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Sleep. Oct 1, 2007; 30(10): 1295–1302.
PMCID: PMC2266281

Sleep Apneas are Increased in Mice Lacking Monoamine Oxidase A

Caroline Real,1 Daniela Popa, PhD,2,3 Isabelle Seif, PhD,1 Jacques Callebert, PharmD, PhD,4 Jean-Marie Launay, PharmD, PhD,4 Joëlle Adrien, PhD,2,3 and Pierre Escourrou, MD, PhD1

Abstract

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.

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.

Keywords: Sleep apnea, serotonin, control of breathing, MAOA, sleep

INTRODUCTION

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 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 5-HT, 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).1417 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.

METHODS

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).

Animals and Experimental Groups

Experiments were performed on mice belonging to the C3H/HeOuJ strain (C3H, control mice) and its transgenic Tg(H2-IFN-β)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-month-old 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 × 20 × 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 and2).2). Group 2 mice had electrodes and were treated with saline and the MAOA inhibitor clorgyline (7 C3H and 8 Tg8; Figs 3 and and4,4, 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 and4,4, 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).

Table 1
MAOA-Deficient (Tg8) Males Show Wild-Type (C3H) Amounts of Vigilance States
Table 2
MAOA-Deficient (Tg8) Males Show Higher Indices of Sleep Apnea Than Wild Type (C3H) Males
Figure 3
Effects of clorgyline and PCPA injections on the amounts of wake, SWS and REMS in wild-type and mutant mice. Clorgyline (10 mg/kg, i.p.) induced a decrease of REMS in C3H mice (n = 7), but not in Tg8 mice (n = 8). Subchronic treatment with PCPA (300 mg/kg ...
Figure 4
Effects of clorgyline and PCPA injections on apnea index during SWS and REMS in wild-type and mutant mice. Breathing was recorded for 6 h in the same experiment as in Figure 3. Apnea indices were analyzed by the paired t-test and are presented as a percentage ...
Figure 5
Subchronic administration of PCPA in Tg8 mice reduced the amounts of serotonin (5-HT) but not of dopamine (DA) and norepinephrine (NE). These three monoamines and two of their metabolites (5-HIAA and HVA) were measured by HPLC from the brains of mice ...

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 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.

Sleep Recording and Scoring

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 slip-ring 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).

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). ...

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.

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 spontaneous (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

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 ...

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).

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 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 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 (5-hydroxyindole-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.

Statistical Analysis

All data were expressed as mean ± standard deviation (SD). Significance was tested by first performing a 3-way analysis of variance (ANOVA) using genotype, treatment, and time as repeated measures. Because the effect of time was not significant in all cases (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 <0.05), it was followed by a one-way ANOVA or Kruskal-Wallis test when appropriate to compare data between genotypes, and the paired t-test to assess the effect of drugs (SigmaStat software, SPSS, Chicago).

RESULTS

Vigilance States

In Group 1 mice (untreated mice, see Methods), Tg8 mutant mice (n = 6) showed the same amounts of wake, SWS and REMS as wild-type C3H mice (n = 7) in the barometric chamber during 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 <0.001) (Figure 3, left side).

In Group 3 mice (5 C3H and 8 Tg8), a 3-day treatment with the 5-HT synthesis inhibitor, PCPA (300 mg/kg, i.p. daily), did not significantly affect vigilance states in any strain (Figure 3, right side).

Sleep Apnea Index

In Group 1 mice (untreated mice), Tg8 mice (n = 7) significantly differed from C3H mice (n = 6) by a greater total (post-sigh and spontaneous) apnea index, which was increased about 3-fold during SWS (F1,12 = 8.5, P = 0.014) and REMS (F1,12 = 5.4, P = 0.04) (Table 2). In SWS, this increase corresponded to higher indices of spontaneous and post-sigh apneas (F1,12 = 7.0, P = 0.02 and F1,12 = 6.2, P = 0.03, respectively). The 2 mouse strains did not differ with respect to overall respiratory frequency, baseline respiratory frequency before apneas, or sigh occurrence indices (data not shown) (Table 2). In REMS, the increase in spontaneous apnea index was close to significance (F1,12 = 4.5, P = 0.057) (Table 2). Similarly, a 5-fold interstrain difference in apnea frequency was observed between C3H (n = 7) and Tg8 (n = 7) mice that underwent no surgery (neither anesthesia nor implantation of electrodes; Group 4 mice) (H(1,13) = 6.6, P = 0.007, data not shown).

Then, apneas were evaluated after treatment with saline and either clorgyline (Group 2 mice) or PCPA (Group 3 mice). In Group 2, after saline treatment, Tg8 mice (n = 8) had a 5-fold higher apnea index than C3H mice (n = 7) in SWS and REMS (H(1,14) = 9.8, P <0.001, and F1,14 = 5.1, P = 0.04, respectively). The MAOA blocker clorgyline (10 mg/kg, i.p.) had a significant effect on apnea indices in SWS and REMS (F1,29 = 6.6, P = 0.02, and F1,28 = 6.1, P = 0.02, respectively) (Figure 4). In C3H mice, clorgyline increased the apnea index in SWS (5.7-fold) and in REMS (9.4-fold) (t = −3.1, P = 0.02, and t = 4.2, P = 0.009, respectively). In SWS, this increase was attributable to an enhancement of both spontaneous and post-sigh apneas (t = −2.7, P = 0.04, and t = −2.9, P = 0.03, respectively, data not shown). In Tg8 mice, clorgyline had no significant effect on apnea index (Fig. 4). Clorgyline-treated C3H and Tg8 mice did not significantly differ in SWS (P = 0.7) nor in REMS (P = 0.08). Clorgyline did not change overall respiratory frequency, baseline respiratory frequency before apneas, or sigh occurrence indices (data not shown).

In Group 3, after saline treatment, Tg8 mice (n = 8) had a higher apnea index than C3H mice (n = 5) in SWS (2.2-fold) and REMS (3.4-fold) (F1,12 = 4.8, P = 0.05, and F1,11 = 7.5, P = 0.02, respectively). With the PCPA treatment (300 mg/kg i.p. daily for 3 days), there was a significant genotype/treatment interaction in SWS and in REMS (F1,25 = 7.0, P = 0.015, and F1,24 = 6.5, P = 0.02) (Fig. 4). PCPA induced a decrease of apnea index in Tg8 mice only, in both SWS (− 67%) and REMS (− 61%) (t = 36.0, P = 0.008, and t = 5.0, P = 0.003, respectively) (Fig. 4) in such a way that apnea indices in Tg8 mice did not differ from those in C3H mice in SWS (P = 0.2) and REMS (P = 0.4). In Tg8 mice, this decrease in SWS was observed for both spontaneous and post-sigh apneas (t = 3.0, P = 0.02, and t = 2.9, P = 0.02, respectively, data not shown). PCPA did not change overall respiratory frequency, baseline respiratory frequency before apneas, or sigh occurrence indices (data not shown).

Neurochemical Analysis

As shown in Figure 5, vehicle-treated Tg8 mice (n = 7) had higher brain concentrations of 5-HT, NE and DA than vehicle-treated C3H mice (n = 4) [(+ 49%, F1,10 = 252.8, P <0.001); (+ 81%, F1,10 = 223, P <0.001); (+ 16%, F1,10 = 44.2, P <0.001)]. Conversely, vehicle-treated C3H mice showed higher brain concentrations of 5-HIAA and HVA [(+ 56%, F1,10 = 69.6, P <0.001); (+ 196%, F1,10 = 284.1, P <0.001)].

Subchronic treatment with PCPA decreased 5-HT and 5-HIAA levels in C3H mice (n = 7) [(− 28%, F1,10 = 120.9, P <0.001); (− 49%, F1,10 = 99.1, P <0.001)] and in Tg8 mice (n = 7) [(− 11%, F1,13 = 41.8, P <0.001); (− 39%, H(1,13) = 48.3, P <0.001)] (Fig. 5). These PCPA effects on the 5-HT system were greater in wild-type mice, and the level of 5-HT in PCPA-treated Tg8 mice remained significantly higher than in vehicle-treated C3H mice (F1,10 = 94.3, P <0.001). Somewhat correspondingly, PCPA decreased the brain concentration of DA in C3H mice (− 10%; F1,10 = 14.9, P = 0.004) but not in Tg8 mice (Figure 5).

DISCUSSION

In the present study, we show that the MAOA gene deletion in Tg8 mice did not affect the mean vigilance state amounts and average durations of states during the light phase in the barometric chamber, but increased the apnea index about 3-fold in SWS and in REMS, as compared with control C3H mice. Acute treatment with the MAOA inhibitor clorgyline decreased REMS in C3H mice and mimicked the effect of the mutation on the apnea index in SWS and REMS. Subchronic treatment with the tryptophan hydroxylase inhibitor PCPA did not significantly modify the vigilance states of both strains but decreased the apnea index of Tg8 mice by 61%-67% during SWS and REMS.

Sleep

It is known that monoamines, particularly 5-HT and NE, have an important role in sleep-wakefulness regulation.18 In particular, 5-HT has a tonic inhibitory influence on the expression of REMS.24 However, even though 5-HT and NE concentrations in brain were increased 1.5- and 2-fold, respectively in MAOA mutants, no sleep-wakefulness differences from C3H mice were observed under baseline conditions during the light phase (10:00 to 16:00). This is in agreement with a previous report.25 To pharmacologically mimic the MAOA mutation, we have injected the MAOA inhibitor clorgyline in C3H mice. In contrast to the MAOA mutation, clorgyline decreased REMS in C3H mice, which is in agreement with previous studies in cats26 and rats.27 As expected, clorgyline did not modify sleep in Tg8 mice.

Such difference between the effects of gene invalidation and acute pharmacological inactivation of the gene coded protein in adults has been shown in other models.13,28 This apparent discrepancy could originate in developmental alterations, because brain concentrations of NE and particularly of 5-HT are elevated from early developmental stages in MAOA mutants (i.e., 5-HT is elevated up to 9-fold during the perinatal period in Tg8 mice, as compared with wild-type C3H mice).14 Alternatively, adaptations at other levels of 5-HT or NE neurotransmission might have occurred, as suggested in the case of constitutive 5-HT receptor knockout mice.24 For example, although acute blockade of 5-HT2A receptor with MDL 100907 produces an increase in SWS in 5-HT2A +/+ mice, 5-HT2A −/− animals express reduced SWS under baseline conditions (and are insensitive to MDL 100907 treatment).13 This result suggests the existence of adaptive mechanisms in these constitutive mutants. A deficit in 5-HT2B receptor function could represent one of these compensatory adaptive phenomena.13 In the same manner, 5-HT1A −/− mutants exhibit functional supersensitivity since acute activation of their 5-HT1B receptor with CP 94253 causes a more pronounced reduction of REMS amounts than in wild-type mice.28

Several studies have demonstrated that sleep is reduced after administration of PCPA, in cats (a single injection of 200 mg/kg is sufficient to reduce sleep to a daily average of less than 10% of the normal sleep time)29 and in rats (an injection of 300 mg/kg reduces sleep by half after 24 h).30 This insomnia is reversed by the serotonin precursor 5-hydroxytryptophan (5-HTP, the synthesis of which is inhibited by PCPA).29 In C3H and Tg8 mice, PCPA had no effect on waking time. This may be attributable to the smaller decrease in cerebral 5-HT in C3H and Tg8 mice (-28% and - 11%, respectively), as compared to PCPA-treated cats and rats.29,30 The variable efficacy of PCPA in reducing 5-HT concentration in different mammalian species and mouse strains, likely resides in the primary structure and expression levels of tryptophan hydroxylase (TPH2 controls brain 5-HT concentrations, for the most part, whereas TPH1 controls 5-HT concentrations in the gut, blood and pineal gland; both enzymes are inhibited by PCPA) and of other molecules involved in 5-HT synthesis, storage, release, uptake, or metabolism.3133

Sleep Apneas

During sleep in C3H mice, post-sigh apneas occurred during SWS, whereas spontaneous apneas occurred during both SWS and REMS, which is in fair agreement with studies in 129/Sv and C57BL/6J mice (using other threshold values).12,13 The same state-related frequency of spontaneous and post-sigh apneas was observed in Tg8 mice. It has been shown that apneas in 129/Sv mice depend on a central mechanism.12 We confirmed this result by recording diaphragmatic EMG in two Tg8 mice and finding no diaphragmatic effort during apneas. Post-sigh apneas are generally thought to be related to hypocapnia following the augmented breath.34 However, mechanoreceptor activation has also been suggested as a possible mechanism.12 The origin of spontaneous central apnea is unknown.

Physiological evidence suggests that 5-HT acts as a ventilatory inhibitor.3,4 In intact anesthetized cats, 5-HTP given alone depresses ventilation, this effect being potentiated by MAO inhibitors and associated with elevation of 5-HT level in the brain.35 In line with these results, Tg8 mice had a greater apnea index than C3H mice in both SWS and REMS. Furthermore, acute administration of clorgyline to C3H mice increased apnea index in SWS and REMS to the level observed in Tg8 mice. Thus, sleep apnea might be related to 5-HT or NE concentrations.

In this regard, chronic treatment with PCPA reduced brain 5-HT concentration by 11% in Tg8 mice (with no effect on total brain NE content) and induced a decrease of apnea index in Tg8 mice to the baseline level observed in C3H mice. These results in Tg8 mice are in agreement with a study indicating that in lightly anesthetized mice, PCPA restores normal respiratory response to hypoxia.17 This rescue effect of PCPA is in agreement with a report that Tg8 mice show weaker respiratory responses to hypoxia and lung inflation than their C3H counterparts, under phenobarbital anesthesia, and that this phenotype is rescued by a 4-day treatment with PCPA.17 Somewhat similarly, in decerebrate cats, PCPA increases ventilation, an effect that is reversed by administration of 5-HTP.36 In awake rats, depletion of cerebral 5-HT with an intracerebroventricular injection of 5,7-dihydroxytryptamine produces hypoventilation, whereas intraperitoneal injection of PCPA or 6-fluorotryptophan (another tryptophan hydroxylase inhibitor) produces hyperventilation, which is alleviated by 5-HTP administration.37 However, we have not found a significant effect of PCPA on apnea index in C3H mice, even though the brain 5-HT level was decreased by 28% in this strain. Thus, it seems that a moderate reduction in 5-HT concentration only reverses the effect of an excess of 5-HT on ventilation (i.e., in Tg8 mice).

In a rat model of central apnea during SWS and REMS, intraperitoneal injection of 5-HT increases apneas during REMS (but not during SWS). This effect is blocked by GR38032F (ondansetron), a 5-HT3 receptor antagonist.10 Because 5-HT does not readily cross the BBB, this observation in rats favors a role of peripheral 5-HT receptors in apnea genesis during REMS in our mouse model. However, we did not assess the effects of the subchronic PCPA treatment on peripheral monoamines in C3H and Tg8 mice. Therefore, there is a need for complementary studies to ascertain the relative role of peripheral versus central aminergic mechanisms in mice.

Another hypothesis for the role of 5-HT in sleep apnea is a developmental effect of its excess in MAOA mice, which could alter some components of the respiratory system during the perinatal period.38 These abnormalities would result in lifelong dysfunction of the respiratory system.17

Norepinephrine ordinarily stimulates ventilation.3,4 Decreasing NE levels in decerebrate cats by injecting α-methyl-tyrosine36 and in awake dogs by adrenal medullectomy39 depresses ventilation. Conversely, increasing brain levels of both 5-HT and NE in decerebrate cats with the MAOA/B inhibitor tranylcypromine stimulates the resting spontaneous breathing.36 Although Tg8 mice have an increase in both 5-HT and NE, our treatment with PCPA decreased their apnea index to the same level as that in C3H mice, even if this treatment decreased only 5-HT, and not NE, brain concentration. Our results do not suggest an important role for NE in central apnea generation.

CONCLUSION AND CLINICAL RELEVANCE

The combined use of gene invalidation and specific pharmacological treatments allowed us to assess the differential contribution of 5-HT to sleep and breathing control in mice. High constitutive concentrations of 5-HT in MAOA mutants did not prevent normal sleep control during the light phase, but MAOA mutants showed an increased apnea index in SWS and REMS. Systemic injections of PCPA reduced these apnea indices and brain 5-HT concentration, without significantly changing the amounts of SWS or REMS and brain NE concentration. Thus, elevated concentrations of 5-HT (in the CNS or periphery) appear to play a major role in the generation of apnea during SWS and REMS. Similarly, in control wild-type mice, acute blockade of MAOA with clorgyline increased sleep apnea indices.

Mechanisms underlying obstructive sleep apnea syndrome (OSAS) in humans and central sleep apnea in mice may be very different. However, it is possible that both central and obstructive apnea at least partly reflect dysregulation of central neural motor control of the respiratory system.40 Our results could explain why pharmacologic interventions aiming at increasing upper airway muscle activity by manipulating endogenous 5-HT in patients with OSAS, have had limited clinical benefit. An optimal level of brain 5-HT may be necessary to maintain both a patent airway and a stable ventilatory drive: abnormally high and low levels may predispose to sleep apnea.

ACKNOWLEDGMENTS

We thank Pauline Robert and Valérie Domergue for dedicated animal care.

This study was supported by the CNRS, INSERM, French Ministry of Research, and University of Paris-Sud.

Footnotes

Disclosure Statement

This was not an industry supported study. The authors have reported no financial conflicts of interest.

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