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Research Report

Effects on sleep of melanin-concentrating hormone (MCH)microinjections into the dorsal raphe nucleus

Patricia Lagosa, Pablo Torteroloa,⁎, Héctor Jantosd, Michael H. Chaseb,c, Jaime M. Montid

aDepartamento de Fisiología, Facultad de Medicina, Universidad de la República, General Flores 2125, 11800, Montevideo, UruguaybWebSciences International, Los Angeles, CA, USAcUCLA School of Medicine, Los Angeles, CA, USAdDepartamento de Farmacología y Terapéutica, Hospital de Clínicas, Montevideo, Uruguay

A R T I C L E I N F O

⁎ Corresponding author. Fax: (5982) 9248784.Abbreviations: CNScentral nervous syste

tromyogram; LDTlaterodorsal tegmental nreceptor type 2; NPOnucleus pontis oralis; PP

0006-8993/$ – see front matter © 2009 Elsevidoi:10.1016/j.brainres.2009.02.010

A B S T R A C T

Article history:Accepted 4 February 2009Available online 20 February 2009

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are locatedin the lateral hypothalamus and incerto-hypothalamic area, and project diffusely throughoutthe central nervous system, including areas that participate in the generation andmaintenance of the states of sleep and wakefulness. Recent studies have shown that thehypothalamicMCHergic neurons are active during rapid eyemovements (REM) sleep, and thatintraventricular microinjections of MCH induce slow wave sleep (SWS) and REM sleep. Thereare particular denseMCHergic projections to the dorsal raphe nucleus (DR), a neuroanatomicalstructure involved in several functions during wakefulness, and in the regulation of sleepvariables. Because of this fact, we analyzed the effect of microinjections of MCH into thisnucleus on sleep and waking states in the rat. Compared to control microinjections, MCH(100 ng) produced a moderate increase in SWS (243.7±6.0 vs. 223.2±8.8 min, p<0.05) and animportant increment in REM sleep (35.5±2.5 vs. 20.8±3.4min, p<0.01) due to an increase in thenumber of REM sleep episodes. The increase of REM sleep was accompanied by a reduction inthe time spent in light sleep and wakefulness. We therefore conclude that the hypothalamicMCHergic system, via its action in the DR, plays an important role in the generation and/ormaintenance of the states of sleep.

© 2009 Elsevier B.V. All rights reserved.

Keywords:NeuropeptideSerotoninHypothalamusREM sleep

1. Introduction

The melanin-concentrating hormone (MCH) was initiallycharacterized as a circulating factor mediating color changein teleost fish (Kawauchi et al., 1983). Thereafter, MCH wasidentified as a 19 amino acid cyclic neuropeptide thatfunctions as a neuromodulator in the rat; MCH was found tobe fully conserved in mammals, including humans (Forray,2003; Shi, 2004; Nahon, 2006; Saito and Nagasaki, 2008). The

m; CSFcerebrospinal fluiduclei; MCHmelanin-conceTpedunculopontine tegm

er B.V. All rights reserved

biological function of MCH is mediated by two G-proteincoupled receptors known as MCHR-1 and MCHR-2. Althoughthe MCHR-2 gene was found to be a pseudo-gene in rodents,this receptor is functional in humans, monkeys, ferrets anddogs (An et al., 2001; Tan et al., 2002).

Neurons that synthesize MCH are located mainly in thelateral hypothalamus and incerto-hypothalamic area, andproject throughout the central nervous system (CNS) (Bitten-court et al., 1992; Mouri et al., 1993; Torterolo et al., 2006). They

; DRdorsal raphe nucleus; EEGelectroencephalogram; EMGelec-ntrating hormone; MCHR-1MCH receptor type 1; MCHR-2MCHental nuclei; REMrapid eye movements; SWSslow wave sleep

.

Fig. 1 – Microinjections were localized in the dorsal raphenucleus. (A) Photomicrograph of a coronal section of arepresentative rat brain at the level of the DR. Themicroinjection site is recognizable by the cannula track andlesion. This 5μm thick sectionwas stainedwith hematoxilinand eosin. (B) Microinjection site localized by Pontamine SkyBlue microinjection. The blue stain is readily observed in theDR. This 40 μm thick coronal section was counterstainedwith Pyronin-y. (C) Schematic showing the location of themicroinjection sites. Each spot corresponds approximately tothe center of the cannula lesion or of the dye deposit, and itsposition was adjusted to the prototypical coronal sections ofPaxinos and Watson (2005). Aq, aqueduct; IC, inferiorcolliculus; dPAG, dorsal periaqueductal gray; DR, dorsalraphe nucleus; mlf, medial longitudinal fascicle. Calibrationbar, 0.5 mm.

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send relatively dense projections to specific areas of thebrainstem that have been involved in sleep and wakingfunctions such as the dorsal raphe nucleus (DR), the locuscoeruleus, the ventral periaqueductal gray and the laterodor-sal and pedunculopontine tegmental nuclei (LDT and PPT);high densities of MCHR-1 are also present in these areas(Bittencourt et al., 1992; Hervieu et al., 2000; Kilduff and DeLecea, 2001; Saito et al., 2001; Jones, 2005; Vanini et al., 2007).

MCH has been involved in the central regulation of feedingand energy homeostasis (Nahon, 2006; Saito and Nagasaki,2008). However, based upon the expression of the immediate-early gene c-fos, which is used as amarker of neuronal activity,recent reports have shown that MCHergic neurons in the ratare active during sleep, predominantly during rapid eyemovement (REM) sleep (Verret et al., 2003; Modirrousta et al.,2005; Hanriot et al., 2007). In addition, there is a large increasein REM sleep after the intraventricular microinjections ofMCH; consequently, it has been suggested that MCH plays animportant role in the control of REM sleep (Verret et al., 2003).It was recently demonstrated in rats, that systemically appliedMCHR-1 antagonists results in a dose-dependent decrease inslow wave sleep (SWS) and REM sleep, together with anequivalent increase in wakefulness (Ahnaou et al., 2008). Inaccordance with the preceding results, MCH knock-out micesleep less than wild-type animals, and in response to fastingexhibited hyperactivity and an exaggerated decrease in REMsleep (Willie et al., 2008). However, these results are difficult toreconcile with the finding that MCHR-1 knock-out mice pre-sent larger amounts of REM sleep in comparison to wild typeanimals (Adamantidis et al., 2008).

In the cat, studies that employed Fos immunoreactivity didnot replicate previous findings that were obtained in rats (Tor-terolo et al., 2006). However, microinjections of MCH into thenucleus pontis oralis (NPO), an executive area for REM sleepgeneration, result in an increment in REM sleep (Torteroloet al., in press).

Since the DR is strongly innervated by theMCHergic systemand plays a key role in waking-related activities (Jacobs andAzmitia, 1992; Dringenberg and Vanderwolf, 1998; Jacobs andFornal, 1999; Monti and Monti, 2000), in the present study weexplored the effects of microinjections of MCH into the DR onwakefulness and sleep in the rat. The administration of MCHinto the DR produced a large increase in the time spent in REMsleep as well as a modest increase in SWS accompanied by adecrease in wakefulness and light sleep. This results supportthe hypothesis that MCHergic neurons promote the sleepstates by modulating the activity of the DR neurons.

2. Results

MCH (50 and 100 ng) and vehicle (as a control) weremicroinjected into the DR in six rats. In all animals, the tipof the cannula was localized in the DR. Photomicrographsshowing examples of the cannula track and lesion, the dyedeposit after the end of the microinjections procedures, aswell as a schematic of the locations of the microinjectionssites are presented in Fig. 1.

The results obtained after recording sessions of 6 hfollowing microinjections of MCH or vehicle into the DR are

summarized in Table 1. Compared with the control vehicle,MCH 100 ng significantly modified the values correspondingto a number of sleep variables. Accordingly, MCH 100 ngincreased significantly REM sleep time from a control value of20.8±3.4 min (5.8% of the total recording time) to 35.5±2.5 min(9.7% of the total recording time). The increment of REM sleeptime amounted to 70.7% of the control value, and was relatedto a greater number of REM episodes (Table 1). The increasednumber of REM episodes after MCH 100 ng administration is

Table 1 – Effects of MCH microinjected into the dorsal raphenucleus on sleep and waking during 6-h polysomnographicrecordings

Control MCH (50 ng) MCH (100 ng)

Wakefulness 63.0±7.7 66.3±4.9 41.7±4.8⁎Light sleep 53.0±5.2 42.2±5.9 39.2±3.7⁎Slow wave sleep 223.2±8.8 227.0±8.8 243.7±6.0⁎REM sleep 20.8±3.4 24.2±1.8 35.5±2.5⁎⁎Number of

REM episodes9.7±1.6 11.2±1.3 14.2±0.9⁎⁎

Mean REMperiod duration

2.2±0.1 2.2±0.2 2.5±0.1

Slow wavesleep latency

3.7±2.0 1.2±0.8 0.7±0.2

REM sleep latency 34.3±7.4 37.8±9.0 35.8±8.7

Sleep stages, mean REM period duration and sleep latencies werequantified in minutes. ⁎p<0.05, ⁎⁎p<0.01; significant statisticaldifference with respect to control.

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readily observed in the hypnograms of representative recor-dings that are shown in Fig. 2.

MCH 100 ng produced, in addition, a small but significantincrease in SWS (9.2% increment of the control value) (Table 1).Besides, MCH reduced the time spent in wakefulness and lightsleep 33.8% and 26% of the control values, respectively. On theother hand, no significant changes in the sleep and wakeful-ness variables were obtained after the administration of MCH50 ng (Table 1).

The charts corresponding to Fig. 3 describe the time theanimals spent in different behavioral states, which wasanalyzed in 2-hour blocks, after MCH 50 and 100 ng or controlmicroinjections. Note that the time spent in the differentbehavioral states along the 6 h of recording following MCHadministration was quite homogeneous. The largest effectmanifested in all the two-hour blocks after MCH 100 ng wasthe significant increment in REM sleep. A decrease in the timespent in light sleep were observed after 50 ng and 100 ngadministration of MCH in the first and the last two-hourblocks of the recordings.

SWS and REM sleep latencies were not significantly affectedbyMCHmicroinjections (Table 1). Of note, MCHmicroinjectionsdid not modify the animals' behavior during wakefulness.

Fig. 2 – Representative hypnograms illustrating theoccurrence of wakefulness and sleep followingmicroinjection of MCH into the dorsal raphe nucleus. Theeffects of the vehicle (A), 50 ng (B) and 100 ng of MCH (C) arepresented. The hypnogram corresponding to the 100 ng doseof MCH depicts a substantial increase in the number of REMsleep episodes.

3. Discussion

In the present study, we examined the hypothesis that theMCHergic system facilitates the generation of REM sleep bymodulating the activity of neurons located in the DR. In orderto test this hypothesis, MCH was microinjected into the DR ofrats and the effects on sleep and waking states were analyzed.MCH produced a marked and dose-dependent increase of thetime spent in REM sleep as well as a modest increase in SWS.Conversely, values corresponding to wakefulness and lightsleep were reduced.

3.1. Technical considerations

The microinjection of receptor ligands into the DR is atechnical approach in which the authors have broad expe-

rience (Monti and Jantos, 1992; Monti et al., 2000, 2002; Montiand Jantos, 2003, 2005, 2006a,b). Although we verified that thecannula tips were confinedwithin the limits of the DR, it is notpossible to discard the diffusion of MCH outside this nucleus.In this respect, it is well-known that the diffusion rate of agiven drug depends upon a number of factors including itsdiffusion coefficient, and the characteristics of the brain regionwhere the microinjection is performed (Nicholson, 1985).However, it should be taken into account that methyleneblue microinjected into the CNS in a 0.2 μl volume diffuses anaverage ratio of 520 μm (Lohman et al., 2005). Because of thesmall doses and volume employed in the present study, weconsider that the diffusion of effective concentrations of MCHoutside the DR, if present, was negligible.

3.2. Anatomical relationship between the MCHergicsystem and the dorsal raphe nucleus

The serotonergic system constitutes one of the most widelydistributed neurochemical systems in the CNS of vertebrates(Jacobs and Azmitia, 1992). Serotonergic neurons project to

Fig. 3 – Effects on sleep andwakefulness of MCHmicroinjections into the dorsal raphe nucleus. Bar charts show the time spentin wakefulness, light sleep, slow wave sleep and REM sleep analyzed in 2-hours blocks. *p<0.05 and **p<0.01 compared withvehicle injection (ANOVA and Dunnett Multiple Comparisons test).

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diverse regions of the CNS, and play an important role inseveral functions such as analgesia, thermoregulation, fee-ding, motor activity, EEG activation, etc. (Jacobs and Azmitia,1992; Wang and Nakai, 1994; Dringenberg and Vanderwolf,1998; Jacobs and Fornal, 1999; Monti and Monti, 2000; Portaset al., 2000). In mammals, the majority of the serotonergicneurons are localized in the DR and send extensive ascendingprojection to the telencephalon and diencephalon (Dahlströmand Fuxe, 1964). These serotonergic neurons exhibit strongneuroplasticity both during development and in adulthood,and both their function and morphology are modified bydifferent neuroactive substances including neuropeptides(Azmitia, 1999, 2007).

GABAergic, dopaminergic, glutamatergic and peptidergicneurons coexist with serotonergic neurons in the DR, but theirfunction is hardly known (Ottersen and Storm-Mathisen, 1984;Mugnaini and Oertel, 1985; Geffard et al., 1987; Sutin andJacobowitz, 1988; Jolas and Aghajanian, 1997).

MCHergic fibers and receptors, as well as MCH-immunoreactive tanycytes are present in the DR (Bitten-court et al., 1992; Hervieu et al., 2000; Kilduff and De Lecea,2001; Saito et al., 2001; Torterolo et al., 2008). These cells arespecialized to absorb substances from the cerebrospinalfluid (CSF), and may tentatively take up MCH from the fourthventricle; these data suggest that the MCHergic system

could influence neuronal activity in the DR not only throughneuronal projections but also by a neurohumoral pathway(Torterolo et al., 2008). Although the presence of the MCHR-1has been documented in the DR, it is still unknown ifserotonergic neurons, or other neuronal types within theDR, actually express these receptors (Hervieu et al., 2000;Kilduff and De Lecea, 2001; Saito et al., 2001). The effects ofMCH on the neurons in the DR are also unknown; however,as was demonstrated in hypothalamic neurons, it is likelythat MCH has an inhibitory effect onto these cells (Gao andvan den Pol, 2001).

3.3. MCH may facilitate REM sleep by inhibition ofserotonergic neurons

Serotonergic neurons of the DR are active during wakefulness,reduce their discharge during SWS and are essentially silentduring REM sleep (REM-off neurons) (McGinty and Harper,1976; Trulson and Jacobs, 1979; Lydic et al., 1983, 1987a,b).Furthermore, serotonin release decreases during REM sleep indiverse brain areas (Portas et al., 2000). Application of 5-HT1A

receptor agonists into the DR facilitates REM sleep occurrence,an effect mediated by the activation of inhibitory somatoden-dritic 5-HT1A autoreceptors (Portas et al., 1996; Monti et al.,2002). In addition, the inhibition of serotonergic neurons of the

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DR by microinjections of the GABA-A agonist muscimol(0.25 mg in 0.5 μl) produces a 67% increase in the REM sleeptime (Nitz and Siegel, 1997). This outcome is similar to theeffect that we obtained following the microinjections of MCH,the only difference is that muscimol increased REM sleep inthe first 2 h after themicroinjection, while the increase in REMsleep elicited by MCH was maintained up to 6 h. It isinteresting to note that in preliminary studies, MCH micro-injected into the DR of cats produced similar effects (Deveraet al., 2007). Because MCH is an inhibitory neuropeptide, wehypothesized that MCHwould facilitate the generation of REMsleep by inhibiting serotonergic neurons.

Based upon the wake-related discharge pattern of seroto-nergic neurons it has been suggested that these neuronspromote wakefulness (McGinty and Harper, 1976; Trulson andJacobs, 1979). There are also strong evidences that theserotonergic neurons of the DR have a key role in the acti-vation of the EEG (Dringenberg and Vanderwolf, 1998). Thefact that approximately one fourth of the DR serotonergicneurons increased their firing rate as much as fivefold inrelation with rhythmic motor activity (Jacobs and Fornal,1999), implies that some serotonergic neurons of the DR areinvolved in one aspect of wakefulness: motor activity.Therefore, it is not unexpected that the inhibition of the DRserotonergic neurons also reduces the time spent in wakeful-ness. In accordance with our results, (Verret et al., 2003)described a decrease of wakefulness after intra-ventricularMCH administration.

3.4. The dorsal raphe nucleus is regulated by MCH:physiological aspects

Experimental evidence suggest that GABAergic neurons areinvolved in the inhibition of serotonergic neurons during REMsleep (Nitz and Siegel, 1997; Maloney et al., 1999; Gervasoniet al., 2000; Torterolo et al., 2000). Since almost 60% of thehypothalamic MCHergic neurons are active during REM sleep(Verret et al., 2003), it would be of interest to test thehypothesis that the DR-projecting MCHergic neurons collabo-rate with the inhibition of the serotonergic neurons duringREM sleep.

Intra-ventricular microinjections of MCH increase REMsleep time up to 200% of control values, and in accordancewith our results, the effect on REM sleep lasted up to 8 h(Verret et al., 2003). However, this increment in the time spentin REM sleep is larger than the one we observed after MCHmicroinjections into the DR. Because the experiments ofVerret et al. (2003) were performed during the dark period, itis likely that the sleep-increasing effect of MCHwould bemoreevident under this condition. In addition, the larger effectobserved following icv microinjections may be due to MCHfacilitation of REM sleep by exerting its actions simultaneouslyon several neuroanatomical loci, such as the noradrenergiclocus coeruleus or mesopontine cholinergic region, that areheavily innervated by MCHergic neurons (Bittencourt et al.,1992; Mouri et al., 1993). The fact that MCH microinjectionsinto the NPO induce REM sleep, and the recent demonstrationthat MCHergic neuronal somata are present in the LDT, acritical REM-sleep inducing zone, are data that support thishypothesis (Rondini et al., 2007; Torterolo et al., in press).

3.5. The dorsal raphe nucleus is regulated by MCH:translational implication

Patients with major depression present a decrement in REMsleep latency, an increase in the time spent in REM sleep andin the duration of the first REM sleep episode, as well as anincrease in the density of eyes movements within episodes ofREM sleep (Adrien, 2002; Benca, 2005). Furthermore, antide-pressive drugs diminish or inhibit the occurrence of REMsleep, and the selective deprivation of REM sleep alleviatesdepressive symptoms (Adrien, 2002; Benca, 2005). Borowskyet al. (2002) showed that MCHR-1 antagonists have antide-pressant properties in animal models (Borowsky et al., 2002).Since the serotonergic system plays a critical role both inmajor depression and suicide behavior (Mann et al., 2001;Kalia, 2005; Berton and Nestler, 2006), disruptions in theinteractions between the MCHergic and the serotonergicsystems likely play an important role in the generation ofmood disorders.

3.6. Conclusions

The present data demonstrate that the modulation ofneuronal activity in the DR by MCH, facilitates the generationof sleep.

4. Experimental procedures

4.1. Animals

Six maleWistar rats, each weighing 350–400 g, were employedin the study. All rats were used in strict accordance with the“Guide to the care and use of laboratory animals” (7th edition,National Academy Press, Washington D. C., 1996). Further-more, the Institutional Animal Care Committee approved theexperimental procedures. In addition, adequate measureswere taken to minimize pain, discomfort or stress of theanimals, and all efforts weremade in order to use theminimalnumber of animals necessary to produce reliable scientificdata.

4.2. Surgical procedures

The animals were anesthetized with sodium pentobarbital(40 mg/kg, i.p.) and prepared for standard polysomnography.Nichrome electrodes (200 μm diameter) were implanted in theskull for recording the frontal and occipital EEG and in theneck muscles to monitor EMG activity; all electrodes weresoldered to a connector. In addition, a guide cannula wasinserted into the DR. The coordinates for the DR were AP 7.8, L0.0 and H −6.4 mm from Bregma (Paxinos and Watson, 2005).The tip of the guide cannula (gauge 26) was placed 2mmabovethe DR to minimize cellular damage at the injection site. Theconnector and cannulawere cemented to the skull with dentalacrylic.

The animals were treated postoperatively for 4 days withan antibiotic (Cefradine, 50.0 mg/kg i.m.). A topical antibioticcream (Neomycin) was also applied to the skin marginssurrounding the implant.

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4.3. Recording and microinjection procedures

The animals were housed individually in a temperature-controlled room (23±1 °C) under a 12-h light/dark cycle (lightswent on at 07.00 am), with food andwater ad libitum. Ten daysafter surgery the animals were habituated during approxi-mately five consecutive days to a soundproof chamber fittedwith slip rings and cable connectors. The endpoints of theadaptation recording sessions were determined when theanimals showed consistent REM sleep time and latencies forat least three consecutive recording sessions. EEG and EMGsignals were amplified, filtered and recorded by a Grass-model8 polygraph.

MCH (50 and 100 ng in 0.2 μl of sterile saline, PhoenixPharmaceutical Inc., Belmont, CA) or vehicle (0.2 μl of sterilesaline) was microinjected into the DR during a period of2 min, with an injection cannula (28 gauge), which extended2 mm beyond the guide cannula. Aliquots for the dosesemployed were prepared and frozen at −20 °C, and thawedimmediately before use. Microinjections were always per-formed during the light phase at approximately 07.30 h.Thereafter, the animals were placed in the recording cham-ber; the recording sessions began 15 min later and lasted for6 h. Each animal received 3 microinjections (vehicle, 50 ngand 100 ng of MCH); only one microinjection was performedduring each recording session and no further experimentswere conducted during the following 3 days. A balanced orderof MCH and control microinjections was always used tomerge the effects of both the drug and the time elapsedduring the experimental protocol.

On completion of the microinjections, we identified theinjection site either by the observation of cannula track andlesion or by the microinjection of Pontamine Sky Blue (0.2 μl)(Fig. 1). The rats were sacrificed with an overdose of penõ-tobarbital, perfused with 4% paraformaldehyde and theirbrains were removed. Thereafter, the brains were cryo-protected in a solution of sucrose 30% and cut in 40 μmcoronal sections with a cryostat, or, were embedded inparaffin and cut in 5 μm sections by a microtome. Selectedsections were stained either with Pyronin-y or with hema-toxilin and eosin.

4.4. Sleep scoring and data analysis

A trained researcher (H.J.) blind to the treatment received bythe animals visually scored the polysomnographic data in 10-second epochs. The predominant activity of each epoch wasassigned to the following categories based in standard criteria:wakefulness, light sleep, SWS, and REM sleep. Latencies forSWS (from the beginning of the recording to the first epoch ofSWS) and for REM sleep (from the first epoch of SWS to REMsleep onset), as well as the number of REM sleep episodes andthe mean duration of the REM sleep episodes were alsodetermined.

All values are presented as mean±S.E.M. (standard error ofthe mean). The statistical significance of the differencebetween controls vs. MCH effects was evaluated usinganalysis of variance (ANOVA) and Dunnett Multiple Compa-risons post-hoc test. The criterion used to discard the nullhypothesis was p<0.05.

Acknowledgments

This study was supported by the “Proyecto de DesarrolloTecnológico— Salud 76/36, Ministerio de Educación y Cultura,República Oriental del Uruguay” grant.

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