Is augmented central respiratory–sympathetic coupling involved in the generation of hypertension?

Respiratory Physiology & Neurobiology 174 (2010) 89–97

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Review

Is augmented central respiratory–sympathetic coupling involved in the generation of hypertension?夽 A.E. Simms a,b , J.F.R. Paton b , A.M. Allen a,c , A.E. Pickering b,∗ a

Department of Physiology, University of Melbourne, Vic., 3010, Australia Department of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK c Florey Neurosciences Institutes, University of Melbourne, Vic., 3010, Australia b

a r t i c l e

i n f o

Article history: Accepted 22 July 2010 Keywords: Hypertension Sympathetic nervous system Respiratory–sympathetic coupling

a b s t r a c t Respiratory modulation of autonomic neural activity, with consequent phasic alteration of cardiac and vascular function, has been observed in many species including humans and is considered an index of cardiovascular health. Whilst many factors contribute to this modulation, including for example baroreceptor reflex feedback, it is accepted that a significant component is derived from an interaction within the central nervous system. Functional links between the brainstem circuitry generating the respiratory rhythm and neurons responsible for generate sympathetic and parasympathetic activity to the cardiovascular system have long been hypothesized, although the detailed understanding of these interactions is incomplete. There are several proposed physiological functions for these interactions including the matching of ventilation to cardiac output and tissue blood flow. However, recent observations suggest that altered central respiratory coupling may play a role in the development of hypertension and in the maintenance of elevated levels of sympathetic vasomotor activity in disease. The focus of this review article is to discuss these observations and place them within the context of current understanding of the neural substrates that might be responsible for respiratory–sympathetic coupling. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The cardiovascular and respiratory systems have closely entwined roles in the delivery of oxygen and nutrients to, and the elimination of CO2 and metabolites from, the cells of the vertebrate organism. Both systems have pumps, the heart and the respiratory musculature respectively, which are under the control of the central nervous system. The respiratory pump consists of skeletal muscle whose contraction depends upon drive from phrenic and intercostal motor neurons in the spinal cord that in turn are driven from respiratory centers in the brainstem. These respiratory neural centers generate a phasic bursting discharge that produces the inspiration–expiration cycle of respiration. In contrast the heart has its own intrinsic pacemaker activity whose rate of discharge is controlled by inputs from the sympathetic and parasympathetic nervous systems. Additionally, the distribution of the cardiac output to different vascular beds is subject to neural regulation by the sympathetic nervous system.

夽 This paper is part of a special issue entitled “Central cardiorespiratory regulation: physiology and pathology”, guest-edited by Thomas E. Dick and Paul M. Pilowsky. ∗ Corresponding author. Tel.: +44 1173317579. E-mail address: [email protected] (A.E. Pickering). 1569-9048/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2010.07.010

The dominant rhythm in the vasculature is the pulse wave generated by the pump cycle of the heart but it has long been appreciated that other rhythms are present, such as those entrained to the respiratory cycle. For example, respiratory sinus arrhythmia (RSA) in heart rate is in large part the product of central modulation of the activity of cardiac vagal motor neurons (Richter and Spyer, 1990). In their seminal observations Traube and Hering showed that there were rhythmic fluctuations in arterial pressure in phase with respiration, subsequently termed Traube–Hering waves (as described in Killip, 1962). Some of the original observations indicated that these waves were not simply a consequence of changes in intrathoracic pressure and it was hypothesized that they may be the product of fluctuations in sympathetic nerve activity (SNA). The first pioneering recordings of SNA clearly demonstrated the presence of bursting discharge in phase with respiration (Adrian et al., 1932)—a finding that has been replicated in many subsequent studies, both in animals (Barman and Gebber, 2000; Czyzyk-Krzeska and Trzebski, 1990; Dick et al., 2004; Habler et al., 1994; Haselton and Guyenet, 1989) and in humans (Badra et al., 2001; Dempsey et al., 2002; Eckberg et al., 1985). In part this respiratory–sympathetic coupling is a result of feedback from peripheral baroreceptors, as venous return varies with intrathoracic pressure, and also a result of the inhibitory influence of activation of pulmonary stretch receptors on the sympathetic activity. However, respiratory oscillations in SNA persist

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after removal of these inhibitory inputs by vagotomy (Adrian et al., 1932; Barman and Gebber, 1980; Habler et al., 1994; Haselton and Guyenet, 1989). Similarly, respiratory oscillations in SNA are strikingly demonstrated in the working heart brainstem preparation (WHBP) which, as a consequence of bilateral pneumonectomy, has no fluctuations in intrathoracic pressure and no feedback from pulmonary stretch receptors (Pickering and Paton, 2006; Simms et al., 2009). These observations indicate a central neural coupling between the respiratory pattern generator and the sympathetic circuits responsible for the generation of the sympathetic outflow. This respiratory–sympathetic coupling underpins the production of Traube–Hering waves which are derived from the respiratory modulation of the sympathetic outflow causing phasic constriction of the arterial tree, particularly in the muscle vasoconstrictor class of sympathetic fibers (Habler et al., 1994; Janig, 2006; Malpas, 1998). In fact these respiratory and cardiovascular neural circuits appear just as closely intertwined within the brainstem as the cardiovascular and respiratory systems are functionally linked in the periphery. It is surely no coincidence that the neurons regulating the generation of respiratory activity and cardiovascular autonomic activity (e.g. cardiac vagal preganglionic and pre-sympathetic motor neurons) are intermingled in overlapping areas within the ventrolateral medulla. With the synaptic connectivity between these neuronal groups, Richter and Spyer (1990) have proposed a ‘common cardiorespiratory oscillator’ for cardiorespiratory synchrony. Some of these neurons may have shared roles in the control and regulation of both the cardiovascular and respiratory systems; leading to a blurring of the textbook boundaries between central respiratory and cardiovascular neural circuitry. 1.1. Functional role of neural cardiorespiratory coupling Central nervous coupling of the respiratory and cardiovascular systems, which intrinsically links ventilation to ‘breath by breath’ variations in cardiac output and distribution of blood flow, which optimizes oxygen delivery and carbon dioxide removal. A particularly elegant demonstration of this principle was provided in dogs where RSA was shown to improve matching of pulmonary perfusion to ventilation with a consequent 50% reduction in intrapulmonary shunt (Hayano et al., 1996). In addition to matching the distribution of cardiac output to the minute ventilation there may be other physiological reasons for the central neural coupling of these systems. For example the coupling may provide a mechanism to reduce fluctuations in blood pressure due to respiratory phase-related changes in venous return. During expiration venous blood flow to the heart is reduced by the relative increase in intrathoracic pressure whereas during inspiration venous return is enhanced due to both the negative intrathoracic pressure and increased abdominal pressure as the diaphragm moves downwards. During expiration when venous return is reduced, the cycle of RSA produces a slowing of heart rate that allows longer ventricular filling time helping to maintain stroke volume and hence blood pressure. This vagally-mediated bradycardia during expiration also assists with coronary blood flow by prolonging diastole and also perhaps by reducing ventricular contractility and producing coronary dilatation. This central neural cardiorespiratory coupling is not only present in mammals but also in reptiles and fish. Turtles show a particularly extreme variation with a two- to four-fold synchronous increase in heart rate and pulmonary blood flow during ventilation compared with breath-hold diving (Wang and Hicks, 1996). This is mediated by neural vasodilation. Similarly in fish, buccal pumping of water across the gills is tightly coupled with cardiac output by

a central neural mechanism (Taylor et al., 2006). The physiological role of respiratory fluctuations in sympathetic activity to the vasculature in mammals is still a subject of some debate; however, it has been suggested to also play a role in the optimal matching of blood delivery to lungs and active muscle groups. This may be particularly important during periods of increased metabolism such as during exercise which produces enhanced respiratory–sympathetic coupling and this may serve to direct blood away from inactive muscle groups towards those whose activity evoked local vasodilation opposes the sympathetic drive (Habler et al., 1994). This role in optimising perfusion is also consistent with the proposal that slow vasomotor oscillations such as Traube–Hering waves can increase vascular conductance and enhance flow to potentially ischemic tissues (reviewed in Nilsson and Aalkjaer, 2003). In addition to the beneficial physiological role outlined above there is evidence to suggest that there may also be a pathological role of respiratory–sympathetic coupling. This was perhaps first noted in the experiments of Harvey Cushing who showed elevated intracranial pressure caused an augmentation of Traube–Hering waves that accompanied the development of severe hypertension (Cushing, 1901). This augmented Traube–Hering wave is reflected in the occurrence of pathological C-waves of intracranial pressure found in patients following traumatic head injury. Evidence is beginning to accumulate that abnormal respiratory–sympathetic coupling may play a pathological role in chronic cardiovascular diseases such as hypertension and heart failure. In sleep apnea (Hoffmann et al., 2004) the respiratory pathology is accompanied by increased levels of SNA and is recognized as a risk factor for hypertension and heart failure (Floras, 2009; Goso et al., 2001). Additionally, heart failure patients with altered respiratory patterns have a higher mortality and poorer prognosis suggesting a role for respiratory–sympathetic interaction in the pathology (Garde et al., 2009). Intriguingly, altered breathing patterns have been demonstrated to induce short-term decreases in blood pressure in patients with essential hypertension (Joseph et al., 2005). The potential role of altered sympatho-respiratory coupling in the generation and pathogenesis of hypertension will form the core topic of this review. 1.2. Central origins of sympathetic vasomotor tone The vasculature receives its sympathetic innervation from a dedicated population of vasomotor sympathetic pre- and postganglionic neurons (Janig, 2006). There is a tonic level of ongoing discharge seen in these sympathetic vasomotor pathways that integrates drives from all levels of the neuraxis. There is some evidence for intrinsic pacemaker-like activity in groups of sympathetic preganglionic neurons (e.g. Logan et al., 1996), however, it is likely that synaptic interactions between neuronal cell groups in the pons, medulla and spinal cord are required to provide basal activity via ongoing glutamatergic input (for review see Guyenet, 2006). Trans-synaptic viral tracing studies indicate that there are a limited number of neuronal cell groups which send monosynaptic projections to the sympathetic preganglionic neurons in the intermediolateral cell column (IML) of the spinal cord (Sly et al., 1999; Strack et al., 1989). These are termed sympathetic premotor neurons and those in the hypothalamic paraventricular nucleus, A5 noradrenergic cell groups of the pons, caudal raphé, rostral ventrolateral medulla (RVLM), rostral ventromedial medulla and white matter of the upper cervical spinal cord provide inputs to multiple levels of the sympathetic neuraxis (Jansen and Loewy, 1997; Strack et al., 1989). This is indicative of widespread influence on sympathetic efferent activity such as might be expected for regulation of vasomotor function. Many studies have demonstrated effects of these neuronal groups on the regulation of sympathetic vasomotor tone however

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the RVLM seems to play a pivotal role. Inactivation or lesion of the RVLM just caudal to the facial motor neurons reduces SNA to spinal levels in anesthetized animals suggesting that this region is a critical source of the ongoing excitatory drive essential for maintenance of sympathetic vasomotor tone (Dampney and Moon, 1980; Guertzenstein and Silver, 1974; Guyenet, 2006; Ross et al., 1984). This region contains two major groups of glutamatergic neurons that project to the IML, one of which expresses all the enzymes required for production of adrenaline and is thus part of the C1 cell group and the other one consists of non-catecholaminergic neurons (Ruggiero et al., 1994; Stornetta et al., 2002). At rest the activity of these neurons is modulated by a GABA-ergic input from the caudal ventrolateral medulla (CVLM) (Cravo et al., 1991; Schreihofer and Guyenet, 2003). Many other nuclei including the nucleus of the solitary tract (NTS), the raphé nuclei, A5, caudal pressor area, parabrachial nucleus the hypothalamic paraventricular nucleus, perifornical lateral hypothalamus, amygdala and cortical regions also project to and influence the activity of RVLM neurons in different situations (reviewed by Guyenet, 2006). The activity of pre-sympathetic RVLM neurons is therefore dependent on the balance of inhibitory and excitatory inputs as well as potentially the intrinsic pacemaker-like properties displayed by these neurons in vitro (Sun et al., 1988). 1.3. Sympathetic over-activity in hypertension The ontogeny of essential hypertension is not fully understood but it is becoming clear that an alteration in SNA is associated with this pathology in both humans and animal models (Guyenet, 2006; Paton et al., 2009). In humans both direct recordings using

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microneurography and measures of noradrenaline spill-over show increased levels of SNA in young hypertensive patients and populations at risk of developing hypertension (Esler, 2000; Grassi, 1998; Julius et al., 1991; Schlaich et al., 2004). In a rat model of essential hypertension, the spontaneously hypertensive (SH) rat, there is a detectable increase in blood pressure at 5 weeks of age with associated increases in noradrenaline release and elevated SNA relative to normotensive control Wistar Kyoto (WKY) rats (Judy and Farrell, 1979; Lundin et al., 1984). Intriguingly, sympathectomy in neonatal SH rats prevents the development of the hypertensive phenotype and the associated vascular and cardiac hypertrophy (Korner et al., 1993; Zicha and Kunes, 1999) raising the possibility that the sympathetic over-activity plays a causal role in the development of hypertension. We have addressed this issue using an in situ perfused preparation of the rat, the working heart brainstem preparation (WHBP, Paton, 1996). We recorded perfusion pressure in SH and WKY rats at three ages—9–16 days, 3 weeks and 5 weeks old (Simms et al., 2009). In these experiments the SH rats had increased vascular resistance compared to age-matched WKY rats at all ages. In the WHBP the flow rate is set and the measured perfusion pressure gives a direct indication of vascular resistance. Thus at equivalent flow rates for each age group the SH rats showed significantly higher perfusion pressures and vascular resistances relative to normotensive animals (p < 0.05, Fig. 1A). Whilst there are no direct observations of this in vivo, our data would suggest that SH rats younger than 5 weeks of age are either already hypertensive, or have decreased cardiac output to maintain a normal arterial pressure in the face of increased total peripheral resistance.

Fig. 1. Perfusion pressure, Traube–Hering waves and thoracic sympathetic nerve activity are amplified in SH rats. Histograms of pooled data from each age group of WKY (open bars) and SH (closed bars) rats showing (A) perfusion pressure (PP), (B) Traube–Hering (TH) waves and (C) respiratory-related sympathetic burst amplitude (peak-trough). (Mean ± SEM, *p < 0.05 compared to WKY (2-way ANOVA), n = 5 per group.) Adapted from Simms et al. (2009).

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1.4. Sympathetic recordings show augmented respiratory coupling in SH rats Our recordings of SNA in the WHBP showed the expected bursting pattern with clear respiratory modulation (Pickering and Paton, 2006). In comparisons of age-matched WKY and SH rats the mean level of thoracic SNA (tSNA) was only elevated in the neonatal SH rats. Notably, however, there was a dramatic enhancement of the respiratory-related modulation of tSNA at all ages compared to WKY rats (Figs. 1C and 2). This was quantified using phrenictriggered averaging of tSNA (as described in Simms et al., 2009). Briefly, the mean phrenic inspiratory burst duration (from onset to the end of the augmenting phase) in each preparation was used as a time base to divide the respiratory cycle. Thus, both the amplitude of the peak to trough and the mean level of tSNA during each respiratory period could be calculated and pooled across preparations (Fig. 2Aii and Bii). Using this approach we showed that at all ages SH rats had significantly larger respiratory-related bursts of tSNA compared to WKY rats particularly during late inspiration (I) or early post-inspiration (PI) (Figs. 1C and 2Aii and Bii). Importantly, we did not find any difference in central inspiratory drive between SH and WKY rats. This augmentation of respiratory–sympathetic coupling in the SH rat remained unchanged even in the absence of input from arterial baroreceptors or peripheral chemoreceptors nor following alterations in central chemoreceptor stimulation. These data strongly suggest a centrally generated change in the interaction

between the central respiratory and sympathetic networks (Simms et al., 2009). In addition to having higher baseline perfusion pressure and increased respiratory-related bursts of tSNA, SH rats had larger Traube–Hering waves (Fig. 1B). Using phrenic cycle triggered averaging we showed that the temporal relationship between phrenic cycle, tSNA and the subsequent change in perfusion pressure was relatively constant, with a latency of 2–3 s between the peak of tSNA and the peak in pressure. Across preparations the average size of Traube–Hering waves was significantly greater in SH rats at all ages compared to age-matched WKY rats (p < 0.05, Fig. 1B). This finding indicates that the augmentation of respiratory modulated SNA seen in SH rats is not the product of an altered recording condition but rather reflects a true amplification of the sympathetic activity reaching the blood vessels. Importantly, subsequent frequency analysis of systolic blood pressure recordings from conscious adult SH and WKY rats, using radiotelemetry, also demonstrate increases in high frequency power (in the frequency range of Traube–Hering waves, Fig. 3) which validates and extends our initial observations in the reduced in situ preparation (Hendy E, Pickering AE, Paton JFR, unpublished observations). 1.5. Can augmentation of Traube–Hering waves cause hypertension? To address this question central respiration was transiently halted for 2–4 min, using a hypocapnic perfusate (2% CO2 ) to induce

Fig. 2. Respiratory-related bursts of thoracic sympathetic nerve activity  are larger in SH rats than WKY rats. Original  traces from 5-week-old WKY (Ai) and SH (Bi) rats showing raw thoracic sympathetic nerve activity (tSNA), integrated tSNA ( tSNA) and integrated phrenic nerve activity ( PNA) over a period of 20 phrenic cycles. Phrenic-triggered averaging of the integrated tSNA signal in the WKY (i) and SH (ii) rats clearly shows the larger inspiratory (I) related burst amplitude in the SH rat. LE, late expiration; I, inspiration; PI, post-inspiration; ME, mid-expiration. Adapted from Simms et al. (2009).

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Fig. 3. Traube–Hering waves are increased in conscious SH rats compared with WKY rats. The high frequency (HF) spectra of systolic blood pressure (SBP) was compared between SH (n = 5) and Wistar (n = 6) rats. The frequency bandwidth used (0.75–3.3 Hz) encompasses the frequency of respiration and thus also the Traube–Hering waves. Note that the reduction in the HF spectra was robust as it occurred in both light and dark phases. Hendy E, Pickering AE, Paton JFR (unpublished observation).

apnea (Fig. 4A and B). On return to normocapnea, with restoration of eupneic phrenic discharge, the increase in perfusion pressure was significantly greater in SH rats than in WKY rats (14.8 ± 1.3 versus 4.5 ± 1.7 mm Hg, n = 6, p < 0.05, Fig. 4C). The rise in perfusion pressure occurred only with the onset of bursting phrenic nerve activity (shown in Fig. 4) and coincided with the reinstatement of respiratory modulation of tSNA and the Traube–Hering waves. The Traube–Hering waves were seen to summate in the SH rats thus driving the increase in perfusion pressure (Fig. 4B and D). Uniquely this experiment indicates that the Traube–Hering wave can drive increase basal arterial pressure through summation, particularly in the SH rat. It also shows that the normal assessment of TH wave amplitude from the ripple on the arterial pressure trace provides an underestimate of their magnitude as it only measures the peak of the iceberg. These data provide evidence that the increased sympathetic burst amplitude drives the production of larger Traube–Hering waves in the SH rats and strongly supports a direct causal link between the augmented respiratory–sympathetic coupling and increased vascular resistance in SH rats.

1.6. Altered phase relationship of sympathetic–respiratory coupling in hypertension Altered respiratory–sympathetic coupling in SH rats was first demonstrated in experiments on anesthetized, vagotomized adult rats where it was noted that the respiratory-related peak of SNA occurred during late inspiration in the SH rat but during PI in agematched WKY rats (Czyzyk-Krzeska and Trzebski, 1990). In the WHBP we observed a very similar shift in timing of the respiratoryrelated SNA bursts that developed with age in the SH rats. In the neonatal period a similar temporal pattern was observed in SH and WKY rats. This pattern was maintained throughout development in the WKY with the peak of sympathetic activity occurring in PI (Fig. 2Aii). By 3 weeks of age the respiratory-related peak occurred in the inspiratory phase in the SH rat and by 5 weeks of age an adult pattern similar to that observed by Czyzyk-Krzeska and Trzebski had developed (Fig. 2Bii). This suggests there is plasticity in the neuronal networks involved in respiratory–sympathetic coupling such that over the first weeks of life in the SH rat there is recruit-

Fig. 4. Restoration of phrenic nerve activity following apnea induces summation of Traube–Hering waves and larger increases in perfusion pressure in SH compared to WKY rats. (A) Grouped data (mean ± SEM) showing the augmented increase in perfusion pressure (PP) with the recovery of eupnoea to WKY rats (*p < 0.05  in SH rats compared  (Student’s t test), WKY n = 6, SHR n = 6). (B) Representative ratemeter record from a 5 week old SH rat showing integrated PNA ( PNA), SNA ( tSNA), and perfusion pressure (PP). A period of apnea, induced with a hypocapnic perfusate (2%CO2 ; grey bar), was followed by restoration of eupnoea with 5%CO2 perfusate. Note the Traube–Hering waves disappear with apnea and recover with eupnoea in both WKY and SH rats. Adapted from Simms et al. (2009).

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Fig. 5. Alterations in sympathetic activity in the late-expiratory period.  (A). Traces, reproduced from Zoccal et al. (2008), showing raw and integrated ( ) abdominal nerve activity (Abd), thoracic sympathetic nerve activity (tSNA) and phrenic nerve activity (PNA) in WHBP from control and chronic intermittent hypoxia (CIH) rats. The grey bars highlight the peak of activity during late expiration (LE) correlated with a peak in tSNA activity in CIH, but not control rats. (B) Example of recordings from the WHBP preparation of 5-week-old WKY and SH rats. The grey bars highlight an example of late-expiratory abdominal activity correlated with an augmented peak in late-expiratory tSNA in the SH which is not present in the WKY. It should be noted that this has been observed in several, but not all SHRs and is not present in all Abd nerves recorded. Simms AE, Paton JFR, Pickering AE, Allen AM (unpublished observations).

ment of inspiratory-related excitatory synaptic drive to produce enhanced coupling. The end result of both altered SNA pattern and increased amplitude of the respiratory-related peak of SNA activity is that vascular tone is increased, reflected in larger Traube–Hering waves which drive arterial pressure higher. This is likely to make a significant contribution to the generation of the hypertensive phenotype. We propose that this heightened central respiratory–sympathetic coupling is pathologically important and may represent a target for therapeutic intervention. Our findings in the young SH rat echo a recent report of changes in SNA seen after chronic intermittent hypoxia which also indicate that there is plasticity in the circuitry generating respiratory–sympathetic outflow (Zoccal et al., 2008). In this report rats were subjected to a 10-day period of chronic intermittent hypoxia, as a model of obstructive sleep apnea induced hypertension. When studied in the WHBP these rats exhibited an enhancement of SNA in the late-expiratory phase of the respiratory cycle which is correlated to enhanced activity to expiratory muscles via the abdominal nerves (Zoccal et al., 2008) (Fig. 5A). In the SH rat (in some, but not all cases) we observed expiratory activation of tSNA and abdominal nerve activity (Fig. 5B). Similarly, when a model of renin-dependent hypertension, the Goldblatt two kidney, one clip model, is studied in the WHBP a dramatic amplification of inspiratory-related tSNA, with emergence of an expiratory-

related peak in tSNA co-incident with the development of hypertension, has also been observed (Sales E, Campos R, Paton JFR, unpublished observation). Although some qualitative distinctions appear in the pattern of SNA modulation in these different rodent models of hypertension with diverse initiating causes, the findings support the hypothesis that respiratory–sympathetic coupling is a centrally generated and potentially plastic phenomenon even in adulthood. In each of these hypertension models there was no evidence of augmentation of inspiratory activity either in terms of amplitude or frequency of phrenic (inspiratory) discharge. However, this measure of phrenic output is only one index of central respiratory drive and may not show changes in the post-inspiratory or stage II expiratory activity in the respiratory network (which are better reflected in recordings of central vagal or abdominal motor nerves respectively). In our recordings of SNA the inspiratory period coincided with a trough in sympathetic nerve activity (as has been noted previously (Habler et al., 1994)) perhaps reflecting an inhibitory or disfacilitatory phase. Certainly the modulated peaks of sympathetic activity seen in these models of hypertension occur at the transition between late inspiratory/post-inspiratory period and in the late-expiratory periods. This suggests that it may be modulation of the coupling between respiratory neurones involved in the generation of post-inspiration and expiration (rather than inspiration) with the sympathetic circuits that mediate the increase in SNA. An aspect worth exploring is how we can define the degree of respiratory modulation of sympathetic activity. There appear to be drives (both excitatory and inhibitory) to the sympathetic network originating in all phases/transitions of the respiratory cycle thus we cannot with certainty identify any section of the averaged, integrated SNA waveform that is not respiratory modulated. Put another way, although the peak to trough cycle amplitude of the phrenic-triggered average SNA clearly reflects respiratory modulation (and generates the TH waves) it does not then follow that the remaining signal is uninfluenced by the level of central respiratory drive. In order to determine the total magnitude of respiratory–sympathetic interaction (and hence exclude an influence of respiratory–sympathetic coupling) a subtraction experiment is needed to “stop” the central respiratory pattern generator cycling to reveal the residual sympathetic activity (e.g. Koshiya and Guyenet, 1996; Simms et al., 2009). This consideration, along with the natural tendency to focus on the inspiratory phrenic drive, may underpin some of the controversy about the dependence of sympathetic augmentation on changes in respiratory drive such as that seen in the acute intermittent hypoxia model (Dick et al., 2007; Xing and Pilowsky, 2010) where there is debate about whether inspiratory facilitation is required for the expression of sympathetic facilitation. Furthermore, as the enhancement of coupling may actually occur downstream of the respiratory network, this would not require any change in the activity within the respiratory pattern generator. In order to explore these ideas further, it is necessary to consider the organisation of the respiratory pattern-generating network and identify the potential loci for respiratory–sympathetic interaction. Moreover, we submit that coupling and altered coupling may not be uniform across all sympathetic nerves which can be controlled differentially (Morrison, 2001; Polson et al., 2007; Yoshimoto et al., 2010). 1.7. Central respiratory pattern-generating network The major sources of respiratory drive are located in three areas in the brainstem: the ventral respiratory column (VRC), the dorsal respiratory column (DRC) and the pontine nuclei. Respiratory activity is thought to be a result of reciprocal inhibitory connections between inspiratory and expiratory brain stem neurons which results in phasic drive to inspiratory and expiratory motor neu-

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rons in the spinal cord (Richter, 1982; Smith et al., 2007; Smith et al., 2009). The classification of respiratory neurons is in accordance with their firing pattern, either decrementing or augmenting and relative to the respiratory cycle (Smith et al., 2009). Thus there are pre-inspiratory (pre-I), early inspiratory (early-I), augmenting inspiratory (aug-I), post-inspiratory (post-I) and augmenting and decrementing expiratory (E) cell types (Bianchi et al., 1995). Animal studies have shown that the VRC plays a central role in the generation of the respiratory pattern and can be divided into four distinct regions with differing functions as follows (Bianchi et al., 1995; Ezure, 1990; Ezure et al., 2003a,b; Jiang and Lipski, 1990; Smith et al., 1991; Smith et al., 2007, 2009): (i) The Bötzinger complex is considered to be the major source of expiratory activity and contains both post-I and E-augmenting and E-decrementing neurons; (ii) The pre-Bötzinger complex is the major source of inspiratory activity and contains both pre-I and early-I interneurons; (iii) The rostral VRC contains premotor I neurons which project to the spinal cord; and (iv) The retroambigual part of the caudal VRC contains E neurons which send excitatory projections to the spinal motor neurons. The respiratory pontine nuclei include the Kölliker-Fuse nucleus and the medial and lateral parabrachial nuclei. These areas also play an important role in the generation of respiratory pattern since they produce both tonic and phasic drive onto the VRC (Dutschmann et al., 2008; Smith et al., 2007). In addition to the VRC and pontine nuclei, the DRC which consists of the intermediate and commissural NTS contains both inspiratory and expiratory neurons (Bianchi et al., 1995; Subramanian et al., 2007) and plays an important modulatory role on respiratory activity at pontine and medullary levels (Bianchi et al., 1995; Bonham and McCrimmon, 1990). A fourth group of neurons named the retrotrapezoid nucleus (RTN), located below the facial nucleus, has been implicated in central chemoreception and can modulate the activity of VRC neurons in response to changes in CO2 /H+ in the blood (Guyenet et al., 2008). Furthermore in vivo and in vitro studies suggest that neurons of the RTN may exhibit a late-expiratory pattern of activity (Abdala et al., 2009; Janczewski and Feldman, 2006; Onimaru et al., 2006). Emerging evidence suggests that two major rhythm generators are present in the medulla with the pre-Bötzinger complex generating inspiration and, under conditions of increased respiratory drive, the RTN generating active expiration (Janczewski and Feldman, 2006; Smith et al., 2009). The interaction between, and modulation of, these rhythmogenic brainstem regions enable distinct respiratory rhythms to be produced under differing physiological conditions (for a review see Smith et al., 2009). Clearly there are multiple potential specific neural outputs from this respiratory network that could deliver phasic excitatory or inhibitory drives to the sympathetic vasomotor network either through local connections in the brainstem or via long-range projections direct to the spinal cord. 1.8. Origins of respiratory–sympathetic coupling Coupling of respiratory and sympathetic outflows, observed as respiratory-linked oscillations in SNA, has been observed in all species studied (see Habler et al., 1994 for review). The evidence indicates a significant component of this is derived from central interactions between the respiratory central pattern generator and nuclei responsible for generating SNA (see Richter and Spyer, 1990). A more recent generalised schema for this respiratory - autonomic coupling has been presented by Janig (Fig. 10.17, Janig, 2006) with partly intersecting networks of ponto-medullary neurons involved in the generation of respiratory and cardiovas-

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cular rhythms with specific outputs from each of these networks to either respiratory or autonomic premotor/motor neurones that generate the phasic drives and patterns of activity. However, the specific interactions between these components that gives rise to respiratory–sympathetic coupling has been difficult to establish. We summarise below some of the possible mechanisms and sites at which this coupling may be manifest. Electrophysiological evidence shows that respiratory modulation of spike activity occurs in “pre-sympathetic” neurons in CVLM, RVLM and in the sympathetic preganglionic neurons (SPN). Identification of the “pre-sympathetic” neurons is predominantly based upon the response to stimulation of the baroreceptor reflex (e.g. inhibiting RVLM neurons and exciting CVLM neurons), neuronal phenotype, axonal projections and anatomical location. It is not yet clear whether any modulation observed is the result of independent, direct inputs from the central respiratory network to spinal preganglionic neurons and/or transmission of input from antecedent cardiovascular neurons e.g. RVLM to sympathetic preganglionic neurons. The barosensitive GABA-ergic neurons of CVLM exhibit a wide range of patterns of respiratory modulation but the commonest pattern is one of an I peak ± post-I depression (Mandel and Schreihofer, 2006) a pattern of activity that is consistent with the pattern of SNA seen in our studies. Individual RVLM neurons also exhibit different patterns of activity entrained with the respiratory cycle such as decreased activity during I, peak of activity during I or post-I (Haselton and Guyenet, 1989; McAllen, 1987; McAllen et al., 2001; Miyawaki et al., 1995). This evidence of respiratory modulated firing supports the hypothesis that central respiratory–sympathetic coupling occurs at the level of the medulla oblongata via the RVLM and CVLM. However, the source of this input from within the respiratory network is not yet determined. Pilowsky and colleagues found that tonic GABA-ergic inputs to RVLM neurons inhibit excitatory inputs from central respiratory neurons (particularly post-I neurons) which normally activate the RVLM pre-sympathetic neurons (Miyawaki et al., 2002). Recent evidence suggests a role for the pontine respiratory neurons during eupneic breathing since transection of the pons to remove the Kölliker-Fuse, parabrachial nucleus and A5 eliminated the respiratory oscillations on SNA (Baekey et al., 2008). In line with the gaps in our knowledge related to the neuronal cell groups involved in respiratory–sympathetic coupling, our understanding of the role of different neurotransmitters or neuromodulators in this coupling remain rudimentary. Recent data support an involvement of the neuromodulator angiotensin II. Our observations in the renin-dependent Goldblatt model of hypertension show early augmentation of respiratory modulation of tSNA (Oliveira Sales E, Campos R, Paton JFR, unpublished data). Further support is derived from experiments in anesthetized rats exposed to a high salt (2%) diet and infusion of angiotensin II for 10–14 days (Toney et al., 2010). These rats have significant increases in blood pressure and splanchnic SNA. Inhibition of angiotensin AT1 receptors in the RVLM significantly decreased the inspiratory-related augmentation of SNA without affecting the strength of phrenic nerve activity. The study also showed that a population of barosensitive, bulbospinal neurons in RVLM which did not display obvious cardiac rhythmicity, had a marked elevation in their discharge rate compared to control rats. These neurons had a clear respiratory rhythmicity with post-I discharge. Together the data suggest a modulatory role for angiotensin II in the RVLM which leads to altered coupling between the respiratory and sympathetic generator circuits. These findings in the brainstem also do not preclude a possible spinal mechanism with changes in either the synaptic strength of inputs reaching the sympathetic preganglionic neurons or alterations in their excitability. There have been reports of alterations in

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the morphology and neurochemical phenotype of sympathetic preganglionic neurons in the SH rat (Powers-Martin et al., 2006). We have begun to explore this possibility with whole cell patch clamp recordings from sympathetic preganglionic neurons in the working heart brainstem preparation of neonatal SH and Wistar rats. These recordings have indicated that the muscle vasoconstrictor class of sympathetic preganglionic neuron have a higher ongoing firing frequency in the SH rat with an augmented respiratory modulation consistent with our findings from whole nerve recordings. Furthermore, there are differences in the intrinsic excitability of these neurons that may in part account for the augmented respiratory–sympathetic coupling and the alteration in burst timing seen in this strain (Paton et al., 2010 and Stalbovskiy AO, Paton JFR, Pickering AE, unpublished observations). 2. Conclusion Our results establish that SNA is elevated in the SH rat very early in development and that this elevation is driven by enhanced coupling with the central respiratory generator. This produces larger Traube–Hering waves that we have shown are capable of summating to produce elevated arterial pressure in the SH rat. The mechanism(s) driving this enhanced coupling remain uncertain but it may be a manifestation of the “Cushing mechanism” with an “at risk” brainstem attempting to maintain its perfusion by driving up arterial pressure (Paton et al., 2009). We review the published work by others demonstrating respiratory modulation of the activity of “cardiovascular” neurons in the CVLM, RVLM and IML and also a reliance upon the pons for manifestation of sympathetic–respiratory coupling. Altered coupling between the central respiratory generator and neurons at any of these sites could lead to increased SNA to trigger the development of hypertension. We predict that altered respiratory–sympathetic coupling occurs in a population of humans who will go on to develop essential hypertension, and possibly other cardiovascular diseases, vessel hypertrophy and consequent deleterious effects in the vasculature. Intriguingly, altered breathing patterns have been demonstrated to induce short-term decreases in blood pressure in patients with essential hypertension (Joseph et al., 2005). We suggest that approaches to reverse pathological respiratory–sympathetic coupling may be potential therapeutic strategies for the treatment of cardiovascular disease and induction of chronic changes in blood pressure. Acknowledgements Original work from the author’s laboratories that is discussed in this manuscript was supported by grant funds from the Australian National Health and Medical Research Council (ID #454432 and #628838) and the British Heart Foundation. JFRP is the recipient of a Royal Society Wolfson Research Merit award. AEP is a Wellcome Trust Senior Clinical Fellow. References Abdala, A.P., Rybak, I.A., Smith, J.C., Paton, J.F., 2009. Abdominal expiratory activity in the rat brainstem-spinal cord in situ: patterns, origins and implications for respiratory rhythm generation. J. Physiol. 587, 3539–3559. Adrian, E., Bronk, D., Philips, G., 1932. Discharges in mammalian sympathetic nerves. J. Physiol. 74, 115–133. Badra, L.J., Cooke, W.H., Hoag, J.B., Crossman, A.A., Kuusela, T.A., Tahvanainen, K.U., Eckberg, D.L., 2001. Respiratory modulation of human autonomic rhythms. Am. J. Physiol. Heart Circ. Physiol. 280, H2674–H2688. Baekey, D.M., Dick, T.E., Paton, J.F., 2008. Pontomedullary transection attenuates central respiratory modulation of sympathetic discharge, heart rate and the baroreceptor reflex in the in situ rat preparation. Exp. Physiol. 93, 803–816. Barman, S.M., Gebber, G.L., 1980. Sympathetic nerve rhythm of brain stem origin. Am. J. Physiol. 239, R42–R47.

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