SLEEP APNEA AND MONOAMINE OXIDASE A
Sleep Apneas are Increased in Mice Lacking Monoamine Oxidase A
Caroline Real1; Daniela Popa, PhD2,3; Isabelle Seif, PhD1; Jacques Callebert, PharmD, PhD4; Jean-Marie Launay, PharmD, PhD4 ; Joëlle Adrien, PhD2,3; Pierre Escourrou, MD, PhD1 Univ Paris-Sud, EA3544, Châtenay-Malabry Cedex, F-92296, Sérotonine et Neuropharmacologie, France; 2Université Pierre et Marie Curie-Paris 6, Faculté de Médecine Pierre et Marie Curie, Site Pitié-Salpêtrière, IFR 70 des Neurosciences, UMR S677, Paris, F-75013, France; 3INSERM, U677, Paris, F-75013, France; 4AP-HP, Hôpital Lariboisière, Service de Biochimie et Université Paris 5, EA 3621, F-75010 Paris, France. 1
Study objectives: Alterations in the serotonin (5-HT) system have been suggested as a mechanism of sleep apnea in humans and rodents. The objective is to evaluate the contribution of 5-HT to this disorder. Design: We studied sleep and breathing (whole-body plethysmography) in mutant mice that lack monoamine oxidase A (MAOA) and have increased concentrations of monoamines, including 5-HT. Measurements and Results: Compared to wild-type mice, the mutants showed similar amounts of slow wave sleep (SWS) and rapid eye movement sleep (REMS), but exhibited a 3-fold increase in SWS and REMS apnea indices. Acute administration of the MAOA inhibitor clorgyline decreased REMS amounts and increased the apnea index in wild-type but
not mutant mice. Parachlorophenylalanine, a 5-HT synthesis inhibitor, reduced whole brain concentrations of 5-HT in both strains, and induced a decrease in apnea index in mutant but not wild-type mice. Conclusion: Our results show that MAOA deficiency is associated with increased sleep apnea in mice and suggest that an acute or chronic excess of 5-HT contributes to this phenotype. Keywords: Sleep apnea, serotonin, control of breathing, MAOA, sleep Citation: Real C; Popa D; Seif I; Callebert J; Launay JM; Adrien J; Escourrou P. Sleep apneas are increased in mice lacking monoamine oxidase A. SLEEP 2007;30(10):1295-1302.
INTRODUCTION
receptor agonistic but 5-HT2,3 receptor antagonistic) effects.7 In a study on sudden infant death syndrome (SIDS), it was shown that in regions of the medulla, the number and density of 5-HT neurons were higher and the density of 5-HT1A receptors labeled with [3H]8-OH-DPAT was lower in SIDS victims than in controls.8 In Sprague-Dawley rats, intraperitoneal administration of 5HT, which does not penetrate the blood-brain barrier (BBB), increases apnea index by at least 250% during rapid eye movement sleep (REMS),9 whereas administration of the 5-HT3 receptor antagonist GR38032F reduces apnea expression during slow wave sleep (SWS) and REMS.10 Similarly, mirtazapine reduces central apnea expression during SWS and REMS.11 This supports the clinical relevance of studying central sleep apneas in rodents, notably because most patients with sleep apnea syndrome exhibit a combination of the 3 types of sleep apneas. During SWS and REMS, wild-type (129/Sv) mice show central apneic episodes, typically accompanied by the disappearance of intercostal electromyogram bursts (absence of respiratory efforts).12 In this mouse, the 5-HT2A receptor inhibitor MDL 100907 decreases the index of central apneas during SWS.13 To further investigate the role of endogenous 5-HT in central sleep apnea, we examined breathing during sleep in transgenic (Tg8) mice that display increased monoamine levels as a result of a genetic lack of monoamine oxidase A (MAOA), an important enzyme in the degradation of monoamines such as 5-HT and norepinephrine (NE).14-17 At the same time, because 5-HT is involved in sleep state regulation,18 we evaluated the effect of this genetic deficiency on vigilance state distribution. We found that sleep apnea indices were significantly higher in Tg8 mice than in their wild-type controls. We also studied sleep and breathing after acute administration of the MAOA inhibitor clorgyline. Finally, we assessed the role of 5-HT in sleep apnea genesis, by lowering 5-HT synthesis with p-chlorophenylalanine, without changing whole-brain NE concentration, in both mutant and wild-type mice. Our results suggest that excess endogenous 5-HT facilitates sleep apnea.
THERE IS A NEED TO CLARIFY NEUROCHEMICAL MECHANISMS UNDERLYING SLEEP APNEA, SUCH AS THE ROLE OF MONOAMINE NEUROTRANSMITTERS, IN the context of pharmacotherapy. Sleep apnea syndrome is estimated to affect 4% of men and 2% of women in the middle-aged work force population in the United States.1 It is associated with reduced daytime vigilance and an increased risk of cardiovascular disease. Sleep apneas are classified into 3 types: obstructive, central, and mixed.2 Obstructive sleep apneas are characterized by repetitive obstructions of the upper airway with increased respiratory effort, whereas central sleep apneas occur in the absence of respiratory effort, and mixed apneas begin as central but continue as obstructive. Current mechanical and surgical therapies carry significant morbidity or discomfort. The monoamine serotonin (5-HT) is known to have an important role in the control of ventilation in humans.3,4 One early report suggested that L-tryptophan, a 5-HT precursor, may have a beneficial effect in the treatment of obstructive (but not central) sleep apnea.5 More recently, fluoxetine and paroxetine, 2 selective serotonin reuptake inhibitors (SSRIs), were demonstrated to benefit some patients with obstructive apnea.6 In the same manner, patients with obstructive apnea syndrome showed therapeutic response to mirtazapine, an antidepressant with serotonergic (5-HT1
Disclosure Statement This was not an industry supported study. The authors have reported no financial conflicts of interest. Submitted for publication February, 2007 Accepted for publication June, 2007 Address correspondence to: C. Real, Sérotonine et neuropharmacologie, EA3544, Faculté de pharmacie, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry cedex, France; Tel: + 33 1 46 83 53 78; Fax: + 33 1 46 83 53 55; E-mail:
[email protected] SLEEP, Vol. 30, No. 10, 2007
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METHODS
on each side of the left eye, and 2 electromyogram (EMG) electrodes were positioned into the neck muscles. All electrodes were fixed to the skull with Super-Bond and acrylic cement (Dentalon Plus, GACD, France), and soldered to a connector also embedded in cement. After surgery, the animals were allowed 7-10 days to recover from surgery before recording.
All the procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (Council Directive # 87-848, October 19, 1987, French Ministry of Agriculture).
Sleep Recording and Scoring
Animals and Experimental Groups
Recordings were performed during the light phase from 10:00 to 16:00. The night before the experiment, animals were placed in the plethysmograph recording chamber (500 mL, 10 cm internal diameter; see next procedure) and connected to the recording cables. To allow freedom of movement for the animal during overnight habituation and during the recording procedure, a slipring was placed at the connection of the electrodes to the lines outside of the plethysmograph. The animal had food and water ad libitum, and ambient temperature was maintained at 24°C. The EEG, EMG, and EOG signals were amplified by an EMBLA system (Medcare, Reykjavik, Iceland) and fed into a computer at a sampling frequency of 200 Hz for neck EMG and 100 Hz for EEG and EOG. Sleep architecture was scored in 5-sec epochs by visual inspection of EEG, EOG, and neck EMG signals (Somnologica2 software, Medcare, Reykjavik, Iceland), using the following criteria: wake was defined by a high-frequency (8-30 Hz) and low-amplitude EEG, high-amplitude neck EMG as well as muscular activity and eye movements on the EOG channel; SWS was defined by a low-frequency (0.25-4 Hz) and high-amplitude EEG, low-amplitude EMG, and no activity on the EOG; and REMS was defined by a mixed-frequency (4-8 and 8-30 Hz) and low-amplitude EEG associated with atonia on the EMG as well as phasic activity and REMs on the EOG (Figure 1).
Experiments were performed on mice belonging to the C3H/ HeOuJ strain (C3H, control mice) and its transgenic Tg(H2-IFNβ)8 strain (Tg8, MAOA-deficient mice). Tg8 mice were obtained by injecting an IFN-β minigene into a one-cell C3H embryo, leading to the insertional deletion of two essential exons of the MAOA gene.14 C3H and Tg8 mice were bred and raised under standard housing conditions in the transgenic animal facility of Paris-Sud University at Châtenay-Malabry, France. We used 2- to 3-monthold C3H and Tg8 males (20-25 g body weight), maintained in a ventilated cabinet with a 12:12-hr light-dark cycle (lights on at 07:00), a temperature of 23 ± 1°C, and food and water available ad libitum. Because of the frequent fighting initiated by Tg8 males,14 both mutant and wild-type males were housed in individual cages (20 x 20 x 30 cm) from the age of 6 weeks. This study examined 80 mice that were assigned into 1 of 5 groups. Group 1 mice had electrodes implanted and did not receive a drug treatment (6 C3H and 7 Tg8; Tables 1 and 2). Group 2 mice had electrodes and were treated with saline and the MAOA inhibitor clorgyline (7 C3H and 8 Tg8; Figs 3 and 4, left side). Group 3 mice had electrodes and were treated with saline and the tryptophan hydroxylase inhibitor PCPA (5 C3H and 8 Tg8; Figs 3 and 4, right side). Group 4 mice received neither surgery nor any treatment (7 C3H and 7 Tg8; data in the text). Group 5 mice received no surgery and were treated with either saline or PCPA (11 C3H and 14 Tg8; Fig. 5).
Measurement of Ventilation by Whole Body Plethysmography Double-chamber whole body plethysmography was used to monitor ventilation,20 the mouse being placed in the barometric chamber (500 mL, 10 cm internal diameter; food and water available), while the other chamber provided the reference pressure. The plethysmograph was placed in a circulating water bath set at 24°C, in a ventilated room with a temperature of 21 ± 1°C and a relative humidity of 55%. Each chamber was continuously flushed with room air at a rate of 700 mL/min; outlet gas was monitored for O2 and CO2 (Elisa Duo, Engström, Danemark). Plethysmographic signals were recorded as changes in the pressure difference between the two chambers by use of a differential pressure transducer. Amplified signals were fed into a computer with EEG, EOG, and EMG signals.
Surgical Procedure Mice were anesthetized with a combination of ketamine and xylazine (100 and 20 mg/kg, respectively) and electrodes (enameled nichrome wire, 150 μm in diameter) were implanted for polygraphic sleep monitoring, as previously described.19 In brief, 2 electroencephalogram (EEG) electrodes were inserted over the right cortex (2 mm lateral and 2 mm posterior to the bregma) and over the cerebellum (at midline, 2 mm posterior to lambda), 2 electrooculogram (EOG) electrodes were located subcutaneously
Table 1—MAOA-Deficient (Tg8) Males Show Wild-Type (C3H) Amounts of Vigilance States Genotype C3H Tg8
Vigilance state (%) Wake 30.4 ± 10.3 36.5 ± 9.1
SWS 65.0 ± 9.6 57.3 ± 9.2
Definition of Apnea Apneas and sighs in SWS and REMS were identified visually. Apneas were defined as a cessation of the plethysmographic signal for at least twice the average respiratory cycle duration calculated over a 10-sec period of visually identified regular breathing within the 20 sec preceding the apnea (Figure 2A).12 Sighs were defined as a respiratory cycle with an amplitude at least 50% higher than the average amplitude calculated over a 10-sec period of regular breathing preceding the sigh.13,19 Apneas were classified as post-sigh (i.e., with a sigh in the preceding 10 sec) or spon-
REMS 4.6 ± 1.7 6.3 ± 2.8
Amounts of vigilance states (mean ± SD) are expressed as percent of total recording time (6-h recording, 10:00 to 16:00). There was no significant difference in state amounts between C3H (n = 6) and Tg8 (n = 7) mice (P > 0.17). SLEEP, Vol. 30, No. 10, 2007
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Table 2—MAOA-Deficient (Tg8) Males Show Higher Indices of Sleep Apnea Than Wild Type (C3H) Males Genotype
Sleep
C3H Tg8 C3H Tg8
SWS
Total 2.6 ± 2.0 8.4 ± 4.5# 4.9 ± 4.9 13.9 ± 8.4#
REMS
Apnea index Spontaneous 0.8 ± 0.9 2.9 ± 1.7# 4.9 ± 4.9 13.3 ± 8.6
Sigh index
Post-sigh 1.7 ± 1.3 4.9 ± 2.9# 0.0 ± 0.0 0.6 ± 1.1
22.5 ± 10.4 26.9 ± 4.4 0.5 ± 1.2 1.5 ± 2.1
Apnea and sigh indices (mean ± SD) are the number of (spontaneous, post-sigh, or total) apneas and sighs per hour of SWS or REMS (6-h recording; 10:00 to 16:00). The mice are the same as for Table 1. #P < 0.05, significant difference between C3H (n = 6) and Tg8 (n = 7) mice.
Neck EMG
EOG EEG
SWS
REMS
Wake
Breathing
10 sec Figure 1—Polygraphic recording of different vigilance states in an untreated C3H mouse over a 90-sec sample period. Vigilance states were classified as wake or SWS or REMS on the basis of the EMG, EOG, EEG, and breathing signals (tracings from top to bottom). Wake was characterized by low amplitude EEG, high EMG and EOG signals, and very unstable breathing. SWS was characterized by high amplitude EEG, low EMG and EOG signals, and very stable breathing. REMS was characterized by low amplitude EEG and EMG, discrete EOG signals, and unstable breathing. Because mouse activity was not videotaped while electrophysiological data were collected, agitated sleep and quiet waking were not scored during the 6-h recording period.
taneous (no sigh in the preceding 10 sec) (Fig. 2A).19 The apnea occurrence index, defined as the number of apneas per hour, was calculated separately for each stage of sleep (SWS or REMS). The sigh occurrence index was defined as the number of sighs per hour. Wild-type and mutant mice did not show apnea during quiet wakefulness, as previously reported for wild-type 129/Sv mice.12 To determine the type of apnea (central or obstructive), we also implanted a pair of electrodes (enameled nichrome wire, 50 μm in diameter) into the motor units of the right costal diaphragm of two Tg8 mice, after a skin incision. The electrodes were then tunneled subcutaneously to the neck, and soldered to the connector17 (Figure 2B).
intraperitoneal (i.p.) injection. For the clorgyline experiment (Group 2 mice), each animal received an injection of vehicle at 09:45 (15 min before baseline recording) and then an injection of clorgyline (10 mg/kg; 2.5 mg/mL)21 48 h later (15 min before final recording). For the PCPA experiment (Group 3 mice), each animal received a daily injection of vehicle for 3 consecutive days at 18:00 (baseline recording the next day) and then a daily injection of PCPA (300 mg salt/kg; 75 mg salt/mL)22 for another 3 consecutive days at 18:00 (final recording the next day). Neurochemical Analysis We used high-performance liquid chromatography (HPLC) to measure monoamines and deaminated metabolites in tissues of 70-day-old C3H and Tg8 males (Group 5 mice) treated for 3 days with either PCPA or vehicle with the same regimen as above. Animals were decapitated 15 h after the 3rd injection and blood was drained from the head. The whole brain (with the pineal gland attached) was dissected out, frozen in isopentane, and stored at -80°C. Frozen brains were ultrasonicated in 4 mL of ice-cold 0.1
Pharmacology We used the MAOA inhibitor clorgyline (N-Methyl-N-propargyl-3-(2,4-dichlorophenoxy)propylamine hydrochloride) and the tryptophan hydroxylase inhibitor PCPA (4-Chloro-DL-phenylalanine methyl ester hydrochloride) (Sigma-Aldrich, Lyon, France), each dissolved extemporaneously in saline and administered by SLEEP, Vol. 30, No. 10, 2007
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A
Sigh Spontaneous apnea
Breathing (au)
0.25
Post-sigh apnea
0
10 sec
-0.25
B
Diaphragmatic EMG Breathing
5 sec Figure 2—(A) An example of the 2 types of apnea is shown on a breathing tracing recorded during SWS in an untreated Tg8 mouse: the spontaneous apnea is characterized by a sudden interruption of flow during quiet breathing, whereas the post-sigh apnea is identified by an interruption of flow preceded by a sigh. A 10-sec reference baseline for both apneas is underlined. (B) An example of diaphragmatic EMG recording during 2 successive central apneas in an untreated Tg8 mouse: during the interruption of the plethysmographic signal, there was no diaphragmatic EMG activity.
N perchloric acid containing disodium EDTA (122 mg/L) and ascorbic acid (8.8 mg/l). Homogenates were aliquoted in triplicate and stored at -80°C. Prior to HPLC analysis, homogenates were briefly centrifuged at 14000 g and supernatants were passed through a Nanosep 10K centrifugal filter (Pall). Then, a 50-μl aliquot of sample was analyzed for serotonin (5-hydroxytryptamine, 5-HT) by fluorometric detection.23 The amounts of catecholamines (dopamine, DA; norepinephrine, NE) and metabolites (5hydroxyindole-3-acetic acid, 5-HIAA; homovanillic acid, HVA) were measured by electrochemical detection on a serial array of coulometric flow-through graphite electrodes (Coularray, ESA). Results were expressed as picograms per milligram of wet tissue.
the light phase, i.e., approximately 35% of wake, 60% of SWS, and 5% of REMS (Table 1). These 2 strains also did not differ in the mean durations of wake, SWS, and REMS bouts (P >0.1, data not shown). In Group 2 mice (7 C3H and 8 Tg8), acute treatment with the MAOA blocker clorgyline (10 mg/kg, i.p.) did not affect wake and SWS, but had a significant effect on REMS amounts (F1,29 = 6.4, P = 0.02). Clorgyline induced a 75% decrease in the amount of REMS in C3H mice (t = 6.4, P 0.05), we performed a 2-way ANOVA on 6-h recording times, with genotype and treatment as factors. In case of significance (P
