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The Paton Lecture |
1 Division of Neuroscience, The Medical School, University of Birmingham, Birmingham B15 2TT, UK
Abstract
In this Paton Lecture I have tried to trace the key experiments that have developed ideas on how the brain regulates the cardiovascular system. It is a personal view and inevitably, owing to constraints on space and time, I have not been able to cover areas such as the nucleus tractus solitarius and cardiac vagal neurones, although I acknowledge that some may consider the story is incomplete without them. Starting with the crucial discovery of vasomotor nerves and ‘vasomotor tone’, the patterns of activity in sympathetic nerves which led to the important idea of central oscillating networks of neurones are described. I discuss how this knowledge has informed current controversies on the origin of vasomotor activity in presympathetic neurones in the ventral medulla, which identify intrinsic pacemaker activity or synaptic input from multiple oscillators as prime mechanisms. I present an emerging view that the role of other regions of the brain, in particular supramedullary sites, has been underplayed. These regions are pivotal for the non-uniform distribution of cardiac output that is unique to each reflex and behavioural state. I discuss the most recent evidence for ‘central command’ neurones that offers a plausible explanation for how these patterns of sympathetic activity are achieved. Finally, I stress the importance of these current ideas to the understanding of pathological changes in sympathetic activity in cardiovascular diseases such as hypertension or congestive heart failure.
(Received 18 August 2006;
accepted after revision 3 October 2006; first published online 9 October 2005)
Corresponding author J. H. Coote: Division of Neuroscience, The Medical School, University of Birmingham, Birmingham B15 2TT, UK. Email: j.h.coote{at}bham.ac.uk
The discovery of vasomotor nerves and vasomotor tone
Following Harvey's seminal treatize De Mortis Cordis (Harvey, 1628) describing the motion of the heart and circulation, the discovery that this was regulated by nerves probably ranks as the next major landmark. In particular, the realization that blood vessels had ‘nervous tone’ was the important feature because it revealed a key evolutionary advance made by vertebrates. Tonic activity in sympathetic nerves enables animals to vary the delivery of blood to different organs on demand whilst optimizing cardiac output. Put another way, it allows stealing of blood from an organ where it is less needed, to supply another organ where it is more needed, so overcoming the limits of total vascular capacity and cardiac output that are imposed by body size.
Henle, in 1840 (discussed by Montastruc et al. 1996), was amongst the first to suggest that nerve fibres controlled the muscle fibres in the wall of blood vessels, and it was Stilling 1840 (cited by Ackerknecht, 1974) who coined the term ‘vasomotor system’ for this innervation. The especially important discovery that these nerves are ‘tonically’ active came from some quite simple experiments. Claude Bernard (1851) in Paris and, independently, Brown-Sequard (1852) in London, measured the temperature change in the rabbit ear whilst also observing the diameter of ear blood vessels, following section of the sympathetic nerve in the neck. This resulted in an increase in ear temperature and ‘reddening’ of the ear. Waller (1854), a Professor of Physiology in Birmingham, UK, went on to show that these changes could be reversed by electrical stimulation of the peripheral cut end of the sympathetic nerve. Thus, the intact sympathetic nerve was clearly responsible for a maintained vasoconstrictor tone. Later, Goltz (1864) observed, in the frog, that destroying the spinal cord resulted in dilatation of mesenteric blood vessels, thus showing that the ‘vasomotor tone’ was dependent on an intact CNS. Some 70 years later, following the development of the oscillograph, Adrian et al. (1932) in Cambridge recorded from a variety of sympathetic vasomotor nerves in rabbits and cats and showed that they all displayed rhythmic voltage changes reflecting the grouping of action potentials in many postganglionic neurones. This activity disappears following pharmacological blockade of ganglia or removal of preganglionic input by section of white rami (Ninomiya et al. 1993); therefore, it originates in the CNS as originally indicated by Goltz (1864).
Rhythmic activity in vasomotor nerves
Most commonly, the discharge of pre- or postganglionic sympathetic nerves is synchronized into bursts locked into a 1:1 relation to the cardiac cycle (2–6 Hz in the cat and dog or up to 8 Hz in the rat) with the magnitude of the bursts waxing and waning with the period of the respiratory cycle (1–2 Hz; McCall & Gebber, 1975; Camerer et al. 1977; Coote, 1988; Janig, 1988; Zhang & Johns, 1996). This synchronization seemed to disappear following section of arterial baroreceptor afferents or removal of respiratory drive. As a consequence, the traditional view was that a population of neurones in the brain randomly generated discharges which were synchronized by powerful inputs from cardiovascular and respiratory sources. The first important departure from this view was made by Green & Heffron (1967) and later by Cohen & Gootman (1970), who subjected the activity of vasomotor nerves in cats to a detailed mathematical analysis. This revealed a strong 10 Hz component in addition to the lower frequency oscillations. These and later studies (Barman & Gebber, 1980; Gebber & Barman, 1985) suggested to some authors that vasomotor activity was more likely to be dependent on central neural networks capable of rhythm generation. An interesting and important debate then arose, and is still on-going, as to whether ‘spontaneous’ activity in vasomotor nerves arises from the intrinsic activity of pacemaker cells or is dependent on dedicated groups of interconnected neurones driven by afferent inputs.
Sympathetic network oscillators
The most persuasive evidence that vasomotor tone is generated by assemblies of interconnected neurones has been provided by studies in Gerry Gebber's laboratory (Gebber, 1980; Gebber & Barman, 1989; Gebber et al. 1994a,b). Using the cat as an experimental model, it was shown that the 2–6 Hz oscillations could be uncoupled from the cardiac cycle but still persisted after removal of arterial baroreceptors. Similar data has been provided for the rat (Barman & Gebber, 1989) and for the dog (Camerer et al. 1977). Subsequently, using time series and spectral analysis of the relationship between discharges in two simultaneously recorded sympathetic nerves, they found that although the activity showed a significant correlation or coherence, there was a shift in the phase angle of the periodic component of the discharges in postganglionic nerves that exit from different ganglia that was not dependent on delays in the path lengths (Barman et al. 1992; Gebber et al. 1994a,b). This suggests that the driving inputs to the different sympathetic nerves arise from seperate pools of central neurones (Gebber, 1980; Barman, 1990). The concept has gained further support from studies in Michael Gilbey's laboratory using a novel technique of micropipette recording from single postganglionic nerves supplying the vasculature of the rat tail (a thermoregulatory organ). It was shown that these neurones exhibit a characteristic rhythm of discharge of 0.4–1.2 Hz which they termed the T-rhythm (Johnson & Gilbey, 1996), a rhythm which they considered to be characteristic of thermoregulatory cutaneous sympathetic nerves of the rat. Thus, sympathetic vasomotor nerves supplying different vascular beds display different basic rhythms, supporting the concept of multiple oscillators (Gebber et al. 1994a; Gilbey, 2001). Further support for the idea of oscillating networks was provided by evidence showing that the fundamental rhythm can be entrained by afferent input from arterial baroreceptors, central respiratory drive and lung inflation, provided that the frequency of the input is close to the uncoupled frequency of the discharge (Gebber et al. 1994a; Zhang et al. 1997; Johnson & Gilbey, 1998; Chong et al. 1999; Staras et al. 2001). This is unlikely to be a characteristic of pacemaker neurones, since they would reset to the same rhythm after each stimulus. In a key experimental analysis of entrainment, Gilbey and colleagues realized that if the pattern of sympathetic discharge is dependent on an oscillator, then it should be possible to force the oscillator to fire at the frequency of a rhythmic external input. Given this, a stable entrainment pattern would only occur over a narrow frequency range and be proportional to the strength of the input. This was beautifully shown (Staras et al. 2001) for the T-rhythm oscillator using somatic afferent nerve stimulation in the rat. This might explain entrainment of the 2–6 Hz rhythm observed in the early recordings of somatosympathetic reflexes in cardiac and renal nerves of the cat (Coote & Downman, 1966; Coote & Perez-Gonzalez, 1970).
Although different oscillations in sympathetic nerve activity most probably reflect the characteristic electrophysiology and organization of specific neural networks, they are also important for the function of the vasomotor system. The linking of synchronized oscillations provides a signal that adjusts the strength of central and ganglionic synapses and so enhances transmission. In addition, we know that throughout the vascular beds there is an optimum pattern and frequency of postganglionic nerve activity for effective activation (Burgess et al. 1999; Ringwood & Malpas, 2001; Grisk & Stauss, 2002). Therefore, I think we should regard these data, which are best explained by multiple oscillating networks of neurones, as an important landmark in our understanding of sympathetic vasomotor control.
Origin of vasomotor tone
Spinal cord. It was natural for many studies to address the question concerning the site of origin of the rhythmic activity in vasomotor nerves. As implied earlier, we can probably rule out sympathetic ganglia, although it is clear that integration of preganglionic input does occur here and that there may be some modification of postganglionic neurone excitability via visceral afferents (Morales et al. 2004). Another possible site is the pools of sympathetic preganglionic neurones (SPN) in the spinal cord. Intracellular recording from these neurones in vivo has proved difficult, but the most detailed study to date, carried out in the cat, showed that the action potentials of all tonically active SPN in the third thoracic segment were preceeded by EPSPs (Dembowsky et al. 1985), confirming earlier more limited studies (Coote & Westbury, 1979; McLachlan & Hirst, 1980). More recent studies in my laboratory suggest that the situation is the same for rat SPN (Lewis & Coote, 1994; Lewis DI & Coote JH unpublished observations). Also, there are numerous intracellular studies of SPN recorded in slices of spinal cord in vitro (Coote, 1988; Brailoiu & Dun, 2004) that in general confirm the importance of synaptic input for the initiation of firing. Such observations are not surprising, since it has long been recognized that rhythmic activity in vasomotor nerves disappears when the spinal cord is isolated from the brainstem, for example by local cooling of the medulla, and is replaced by irregular and unsynchronized activity. It reappears on rewarming (Coote & Downman, 1966). It is also of note that in spinal animals, the highly synchronized response to somatic afferent stimulation becomes fractionated, decaying in strength at spinal segments remote from the afferent input (Coote & Downman, 1966; Coote et al. 1969). The importance of supraspinal afferents is well illustrated by the observation that rhythmic firing of single SPN can be induced by locally applied catecholamines or by 5-HT in anaesthetized rats (Coote & Lewis, 1995; Lewis & Coote, 1996). Similarly, in spinalized rats, application of 5-HT to the L1 segment of the spinal cord was shown to induce a T-rhythm in the sympathetic nerve supplying the rat tail vessels (Marina et al. 2006). Thus, synchronicity and rhythmicity of firing in pools of vasomotor neurones in the spinal cord appear mainly to depend on an input from supraspinal neurones (Shen et al. 1994; Marina et al. 2006; Fig. 1).
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Medulla oblongata. The importance of the medulla for the regulation of vasomotor tone has long been recognized since the studies of Dittmar (1870, 1873) and Owsjannikow (1871) carried out in Carl Ludwig's laboratory in Germany. They found, by way of transection, that the patency of a region in the ventral medulla was essential for the maintenance of arterial blood pressure. Despite numerous studies over the intervening years, the exact location of the neurones that were responsible was not identified until Pedro Guertzenstein & Ann Silver (1974), working in Cambridge, used a novel approach by applying the inhibitory amino acid glycine to the exposed ventral surface of the medulla of cats. Glycine placed on a small area, bilaterally, between the caudal edge of the trapezoid body and the exit of the hypoglossal nerve caused a profound fall in blood pressure. Subsequent neuroanatomical retrograde tracing studies revealed that within the medulla close to this region there were reticulospinal neurones projecting to the sympathetic columns in the spinal cord (Amendt et al. 1978; Dampney et al. 1982; Caverson et al. 1983; Reis et al. 1984; Ross et al. (1984) which made direct monosynaptic connections with SPN (Zagon & Smith, 1993). This region, first identified in the rat by Reis and colleagues, became known as the rostral ventrolateral medulla (RVLM; Ross et al. 1981). Activation of these neurones by miroinjection of an excitatory amino acid increased blood pressure (Dampney et al. 1982; Caverson et al. 1983) and sympathetic vasomotor activity (Morrison et al. 1988) and monosynaptically excited SPN (Deuchars et al. 1995). These data appear to support the old concept of a ‘medullary vasomotor centre’ much heralded by textbooks of physiology. Are these RVLM–spinal vasomotor neurones indeed independent generators of vasomotor tone?
An attempt to answer this question was led by a series of studies in Patrice Guyenet's laboratory in the USA. Extracellular recordings from neurones in the RVLM of the rat that were identified antidromically as projecting to the spinal cord showed that they were tonically active with firing rates in the range of 5–40 spikes s–1. This activity had cardiac cycle-related rhythmicity, and it was markedly decreased or increased by arterial baroreceptor activation or deactivation, respectively, as well as showing respiratory cycle modulation (Brown & Guyenet, 1985; McAllen, 1987; Haselton & Guyenet, 1989). A parallel discovery that sympatho-inhibitory effects of arterial baroreceptors was prevented by blocking GABA receptors with application of bicuculline, either by microinjection into RVLM (Willette et al. 1984) or directly in the vicinity of single RVLM neurones (Sun & Guyenet, 1985), or by placing bicuculline on the ventral surface of the brain in the region of RVLM (Yamada et al. 1984), reinforced the idea that the mechanism responsible for vasomotor tone generation lay within this group of spinally projecting neurones.
As pointed out by Guyenet & Stornetta (2004), however, there are two plausible hypotheses that could explain the tonic activity of RVLM neurones. Either it results from synaptic driving by neurones located elsewhere in the brain, or it really is generated in the RVLM by intrinsic pacemaker activity in the RVLM neurones.
Pacemaker presympathetic neurones
Studies in Guyenet's laboratory (Sun & Guyenet, 1986, 1987; Koshiya et al. 1993) appeared to indicate that synaptic drive to RVLM vasomotor neurones was strongly dependent on activation of glutamate receptors. Block of these receptors, however, did not significantly reduce the tonic discharge of these neurones. Therefore, this favoured the idea that tonic activity was being generated intrinsically by pacemaker neurones. Indeed, RVLM neurones with both regular and irregular discharge, which persists after removing synaptic input by blockade of excitatory or inhibitory amino acid receptors or during perfusion with low-Ca2+ high-Mg2+ solution, have been recorded intracellularly in brain slices. Also, the action potentials in such neurones were not preceded or triggered by EPSPs, and the regular discharge was reset by an experimentally introduced stimulus and was abolished by membrane hyperpolarization with no residual EPSPs. The cells also displayed other membrane properties that could contribute to intrinsic oscillating depolarization (Sun et al. 1988a,b; Lewis & Coote, 1993; Li et al. 1995; Kangrga & Loewy, 1995).
However, such ‘pacemaker-like’ neurones have only been recorded in vitro in isolated medulla and spinal cord preparations removed from immature rats and maintained at 30–34°C. They are not observed in the anaesthetized adult rat, in which intracellular recordings in vivo of identified RVLM–spinal vasomotor neurones reveal irregular low-frequency action potentials preceded by EPSPs (Lipski et al. 1996). Significantly, the discharge characteristics of these RVLM neurones recorded in vivo are in accord with the synaptic drive observed in sympathetic preganglionic neurones, which display irregular EPSPs and a low-frequency (< 2 Hz) discharge (Coote & Westbury, 1979; McLachlan & Hirst, 1980; Dembowsky et al. 1985).
The pacemaker hypothesis is also lacking as an explanation because it fails to account for the basic rhythmic oscillations of activity in RVLM–spinal neurones, which are correlated with similar activity in postganglionic sympathetic nerves (Barman & Gebber, 1980; Gebber, 1980), nor does it easily explain the non-uniformity of sympathetic activity in various vasomotor nerves (Ninomiya & Irisawa, 1975; Meckler & Weaver, 1985; Janig, 1988; Michaelis et al. 1993; Habler et al. 1994a).
In the unchallenged adult animal, therefore, synaptic drive is the most likely source of tonic activity in RVLM neurones. However, before entirely dimissing ‘pacemaker neurones’ as a phenomenon associated with reduced and immature preparations, a word of caution is needed. It is wise to entertain the possibility that conditions favouring the activation of selective inward currents, which have been shown to underlie beating activity in RVLM neurones recorded in slices of medulla in vitro where much of the synaptic input has been removed, could be induced by specific slow synaptic inputs (angiotensin, vasopressin) in response to challenging environmental stimuli.
Brainstem network oscillators
The major evidence that RVLM–spinal activity is dependent on synaptic drive has come from the landmark studies of rhythm generation by Gebber and colleagues. By focusing on the basic rhythms of vasomotor activity, to which Koepchen (1962, 1980) had previously drawn attention, a very plausible explanation of tone generation has arisen. According to Gebber and Barman, a significant source of the drive to RVLM is from specific groups of neurones lying in the adjacent reticular formation in the lateral tegmental field (LTF), dorsal to RVLM neurones (Orer et al. 1999; Barman et al. 2000). In the anaesthetized cat, these LTF neurones appear to be synaptically linked to RVLM neurones and display a 2–6 Hz rhythm, which is entrained by baroreceptor input and is significantly correlated with the oscillation of activity in postganglionic vasomotor nerves (Gebber & Barman, 1985; Orer et al. 1999). Importantly, cross-correllograms showed that LTF neurones fired significantly earlier than RVLM–spinal neurones and both discharged before the slow wave of postganglionic nerve activity (Barman & Gebber, 1987; Orer et al. 1999). In confirmation, similar delays for evoked responses were observed following stimulation of LTF neurones, supporting the conclusion that the sequence of events commenced in LTF neurones and travelled via RVLM–spinal neurones to activate the pre- and postganglionic neurones (Barman & Gebber, 1987). A final weighty piece of evidence was that blockade of synaptic drive from LTF neurones by microinjection of an excitatory amino acid antagonist, localized to the LTF, caused a profound reduction in postganglionic vasomotor activity (Barman et al. 2000). It was also shown that the 10 Hz rhythm arises from a distinct group of pontomedullary neurones separate from the LTF (Zhong et al. 1992; Orer et al. 1996).
Therefore, there is strong evidence in the cat that synaptic drive to RVLM-spinal neurones rather than ‘pacemaker neurones’ plays a significant role in generating the oscillating rhythms associated with vasomotor tone (Fig. 1). There is some concern that organization in the rat is different. However, apart from a 10 Hz rhythm, the other oscillations have been demonstrated in the rat (Barman & Gebber, 1989; Persson et al. 1992; Zhang et al. 1997) even after baroreceptor denervation. Also, it is well recognized that there are many neurones in the LTF close to RVLM in the rat that display a cardiac-related rhythm and are strongly baroreceptor sensitive and that do not project to the spinal cord (e.g. Bertram & Coote, 2001), so these may be equivalent to cat LTF neurones although this still needs to be demonstrated.
The foregoing arguments have not addressed the question of whether vasomotor tone is entirely dependent on RVLM–spinal neurones. Most studies have been performed in anaesthetized or decerebrate animals, in which a contribution from the many supramedullary–spinal projections is likely to be depressed or absent. We are now aware from studies of homeostasis or behavioural states that groups of hypothalamic neurones can directly influence sympathetic vasomotor neurones in the spinal cord (Coote, 2004). These are discussed in the following section.
Central and reflex motor patterns
As referred to earlier, perhaps the most important value of vasomotor tone is that it provides the capability to respond optimally to environmental challenges by enabling the fine tuning of the distribution of cardiac output according to where it is most needed. A feature of paramount significance to the fine tuning of cardiac output is that its distribution needs to be precisely co-ordinated with ventilation. The interaction between the two control systems is clearly expressed by the respiratory-dependent modulation of sympathetic vasomotor activity. There is still much to learn about this co-ordination, and there are some puzzling features. The extent of respiratory modulation of vasomotor activity varies between sympathetic nerves and between species. In the cat and probably also in humans, it is stronger in muscle vasoconstrictors than cutaneous vasoconstrictors, but it is similar for both outflows in the rat (Habler et al. 1994a,b). There are also differences in the modulation of vasoconstrictor outflows to different organs during the phases of respiration. It is still unclear where the influence of central respiratory drive is injected into the rhythmic pattern of vasomotor discharge. However, the synaptic interaction between central respiratory drive and vasomotor neurones probably takes place mainly at the level of the medulla (Richter & Spyer, 1990), but in view of the different characteristics described for vasomotor nerves supplying different vascular beds, the nature of the coupling must vary with the function of the vasomotor neurone. It is unclear whether coupling between central respiratory drive neurones and vasomotor neurones also occurs at the level of the spinal cord. Further discussion of this highly important topic is beyond the scope of this article, and the interested reader should refer to Richter & Spyer (1990), Habler et al. (1994b) and Janig (1996).
The fine adjustments of the distribution of cardiac output are further exemplified by the various combinations of activated and inhibited discharge in different sympathetic nerves that are each unique to a specific state-dependent behaviour like arousal or sleep, or to responses like diving, exercise or sex or to homeostatic responses like thermoregulation, energy balance and hydration. This is also clearly shown in Janig's landmark studies (see reviews Janig, 1988, 1996) of the characteristic discharge and reflex changes elicited in muscle sympathetic vasomotor nerves compared with those evoked in cutaneous sympathetic vasomotor nerves, by stimulation of specific types of skin receptors (Habler et al. 1994a). The reflex responses are characteristically different for each sympathetic pathway, although interestingly the difference is lost when connections between the brain and spinal cord are severed (Janig, 1996).
Insights into the means by which the central nervous system organizes patterns of sympathetic nerve activity were first gained from studies of arousal or the alerting response. The seminal studies of Hess & Brugger (1943) suggested that structures in the hypothalamus were mainly responsible for integration of the somatic and visceral components of the alerting response. This led Hilton and colleagues to conduct detailed mapping of sites in the brain at which electrical stimulation elicited the same pattern of alerting. They demonstrated that the alerting response was accompanied by increased skeletal muscle blood flow and cardiac output, with vasoconstriction in many other vascular beds, and could be elicited from specific sites in amygdala, hypothalamus or midbrain in anaesthetized and conscious animals (Abrahams et al. 1960, 1964; Hilton & Zbrozyna, 1963). The details were confirmed in a number of species (Smith et al. 1980; Tan & Dampney, 1983; Yardley & Hilton, 1986). This led Hilton to suggest that control of cardiovascular function was not by some hypothetical vasomotor centre but was by neurones dispersed along the neuraxis (Hilton, 1982), a view that subsequent evidence has modified considerably. In my opinion, however, these studies were an important landmark because they revealed the capability of specific suprabulbar sites to elicit a complete pattern of autonomic and somatic activity, as well as the associated behaviour. The efferent pathway of the cardiovascular components of the alerting response depends on a synaptic relay in the RVLM (Schramm & Bignall, 1971; Hilton et al. 1983; Lovick, 1985; Dean & Coote, 1986), suggesting that there is a hierarchical organization of CNS regions controlling vasomotor activity. This apparent dependence on RVLM for vasomotor responses was further emphasized by studies showing that sympathetic reflexes such as those elicited by baroreceptors, peripheral chemoreceptors or somatic afferents were prevented by interfering with synaptic transmission in the RVLM. This seemed to favour the idea that all cardiovascular responses are entirely dependent on synaptic relays in the RVLM. Such an interpretation needs to be viewed with some caution because, in anaesthetized animals, afferent transmission to suprabulbar regions of the brain may be blocked (Abrahams et al. 1962). Furthermore, there is good evidence that cardiovascular reflexes are significantly altered by removal or pharmacological block of supramedullary regions (Korner, 1971; Olivan et al. 2001; Reddy et al. 2005). It has also become clear in the last 30 years that there are several groups of neurones at cardiovascular sites in the hypothalamus that project to spinal sympathetic vasomotor neurones after bypassing the RVLM (Coote, 2004).
Neuroanatomical basis for non-uniform vasomotor responses
The ability of the brain to selectively influence different vascular beds indicates that vasomotor neurones are organotopically characterized. This was first indicated by Langley (1897), who showed that the contribution to control of different visceral structures, in the head of cats, supplied by sympathetic preganglionic nerves exiting from different spinal segments and that relay in the superior cervical ganglion, remained largely unchanged following cervical nerve regeneration after complete section. This suggests a functional specificity of neuronal location. Modern neuroanatomical retrograde tracing methods using fluorescent labels or horseradish peroxidase have confirmed this. Such studies have clearly shown that target organ-specified preganglionic neurones are located in specific spinal cord segments (Strack et al. 1988; Pyner & Coote, 1994a) and are confined to discrete longitudinally arranged columns within the lateral horn of the thoracic–lumbar cord (Janig & McLachlan, 1986; Pyner & Coote, 1994b).
A viscerotopic organization is also apparent amongst the presympathetic neurones of the RVLM in the cat, although it may not be as discrete as that of preganglionic neurones in the spinal cord. Microinjections of small volumes of excitatory amino acids have been shown to selectively affect different vascular beds or activity in different sympathetic nerves, depending on the location of the injection within the RVLM (Lovick, 1987; Dampney et al. 1987; Dampney & McAllen, 1988; Ootsuka & Terui, 1997; McAllen et al. 1995). These studies have so far not been replicated in the rat. However, the arrangement is further supported by neuroanatomical studies in the rat showing that terminals of RVLM neurones, anterogradely labelled by discrete injections of biotin dextran amine, are confined to specific groups of prelabelled target organ-specified preganglionic neurones in the thoracic spinal cord (Pyner & Coote, 1998). Such an organotypic arrangement of the RVLM spinal projection is supported by functional studies in cats using power spectral analysis of nerve activity. These studies reveal significant differences in the dominant oscillations in simultaneously recorded pairs of vasomotor nerves (Gebber et al. 1994a,b), suggesting that different network oscillators influence separate populations of neurones. A discrete organization of target organ-specific vasomotor neurones is also observed for premotor neurones supplying the tail vessels and brown fat tissue of rats and for neurones supplying the blood vessels of the ear in rabbits. These thermoregulatory presympathetic neurones lie in the mid-line raphe nuclei in rats (Morrison, 2001; Tanaka et al. 2002; Korsak & Gilbey, 2004; Ootsuka et al. 2004) or medial to RVLM in rabbits (Ootsuka et al. 2004; Koganezawa & Terui, 2005). A discrete spinal projection does not rule out that in addition there could be collateral innervation amongst the same medullary neurones (Zagon, 1993), which may, for example, contribute to control of skin blood flow and brown fat (Morrison, 2001).
Despite this strong representation of presympathetic neurones in the lower brainstem, non-uniform functional patterns of sympathetic vasomotor activity cannot be naturally evoked in the absence of suprabulbar regions. Indeed, intercollicular transection has long been known to lead to gross alteration of reflexes such as baroreceptor, peripheral chemoreceptor and nasopharyngeal reflexes (Korner et al. 1969; Korner, 1971; White et al. 1976; Olivan et al. 2001; Reddy et al. 2005).
There is now much evidence that groups of neurones in the hypothalamus play a significant integrative role in determining different vascular responses.
The hypothalamic control of sympathetic outflow
Although the hypothalamus has for a long time been viewed as the ‘master’ of the autonomic nervous system, the landmark demonstrations considered below, showing that groups of hypothalamic neurones project directly to the spinal cord, have considerably changed ideas on how control by neurones in this region of the brain is achieved.
Particular attention was first drawn to the existence of a direct projection of fibres from the hypothalamus to the autonomic regions of the spinal cord by Smith (1965), although the idea was not new (Beattie et al. 1930). Smith (1965) made electrolytic lesions at sites in the hypothalamus of the dog, cat and rat, which on prior stimulation had elicited pressor and cardioacceleratory responses. Following recovery times of 1 week to 1 month, the brains were removed, and the Nauta stain was used to determine patterns of degeneration. This showed many degenerating fibres in the intermediate grey matter of the thoracic cord, but the study failed to excite interest. However, the advent of better neuroanatomical tracing techniques led, in the late 1970s, to a revival of interest. Anterograde neuronal labelling studies, initially by Buijs (1978) and later by Sawchenko & Swanson (1982), complemented by similar studies at a number of laboratories worldwide, revealed that there are several diencephalic groups of neurones with axons projecting directly to the spinal cord and terminating in the sympathetic lateral horn (see Coote, 2004 for review). Studies of one particular nucleus, the paraventricular nucleus (PVN), have been especially enlightening, and I will use this as an example to illustrate a basic model for CNS vasomotor control.
The paraventricular nucleus and cardiovascular control
Detailed studies, using anterograde or retrograde dye labelling of the small parvocellular neurones of the PVN, have revealed that many have extra hypothalamic projections. Some of these directly innervate sympathetic preganglionic neurones in the spinal cord, such as those targeted to the superior cervical ganglion, stellate ganglion or adrenal medulla (Hosoya et al. 1995; Ranson et al. 1998; Motawei et al. 1999). These studies established, beyond doubt, that certain PVN–spinal neurones control sympathetic activity. A key question still remained as to whether these PVN neurones regulated cardiovascular end organs. The answer was provided by a series of elegant studies, initiated by Loewy, which utilized the property of a strain of pseudorabies virus to retrogradely transport across synapses after injection into peripheral organs (see Strack & Loewy, 1990). Studies by several groups revealed that the PVN neurones form part of neuronal pathways to the heart, kidney and other autonomic end organs (Strack et al. 1989; Schramm et al. 1993; Ter Horst et al. 1993; Jansen et al. 1995b; Smith et al. 1998; Huang & Weiss, 1999).
Despite these discoveries, it has still been difficult to escape from the idea that vasomotor control is dominated by the medulla and that supramedullary regions are part of a hierarchical organization of cardiovascular control. Therefore, there remained the question of the extent to which the supramedullary projections are independent. This was tackled by two studies of PVN neurones (Shafton et al. 1998; Pyner & Coote 2000). Use of retrograde tracing of two different dyes, one injected into the lateral cell column of the spinal cord and one into RVLM, revealed three classes of neurone. There are PVN neurones that project to RVLM neurones, PVN neurones that project to spinal cord and branch to innervate RVLM and PVN neurones that project only to the spinal cord. This third group is substantial (> 70%; Pyner & Coote, 2000), thus implying that there is a potential for parallel processing in cardiovascular regulation by the brain.
Amongst the PVN–spinal projection are neurones expressing either arginine vasopressin (25–40%) or oxytocin (20–30%) and, to a lesser extent, enkephalin or dopamine (Sawchenko & Swanson, 1982; Cechetto & Saper, 1988; Hallbeck & Blomqvist, 1999; Huang & Weiss, 1999). There is also substantial evidence that vasopressin and oxytocin act as neurotransmitters, both directly exciting sympathetic preganglionic neurones via their respective receptors (V1a in the case of vasopressin; Coote, 2004; Yang et al. 2002). Interestingly, no immunohistochemical study has so far attempted to identify glutamate in the PVN–spinal projection, although sympatho-excitatory effects of stimulating PVN neurones can be blocked selectively at the spinal level by the glutamate antagonist kynurenic acid (Yang et al. 2002). Whether this results from a direct spinal pathway or from PVN activation of RVLM–spinal neurones, many of which are known to be glutamatergic, or results from activation of other groups of neurones lying close to PVN is unresolved.
The PVN and plasma volume regulation
The extensive knowledge of PVN central projections has provided a sound basis for examining the critical question regarding its functional role and its relation to the RVLM. There are now substantial data indicating that at least some of the PVN parvocellular spinal projection is involved in plasma volume regulation. Lesions that destroy a substantial portion of PVN–spinal neurones reduce the renal response to blood volume expansion (Lovick et al. 1993; Haselton et al. 1994). Paraventricular nucleus–spinal neurones are activated or inhibited following stimulation of cardiac afferents or arterial baroreceptors (Lovick & Coote, 1988a,b), or by circulating atrial natriuretic factor (ANF; Lovick & Coote, 1989) and excited by hyperosmotic solutions injected into the blood supplying the brain via the internal carotid artery (Chen & Toney, 2001; Coote & Yang, 2005). Furthermore, stimulation of volume receptors by distending the right atrial–caval junction with a balloon catheter or by plasma volume expansion activates the early gene c-fos in PVN parvocellular neurones (Deng & Kaufman, 1995; Badoer et al. 1997; Pyner et al. 2002). Evidence that PVN–spinal vasomotor neurones transmit these afferent signals directly to spinal vasomotor neurones is provided by the demonstration that an increase in renal sympathetic nerve activity in response to a mild venous haemorrhage or to intracarotid hyperosmotic solution is significantly reduced by blocking the PVN–spinal vasopressin influence by application of V1a antagonist to the spinal neurones (Coote & Yang, 2005; Yang & Coote, 2006; Antunes et al. 2006).
Central mechanisms initiating the volume expansion reflex
These data support the idea that the PVN parvocellular–spinal neurones are a target for afferent signals related to fluid balance which are then translated into appropriate sympathetic vasomotor nerve responses. The vasomotor response to volume expansion, mimicked by distension of the venous atrial juctions, is especially interesting because it comprises an inhibition of renal sympathetic activity (see Coote, 2005) and, simultaneously, a sympathetically mediated increase in heart rate, as first described by Bainbridge (1915). Thus, the atrial receptors elicit a unique non-uniform pattern of sympathetic activity to the heart and kidney as part of the defence against plasma volume load. How then does the PVN achieve this? A clue was provided by studies in Kaushik Patel's laboratory showing that renal sympathetic nerve activity was under tonic inhibitory constraint mediated by GABA neurones (Zhang & Patel, 1998). This is determined in part by an action of GABA on PVN–spinal vasopressin neurones, since an increase in renal sympathetic activity elicited by microinjection of bicuculline into the PVN is significantly reduced by spinal application of a V1a antagonist (Yang et al. 2002). It was subsequently shown that a volume reflex inhibition of renal sympathetic activity was abolished by microinjection of the GABAA antagonist bicuculline into the PVN (Yang & Coote, 2003; see Coote, 2005 for discussion of this). Therefore, the evidence shows that the PVN influence can determine the pattern of renal sympathetic activity largely independent of the RVLM (Fig. 1).
This is only part of the story, however, since the atrial receptor reflex may also elicit a sympathetic nerve-dependent increase in heart rate. In this regard, our preliminary studies show that a PVN sympathetically elicited increase in heart rate is selectively blocked by an oxytocin antagonist applied to the upper thoracic spinal cord and is not affected by a V1a antagonist (Yang et al. 2004). This selective influence of the two peptides is in accord with the observation by Yashpal et al. (1987) that intrathecal oxytocin applied to the upper thoracic spinal cord caused an increase in heart rate with no effect on blood pressure. Also, there is electrophysiological evidence that sympathetic preganglionic neurones in the upper thoracic cord are directly excited by oxytocin (Desaules et al. 1995), whereas sympathetic neurones in the lower thoracic cord are not selectively influenced (Sermasi & Coote, 1994). In line with this, neuroanatomical studies show that sympathetic neurones in the upper thoracic segments are innervated by oxytocin-containing fibres, most probably originating from the PVN (Hosoya et al. 1995), whereas such fibres appear to avoid many sympathetic preganglionic neurones in the lower thoracic cord (Appel & Elde, 1988).
Therefore, it seems entirely possible that afferent signals from atrial receptors reach PVN–spinal oxytocin neurones to increase heart rate, as well as directly activating GABA neurones synaptically coupled to PVN vasopressin neurones projecting to spinal renal sympathetic neurones (see Coote, 2005). If such a scheme is in any way plausible, then we might expect to find sites in PVN from which the relevant functional pattern of sympathetic activity is elicited, and this was shown by Deering & Coote (2000), who recorded from several sympathetic nerves simultaneously in the rabbit.
Concept of ‘command neurones’
A persuasive explanation for these data is that the afferent signal converges on command neurones that have the required wiring for the basic pattern of response (Coote, 2004). There is neuroanatomical evidence that appears to support this arrangement. Using the technique of transynaptic labelling of a chain of neurones by the transynaptic transfer of a virus, Arthur Loewy's group injected two different genetically engineered forms of a virus into separate cardiovascular end organs, the heart and adrenal gland. They showed that, while many brain neurones were singularly labelled, indicating a target organ specificity, a few neurones were double labelled, indicating they had a direct line to more than one target organ (Jansen et al. 1995a). It was suggested that these could be ‘command neurones’ that initiate a pattern of sympathetic activity as part of a behavioural response, such as the defence/arousal response.
This is a provocative and potentially fruitful concept, which readily explains how all reflex and behavioural non-uniform patterns of sympathetic nerve activity to cardiovascular end organs may be elaborated. Thus, the results of these studies and those of others on the role of the PVN in blood volume control may serve as an important model for understanding brain control of the cardiovascular system (Fig. 1). Strong support for such a model is also emerging from recent studies of vasomotor neurones involved in thermoregulation (Oosuka et al. 2004; Tanaka et al. 2002; Koganezawa & Terui, 2005).
Up to now, a dominant role in the generation of vasomotor tone has been attributed to RVLM–spinal neurones. It is clear that these neurones convey afferent signals from many sources (Guyenet & Stornetta, 2004), but studies done under anaesthesia are likely to have obscured the role of supramedullary sites (by Abrahams et al. 1962). Up to now, the extent of their influence in the absence of RVLM–spinal activity has never been tested. It seems to me that this is a key question for future studies.
The brain and cardiovascular disorders
Interestingly, the significance of supramedullary control becomes more apparent in cardiovascular disorders such as hypertension and heart failure. In some models of hypertensive disease, where there is evidence of increased sympathetic vasomotor tone, lesions of or inhibition of PVN neurones reduces blood pressure (Goto et al. 1981; Ciriello et al. 1984; Allen, 2002). Intriguingly, the evidence for an upregulation of RVLM neurone activity in these models is either equivocal or contradictory, so it may be that PVN projections that go directly to the spinal cord play a major role.
In heart failure, a characteristic feature is fluid retention, and this is accompanied by increased sympathetic nerve activity (Leimbach et al. 1986), especially to the heart (see Dampney et al. 2002; Weiss et al. 2003). This suggests that there is an inability of cardiovascular receptors to reflexly inhibit the raised sympathetic activity. Although there may be some impairment of the sensitivity of peripheral receptors (Mancia et al. 2006), there is a growing body of evidence pointing towards a central nervous abnormality. In a rat model of heart failure, produced by coronary artery ligation, the renal sympatho-inhibition in response to acute volume expansion is blunted. This is accompanied by an increase in PVN pre-autonomic neurone activity and by a reduced endogenous GABA-mediated inhibition of renal sympathetic activity (Zhang et al. 2002; Li & Patel, 2003). The physiological consequences of this poorer reflex regulation of sympathetic excitability are that sympathetic nerve activity to the heart and blood vessels increases (Mancia et al. 2006). A key question is what initiates this sequence of damaging events? Is it a signal from the heart to the brain that results in the CNS changes or are there changes occurring first in the supramedullary regions of the brain? These questions remain to be answered.
Conclusion
In this lecture, I have chosen key discoveries arising from studies over almost 200 years that have led to current concepts of cardiovascular control and which are informing a general understanding of brain action. I have tried to show how the advance of knowledge has built on enlightened and careful experimentation using integrative approaches on experimental animals as models. The discovery of vasomotor nerves that displayed a continuous rhythmic activity was a major landmark, but it required many contributions. Somehow, its significance to survival amongst the vertebrates became lost in the intense search for its site of origin, which became known as the vasomotor centre. The elegant studies identifying RVLM–spinal vasomotor neurones, although of no less importance, unfortunately continued to cloud the issue. It has taken studies over the last 30 years or so to show that other groups of neurones, particularly in the hypothalamus, project to synapse directly with sympathetic preganglionic neurones in the spinal cord. These studies have shown that RVLM neurones are not alone in directly controlling sympathetic vasomotor outflow. Using the hypothalamic PVN as an example, I have tried to illustrate that the tonic activity of vasomotor nerves allows the PVN to initiate non-uniform patterns of sympathetic nerve activation, so that delivery of blood can be directed to organs where it is most needed. I show how this has led to the concept of ‘command neurones’ and that the new understanding has directed interest to changes in the brain as a cause of alterations in sympathetic activity in cardiovascular disorders such as hypertension and congestive heart failure.
Footnotes
This lecture was given at the joint international meeting of The Physiological Society and FEPS at the University of Bristol, UK on 21st July 2005.
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