Effects of 5-hydroxytryptamine ascending pathways of dorsal raphe nuclei and habenular nucleus on the respiration and blood pressure of rats

Obstructive sleep apnea syndrome (OSAS) was a disease of breath obstacle happened in the process of sleep. The central mechanism of OSAS has not yet been fully elucidated.1 Most of studies focused on raphe nuclei and 5-hydroxytryptamine (5-HT), and showed that brain serotonergic activity might be decreased in OSAS.2,3 It is well known that the dorsal and medial raphe nuclei provide almost all the serotonergic innervation to the forebrain. A previous study evaluated the stimulation of the dorsal raphe nuclei (DRN) in the rat inducing mainly pressor and sympathoexcitatory responses.4 The habenular nucleus (Hb) located on the posterior medial surface of the caudal thalamus plays a pivotal role in linking various limbic forebrain areas with the midbrain neuromodulatory centers such as the interpeduncular nucleus, substantia nigra pars compacta, ventral tegmental area, and dorsal raphe nucleus.5,6 Hb is involved in a broad array of functions including the regulation of sleep,7 the control of respiration and of blood pressure.8,9 The respiratory alteration induced by stimulation of Hb is similar to OSAS.8 Therefore Hb may participate in the regulation of ascending pathways of DRN for respiration and blood pressure in rats. The aim of this study was to investigate the effect of respiratory and blood pressure induced by DRN-Hb ascending pathways, and to explore the possible role of Hb in the happening of OSAS. METHODS Animals A total of 44 adult Wistar rats (either sex weighing 220—260 g) were used. Animals were maintained under standard conditions ((22 ± 1) °C, 40%-50% humidity, food and water available continuously). Anesthesia was produced with urethane (1.2 g/kg, i.p.) Supplementary doses of anesthetic (0.2 g/kg, i.p.) were administered when necessary, as indicated by the pupil size, paw pinch reflexes and unstable blood pressure. All of the surgical and animal care methods conformed to the Guidelines for the Use of Animals of the International Brain Research Organization. Respiration and blood pressure recording A tension transducer was linked to the skin of the chest surface for recording respiratory movement. The femoral artery was cannulated with a polyethylene tube filled with 1% heparin natrium for continuous measurement of arterial pressure. The arterial catheter was connected to a strain gauge transducer. Respiration and the pulsatile blood pressure were monitored and recorded simultaneously on computer installed MedLab software (Nanjing, China). Electrical stimulation of DR The head of the animal was fixed in a stereotaxic apparatus (VDT-1, China), and a small craniotomy was performed over DR in order to allow the insertion of the electrode. The stereotaxic coordinates of DR were 7.5 mm posterior to the bregma, 0 mm lateral to the midline, and 5.0 mm below the brain surface, according to the atlas of Paxinos and Watson.10 A circular electrode (0.4 mm in outside diameter, 0.2 mm in inside diameter) was inserted into DR according to the above-mentioned stereotaxic coordinates. DR was excited by electrical stimulation with current intensity of 0.03 mA—0.05 mA, duration of 0.3 ms and frequency of 50 Hz for 5 seconds (stimulator, SEN-7103, Japan). The electrode resistance was 40—60 kΩ. Microinjection in Hb A stainless steel guide cannula (0.4 mm in outside diameter, 0.2 mm in inside diameter) attached to a 1 μl microsyringe was positioned into Hb. The stereotaxic coordinates of Hb were 3.3 mm posterior to the bregma, 0.5 mm lateral to the midline, and 4.0 mm below the brain surface.10 Lidocaine (2 %, 0.3 μl, Sigma, USA) or physiological saline (0.3 μl) was injected respectively into Hb during a period of 2 minutes. The method of 5-HT (3 μg, 0.3 μl, Sigma) microinjection is same as that of lidocaine. Lidocaine and 5-HT were dissolved in physiological saline, respectively. Histological examination At the end of each experiment, 2% pontamine sky blue (0.3 μl) was injected into Hb to mark the final injection site. At the end of the recording period the position of the stainless steel electrode was marked by passing a DC current of 200 μA for 30 seconds (stainless steel electrode anode); a small deposit of iron was made at the stimulus site in this manner. After the animals was anesthetized deeply with an overdose of barbiturate, the brains were rapidly removed and fixed in 10% solution of formalin for about 7 days. Coronal frozen sections (40 μm) including in the region of the Hb and DRN were cut for histological identification of the injection site. The location of each of the effective sites was determined with the aid of a stereotaxic atlas of the rat brain.10 The data from one inaccurate orientation was excluded from the results. Statistical analysis All data were presented as mean ± standard deviation (SD). The paired Student t test was used for statistical comparisons, SPSS 10.0 was used in this study. P value less than 0.05 was considered statistically significant. RESULTS Effect of DR electrical stimulation on respiratory rate and blood pressure Experiments were performed on the 14 spontaneously breathing rats. Electrical stimulation of eight rats' DR caused an increase in respiratory rate and blood pressure. Breath rhythm was regular and chest movement range has no obvious change. The respiratory rate increased from (85.8 ± 10.4) breaths/min to (214.8 ± 43.7) breaths /min (P<0.001) and blood pressure increased from (90.2 ± 6.8) mmHg to (114.3 ± 10.5) mmHg (P<0.05). Histological examination showed that stimulating electrode tip was in DR. Six other rats' DR was stimulated by applying the same stimulation parameter when electrode tip wasn't in DR, no significant respiratory or blood pressure changes. Effect of microinjection of lidocaine into bilateral Hb on respiratory rate and blood pressure Seventeen rats (10 for the microinjection of lidocaine and 7 for the microinjection of physiological saline) were used. When lidocaine were microinjected into both sides of ten rats' Hb, the increasing effect of the respiratory rate induced by stimulation of DR was strengthened obviously in 5 minutes after injection (56.8 % excited, P<0.05, Table 1). However, the pressor effect induced by stimulation DR was alleviated (62.1% blockade, P<0.01). Both the increase and decrease effects were recovered to baseline level 90 minutes after injection lidocaine. Respiratory rhythm and chest movement range were in order and they were not obviously changed during the period of electrical stimulation of DR and lidocaine microinjection in Hb.Table. 1: Effect of microinjection of lidocaine into bilateral Hb on the changes of respiratory rate and blood pressure elicited by stimulating DRThe other seven rats were microinjected physiological saline into both sides of Hb. As shown in Table 2, an increase data in respiratory rate and blood pressure caused by electrical stimulation of DR did not differ significantly (P>0.05) before and after microinjection saline.Table. 2: Effect of microinjection of physiological saline into bilateral Hb on the changes of respiratory rate and blood pressure elicited by stimulating DREffect of microinjection of 5-HT into Hb on respiratory rate and blood pressure Experiments were performed on 13 rats (7 for the microinjection of 5-HT and 6 for the microinjection of physiological saline). 5-HT (3 μg, 0.3 μl) microinjection into Hb of seven rats caused decreased respiratory rate and increased blood pressure. The respiratory rate gradually decreased after injection from (88.5 ± 7.2) breaths/min to (76.4 ± 10.03) breaths/min and decreased to minimum 4—6 min after injection (P<0.01). The blood pressure immediately increased after injection from (79.7 ± 12.8) mmHg to (91.0 ± 7.1) mmHg and increased to maximum 2—3 minutes after injection (P<0.05). Above changes recovered to the normal level 10—15 minutes after injection. The microinjection of physiological saline (0.3 μl) into Hb of six rats caused no significant changes in the respiratory rate and blood pressure (P>0.05). DISCUSSION More and more investigators have paid attention to OSAS in recent years. Previous studies showed that 5-HT released by raphe may play an important role in initiation of OSAS3 and there was a relationship between DRN and OSAS.11 In central nervous system serotonin nerve cells concentrate in midbrain raphe. 5-HT is the main putative transmitters in respiratory control.12 Lee et al13 demonstrated that dorsal raphe had synapse connection with respiratory center and pontine reticular related to REM. So raphe nuclei are the site of 5-HT production and relate to the regulation of sleep and respiration. In this experiment, electrical stimulation of DRN caused an increase in respiratory rate and blood pressure. When lidocaine was microinjected into both sides of Hb, the respiratory rate increasing effect induced by stimulation of DRN obviously increased; however, the pressor effect induced by stimulation DRN was weakened. All data showed that DRN modulated the respiratory movement and cardiovascular activity. The respiratory rate increasing effect and pressor effect induced by stimulation of DRN are mediated by Hb. Because microinjection of lidocaine into the Hb in rats strengthened the respiratory rate increasing effect induced by stimulation of DRN and alleviated the pressor effect induced by stimulation DRN. We considered that serotonergic neuron were suppressed by lidocaine. To test this hypothesis, we microinjected 5-HT into the Hb of rats; the respiratory rate decreased and blood pressure increased. These results supported the hypothesis that Hb mediated respiratory and cardiovascular effect induced by DRN and 5-HT is one of the neurotransmitters involved in respiration and cardiovascular effect. Hb serves an important relay function, conveying limbic forebrain inputs to midbrain structures.14 Hb is concerned with many regulation of physiological functions, including cardiovascular activities, respiratory movement and sleep. It was reported that there is a dense serotonin terminal plexus from raphe nuclei in Hb. Hb has an influence on the serotonergic efferent neuron.15 It was suggested that there was the morphological and functional connection between Hb and raphe nuclei and 5-HT was involved in the feedback pathway. Hb influences respiration8 and the regulation of 5-HT ascending pathways of dorsal raphe. So we also can hypothesize that Hb may be an important centre during OSAS initiation and modulation, which provides a new theoretical basis for the studying of OSAS.