5-hydroxytryptamine-mediated apnea caused by the habenular nucleus＊☆

Min Wang, Weihong Lin, Jinghua Wang, Min Huang, Chunyong Wang, Mingxian Li,Shao Wang

1Department of Pneumology, First Hospital of Jilin University, Changchun 130021, Jilin Province, China

2Department of Neurology, First Hospital of Jilin University, Changchun 130021, Jilin Province, China

3Department of Physiology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, Jilin Province, China

4School of Public Health, Jilin University, Changchun 130021, Jilin Province, China

lNTRODUCTlON

An increasing number of recent studies have clearly demonstrated that obstructive sleep apnea syndrome (OSA) is associated with cardiovascular complications and even sudden death, and is an independent risk factor for hypertension and stroke, and non-fatal cardiovascular events[1]. There is a high incidence of these events among the OSA population, and OSA treatment has been shown to reduce cardiovascular morbidity[2-3]. However, the pathogenesis of this syndrome remains unclear and no satisfactory treatment or clinical measures are available.

The onset of OSA is closely associated with an altered respiratory drive in the central nervous system, and dilator muscle function in the upper respiratory tract. The genioglossus muscle is the most important dilator muscle in the upper airway[4-5]. Genioglossus electromyogram (EMG) has revealed that the myoelectric activity is significantly decreased in animal models of OSA subjected to intermittent hypoxia[6].

5-hydroxytryptamine (5-HT) is reported to be the most dominant neurotransmitter influencing the hypoglossal nerve-mediated genioglossus activity[7](5-HT molecular structure is shown in supplementary Figure 1 online). 5-HT in vivo is mainly derived from the nucleus raphe magnus, in which the neurons are the main nuclei contributing to 5-HT synthesis and release in the central nervous system[8-9]. 5-HT activity and respiratory movements are modulated by the central nervous system. The involved nerve nucleus group is the habenular nucleus (Hb)and rapheal nucleus, in addition, rapheal nucleus release of 5-HT is modulated by the Hb in a continuous manner. Hb excitation can cause difficulty in respiration because of Hb’s inhibiting effect on the raphe[10]. However, it is unknown if stimulation of the Hb can cause long-term respiration cessation.

Yu et al[11]have shown that Hb may play a role in the pathogenesis of OSA, because the stimulation of Hb changes the respiratory movement in rats, which proves the relationship between Hb excitement and respiratory movement. However, no causality was determined. The present experiments presumed that 5-HT reduction is the main cause of OSA. Hb participates in the pathogenesis of OSA, by controlling 5-HT release. Therefore, 5-HT in the peripheral blood of the experimental animals with apnea should theoretically be at a lower level.

In the present study the Hb in rats was excited by an injection of L-glutamate, thus producing apnea, and genioglossus EMG changes were observed. Then, 5-HT levels in the peripheral blood of apnea-induced animals were examined. This study aimed to elucidate the following issues: (1) to determine if 5-HT release from the raphe nuclei is reduced following Hb excitation; (2) to examine if genioglossus EMG performance is abnormal and coincident with 5-HT reduction. The relationship between the Hb, raphe nuclei, 5-HT, and changes in respiration were elucidated. We show that both 5-HT and the neurotransmitter associated with OSA signal transduction may mediate genioglossus myoelectric activity and regulate breathing apnea, and that Hb is the key nucleus regulating this signaling pathway.

RESULTS

Quantitative analysis and grouping of experimental animals

One hundred and ten adult Wistar rats were randomly selected into the experiment. Except infection, hemorrhage, death and missing due to inaccuracy location of Hb position, sixty rats were entered into the final analysis.

L-glutamate stimulation of Hb group (artificial cerebrospinal fluid prepared L-glutamate solution was injected),and a control group (artificial cerebrospinal fluid was utilized to stimulate Hb). Each group contained 30 rats.

L-glutamate stimulation of Hb on respiration

After L-glutamate (0.3 μL, 10 mmol/L) was injected into the Hb, 15-30 minutes later the rats began to experience apnea. The apnea time was 2-10 seconds in duration.

The genioglossus myoelectric activity was significantly suppressed and showed significantly reduced EMG amplitude. The respiratory rhythm decreased, or even disappeared, and the EMG integral (92.44 ± 1.83)% was significantly lower than the control group (P ＜ 0.01; Figure 1). This stimulation was repeated in the same rats,and similar results were measured. In our study, the respiratory movement curve and genioglossus EMG were simultaneously recorded.

Stimulation of Hb on peripheral blood 5-HT levels

Peripheral blood 5-HT levels were detected following stimulation of the Hb. The results show that 5-HT levels in the peripheral blood were significantly lower than the control group (1.68 ± 0.09 μmol/L vs. 0.36 ± 0.20 μmol/L;P ＜ 0.05).

DlSCUSSlON

The results showed that stimulation of the Hb caused respiration abnormalities and apnea, suppressed genioglossus discharge, reduced shrinkage, and reduced peripheral blood 5-HT levels. These results are all consistent with OSA symptoms. The hypoglossal nerve controls the genioglossus. Hypoglossal neurons are modulated by 5-HT released from the raphe nuclei[7]. This release of 5-HT can increase neuronal activity in the upper airway, resulting in expansion of the upper airway.

Therefore, 5-HT is a neurotransmitter that aids in the regulation of the genioglossus and mediates the pathogenesis of OSA.

Figure 1 Breathing level and genioglossus (GG) cell discharge curve in the stimulation group and control group.The first curve represents the respiratory motion curve, the second curve is the electromyogram (EMG) amplitude curve of the GG muscle, and the third curve is the EMG integral curve.

L-glutamate only excites neuronal bodies and has no impact on the crossing nerve fibers. In this study,L-glutamate stimulation of the Hb excited the cell bodies,and strengthened the inhibition of the raphe nuclei[10].

Genioglossus cell discharge was significantly suppressed, EMG amplitude was significantly lowered, and EMG integral was markedly reduced, which are objective signs of OSA associated symptoms (weakened respiratory motion) caused by stimulation of the Hb. Together these findings agree with genioglossus EMG observations in OSA patients by Eckert et al[12-13].

The present experiment showed that stimulation of the Hb induced apnea, at a duration ranging from 2-10 seconds, while an equal dose of artificial cerebrospinal fluid was not able to produce any changes in breathing movements. This evidence indicates the stimulation of Hb neurons. The average apnea time was 2.5 seconds, which was consistent with the standards set by Carley et al[14].

Endogenous 5-HT is mainly derived from the tail end of raphe nuclei in the central nervous system. The source of 5-HT input to the hypoglossal motor nucleus is the raphe neurons of the medial medulla. The raphe neurons are at the highest activity level for a sober person, but it gradually decreases in sleep. Thus, the lost excitability which is caused by losing stable input from 5-HT to hypoglossal motor, makes diaslatic muscle of upper respiratory tract present an unstable contraction in sleep[15].

5-HT is a powerful moderator for the activity of hypoglossal motor and it increases the excitability of hypoglossal motor[16]. The GG consists of a pair of fan-shaped muscles that lie on either side of the midline of the tongue. It is well established that the activity of the hypoglossal nerve (which innervates the genioglossus muscle) is moduled by 5-HT[7]. Genioglossus are the main diaslatic muscle of upper respiratory tract controlled by hypoglossal motor. It has been reported that decreased 5-HT in rapheal nuclei contributes to OSA[9,17].

Hbenular nucleus control the raphe neurons[18].

Hb excited by chemical stimulation can enhance the suppression of the raphe nuclei. A reduction in 5-HT release can weaken dilator muscle contraction, causing the airway to become narrow, resulting in breathing difficulties and even apnea. This phenomenon is produced by the stimulation of the Hb, and thus shows a causal relationship between the Hb and OSA.

In the present study, 5-HT in peripheral blood was significantly reduced following stimulation of the Hb, which means that the release of endogenous 5-HT decreased,and this was the result of Hb excitement. 5-HT levels were significantly lower in the stimulated rats. No such change was observed in the control group. Thus, it is plausible that the stimulation of Hb decreased the levels of 5-HT released from the raphe nuclei, and due to the reduced 5-HT release, genioglossus discharge and contraction decreased, causing the OSA breathing pattern resulting from Hb excitement. Accordingly, the pharyngeal genioglossus contraction weakened. Analysis of peripheral blood 5-HT levels was utilized in this study to reflect the 5-HT changes following stimulation of Hb, and to further elucidate the regulatory mechanism underlying 5-HT on the genioglossus, and especially the role of the Hb.

The present study confirmed that the hypothalamic Hb regulated sleep and was the basis for the breathing disorders. Genioglossus muscle, controlled by the hypoglossal motor neurons, can contract via 5-HT release from the raphe nuclei. Thus, the upper airway was maintained in an open position. Stimulation of Hb resulted in apnea, similar to OSA that was mediated by 5-HT released from the raphe nuclei. Therefore, it appears that Hb excitement is the underlying cause of respiratory disorders in the whole conduction pathway.

Animal models of OSA can be established by stimulation of the Hb. Its core element is reduced 5-HT levels, which is in line with the initial hypothesis on the central mechanism underlying OSA onset and blockage of the habenular nucleus can eliminate apnea[19]. In this study,the total content of 5-HT in peripheral blood was roughly observed, but future experiments will be required to conclusively determine the role and action of 5-HT subtypes.

MATERlALS AND METHODS

Design

Randomized controlled animal experiments.

Time and setting

Experiments were performed in 2006 in the Department of Physiology, Norman Bethune College of Medicine, Jilin University, China.

Materials

One hundred and ten healthy adult Wistar rats,weighing 200-280 g, irrespective of gender, were purchased from the Experimental Animal Center of Jilin University, China (license: SYXK (Jilin) 2003-0001). Rats were housed in standard cages. Food and water was provided twice per day. The animals were maintained at room temperature (22 ± 2)°C with 12-hour light-dark cycle. Experimental treatments of animals were in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, issued by the Ministry of Science and Technology of the People’s Republic of China[20].

The experiment was given full approval of Jilin University Animal Ethics Committee, China.

Methods

Anesthesia

Rats were treated with 20% urethane (6 mL/kg) via intraperitoneal injection. The effects of light anesthesia were ascertained by the sensitivity of the corneal reflex,bent knee reflex after clamping the hind legs, but the animals should not have any spontaneous activity throughout the experiment[21]. Anesthetic agents were supplemented at 1/5 of the initial dose. The rectal temperature was maintained at 36°C using a thermal pad.Experiments were conducted in a quiet environment,with soft-lighting conditions.

Intervention

L-glutamate stimulation of the Hb group: L-glutamate(Beijing Dingguo Biotechnology Development Co., Ltd.,Beijing, China) was prepared using 10 mmol/L artificial cerebrospinal fluid at pH 7.4, and 0.3 μL volume was microinjected within 2-3 minutes. Control group: Hb was stimulated using artificial cerebrospinal fluid (10 mmol/L,including NaCl 119 mol/L, KCl 3.3 mol/L, CaCl21.3 mol/L,MgCl21.2 mol/L, Na2HPO40.5 mol/L, NaHCO321.0 mol/L,glucose 3.4 mol/L; pH 7.4), and a 0.3 μL volume of this solution was injected into the Hb in 2-3 minutes.

L-glutamate stimulation of Hb

Rats in the stimulation group and control group were fixed using an ear rod in a stereotaxic apparatus (Takahashi Company, Date City, Japan). The scalp was in-cised to expose the temporal bone. Hb coordinates were AP: -3.2, L: 0.3-0.6, H: 3.8-4.2. While in stable respiratory condition, a glass microelectrode was inserted in the rats, with direct connection to a microinjection apparatus.

Using a microelectrode propeller (Narishige, Tokyo, Japan), 0.3 μL of 10 mmol/L L-glutamate (pH 7.4) was injected into the Hb nuclei. The injection time lasted at least 3 minutes to avoid an increase in the intracranial pressure that could potentially affect neuronal function.

Breathing changes in the rats were closely observed before and after injection. The control group was stimulated with artificial cerebrospinal fluid, rather than L-glutamate.

Recording respiratory movements and genioglossus EMG

In our study, the respiratory movement curve and genioglossus EMG were simultaneously recorded. The rat abdomen was connected to the tension amplifying apparatus. The signal output was to the BL-420E biology experimental system (Chengdu Technology & Market Co.,Ltd., Chengdu, Sichuan, China), which traced the rat respiratory movement curve[22]. The EMG activity of the GG was monitored via fully insulated copper wires(1.0 mm diameter) implanted at the proximal end of the GG through the skin with 27-gauge needles. A reference electrode was positioned subcutaneously on the left ear helix. The data were digitized in 0.3 second epochs(sampling frequency 60 Hz) and recorded by computer.Genioglossus EMG was partially integrated.

Determination of peripheral blood 5-HT levels

The occurrence of apnea was confirmed after the Hb was stimulated. One mL of venous blood was placed in a test tube, and 26 μL 7.5% EDTA was used as anticoagulant. The sample was centrifuged at 3 000 r/min for 10 minutes, and then the supernatant was retained for further use. Venous blood samples were thawed prior to the determination of 5-HT. Plasma 5-HT content was detected using the Serotonin RIA kit (Labor Diagnostika Nord GmbH & Co. KG, Germany) and measured with a HTEC-500 fluorescence spectrophotometer (EHbom Corporation, Japan).

Statistical analysis

The experimental measurement data were expressed as mean ± SD. The data were tested in line with normal distribution. Completely randomized single-factor analysis of variance was conducted using SPSS13.0 statistical software (SPSS, Chicago, IL, USA). SNK-q test was utilized for pairwise comparisons among multiple groups. A level of P ＜ 0.05 was considered a statistically significant difference.

Author contributions:Min Wang, Weihong Lin, Jinghua Wang,and Min Huang provided and integrated the data. Shao Wang and Mingxian Li were responsible for the study proposal and design. Shao Wang validated the studies and the manuscript.Jinghua Wang and Min Huang analyzed the data. Mingxian Li drafted the manuscript. Chunyong Wang was responsible for statistical analysis. Min Huang provided technical or informational support.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by the National Natural Science Foundation of China, No. 30270502.

Ethical approval:The experiment was given full approval of Jilin University Animal Ethics Committee, China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 20, 2011 after selecting the “NRR Current Issue” button on the page.

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