire in bursts during PS, but nothing is known about the activity of the other DA cell groups in relation to waking or PS. To fulfil this gap, we used a protocol in which rats were maintained in continuous W for 3h in a novel environment, or specifically deprived of PS for 3 days with some of them allowed to recover from this deprivation. A double immunohistochemical labeling with Fos and tyrosine hydroxylase was then performed. DA neurons in the substantia nigra (A9) and ventral tegmental area (A10), and its dorsocaudal extension in the periaqueductal gray (A10dc), almost never showed a Fos-immunoreactive nucleus, regardless of the experimental condition. The caudal hypothalamic (A11) group showed a moderate activation after PS deprivation and novel environment. During PS-recovery, the zona incerta (A13) group contained a significant number and percentage of double-labeled neurons. These results suggest that some DA neurons (A11) could participate in waking and/or the inhibition of PS during PS deprivation whereas others (A13) would be involved in the control of PS.

Brain serotonergic circuitries.

Brain serotonergic circuitries interact with other neurotransmitter systems on a multitude of different molecular levels. In humans, as in other mammalian species, serotonin (5-HT) plays a modulatory role in almost every physiological function. Furthermore, serotonergic dysfunction is thought to be implicated in several psychiatric and neurodegenerative disorders. We describe the neuroanatomy and neurochemistry of brain serotonergic circuitries. The contribution of emergent in vivo imaging methods to the regional localization of binding site receptors and certain aspects of their functional connectivity in correlation to behavior is also discussed. 5-HT cell bodies, mainly localized in the raphe nuclei, send axons to almost every brain region. It is argued that the specificity of the local chemocommunication between 5-HT and other neuronal elements mainly depends on mechanisms regulating the extracellular concentration of 5-HT the diversity of high-affinity membrane receptors, and their specific transduction modalities.

Role of the melanin-concentrating hormone neuropeptide in sleep regulation.

Melanin-concentrating hormone (MCH), a neuropeptide secreted by a limited number of neurons within the tuberal hypothalamus, has been drawn in the field of sleep only fairly recently in 2003. Since then, growing experimental evidence indicates that MCH may play a crucial role in the homeostatic regulation of paradoxical sleep (PS). MCH-expressing neurons fire specifically during PS. When injected icv MCH induces a 200% increase in PS quantities in rats and the lack of MCH induces a decrease in sleep quantities in transgenic mice. Here, we review recent studies suggesting a role for MCH in the regulation of the sleep-wake cycle, in particular PS, including insights on (1) the specific activity of MCH neurons during PS; (2) how they might be controlled across the sleep-wake cycle; (3) how they might modulate PS; (4) and finally whether MCH might take part in the expression of some symptoms observed in primary sleep disorders.

Localization of the brainstem GABAergic neurons controlling paradoxical (REM) sleep.

Paradoxical sleep (PS) is a state characterized by cortical activation, rapid eye movements and muscle atonia. Fifty years after its discovery, the neuronal network responsible for the genesis of PS has been only partially identified. We recently proposed that GABAergic neurons would have a pivotal role in that network. To localize these GABAergic neurons, we combined immunohistochemical detection of Fos with non-radioactive in situ hybridization of GAD67 mRNA (GABA synthesis enzyme) in control rats, rats deprived of PS for 72 h and rats allowed to recover after such deprivation. Here we show that GABAergic neurons gating PS (PS-off neurons) are principally located in the ventrolateral periaqueductal gray (vlPAG) and the dorsal part of the deep mesencephalic reticular nucleus immediately ventral to it (dDpMe). Furthermore, iontophoretic application of muscimol for 20 min in this area in head-restrained rats induced a strong and significant increase in PS quantities compared to saline. In addition, we found a large number of GABAergic PS-on neurons in the vlPAG/dDPMe region and the medullary reticular nuclei known to generate muscle atonia during PS. Finally, we showed that PS-on neurons triggering PS localized in the SLD are not GABAergic. Altogether, our results indicate that multiple populations of PS-on GABAergic neurons are distributed in the brainstem while only one population of PS-off GABAergic neurons localized in the vlPAG/dDpMe region exist. From these results, we propose a revised model for PS control in which GABAergic PS-on and PS-off neurons localized in the vlPAG/dDPMe region play leading roles.

Noradrenergic neurons expressing Fos during waking and paradoxical sleep deprivation in the rat.

Noradrenaline is known to induce waking (W) and to inhibit paradoxical sleep (PS or REM). Both roles have been exclusively attributed to the noradrenergic neurons of the locus coeruleus (LC, A6), shown to be active during W and inactive during PS. However, the A1, A2, A5 and A7 noradrenergic neurons could also be responsible. Therefore, to determine the contribution of each of the noradrenergic groups in W and in PS inhibition, rats were maintained in continuous W for 3h in a novel environment or specifically deprived of PS for 3 days, with some of them allowed to recover from this deprivation. A double immunohistochemical labeling with Fos and tyrosine hydroxylase was then performed. Thirty percent of the LC noradrenergic cells were found to be Fos-positive after exposure to the novel environment and less than 2% after PS deprivation. In contrast, a significant number of double-labeled neurons (up to 40% of the noradrenergic neurons) were observed in the A1/C1, A2 and A5 groups, after both novel environment and PS deprivation. After PS recovery and in control condition, less than 1% of the noradrenergic neurons were Fos-immunoreactive, regardless of the noradrenergic group. These results indicate that the brainstem noradrenergic cell groups are activated during W and silent during PS. They further suggest that the inhibitory effect of noradrenaline on PS may be due to the A1/C1, A2 and to a lesser degree to A5 neurons but not from those of the LC as previously hypothesized.

Characterization of the melanin-concentrating hormone neurons activated during paradoxical sleep hypersomnia in rats.

Although the main nodes of the neuronal network that regulate paradoxical sleep (PS), also called rapid eye movement sleep, have been identified in rodents, it still needs to be more thoroughly described. We have recently shown that 58% of a hypothalamic neuronal population, the melanin-concentrating hormone (MCH) neurons, are activated after a PS hypersomnia and that MCH, when injected intracerebroventricularly, induces a dose-dependent increase in PS. This suggests that MCH plays a role in PS regulation. Two subpopulations of MCH neurons have been distinguished neurochemically, one that coexpresses cocaine and amphetamine-regulated transcript (CART) and sends ascending projections to the septum and the hippocampus, the other, the non-CART MCH neurons, send descending projections to the lower brainstem and the spinal cord. In order to better characterize the PS-activated MCH neurons it is interesting to determine whether they belong to the first, the second, or both subgroups. We therefore undertook an MCH, CART, and Fos triple immunolabeling study in PS hypersomniac rats. We showed that the MCH neurons activated during PS are part of both subpopulations since we found CART and non-CART MCH-activated neurons. Based on these results and the literature, we propose that MCH could be involved in memory processes and in the inhibition of muscle tone during PS.

A comparison of in vivo and in vitro neuroimaging of 5-HT 1A receptor binding sites in the cat brain.

To validate the cat as a suitable model for positron emission tomography imaging (PET) and to gain further knowledge on the anatomical distribution of the serotonin-1A receptor (5-HT 1A) in the feline brain, we used PET with [18F]MPPF and in vitro autoradiography with [3H]MPPF, [3H]8-OH-DPAT and [3H]paroxetine. PET radioactivity curves with [18F]MPPF were very reproducible in anaesthetized cats, with the highest radioactivity uptakes recorded in the hippocampus, cingulate cortex, septum, infralimbic cortex and raphe nucleus, whereas the lowest were found in the cerebellum. [3H]8-OH-DPAT binding displayed a comparable, albeit lower, regional distribution than with [3H]MPPF. Autoradiography also revealed the presence of 5-HT 1A receptor binding sites in the cortex and in the interpeduncular nucleus, due to its greater sensitivity and spatial resolution compared with PET imaging. The cat constitutes an interesting experimental model for PET imaging, as many physiological concepts have been well established with this animal. Our study also shows the advantages of combining complementary neuroimaging techniques such as in vivo PET imaging and in vitro autoradiography to visualize the distribution of the 5-HT 1A receptors.

Localization of the neurons active during paradoxical (REM) sleep and projecting to the locus coeruleus noradrenergic neurons in the rat.

Locus coeruleus (LC) noradrenergic neurons are active during wakefulness, slow their discharge rate during slow wave sleep, and stop firing during paradoxical sleep (PS). A large body of data indicates that their inactivation during PS is due to a tonic GABAergic inhibition. To localize the neurons responsible for such inhibition, we first examined the distribution of retrogradely and Fos double-immunostained neurons following cholera toxin b subunit (CTb) injection in the LC of control rats, rats selectively deprived of PS for 3 days, and rats allowed to recover for 3 hours from such deprivation. We found a significant number of CTb/Fos double-labeled cells only in the recovery group. The largest number of CTb/Fos double-labeled cells was found in the dorsal paragigantocellular reticular nucleus (DPGi). It indeed contained 19% of the CTb/Fos double-labeled neurons, whereas the ventrolateral periaqueductal gray (vlPAG) contained 18.3% of these neurons, the lateral paragigantocellular reticular nucleus (LPGi) 15%, the lateral hypothalamic area 9%, the lateral PAG 6.7%, and the rostral PAG 6%. In addition, CTb/Fos double-labeled cells constituted 43% of all the singly CTb-labeled cells counted in the DPGi compared with 29% for the LPGi, 18% for the rostral PAG, and 10% or less for the other structures. Although all these populations of CTb/Fos double-labeled neurons could be GABAergic and tonically inhibit LC neurons during PS, our results indicate that neurons from the DPGi constitute the best candidate for this role.

Paradoxical (REM) sleep genesis: the switch from an aminergic-cholinergic to a GABAergic-glutamatergic hypothesis.

In the middle of the last century, Michel Jouvet discovered paradoxical sleep (PS), a sleep phase paradoxically characterized by cortical activation and rapid eye movements and a muscle atonia. Soon after, he showed that it was still present in "pontine cats" in which all structures rostral to the brainstem have been removed. Later on, it was demonstrated that the pontine peri-locus coeruleus alpha (peri-LCalpha in cats, corresponding to the sublaterodorsal nucleus, SLD, in rats) is responsible for PS onset. It was then proposed that the onset and maintenance of PS is due to a reciprocal inhibitory interaction between neurons presumably cholinergic specifically active during PS localized in this region and monoaminergic neurons. In the last decade, we have tested this hypothesis with our model of head-restrained rats and functional neuroanatomical studies. Our results confirmed that the SLD in rats contains the neurons responsible for the onset and maintenance of PS. They further indicate that (1) these neurons are non-cholinergic possibly glutamatergic neurons, (2) they directly project to the glycinergic premotoneurons localized in the medullary ventral gigantocellular reticular nucleus (GiV), (3) the main neurotransmitter responsible for their inhibition during waking (W) and slow wave sleep (SWS) is GABA rather than monoamines, (4) they are constantly and tonically excited by glutamate and (5) the GABAergic neurons responsible for their tonic inhibition during W and SWS are localized in the deep mesencephalic reticular nucleus (DPMe). We also showed that the tonic inhibition of locus coeruleus (LC) noradrenergic and dorsal raphe (DRN) serotonergic neurons during sleep is due to a tonic GABAergic inhibition by neurons localized in the dorsal paragigantocellular reticular nucleus (DPGi) and the ventrolateral periaqueductal gray (vlPAG). We propose that these GABAergic neurons also inhibit the GABAergic neurons of the DPMe at the onset and during PS and are therefore responsible for the onset and maintenance of PS.

Cholinergic and noncholinergic brainstem neurons expressing Fos after paradoxical (REM) sleep deprivation and recovery.

It is well accepted that populations of neurons responsible for the onset and maintenance of paradoxical sleep (PS) are restricted to the brainstem. To localize the structures involved and to reexamine the role of mesopontine cholinergic neurons, we compared the distribution of Fos- and choline acetyltransferase-labelled neurons in the brainstem of control rats, rats selectively deprived of PS for approximately 72 h and rats allowed to recover from such deprivation. Only a few cholinergic neurons from the laterodorsal (LDTg) and pedunculopontine tegmental nuclei were Fos-labelled after PS recovery. In contrast, a large number of noncholinergic Fos-labelled cells positively correlated with the percentage of time spent in PS was observed in the LDTg, sublaterodorsal, alpha and ventral gigantocellular reticular nuclei, structures known to contain neurons specifically active during PS. In addition, a large number of Fos-labelled cells were seen after PS rebound in the lateral, ventrolateral and dorsal periaqueductal grey, dorsal and lateral paragigantocellular reticular nuclei and the nucleus raphe obscurus. Interestingly, half of the cells in the latter nucleus were immunoreactive to choline acetyltransferase. In contrast to the well-accepted hypothesis, our results strongly suggest that neurons active during PS, recorded in the mesopontine cholinergic nuclei, are in the great majority noncholinergic. Our findings further demonstrate that many brainstem structures not previously identified as containing neurons active during PS contain cholinergic or noncholinergic neurons active during PS, and these structures may therefore play a key role during this state. Altogether, our results open a new avenue of research to identify the specific role of the populations of neurons revealed, their interrelations and their neurochemical identity.

A role of melanin-concentrating hormone producing neurons in the central regulation of paradoxical sleep.

BACKGROUND: Peptidergic neurons containing the melanin-concentrating hormone (MCH) and the hypocretins (or orexins) are intermingled in the zona incerta, perifornical nucleus and lateral hypothalamic area. Both types of neurons have been implicated in the integrated regulation of energy homeostasis and body weight. Hypocretin neurons have also been involved in sleep-wake regulation and narcolepsy. We therefore sought to determine whether hypocretin and MCH neurons express Fos in association with enhanced paradoxical sleep (PS or REM sleep) during the rebound following PS deprivation. Next, we compared the effect of MCH and NaCl intracerebroventricular (ICV) administrations on sleep stage quantities to further determine whether MCH neurons play an active role in PS regulation. RESULTS: Here we show that the MCH but not the hypocretin neurons are strongly active during PS, evidenced through combined hypocretin, MCH, and Fos immunostainings in three groups of rats (PS Control, PS Deprived and PS Recovery rats). Further, we show that ICV administration of MCH induces a dose-dependent increase in PS (up to 200%) and slow wave sleep (up to 70%) quantities. CONCLUSION: These results indicate that MCH is a powerful hypnogenic factor. MCH neurons might play a key role in the state of PS via their widespread projections in the central nervous system.

Neurokinin NK1- and NK3-immunoreactive neurons in serotonergic cell groups in the rat brain.

Interactions are known to occur in the brain between serotonin (5-HT) and substance P (SP). To investigate whether SP can directly influence serotonergic neurons, double immunohistochemical labelings were performed on rat brain sections with NK1 or NK3 affinity-purified antibodies and a 5-HT monoclonal antibody. It was found that the vast majority of serotonergic cell bodies do not colocalize NK1 or NK3 labeling. Only in the central linear nucleus and ventral part of the dorsal raphe nucleus were a few serotonergic neurons double-labeled for NK1 receptors (15 and 0.8% of serotonergic neurons, respectively). It is suggested that serotonergic neurons are not major direct targets for SP in the rat brain.