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1 Division of Pulmonary Critical Care and Sleep Medicine, Department of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4941, USA
Abstract
The respiratory system expresses multiple forms of plasticity, defined as alterations in the breathing pattern that persist or develop after a stimulus. Stimulation of breathing with intermittent hypoxia (IH) elicits long-term facilitation (LTF), a type of plasticity in which respiratory motor activity progressively increases in anaesthetized animals, even after the stimuli have ceased and blood gases have normalized. It is unknown whether the sympathetic nervous system similarly expresses IH-induced plasticity, but we predicted that IH would evoke LTF in sympathetic nerve activity (SNA) because respiratory and sympathetic control systems are coupled. To test this idea, we recorded splanchnic (sSNA) and phrenic nerve activities (PNA) in equithesin-anaesthetized rats. Animals were exposed to 10 45 s episodes of 8% O2–92% N2, separated by 5 min intervals of 100% O2, and recordings were continued for 60 min following the last hypoxic exposure. Cycle-triggered averages of integrated PNA and sSNA from periods preceding, and 5 and 60 min following the hypoxic stimuli were compared. Intermittent hypoxia significantly increased both sSNA and PNA. Treatment with methysergide (3 mg kg–1, I.V.) 20 min before the intermittent hypoxic exposures prevented the increases in integrated PNA and sSNA 60 min after IH, indicating a role of serotonergic pathways in this form of plasticity. No increases in PNA and sSNA occurred at comparable times (60 and 120 min) in rats not exposed to hypoxia. The increased sSNA was not simply tonic, but was correlated with respiratory bursts, and occurred predominantly during the first half of expiration. These findings support the hypothesis that sympathorespiratory coupling may underlie the sustained increase in SNA associated with the IH that occurs during sleep apnoea.
(Received 12 September 2006;
accepted after revision 15 November 2006; first published online 30 November 2006)
Corresponding author T. E. Dick: Biomedical Research Building BRB B55, 10900 Euclid Avenue, Cleveland, OH 44106-4941, USA. Email: ted3{at}po.cwru.edu
Patients with sleep apnoea can develop hypertension, which is associated not only with elevated sympathetic nerve activity (SNA), but also with increased cardiorespiratory sensitivity to hypoxia (Cutler et al. 2004a,b; Leuenberger et al. 2005). These changes in autonomic control may reflect forms of activity-dependent plasticity analogous to those previously described for respiratory control (Bach & Mitchell, 1996; Coles & Dick, 1996; Powell et al. 1998; Poon & Siniaia, 2000; Ling et al. 2001; Mitchell et al. 2001; Mitchell & Johnson, 2003). In sleep apnoeic patients, exposure to repeated bouts of hypoxia leads to increased sympathetic activity, as recently observed in recordings of muscle SNA (Cutler et al. 2004a,b). Further, even healthy humans exhibit sustained sympathetic activation and a transient elevation of blood pressure after single (Xie et al. 2001) or repetitive exposures to hypoxia (Leuenberger et al. 2005). We hypothesize that these hypoxia-induced increases in sympathetic activity and hypoxic sensitivity are co-ordinated, and caused by interactions between the respiratory and sympathetic control systems (Huang et al. 1988; Lahiri et al. 1991; Koshiya & Guyenet, 1996; Dick et al. 2004; Mandel & Schreihofer, 2006).
Various mechanisms, which have been identified primarily in rodent models, contribute to the sustained alterations of the respiratory pattern that occur following exposure to hypoxia (Coles & Dick, 1996; Powell et al. 1998; Dick & Coles, 2000; Poon & Siniaia, 2000). After a single brief exposure to hypoxia in anaesthetized or decerebrate preparations, diaphragmatic motor amplitude increases, while respiratory frequency (FR) decreases. These posthypoxic changes are referred to as short-term potentiation and posthypoxic frequency decline, respectively. These responses are neurally mediated, because they can be replicated by stimulation of the carotid sinus nerve, in the absence of changes in blood gases (Hayashi et al. 1993), and they constitute distinct forms of ‘activity-dependent’ plasticity, because they depend on the persistent activity in the chemoreflex pathway (Wagner & Eldridge, 1991). For instance, posthypoxic frequency decline may be due to increased activity of the ventrolateral pontine neurones that are activated during, and remain activated after, hypoxia (Dick & Coles, 2000).
Repeated exposure to hypoxia induces an additional type of plasticity in the respiratory motor system, referred to as long-term facilitation (LTF; Millhorn et al. 1980a; Eldridge & Millhorn, 1986; Bach & Mitchell, 1996; Ling et al. 2001; Mitchell et al. 2001; Mitchell & Johnson, 2002). Long-term facilitation is expressed in anaesthetized rodents as a sustained increase in respiratory motor activity, which increases progressively following the last hypoxic exposure and remains elevated for at least an hour (Millhorn et al. 1980a; Eldridge & Millhorn, 1986; Mitchell et al. 2001). Respiratory LTF is thought to be mediated by serotonin, because pretreatment with methysergide, a serotonergic antagonist, blocks its occurrence (Millhorn et al. 1980a,b; Bach & Mitchell, 1996).
Persistent posthypoxic sympathetic activity may be related to its entrainment with respiration. Indeed, in the absence of respiratory rhythmogenesis, even though hypoxaemia evokes increased SNA, it does not evoke any short-term potentiation or post-stimulus effects (Huang et al. 1988; Lahiri et al. 1991; Koshiya & Guyenet, 1996). In contrast, when formation of the breathing pattern is intact, we noted that the respiratory-modulated pattern of SNA is enhanced following hypoxia, indicating that a single, brief exposure to hypoxia leads to augmented sympathorespiratory coupling (Dick et al. 2004).
In the present study, we hypothesized that intermittent hypoxic exposures would induce LTF in SNA. To test this possibility, we used a rodent model of hypoxia-induced respiratory modulation of SNA established previously, which exhibits robust ventilatory and sympathetic hypoxic responses as well as plasticity in the respiratory pattern (Dick et al. 2004). This model is advantageous because both splanchnic SNA (sSNA) and PNA have an excitatory response to hypoxia (Strohl et al. 1997; Dick et al. 2004) and are isolated from modulatory sensory influences (Dick et al. 2004). In addition, the time domains of its respiratory modulation, which include posthypoxic short-term changes in pattern and entrainment, are well defined (Powell et al. 1998; Dick et al. 2004). We used cycle-triggered histograms (Dick et al. 2004) of sSNA to assess expression of LTF, and also tested whether serotonin receptors are involved in mediating LTF expression in SNA, as has been described for LTF in the respiratory system, by using pharmacological blockade of serotonin receptors.
Methods
General procedures
The methods were similar to those published previously (Coles & Dick, 1996; Dick et al. 2004). Briefly, adult male rats (Sprague–Dawley, obtained from Zivic Miller (Pittsburgh, PA, USA), n = 20, 320–470 g) were anaesthetized with an intraperitoneal injection of equithesin (30 and 133 mg kg–1 sodium pentobarbitone (Sigma, St Louis, MO, USA) and chloral hydrate (Sigma), respectively). We evaluated the anaesthetic level by testing the withdrawal reflex before initiating surgery. Surgical procedures and experimental protocols followed NIH guidelines and were approved by the Institutional Animal Care and Use Committee.
We cannulated a femoral artery to monitor blood pressure, a femoral vein to administer pharmacological agents, and the trachea to ventilate the animal. After cannulae were implanted, the animals were positioned in a stereotaxic frame. The left phrenic and splanchnic sympathetic nerves were isolated and transected. When these nerves were ready to be mounted on bipolar recording electrodes, we injected the animals with a neuromuscular blocking agent (Pavulon (Sigma), pancuronium bromide, 0.1 mg (100 g body weight)–1 h–1, I.V.); ventilated them with 100% O2, performed a pneumothoracotomy and transected their cervical vagi bilaterally. Pneumothoracotomy reduced chest wall movement, minimizing rhythmic afferent input arising from joint receptors and stabilizing the splanchnic sympathetic nerve recordings. Vagotomy eliminated pulmonary stretch receptor input. Thus, we removed sensory input which would entrain sympathetic and respiratory rhythms with the ventilator. We ventilated the animals with 100% O2 to minimize excitatory chemosensory input before, between and after hypoxic exposures, to minimize hypoxaemia in poorly perfused regions, to ensure that arterial partial presssure of CO2 (PaCO2) was the primary ventilatory stimulus and to maximize the magnitude of the step change between hypoxic exposures. We recorded blood pressure, airflow, end-tidal partial pressure of CO2, raw and integrated PNA and sSNA on a chart recorder (Astro-Medical Dash 8), and integrated PNA and sSNA on computer (Gateway 500 equipped with a National Instruments A/D board (Austin, TX) and LabView software (Labview Software, Austin, TX, USA)). After continuously from the blood pressure and nerve recordings an neuromuscular blockade, we evaluated anaesthetic level placing the cannalae and especially after, at least hourly, by recording the cardiorespiratory response to a painful paw pinch. If mean arterial blood pressure fluctuated by 10% or if there was the slightest response to painful stimuli, then anesthesia was supplemented as needed by administering a tenth of the initial dose intravenously. Before starting the experiment, we corrected for the postsurgical acidic pH with a 1 ml intravenous injection of 8.4% sodium bicarbonate solution. For the group, the initial arterial pH was 7.36 ± 0.04 and the initial blood gas values had a hyperoxic arterial partial pressure of O2 because the animals were ventilated with 100% O2, and PaCO2 was 39.2 ± 1.5 mmHg, approximately 8 mmHg above the apnoeic threshold (31 mmHg found in a subset of animals). Under these conditions, the hypoxic response was robust. Even though the hypoxic exposure was poikilocapnic, the hypocapnia associated with hypoxia was minimized by short duration of hypoxia. When animals were being ventilated with hyperoxia, eucapnia was maintained by adjusting the ventilatory rate. Rectal temperature was maintained at 37 ± 0.5°C throughout the experiment by a servocontrolled recirculating water blanket and infrared lamps. Animals were killed after the experiment with an intravenous injection of the anaesthetic at a dose equivalent to that administered intraperitoneally to induce anaesthesia. Animals were removed from the ventilator upon cessation of the heart beat and/or when arterial blood pressure dropped to below 10 mmHg.
Experimental protocol
Once PNA and sSNA were being recorded and arterial blood gas levels were corrected, one of three protocols was initiated. In protocol 1 (Fig. 1), animals (n = 8) were exposed to intermittent hypoxia (IH), consisting of 10 exposures to a hypoxic gas mixture separated by 5 min intervals of ventilation with 100% O2. The hypoxic gas mixture was 8% O2 in 92% N2, and the duration of each exposure was 45 s. End-tidal partial pressure of CO2 decreased by < 2 mmHg durng an exposure. Animals were ventilated with 100% O2 before and after IH. Recordings continued for 60 min following IH. Protocol 2 was identical to protocol 1 except that animals (n = 5) received an injection of methysergide maleate (3 mg kg–1 dissolved in 0.9% NaCl, I.V.) 20 min before the onset of the hypoxic exposures. In protocol 3, which served as a time control, PNA and sSNA were similarly recorded for 2 h, the duration of the repeated hypoxic exposures, but the animals (n = 7) were not exposed to a hypoxic gas mixture. Blood gases were also measured and, if necessary, adjusted at the end of experiments, either 60 min after the 10th hypoxic exposure (protocols 1 and 2) or 120 min after starting the recording (protocol 3).
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To test our hypothesis, we compared the coupling patterns between PNA and sSNA preceding the hypoxic exposures (baseline) with those made at 5 and 60 min after the hypoxic exposures. Cycle-triggered averages (CTAs) were constructed for PNA and sSNA to increase the signal-to-noise ratio of sSNA that was time locked to the respiratory cycle (Dick et al. 2004). The reference point (time zero) for the CTAs was the time of phase transition between inspiration (I) and expiration (E). The target, or analog signal, was electronically integrated (Paynter Filter, 50 ms time constant; CWE, Inc., Ardmore, PA, USA), and was sampled and summed at 200 Hz. Time parameters for sampling before and after the triggering event were set so that the signals occurring during the interval beginning 200 ms prior to the onset of I and ending 50–100 ms after the onset of the next I were averaged.
We used standard criteria to determine the respiratory phases (see Figs 1 and 3; Dick et al. 2004). The onset of PNA was identified as the value 10% above the baseline value for PNA, and on the positive slope of the integrated PNA signal. The onset of the next burst was used as the end of the cycle. We averaged PNA and sSNA for 2 min before the hypoxic exposures to determine the pattern of ‘baseline’ synchronization of sSNA and PNA, and compared the baseline averages to those obtained for 2 min periods at 5 and 60 min after the hypoxic exposures. The cycles analysed were consecutive. The total number of averaged cycles depended on the respiratory frequency and was > 50 cycles.
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To test for significant differences in the magnitude of sSNA and its c.v., and PNA before and after IH, we applied a two-way analysis of variance (ANOVA) for repeated measures. Parameters showing significant differences were subjected to the Student–Newman–Keuls test to identify specific differences. To test for significant correlations among sSNA, tI, tE and PNA, we performed linear regressions of sSNA in the first and second half of each phase against tI, tE and PNA. Significance was accepted as P
0.05. Data are presented as means ±
S.D. except where indicated.
Results
Activity before and after 10 hypoxic exposures
Exposure to IH elicited sustained increases in PNA and sSNA (Figs 1–4) but without a significant change in arterial blood pressure (Table 1). These increases were not associated with significant changes in blood gases, which were maintained by the ventilator (values before and 1 h after IH were, respectively: PaCO2, 39.2 ± 1.2 and 40.7 ± 1.1 mmHg; and pH, 7.36 ± 0.04 and 7.37 ± 0.05). For both integrated PNA (Figs 1–4) and integrated SNA (Figs 2–4), both amplitudes and areas under the curves increased significantly after IH and remained increased for at least 60 min (Figs 1–4). Integrated sSNA values at both 5 and 60 min after IH were significantly greater than those before IH (Fig. 4).
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Effect of serotonergic blockade on IH-evoked activity
Systemic administration of methysergide (3 mg kg–1, I.V.) attenuated the sustained IH-induced increases in sSNA and PNA (Figs 5 and 6) and did not change arterial blood pressure (Table 1). Despite a transient vasodilatation elicited by methysergide (Fig. 5B), the acute and short-term responses of sSNA and PNA to brief hypoxia were similar before (Fig. 5A) and after methysergide administration (Fig. 5C), but the long-term response to hypoxia was attenuated. One hour following IH, integrated PNA and sSNA were unchanged versus baseline for the group (Fig. 6). Similarly, burst timing was not significantly different before and after IH. Also, the distribution of sSNA was not significantly different in the respiratory phases, consistent with the absence of IH-induced change in total activity following methysergide treatment (Fig. 6).
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In animals subjected to continuous recording in the absence of IH, no significant changes in PNA, sSNA and arterial blood pressure occurred (Figs 7 and 8). In the example (Fig. 7), PNA decreased 47%, sSNA increased 42% and FR decreased 6% during the 2 h recording period, but for the group (Fig. 8), the areas of integrated PNA (–13 ± 48%) and sSNA (18 ± 44%) were not significantly different from baseline. The percentage changes in the areas of integrated PNA and sSNA were not correlated (r2 = 0.47), and similar changes in integrated sSNA occurred during each phase of the respiratory cycle, indicating that IH-induced effects (Figs 1–5) were not attributable to other time-dependent changes occurring in this model.
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The increase in sSNA was respiratory phase specific, and appeared to be coupled with the increase in respiratory motor activity (Figs 2, 3 and 4). To test the strength of this relationship, we determined the correlation between the amplitudes (Fig. 9A) and areas (not shown) of integrated sSNA and PNA responses. In the example (Fig. 9A), sSNA during each half-phase was correlated significantly with PNA, but the relationship was strongest (i.e. the correlation coefficient was greatest) between sSNA and PNA during the first half of expiration. We averaged and compared the regression coefficients obtained in all animals by correlating sSNA for each half-phase to PNA (Fig. 9B). For the group, the regression coefficient between PNA and sSNA during the first half of expiration was significantly greater than those occurring during the other half-phases.
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The major findings in the present study are that IH evoked sustained increases in both sSNA and PNA, and the increase in sSNA occurred predominantly during the first half of expiration. As found earlier for respiratory LTF (Bach & Mitchell, 1996; Millhorn et al. 1980a,b), the IH-induced increase in SNA was blocked by pretreatment with methysergide, indicating a role of serotonergic pathways in mediating the response. Together, these findings support the hypothesis that the sustained increase in SNA caused by IH in our model is a form of sympathetic LTF that involves cardiorespiratory coupling mechanisms. By extension, we interpret these findings to suggest that similar sympathorespiratory coupling mechanisms may be involved in mediating the sustained increase in SNA evoked acutely in humans by IH, possibly contributing to the clinically observed hypertension that is commonly associated with IH in sleep apnoea patients.
The observed changes in sympathetic activity after IH contrast with those we have previously found to be evoked by a transient exposure to hypoxia. During such a posthypoxic FR decline, sympathetic motor activity, like FR, decreased sharply and then gradually increased to baseline values (Coles & Dick, 1996; Dick et al. 2004). In contrast, respiratory motor activity remained elevated and gradually decreased to baseline. Also, during this time, the sSNA became more entrained with respiration, as indicated by a significant decrease in the variability of sSNA from cycle to cycle (Dick et al. 2004). The IH-induced increase in sSNA was also respiratory modulated, because increased SNA during the first half of expiration was highly correlated with the increase in amplitude of PNA. However, the increase in sSNA appeared much greater than that of PNA. While this may be due to a respiratory-modulated inhibitory mechanism (Mandel & Schreihofer, 2006), we cannot exclude non-respiratory influences (Huang et al. 1988; Lahiri et al. 1991; Koshiya & Guyenet, 1996). Thus, the dynamic properties of sSNA include an increase in its entrainment with the respiratory pattern after a single brief hypoxic exposure and an increase in its magnitude following IH. Further, the changes in patterning and magnitude of sSNA following single and repetitive exposures to hypoxia, respectively, coincided with increased respiratory motor activity.
In the case of ventilatory LTF, its incidence and magnitude are influenced by several variables, including the pattern of hypoxic exposures, as well as the strain, age, gender and state of consciousness of the animal. In the present experimental model, the magnitude of respiratory LTF is robust. Several factors contribute to the robustness of this response, including: (1) the exposures were brief (45 s), repetitive (n = 10) and severe (decreasing from 100 to 8% O2); (2) this strain of rat is highly responsive (Strohl et al. 1997); (3) we used young adult males, known to be particularly responsive (Mitchell et al. 2001; Zabka et al. 2001); and (4) animals were anaesthetized and vagotomized, a state facilitating the expression of LTF (Mitchell et al. 2001; McGuire et al. 2002, 2003, 2004). This animal model of IH was developed to investigate the pathophysiology associated with sleep apnoea (Fletcher et al. 1992a,b). We modified Fletcher's protocol to address whether the mechanism of increased SNA could be related to the sustained increase in PNA evoked by acute IH, in particular to determine whether the increase in sSNA was related to respiratory phases, as opposed to simply tonic, and whether the increase in sSNA depended on functional serotonergic receptors.
This was an initial set of experiments to identify and characterize LTF in sympathetic motor activity. Therefore, we adopted the strategies that facilitated the development of respiratory motor LTF, as well as duplicating the initial experiments that characterized ventilatory LTF. Anaesthesia and vagotomy have been found to unmask serotonergic influences on respiratory motor activity, at least of the upper airway (Sood et al. 2006). In conscious vagally intact animals, ventilatory LTF is expressed primarily as an increase in respiratory frequency rather than in motor amplitude. In the original experiments, ventilatory LTF was followed for at least an hour after cessation of the repetitive hypoxic exposures and was blocked by I.V. injections of methysergide (Millhorn et al. 1980a,b). These aspects of ventilatory LTF were replicated in this set of experiments describing sympathetic LTF. However, we shortened the exposures from 5 min to 45 s and increased the number from three to 10 exposures to maximize the transitions from low to high oxygen levels and to minimize the effect of hypoxia on metabolism and CO2 production. The exposures were poikilocapnic rather than iso- or hypercapnic because hypoxia is the primary stimulus for developing ventilatory LTF (Fletcher et al. 1995).
Based on the present findings, our hypothesis is that the same variables that strongly influence respiratory LTF, listed above, also affect LTF of sympathetic activity. One feature of particular potential relevance to the clinical pathophysiology of sleep apnoea is the role that the pattern of hypoxic exposures has in evoking sympathetic LTF, and its relationship to the differential expression of hypertension in sleep apnoeic patients. Acute IH increases human muscle SNA (Cutler et al. 2004a,b; Leuenberger et al. 2005). In awake and healthy humans, breathholding for 20 s of each minute over a 30 min period increased muscle SNA when the subjects inspired a hypoxic gas mixture (10% O2) immediately preceding the apnoea, but not when the held breaths contained room air (Leuenberger et al. 2005). Similarly, in awake sleep apnoeic patients who inspired a hypoxic gas mixture for 30 s of each minute for a period of 20 min, muscle SNA remained significantly elevated for 3 h after IH (Cutler et al. 2004a,b). These data support the concept that acute IH is effective in recruiting SNA that is sustained for long periods after the stimulus. However, a single prolonged (20 min) exposure to isocapnic hypoxia (to an arterial O2 saturation of 77–87%) also induced a long-lasting increase in muscle SNA in humans (Xie et al. 2001). This is distinctly different from ventilatory LTF because IH is more effective than continuous hypoxia in evoking ventilatory LTF in animals (Baker & Mitchell, 2002; Prabhakar & Kline, 2002; Peng & Prabhakar, 2004) and humans (Babcock & Badr, 1998; Aboubakr et al. 2001; Babcock et al. 2003); however, prolonged stimulation (10 min) of the raphé can induce phrenic LTF (Millhorn, 1986). Further research is necessary to determine whether SNA that is activated by continuous rather than intermittent hypoxia is tonic or respiratory modulated and whether it can be blocked by methysergide. However, unlike ventilatory LTF, various patterns of acute hypoxic exposures can evoke sustained sympathetic activity in humans.
In patients with sleep apnoea, hypertension associated with increased SNA is a commonly occurring comorbidity. Even though hypercapnia and arousal also result from apnoea during sleep, animal studies suggest that the hypoxia is the critical variable for increased SNA (Fletcher et al. 1995). In rats, chronic IH (for 7 h of each day for 35 days) produced increases in SNA and blood pressure that were sustained throughout the day (Fletcher et al. 1992a,b), which was not increased further by adding CO2 to the inhaled hypoxic gas (Fletcher et al. 1995). Intact peripheral chemoreceptors, which are primarily sensitive to hypoxia, are necessary for the elevation in blood pressure to be manifested (Fletcher et al. 1992b), and increased sensory input from the carotid body may contribute to the sustained hypertension observed in that model (Peng et al. 2003). In animals conditioned by chronic IH, the carotid body also expresses sensory LTF, manifested as increased activity accompanied by increased responsiveness to hypoxia (Peng et al. 2003). Together, the available evidence strongly supports the hypothesis that while acute IH evokes increased SNA, chronic IH elicits additional autonomic responses that more completely mimic the pathophysiology of sleep apnoea in humans. These findings also raise the critical question of whether, and how, the mechanisms that contribute to IH-induced sympathetic LTF may also be involved in the mechanisms that relate to the sustained hypertension that accompanies chronic IH.
The present finding that sympathetic LTF is correlated with respiratory LTF provides insight into the possible mechanisms involved. Serotonin is essential for both respiratory (Millhorn et al. 1980a,b) and sympathetic (present study) LTF, which suggests the involvement of brainstem and spinal mechanisms in these forms of neural plasticity. This view is supported by several additional lines of evidence. For example, hypoxia activates medullary raphé neurones (Erickson & Millhorn, 1994), and stimulation of neurones in the raphé obscurus leads to serotonergic dependent excitation of respiratory motoneurones (Millhorn, 1986; Holtman et al. 1986), while injection of methysergide, a serotonergic antagonist, blocks LTF (Millhorn et al. 1980a,b; Bach & Mitchell, 1996; Baker-Herman & Mitchell, 2002). Ensemble recordings from medullary and raphé neurones revealed that repetitive hypoxia increased short-time scale synchrony among raphé activities (Morris et al. 1996a,b; Morris et al. 2001), an effect which would favour increased effectiveness of neural signals converging at a common target by enhancing the temporal summation of synaptic potentials (Morris et al. 2001). These studies indicate that plasticity occurs at other levels than the motoneuronal pools. Nevertheless, ventilatory LTF can be blocked by interventions in the phrenic motor nucleus. For instance, serotonin release intrathecally can also evoke synthesis of proteins, including brain-derived neurotrophic factor (BDNF) locally in the spinal cord (Baker-Herman et al. 2004). Intrathecal blockade of serotonergic receptors or BDNF synthesis prevents LTF evoked in PNA (Baker & Mitchell, 2002; Baker-Herman et al. 2004). If IH also evokes synthesis and release of BDNF in the brainstem, then BDNF may play a similar role at multiple levels of the neuraxis and mediate increased short-term synchrony between raphé neurones (Fujisawa et al. 2004). Finally, although the evidence strongly favours the involvement of brainstem–spinal cord interactions in the development of IH-induced LTF, cerebellar pathways also appear to be involved, because repetitive hypoxia failed to elicit LTF in cerebellectomized animals (Hayashi et al. 1993). Considering the potential clinical relevance of IH-induced sympathetic LTF to the pathophysiology of human sleep apnoea, a critical goal of further research will be to determine the extent to which sympathetic and ventilatory LTF responses may be mediated by similar neuromodulators and neural pathways.
In summary, we evaluated the properties of SNA plasticity evoked by IH in the presence of the respiratory pattern and in the absence of pulmonary stretch receptor afferent feedback, and found a strong correlation between the increase in sSNA during the first half of expiration and the magnitude of LTF in PNA. Together, these findings indicate that exposure to IH elicits enhanced cardiorespiratory coupling. These results raise the possibility that a similar phenomenon may contribute to the altered cardiorespiratory patterning observed in conscious human subjects following repeated hypoxia, and further suggest that IH-induced sympathetic LTF may contribute to the pathogenesis of SNA elevation and hypertension that commonly accompany human sleep apnoea, representing a potentially significant source of morbidity and mortality in patients having this disorder.
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Acknowledgements
The authors wish to thank Flamur Semaj and Ying-Jie Peng for their assistance in data acquisition and analysis. We also gratefully acknowledge the support of NIH funding through HL-25830 and HL-63042.
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) of expiration (Exp), and for integrated PNA (middle graph), were significantly greater than those at baseline, both immediately and 60 min after the acute IH (asterisks). Splanchnic SNA during the second half of Exp was greater than baseline only at 60 min after IH (asterisk). In contrast, sSNA during the first and second halves of Inspiration (Insp), as well as the durations of inspiration (tI) and expiration (tE; right graphs) were unchanged from their respective baselines.
) was highly and significantly correlated to that of PNA (r2
= 0.91). The correlation coefficient for the other half-phases (r2
= 0.79 for 1st half of I, 0.85 for 2nd half of I, and 0.68 for 2nd half of E). In B, the mean correlation coefficient for 1st half of E (filled bar) was significantly greater than that for the other phases (open bars; pairwise multiple comparison procedure used was Student–Newman–Keuls test).