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Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We hypothesized that the 5-hydroxytryptamine (5-HT) active drugs ketanserin and 5-carboxamidotryptamine (5-CT) would modulate time-dependent hypoxic phrenic and hypoglossal responses, including 1) short-term hypoxic response, 2) posthypoxia frequency decline (PHFD), and 3) long-term facilitation (LTF) of respiratory motor output. Phrenic and hypoglossal nerve activities were recorded in urethan-anesthetized, paralyzed, vagotomized, and artificially ventilated rats pretreated either with ketanserin (5-HT2A/C antagonist; 2 mg/kg iv), 5-CT (5-HT1A/B agonist; 10 µg/kg iv), or saline (sham). Rats were exposed to three 5-min episodes of hypoxia [fractional inspired O2 (FIO2) = 0.11], separated by 5 min of hyperoxia (FIO2 = 0.5). During hypoxia, ketanserin augmented phrenic but not hypoglossal burst amplitude; 5-CT had no effect. Both drugs accentuated PHFD. Ketanserin blocked phrenic LTF; hypoglossal LTF was not apparent, even in sham-treated rats. 5-CT reversed LTF, resulting in a long-lasting depression of phrenic burst frequency and amplitude without effect on hypoglossal burst amplitude. The data suggest that 1) 5-HT2A/C receptor activation modulates the short-term hypoxic phrenic response and PHFD and is necessary for LTF; and 2) 5-CT may affect time-dependent hypoxic ventilatory responses by reducing serotonin release via 5-HT1A/B autoreceptor activation.
respiratory control; serotonin; phrenic nerve; hypoglossal nerve; plasticity; modulation; episodic stimulation
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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EPISODIC HYPOXIA elicits complex, time-dependent
ventilatory responses in rats as in other mammals (19, 36). Each of
these time-dependent responses results from a unique neural mechanism, distinguishable on the basis of time course, ventilatory pattern change, and neurochemical involvement (36). Within a brief hypoxic exposure (e.g., 5 min), the short-term response results from the contributions of at least three mechanisms: the acute response, short-term potentiation, and short-term depression (14, 36). Five to
ten minutes after hypoxia, a manifestation of short-term depression,
referred to as posthypoxia frequency decline (PHFD), is observed in
rats (1, 9). Posthypoxia frequency decline is attenuated by prior
hypoxic exposures and systemic pretreatment with
2-adrenergic receptor or
N-methyl-D-aspartate
receptor antagonists (2, 4, 10). After successive hypoxic episodes
(e.g., 15-60 min), long-term facilitation is revealed (LTF; 3, 14, 19). LTF is attenuated by methysergide, a nonselective serotonin
receptor antagonist, by serotonin depletion with
para-chlorophenylalanine, or by the neurotoxin
5-hydroxytryptamine (5-HT; 3, 32). Although these data indicate that
LTF requires activation of serotonin receptors, the specific subtypes
of receptors involved are unknown. Because serotonin is involved in
many aspects of ventilatory control (for review, see Refs. 6, 7, 30)
and hypoxia activates raphe serotonergic neurons (16, 40), we
investigated the effects of pharmacological agents known to interact
with 5-HT1A/B or
5-HT2A/C receptors on
time-dependent mechanisms of the ventilatory response to episodic hypoxia.
In neonatal rat in vitro preparations, 5-HT increases phrenic and hypoglossal motoneuron excitability via activation of 5-HT2A/C receptor subtypes (29, 34). Thus we predicted that pretreatment with a 5-HT2A/C receptor antagonist would reduce respiratory motoneuron excitability, thereby reducing respiratory motor output (amplitude) during the short-term hypoxic ventilatory response. Because it has been hypothesized that LTF results, at least in part, from 5-HT2A/C receptor activation (3, 30), we further predicted that a selective 5-HT2A/C receptor antagonist would abolish LTF.
5-HT1A and 5-HT1B receptors are localized on the soma and terminals of raphe serotonergic neurons (30, 22, 44). Activation of these presynaptic autoreceptors regulates (inhibits) raphe neuron activity, as well as 5-HT synthesis and release (21, 43, 44). Thus we hypothesized that, similar to a 5-HT2A/C antagonist, a 5-HT1A/B receptor agonist would reduce the short-term hypoxic ventilatory response and LTF.
Serotonin also exerts complex effects on medullary neurons involved in respiratory rhythmogenesis (26, 27, 28; for review, see Refs. 6, 7), raising the possibility that serotonin receptor activation also contributes to PHFD. However, the specific actions of 5-HT2A/C antagonist and 5-HT1A/B agonists on PHFD are difficult to predict.
The specific objectives of this study were to investigate the effects of ketanserin, a 5-HT2A/C receptor antagonist, and 5-carboxamidotryptamine (5-CT), a 5-HT1A/B receptor agonist on 1) the short-term response during isocapnic hypoxia, 2) PHFD, and 3) LTF of respiratory motor output after episodic hypoxia. These effects were measured in both phrenic and hypoglossal inspiratory motor output because of their significance in generating ventilation (phrenic) and upper airway patency (hypoglossal), respectively. Parts of this study were reported previously in abstract form (23, 24).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiments were performed on 33 adult male Sprague-Dawley rats (Harlan, Madison, WI), weighing between 290 and 519 g (mean 419 g). Changes in respiratory motor output were measured during and after episodic isocapnic hypoxia in four groups of rats pretreated with intravenous saline or serotonergic drugs. Responses were compared with a time control group (without hypoxia). All procedures were approved by the University of Wisconsin animal care and use committee.
Animal Preparation
Methods used to assess neural correlates of respiratory activity have been described in detail by Bach and Mitchell (3). Briefly, rats were anesthetized initially with isoflurane (2.5-3.0% in 50% O2, balance N2) and then slowly converted to urethan anesthesia (1.6 g/kg iv) over a period of 15-20 min. The plane of anesthesia was assessed regularly by testing corneal reflexes and blood pressure responses to toe pinch; supplemental urethan was given as necessary to prevent blood pressure responses to toe pinch before and after the rats were paralyzed (see below). One hour after induction of anesthesia, an intravenous infusion of sodium bicarbonate (5.0%) and lactated Ringer solution (50:50, 1.7 ml · kg
1 · h1)
was initiated to maintain acid-base balance.
Rats were prepared with a tracheostomy to allow for artificial ventilation (rodent respirator, model 683; Harvard Apparatus, South Natick, MA) and tracheal pressure measurement (Statham pressure transducer, P23-id). The lungs were hyperinflated roughly once per hour to prevent alveolar atelectasis. The rats were vagotomized bilaterally in the midcervical region to prevent entrainment of respiratory motor output with the ventilator and then paralyzed (pancuronium bromide, 2.5 mg/kg iv) to prevent spontaneous breathing efforts. End-tidal CO2 was monitored with a flow-through capnograph (Novametrix; Wallingford, CT) with sufficient response time (<75 ms) to measure rat end-tidal PCO2. Values obtained from this capnograph closely approximate arterial PCO2 (PaCO2; usually within 1-2 mmHg). Blood samples were drawn from a catheterized femoral artery to determine blood gases and pH (ABL-330; Radiometer, Copenhagen, Denmark). Blood gas and pH values were corrected to the measured rectal temperature of the rat. Blood pressure was monitored at the femoral artery (Statham pressure transducer, P23-id). Rectal temperature was maintained between 37 and 38°C with a heated pad.
Phrenic and hypoglossal nerves were isolated unilaterally, using a left dorsal approach, cut distally, and desheathed. The nerves were submerged in mineral oil and placed on bipolar silver recording electrodes. Nerve activity was amplified (gain = 10 k; CWE BMA-931 Bio amp; Ardmore, PA), band-pass filtered (100 Hz-5 kHz), full-wave rectified, and fed to a moving averager (CWE MA-1000; time constant 100 ms) before being digitized, recorded, and analyzed with computer software developed in our laboratory.
Experimental Groups
Once the rat preparation was complete, at least 60 min were allowed for the electroneurograms and arterial blood pressure to stabilize under hyperoxic and normocapnic conditions [fractional inspired O2 (FIO2) = 0.5; arterial PO2 (PaO2) > 150 mmHg; PaCO2 ~2-3 mmHg above CO2 apneic threshold; Tables 1 and 2]. Initial (predrug treatment) nerve activity was achieved by manipulating inspired CO2 and respiratory pump rate and/or volume while monitoring end-tidal CO2 levels until both phrenic and hypoglossal nerve activity attained low but stable levels of activity. The four experimental groups are as follows.
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Group 1: control rats. Due to the long duration of experiments, we conducted time-control experiments to ensure that the changes in respiratory motor output were not related to time-dependent changes in blood pressure, anesthesia, repeated blood sampling, or other factors. Thus respiratory motor output was monitored continuously for 1.5 h in rats (n = 7) that received neither intravenous drug injection nor episodic hypoxia but in which the same number of blood samples was taken (see below).
Group 2: sham (saline injected) rats. Rats (n = 13) were injected with 0.3 ml of saline, followed by a saline flush of 0.5 ml 25 min before the onset of an episodic hypoxia protocol. The data obtained from this group served as a reference to assess the effects of the drug injections on the time-dependent hypoxic responses.
Group 3: ketanserin. To assess the requirement for 5-HT2A/C receptor activation in the time-dependent responses to episodic hypoxia, this group of rats (n = 7) received a slow (~1 min) intravenous injection of ketanserin tartrate (concentration: 2 mg/ml; dose: 2 mg/kg), a high-affinity 5-HT2A/C receptor antagonist [5-HT2A: affinity value (Ki) range = 0.4-3.1 nM; 5-HT2C: Ki range = 28-98 nM; Ref. 44].
Group 4: 5-CT. The effects of 5-HT1A/B receptor activation on time-dependent changes in respiratory motor output during and after episodic hypoxia were assessed in rats (n = 6) that had received a slow (~1 min) intravenous injection of 5-CT (concentration: 20 µg/ml; dose: 10 µg/kg). Among other effects, 5-CT reduces 5-HT release by stimulating 5-HT1A (somatodendritic) and 5-HT1B (terminals) autoreceptors on serotonergic raphe neurons (21, 39, 44). 5-CT is a high-affinity, nonselective 5-HT1 receptor agonist that binds to both 5-HT1A and 5-HT1B receptor subtypes with nanomolar affinity (5-HT1A: Ki = 0.21 nM; 5-HT1B: Ki = 5.1 nM; Ref. 44). The very high affinity of 5-CT for 5-HT1 receptors allowed us to use low doses, thereby minimizing agonist effects at 5-HT2A/C receptors (5-HT2A: Ki = 19,952 nM: 5-HT2C: Ki = 630 nM; Ref. 44).
To ensure that the doses used were adequate for the duration of the experimental protocol, cardiorespiratory variables were monitored in one animal (in each group) after each drug administration at that particular dose for 1.5 h. Variability outside that measured in the time-control group was deemed unacceptable, and the dose was modified accordingly. All drugs were obtained from Research Biochemical International (Natick, MA) and were dissolved in saline.
Experimental Protocol
Each injection was followed by 0.5 ml injection of saline to ensure complete drug delivery. The experiment began when the neural correlates of respiratory activity and blood pressure were stable (protocols typically commenced 20-30 min postinjection). The protocol began with an arterial blood sample [0.3 ml drawn into a 0.5-ml heparinized glass syringe; unused blood (~0.1 ml) was returned to the animal]. All subsequent blood gas values were compared with this initial baseline value. Baseline nerve activity was recorded, followed by three 5-min episodes of isocapnic hypoxia (FIO2 = 0.11), separated by 5 min of hyperoxia (FIO2 = 0.5). Isocapnia was maintained during hypoxia to minimize the occurrence of hypocapnia, which could be a confounding factor in our stimulation protocol. To minimize blood sampling, the PaO2 was measured only during the first bout of hypoxia. The arterial blood gas values shown in Table 2 indicate that the levels of hypoxemia were similar in all groups (P > 0.05). Throughout the protocol, relative isocapnia was actively maintained by monitoring end-tidal CO2 and adjusting inspired CO2 accordingly. Arterial blood samples were also taken at 30 and 60 min posthypoxia to assure that PaCO2 was within 1 mmHg of the baseline value during data collection (Table 1). On average, the net blood volume removed during a typical experiment ranged between 0.8 and 1.0 ml. Rats in which PaCO2 deviated from baseline by >1 mmHg were not included in the analysis. Therefore, changes in PaCO2 are unlikely to be responsible for the LTF observed after episodic hypoxia (3). At the end of the protocol, the response to elevated levels of inspired CO2 (end-tidal CO2 = 90-95 mmHg) was recorded to obtain a measure of maximal (or at least a standardized "hypercapnic control") nerve activity. Euthanasia by a large urethan overdose (intravenously) terminated the experiment.Data Analysis
Peak amplitude and frequency (bursts/min) of phrenic and hypoglossal nerve activity were averaged over a minimum of 50 bursts for each recorded data point. Averaged amplitude data were then normalized as a percentage change from the baseline (prehypoxic) activity and as change from baseline, expressed as the percentage of the maximum (CO2 stimulated) nerve activity. The latter form of normalization obviates concerns about expressing data in terms of the percentage increase above an arbitrary (low) baseline value (18). To assess PHFD, burst frequency recorded after the first hypoxic episode was averaged in 20-s bins. The results were analyzed statistically with a two-way ANOVA (Sigmastat, Jandel Scientific) followed by pairwise comparisons using a Bonferroni correction (P < 0.05); a repeated-measures design was used when appropriate.| |
RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of Drugs on Baseline Activity
Saline pretreatment (sham) to mimic the potential effects of volume loading had no persistent effect on respiratory motor output or on blood pressure (Tables 3 and 4). Similarly, baseline phrenic and hypoglossal activities were unaffected by ketanserin administration (Table 3). However, the 5-HT1A/B receptor agonist 5-CT enhanced phrenic burst amplitude (24%) and frequency (14 breaths/min), but had no effect on hypoglossal burst amplitude (Table 3). Hypoxic protocols commenced only after arterial blood pressure and nerve activities had stabilized to this new baseline value, typically 20-30 min postinjection.
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Although ketanserin and 5-CT had different effects on phrenic nerve amplitude and frequency (Table 3), both drugs elicited hypotension (Table 4). Nevertheless, there were no systematic differences in time-dependent blood pressure responses during or after episodic hypoxia that could account for the observed effects on respiratory nerve activity.
To minimize normalization artifacts caused by variable baseline nerve activities, data were expressed both as a percentage change from baseline and as a change from baseline expressed as a percentage of the maximal CO2-stimulated response (%maximum). Because all results were quantitatively similar, regardless of the normalization used, only data expressed as percentage change from baseline are presented, with the exception of Table 3, where the effects of drug administration on baseline nerve activity are expressed as percentage maximum. The effects of pharmacological agents on each time-dependent mechanism of the hypoxic ventilatory response will be described in their temporal sequence.
Short-Term Hypoxic Ventilatory Response
Phrenic and hypoglossal burst amplitude both increased during hypoxia (Fig. 1, A and B, respectively). Phrenic and hypoglossal burst amplitude responses were significantly greater in all three hypoxic groups versus the time control group. However, the increase in phrenic burst amplitude during hypoxia was greater in ketanserin-treated rats than in sham-treated rats (Figs. 1A and 2, A and B). Pretreatment with 5-CT did not affect phrenic burst amplitude during hypoxia (Figs. 1A and 2C). Furthermore, none of the drugs affected hypoglossal burst amplitude during hypoxia (Fig. 1B).
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During hypoxia, burst frequency was increased significantly from baseline or time controls in sham-treated rats only (Fig. 1C). Both drug treatments attenuated the frequency response to short-term hypoxia relative to the sham-treated group (Figs. 1C and 2).
In rats pretreated with saline or ketanserin, arterial blood pressure decreased during hypoxia (Table 4). In contrast, hypoxia had no significant effect on arterial blood pressure in the 5-CT group (Table 4).
Posthypoxia Frequency Decline
The period immediately after the first hypoxic episode was characterized by a decrease in respiratory burst frequency below prehypoxic baseline values. This PHFD was sustained for the duration of the posthypoxic interval (5 min). Burst frequency was significantly lower posthypoxia in sham rats versus time controls (Fig. 3). Pretreatment with either 5-CT or ketanserin augmented PHFD in comparison to sham-treated rats (Fig. 3, A and B, respectively).
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Some rats displayed a marked decrease in arterial blood pressure during hypoxia, with a sharp return toward prehypoxic values at the onset of hyperoxia. However, there was no consistent trend in the relationship between changes in arterial blood pressure and the observed PHFD in different groups. It does not appear, therefore, that changes in arterial blood pressure regulation can account for changes in PHFD (relate Table 4 and Fig. 3).
Long-Term Facilitation
After episodic hypoxia, integrated phrenic nerve activity in sham-treated rats progressively increased above baseline (Figs. 2 and 4A). Mean phrenic burst amplitude was increased by 37 ± 6% at 60 min posthypoxia and was significantly greater than in time controls (change from baseline:
2 ± 4%; Fig.
4A).
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In contrast, sham-treated rats did not exhibit LTF of hypoglossal nerve
activity after episodic hypoxia. One hour posthypoxia, hypoglossal
burst amplitude was not significantly different from nonhypoxic time
controls (sham: 19 ± 11%; control: 6 ± 4%; Fig. 4B). Sixty minutes posthypoxia, the
change in burst frequency (from baseline) of the sham-treated rats (6 ± 2 bursts/min) was not significantly different from time controls
(2 ± 2 bursts/min; Fig. 4C).
Thus LTF was not evident in burst frequency (Fig. 2 and
4C).
Pretreatment with ketanserin blocked LTF of phrenic burst amplitude. Sixty minutes posthypoxia, the change in integrated phrenic amplitude in ketanserin-treated rats (change from baseline: 0.8 ± 9.7%) was significantly lower than in sham-treated animals but not different from time controls (Fig. 4A). Ketanserin administration had no significant effect on hypoglossal burst amplitude (change from baseline: 17 ± 15%; P = 0.17; Fig. 4B) or on the change in burst frequency measured 60 min posthypoxia (7 ± 2 bursts/min; Fig. 4C).
In rats pretreated with 5-CT, phrenic burst amplitude and frequency
decreased after the last hypoxic episode and remained depressed for the
duration of the study (Fig. 2C). One
hour posthypoxia, phrenic burst amplitude was significantly lower
(36 ± 10%) than in either sham-treated or time control rats
(Fig.
5A).
5-CT had no significant effects on hypoglossal burst amplitude after
episodic hypoxia (12 ± 18%;
P = 0.098; Fig.
5B). Moreover, rats pretreated with
5-CT exhibited a 9 ± 1 bursts/min decrease in burst
frequency at 60 min posthypoxia (Fig.
5C).
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Arterial blood pressures of sham-treated rats progressively decreased with time; the value measured 60 min posthypoxia (89 ± 7 mmHg) was significantly lower than its baseline (111 ± 6 mmHg; Table 4). Although this was the only experimental group in which blood pressure measured 1 h posthypoxia was significantly different from prehypoxic baseline values (Table 4), there were no differences in the trend with time among experimental groups.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We investigated the effects of two serotonergic drugs, ketanserin and 5-CT, on time-dependent hypoxic phrenic and hypoglossal responses in rats including 1) the short-term hypoxic response, 2) PHFD, and 3) LTF after episodic hypoxia. The 5-HT2A/C receptor antagonist ketanserin affected all three time domains of the hypoxic response, accentuating the short-term hypoxic response and PHFD and abolishing LTF. On the other hand, the 5-HT1A/B receptor agonist 5-CT had no effect on the short-term hypoxic response or PHFD but reversed LTF, revealing an unexpected long-term depression. Collectively, these data suggest that both 5-HT1A/B and 5-HT2A/C receptors modulate phrenic and hypoglossal nerve responses to hypoxia in complex ways.
Critique of Methods
Serotonin affects many types of neurons throughout the neuraxis (22), making it difficult to determine the specific site(s) of action of drugs injected intravenously. Moreover, drugs used in the present study likely interact (to a lesser, although unknown, extent) with other 5-HT receptor subtypes (21, 44) or even other neurotransmitter systems. For example, high doses of ketanserin can block1-adrenergic receptors (41).
The selectivity of any drug for designated 5-HT receptors is limited
and difficult to assess after systemic injections, because selectivity
is often determined in very reduced preparations with concentrations
much lower than those typically used in vivo. Moreover, 5-HT active drugs can affect other neurotransmitter systems either directly (i.e.,
acting on nonserotonergic receptors) or indirectly, owing to the
extensive interactions between serotonergic raphe neurons and other
neurotransmitter systems (e.g., dopaminergic or noradrenergic). Nevertheless, the approach used in the present study allows an initial
screening of potential receptor contributions to long-lasting changes
in complex integrative behaviors such as the time-dependent ventilatory
responses to hypoxia.
Although possible, it does not appear that baroreflexes or other blood pressure-related effects (e.g., changes in cerebral blood flow and consequent effects on medullary CO2/pH) account for the phrenic or hypoglossal responses observed in this study. There were no trends in blood pressure consistent with observed changes in respiratory motor output. Moreover, blood pressure changes similar to those reported here have little effect on hypoglossal or phrenic motor output in urethan-anesthetized and vagotomized rats (37; K. B. Bach and G. S. Mitchell, personal communication).
Serotonergic drugs can cause acute changes in "eupnic" respiratory motor output (37; present study). Although not the major thrust of this paper, these effects cannot be ignored because they may alter the input-output relationship of the respiratory control system and thus bias the responses being studied (13). We have addressed this potential shortcoming in several ways. First, we minimized potential normalization artifacts that could arise from differences in baseline nerve activity by using two different methods of data normalization (i.e., %baseline and %maximum). Second, for each drug, we adjusted baseline (postdrug) nerve activity of one animal to its predrug level by controlling inspired CO2 before the experimental protocol (data not shown). The results obtained from these rats were qualitatively and quantitatively similar to the mean response for each group, suggesting that the drug-induced shifts in baseline activity played no role in the experimental outcome.
Baseline Activity and Short-Term Hypoxic Response
Serotonin increases phrenic and hypoglossal motoneuron excitability via 5-HT2A/C receptor subtypes (29, 34), and activation of brain stem 5-HT2A/C receptors has been shown to increase respiratory frequency in vitro (29) and in vivo (38). The latter effect of 5-HT2 receptor activation is consistent with the reduced hypoxic frequency response in ketanserin-treated rats. However, the greater increase in phrenic burst amplitude during hypoxia in this group is inconsistent with known 5-HT2A/C receptor actions on respiratory motoneurons (i.e., increased excitability). However, because ketanserin-treated rats had the lowest blood pressure during hypoxia, the possibility that the enhanced phrenic response in this group is related to the pronounced hypotension observed under these experimental conditions cannot be ruled out.The effects of 5-CT injection on baseline nerve activity were qualitatively similar to acute effects reported after administration of 8-hydroxy-2dipropylaminotetralin (8-OH-DPAT), a selective 5-HT1A agonist. The lack of significant change in hypoglossal burst amplitude after 5-CT injection is consistent with the findings of Okabe and Kubin (35) and support their interpretation that 5-HT1A receptors exert minimal effect on these cranial motoneurons. Moreover, increased phrenic burst amplitude and frequency typically follow systemic injection (37) or in vitro bath application of 8-OH-DPAT on neonatal rat (34) or mice (20) preparations, effects consistent with increased phrenic burst amplitude and frequency after 5-CT administration. However, these data should be interpreted cautiously because 5-HT1A receptor activation inhibits phrenic motor output as well as the activity of medullary respiratory neurons in cats (27). Moreover, although 5-HT1A receptors may exert minimal effect on hypoglossal motoneurons, 5-HT1B receptors may be implicated in the control of hypoglossal discharge (35).
Posthypoxia Frequency Decline
Although the physiological significance of PHFD is unclear it may be part of an adaptive mechanism aimed at minimizing the energy expenditure required to provide sufficient O2 supply during sustained hypoxia (9). Noradrenergic neurons are important modulators of respiratory rhythm (1, 8, 11, 15), leading to the hypothesis that noradrenergic mechanisms contribute to PHFD (4, 5). The present data suggest, however, that serotonin may also play an important role in PHFD.Medullary 5-HT2A/C receptors modulate respiratory rhythm (29, 38). The marginally larger PHFD in ketanserin-treated rats is consistent with these reports and suggests that stimulation of 5-HT2A/C receptors may help maintain respiratory rhythm after hypoxia (i.e., offset PHFD). However, we cannot rule out the possibility that the pronounced hypotension observed during hypoxia in ketanserin-treated animals contributed to this effect. Although difficult to explain, the enhancement of PHFD by 5-CT suggest that 5-HT1 receptors are also important modulators of this time-dependent hypoxic response.
Long-Term Facilitation
Long-term facilitation is a form of activity-dependent memory of respiratory motor activity that requires serotonin receptor activation for its manifestation (3, 30-32). 5-HT2 receptor activation augments phrenic (12, 29, 34) and hypoglossal (25) motoneuron excitability. Thus it has been hypothesized that the long-lasting enhancement of inspiratory motor output after episodic hypoxia is mediated largely by activation of 5-HT2A/C receptors located on respiratory motoneurons (3, 30). The observation that ketanserin administration blocked phrenic LTF further supports this hypothesis.We initially hypothesized that 5-CT would block LTF by reducing
serotonin release via 5-HT1
autoreceptor activation. Therefore, the reversal of LTF, resulting in a
long-lasting depression of phrenic motor output in 5-CT-treated rats,
was unexpected. These data are difficult to explain because neither
methysergide (3) nor ketanserin (present study) had similar effects on
burst amplitude or frequency after episodic hypoxia in normal animals.
5-CT has the highest affinity for
5-HT1A/B receptor subtypes,
whereas methysergide and ketanserin have the greatest affinity for
5-HT2A/C receptors (21, 44). Thus
5-CT may have acted on 5-HT1
autoreceptors to diminish serotonin release, thereby revealing
long-lasting inhibitory neuromodulatory mechanisms affecting phrenic
motor output. Although the mechanisms mediating the long-lasting
depression of respiratory motor output in 5-CT-treated rats are
unknown, the response is similar to the
2-dependent long-term
depression of respiratory activity that follows episodic hypercapnia
(5). Hypercapnia-induced long-term depression of phrenic motor output affects phrenic burst amplitude and burst frequency, lasts for >1 h, and requires the activation of
2-adrenoceptors (5). Thus our
data lend further support to the hypothesis that LTF reflects a balance
between excitatory (serotonergic) and inhibitory (noradrenergic)
mechanisms activated differentially by hypoxia and hypercapnia (3, 5).
Heterogeneity of Serotonergic Modulation of Phrenic and Hypoglossal Motor Output
Several observations support the hypothesis that serotonin exerts differential effects on discrete motoneuron pools (17, 33). Specifically, the ketanserin-induced enhancement of phrenic, but not hypoglossal, burst amplitude responses to hypoxia and the long-lasting depression of phrenic (but not hypoglossal) burst amplitude after 5-CT each demonstrate a degree of heterogeneity between phrenic and hypoglossal motor output. Understanding the precise anatomic and neurophysiological mechanisms underlying such differences in respiratory motor output may provide valuable insight into diseases related to upper airway function, such as obstructive sleep apnea (17).Perspectives
Our results suggest that 5-HT1A/B and 5-HT2A/C receptors contribute to a balance between inhibitory and facilitatory serotonergic neuromodulation, imparting a degree of plasticity to the respiratory control system. 5-HT1A/B receptors may play an important role in this balance, acting at least in part by autoinhibitory receptors on raphe neurons, controlling the release of serotonin. Moreover, our results support the hypothesis that 5-HT2A/C receptor activation is necessary for LTF (3, 30). Although our understanding of the mechanisms underlying LTF is far from complete, we hypothesize that repetitive stimulation of 5-HT2A/C receptors on motoneurons plays a key role in a cascade of events increasing respiratory motoneuron excitability for a prolonged (hours) duration.| |
ACKNOWLEDGEMENTS |
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We thank Dr. K. B. Bach for reading earlier versions of this manuscript and B. Hodgeman for help in preparing this manuscript for publication.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grants HL-36780 and HL-53319 and a Centennial postdoctoral fellowship from the Medical Research Council of Canada (to R. Kinkead).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. Kinkead, Unité de Recherche de Pédiatrie, Centre Hospitalier Universitaire de Québec, Pavillon Saint-François d'Assise, 10 rue de l'Espinay, D0-707, Québec QC, G1L 3L5, Canada (E-mail: richard.kinkead{at}crsfa.ulaval.ca).
Received 17 September 1998; accepted in final form 10 May 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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, n = 7),
5-carboxamidotryptamine (5-CT; 
, n = 6), or saline (sham treated, 
, n = 13). A: pretreatment with ketanserin
enhances phrenic burst amplitude during hypoxia. Pretreatment with 5-CT
had no significant effect on magnitude of response.
B: neither pharmacological treatment
affected hypoglossal response to hypoxia. Integrated phrenic and
hypoglossal burst amplitudes are expressed as %change from baseline
(prehypoxia). C: burst frequency
response to hypoxia, expressed as change from prehypoxic (baseline)
value. Frequency response was reduced by ketanserin and 5-CT. Data
presented as means ± SE. * Significantly different from the
hyperoxic "time" control value (
,
n = 7;
P < 0.05); 
significantly
different from sham-treated group (P < 0.05).
Frequency values are
expressed relative to prehypoxic (baseline) frequency. Data presented
as mean ± SE. * Significantly different from time control
group (
