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Immunoneutralization of melanin-concentrating hormone (MCH) in the dorsalraphe nucleus: Effects on sleep and wakefulness

Patricia Lagos, Pablo Torterolo, Hector Jantos, Jaime M. Monti

PII: S0006-8993(10)02540-0DOI: doi: 10.1016/j.brainres.2010.11.027Reference: BRES 40990

To appear in: Brain Research

Accepted date: 7 November 2010

Please cite this article as: Patricia Lagos, Pablo Torterolo, Hector Jantos, Jaime M.Monti, Immunoneutralization of melanin-concentrating hormone (MCH) in the dor-sal raphe nucleus: Effects on sleep and wakefulness, Brain Research (2010), doi:10.1016/j.brainres.2010.11.027

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Immunoneutralization of melanin-concentrating hormone (MCH) in

the dorsal raphe nucleus: effects on sleep and wakefulness

Patricia Lagos1, Pablo Torterolo1*, Héctor Jantos2 and Jaime M. Monti2

1. Department of Physiology, School of Medicine, University of the Republic,

Montevideo, Uruguay. 2. Department of Pharmacology and Therapeutics, School of

Medicine, Clinics Hospital, Montevideo, Uruguay

Number of text pages: 24

Number of figures: 2

Number of tables: 1

* Please address correspondence to:

Dr. Pablo Torterolo

Departamento de Fisiología, Facultad de Medicina, Universidad de la República.

General Flores 2125, 11800, Montevideo, Uruguay. Email: ptortero@fmed.edu.uy

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Abstract

Hypothalamic neurons that utilize melanin-concentrating hormone (MCH) as a

neuromodulator exert a positive control over energy homeostasis, inducing feeding and

decreasing metabolism. Recent studies have shown also that this system plays a role in

the generation and/or maintenance of sleep.

MCHergic neurons project to the serotonergic dorsal raphe nucleus (DR), a

neuroanatomical structure involved in several functions during wakefulness (W), and in

the regulation of rapid-eye movements (REM) sleep. Recently, we determined the

effect of MCH microinjected into the DR on sleep variables in the rat. MCH produced

a marked increment of REM sleep whereas slow wave sleep (SWS) showed only a

moderate increase.

In the present study, we analyze the effect of immunoneutralization of MCH in

the DR on sleep and W in the rat. Compared to the control solution, microinjections of

anti-MCH antibodies (1/100 solution in 0.2 µl) induced a significant increase in REM

sleep latency (31.2 ± 7.1 vs. 84.2 ± 24.8 minutes, p < 0.05) and a decrease of REM

sleep time (37.8 ± 5.4 vs. 17.8 ± 2.9 minutes, p < 0.05) that was related to the reduction

in the number of REM sleep episodes. In addition, there was an increase of total W

time (49.8 ± 4.6 vs. 72.0 ± 5.7 minutes, p < 0.01). Light sleep and SWS remained

unchanged. The intra-DR administration of a more diluted solution of anti-MCH

antibodies (1/500) or rabbit pre-immune serum did not modify neither W nor REM

sleep variables.

Our findings strongly suggest that MCH released in the DR facilitates the

occurrence of REM sleep.

Section: 6. Regulatory systems

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Keywords: neuropeptide, serotonin, hypothalamus, REM sleep.

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Abbreviations

DR, dorsal raphe nucleus.

EEG, electroencephalogram.

EMG, electromyogram.

LDT, laterodorsal tegmental nucleus.

MCH, melanin-concentrating hormone.

MCHR-1, MCH receptor type 1.

MCHR-2, MCH receptor type 2.

REM, rapid-eye movement.

SWS, slow wave sleep.

W, wakefulness

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1. Introduction

Classical studies established that the posterior region of the lateral hypothalamus

is involved in the control of behavioral states (Von Economo, 1930; Nauta, 1946).

Within this and adjacent areas of the hypothalamus there are neurons that synthesize

melanin-concentrating hormone (MCH) that project throughout the central nervous

system (Bittencourt et al., 1992; Mouri et al., 1993; Torterolo et al., 2006).

MCH is a 19 amino acid cyclic neuropeptide that functions as a neuromodulator;

its biological functions are mediated by two G-protein coupled receptors known as

MCHR-1 and MCHR-2 (Saito and Nagasaki, 2008). Interestingly, it has been found

that the MCHR-2 is not functional in rodents (Tan et al., 2002). The MCHergic system

has been studied in detail in relation to the control of feeding and energy homeostasis

(Qu et al., 1996; Shimada et al., 1998; Ludwig et al., 2001; Nahon, 2006; Saito and

Nagasaki, 2008). In this respect, it has been determined that MCHR-1 antagonists exert

a control over experimental obesity (Borowsky et al., 2002; Handlon and Zhou, 2006;

Rivera et al., 2008).

MCHergic neurons send dense projections to the dorsal raphe nucleus (DR)

(Bittencourt et al., 1992; Torterolo et al., 2008), a brainstem region involved in the

regulation of wakefulness (W) and sleep (Jacobs and Azmitia, 1992). Serotonergic

neurons of the DR are active during W, reduce their discharge during slow wave sleep

(SWS) and become virtually silent during rapid-eye movements (REM) sleep (REM-off

neurons) (McGinty and Harper, 1976; Trulson and Jacobs, 1979; Lydic et al., 1983,

1987a, b). The neuronal release of serotonin in diverse brain areas is also maximal

during W and decreases to its minimal level during REM sleep (Portas et al., 2000). It

has been proposed that the decrease of serotonergic neuronal activity of during REM

sleep has a “permissive” effect on neuronal systems involved in the induction and

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maintenance of this behavioral state. In other words, the inhibition of serotonergic

neurons would be a prerequisite for the generation of REM sleep (McCarley, 2007).

Based upon the wake-related discharge pattern of the serotonergic neurons, it has been

proposed that these neurons promote W (McGinty and Harper, 1976; Trulson and

Jacobs, 1979; Lydic et al., 1983, 1987a, b). There is also evidence showing that these

neurons participate in the electroencephalogram (EEG) activation and in rhythmic

motor activity during W (Dringenberg and Vanderwolf, 1998; Jacobs and Fornal, 2008).

The DR contains a large number of MCH-labeled fibers, MCHR-1 containing

neurons and MCH-labeled tanycytes (Bittencourt et al., 1992; Hervieu et al., 2000;

Kilduff and De Lecea, 2001; Saito et al., 2001; Torterolo et al., 2008); the latter cells are

specialized in transporting neuroactive substances from the cerebrospinal fluid to the

brain parenchyma (Torterolo et al., 2008). Recently, we demonstrated that the

microinjection of MCH into the DR during the light phase increases REM sleep and, to

a lesser extent, SWS (Lagos et al., 2009). These effects were accompanied by a

reduction of W. In the present study we further examine the role of MCH in the DR by

means of the immunoneutralization of the neuropeptide. The microinjection of anti-

MCH antibodies into the DR induced a marked decrease of REM sleep and an

increment of W. These results support the proposal that MCH released in the DR

facilitates the occurrence of REM sleep.

2. Results

Anti-MCH antibodies (1/100 and 1/500 solutions) and vehicle (as control) were

microinjected into the DR of six rats. In a separate set of control experiments (6 rats)

saline and rabbit pre-immune serum were also microinjected into the DR. In all the

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animals, the tip of the cannula was located within the DR. A representative example of

the microinjections site is shown in Figure 1.

The results obtained during 6-h sessions following the microinjection of anti-

MCH antibodies or vehicle into the DR are summarized in Table 1. Compared to the

control vehicle, anti-MCH antibodies (1/100) decreased significantly REM sleep time

from a control value of 37.8 ± 5.4 min (10.5 % of the total recording time) to 17.8. ± 2.9

min (4.9 % of the total recording time). The suppression of REM sleep was related to a

reduction of the number of REM sleep episodes, whereas the duration of these episodes

was not affected. The microinjections of the antibodies also increased markedly the

REM sleep latency. Furthermore, the administration of anti-MCH antibodies (1/100)

produced a significant increase of W. On the contrary, light sleep, SWS and SWS

latency were not affected by the treatment. The 1/500 solution of anti-MCH antibodies

did not significantly modify sleep variables (Table 1). Hypnograms of representative

recordings following control or anti-MCH antibodies microinjections (1/500 and 1/100)

are shown in Figure 2. The increment in the REM sleep latency and the decrease in the

number of REM episodes after anti-MCH antibodies (1/100) administration are readily

observed.

Figure 1, 2 and Table 1 approximately here.

The charts corresponding to Figure 3 describe the time the animals spent in

different behavioral states after control, anti-MCH antibodies 1/500 or 1/100

microinjections. In all the 2-h blocks REM sleep was reduced and W increased after

anti-MCH antibodies 1/100. In contrast, the 1/500 solution of the anti-MCH antibodies

did not significantly affect sleep variables in any of the 2-h blocks.

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It should be pointed out that microinjection of anti-MCH antibodies did not modify the

animals' behavior during W. Moreover, the EEG and the electromyogram (EMG) did

not show atypical activity during either sleep or W.

Figure 3 approximately here.

The results of the intra-DR administration of rabbit pre-immune serum and

saline for the 6-h recording sessions are exhibited in Table 2. In contrast to anti-MCH

antibodies, pre-immune serum did not affect neither W nor REM sleep variables. On

the contrary, pre-immune serum produced only a slight increase in SWS (4.4% above

control values), which was evident only in the first 2 hs block (data not shown).

Table 2 approximately here

3. Discussion

The present report examined the role of MCH in the regulation of sleep in the

DR. For this purpose, anti-MCH antibodies were microinjected into the DR of animals

prepared for chronic sleep recordings. The immunoneutralization of MCH increased

the REM sleep latency and produced a large and dose-dependent reduction of the time

spent in REM sleep, while SWS or light sleep were not affected. The decrease in REM

sleep time was due to a reduction in the number of REM sleep episodes. In contrast,

REM sleep episodes maintained their physiological duration, suggesting that MCH

regulates the opportunity to generate REM sleep in the DR but does not affect its

mechanism of generation.

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Recently, Lagos et al., (2009) demonstrated that microinjections of MCH into

the DR markedly increases REM sleep time but only slightly affects the time spent in

light sleep and SWS. In the present study we have demonstrated that the neutralization

of the effects of endogenous MCH by specific antibodies within the DR alters the

physiological generation of REM sleep.

3.1. Technical considerations

Although the authors have broad experience with the microinjections of

neuroactive substances into the DR (Monti and Jantos, 2003, 2005, 2006a, b; Lagos et

al., 2009), the diffusion outside the DR cannot be fully discarded. However, because of

the high molecular size of the anti-MCH antibodies, their diffusion to neighboring

structures should be minor.

An approach to antagonize the effect of MCH is its immunoneutralization. This

strategy has been widely used either for studies of the MCHergic system or for other

hypothalamic peptides (Stanley et al., 1992; Lambert et al., 1993; Roky et al., 1994;

Yamada et al., 2000; Matsuda et al., 2007). We previously employed the same anti-

MCH polyclonal antibodies in immunohistochemical studies performed in cats and

guinea pigs tissues with optimal and reproducible results (McGregor et al., 2005;

Torterolo et al., 2006; Torterolo et al., 2008).

The following evidences strongly suggest that the effects obtained after anti-

MCH antibodies microinjections were specific for the neutralization of the endogenous

MCH. First, the effects were dose-dependent. Secondly, light sleep and SWS, which

are easily disturbed by unspecific manipulations of the animal, were intact after the

treatment with the antibodies. Furthermore, the changes observed on sleep and W were

opposite to those observed after MCH microinjections into the same loci (Lagos et al.,

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2009). In addition, we microinjected pre-immune serum into the DR in order to discard

unspecific effect of the anti-MCH antibody (Kokkotou et al., 2008); in contrast to MCH

immunoneutralization, this serum did not produce any effect neither in W nor REM

sleep variables. Finally, in a previous report we have microinjected MCH and the same

anti-MCH antibody (at the same doses) into the DR of rats, to determine their

behavioral effects on the forced swim test and open field test (Torterolo et al., 2009b;

Lagos et al., 2010); it is remarkable that MCH and the neutralization of MCH by the

antibodies also had opposite effects on these tests.

Regulatory peptidergic axons make synaptic contacts with large synaptic cleft

(diffuse synapses); neuropeptides are also released at extrasynaptic regions (Zupanc,

1996). These anatomical features may facilitate the access of the anti-MCH antibodies

microinjected into the DR to neutralize the physiologically released MCH.

Several antagonists for the MCHR-1 have been synthesized in recent times

(Rivera et al., 2008); it would be important to compare the results of the

immunoneutralization with the effect of MCHR-1 antagonists applied into the DR.

3.2. MCH facilitates the generation of REM sleep

There is experimental evidence supporting the proposal that the MCHergic

system is involved in the control of behavioral states. The use of Fos immunoreactivity

as a marker of neuronal activity, has shown that MCHergic neurons of the rat are active

during sleep, mainly during REM sleep (Verret et al., 2003; Modirrousta et al., 2005;

Hanriot et al., 2007). Recently, identified MCHergic neurons have been recorded along

the waking-sleep cycle (Hassani et al., 2009). These neurons are almost silent during W

increase their firing rate during SWS and reach their maximal activity during REM

sleep. Besides, intraventricular microinjections of MCH produce a large increase in

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REM sleep while SWS is only moderately augmented (Verret et al., 2003). In contrast,

systemic administration of MCHR-1 antagonists decrease SWS and REM sleep

(Ahnaou et al., 2008). In agreement with the preceding results, MCH knock-out mice

sleep less than wild-type animals, and exhibit hyperactivity and an abnormal decrease of

REM sleep in response to fasting (Willie et al., 2008).

Experimental findings also suggest that the DR is not the solely site where MCH

regulates sleep. In fact, MCH microinjections into the nucleus pontis oralis of the cat

also facilitates the generation of REM sleep (Torterolo et al., 2009a). The MCHergic

neurons also innervate profusely waking and sleep-related regions such as the

laterodorsal and pedunculopontine tegmental nuclei (LDT-PPT), tubero-mammilar

nucleus or the basal forebrain (Bittencourt et al., 1992). The MCHergic neurons may

also promote sleep regulating in tandem the activity of all these regions.

3.3. Cellular effects of MCH in the dorsal raphe nucleus

MCH receptor stimulation activates the Gi/o intracellular pathway in several cell

lines (Lembo et al., 1999). In this respect, it has been described that MCH reduces both

excitatory and inhibitory synaptic events in cultured neurons of the lateral hypothalamus

(Gao and van den Pol, 2001). Furthermore, MCH has been shown also to inhibit action

potential generation and glutamatergic synaptic transmission in hypocretinergic neurons

recorded in hypothalamic slices (Rao et al., 2008). However, the cellular effects of

MCH in the DR have not been characterized to date.

Serotonergic, GABAergic, dopaminergic, glutamatergic and peptidergic neurons

have been recognized in the DR (Lowry et al., 2008). In addition, afferents that utilize

different neurotransmitters that are involved in the regulation of sleep and waking reach

the DR (Lowry et al., 2008). Pertinent to our topic, MCHergic fibers, MCH-

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immunoreactive tanycytes, and MCHR-1 containing neurons are present in the DR

(Bittencourt et al., 1992; Hervieu et al., 2000; Kilduff and De Lecea, 2001; Saito et al.,

2001; Torterolo et al., 2008).

Previous studies have shown that selective activation of the somatodendritic 5-

HT1A receptor or direct administration of GABAA receptor agonists into the DR results

in the inhibition of the serotonergic neurons and in an increment in REM sleep time

(Portas et al., 1996; Nitz and Siegel, 1997; Monti et al., 2002). The finding that

immunoneutralization of MCH in the DR suppresses REM sleep, suggests that under

physiological conditions the serotonergic neurons are inhibited by MCH. Considering

that the serotonergic neurons play a role in W (Dringenberg and Vanderwolf, 1998;

Jacobs and Fornal, 2008), the increment of W that we described could also be explained

by the neutralization of the inhibitory MCHergic regulation of the serotonergic neuronal

activity. However, it is not known whether the MCHR-1 is expressed in serotonergic

neurons (direct inhibitory effect), in interneurons that facilitate the serotonin release

(indirect inhibitory effect), or in presynaptic terminals that facilitate serotonin release

((indirect inhibitory effect). Further studies are needed to resolve this issue.

3.4. Conclusions

The present data indicate that the MCHergic neurons modulate neuronal activity

in the DR. The inactivation of the physiologically released MCH induced an increase of

W and a reduction of REM sleep.

4. Experimental procedures

4.1. Animals

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Twelve male Wistar rats, each weighing 350-400 g, were employed in 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). Furthermore,

the Institutional Animal Care Committee approved the experimental procedures. In

addition, adequate measures were taken to minimize pain, discomfort or stress of the

animals, and all efforts were made in order to use the minimal number of animals

necessary to produce reliable scientific data.

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 the skull for recording the frontal and occipital EEG and in the neck

muscles to monitor EMG activity; all electrodes were soldered to a connector. In

addition, a guide cannula was inserted into the DR. The coordinates for the DR were

AP 7.8, L 0.0 and H -5.8 mm from Bregma (Paxinos and Watson, 2005). The tip of the

guide cannula (gauge 26) was placed 2 mm above the DR to minimize cellular damage

at the injection site. The connector and cannula were cemented to the skull with dental

acrylic.

The animals were treated postoperatively for 4 days with an antibiotic

(Cefradine, 50 mg/kg i.m.). A topical antibiotic cream (Neomycin) was also applied to

the skin margins surrounding the implant.

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 (lights went on at 07.00 am), with food and water ad

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libitum. Ten days after surgery the animals were habituated during five consecutive

days to a soundproof chamber fitted with slip rings and cable connectors. The

endpoints of the adaptation recording sessions were determined when the animals

showed consistent REM sleep time and latencies for at least three consecutive recording

sessions. EEG and EMG signals were amplified, filtered and recorded by a Grass-

model 8 polygraph.

Rabbit polyclonal anti-MCH primary antibodies were obtained from Phoenix

Pharmaceuticals Inc. (# H-070-47) and diluted in saline at concentrations of 1/100 and

1/500. Aliquots were prepared, frozen at -20 oC, and thawed immediately before use.

Saline and rabbit pre-immune serum was used as control (Kokkotou et al., 2008).

The solutions of anti-MCH antibodies (0.2 µl), pre-immune serum (0.2 µl) or

vehicle (0.2 µl of sterile saline) were microinjected into the DR during a period of 2

minutes with an injection cannula (28 gauge), which extended 2 mm beyond the guide

cannula. Microinjections were always performed during the light phase at

approximately 07.30 h. Thereafter, the animals were placed in the recording chamber;

the recording sessions began 15 minutes later and lasted for 6 hours.

In the first set of experiments each animal received 3 microinjections (vehicle,

1/100 and 1/500 solutions of the anti-MCH antibodies); only one microinjection was

performed during each recording session and no further experiments were conducted

during the following 3 days. A balanced order of anti-MCH antibodies and control

microinjections was always used to merge the effects of both the drug and the time

elapsed during the experimental protocol. In the second set of experiments each animal

received 4 microinjections (2 saline and 2 pre-immune serum).

On completion of the microinjections series we microinjected of Pontamine Sky

Blue (0.2 µl) into the DR. The rats were sacrificed with an overdose of pentobarbital,

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perfused with 4% paraformaldehyde and their brains were removed. Thereafter, the

brains were cryoprotected in a solution of sucrose 30% and cut in 150 µm-thick sections

by a vibratome. We identified the injection site by the observation of the Sky Blue

deposit, the cannula track and lesion (Figure 1).

4.4. Sleep scoring and data analysis

A trained researcher (H.J.) blind to the treatment received by the animals

visually scored the polysomnographic data in 10 seconds epochs. The predominant

activity of each epoch was assigned to the following categories based in standard

criteria: W, light sleep, SWS, and REM sleep. Latencies for SWS (from the beginning

of the recording to the first epoch of SWS) and for REM sleep (from the first epoch of

SWS to REM sleep onset), as well as the number of REM sleep episodes and the mean

duration of the REM sleep episodes were also determined.

All values are presented as mean ± S.E.M. (standard error of the mean). The

statistical significance of the difference between controls versus anti-MCH antibodies

effects was evaluated using analysis of variance (ANOVA) and Dunnett Multiple

Comparisons post-hoc test, while between saline versus pre-immune serum the effects

were evaluated using the two-tailed paired Student t test. The criterion used to discard

the null hypothesis was p < 0.05.

Acknowledgments

This study was supported by the "Proyecto de Desarrollo Tecnológico - Salud

76/36, Ministerio de Educación y Cultura, República Oriental del Uruguay" grant.

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Figure Legends

Figure 1. The microinjection sites were localized in the dorsal raphe nucleus. The

photomicrographs show a coronal section at the level of the DR of a representative rat,

approximately -8.15 from Bregma according to Paxinos and Watson (2005) atlas. The

Pontamine Sky Blue deposit indicates the microinjection site (the deposit is showed at

higher magnification in the inset). Aq, aqueduct; IC, inferior colliculus; DR, dorsal

raphe nucleus; ml, medial lemniscus; mlf, medial longitudinal fasciculus; NPO, nucleus

pontis oralis, PAG, periaqueductal gray. Py, pyramidal tract. Calibration bars, 2 mm;

inset, 1 mm.

Figure 2. Representative hypnograms illustrating the occurrence of wakefulness

and sleep following microinjection of anti-MCH antibodies into the dorsal raphe

nucleus. The effects of control (A), 1/500 anti-MCH antibody (B) and 1/100 anti-MCH

antibody (C) microinjections are presented. The hypnogram corresponding to the 1/100

solution of the antibody depicts a substantial decrease in the number of REM sleep

episodes and a large increase in the REM sleep latency. REM, REM sleep; SWS, slow

wave sleep; LS, light sleep; W, wakefulness. The microinjections volume was 0.2 µl.

Figure 3. Effects on sleep and wakefulness of anti-MCH antibodies

microinjections into the dorsal raphe nucleus. Bar charts show the time spent in

wakefulness, light sleep, slow wave sleep and REM sleep analyzed in 2 hours blocks. *

P < 0.05 and ** P < 0.01 compared with vehicle microinjections (ANOVA and Dunnett

Multiple Comparisons test). The microinjections volume was 0.2 µl.

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Research HighlightsImmunoneutralization of MCH in the dorsal raphe nucleus suppresses REM sleep time and increases wakefulness.

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Table 1. Effects of anti-MCH antibodies microinjected into the dorsal raphe nucleus on sleep and waking during 6-h polysomnographic recordings

Control anti-MCH (1/500) anti-MCH (1/100)Wakefulness 49.8 ± 4.6 54.7 ± 5.6 72.0 ± 5.7 **Light sleep 49.8 ± 3.4 46.8 ± 5.8 51.2 ± 5.8

Slow wave sleep 222.3 ± 6.8 228 ± 8.9 219 ± 6.8REM sleep 37.8 ± 5.4 26.0 ± 4.2 17.8 ± 2.9*

Number of REMepisodes

15.8 ± 1.5 13.2 ± 2.1 8.5 ± 1.1*

Mean REM periodduration

2.3 ± 0.2 2.0 ± 0.1 2.1 ± 0.2

Slow wave sleep latency

1.7 ± 0.8 0.5 ± 0.2 3.7 ± 2.5

REM sleep latency 31.2 ± 7.1 32.5 ± 2.0 84.2 ± 24.8*Sleep stages, mean REM period duration and sleep latencies were quantified in minutes. *p < 0.05, **p < 0.01; significant statistical difference with respect to control.

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Table 2. Effects of rabbit pre-immune serum microinjected into the dorsal raphe nucleus on sleep and waking during 6-h polysomnographic recordings

Control (saline) Pre-immune

serumWakefulness 62.7 ± 12.6 55.8 ± 4.0Light sleep 39.7 ± 5.0 41.4 ± 4.7

Slow wave sleep 226.3 ± 10.2 236.3 ± 6.3*REM sleep 31.3 ± 6.1 26.7 ± 3.8

Number of REMepisodes

13.0 ± 1.7 12.3 ± 0.8

Mean REM periodduration

2.3 ± 0.3 2.2 ± 0.2

Slow wave sleep latency

4.5 ± 2.3 1.8 ± 0.9

REM sleep latency 26.2 ± 2.1 32.5 ± 8.3Sleep stages, mean REM period duration and sleep latencies were quantified in minutes. *p < 0.05, paired two-tailed Student t test.

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Fig. 1

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Fig. 2

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Fig. 3

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