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CARDIAC, RENAL, AND RESPIRATORY INTEGRATION
Division of Sleep Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
Submitted 14 August 2003 ; accepted in final form 6 October 2003
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ABSTRACT |
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1 h after systemic injection of methysergide (1 mg/kg ip), ketanserin (1 mg/kg), or clozapine (1.5 mg/kg). Resting ventilation, metabolic rate, and hypoxic ventilatory response (HVR) were unchanged, but LTF (18% above baseline) was eliminated by each drug. In CIH-treated rats, LTF was also measured twice, before and 8 h after CIH. Vehicle, methysergide, ketanserin, or clozapine was injected 1 h before the second measurement. Neither resting ventilation nor metabolic rate was changed after CIH and/or any drug. HVR was unchanged after methysergide and ketanserin but reduced in four of seven clozapine rats. The CIH-enhanced LTF (28%) was abolished by methysergide and clozapine but only attenuated by ketanserin (to 10%). Collectively, these data suggest that ventilatory LTF requires 5-HT2 receptors and that the CIH effect on LTF requires non-5-HT2 serotonin receptors, probably 5-HT6 and/or 5-HT7 subtype(s). respiratory control; plasticity; 5-HT antagonists
Pretreatment with chronic intermittent hypoxia (CIH) has been shown to enhance hypoxia-induced LTF (17, 23, 32). In a previous study, CIH (5 min 11-12% O2-5 min air, 12 h/night, 7 nights) greatly enhanced the magnitude of phrenic LTF (17). This result has been confirmed in a recent study, which used AIH and CIH protocols mimicking the episodes of hypoxic apnea that often occur in patients with obstructive sleep apnea (OSA; 32). These experiments were carried out on anesthetized, paralyzed, vagotomized, and artificially ventilated rats. Our recent study (23), conducted on conscious, freely behaving rats, showed that CIH converted an ineffective protocol to an effective one in eliciting LTF, increased LTF magnitude, and prolonged LTF duration, thus extending the conclusion that CIH enhances LTF to a preparation with no potential confounding effects of anesthesia, surgery, and restraint.
Activation of serotonin receptors is required for the manifestation of LTF, as LTF of phrenic or hypoglossal nerve activity can no longer be elicited by CSN stimulation or AIH after application of the broad-spectrum serotonin receptor antagonist methysergide (1, 3, 17, 26). More specifically, phrenic LTF was abolished by the 5-HT2 receptor antagonist ketanserin (16, 17). The CIH-enhanced phrenic LTF is also eliminated by methysergide but only attenuated by ketanserin, suggesting that although the CIH-induced enhancing effect on LTF also depends on serotonergic mechanisms, this CIH effect requires non-5-HT2 serotonin receptors (17). All these pharmacological studies were conducted on anesthetized animals. As far as we are aware, the involvement of serotonergic mechanisms in either LTF per se or the CIH effect on LTF has not been studied in any awake preparation. In addition, which specific serotonin receptor subtype(s) is required for the CIH effect on LTF is unknown.
The present study was thus designed to examine the effects of methysergide, ketanserin, and clozapine (a serotonin 5-HT2,6,7 receptor antagonist) on both ventilatory LTF and the CIH effect on ventilatory LTF in conscious, freely behaving rats. We hypothesized that the two conclusions stated above (LTF per se requires 5-HT2 receptors and the CIH effect on LTF requires non-5-HT2 serotonin receptors; 17) could be verified in an awake animal model and that the CIH effect on ventilatory LTF required 5-HT6 and/or 5-HT7 receptors.
A portion of this work has appeared in abstract form (20).
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METHODS |
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Ventilatory and Metabolic Measurements
Respiratory ventilation was measured by use of a custom-made 3-liter whole body flow-through plethysmograph (Buxco Electronics, Sharon, CT). Individual freely behaving awake rats were placed in the precalibrated plethysmographic chamber connected via a controlled leak to a reference chamber. The atmosphere within the animal chamber was maintained with air flowing through the chamber at a rate of 3 l/min. A bias flow was connected to an aerosol port of the chamber to maintain the O2 concentration within the animal chamber. A custom-made computer software system (Biosystem XA, Buxco Electronics) monitored the output of a differential pressure transducer (TRD5100) connected between the animal and reference chambers. This software system provided a breath-by-breath display of minute ventilation (VE), tidal volume (VT), and breathing frequency (fR) before, during, and after the AIH used to induce LTF.
The computer continuously monitored the output of the CO2 (Servomex Transducers, Norwood, MA) analyzer sensing alternately inspired and expired gases. With the known flow rate, the measurements of the CO2 gas concentration were used to determine the CO2 production (VCO2) that defines metabolic rate in the present study. Metabolic rate (VCO2) was also measured before (baseline), during, and after the AIH stimulus protocol in each experiment. Body temperature was measured before and immediately after each experiment with a rectal thermometer.
CIH Treatment
Rats were housed in normal rat cages and were given food and water ad libitum. These cages were placed into a custom-made Plexiglas chamber. This chamber was flushed with alternating mixtures of N2, O2, and air to achieve quasi-square wave intermittent hypoxia of 11-12% O2 for 5 min and normoxia for 5 min, in which the targeted hypoxia or normoxia levels were reached within 30 s. Gas mixtures were flushed at a rate sufficient to maintain chamber CO2 levels below 0.5%. This intermittent hypoxia was repeated for 12 h a night (from 6:00 PM to 6:00 AM, when rats are in their active period) for 7 consecutive nights. The chamber was flushed with air or open to room air during the remaining 12 h each day, and temperature within the chamber was maintained at 20-22°C.
Serotonin Receptor Antagonists
The drugs used in the present study include methysergide maleate {(-)-9,10-didehydro-N-[1-(hydroxymethyl)propyl]-1,6-dimethyl-ergoline-8-carboxamide maleate}, which is a broad-spectrum serotonin 5-HT1,2,5,6,7 receptor antagonist with partial agonist effects on 5-HT1A,1B,1D receptors (13), ketanserin tartrate {3-[2-[4-(4-flurobenzoyl)-1-piperidinyl]ethyl]-2,4(1H,3H)-quinazolinedione tartrate}, a specific 5-HT2 receptor antagonist with affinity also for 
-adrenergic receptors and histamine H1 receptors (15), and clozapine {(8-chloro-11-[4-methyl-1-piperazinyl]-5H-dibenzo[b,e][1,4]-diazepine)}, a 5-HT2,6,7 antagonist (24). They are all widely used serotonin receptor antagonists (RBI Sigma) and easily cross the blood-brain barrier. Methysergide and ketanserin were dissolved in saline, and clozapine was dissolved in 30% methanol in saline.
Experimental Procedures
Measurement protocol. Rats were placed in the plethysmographic chamber and allowed to adapt to the chamber for 1 h. Ventilatory LTF was elicited by five episodes of 5-min poikilocapnic hypoxia (10% O2), separated with 5-min intervals of normoxia. VE was measured before (over 10 min), during, and after this AIH stimulus protocol (see below) to determine resting VE (baseline), hypoxic ventilatory response (HVR), and ventilatory LTF, respectively. In the animal chamber, the shift from normoxia to the target hypoxia level (10% O2) took <1 min and the shift from hypoxia to normoxia took <30 s. During these hypoxic and normoxic episodes, only the final 2 min of data for each episode was included in the analysis. The HVR is defined as an increase from baseline to hypoxic VE, normalized to a percentage of the baseline. After the last hypoxia episode, VE was measured at 15-min intervals (i.e., 15, 30, 45, 60, 75, and 90 min) with each value representing a 5-min average (e.g., the 15 min posthypoxia value is an average of the data collected between 15 and 20 min). Ventilatory LTF is defined as an increase from baseline in posthypoxia VE, normalized to a percentage of the baseline.
Untreated rats (i.e., animals not exposed to CIH). The ventilatory and metabolic measurement protocol was conducted twice on the same rat. The first measurement was initiated 1 h after systemic (ip) injection of the vehicle (1 ml saline, n = 12 or 30% methanol in saline, n = 7) in each rat (total n = 19). One or two days later, the second measurement was initiated 30-60 min after systemic injection of methysergide (1 mg/kg, 1 ml, n = 6), ketanserin (1 mg/kg, n = 6), or clozapine (1.5 mg/kg, n = 7). Thus the effect of the serotonergic antagonism on ventilatory LTF was examined in the same rats. All measurements were made at about 2:00 PM. The vehicle effect was examined on four separate rats (saline, n = 2; 30% methanol in saline, n = 2). The measurement was also made twice on the same rats and separated by 1 day, the first measurement with no injection and the second measurement with vehicle injection. There was no difference between the two measurements in either ventilatory or metabolic parameters. Also, these separate results were not different from the above-mentioned control values (i.e., the first measurements with vehicle injection).
CIH-treated rats. The measurement protocol was conducted twice on the same rat. The first measurement was made before CIH (total n = 31). The second measurement was made 8 h after CIH. Vehicle (saline, n = 6; 30% methanol in saline, n = 6), methysergide (1 mg/kg, n = 6), ketanserin (1 mg/kg, n = 6), or clozapine (1.5 mg/kg, n = 7) was injected (ip) 30-60 min before the second measurement. Thus the effect of CIH on ventilatory LTF was examined in the same rats. However, unlike in the untreated rats, the effect of the serotonergic antagonists on the CIH-enhanced LTF was not examined in the same rats (i.e., vehicle and drug were injected in separate groups). The measurements were also made at about 2:00 PM.
Data Analysis
Ventilatory (VE, VT, and fR) and metabolic (VCO2) parameters were measured following a strict rule as previously described (19, 23). Briefly, these parameters were measured in rats when they were observed to be awake and in a quiet state. Data recorded when the rats were moving or asleep were rejected from the analysis. This rejection was done blindly, i.e., values were unknown when these data were rejected. Our criteria for data acceptance are the combination of 1) open eyes, 2) no body movement, and 3) a normal breathing pattern displayed continuously on the computer screen. Furthermore, an additional rejection algorithm was included in the computer software breath-by-breath analysis that allows for further rejection of artificial breaths. These strict rejection criteria helped to generate more consistent results. Baseline VE in the present study was very consistent between and within experimental groups.
To facilitate analysis, LTF is numerically expressed as a magnitude and duration as previously described (19, 23). Briefly, the LTF magnitude was determined by the average of the first three VE values recorded at 15, 30, and 45 min posthypoxia and expressed as a percentage increase above the baseline value. The LTF magnitude was thus calculated by the equation %[(VE15 + VE30 + VE45)/3 - baseline]/baseline. The LTF duration was defined as the last posthypoxia time point at which VE was significantly higher than baseline. This three-value average method and the definition of LTF duration were also used in assessing the magnitude and duration of both fR LTF and VT LTF.
The LTF duration for each group and the between-group differences in baseline and the individual posthypoxia VE (all absolute values) were determined by use of a two-way ANOVA with repeated measures (SigmaStat version 2.0, Jandel, San Rafael, CA) followed by Student-Newman-Keuls post hoc tests. Only baseline and posthypoxia data were included in this analysis (data recorded during AIH not included). All other differences were determined by use of a one-way ANOVA, including the between-group differences in ventilatory, VT, and fR LTF (percentage values calculated by the 3-value average method) and the between/within-group differences in HVR and metabolic rate values. P < 0.05 was considered significant. All values are expressed as means ± SE.
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RESULTS |
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In baseline condition, ventilation (VE), metabolic rate (VCO2), and ventilation relative to metabolic rate (VE/VCO2) were not significantly changed after CIH treatment and/or systemic injection of any drug (methysergide, ketanserin, or clozapine), and this was the case for VCO2 and VE/VCO2 during (10% O2) hypoxia as well (all P > 0.18; Table 1, Fig. 1). The hypoxic VE and the hypoxic ventilatory response (HVR) to 10% O2 was similar among five episodes of poikilocapnic hypoxia (all P > 0.92) and therefore averaged in all groups. In untreated rats (Fig. 2A), the average HVR was not significantly changed after methysergide (P = 0.993), ketanserin (P = 0.126), or clozapine (P = 0.062), although the hypoxic VE was significantly lower after clozapine (P < 0.05; Table 1). In CIH-treated rats (Fig. 2B), the HVR was not significantly changed in methysergide or ketanserin group (both P > 0.14) but was reduced in vehicle and clozapine groups (both P < 0.05), relative to the pretreatment value. The HVR was also lower in clozapine group vs. vehicle, methysergide, or ketanserin group (all P < 0.05). The hypoxic VE results were consistent with the HVR results in CIH-treated rats (Table 1; Fig. 2). The HVR reduction in the clozapine group was mainly due to decreases in four of seven rats, as HVR in the other three rats was not different from the vehicle value. Collectively, these results indicate that CIH treatment and/or drug (methysergide, ketanserin, or clozapine) has little effect on baseline VE, metabolic rate, and HVR, except that CIH slightly reduces HVR (to 10% O2) and that clozapine reduces HVR in most CIH-treated rats.
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Effect of Serotonin Antagonists on Ventilatory LTF
In untreated rats, the AIH protocol (5 episodes of 10% O2) induced a ventilatory LTF with a magnitude of 18.4 ± 0.2% (calculated by the 3-value average method) and a duration of 45 min (Fig. 3A). Systemic (ip) injection of serotonin receptor antagonist methysergide (5-HT1,2,5,6,7, 1 mg/kg), ketanserin (5-HT2, 1 mg/kg), or clozapine (5-HT2,6,7, 1.5 mg/kg) abolished this LTF (2.6 ± 0.4%, 1.7 ± 0.7%, or 2.0 ± 0.7%, respectively; all P < 0.05; Fig. 3). Ventilatory LTF in untreated rats was mainly due to a fR LTF with little VT LTF, and these antagonists abolished ventilatory LTF via eliminating this fR LTF (Fig. 4). These data indicate that methysergide, ketanserin, or clozapine abolishes ventilatory LTF mainly by eliminating the posthypoxia fR increase and strongly suggest that ventilatory LTF requires activation of 5-HT2 serotonin receptors.
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Effect of Serotonin Antagonists on the CIH-Enhanced Ventilatory LTF
Before CIH, the AIH protocol induced a pre-CIH ventilatory LTF (18.3 ± 0.2%, 45 min duration) similar to the untreated rats (Fig. 3B). About 8 h after CIH, the same AIH protocol induced a CIH-enhanced LTF with a greater magnitude (28.3 ± 0.6%; P < 0.05) and a longer duration (75 min) in vehicle group (n = 12). Systemic injection of methysergide (1 mg/kg, n = 6) and clozapine (1.5 mg/kg, n = 7) eliminated this CIH-enhanced LTF (1.2 ± 0.1% and 2.5 ± 0.4%, respectively; both P < 0.05), but ketanserin (1 mg/kg, n = 6) only attenuated it (to 9.9 ± 0.6%; P < 0.05; Fig. 3). In the three clozapine rats, whose HVR was similar to the vehicle value (see Baseline, Metabolic Rate, and Hypoxic Ventilatory Response), clozapine also completely abolished the CIH-enhanced LTF just like the other four rats, suggesting that clozapine did not eliminate this CIH-enhanced LTF by simply reducing the magnitude of the HVR. The CIH-enhanced ventilatory LTF was due to a large fR LTF and a small VT LTF, but the CIH-induced enhancement of ventilatory LTF (i.e., the difference between LTF per se and CIH-enhanced LTF) was mainly due to an increase in the VT LTF with a possible, slight increase in the fR LTF (insignificant, Fig. 4). These data indicate that methysergide and clozapine abolish CIH-enhanced ventilatory LTF by reducing both fR and VT LTF and that ketanserin attenuates CIH-enhanced ventilatory LTF mainly by reducing fR LTF (Fig. 4).
It is probably more than just a coincidence that numerically adding the LTF magnitude in the CIH+ketanserin group (9.9%) to that of the pretreatment group (18.3%) approximately equals to the CIH-treated group value (28.3%; Fig. 3B). This is also true in the absolute VE values due to the similar baseline (resting VE) in these three groups (Table 1). In addition, the LTF duration in these three groups also appears to have such an additive relation (i.e., 30 + 45 = 75 min). These results indicate that ketanserin abolishes ventilatory LTF per se but not the CIH-induced enhancement of ventilatory LTF, suggesting that 5-HT2 receptors are not required for the CIH effect on ventilatory LTF. Because both methysergide and clozapine, which presumably antagonized 5-HT2, 5-HT6, and 5-HT7 receptors, eliminated the CIH-enhanced ventilatory LTF, these data also suggest that the CIH-induced enhancing effect on ventilatory LTF requires 5-HT6 and/or 5-HT7 receptors.
Absolute VE Values
The above statements based on the analysis of the percentage VE data (Fig. 3) were well supported by the analysis of absolute VE values (2-way ANOVA). The resting VE (baseline) and posthypoxia VE, before and after each drug and/or CIH treatment, are all presented in Fig. 1 (VE data during AIH not included). There was a significant interaction between group and time factors in VE [F(18,264) = 79.8; P < 10-6] of the four groups associated with methysergide (Fig. 1A), and the between-group differences in those individual posthypoxia VE were also consistent with the above percentage results (Fig. 3). There was also a significant interaction of VE in the ketanserin groups [F(18,264) = 38.3; P < 10-6; Fig. 1B] and the clozapine groups [F(18,279) = 48.9; P < 10-6; Fig. 1C]. Their between-group differences in the posthypoxia VE were also consistent with the percentage results (Fig. 3). Note that unlike in the methysergide and clozapine cases, ventilatory LTF was still present at 15 or 30 min posthypoxia in CIH-treated rats after systemic injection of ketanserin (Fig. 1), suggesting that this CIH effect on LTF requires non-5-HT2 serotonin receptors.
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DISCUSSION |
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Methodological Consideration
The anesthetized preparation has many advantages, e.g., blood gases and body temperature can be continuously monitored and well controlled. However, in these experiments, the critical baseline of the phrenic nerve activity is artificially set at an end-tidal CO2 partial pressure 3 Torr above the CO2-apneic threshold. Although this is a widely accepted method in LTF studies to standardize baseline phrenic activity for comparison among different rats, it sometimes confounds results quantitatively. In the study by Ling et al. (17) using an anesthetized animal, the average CO2-apneic threshold, and thus the baseline CO2 level, was 6 Torr lower in CIH-treated vs. control rats. This difference could confound the assessment of (overestimate) both the phrenic response to hypoxia and phrenic LTF because both are expressed as values normalized to baseline (cf. Ref. 23). The input-output relationship of the respiratory central neural circuits is also nonlinear, e.g., the phrenic response to a constant hypoxic stimulus is progressively increased in magnitude as the prestimulus level of respiratory activity is reduced (7). In addition, the drugs used in that study affected the baseline, thus further complicating the LTF assessment. In fact, the hypoxic phrenic response was greatly enhanced after CIH in anesthetized rats (17), but the HVR was not enhanced after CIH in awake rats (23). This discrepancy in the results between preparations was one of the major reasons why we wanted to conduct the present study on awake animals.
The use of barometric plethysmography to measure VE and metabolic rate in awake rats provided a more natural and physiological way to investigate the serotonergic mechanisms involved in LTF per se and the CIH effect on LTF. As a result, confounding issues related to anesthesia, surgery, and restraint could be eliminated, and the metabolism could also be taken into consideration. In the present study, resting VE and metabolic rate were barely changed by CIH treatment and/or any of the drugs, thereby simplifying the data analysis and eliminating the potential confounding effect of baseline changes on assessment of LTF and HVR values.
The present study was conducted according to a within-subject experimental design, i.e., the measurement protocol was conducted twice on the same untreated and CIH-treated rats. The drug effects on ventilatory LTF as well as the CIH effect on LTF were examined in the same rat group. However, the drug effects on the CIH-enhanced LTF were examined in separate groups (i.e., vehicle and drug were injected into different CIH-treated groups) in an attempt to avoid a possible confounding time effect on the CIH effect on LTF, which has been shown to decline gradually with time (23). This experimental design plus the strict rule (i.e., measurement only during quiet wakefulness) improved consistency of our result by reducing data variability. There was no confounding carryover effect in this design because no difference in either ventilatory or metabolic parameters was found between multiple measurements in our previous time-control experiments (23). It has also been shown that 1 day or even several hours after the first measurement of ventilatory LTF had no carryover effect on the second one (21, 30).
Serotonin Receptor Subtypes
The serotonin receptor antagonists methysergide and ketanserin have been often used in studies on serotonergic mechanisms underlying respiratory LTF. Methysergide is reported to block 5-HT1,2,5,6,7 receptors and thus is a broad-spectrum 5-HT receptor antagonist, which has been used in anesthetized LTF studies through systemic (both iv and ip) injection (1, 17, 26) or intrathecal application (3). However, methysergide has been shown to increase the baseline phrenic burst amplitude in anesthetized rats (1, 3). On the other hand, ketanserin (a specific 5-HT2 receptor antagonist) appears to reduce baseline phrenic activity in anesthetized rats (11; Y. Zhang, M. McGuire, and L. Ling, unpublished observations). In the present study, both methysergide and ketanserin produced little change in resting VE measured at 30-60 min after the drug administration. The reason why there are different effects on baseline is unclear. However, this might relate to ventilatory compensatory mechanisms maintaining proper blood-gas homeostasis in an unanesthetized preparation.
Clozapine, an atypical antipsychotic in the treatment of schizophrenia (4), has been reported to block serotonin 5-HT2,6,7 subtype receptors (24). However, there is also evidence suggesting that clozapine acts on other types of neurotransmitter receptors (e.g., histaminergic, dopaminergic and adrenergic receptors; 6, 24), thus challenging our interpretation that the CIH effect on LTF requires 5-HT6 and/or 5-HT7 receptors. We strongly argue against the possibility that clozapine might eliminate the CIH-enhanced LTF via its action on 5-HT2 as well as those nonserotonin receptors. It has been shown that the dopamine-norepinephrine antagonist -methyltyrosine had no effect on LTF (26). In addition, methysergide, which has little effect (low affinity) on any subtype of histaminergic, dopaminergic, and adrenergic receptors (14), completely abolishes LTF per se and the CIH-enhanced LTF in both anesthetized (17) and awake animals of the present study. These data suggest that the CIH effect on LTF does not depend on histaminoceptors, dopaminoceptors, and/or adrenoceptors. Therefore, it is likely that clozapine abolishes ventilatory LTF per se by its action on 5-HT2 receptors and abolishes the CIH-induced enhancing effect on LTF by its action on 5-HT6 and/or 5-HT7 receptors. The fact that clozapine completely abolishes ventilatory LTF also further confirms the conclusion (i.e., LTF per se requires 5-HT2 receptors) based on the ketanserin data in both anesthetized and awake animals.
In pilot experiments, we tried using ritanserin, a relatively more selective serotonin (5-HT2,6,7) receptor antagonist. However, systemic injection of this drug (1 mg/kg) produced a significant, behavioral effect, e.g., the rat became agitated and unable to settle down for 3 h. A lower dose (0.5 mg/kg) was also tried, but it could not completely block LTF while still having the behavioral effect on baseline (although reduced). As a result, we replaced it with clozapine, which had less effect on behavior, allowing measurement of the critical baseline VE according to the strict rule (see METHODS).
A limited number of preliminary experiments were also conducted to briefly assess an appropriate dose for each drug. Four different doses (0.5, 1, 2, 4 mg/kg) were chosen for each drug but each dose was only tested in one rat for each drug (total n = 12). Our criteria for a proper dose were 1) causing little behavioral effect, 2) having little effect on baseline and HVR, and 3) completely blocking ventilatory LTF. Thus we intended to choose a drug dose as high as possible to antagonize most of the serotonin receptors, but having little effect on behavior, baseline, and HVR. Both methysergide and ketanserin eliminated ventilatory LTF at all four doses. The 1 mg/kg concentration was selected for these two drugs because it caused less behavioral effect than the higher doses and had little effect on baseline and HVR. For clozapine, 1, 2, and 4 mg/kg doses all eliminated LTF, but 0.5 mg/kg only partially blocked LTF. The dose of 1.5 mg/kg was selected for clozapine because it had less behavioral effect than 2 and 4 mg/kg.
There is a weak but significant correlation between hypoxic phrenic response and LTF magnitude (10), suggesting that the size of the hypoxic response may somehow determine LTF magnitude. In the present study, clozapine significantly reduced HVR in four of seven CIH-treated rats. However, we do not believe that clozapine eliminated ventilatory LTF through reducing hypoxic responsiveness because LTF in three other clozapine rats, whose HVR was similar to the vehicle value, was also completely abolished. In addition, a recent study from our lab demonstrated that intermittent phrenic-inhibitory vagus nerve stimulation paradoxically elicited phrenic LTF, suggesting that elicitation of LTF can be totally dissociated from inspiratory or phrenic augmentation (let alone the magnitude of HVR) during the elicitation (35).
Potential Mechanism
Substantial progress has been made in recent years regarding where and how LTF is generated (cf. Ref. 27). Briefly, the carotid chemoafferents, activated by intermittent hypoxia or CSN stimulation, stimulate the raphe nuclei. Released serotonin from the raphe serotonergic neurons activates 5-HT2 receptors on the phrenic motoneurons, which initiates a series of intracellular signaling events, leading eventually to LTF. These concepts are supported by many studies. However, LTF has also been reported to be present in the carotid chemoafferents (31, 33) and some medullary neurons (29). Therefore, the exact, major anatomic location(s) or cellular and neuronal mechanisms underlying LTF are still not clear (cf. Refs. 8, 10, 27).
Where and how CIH enhances LTF is even less clear. LTF per se and the CIH effect on LTF are similar in some aspects, e.g., both are intermittent hypoxia-induced ventilatory plasticity and both are serotonin dependent. However, these two forms of plasticity are different in at least three aspects: 1) elicitation protocol (AIH vs. CIH), 2) the role of 5-HT2 receptors (required for LTF but not the CIH effect), and 3) their persistence period (LTF lasting minutes/hours vs. the CIH effect lasting days; 23). It is unlikely that the CIH effect on LTF is mediated by the changes in hormones (e.g., epinephrine and norepinephrine) or blood gases and pH, as most data were collected 8 h or even 3 days after CIH (23), and this CIH effect on LTF can be eliminated by serotonin receptor antagonism (17) or greatly attenuated by NMDA receptor blockade (22). We speculate that CIH repeatedly activates the neural networks, leading to an activity-dependent upregulation of serotonin receptors on the phrenic motoneurons. These newly synthesized serotonin receptors (mainly of 5-HT6 and/or 5-HT7 subtype) increase the overall efficacy of the serotonergic synaptic transmission, which then augments the intracellular signaling events, thus enhancing a subsequent LTF when it is evoked. There has been evidence suggesting that this CIH effect is primarily a central mechanism (17). However, this interpretation of the data might also be confounded because of the artificial baseline setting (see Methodological Consideration). Further studies using microinjection of drugs into the phrenic motor nucleus region would help to clarify this issue.
A recent study (32), using stimulus protocols that mimic hypoxic episodes of OSA patients, shows that AIH elicits phrenic LTF and that pretreatment with CIH also enhances this phrenic LTF in anesthetized rats. In addition, this CIH effect on LTF is eliminated by systemic injection of a potent scavenger of 
radicals before CIH, suggesting that reactive O2 species (ROS), especially the radicals, play a role in this CIH effect (32). It has also been shown that this form of CIH enhances (or reveals) LTF of the carotid chemoafferent activity (31, 33). Collectively, these data suggest that the CIH effect on LTF may require ROS as well as serotonergic mechanisms and that the carotid body may be an important location for the CIH effect. However, caution should be used for such a generalization because the CIH effects induced by different protocols might use different mechanisms.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Significant differences from all other 3 groups at that specific time point.
significant difference between C and T+drug groups. Difference between C+drug and T groups is significant in all 6 pairs but not indicated for clarity.