Melanin-concentrating hormone (MCH) in the median raphe nucleus: Fibers, receptors and cellular effects.

Serotonergic neurons of the median raphe nucleus (MnR) and hypothalamic melanin-concentrating hormone (MCH)-containing neurons, have been involved in the control of REM sleep and mood. In the present study, we examined in rats and cats the anatomical relationship between MCH-containing fibers and MnR neurons, as well as the presence of MCHergic receptors in these neurons. In addition, by means of in vivo unit recording in urethane anesthetized rats, we determined the effects of MCH in MnR neuronal firing. Our results showed that MCH-containing fibers were present in the central and paracentral regions of the MnR. MCHergic fibers were in close apposition to serotonergic and non-serotonergic neurons. By means of an indirect approach, we also analyzed the presence of MCHergic receptors within the MnR. Accordingly, we microinjected MCH conjugated with the fluorophore rhodamine (R-MCH) into the lateral ventricle. R-MCH was internalized into serotonergic and non-serotonergic MnR neurons; some of these neurons were GABAergic. Furthermore, we determined that intracerebroventricular administration of MCH induced a significant decrease in the firing rate of 53 % of MnR neurons, while the juxtacellular administration of MCH reduced the frequency of discharge in 67 % of these neurons. Finally, the juxtacellular administration of the MCH-receptor antagonist ATC-0175 produced an increase in the firing rate in 78 % of MnR neurons. Hence, MCH produces a strong regulation of MnR neuronal activity. We hypothesize that MCHergic modulation of the MnR neuronal activity may be involved in the promotion of REM sleep and in the pathophysiology of depressive disorders.

Melanin-concentrating hormone does not modulate serotonin release in primary cultures of fetal raphe nucleus neurons.

Melanin-concentrating hormone (MCH) is a neuropeptide present in neurons located in the hypothalamus that densely innervate serotonergic cells in the dorsal raphe nucleus (DRN). MCH administration into the DRN induces a depressive-like effect through a serotonergic mechanism. To further understand the interaction between MCH and serotonin, we used primary cultured serotonergic neurons to evaluate the effect of MCH on serotonergic release and metabolism by HPLC-ED measurement of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels. We confirmed the presence of serotonergic neurons in the E14 rat rhombencephalon by immunohistochemistry and showed for the first time evidence of MCHergic fibers reaching the area. Cultures obtained from rhombencephalic tissue presented 2.2 ±â€¯0.7% of serotonergic and 48.9 ±â€¯5.4% of GABAergic neurons. Despite the low concentration of serotonergic neurons, we were able to measure basal cellular and extracellular levels of 5-HT and 5-HIAA without the addition of any serotonergic-enhancer drug. As expected, 5-HT release was calcium-dependent and induced by depolarization. 5-HT extracellular levels were significantly increased by incubation with serotonin reuptake inhibitors (citalopram and nortriptyline) and a monoamine-oxidase inhibitor (clorgyline), and were not significantly modified by a 5-HT1A autoreceptor agonist (8-OHDPAT). Even though serotonergic cells responded as expected to these pharmacological treatments, MCH did not induce significant modifications of 5-HT and 5-HIAA extracellular levels in the cultures. Despite this unexpected result, we consider that assessment of 5-HT and 5-HIAA levels in primary serotonergic cultures may be an adequate approach to study the effect of other drugs and modulators on serotonin release, uptake and turnover.

MCH levels in the CSF, brain preproMCH and MCHR1 gene expression during paradoxical sleep deprivation, sleep rebound and chronic sleep restriction.

Neurons that utilize melanin-concentrating hormone (MCH) as neuromodulator are located in the lateral hypothalamus and incerto-hypothalamic area. These neurons project throughout the central nervous system and play a role in sleep regulation. With the hypothesis that the MCHergic system function would be modified by the time of the day as well as by disruptions of the sleep-wake cycle, we quantified in rats the concentration of MCH in the cerebrospinal fluid (CSF), the expression of the MCH precursor (Pmch) gene in the hypothalamus, and the expression of the MCH receptor 1 (Mchr1) gene in the frontal cortex and hippocampus. These analyses were performed during paradoxical sleep deprivation (by a modified multiple platform technique), paradoxical sleep rebound and chronic sleep restriction, both at the end of the active (dark) phase (lights were turned on at Zeitgeber time zero, ZT0) and during the inactive (light) phase (ZT8). We observed that in control condition (waking and sleep ad libitum), Mchr1 gene expression was larger at ZT8 (when sleep predominates) than at ZT0, both in frontal cortex and hippocampus. In addition, compared to control, disturbances of the sleep-wake cycle produced the following effects: paradoxical sleep deprivation for 96 and 120 h reduced the expression of Mchr1 gene in frontal cortex at ZT0. Sleep rebound that followed 96 h of paradoxical sleep deprivation increased the MCH concentration in the CSF also at ZT0. Twenty-one days of sleep restriction produced a significant increment in MCH CSF levels at ZT8. Finally, sleep disruptions unveiled day/night differences in MCH CSF levels and in Pmch gene expression that were not observed in control (undisturbed) conditions. In conclusion, the time of the day and sleep disruptions produced subtle modifications in the physiology of the MCHergic system.

Melanin-concentrating hormone control of sleep-wake behavior.

The melanin-concentrating hormone (MCH) is a 19 aminoacid peptide found in mammals predominantly in neurons located in the lateral hypothalamus and incerto-hypothalamic area. The biological function of MCH is mediated by two G-protein-coupled receptors known as MCHR1 and MCHR2, although the latter is expressed only in carnivores, primates and man. The MCHR1 couples to Gi, Gq and Go proteins, with Gi leading to the inhibition of both excitatory and inhibitory synaptic events. Within the central nervous system (CNS) MCH participates in a number of functions including sleep-wake behavior. In this respect, MCHergic neurons project widely throughout the CNS to brain regions involved in the regulation of behavioral states. MCHergic neurons are silent during wakefulness (W), increase their firing during slow wave sleep (SWS) and still more during REM sleep (REMS). Studies in knockout mice for MCH (MCH(-/-)) have shown a reduction in SWS and an increase of W during the light and the dark phase of the light-dark cycle. Moreover, in response to food deprivation a marked reduction in REMS time was observed in these animals. Conflicting effects on sleep variables have been reported in MCHR1(-/-) mice by different authors. The i.c.v. administration of MCH increases REMS and SWS in the rat. In addition, an enhancement of REMS has been described following the microinjection of the neuropeptide into the nucleus pontis oralis of the cat, while its infusion into the dorsal raphe nucleus (DR) and the basal forebrain (horizontal limb of the diagonal band of Broca) is followed by an increase of REMS and a reduction of W in the rat. Immunoneutralization of MCH in the DR augmented W and suppressed REMS in the rat, as did the s.c. injection of selective MCHR1 antagonists. The robust REMS-inducing effect of MCH is likely related to the deactivation of monoaminergic, orexinergic, glutamatergic, cholinergic (W-on) and GABAergic (REM-off) neurons involved in the generation of W and the inhibition of REMS. On the basis of preclinical studies, it can be proposed that selective MCHR1 receptor agonists could constitute potential therapeutic modalities in the arsenal of insomnia pharmacotherapy. Due to the lack of adequate animal models, the role of the MCHR2 on sleep is still unknown.