Ventilatory Action of the Hypaxial Muscles of the Lizard Iguana Iguana: A FuncTion Of Slow Muscle

Recent observations suggest that lizards are unable to run and breathe at the same time (Carrier, 1987a). In the four species that have been studied (Iguana iguana, Ctenosaura similis, Varanus exanthematicus and Varanus salvator), lung ventilation is clearly disrupted at speeds greater than a slow walk. As speed increases above that of a walk, breath volume declines rapidly. The faster these lizards run the less they breathe. This is surprising given that the energetic cost of locomotion increases with speed (Bennett, 1982), and it contrasts with the enhanced ventilation that accompanies locomotion in birds and mammals. In flying birds and bats, and running mammals, lung ventilation not only increases with speed, but is phase-locked to the locomotor cycle (Suther et al. 1972; Thomas, 1981; Butler, 1982; Hornicke et al. 1983; Bramble & Carrier, 1983; Baudinette et al. 1987; Jenkins et al. 1988; Bramble, 1989). In birds and mammals, the locomotor movements may actually facilitate breathing. Thus, diminished breathing in running lizards is an unexpected finding.

A physical conflict between the locomotor and ventilatory systems may be responsible for the reduced breathing in running lizards (Carrier, 1987a,b). The actions of the ventilatory muscles might differ, in an opposing fashion, from the actions of the locomotor muscles. If this were true, locomotion and ventilation would place conflicting demands on the thorax, limiting the capacity for ventilation whenever the animals walked or ran.

The possibility of a physical constraint on simultaneous running and breathing cannot be addressed directly because very little is known of how lizards ventilate their lungs. Lizards are known to be aspiration breathers (Milsom, 1984). That is, they draw air into their lungs by actively deforming the walls of the thoracic cavity to create a subatmospheric pressure. However, the axial muscles responsible for these ventilatory movements have not been identified in lizards. In contrast, the muscles responsible for ventilation have been studied extensively in mammals (De Troyer & Loring, 1986) and have received limited attention in turtles (Gans & Hughes, 1967; Gaunt & Gans, 1969), crocodilians (Naifeh et al. 1970; Gans & Clark, 1976) and birds (Kadono et al. 1963; Fedde et al. 1964a,b). Hence, Lepidosauria (i.e. squamates and Sphenodom, Gauthier et al. 1988) is the one remaining group of amniotic tetrapods for which the basic mechanism of lung ventilation has not been determined.

This study used electromyography to identify those muscles responsible for ventilation in the lizard, Iguana iguana. The aim was to increase our understanding of how lizards breathe and to provide a basis for further investigations of locomotor-ventilatory coupling in lizards. During the course of the investigation, questions arose concerning the contractile physiology of the muscles responsible for ventilation. Consequently, in addition to characterizing the activity patterns of the ventilatory muscles, data are presented that are relevant to the function of slow muscle.

The experiments in this study were carried out on 12 adult green iguana (Iguana iguana, 600–1735 g) that were obtained from local animal suppliers and by courtesy of Dr Dagmar Werner of the Smithsonian Tropical Research Institute. They were housed in large cages with a photothermal gradient on a 12h:12h light: dark photoperiod, and were fed a diet of Romaine lettuce and Iguana Chow (Zeigler Bros, Inc.).

For experiments requiring surgery, the lizards were anaesthetized by an initial intramuscular injection of 140 mg of Ketamine per kilogram body mass, followed by one-third the initial dosage, as needed. Throughout the various experiments body temperature of the lizards was monitored with a cloacal thermometer and maintained at 30–35 °C.

Thoracic pressure was measured in anaesthetized animals, with a Statham PM5 pressure transducer. To accomplish this, an incision was made in the skin of the throat and the muscles overlying the trachea were reflected. The trachea was then cannulated with a small-diameter tube (<one-third the diameter of the trachea) the end of which was advanced into the lung.

Inspiratory air flow was monitored in both anaesthetized and fully active animals, with a thermistor flow meter (Thomas, 1981). This provided simultaneous and independent measurements of the direction of air flow and tidal volume. The probe of the flow meter was attached over one of the external nares and did not appear to annoy the animals in any way (Carrier, 1987a).

Changes of thoracic pressure resulting from contraction of individual muscles were measured in four anaesthetized animals. In these animals, the skin on one side of the trunk was reflected. Starting with the most superficial layers, successive muscles were stimulated directly (1-ms square wave pulses between 0·2 and 1·0 V, just above threshold level) with pin electrodes held on the surface of the muscle. Low voltages were used in an effort to restrict contraction to those muscle fibres closest to the stimulation site. Although the observed changes in thoracic pressure were quite small, the direction of pressure change could be recorded. A response profile for each muscle was obtained by moving the electrode from site to site.

Slow muscle can be differentiated from twitch muscle by its response to immersion in depolarizing solutions and by its pattern of innervation, although there are exceptions (Lannergen, 1979; Johnston, 1985). In depolarizing solutions, twitch muscle either does not contract or contracts and then relaxes quickly. In contrast, sustained depolarization of slow muscle results in a prolonged contraction, lasting many minutes (Morgan & Proske, 1984). Differences in innervation are equally distinct. Twitch muscle fibres generally have a single, centrally located endplate, whereas slow fibres have multiple, small endplates spaced at 0·2-to 2 mm intervals along the cell (Morgan & Proske, 1984).

The occurrence of slow fibres in the intercostal muscles was tested by measuring isometric contractile properties. Anaesthetized animals were killed and small segments of internal or external intercostal muscle were removed from the fourth intercostal space (i.e. between sternal ribs 2 and 3). Small portions of the two ribs were removed with the muscle segments. One rib segment was anchored to the bottom of a 500-ml constant-temperature (40° ±l°C) bath of Ringer’s solution (155 mmol I⁻¹ NaCl, 4 mmol 1⁻¹ KC1, 2 mmol 1⁻¹ CaCl₂, 2 mmol 1⁻¹ phosphate buffer, pH7·2). The other end of the muscle was anchored with surgical silk (00 gauge) to a Cambridge Technology 300H Servo muscle lever. Output of the transducer was passed to a Honeywell 117 d.c. amplifier, reduced to one-tenth the original voltage and then recorded on a Gould Brush 481 recorder and stored on magnetic tape in a Honeywell 5600 medium band-path tape recorder.

Contractile properties were measured after 5–10 min of thermal equilibration. The muscles were first stimulated through two aluminium plate electrodes using a Grass S44 stimulator. Twitch contractions were elicited with single pulses of 1-ms duration and 40-80 V. The capacity of the intercostals to contract tonically was then measured by depolarizing the cells with high extracellular potassium. The Ringer’s bath was drained and quickly replaced by Ringer’s solution with 155 mmol 1⁻¹ KC1 and no NaCl. Tensions are reported as g cm⁻² of cross-sectional area. Cross-sectional area was estimated by dividing muscle mass by muscle length.

The distribution of motor endplates in the hypaxial muscles was determined by staining whole muscles with the cholinesterase stain of Karnovsky & Roots (1964). Freshly dissected whole muscles were pinned at resting length in an incubation chamber. The muscles were immersed in the acetylthiocholine iodide medium for 2–4h and then placed in 1 % ammonium sulphate for 1–2min.

The axial muscles responsible for ventilation were determined electromyographically. The hypaxial muscles are thin sheets and in some cases very difficult to reach surgically. Consequently, a variety of barbed bipolar and patch electrodes (Loeb & Gans, 1986) were employed. In one set of experiments the animals were anaesthetized, the skin and external oblique were reflected on one side of the body and up to 34 bipolar electrodes (75 μm diameter stainless-steel wire, Teflon coated) were inserted in the intercostal and underlying muscle layers. Bared electrode tips 0·5 mm long were placed approximately 1–2mm apart within the muscle. These electrodes were monitored four at a time, along with thoracic pressure and air flow in the anaesthetized animal.

In a separate set of experiments, patch electrodes were implanted surgically throughout the hypaxial musculature. Patch electrodes have two advantages when recording from successive thin muscle layers. First, they can be positioned and held in place between muscle layers that are much too thin to contain a standard barbed bipolar electrode. Second, they provide electrical insulation from one direction, thus greatly reducing the effects of cross-talk from other muscles. Bared electrodes were approximately 1·0 mm long and spaced 1·0 mm apart on 5- ta 10-mm square patches of Dow Corning 501–1 Silastic reinforced sheeting.

Electrode wires passed percutaneously to exit points along the midline of the back. At the exit point the wires were glued to the skin and soldered to gold connector pins. The animals were allowed to recover from the anaesthesia, and muscle activity and air flow were monitored during both quiet breathing and heavy breathing induced by vigorous locomotor activity.

The EMG signals were passed to Tektronix FM 122 preamplifiers, amplified 1000 times and filtered below 8 Hz and above 10 kHz. Signals were then passed to Honeywell 117 d.c. amplifiers, and simultaneously stored on a Honeywell 5600 tape recorder and printed out on a Gould chart recorder. To provide a comparison of the amplitude of the EMG activity and tidal ventilation, some recordings were digitized by an IBM AT microcomputer through a Keithley 570 analog-to-digital converter. Data were collected at 2500 Hz, and analysed with a program that recorded the number and amplitude of individual spikes (Beach et al. 1982). Multiplication of the number and average amplitude of the spikes occurring in a given interval provided a measure of the EMG activity.

Fast Fourier transformations (FFT) were performed on selected electromyograms to determine the range of dominant frequencies. Data were digitized at 2500 Hz, maintaining a frequency sensitivity of 1250 Hz based on Nyquist sampling criteria. The Cooley & Tukey (1965) algorithm was used in a program that calculates an FFT using a sample size of any power of two.

The axial musculoskeletal system of Iguana iguana is described elsewhere (Carrier, 1988). Only those muscles and bones that play a role in lung ventilation are noted here.

The anterior 10 ribs enclose the thoracic cavity (Fig. 1). The first three thoracic ribs lack costal cartilages and do not articulate with the sternum. The next seven ribs (sternal ribs) are composed of a bony element that articulates with the vertebrae and a costal cartilage that articulates with the sternum. The vertebral segments extend laterally and ventrally to the mid-body wall and have a circular cross-section. In contrast, the costal cartilages are composed of fibrocartilage that is readily deformable.

The external intercostal muscle runs from rib to rib in the dorsal-lateral thorax (Fig. 1). The muscle extends dorsoventrally, from the vertebral centra to just below the bend in the costal cartilage. Fibres run anteriorly and dorsally at an angle of 30–40° from the horizontal. The muscle becomes progressively thinner Jentrally.

The internal intercostal muscle lies medial to the external intercostal and occupies a more ventral position in the thorax (Fig. 1). Fibres run from rib to rib, directed anteriorly and ventrally. The muscle extends dorsoventrally from the mid-portion of the vertebral ribs to the ventral aspect of the costal cartilages. Dorsal to the bend in the costal cartilages the fibres lie at an angle of about 60° to the horizontal. Fibres below the bend have a more shallow orientation of about 20° to the horizontal. Posterior to the last true rib a slip of this muscle extends dorsally to attach to the lumbodorsal fascia.

The transversalis is the innermost muscle of the lateral body wall (Fig. 1). It is quite thin, but continuous from girdle to girdle. Dorsally, it attaches at the same sites as the internal oblique: lumbodorsal fascia and the mid-portion of the vertebral ribs. The fibres run ventrally and slightly caudally to insert on the dorsal aspect of the rectus abdominus and in the thoracic region on the ventral-most portions of the costal cartilages.

The retrahentes costarum muscle is confined to the dorsal thoracic region, deep to the intercostal musculature (Fig. 1). Fibres attach to the ventral aspect of the centra of the vertebrae. They course anteriorly and ventrally to insert on the mid portion of the vertebral element of each sternal rib.

Green iguanas are aspiration breathers. They pump air out of their lungs by increasing thoracic pressure above atmospheric (Fig. 2). They then fill their lungs by the generation of a subatmospheric thoracic pressure. Pressure changes during regular breathing were of quite low magnitude, generally less than 98 Pa (1 cmH₂O). However, thoracic pressures could be much higher (621 ± 37·3 Pa in a 1060 g individual) when the lungs were filled during routine periods of apnoea.

The ribs could be seen to swing posteriorly during expiration and anteriorly during inspiration. Manual simulation of these rib displacements produced changes in thoracic pressure in anaesthetized animals or fresh cadavers. Anterior rotation of the ribs decreased thoracic pressure, whereas posterior rotation increased pressure. This held for each of the seven sternal ribs.

In anaesthetized iguanas, direct unilateral stimulation of small portions of individual axial muscles produced changes in thoracic pressure that were muscle- and/or site-specific. The direction and amplitude of these changes of pressure were largely independent of the phase of ventilation. Most thoracic muscles increased thoracic pressure when they were stimulated individually. Stimulation of those muscles that lie lateral to the ribs (i.e. external oblique and iliocostalis) as well as those medial to the ribs (i.e. internal oblique, transversalis, retrahentes costarum) increased thoracic pressure. The external oblique, iliocostalis and retrahentes costarum appeared to effect this increase in thoracic pressure by rotating the vertebral portion of the ribs caudally. However, stimulation of the thoracic portions of the internal oblique and transversalis complex could be seen to pull the costal cartilages medially, actually deforming these flexible skeletal elements. In these five muscles stimulation produced increased thoracic pressure, no matter which portion of the muscle was activated.

In contrast, stimulation of the intercostal muscles produced either increased or decreased thoracic pressure, depending on the site of stimulation. For any given intercostal segment, separate stimulation of the external or internal intercostal muscles usually had the same effect on thoracic pressure, and this response was largely independent of the phase of ventilation. Stimulation of anterior segments decreased thoracic pressure, whereas stimulation of posterior segments increased thoracic pressure (Fig. 3). However, there was a significant amount of variation among individuals. In some, decreased thoracic pressure resulted from activation of only the anterior three intercostal segments, whereas in others activation of the anterior five segments decreased thoracic pressure.

Those appendicular muscles which originate on the ribs also influenced thoracic pressure. Stimulation of the pectoralis and latissimus dorsi increased thoracic pressure, whereas stimulation of the serratus ventralis decreased it.

Muscle fibres of both the external and internal intercostal muscles displayed two distinct patterns of innervation (Fig. 4). The lateral portion of each muscle was composed entirely of fibres innervated by a single, centrally located motor endplate (Fig. 5A). The endplates of these singly innervated fibres were large and compact and stained darkly. They displayed the typical en plaque configuration of twitch fibres (Hess, 1970). In contrast, the medial portion of each muscle was composed predominantly of fibres that were multiply innervated by small motor endplates. Five to eight endplates were spaced at intervals of approximately 1–2 mm along the fibre (Fig. 5B). These motor terminals were composed of many small expansions of the nerve ending, and displayed the typical en grappe configuration, generally associated with slow fibres. Interspersed among these multiply innervated fibres were a few singly innervated fibres. In summary, both the external and the internal intercostal muscles were composed of a lateral portion of singly innervated fibres and a medial portion of predominantly multiply innervated fibres.

Three other hypaxial muscles, the external oblique superficialis, transversalis and retrahentes costarum, also contained multiply innervated fibres. As in the intercostal muscles, these three muscles were stratified, with the multiply innervated fibres located only on the medial side. The internal oblique and the rectus abdominus contained only singly innervated fibres. The epaxial muscles and the quadratus lumborum also appeared to be composed of singly innervated fibres, but only superficial surfaces of these muscles were examined.

The isometric contractile properties of the external and internal intercostal muscles, between sternal ribs 2 and 3, are summarized in Table 1. Segments of both the external and internal intercostal muscles underwent a tonic contraction when immersed in a bath of Ringer’s solution of high potassium content. This was a prolonged contraction in which the muscle took 1–3 min to reach maximum tension and 10–15 min to reach half-relaxation. Maximum forces generated during these tonic contractions were 0·5–3 times the forces produced in twitch contractions by the same samples. The tonic force varied, depending on the site from which the segment was taken. Segments from the most ventral part of the internal intercostal showed a much lower tonic response than segments of either muscle taken from the lateral body wall. This is consistent with the distribution of multiply innervated fibres in the internal intercostal (Fig. 5).

Preliminary attempts to record muscle activity during lung ventilation were largely unsuccessful. Occasionally, low-amplitude activity was recorded during expiration from one or more of the deep hypaxial muscles, but none of the hypaxials displayed any activity during inspiration. This posed a problem because the ribs move anteriorly during inspiration. Analysis of fibre orientations of the various hypaxial muscles indicated that the only muscles mechanically capable of moving the ribs anteriorly were the intercostals. However, repeated attempts to record electrical activity from the intercostals suggested that they did not play a role in any phase of ventilation.

The apparent necessity of intercostal involvement in inspiration, combined with the lack of EMG activity, led to the suggestion that inspiration might be produced by slow muscle fibres. Because slow muscle fibres do not generate action potentials, their activity is relatively difficult to monitor with standard electro myography. The electrical activity of slow fibres can be recorded with extracellular techniques (Hetherington & Lombard, 1983), but the amplitude and frequency of these signals are much lower than those generated by twitch fibres that propagate action potentials. Consequently, to address the possibility that slow fibres were involved in ventilation, the distribution of twitch and slow fibres in the hypaxials was first determined as described above, and then activity was recorded from patch electrodes positioned directly over regions composed predominantly of multiply innervated fibres. Additionally, the low-pass filter of the preamplifiers was set at 8 Hz so as not to filter out the lower-frequency signals of slow fibres. This gave positive results.

Active inspiration appeared to be powered solely by the two intercostal muscles. Multiply innervated fibres on the medial side of both the external and internal intercostal muscles from the anterior rib segments (1–4) were active throughout the inspiratory phase (Fig. 6). In contrast, patch electrodes placed on the lateral side, over the singly innervated fibres, of these same segments or on either side of the more posterior segments showed no activity during ventilation. During inspiration, electrical activity in the anterior intercostals was bilaterally symmetrical (Fig. 6C), and positively correlated with breath amplitude (Fig. 7). Thus, deeper inspiration required greater muscular activity from the intercostal muscles.

In contrast, neither intercostal muscle was active during expiration. Expiration was associated with activity in the retrahentes costarum (Fig. 6A) and in the internal oblique-transversalis complex (Fig. 6B). Activity of the inspiratory and expiratory muscles showed little or no temporal overlap.

Most of the axial muscles were not involved in lung ventilation. The epaxial muscles and the greater portion of the hypaxial musculature displayed no electromyographic activity, even during the most vigorous ventilation. In particular, the rectus abdominus, external oblique, quadratus lumborum and the major portion of both intercostal muscles were silent during ventilatory activity. Although the multiply innervated fibres of the medial surfaces of the anterior intercostal segments were active during inspiration, analogous fibres in the more posterior rib segments (5–9) were not (Fig. 8).

The initiation and cessation of muscular activity was closely correlated with the phases of ventilation (Fig. 9). Activity in the two intercostal muscles began roughly 100–230 ms before the start of inspiratory air flow, ending some 120–250 ms before inspiration stopped. The two expiratory muscles became active 40–150 ms before expiratory air flow began, activity ceasing 50–110 ms before the end of expiration.

The relatively long delays between muscle activity and the initiation and cessation of air flow appeared to result from a slow development of muscular force. The long delays were not a result of the response time of the recording equipment. The response time of the flow direction circuitry was of the order of a few milliseconds (compare the change in flow direction to the change of sign of thoracic pressure in Fig. 2). Furthermore, during rapid breathing (e.g. Fig. 6), the glottis was held open and did not affect air flow. This suggests a relatively long interval between activation of the ventilatory muscles and the generation of forces sufficient to affect thoracic pressure.

The amplitude and frequency of the electrical signals generated by the intercostal muscles varied dramatically, depending on the type of movement the animal engaged in. Fig. 10 compares an electromyogram recorded from a patch electrode on the internal intercostal muscle during inspiration to one recorded from the same electrode while the animal changed the position of its body. The maximum peak-to-peak amplitude of the inspiratory signal was less than 0·2 m W whereas the maximum amplitude of the postural signal was roughly 1·2 mV. Fourier transformations revealed differences in the frequencies of the two signals. The dominant frequencies during ventilation were below 100 Hz, with greatest activity around 30 Hz. These low frequencies were also produced during postural or locomotor movements. However, the dominant frequencies during postural adjustments were much higher, ranging from 100 to 700 Hz.

The basic pattern of ventilatory air flow in lizards has been described for a number of species (Gans & Clark, 1978; Cragg, 1978; Milsom, 1984). Green iguanas conform to these published accounts. In resting lizards, ventilation begins with expiration, which is followed immediately by inspiration. Usually a single ventilatory cycle, or a few cycles, will be followed by a prolonged period of breath holding. This periodic pattern of breathing has been shown to minimize the work of breathing in the Tokay gecko (Milsom & Vitalis, 1984; Milsom, 1984). When metabolic demand is increased, as it is after a bout of exercise, periods of breath holding are less common and may be absent (Milsom, 1984; Carrier, 1987a).

In green iguanas, inspiration results from the production of a subatmospheric pressure in the thoracic cavity. Several observations indicate that the reduced thoracic pressure depends on activity of the anterior intercostal muscles. First, fibre orientation and direct electrical stimulation suggest that the intercostals are the only hypaxial muscles that are mechanically capable of moving the ribs craniolaterally, and thereby expanding thoracic volume. Second, electromyographic recordings show that both the external and the internal intercostals from the anterior intercostal segments are active throughout the period of inspiratory air flow. In contrast, the intercostal muscles from the four posterior intercostal segments and the other hypaxial muscles are not active during inspiration. Third, the level of EMG activity recorded from the anterior intercostal muscles is positively correlated with the depth of inspiration.

The electromyographic signals produced during inspiration are lower in amplitude and frequency than the signals produced during postural or locomotor movements of the trunk. The low-amplitude, low-frequency signals resemble those produced by the slow myotomal muscle of elasmobranch fishes (Bone, 1966) and the slow opercularis muscle of frogs (Hetherington & Lombard, 1983). Such signals have been suggested to be characteristic of the junctional potentials produced by slow (non-twitching) fibres (Roberts, 1969; Fetcho, 1987).

Junctional potentials of slow muscle fibres have been compared to action potentials of fast fibres in the lizard, Tiliqua nigrolutea (Proske & Vaughan, 1968) and the snake Thamnophis sp. (Ridge, 1971). In these species, intracellular recordings show that the amplitudes of the junctional potentials of slow fibres are roughly 6–10 times lower than the amplitudes of action potentials (10–20 mV compared to 120mV). Furthermore, the time of half-decay of potentials is 10–20 times longer in slow fibres than in fibres that propagate action potentials (40–70 ms compared to 3–10ms). These differences seem to provide an explanation for the two types of electrical signals produced by the intercostal muscles. The electromyographic signals produced during inspiration have amplitudes and frequencies that are consistent with intracellular recordings of junctional potentials of slow fibres. In constrast, signals produced during postural movements appear to be recordings of action potentials.

Several additional observations suggest that inspiration is produced by slow fibres rather than twitch fibres. First, electrical activity can only be recorded from the multiply innervated side (i.e. medial) of either intercostal muscle. Second, these multiply innervated fibres produce a tonic contraction when immersed in a depolarizing solution. Finally, the latency period between activation of the muscles and detectable changes in thoracic pressure is longer than would be expected for twitch fibres. Thus, although inspiration is a phasic activity, it is produced by tonic (i.e. slow) muscle fibres.

During expiration, electromyographic activity was recorded from only the retrahentes costarum and the internal oblique-transversalis complex. This activity was consistently of low frequency and amplitude. The low-frequency (<50Hz) signals indicate that slow, and not twitch, muscle fibres are responsible for expiration. The internal oblique, however, is composed entirely of twitch fibres. Therefore, the EMG signals recorded from the internal oblique-transversalis complex must have been produced by the transversalis.

Two additional observations indicate that the ventilatory action of the retrahentes costarum and transversalis is to decrease thoracic volume. First, their fibre orientation is such that contraction will act to decrease thoracic volume. The retrahentes costarum decreases thoracic volume by pulling the vertebral ribs posteriorly. In contrast, because the ventral body wall is more compliant than the dorsal body wall, contraction of the transversalis pulls the deformable costal cartilages medially. Second, direct electrical stimulation of either of these muscles produces an increase in thoracic pressure. Thus, in green iguanas, the transversalis and retrahentes costarum are the main agonists of expiration.

In addition to the retrahentes costarum and transversalis muscles, four other hypaxial muscles could participate in exhalation. The orientation of their fibres and direct electrical stimulation indicate that the external and internal obliques and both intercostals are mechanically capable of decreasing thoracic volume. Indeed, these muscles are active when the animals cough (D. R. Carrier, unpublished observations). However, extensive electromyographic monitoring provided no direct evidence of activity in these muscles during regular expiration.

The ventilatory action of the intercostal muscles remains controversial (see Otis, 1986; De Troyer et al. 1985). The most widely held notion of how the intercostals produce lung ventilation was first proposed by Hamberger (1727). He realized that the caudoventral orientation of the external intercostal gives its caudal insertions longer moment arms around the centre of rotation of the ribs than the cranial insertions. Hence, when the muscle contracts, the moments tending to move the ribs anteriorly will be greater than those tending to rotate the ribs posteriorly. In contrast, the caudodorsal orientation of the internal intercostal results in larger moments tending to rotate the ribs posteriorly than those tending to rotate them anteriorly. Thus, Hamberger suggested that the external intercostals should increase thoracic volume and be inspiratory in function, whereas the internal intercostals should deflate the thorax. Experimental support for this hypothesis has been provided by work on mammals (Bronk & Ferguson, 1935; Taylor, 1960).

However, there is increasing evidence that the two intercostal muscles do not display the antagonistic pattern predicted by Hamberger. In mammals (Gesell, 1936) and birds (Kadono et al. 1963; Fedde et al. 1964a), anterior portions of external and internal intercostals are active during inspiration, whereas posterior portions of the two muscles are active during expiration. In green iguanas, this study found that anterior portions of both intercostal muscles are active during inspiration, but neither is involved in expiration. During ventilation in all these animals, the external and internal intercostal muscles act as synergists to pump air in and out of the lungs. Thus, in contrast to Hamberger’s model, the different fibre orientations of the two intercostal muscles seem of little significance to ventilatory function.

Recent experiments in dogs suggest that the ventilatory action of the intercostal muscles is determined by the relative resistance of the ribs to anterior versus posterior displacement (De Troyer et al. 1985). In dogs, separate stimulation of the external and internal intercostal muscles causes similar displacement of the ribs. When either of these two muscles is stimulated at low thoracic volume there is a net anterior displacement of the ribs. In contrast, at high thoracic volume, stimulation causes a net posterior displacement of the ribs. De Troyer et al. (1985) concluded that the relative anterioposterior stiffness of the body wall is more important to intercostal action than their respective fibre orientations.

The present study shows that, in green iguanas, stimulation of either external or internal intercostal muscles from anterior segments decreases thoracic pressure, whereas stimulation of either muscle from posterior segments increases thoracic pressure. These observations are consistent with the findings in dogs, and are inconsistent with the older literature.

Muscle fibres of the striated muscles of vertebrates can be divided into two broad categories: twitch and slow (Hess, 1970; Morgan & Proske, 1984). A certain level of confusion exists in the literature because twitch fibres are also divided into fast- and slow-contracting categories. In addition, there are also intermediate fibres which display characteristics of both slow and twitch fibres (Lannergren, 1979; Morgan & Proske, 1984; Johnston, 1985). However, slow fibres are a distinct class that differ from twitch fibres in internal structure, pattern of innervation and contractile physiology. The physiology of slow fibres differs from that of twitch fibres in three principle ways (Morgan & Proske, 1984). First, slow fibres are unable to propagate an action potential. Thus, they cannot be stimulated by a single pulse and do not twitch. Second, slow fibres have a much slower isotonic shortening speed. Third, when immersed in depolarizing solutions, slow fibres contract tonically, maintaining tension for prolonged periods (i.e. minutes). Twitch fibres, in contrast, contract only briefly or do not contract at all. These differences in contractile physiology suggest that slow and twitch muscle might serve separate functions.

Unfortunately, the function of slow muscle has received only limited attention. Studies of contractile and energetic properties led early workers to suggest that slow muscle is ideally suited to the maintenance of body posture (Fulton, 1926). Slow muscle shortens very slowly, can maintain tension for prolonged periods with only minimal energy expenditure (Kuffler & Williams, 1953) and is reported to be grossly inefficient at performing isotonic work (Goldspink et al. 1970). These characteristics suggest that slow muscle is ill-suited to the production of active movements. However, in several cases, slow muscle has been shown to contract phasically to produce isotonic work. Among these are slow swimming in fish (Bone, 1978) and aerial hearing or possibly buccal pumping in frogs (Hetherington & Lombard, 1983). The present study shows that green iguanas use slow fibres of the hypaxial muscles to pump air in and out of their lungs.

The contractile physiology of slow muscle may explain why it is used to ventilate the lungs of lizards. First, in tetrapods, breathing is a relatively slow process. Only during thermoregulatory panting (Richards, 1970) or during locomotion in birds and mammals (Butler, 1982; Bramble & Carrier, 1983) do ventilatory frequencies exceed 2Hz in animals heavier than 100g (Lasiewski & Calder, 1971; Stahl, 1967). Ectotherms breathe particularly slowly. The highest ventilatory frequencies of lizards occur immediately following vigorous activity and usually do not exceed 0-5Hz (Carrier, 1987a). These low frequencies indicate that ventilation could be accomplished by slow fibres. Second, contrary to the observations of Goldspink et al. (1970), there is evidence that slow-contracting muscle can be very efficient at doing work (Woledge, 1968; Morgan & Proske, 1984). Finally, ventilatory muscles must be nonfatigable. Although there appear to be no studies of the relative fatigability of slow muscle, the low metabolic rate of slow muscle (Morgan & Proske, 1984) suggests that it should be highly resistant to fatigue. Thus, slow muscle appears to be well suited to the requirements of aspiration breathing in lizards.

The possibility remains that the muscle fibres which ventilate the lungs are capable of propagating action potentials. That is, in addition to contracting in a slow graded manner, these fibres might also be able to twitch. Such intermediate fibres have been identified in the toad Xenopus laevis (Lannergren, 1979). The present study did not directly investigate whether the multiply innervated fibres associated with ventilation could twitch. The important point, however, is that the hypaxial muscles do not propagate action potentials during lung ventilation.

Slow and twitch fibres in the intercostal muscles of iguanas appear to have different functions. Fourier transformations of the EMG signals from the intercostal muscles show that during ventilation the intercostal muscles produce only low-frequency signals. As suggested above, such low-frequency signals appear to be characteristic of the junctional potentials produced by slow fibres. In contrast, postural changes (i.e. locomotor movements) are associated with much higher frequency EMG signals in these same muscles. Such high-frequency signals (i.e. 100-700Hz) are typical of the electrical activity of muscle fibres that propagate action potentials (i.e. twitch fibres). These observations suggest that the intercostal muscles are important to both ventilation and locomotion, but that different categories of fibres are used to produce these movements.

Slow muscle fibres are widely distributed in both the axial and appendicular muscles of most ectothermic tetrapods (Hess, 1970; Guthe, 1981; Morgan & Proske, 1984). The extent to which slow muscle is used by these animals to produce active movements deserves further investigation. If slowly contracting muscle is particularly efficient at doing work, as Woledge (1968) suggests, then slow muscle might be employed much more commonly than we now realize in behaviour that requires slow movements. The fact that slow muscle is generally not associated with active movements may be more a reflection of the frequency at which our electrical generators create alternating current than biological reality. Biologists attempting to record electrical signals from active muscles typically set their high-pass filters at 80 or 100Hz. This effectively removes the ubiquitous 50 or 60 Hz noise originating from the electrical systems in modern buildings. Unfortunately, filtering below 100 Hz also eliminates the low-frequency signals produced by contracting slow muscle. In many electromyographic studies, the activity of slow muscle may simply have been hidden.

I thank Joe R. Fetcho, Carl Gans, Richard I. Hume, Richard L. Marsh, John M. Olson and Peter A. Pridmore for discussions that were crucial to the development of this investigation. I also thank William R. Dawson, William K. Milsom and Paul W. Webb for comments on early versions of the manuscript. Dagmar Werner of the Smithsonian Tropical Research Institute donated eight adult green iguanas. Chris Young wrote the program that performed fast Fourier transformations. Bruce M. Carlson and Sally J. Schroeter provided advice on histology. Special appreciation is extended to Carl Gans for his advice and support throughout this investigation. Financial support was provided by the Graduate School of The University of Michigan and by NSF DEB 8509490 to C. Gans.