| Central chemoreceptors ( CCRs)play an important role in rapidly adjusting the acid-base balance and in maintaining the stabilization the internal environment. Although researchers have located the CO2/H+ -sensitive parts that distributed mainly in the dorsal and ventral brainstem as well as in the raphe and locus coeruleus, how and by which mechanisms these nucleus sense the changes of CO2/H+ in cerebrospinal fluid and subsequently regulate the pulse released by respiratory centre are still riddles to us. An initial report referred that the depolarization of CO2/H+-sensitive neurons were resulted from the inhibition of a kind of 'background'or 'leak'potassium channel. The different currents of this kind of K+ channel might be the molecular basis of CCRs responding to chemical stimuli. TASK-1 (TWIK-related acid-sensitive K+ channel-1; TWIK, for tandem P domains in a weak inwardly rectifying K+), a two-pore potassium 'leak'channel, is such a open rectifier that is sensitive to the physiological extracellular pH. This kind of ion channel is expressed in many brainstem neurons and is regulated by some neurotransmitters such as 5-HT, NE and SP, which contribute to the breathing regulation. Thus, TASK-1 might be the molecular chemoreceptors existing in the central nervous system and raise the breathing activation by increasing the excitement of breathing-related neurons. Sleep apnea syndrome(SAS)is a kind of disease that seriously affects the health of human beings. Recent reports have found the occurrence of sleep apneas might result from the destabilization of central ventilatory control followed by the abnormity of the sensitivity of CCRs. Considered the properties and the expression of TASK-1 in central nervous system, especially its highly expression in hypoglossal nucleus that innerve the genioglossus whose dysfunction is an important factor contributing to the happening of obstructive sleep apnea, we speculate whether its abnormity of quantity and function take part in the pathogenesis of SAS. So, our studies will firstly systemically observe the expression of TASK-1 mRNA in brainstem breathing-related neurons and try to test the properties of its channel currents, so as to primarily evaluate the possible relationship between TASK-1 and the central respiratory control in light of the anatomical distribution and the electrophysiological properties owned by TASK-1 channel. Then we try to examine whether the expression of TASK-1 participates the occurrence of sleep apneas by analyzing the quantity of TASK-1 mRNA and the apnea index (AI), which also can help us to understand the function of TASK-1 channel in the mechanism of central respiratory control. 1 Expression of TASK-1 channel mRNA in breathing-related neurons of the rat brainstem In order to validate on anatomical distribution that TASK-1 channel is a kind of molecular chemoreceptor existing in the central nervous system, we used digoxigenin-labeled oligonucleotide probe in situ hybridized with the brainstem slices to systemically examine the expression of TASK-1 mRNA in brainstem breathing-related neurons. The neurons we focused on in the experiments include the putative H+/CO2-sensitive central chemoreceptors(CCRs), the classical respiratory groups of the dorsal and ventral respiratory groups in the medullar oblongata and the pontine respiratory group, the pre-B?tzinger complex (pre-B?tC) that related to the genesis of breathing rhythm, and the pedunculopontine tegmental nucleus ( PPN ) and the intertrigeminal region(ITR)which take part in the breathing regulation. The results suggested that TASK-1 mRNAs were expressed in all of the CCRs, including the nucleus tractus solitarius (NTS) and dorsal motor nucleus of vagus (DMV) in dorsal medulla (DM); the retrotrapezoid (RTN), the nucleus ambiguus of ventral respiratory group and pre-B?tC in the ventrolateral medulla (VLM); the caudal medullary raphe; and the locus coeruleus (LOC). TASK-1 mRNA also highly expressed in the dorsal andventral respiratory groups but weakly expressed in the pontine respiratory group, PPN and IRT. The fact that TASK-1 mRNA was widely located in the brainstem breathing-related neurons clewed that TASK-1 channel might take part in the central breathing regulation by some unknown mechanism. The high expression in dorsal and ventral respiratory groups but weakly in pontine respiratory group seemed that TASK-1 channel might mainly contribute to the sensitivity of central H+/CO2 and less to the breathing control of frequency and amplitude accomplished by pons. 2 TASK-1, an Acid-sensitive background K+ channel A significant character of TASK-1 is the sensitivity of its channel current to the physiological extracellular pH, which suggests that TASK-1 might be a molecular chemoreceptor existing in the respiratory centre. But, whether the function of this channel has the same stability and whether its dysfunction can cause the disease are remain unclear. In order to examine its function in later studies, we established a sample method to observe the acid-sensitive character of TASK-1 channel. Because the genioglossus innerved by hypoglossal nucleus (HN) play an important role in maintaining the upper airway patency and TASK-1 was highly expressed in the HN, studying the membrane currents in HN will be helpful to understand the mechanism of sleep apnea. The blind whole-cell patch clamp recording technique with voltage clamp mode was used to record the current of TASK-1 channel in HN within alive brain slices of 300μm. After the voltage clamp mode of whole-cell recording was set up, judged by traces of the screen, the membrane currents were recorded in response to a hyperpolarizing ramp (-60 to -130 mV) and the -10 mV steps. Then the pH of perfusion liquid was changed to 6.4 and 8.4 respectively, and the procedure of ramp and step were managed again to record membrane currents in different extracellular pH states. The membrane currents we recorded from eight cells were all outward of about 200～350 pA. The reversal potential obtained by drawing the I-V curveswas about -90 mV. The acidification of the external solution resulted in the inward direction of currents, whereas alkalization induced a corresponding change of outward direction. The pH sensitivity of these currents was the same determined by titration with solutions of different pH (from pH 6.4 to 8.4) in internal HEPES under the concentration of both 10 mM and 25 mM. We concluded from the results that the membrane currents recorded from HN were TASK-1, which were very sensitive to the physiological extracellular pH. And the method used in the experiment was a simple and feasible approach to record TASK-1 channel. 3 Analysis of sleep structure and sleep apneas in Sprague-Dawley rats Clinical reports demonstrated that the occurrences of both obstructive sleep apnea (OAS)and central sleep apnea (CSA)involved the mechanism of abnormal respiratory drive. Considering that the obstruction of upper airway will affect the observation of central respiratory drive, it is necessary to establish an animal model of CSA through which further studies could be done. It seems that the Sprague-Dawley(SD)rat might be the suitable animal for studying central mechanism of sleep apnea because they have following characters including the similar electroencephalogram (EEG) to human beings, the long sleep time and especially the phenomenon of apneas happened in sleep time. So, in this section we try to evaluate the feasibility of SD rat being an animal model on investigating the central mechanism of sleep apnea by analyzing the sleep structure and the apneas occurring in both awake and sleep states. Each of freely moving adult SD rat was monitored for 6 hours. During the time, a multi-channel amplifier recorded the electrical signals of cortical EEG, neck electromyogram (EMG) and the breathing transformed from a micro-pressure transducer. Waves of these signals were displayed on the screen and stored in the disc synchronously. Data were analyzed by a computer-person mutual pattern in order to distinguish the awake/sleep states and different kinds of sleep apneas. Finally, results were dealt with by SAS software.The ratios of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep to total sleep time were (83.8±7.1)and(16.2±7.1)%, respectively. All of the 26 adult SD rats appeared apneas during sleep time, and the mean sleep apnea index (AI) was (11.5±4.6) events per hour. Apneas were occurred more often in REM sleep than in NREM sleep, with the AI of (9.8±4.6)and(21.8±10.9)events per hour. Furthermore, post-sigh apneas were predominant in NREM sleep and spontaneous apneas in REM sleep respectively(P<0.001). From the results, we concluded that the sleep structure of SD rats was similar to human beings, and the characters of sleep apneas were comparative to human patients with sleep apneas. Therefore, SD rat was proved to be a promising natural animal model for studying the central mechanism of sleep apnea. 4 Analysis of the correlation between the sleep apnea index and the expression quantity of TASK-1/TASK-3 mRNA Recent researches have reported that the occurrence of sleep apnea might result from the abnormity of the sensitivity in CCRs, which tempt us to assume that the unusual expression of TASK-1 channel might be related to the happening of sleep apneas. TASK-3, another member of TASK family, is very possible to form the heterodimers with TASK-1 in cell membrane and has some functional relationship with TASK-1. So we tried to analyze the correlation between the sleep AI and the expression quantity of TASK-1/TASK-3 mRNA, by which we might find some clues on the pathogenesis of the respiratory-control-disordered diseases. After AIs were calculated based on the six hours'monitoring for both sleep and breathing, RNAs extracted from the lower brain stems of 25 rats were prepared. The method of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the mRNA expression of both TASK-1 and TASK-3, and then we analyze the correlation between AIs and the quantity of TASK-1 and TASK-3 mRNAs. The AIs of these 25 rats were between 4.36～24.7 times/hr with theaverage of 11.8±5.05 times/hr. The quantity of TASK-1 mRNA and TASK-3 mRNA was closely correlated with the coefficient of 0.603,P < 0.01。AI and both the TASK-1 mRNA quantity and the ratio of TASK-1 and TASK-3 mRNA quantity were closely correlated with the coefficient of 0.452 and 0.548, respectively, P < 0.01。AI in both NREM and REM states were correlated with the ratio of TASK-1 and TASK-3 mRNA quantity, and the coefficient were 0.449 and 0.453,respectively, P < 0.05。It could be concluded from the results aforementioned that there might exist some internal relationship between the TASK-1 and TASK-3, and the occurrence of sleep apneas are related with the expression of TASK-1 and TASK-3 channel, especially the TASK-1. The different sleep states seemed no impact on the occurrence of sleep apnea affected by these two channels. 5 Influence of different hypoxia on expression of TASK-1 and TASK-3 mRNA In order to make sure the causality of the sleep apnea and the expressions of TASK-1 and TASK-3 channel, we examined the quantity of TASK-1 and TASK-3 mRNA of lower brainstems in both continuous hypoxia rats (CH) and the intermittent hypoxia with hypercapnia (IHH) rats. To make CH rat models, 10 rats were put into the isobaric hypoxia cabin and the concentration of oxygen was maintained at (10.0±0.5)%,2 hours/day for 8 days. For IHH rat models, another 10 rats were put into 1000ml wide-mouth bottles respectively for 1.5 min and then bottles were ventilated with fresh air for 2 min, 2 hours/day for 8 days. Control group was comprised of 10 rats without any kind of hypoxia stimulus and normally fed for 8 days. After the experiments, the venous blood samples and the lower brainstem of these three groups'rat were taken. And then, the values of red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) in blood samples as well as the quantity of the TASK-1 and TASK-3 mRNA of these three groups were analyzed. There were significant difference of RBC, HGB and HCT between the CH group and the control group, the IHH group and the control group(both P< 0.05). RBC had no difference between the CH group and the IHH group, but the HGB and HCT were significant higher than those of IHH group(P < 0.05), which suggested that the effect of CH on the rat body was much severe than that of IHH. About the quantity of mRNA extracted from the lower brain stems, there were no significant differences of TASK-1 and TASK-3 as well as the ratio of TASK-1 and TASK-3 between each two of three groups. The result indicated that rat models of IHH and CH were successfully duplicated and that the hypoxia could not increase the expression of TASK-1 and TASK-3 mRNA. Considered together with the results of part four, the occurrence of sleep apnea might result from the increase expression of TASK-1 channel by unknown mechanism. Conclusion: Recently found TASK-1 channel is an open rectified potassium channel that distributed widely in the central nervous system (CNS), which is very sensitive to physiological extracecullar pH and is modulated by many kinds of neurotransmitters, such as 5-HT, NE and substance P, etc. In this study, we tried to primarily test that TASK-1 channel might be involved in the pathogenesis of sleep apnea as a molecular chemoreceptor existing in the CNS. Results of the study were as follows: 1. TASK-1 mRNA was highly expressed in the brainstem CCRs as well as the dorsal and ventral respiratory groups of oblongata, but was weekly expressed in the pontine respiratory group, PPN and ITR. 2. The current of TASK-1 channel recorded from the HN was an outside "background"potassium current, which was very sensitive to the pH changes of the artificial cerebrospinal fluid. 3. The sleep AI was closely related to the quantity of TASK-1 mRNA according to the analysis of sleep apnea and TASK-1 mRNA of lower brainstem in SD rat. 4. Both of continuous hypoxia and intermittent hypoxia with hypercapnia (IHH) could not influence the expression of TASK-1 mRNA. It can conclude that TASK-1 channel might be a molecular...
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