I. INTRODUCTION

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This review explores the mechanisms of central control and coordination of the respiratory and cardiovascular systems in vertebrates. Animals have evolved sophisticated control mechanisms enabling them to match their rates of oxygen uptake to their rates of aerobic metabolism. As the relative effectiveness of respiratory gas exchange over lungs or gills is determined not only by their physical dimensions but also by the rates and patterns of their ventilation and perfusion, it is essential that these latter components are controlled, both individually and in relation to one another. It has long been recognized that the overall rates of flow of air or water and of blood over the respiratory surfaces are matched according to their respective capacities for oxygen so that the ventilation-to-perfusion ratio varies from ~1 in air-breathers to 10 or more in water-breathers, with the bimodal, air/water-breathers among the lungfishes and amphibians showing variable ratios (286, 498). However, these overall ratios ignore the pulsatile and sometimes intermittent nature of both ventilation and perfusion. Careful study of respiratory and cardiac rhythms often shows them to be temporally related in ways that may optimize respiratory gas exchange. Control of these cardiorespiratory interactions resides in the central nervous system that integrates afferent inputs from a range of central and peripheral receptors and coordinates central interactions between pools of neurons generating the respiratory rhythm and determining heart rate variability. Thus, although the absence of a heart beat signifies death, a high and unvarying heart rate can indicate incipient brain death.

Many aspects of the brain circuitry of this remarkably sensitive system seem to have been highly conserved throughout evolution. Thus the regulatory mechanisms that operate in the central nervous systems of lower chordates such as the elasmobranch fishes show a remarkable degree of homology with those that operate in mammals, including humans (606). Homology literally means "of the same essential nature" having affinity of structure and origin. In this review, we examine apparent homologies in the cardiorespiratory control system of vertebrates, in terms of the location and phenotype of the neuronal substrate, the pattern of central nervous system (CNS) connections, the development and conservation of fundamental rhythms of nerve discharge, and neuroeffector mechanisms. Of course, we are aware that a strictly phylogenetic approach to comparative physiology is inappropriate, since parallel evolution can result in clear homologies of structure and function in distantly related species. Accordingly, we have emphasized the topographical similarities and their apparent evolution and treated the functional role of central nervous connections separately, while drawing parallels with their structural bases. We have not attempted to draw a phylogenetic tree for control of cardiorespiratory function; rather, we have explored its evolutionary roots. We show that there are considerable similarities in the topography and functional characteristics between groups of neurons in the hindbrain and spinal cord of the different vertebrate groups. However, there are also significant differences, the problem then being to know when it is reasonable to generalize and when not.

There are important differences in the construction of the respiratory and cardiovascular systems in vertebrates, related to their modes of respiration. Fish typically propel water unidirectionally over the gills, using ventilatory muscles that operate around the jaws and skeletal elements in the gill arches lining the pharynx. Adult amphibians, which lack a diaphragm, retain the buccal force pump for tidal lung ventilation; their larvae are aquatic gill-breathers. Thus, in fish and amphibians, the major respiratory muscles are cranial muscles, innervated by motoneurons with their cell bodies in the brain stem. Reptiles retain an elaborate buccal, hyoidean force pump, but ventilate the lungs primarily with a thoracic aspiratory pump, although they typically lack a diaphragm. Mammals have aspiratory lungs, and ventilation is accomplished by coordinated contractions of diaphragmatic, intercostal and/or abdominal muscles innervated from the spinal cord, with only some accessory respiratory muscles (e.g., for control of the glottis) innervated by cranial nerves. Consequently, medullary respiratory neurons send axons down the spinal cord to innervate spinal motoneurons. The respiratory system in birds resembles that of mammals, except that they lack a diaphragm and the lungs are ventilated by volume changes in the air sacs.

The cardiovascular system is undivided in a typical fish, with the heart delivering blood into the branchial vasculature and an arterioarterial respiratory route conducting blood directly from the gills to the systemic circuit. A parallel arteriovenous route through the branchial circulation is probably nutritive, rather than constituting a functional shunt past the respiratory route. In contrast, mammals and birds have a completely divided circulatory system, with separate pulmonary and systemic circuits. Air-breathing fish, amphibians, and most reptiles have more or less incompletely divided circulatory systems, allowing differential perfusion of the pulmonary circuit. This ability may be an essential component of their intermittent patterns of ventilation, often associated with periods of submersion. Amphibians may, in addition, utilize bimodal respiration. Larval amphibians possess gills, often in combination with developing lungs, while adult amphibians can switch between cutaneous and lung breathing (e.g., during graded hypoxia or submersion) so that the distributing effect of vascular mechanisms are of paramount importance.

Despite these major differences in the construction and mode of operation of their respiratory and cardiovascular systems, evidence is accumulating that the vertebrates share some important similarities in the mechanisms of central generation of the respiratory rhythm, control of the cardiovascular system and, more specifically in the present context, in the central nervous and reflex generation of cardiorespiratory interactions. The central theme of this review is the evolution of the mechanisms of integration and coordination that match blood flow to ventilatory movements, a relationship probably fundamental to the success of vertebrates. Accordingly, we address such questions as the origin and nature of tonic nervous activity to the heart, to blood vessels, and to the airways. It may be that our review of the evolutionary relationships between cardiorespiratory control systems in vertebrates will illuminate our current inadequate understanding of the fundamental mechanisms underlying the observed interrelationships between respiratory control and cardiac control.

Knowledge of this complex area is of course dominated by the results of medically oriented research on mammals. To thoroughly review the mammalian literature is beyond the scope and length constraints of the current account. Instead, reference will be made in the relevant sections to recent extensive reviews. Readers requiring a more detailed account of the mammalian literature thus have points of access to that debate, without unduly lengthening the current review, or unbalancing it in relation to the available information from "lower" vertebrates. Each aspect of the review, therefore, begins with a summary of our current understanding of the extensive mammalian literature. This then underpins the subsequent comparative survey of the other vertebrate groups, considered in turn from fish, through amphibians and reptiles to birds, in relation to our more thorough understanding of the mammalian pattern. The treatment of each group is necessarily uneven because of the limitations on our knowledge so that the sections on "fish" are sometimes divided between elasmobranchs and teleosts and sometimes not. It must be emphasized here that, unlike the mammals and birds, the so-called "lower vertebrate" groups have a complex phylogeny; that is to say that fish, amphibian, or reptile is an umbrella term describing very diverse groups of animals, some relatively little studied. Because the respiratory and cardiovascular systems and their innervation in the lower vertebrates are less well known than those of mammals, some brief descriptions of selected examples are included to illuminate the account of the mechanisms of their control.

Some consideration of the mechanisms of ventilation and of the generation of the respiratory rhythm in the CNS is a necessary prelude to a review of the control of cardiorespiratory interactions. Consequently, a very brief overview of this area in mammals leads to a comparative account of our more limited understanding of the mechanisms in lower vertebrates, which includes descriptions of their patterns of ventilation and their origins in the brain stem, plus a consideration of the factors determining the onset and frequency of bouts of intermittent breathing in air-breathing fish, amphibians, and reptiles.

We then describe the innervation of the reflexogenic areas supplying the cardiovascular and respiratory systems and implicated in the generation of cardiorespiratory interactions, including central and peripheral chemoreceptors, arterial baroreceptors, and mechanoreceptors supplying the respiratory system. There follows a review of the evidence for functional chemoreceptors and mechanoreceptors in fish, including air-breathing fish, and in amphibians, which considers the developing roles for central chemoreceptors, lung stretch receptors, and arterial baroreceptors as the vertebrates evolved from primarily water-breathing to facultative and then to obligate air-breathing forms.

A review of the efferent innervation of the cardiovascular and respiratory systems is initiated by consideration of the cranial autonomic outflow. Beginning with a detailed description of the central locations of vagal preganglionic neurons (VPN) in mammals, which emphasizes the importance of the nucleus ambiguus (nA), a comparative account of the central origins of vagal efferents innervating the cardiovascular and respiratory systems in lower vertebrates follows. This considers evidence of a developing role in the control of cardiorespiratory interactions for neurons relocated from the dorsal motor nucleus of the vagus (DVN) into the nA. Description of the sympathetic innervation of the cardiorespiratory system explores evidence for the existence of functionally organized specific groups of cells, including the possible functional importance of the arrangement of their dendritic fields, in the control of heart rate, vasomotor control, and the control of airway resistance.

The review culminates in a consideration of the central control of cardiorespiratory interactions in mammals, with a necessarily less detailed comparison of the mechanisms in lower vertebrates. This account begins with a consideration of control of the heart then progresses to a review of the role of central interactions and reflex inputs in the generation of cardiorespiratory modulation of heart rate, vasomotor tone, and control of the airways in mammals. Discussion of the role of neurons and their connections within the nucleus of the solitary tract (NTS) and the ventrolateral medulla in the generation of cardiorespiratory interactions is followed by a consideration of the generation of respiratory oscillations in sympathetic cardiovascular neurons. A comparative account of the central and peripheral interactions resulting in cardiorespiratory synchrony in fish is followed by consideration of the interactions responsible for control of the cardiorespiratory responses of intermittent breathers among the amphibians, reptiles, and diving birds.

We conclude the review with a summary of the apparent evolutionary changes in the control systems described in lower vertebrates, toward the more fully investigated systems in mammals, which attempts to identify areas that merit the urgent attention of comparative physiologists. The identification of these areas is made in the knowledge that comparative studies are becoming ever harder to fund from the agencies that support academic research. It is our task to emphasize that such studies are not only of great intrinsic interest but can further illuminate our understanding of mammalian, and therefore human, systems.

	II. PATTERNS OF VENTILATION AND CENTRAL RESPIRATORY PATTERN GENERATION

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Mammals characteristically display continuous, rhythmic, aspiratory breathing to maintain their relatively high rates of oxygen uptake and carbon dioxide excretion. Exceptions are the fetus and neonate which often show intermittent cycles of breathing related to sleep states (260) and diving or hibernating mammals which suspend or markedly reduce breathing and heart rates for varying periods but otherwise show typical cardiorespiratory control mechanisms. Patterns of ventilatory mechanics are defined solely in terms of the time spent in inspiration and expiration and the rate of air flow. Combinations of these variables produce the more familiar components of breathing, namely, frequency, tidal volume, and minute ventilation. From neurophysiological data, the mammalian ventilatory cycle has been divided into three distinct neural phases in which each phase reflects a "state" of the oscillating network rather than a particular configuration of the motor output. In other words, a cycle phase in this context means a recurring episode when one or more groups of neurons in the network discharge a characteristic pattern of action potentials (528, 529). These phases have been defined as inspiration, postinspiration (passive expiration), and expiration (active expiration). The postinspiratory phase is a period of inspiratory braking, which is also referred to as the first stage of expiration (EI) (364, 528).

Pattern is more complex in arrhythmic or episodic breathers, such as the amphibians and reptiles, where the components of breathing frequency also include number of breaths per episode and an apneic or nonventilatory period of variable duration. Kogo and Remmers (364) have recently discussed the similarity of the respiratory phases between amphibians and mammals. Their intra- and extracellular recordings of respiratory neurons in bullfrogs provide solid evidence to argue that lower vertebrates also have a three-phase respiratory cycle. According to their analysis, the first phase is expiration, and it occurs when the glottis is first opened. This is then followed by inspiration, which is produced by the brisk activation of the buccal levators to push air back into the lungs. The last phase is a period of breath holding, during which neurons other than those involved in the production of the two other phases were shown to be active. This phase corresponds to the postinspiratory phase described previously for mammals. They conclude their discussion by stating that this analysis is consistent with that of Pack et al. (487), who suggest that lungfish, which also have a buccal force pump, have a postinspiratory phase.

The mechanisms underlying respiratory rhythmogenesis in mammals are only now being resolved (67, 529, 585), and even less is known about respiratory rhythmogenesis in nonmammalian species. Recordings from isolated brain stem-spinal cord preparations in lamprey (539), bullfrog (427), and turtle (178) have shown rhythmic respiratory-related discharges in spinal and cranial motoneurons. Because these preparations can produce a respiratory rhythm in the absence of afferent feedback (with the possible exception of input from central oxygen chemoreceptors, when present) it would appear that a central respiratory pattern generator is present in all vertebrates. At the same time, because it is possible to eliminate breathing by artificially meeting the convective requirements of an animal (e.g., external membrane lung, unidirectional gill or lung ventilation; for review, see Ref. 440), it would appear that the CPG requires some external stimulus to trigger respiratory events.

The neural substrate responsible for respiratory rhythm generation and mediation of respiratory reflexes lies within the brain stem of mammals. Groups of respiratory premotoneurons and neurons innervating upper airway muscles are found in the caudal medulla near the nA and the Bötzinger complexes. In addition, at least one site of respiratory rhythmogenesis has been identified, in neonatal mammals, in the "pre-Bötzinger" complex which is situated in the reticular formation of the rostral medulla, at the level of the hypoglossal nuclei (585). These outflows probably derive, in an evolutionary sense, from the branchial motoneurons of more primitive, gill-breathing vertebrates that retain their primary roles in respiratory rhythm generation in present-day fish and larval amphibians. Accordingly, the reticular formation is thought to be the site both of the primary respiratory rhythm generator in fish and amphibians and of the respiratory and suckling rhythms in neonatal mammals.

Because the detailed organization of central respiratory control in mammals has been exceedingly well reviewed recently (67, 200, 529), a brief synopsis, for comparison with nonmammalian vertebrates, will be sufficient here. Two models have been proposed to try to explain respiratory rhythmogenesis in mammals. One proposes that the central respiratory rhythm generator consists of burster or pacemaker neurons, which show spontaneous rhythmic oscillations in membrane potential in the absence of synaptic inputs or alternatively require a tonic excitatory input before they exhibit rhythmic oscillatory activity. The second postulates that respiratory rhythm is produced by neural networks that exhibit oscillatory behavior due to synaptic interactions alone. Indeed, although pacemaker-like neurons have been identified in the pre-Bötzinger region in neonatal mammals in vitro, when sensory input was removed (585), most recently Richter (529) has argued for a hybrid of these two in vivo, whereby under normal conditions of sensory input, the synaptic interactions between respiratory neurons override the effects of pacemaker inputs. In their recent review of the literature on the central control of breathing in mammals, Bianchi et al. (67) have proposed that respiratory rhythm is not generated by a single conditional pacemaker process. Their argument was based on the assumption that brain stem respiratory activity results from the sequential activation of many populations of neurons to produce a three-phase motor act (breathing) in which each process is conditioned by the previous one and initiates the next. An alternate view would be that the coordination of the groups of respiratory neurons would be performed by a different entity. This entity would be responsible for processing the relevant sensory signals and would ensure precise spatial and temporal pattern of muscle activation during each breath so that the respiratory system meets the demand of the organism. It is to help understand the relationship between respiratory rhythm and pattern that the concept of a central respiratory pattern generator has emerged (203, 439). Because the mechanisms underlying the generation of central respiratory rhythms are not the prime subject of this review, central pattern generation will be referred to nonspecifically and the generator designated as the CPG.

Spontaneous bursts of respiration-related activity have been recorded from the isolated brain stem of the lamprey. Recording sites included respiratory motor nuclei in the caudal half of the medulla, innervating the VIIth, IXth and Xth cranial nerves and sites near the trigeminal (Vth) motor nuclei, in the rostral half of the medulla (538, 541, 622). Periodic bursts of small spikes recorded from the rostral medulla, at the levels of the V motor nuclei, continued after isolation of the isthmic-trigeminal region by transections and occurred before bursts recorded from the IX and X cranial nerve roots. Electrical stimulation of this area excited respiratory motoneurons monosynaptically and could entrain or reset the respiratory rhythm. These observations suggest that the motor pattern for respiration is at least partly generated and coordinated in the rostral half of the medulla in the lamprey and is transmitted to respiratory motoneurons through descending pathways (539).

Water contains less oxygen per unit volume than air and yet is considerably more dense and viscous. Consequently, fish have to work relatively hard to extract sufficient oxygen from water and normally exhibit continuous rhythmical breathing movements of the buccal and septal or opercular pumps. Fish use cranial muscles for gill ventilation. These are innervated by a dorsal group of cranial nerves exiting from the brain stem and termed the branchial nerves (536). This series of nerves contains sensory fibers and in most cases visceral motor components (Fig. 1). The nerves innervating respiratory muscles include the trigeminal Vth which provides the major innervation to the mouth region of all vertebrates, including the maxillary branch to the upper jaw and mandibular branch to the lower jaw, responsible for motor control of the jaw-closing muscles. Jaw opening is passive in routine aquatic ventilation (31, 285). The facial VIIth nerve provides the hyomandibular branch to the branchial muscles in the hyoid arch, including the levator hyoidei and, in teleosts, the opercular muscles. The glossopharyngeal IXth and the vagal Xth cranial nerves innervate the gill arches and in particular provide afferent innervation of the mechanoreceptors and chemoreceptors important in ventilatory control and efferent innervation to intrinsic respiratory muscles in the gill arches. These branchial nerves have their efferent cell bodies and afferent sensory projections located dorsomedially in the brain stem, close to the fourth ventricle, in a rostrocaudally sequential series (607; Fig. 2).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Diagram of distribution of components of cranial nerves involved in control of ventilation in a shark (Squalus). Roman numerals refer to cranial nerves: V1, ophthalmic ramus of trigeminal nerve; V2, maxillary division; V3, mandibular division of trigeminal; VII Pal, palatine ramus of facial nerve; X, vagus nerve supplying branchial branches to gill arches 2-5, cardiac and visceral branches; XII, trunk of conjoined occipital and anterior spinal nerves to form hypobranchial nerve innervating ventral muscles inserted on pectoral column and used in feeding and forced ventilation; hm, hyomandibular; lat, lateral line trunk; occ n, occipital nerves; s op, superficial ophthalmic; S, position of spiracle; sp n, anterior spinal nerves; 1-5, position of gill slits. [From Taylor et al. (613).]

View larger version (39K): [in this window] [in a new window]   Fig. 2. Right: schematic diagram of a dorsal view of hindbrain of dogfish, Scyliorhinus canicula, to show distribution of motonuclei (indicated by hatched areas) supplying efferent axons to cranial nerves innervating respiratory muscles and heart. These are adductor mandibulae nucleus of cranial nerve V, supplying closing muscles in jaw; facial motor nucleus of VII, supplying muscles of jaw and spiracle; glossopharyngeal nucleus of IX, supplying first gill arch; dorsal motor nucleus of vagus Xm, which is divided rostrocaudally into separate subnuclei innervating branchial arches 2-4 (Xm 1-3), branchial arch 5 plus branchial cardiac nerve (Xm4), and heart plus anterior regions of gut (X vis). Lateral nucleus of vagus (Xl) supplies axons solely to branchial cardiac nerve. Hypobranchial nucleus (hy) supplies axons to feeding muscles via occipital nerve XI and anterior spinal nerves (not shown). Other labels indicate cerebellum that overhangs medulla and is outlined by heavy line, a cerebellar auricle (aur) octavus nerve (VIII) and spinal canal (spc). Obex marks position where roof of 4th ventricle is devoid of nervous tissue rostrally and covered by choroid plexus. Left: a diagrammatic transverse section (T.S.) taken at obex; through medulla of dogfish (indicated by divided line on right panel) to show vertical and horizontal disposition of vagal and hypobranchial nuclei. Labels indicate a vagal rootlet (X), dorsal motor nucleus of vagus (Xm), lateral vagal nucleus (Xl), vagal sensory nucleus (Xs), hypobranchial nucleus (hy) and sulci in 4th ventricle (4th V), namely, sulcus medianus inferior (smi), sulcus intermedius ventralis (siv), and sulcus limitans of His (slH). [From Taylor (608).]

Rhythmic ventilatory movements continue in fish after brain transection to isolate the medulla oblongata, although changes in pattern indicate that there are influences from higher centers (563). Central recording and marking techniques have identified a longitudinal strip of neurons with spontaneous respiration-related bursting activity, extending dorsolaterally throughout the whole extent of the medulla (36, 564, 565, 636). These neurons make up elements of the trigeminal Vth, facial VIIth, glossopharyngeal IXth, and vagal Xth motor nuclei, which drive the respiratory muscles, together with the descending trigeminal nucleus and the reticular formation (Fig. 1). All the motor nuclei are interconnected, and each receives an afferent projection from the descending trigeminal nucleus and has efferent and afferent projections to and from the reticular formation (29). The intermediate facial nucleus, which receives vagal afferents from the gill arches that innervate a range of tonically and physically active mechanoreceptors (164) as well as chemoreceptors (607), projects to the motor nuclei (34). Finally, areas in the midbrain such as the mesencephalic tegmentum have efferent and afferent connections with the reticular formation (33, 335). The respiratory rhythm apparently originates in a diffuse respiratory pattern generator in the reticular formation, and this remains functional under anesthesia (29).

Some fish will exhibit episodic breathing patterns when exposed to particular environmental conditions such as hyperoxia. Carp were shown to possess a group of neurons with phase-switching properties, situated in the dorsal tegmentum at the level of the caudal midbrain. This group of respiratory rhythmic neurons (termed type A neurons) do not sustain continuous respiration but appear to play a key role in the control of episodic breathing. Indeed, type A neurons fire just before the onset of a breathing bout during intermittent respiration. Furthermore, stimulation of this area of the brain stem, during a ventilatory pause, brings forward the onset of the next breathing bout (334).

Central recordings from the medulla oblongata of the carp suggested that adjacent neurons have different firing patterns (30). These authors identified the target muscle for individual motoneurons by simultaneous recordings of neuronal activity and electromyograms (EMG) from the respiratory muscles. In contrast, retrograde intra-axonal transport of horseradish peroxidase (HRP) along nerves that innervate the respiratory muscles revealed that in the brain stem of elasmobranchs the neurons in the various motor nuclei are distributed in a sequential series (607). Recordings of efferent activity from the central cut ends of the nerves innervating the respiratory muscles of the dogfish Scyliorhinus canicula (52) and the ray Raia clavata (E. W. Taylor and J. J. Levings, unpublished data) have revealed that the branches of the Vth, VIIth, IXth, and Xth cranial nerves fire sequentially in the order of the sequential rostrocaudal distribution of their motonuclei in the brain stem and rostral spinal cord. The resultant coordinated contractions of the appropriate respiratory muscles may relate to their original segmental arrangement before cephalization, an arrangement which is retained in the hindbrain of the fish in the sequential topographical arrangement of the motor nuclei, including the subdivisions of the vagal motonucleus (Fig. 2). This traditional view of the origin of the jaws and visceral arches and their innervation (161) has recently been questioned on the basis of developmental studies of the role of neural crest cells (215). These suggest a separate origin for the jaws as feeding structures, independent of the visceral arches, which combined ventilation with filter-feeding, a view supported by study of marker genes (586). A possible evolutionary antecedent of the jaws may be the velum of filter feeding protochordates or larval cyclostomes (M. A. Smith, personal communication).

Both elasmobranchs and teleosts can recruit an additional group of muscles into the respiratory cycle to provide active jaw occlusion. These are derived phylogenetically from the forward migration of four anterior myotomes (the hypaxial muscles) to form a complex ventral sheet of muscle, inserted between the pectoral girdle, the lower jaw, and the ventral processes of the hyoid and branchial skeleton. They are associated primarily with suction feeding and ingestion in water-breathing fishes (285) but can be recruited into the respiratory cycle during periods of vigorous, forced ventilation such as may occur following exercise or deep hypoxia (32, 285). These muscles are innervated by the hypobranchial nerve, which contains elements of the occipital nerves and the anterior spinal nerves (Figs. 1 and 2). The hypobranchial nerve in fish is the morphological equivalent of the hypoglossal nerve that innervates the muscles of the tongue in reptiles, birds, and mammals. These muscles are utilized in suckling by infant mammals, an activity likely to require its own central oscillator, which is thought to reside in the reticular formation.

In the dogfish and ray, rhythmic opening and closing of the mouth occurs during ingestion of food, implying the central generation of a feeding rhythm (391). The neural mechanisms operative in the control of masticatory rhythms in fish remain unexplored, although it has been argued that the respiratory and feeding rhythms in fish are generated by separate groups of interneurons (32). It is now well established that in mammals the masticatory rhythm is generated in the hindbrain, in the reticular formation (481), and the same has been suggested for birds (181). It is interesting, in this regard, that the CPG in fish is thought to reside in the reticular formation (29).

Preliminary studies on dogfish, in which simultaneous recordings were made of efferent activity in the central cut end of a branchial branch of the vagus and of a branch of the hypobranchial nerve in decerebrate, paralyzed fish, confirmed that the hypobranchial nerve is inactive during normal fictive ventilation (Taylor and Levings, unpublished data). Short sequences of bursting activity were elicited in the silent hypobranchial nerve by activation of tongue mechanoreceptors and skin stretch receptors on the jaw (stimuli associated with feeding). Periods of spontaneous, respiration-related bursting activity could be elicited by stimulation of gill proprioreceptors and chemoreceptors (this latter response to experimental oxygen deprivation) and by intravenous injection of norepinephrine, which increases ventilation in dogfish, possibly due to central stimulation of respiratory neurons (517, 615). The mechanisms involved in recruitment of hypobranchial motoneurons into the respiratory rhythm have not been studied.

Air-breathing fish retain gills, ventilated by cranial muscles, for the uptake of a variable proportion of their oxygen requirements, dependent on species and conditions, and excretion of most of their carbon dioxide. Gulping of air is achieved through the action of the same muscles in all air-breathing fish. These are elements of the jaw musculature, innervated by cranial nerve V, together with hypobranchial musculature, identified by Liem (396, 397) such as the geniohyoideus and sternohyoideus muscles. The combined action of jaw and hypobranchial muscles in the generation of feeding or air-gulping, independently of the visceral arches, may derive from their separate embryological and evolutionary origins (586). Liem (397) described the sequence of events associated with air-breathing in the primitive actinopterygian, the bowfin (Amia calva), a fish that utilizes a well-vascularized swimbladder as an air-breathing organ (ABO), and suggested that the action of air-breathing would require little change in the pattern of neural control required for suction feeding and/or coughing, with the exception of control over glottal opening. Brainerd (82) has suggested separate origins for air-pumping mechanisms in actinopterygian fishes (derived from the suction feeding/coughing pumps) and sarcoptergian lung fish and amphibians (the branchial irrigation pump). However, both pumps utilize the same sets of muscles and possibly the same central oscillators. In the bowfin, there appear to be two types of air breath, one that involves exhalation followed by inhalation (designated "type I" air breaths by the authors) and one that simply involves inhalation ("type II" air breaths), and it is suggested that type I breaths are respiratory in nature, whereas type II breaths have a buoyancy-regulating function (266).

Reorganization of the CNS associated with the evolution of air-breathing has been poorly studied in fish. It has been suggested that the African lungfish (Protopterus aethiopicus) possesses two separate central rhythm generators, one for gill ventilation and the other for air-breathing (205). With regard to actinopterygian, air-breathing fishes, there is probably a CPG for gill ventilation located in the reticular formation of the hindbrain, similar to that of water-breathing fish (29). In the bowfin, catecholamine infusion stimulates gill ventilation, apparently via a central mechanism, but has no effect on air-breathing in normoxia or hypoxia (422, 423), indicating that central sites controlling gill ventilation and air-breathing are pharmacologically and possibly spatially different. The central sites responsible for control of air-breathing reflexes in fish are still unknown. Some authors have suggested that air-breathing is critically dependent on afferent feedback (568, 575, 577) and, as stated above, is simply a reorganization of coughing and suction-feeding movements requiring relatively little neural reorganization (397, 575, 577). In the bowfin, spectral analysis indicates that there is an inherent rhythmicity to type I (i.e., respiratory-related) air-breathing, both in normoxia and hypoxia (267). These authors suggest, however, that this periodicity may be driven by changes in blood oxygen status that occur during the interbreath interval, rather than by a CPG for air-breathing.

Amphibian tadpole larvae have gills ventilated by activity in cranial muscles, with branchial performance comparable to teleost fish, but carry out a large proportion (60%) of respiratory gas exchange over their permeable skin. As development proceeds, the lungs assume increasing importance in oxygen uptake, although the skin remains the major exchange surface until metamorphosis is nearly complete (91). In adult amphibians, most oxygen is taken up from the lungs, ventilated by the buccal cavity, but the skin retains a predominant role in the excretion of carbon dioxide (80%, r = 7.5).

The sequence of air flow in the breathing cycle of lungfish and amphibians such as bullfrogs is similar. Unlike air-breathing fish, which must open their mouth to aspirate ambient air into their buccal cavity at the onset of the breathing cycle, frogs aspirate air via nostrils. Even though this modification imposes a slight resistance to gas flow, it eliminates the energy expenditure associated with gulping air at the water surface (213, 214). Lung ventilation usually occurs episodically in bullfrogs. A breathing cycle begins by activation of the buccal depressor muscles that brings buccal pressure below ambient, and air is aspired into the buccal cavity via the nostrils. The laryngeal dilator muscles then contract to open the glottis, and this allows outflow of pulmonary gas that exits by the nostrils. Subsequent closure of the nostrils coincides with a brisk contraction of the buccal levators, which pushes the bolus of gas through the glottis and into the lungs. The glottis then closes, and the inflated lung is held at a positive pressure. Lung inflation cycles, in which a series of inhalations occur without an intervening expiratory phase, are associated with experimental hypoxia or hypercapnia (640).

Typically, after a bout of lung breathing, there follows a series of elevations and depressions of the floor of the buccal cavity, called buccal oscillations. These small-amplitude, low-pressure buccal movements may help flush the buccal cavity from the previous expiration, before the next air breath (166, 168, 213, 214, 648), although their primary role may be olfaction (650). It has been suggested that they may be remnants of the mechanisms of gill ventilation used by the premetamorphic tadpole stages, and homologous to gill ventilations in fish, and that their rhythm may reflect vestiges of the central rhythm generator for gill ventilation (209, 395, 487, 576). Buccal oscillations and lung ventilations are produced by the same muscles. The primary difference between these two events is the force of the contraction and the positions of the glottis and nares. Lung ventilations are associated with more forceful contractions with the glottis open and nares closed; buccal oscillations are associated with less forceful contractions with the nares open and the glottis closed. In resting animals, buccal oscillations occur more or less continuously and are interrupted by periodic lung ventilations, which normally occur at a time when another buccal oscillation would have been initiated. Regardless of the level of respiratory drive, there appears to be an intrinsic rhythm to lung inflation events, increasing respiratory drive simply appears to result in this rhythm being expressed a greater percentage of the time.

These observations suggest at least two possible scenarios. The first is that there is a single central rhythm generator whose output is integrated with inputs from higher centers and peripheral feedback (mechano- and chemoreceptors) at two distinct pattern generators. At low levels of respiratory drive, only output from the pattern generator driving buccal oscillations is produced, but as respiratory drive increases, output is generated from the pattern generator driving lung inflation, which leads to the increase in the force of buccal contraction and the switch in the state of the nares and glottis. The other possibility is that there are two distinct rhythm generators, with expression of the lung rhythm being conditional upon a higher level of central and/or peripheral receptor input. However, the fact that lung ventilation always occurs at a time when a buccal oscillation would otherwise have occurred suggests that if there are separate rhythm generators, they are entrained to a large degree. Kinkead (354) has described some circumstantial evidence for the existence of two central respiratory rhythm generators in the bullfrog. Hypercapnia had no effect on the frequency of lung inflations but reduced both the occurrence of buccal oscillations and their instantaneous frequency when they did occur. This might suggest that there are separate rhythms for lung inflation and buccal oscillation, which can be uncoupled.

Recently, a number of investigators have used in vitro preparations of the larval or adult anuran brain stem to examine the mechanisms of respiratory rhythmogenesis (209, 427-429, 491). Recordings of fictive breathing in isolated brain stem preparations revealed spontaneous neural output from the roots of cranial nerves V, VII, X, and XII. However, these bursts were synchronous, implying that the spatiotemporal relationships between bursts of activity in these nerves in the intact animal rely on feedback from peripheral receptors. Microinjections of glutamate into rostral areas of the bullfrog brain stem, near the VII motor nucleus, caused a brief excitation of fictive breathing (427). Interestingly, this area corresponds to the pre-Bötzinger area of the reticular formation in the mammalian brain stem, considered to be the primary site for respiratory rhythmogenesis in the neonate (e.g., Ref. 523). The CPG in fish is thought to reside in the reticular formation (29). Other pharmacological investigations support the suggestion that the neural networks associated with respiratory rhythmogenesis may be well conserved during vertebrate evolution (640).

Extracellular recording from in vitro brain stem as well as spinal cord preparations of Rana catesbeiana tadpoles and adults revealed that it is possible to manipulate the two types of neural activity associated with buccal or lung breathing independently, using pharmacological agents (209, 428, 487, 585, 637). Superfusion of an in vitro brain stem-spinal cord preparation from the bullfrog tadpole with chloride-free saline eliminated the rhythmic bursts associated with gill ventilation while augmenting lung bursts, indicating that the former arise from a GABAergic, network-type rhythm generator, whereas lung ventilatory rhythms arise from pacemaker cells (209). This apparent discrimination is of interest in comparison to the situation described in fish, where gill ventilation may depend on pacemaker cells located in the reticular formation, and in adult mammals, where lung ventilation may be dependent on activity in neural networks. The evolution/development of air-breathing rhythms may have required a new motor pattern in the CNS rather than one that evolved from progressive modification of the branchial rhythm generator (354, 577). This may have evolved from the generator for the feeding rhythm that can be recruited by the respiratory CPG during forced ventilation in fish or when air-breathing fish gulp air at the water surface, as described above.

A recent report by Brainerd and Monroy (83) described activity in hypaxial muscles during exhalation in salamanders, possibly representing a primitive condition, intermediate between the buccal force pump of fish and the thoracic/abdominal aspiration pump of reptiles, birds, and mammals. Although similar data are not available for anuran amphibians, which may have lost this function, these data raise important considerations regarding the evolution of the control of ventilation in amphibians, which imply that descending fibers from the brain stem, innervating spinal motoneurons can have important roles in some species, anticipating their roles in the supposedly more advanced tetrapods.

Amphibians often breathe intermittently, with bouts of ventilation interrupted by quiescent periods or, in aquatic species or stages, submersion. Intermittent breathing patterns are common in lower vertebrates, such as reptiles and amphibians, and contrast with the continuous breathing patterns of nondiving birds and mammals in their apparent lack of constancy and intrinsic rhythm. Many researchers have ascribed the genesis of breathing episodes in amphibians and reptiles to the inherent oscillations of blood oxygen and/or CO₂/pH levels associated with intermittent breathing, rather than to the action of a "mammalian-type" central control mechanism. In this model, lung ventilations are induced when a certain arterial PO₂ (Pa_O2) or arterial PCO₂ (Pa_CO2) threshold is reached and breathing ceases when the blood gas values have been brought back within a certain range (79, 568, 649). The observation that breathing is completely suppressed when convective requirements are met by unidirectional ventilation (354, 357, 580, 649) indicates that lung ventilation is conditional upon a minimal stimulatory input (439, 576, 580, 649). However, these experiments were conducted with some degree of lung inflation, which may have overridden peripheral chemoreceptor drive. Preliminary evidence from experiments on decerebrate, paralyzed, and unilaterally ventilated toads suggests that pulmonary stretch receptor inputs may be important in the initiation of breathing. When fictive ventilation had been suppressed by unilateral ventilation, it was induced by lung deflation (640). Clearly, chemoreceptor and lung mechanoreceptor inputs influence the respiratory CPG, but their central interactions are unknown.

Several studies suggest that episodic breathing does not necessarily reflect the phasic nature of afferent chemoreceptor or mechanoreceptor inputs. Unidirectionally ventilated toads (580, 640, 649) can still display episodic breathing or fictive ventilation, although this experimental procedure has been assumed to maintain blood gases constant, and in paralyzed animals, lung distension constant, and thus produces only tonic chemoreceptor and mechanoreceptor input. These data imply that the mechanisms underlying episodic breathing may be an intrinsic property of the central respiratory control system, a view which seems confirmed by the observation that the motor output from a brain stem-spinal cord preparation of the bullfrog was episodic, in the absence of any possible feedback from the periphery (354). The central generation of these episodic breathing patterns has been localized to the nucleus isthmi in the brain stem of the bullfrog (354, 356). This mesencephalic structure is the neuranatomical equivalent of the pons in mammals, which contributes to the control of breathing pattern (202).

Whether episodic air-breathing is generated by central or peripheral mechanisms, it is vulnerable to inputs from centers higher in the CNS. In their recent review, Burggren and Infantino (90) described how adult male newts reduced air-breathing frequency to maximize time for courtship behavior toward females during the breeding season. Foraging or searching for prey can impact on surfacing behavior in amphibians. Larval salamanders supplied with benthic food showed reductions in buoyancy (which reflects degree of lung inflation) and frequency of air breaths compared with plankton feeders. These larvae also reduced air-breathing frequency during daylight hours, presumably to reduce the risk of aerial predation. A similar interpretation was placed on the very different periods of surface breathing between day and night in Xenopus laevis (288).

Lizards, in common with all other reptiles (except some crocodilians), lack a diaphragm. However, unlike modern amphibians, they do have ribs, and lung ventilation has long been considered to be generated by intercostal muscles acting on the rib cage, with a primitive buccopharyngeal or gular pump, like that described in amphibians, utilized primarily for olfaction. As lizards run in a serpentine manner, employing segmental muscles from the body wall, it was asserted by some investigators that they are unable to breathe while running. Recently these views have been questioned. Whole animal plethysmography, together with recordings of EMG from respiratory muscles, in the agamid lizard, Uromastyx microlepis, revealed that the prevailing mode of ventilation in the lightly anesthetized animal involved the intercostal muscles in triphasic lung inflation and deflation, with both passive and active expiratory stages, interrupted by periods of breath-hold (7). However, an alternative mode of ventilation involved a gular pump that alternated with the costal pump. After a short passive expiration, a bout of buccal pumping caused a progressive increase in lung volume, followed by breath-hold (Fig. 3). Gular pumping commenced as lightly anesthetized lizards were warmed from 30 to 35°C, as part of their normal daily cycle of temperature variation, and could be induced by tactile stimulation of conscious lizards. A parallel study, using X-ray imaging of varanid lizards, Varanus exanthematicus, has revealed that when at rest they rarely used an accessory gular pump. However, during recovery from exercise, all animals used gular pumping in addition to a costal pump, with between one and five gular pumping movements following costal inspiration. These clearly caused lung inflation, with caudal translation of the visceral mass (84). More recently, these lizards have been shown to employ gular pumping when walking, thus overcoming the supposed mechanical constraint on active lung ventilation during exercise (486).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Patterns of ventilation in lightly anesthetised agamid lizard, Uromastyx microlepis. Animal was inserted into a whole body plethysmograph to record lung volume changes. Activity in thoracic and gular pumps were recorded as electromyograms (EMG) from appropriate muscles. Recordings from intercostal muscles in thorax included electrocardiogram (ECG). Blood pressure was recorded from a cannulated femoral artery. Traces were from below: 1) lung ventilation recorded as pressure changes in plethysmograph (increased pressure signifies lung inflation); 2) EMG from intercostal muscles, together with ECG; 3) EMG from geniohyoid muscle; 4) EMG from sphincter colli muscle; 5) blood pressure. A series of thoracic aspiratory pumping movements, which inflated lungs, terminated in a bout of gular pumping, which also resulted in lung inflation. A second bout of gular pumping was seemingly initiated by a single thoracic breathing movement. Heart rate appeared unaffected, and variations in blood pressure did not appear to refer to breathing movements. (From M. Al-Ghamdi J. F. X. Jones, and E. W. Taylor, unpublished data.)

The existence of anatomically and functionally separate thoracic and gular respiratory pumps in lizards would seem to require separate sites of central respiratory rhythm generation. However, this interesting possibility remains unexplored. Putative sites of respiratory pattern generation, having similarities in neural organization and activation to those extensively documented for mammals, have been described for turtles (603). However, the direct contribution of these populations of neurons and their potential integration of sensory information in determining the generation of respiratory movements remain unclear (439). In turtles, the basic output of the CPG is episodic, even under experimental conditions when all sensory feedback appears tonic (178). Experiments performed on reptiles demonstrated that mild anesthesia and brain stem section at the level of the rostral rhombencephalon (metencephalon) abolish these breathing episodes, i.e., the animals now breathe in an uninterrupted fashion (461-463). Vagotomy also affects the breathing pattern by reducing the number of breaths per episode in crocodilians (461-463). It is interesting to note, however, that vagotomy had no effect on the breathing pattern when it was performed after episodic breathing had been abolished by a caudal midbrain transection (463).

As in amphibians, it has been suggested that the initiation of bouts of discontinuous breathing may owe more to thresholds for stimulation of central and peripheral chemoreceptors than to patterns dictated by a central rhythm generator (439). This may enable the flexibility of response essential for an ectothermic vertebrate, since the thresholds for stimulation will vary with temperature, in accordance with the animal's oxygen demand. However, unidirectionally ventilated alligators display episodic breathing (179) so that centrally generated rhythmicity may have a role in its initiation.

Birds, like their endothermic relatives the mammals, typically breath continuously and rhythmically, to supply their high demand for oxygen, thus sustaining their high metabolic rate. In both groups, ventilation is cyclic, with air sucked into the lungs during inspiration and expelled at expiration. However, birds do not have a diaphragm, and lung volume appears to vary little over the respiratory cycle. Instead, tidal volume is taken up by thin-walled, highly extensible air sacs. Respiratory gas exchange takes place over the walls of the well-vascularized parabronchi in the lung, which, because of the unique structure of the respiratory apparatus, are ventilated unidirectionally. The walls of the parabronchi bear air capillaries, the functional equivalent of mammalian alveoli, which are in intimate contact with blood capillaries, providing highly effective exchange conditions between blood and air, described as cross-current flow (553).

The respiratory rhythm in birds is assumed to arise from a CPG, evidenced by the virtually constant periods of inspiration and expiration recorded from birds at various levels of ventilatory output (440). Other respiratory variables, such as tidal volume and interbreath interval, do vary, presumably under the influence of inputs from central and peripheral receptors. Breathing hyperoxic gas mixtures reduces ventilation in birds, implying a chemoreceptive drive to ventilation in normoxia (553). Diving birds can show prolonged apneas, associated particularly with forced submersion or extended "escape" dives, during which stimulation of water receptors in the airways overrides respiratory drive. Control of the complex suite of reflex cardiorespiratory responses shown by diving birds to forcible submersion in the laboratory (apnea, profound bradycardia, and marked increase in peripheral resistance) and their very different responses during telemetered natural dives have been comprehensively reviewed (98, 589).

The pools of neurons in the medulla that generate the patterned activity driving the respiratory muscles in birds appear to resemble those described in mammals (153). A pneumotaxic center, similar in location and functional characteristics to that previously described in mammals, has been postulated to exist in dorsal mesencephalic regions of the brain (553). Sections caudal to this region abolish rhythmic respiratory activity that can, however, be reinstated by rhythmic electrical stimulation of the vagus nerves. The normal respiratory period in birds may be set by cyclical changes in lung CO₂ levels. In a unidirectionally ventilated bird preparation, when insufflated CO₂ levels were raised to stimulate spontaneous breathing cycles, then periodically varied around this level, the respiratory movements of the bird were found to lock onto the imposed fluctuations in CO₂ (553).

It has long been recognized that lung ventilation may be coordinated with wing beat in birds. Compressive effects of wing upstroke and expansive effects of downstroke may assist airflow through the lung, in coordination with activity in respiratory muscles. The correspondence between the two rhythms varies from a ratio of 1:1 in crows and pigeons up to 5:1 (wing beats per breath) in ducks and pheasants (95). Bats, as flying mammals, show similar patterns of coordination and also share a relatively large heart and increased blood oxygen capacity with their flying cousins, the birds. Respiratory frequency increases immediately upon take off in pigeons, indicating that a combination of central and peripheral nervous mechanisms, as well as mechanical considerations, is likely to be influencing the relationship. Stimulation of ventilation with CO₂ during flight did not alter the phasic coordination patterns between respiratory and wingbeat cycles in either pigeons or magpies (77), suggesting that neural interactions between control centers in the CNS are important. A potent influence of locomotor centers in the brain stem upon respiratory center motor output (or vice versa) in geese and ducks has been demonstrated by Funk et al. (207, 208). Their studies on decerebrate geese indicated that, in the absence of feedback from flapping wings, there was a predominantly 1:1 ratio between the two motor outputs, implying direct recruitment of one by the other. The various patterns of coordination seen in free-flying birds clearly require feedback from peripheral receptors.

	III. AFFERENT INNERVATION OF THE CIRCULATORY AND RESPIRATORY SYSTEMS

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The activity of the different types of sensory receptors in the cardiovascular system and the airways of mammals has been described in several reviews (123, 489, 490, 544). In addition, the cardiovascular and respiratory responses evoked in mammals by stimulation of arterial chemoreceptors have been reviewed very recently (138, 415). Accordingly, the well-known characteristics of these mammalian sensors and the responses they engender are not described here but are referred to, for comparison, in the descriptions of their equivalents in nonmammalian vertebrates, in which their roles are still not yet fully understood.

The central projections from the various reflexogenic sites in the mammalian cardiorespiratory system are, however, of direct relevance to the current account. A wide variety of afferent fibers transmitting sensory information arises from the heart, vascular, and ventilatory systems of mammals. Arterial baroreceptors are located in the walls of the carotid sinus and aortic arch while arterial chemoreceptor afferents are located in the carotid and aortic bodies, and probably elsewhere in the circulation. Both the atria and ventricles of the heart contain mechano- and chemoreceptive afferents in their walls. Within the respiratory system there is a wide variety of sensory afferents (both mechano- and chemosensitive) throughout the respiratory tract, from the nasal cavity to the alveolar walls. These circulatory and respiratory afferents include both myelinated and unmyelinated nerve fibers and are located mainly in the trigeminal, glossopharyngeal, and vagus nerves. In addition, activity in several types of somatic afferent can have actions on either or both the respiratory and cardiovascular systems. In general, in both systems, the different afferents can be split into those involved in homeostasis, which monitor ongoing activity, whereas others, involved in defensive type reflexes, are only activated by more aversive types of stimuli (120, 121).

Afferents from receptors in the cardiorespiratory system, travelling in the cranial nerves, terminate in the brain stem, in the NTS, and, to some extent, in the trigeminal nucleus. These make multiple synapses in distinct regions of the NTS and show a large amount of overlap in their terminal fields. This allows convergence of input onto postsynaptic neurons in the NTS and may form part of the neural substrate by which various afferent inputs are integrated into physiological response patterns, since it is well known from reflex studies that simultaneous activation of several afferent inputs may interact in either a positive or negative manner (see Ref. 121). At least some of these interactions occur as the afferent information arrives at the CNS. There is some evidence for polysynaptic convergence and interactions of afferent inputs on postsynaptic NTS neurons, but the extent of these interactions is still a matter of debate and has been discussed in detail previously (121, 328).

The topography of these central terminations has been studied by a variety of techniques. The earliest studies have been summarized and discussed previously (328). More detailed information has now become available and will form the basis of this description. Histological studies employing degeneration (129), or more recently anterograde axonal transport of neuronal markers (337), have demonstrated that vagal afferents terminate predominantly in the caudal two-thirds of the NTS, whereas glossopharyngeal afferents terminate in the rostral two-thirds, overlapping in the region around obex. In addition, there was a certain degree of topography of termination within the different subnuclei of the NTS, based on organ of innervation (118, 204, 337). Although there are different degrees of input to the different subnuclei of the NTS, there is no clear anatomical separation between the terminations of afferent fibers from the respiratory or circulatory systems.

These histological studies give little information about the function of the visualized afferents, a major restraint since both the vagus and glossopharyngeal nerve contain a large number of functionally different afferent fibers. Electrophysiological techniques have been used to map terminations of afferents, whose function had been identified (Fig. 4). This antidromic mapping technique has been used to delineate the terminal fields of slowly adapting (176) and rapidly adapting (156) pulmonary stretch receptor afferents, arterial baroreceptor and chemoreceptor afferents (174), and bronchial and pulmonary C-fiber afferents (370). Slowly adapting pulmonary stretch receptor afferents terminate rostral to obex, mainly in the ipsilateral medial subnucleus with some innervation of the lateral and ventrolateral subnuclei. This latter region is the location of the dorsal respiratory group (67, 188, 189). In contrast, rapidly adapting pulmonary stretch receptor afferents terminate more caudally, rostral and caudal to obex, mainly in the ipsilateral commissural nucleus, with less dense innervation of the medial and ventrolateral subnuclei and the contralateral commissural nucleus. Bronchial and pulmonary C-fiber afferents only project to medial regions of the NTS spanning the obex region. Unlike myelinated pulmonary afferents, there are no terminations in the lateral, ventrolateral, or ventral subnuclei of the NTS. Caudal to obex, the terminal fields are localized to the dorsal part of the commissural nucleus. This projection of C-fiber afferents is not dissimilar to that of arterial chemoreceptor afferent fibers that terminate in the medial and dorsomedial NTS and in the commissural nucleus (320).

View larger version (73K): [in this window] [in a new window]   Fig. 4. Summary of major regions of termination within nucleus tractus solitarius (NTS) of cat cardiovascular and pulmonary afferents as determined by antidromic mapping studies. Relative density of ipsilateral () and contralateral () regions of termination is denoted by number of dots, and most extensive regions of termination are shaded. [Modified from Jordan (321).]

Although antidromic activation can delineate the terminal regions of functionally identified afferent fibers, there are limitations when the question of the fine branches is addressed. The organization of preterminal processes and distribution of synaptic boutons for single pulmonary stretch receptor afferents (both slowly and rapidly adapting) has been described (338, 339) by microinjecting an HRP conjugate into axons impaled in the solitary tract, allowing direct visualization of the terminal fields of the labeled afferents. The intermediate, ventral, ventrolateral, and interstitial nuclei were the only regions of the NTS receiving terminals of slowly adapting receptor afferents, whereas rapidly adapting receptor afferents terminated in the intermediate, dorsal, and dorsolateral subnuclei more caudally. Similar studies (55, 557) have shown that laryngeal afferents terminate mainly in the ventral and ventrolateral NTS, with some projections to the interstitial, dorsolateral, medial, and dorsomedial nuclei.

Little is known about the postsynaptic neurons activated by stimulation of bronchial or pulmonary C-fiber afferents, although neurons in the commissural and caudal part of the medial NTS can be activated by stimulation of C fibers in pulmonary branches of the vagus (58). Although some of these neurons also received input from nonmyelinated afferents arising in the heart, they never received input from myelinated afferents, from either the heart or lungs (58). In recent studies we have confirmed that some neurons with these same properties are indeed activated when phenylbiguanide is injected into the right atrium (315, 317), whereas inspiratory neurons in the ventrolateral NTS are inhibited by this stimulus (314). Finally, arterial baroreceptor terminals are restricted to the ipsilateral NTS, rostral to the obex. The dorsolateral and dorsomedial subnuclei are the most often innervated, and the commissural nucleus also received some innervation (318). The central terminations of afferents arising in the heart have not been studied in such detail, but type A atrial receptor afferents have been shown to terminate in the dorsolateral and ventrolateral subnuclei (S. Donoghue and D. Jordan, unpublished observations).

In many species, activation of receptors in different parts of the upper respiratory tract evokes similar cardiorespiratory responses (see Ref. 135). In dogs, cats, and monkeys, stimulation of afferents in the superior laryngeal nerve (SLN) or nasal mucosa results in apnea, bradycardia, and vasoconstriction. The trigeminal nucleus is one site where convergence of such afferent information may take place, since it receives afferent input from some vagal and glossopharyngeal fibers (351, 626) and SLN (258, 599). Indeed, some SLN fibers bifurcate, one branch terminating in the NTS and the other in the rostral trigeminal nucleus (114). Neurons in both the rostral and caudal sensory trigeminal nuclei have been reported to receive a convergent visceral and somatic inputs from stimulation of the SLN, glossopharyngeal nerves, tooth pulp, and cutaneous facial mechanoreceptors (282, 561). In addition, Jordan and Wood (332) reported a group of neurons in the rostral trigeminal nucleus that were activated by SLN stimulation and mechanical stimulation of the nasal mucosa but were unaffected by tactile stimulation of other parts of the face. Finally, stimulation of trigeminal afferents has been shown to evoke a short latency response in vagal nerves (238).

Clearly, the neurons of the NTS and trigeminal nucleus are not simple relay stations. Integration between different afferent inputs can occur here, and there is some degree of functional organization within these nuclei. Unfortunately, such detailed information is not available from the other vertebrate groups, where similar studies have yet to be performed.

B.  Fish

1.  Chemoreceptors

Oxygen-sensitive chemoreceptors exert dominant control over cardiorespiratory reflexes in fish. The typical response to ambient hypoxia is a reflex bradycardia and increased ventilatory effort (607). Many studies support the existence of peripheral oxygen receptors on or near the gills of fish, and these were recently reviewed (93). However, the precise anatomical sites and functional properties of these peripheral chemoreceptors in fish remain uncertain. Saunders and Sutterlin (551) observed an increase in "breathing amplitude" in the sea raven when the dorsal aorta was perfused with hypoxic blood, and also when perfusing the dorsal aorta with normoxic blood during ambient hypoxia, which they regarded as evidence for both central and peripheral sites of oxygen receptor activity. In the sturgeon, cyanide stimulated ventilation, both when added to inspired water and when injected intra-arterially, indicating the presence of oxygen receptors sensitive to both internal and external milieu (425). In contrast, Eclancher and Dejours (182) observed a ventilatory and cardiac response only to an intravascular injection of cyanide; no response was evident to cyanide in the ventilatory water stream of teleosts, indicating that the PO₂ receptors are located internally. Daxboeck and Holeton (159) found that irrigation of the anterior region of the respiratory tract of the trout with hypoxic water caused a reflex bradycardia but no change in ventilation, whereas McKenzie et al. (425) found that cyanide added to the water stimulated a transient bradycardia in the sturgeon, whereas intra-arterial infusion was without effect on heart rate, implying that different receptors are involved in the induction of the two overt responses to hypoxic exposure. Ventilation rate in trout varied inversely with blood oxygen content, independently of partial pressure, indicating that arterial receptors respond to rate of delivery of oxygen to the receptor site (511). There is some evidence for receptor sites outside the branchial apparatus, including the proposed existence of venous oxygen receptors in fish (50, 617). Alternatively, Bamford (36) concluded that the most important site of oxygen detection in the trout is the brain.

The gill arches in fishes are innervated by cranial nerves IX and X, and it is these nerves that innervate the carotid and aortic bodies of mammals. Bilateral section of IX and X abolished the hypoxic bradycardia in the trout (584) but did not in elasmobranchs (547). Butler et al. (103) found it necessary to bilaterally section cranial nerves V, VII, IX, and X to abolish the hypoxic bradycardia in the dogfish and concluded that the oxygen receptors are distributed diffusely in the orobranchial and parabranchial cavities. Laurent et al. (384) recorded oxygen chemoreceptor activity from branches of cranial nerve IX innervating the pseudobranch in the tench. This organ is derived from the spiracle, which is open in elasmobranchs, and because it receives arterialized blood flowing from the gills, it is ideally suited to monitor blood oxygen levels. Although Smith and Davie (583) concluded that oxygen receptors were innervated by the IXth cranial nerve in the salmon, bilateral denervation of the pseudobranch in the trout had no effect on the changes in ventilation volume after exposure to hypoxia and hyperoxia (514). Afferent activity has been recorded from the branchial branch of the vagus innervating the first gill arch of tuna and trout (93, 443). Receptors that increased their rate of discharge in response to a decrease in the rate of perfusion or oxygen level of the perfusate also responded to ambient hypoxia. Fibers responding to hypoxic water showed an exponential increase in rate of discharge to decreasing external oxygen partial pressure, with a sensitivity similar to that exhibited by mammalian carotid body chemoreceptors (93).

Although fish have been shown to respond to hypercapnia, there is no clear evidence of a role for central chemoreceptors in the control of ventilation in fish (93, 232, 512). Although hypercapnic acidosis stimulated ventilation in channel catfish, Ictalurus punctatus (92), the response was abolished by branchial denervation, indicating that it resulted from stimulation of peripheral chemoreceptors, innervated by cranial nerves IX and X. These may be the same receptors that respond to oxygen. Mammalian carotid and aortic receptors respond both to oxygen and CO₂/pH (571).

Similarly, there is no evidence that chemoreceptor stimulation produces behavioral arousal in fish similar to the visceral alerting response that accompanies the stimulation of carotid chemoreceptors in mammals (415, 416). In fact, the unrestrained dogfish responds to environmental hypoxia with a reduction in activity, which remains suppressed throughout the hypoxic period, despite an increase in circulating catecholamines (431). This is analogous to the "playing dead" response shown by many animals, including some mammals (see Ref. 319), and would seem to be the opposite of a defense or alerting response. The absence of clear visceral alerting or baroreceptor responses in dogfish (see sect. IIIB2) precludes their interference in chemoreceptor-induced changes in ventilation or heart rate.

The respiratory muscles in fish contain length and tension receptors, in common with other vertebrate muscles, and the gill arches bear a number of mechanoreceptors with various functional characteristics. Satchell and Way (550) characterized mechanoreceptors on the branchial processes of the dogfish, and Sutterlin and Saunders (598) described receptors on the gill filaments and gill rakers of the sea raven. De Graaf and Ballintinjn (163, 164) described slowly adapting position receptors on the gill arches and phasic receptors on the gill filaments and rakers of the carp. They interpreted their function as maintenance of the gill sieve and detection of and protection from clogging or damaging material. Mechanical stimulation of the gill arches is known to elicit the "cough" reflex in fish (e.g., Ref. 547) and a reflex bradycardia (430, 604). These mechanoreceptors will be stimulated by the ventilatory movements of the gill arches and filaments, but there is no direct evidence that they contribute to respiratory control on a breath-by-breath basis (93). Stimulation of branchial mechanoreceptors by increasing rates of water flow may be the trigger for the cessation of active ventilatory movements during "ram ventilation" in fish (306, 511).

Despite the early recordings of apparent pressoreceptor responses in elasmobranch fish (e.g., Ref. 293), evidence for the involvement of baroreceptors in vasomotor control in fish remains contentious. The evolution of a role for baroreceptor afferents and for vasomotor control, exercised via the sympathetic nervous system, in control of the cardiovascular system, may be associated with the evolution of air-breathing. The gills of fish are supported by their neutral buoyancy in water. Ventilation of the gills generates hydrostatic pressures that fluctuate around, but predominantly above, ambient. Arterial blood pressures in the branchial circulation of fish and the pressure difference across the gill epithelia are relatively low, despite the fact that the highest systolic pressures are generated in the ventral aorta, which leaves the heart to supply the afferent branchial arteries. Consequently, the need for functional baroreceptors in fish is not clear.

Increased arterial pressure has been shown to induce a bradycardia in both elasmobranchs (409, 410) and teleosts (457). However, in both cases, the increase in pressure required to cause a significant reduction in heart rate was relatively high (10-30 mmHg), and dogfish seem not to control arterial pressure after withdrawal of blood (50, 595). In teleosts, injection of epinephrine, which raised arterial pressure, caused a bradycardia, abolished by atropine (516), whereas low-frequency oscillations in blood