Laser vibrometric studies of sound-induced motion of the body walls and lungs of salamanders and lizards: implications for lung-based hearing.

A laser Doppler vibrometer was used to measure the acoustic responses of different body surfaces of several species of salamanders and lizards. The lateral body wall over the lung displayed sound-induced motion up to 30 dB greater than the lateral head surface from 300-1,000 Hz in salamanders and from 200-2,500 Hz in lizards. The lateral body wall of lungless plethodontid salamanders showed no such enhanced motion to sound. The lateral body wall of lizards was more responsive than their tympanum to sound frequencies below about 1,250-2,000 Hz. The frequency of the peak response of lizard body walls matched the resonant frequency of a Helmholtz resonator with the volume and dimensions of their lungs. In contrast, the frequency of peak response of salamander body walls was well below the resonant frequencies calculated for both Helmholtz resonators and closed tubes with the dimensions and volumes of their lungs. Nonetheless, filling the lungs with saline dramatically reduced the responsiveness of the lateral body walls of both the lunged salamanders and the lizards. As previously demonstrated in anuran amphibians, the lateral body wall and lungs of salamanders and lizards may function in sound reception, especially at relatively low frequencies.

Biomechanical and neurophysiological studies on audition in eared and earless harlequin frogs (Atelopus).

Tissue displacement of various body surfaces and the auditory midbrain sensitivities to sound were measured in Atelopus species with or without a tympanic middle ear ("eared" and "earless", respectively). Tissue displacement (vibration) of body regions was measured by laser Doppler vibrometer. The body wall directly overlying the lung is most dramatically displaced by sound pressure in all species tested. The otic (lateral head) region showed low displacement in earless species, but significant displacement to high-frequency sound in eared species. Peak tissue displacement of the body wall occurred within the frequency range of each species' advertisement vocalization. Peak tissue displacement of the otic region of the eared species also occurred within these frequencies. Multi-unit neurophysiological recordings of the auditory midbrain (torus semicircularis) also were obtained. Auditory sensitivity curves showed three distinct regions of sensitivity at low, middle, and high frequencies, the latter located within the frequency range of each species' advertisement vocalization. The correlation between auditory midbrain sensitivity and tissue displacement of the body wall region at advertisement vocalization frequencies, suggests that the body wall/lungs serve as the route of sound transfer to the inner ear in earless species and possibly in the eared species as well.

Semaphoring in an earless frog: the origin of a novel visual signal.

Social communication in anuran amphibians (frogs and toads) is mediated predominantly by acoustic signals. Unlike most anurans, the Panamanian golden frog, Atelopus zeteki, lacks a standard tympanic middle ear and appears to have augmented its communicatory repertoire to include rotational limb motions as visual signals, referred to here as semaphores. The communicatory nature of semaphoring was inferred from experimental manipulations using mirrored self-image presentations and nonresident introductions. Male frogs semaphored significantly more when presented with a mirrored self-image than with a nonreflective control. Novel encounters between resident males and nonresident frogs demonstrated that semaphores were used directionally and were displayed toward target individuals. Females semaphored frequently and this observation represents a rare case of signaling by females in a typically male-biased communicatory regime. Semaphore actions were clearly linked to a locomotory gait pattern and appear to have originated as an elaboration of a standard stepping motion.

The middle ear muscle of frogs does not modulate tympanic responses to sound.

The effect of the opercularis (= middle ear) muscle on the acoustic responsiveness of the tympanic middle ear of anuran amphibians was studied using laser vibrometric measurements of tympanic responses to sound. Removal of the muscle or direct stimulation of denervated muscles had no measurable effects on tympanic responses to sound in either American bullfrogs (Rana catesbeiana) or green treefrogs (Hyla cinerea) at any frequency or at any sound-pressure level studied. These results suggest that, contrary to proposed hypotheses, the opercularis muscle of the anuran middle ear is not capable of modulating the responsiveness of the tympanic middle ear. Instead, the opercularis system most likely functions as an independent system involved in acoustic reception.

Distribution and morphology of motoneurons innervating different muscle fiber types in an amphibian muscle complex.

The distribution and morphology of motoneurons innervating specific types of muscle fibers in the levator scapulae superior (LSS) muscle complex of the bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) were studied by retrograde labelling with cholera toxin-conjugated horseradish peroxidase (CT-HRP). The LSS muscle complex in both of these amphibians has a segregated pattern of muscle-fiber types (tonic; fast oxidative-glycolytic twitch [FOG]; fast glycolytic twitch [FG]) along an anteroposterior axis. The entire motor pool was labelled by injection of CT-HRP into the whole LSS muscle complex. The motoneurons innervating specific fiber types were labelled by injection of CT-HRP into certain muscle regions. The organization of the motoneuron pool of the LSS complex of both species was arranged in two columns--one ventrolateral and one medial. In bullfrogs, the ventrolateral column contains motoneurons innervating FG and tonic fiber types and the medial column contains motoneurons innervating FOG fiber types. In tiger salamanders, the ventrolateral column contains motoneurons innervating FG fiber types and the medial column contains motoneurons innervating FOG and tonic fiber types. The different motoneuron types also have different soma sizes and patterns of dendritic arborization. In both species, FG motoneurons are the largest, whereas FOG motoneurons are intermediate in size and tonic motoneurons are the smallest. In bullfrogs, the main dendrites of FG motoneurons extend into the dorsolateral and the ventrolateral gray matter of the spinal cord, whereas the dendrites of FOG motoneurons extend into the ventral and medial cord. In the tiger salamander, dendrites of FG motoneurons extend into the ventrolateral spinal cord and dendrites of the FOG motoneurons extend more generally into the ventral cord. Dendrites of tonic motoneurons in both amphibians were small and short, and difficult to observe. These results establish that motoneurons innervating different types of muscle fibers in the LSS muscle complex are segregated spatially and display consistent morphological differences.

The effects of body size on functional properties of middle ear systems of anuran amphibians.

Both tympanic and nontympanic pathways of sound reception are utilized by anuran amphibians. The relationship between body size and the acoustic responsiveness of various body surfaces that may serve as pathways for sound reception in anurans was analyzed. The motion of the different surfaces (tympanum, lateral body wall, lateral head surface, and dorsal shoulder surface) produced by sound was measured with a laser vibrometer in anuran species. The frequency response and amplitude levels of motion of these body surfaces clearly were linked with size. In all animals, nontympanic surfaces were most responsive to low frequencies, and the tympanum was most responsive to high frequencies. However, the responsiveness of nontympanic surfaces was greater, and extended to higher frequencies, in small anurans. In the smallest animals studied, nontympanic surfaces were often more responsive than the tympanum up to frequencies as high as 2500 Hz. In larger anurans, nontympanic responsiveness tended to decrease, and tympanic responsiveness tended to increase. In the largest animals studied, the tympanum was the most responsive surface at all except very low frequencies below about 200-300 Hz. These results suggest that small anurans can utilize nontympanic pathways for effective sound reception over a broad frequency range, whereas large anurans are more restricted to using a standard tympanic middle ear for hearing. This effect of body size on the utility of nontympanic sound reception may explain evolutionary patterns of tympanic ear reduction and loss observed in several small species of anurans.

Biomechanics of vibration reception in the bullfrog, Rana catesbeiana.

The opercularis system (OPS) of amphibians consists of an opercularis muscle that connects the shoulder girdle skeleton to the operculum, a movable element in the oval window of the otic capsule. The role of the OPS in reception of vibrations was examined in bullfrogs (Rana catesbeiana) tested in various postures that manipulated differential motion between the shoulder girdle (the origin of the opercularis muscle) and skull (including the inner ear). Amplitude and phase relationship of motions of the suprascapular cartilage of the shoulder girdle and the posterior skull were also measured during these tests. 1. Microphonic responses to vertical vibrations from 25-200 Hz were typically highest when frogs were in a normal, sitting posture with the head held off the vibrating platform. Responses from animals in which the head directly contacted the platform were often less (by up to 10 dB at certain frequencies). Responses from all test positions were highest at lower frequencies, especially between 50-100 Hz. 2. Suprascapular accelerations were typically highest in the normal, sitting posture, and at lower frequencies (50-75 Hz) were often greater than that of the vibrating platform by up to 8 dB. The shoulder girdle skeleton of the bullfrog is therefore readily affected by vertical substrate motion. 3. The amplitude of microphonic responses in the different test postures did not correspond well with head acceleration. Rather, response amplitude corresponded best with the absolute difference between shoulder and head motion. For example, in the normal posture, suprascapular motion was much greater than head motion, and responses were relatively high. If only the head was vibrated, head motion was high and shoulder motion low, and responses also were relatively high. If the head and body were vibrated together, their motions were similar, and responses to the same platform accelerations were often reduced. Phase differences between shoulder and head motions were small at the frequencies examined and may be of little functional significance. The importance of differences in shoulder and head motion suggests that the resulting differential motion of the operculum and inner ear fluids can produce waves that stimulate appropriate end organs (such as the saccule). 4. Removal of the opercularis muscle reduced responses up to 18 dB at certain frequencies in some of the test postures. The most significant reductions were observed in those postures with a significant difference between shoulder and head motion (such as the normal posture).(ABSTRACT TRUNCATED AT 400 WORDS)

The physiological basis of slow locomotion in chamaeleons.

The African chamaeleon, Chamaeleo senegalensis, will not move faster than approximately 0.1 m/second at 23 degrees C, whereas the lizard Agama agama, like most lizards its size, runs at speeds more than 10X as fast. To account for this difference, we measured various physiological parameters of the iliofibularis muscle of both lizards. The maximum speed of tetanic contraction of unloaded Chamaeleo muscle was half as fast as that of Agama muscle (2.5 vs. 5.8 resting lengths per second). Heavily loaded Chamaeleo iliofibularis contracted at nearly 1/4 the speed of Agama muscle. Time to peak isometric twitch tension and time to half relaxation were twice as long in Chamaeleo as in Agama (122 vs. 58 msec, and 168 vs. 81 msec). Much more of the Chamaeleo muscle consisted of tonic muscle fibers, and the Chamaeleo muscle, compared to Agama muscle, showed physiological evidence of having a significant amount of tonic fibers (potassium contracture and high tetanus to twitch ratios). Finally, the myofibrillar ATPase activity of the Chamaeleo muscle was 1/3 that of Agama muscle. Thus, these results show that the slow locomotion of old world chamaeleons can, in part, be explained by the physiology, biochemistry, and fiber-type distribution of their muscles.

Physiological features of the opercularis muscle and their effects on vibration sensitivity in the bullfrog Rana catesbeiana.

The amphibian opercularis muscle connects a movable otic element (the operculum) to the pectoral girdle and can act in reception of ground vibrations. Various physiological parameters of the opercularis muscle of the bullfrog Rana catesbeiana were measured and compared with similar measurements on the iliofibularis muscle of the hindlimb. The opercularis muscle is a very slowly contracting muscle, with a Vmax of 1.81 muscle lengths s-1 compared to a Vmax of 6.24 muscle lengths s-1 for the iliofibularis muscle. The opercularis muscle develops tension slowly, taking about 10 s to attain maximum isometric tension when stimulated at 100 Hz. The muscle can retain high levels of tension for several minutes, and following stimulation has a time to half-relaxation of about 4-6 s. The slow velocity of contraction, slow rate of tension development, fatigue-resistance and slow rate of relaxation of the opercularis muscle support morphological evidence that it consists mostly of tonic muscle fibres. Experiments were also made to examine the effects of muscle tension on reception of ground vibrations as measured by inner ear microphonics. Severing the nerve supplying the opercularis muscle produced slight decreases of no more than 2 dB in responses to vibrations from 25 to 200 Hz. Artificial stimulation of the opercularis muscle after severing the nerve supplying the muscle increased responses to vibration across the entire frequency range. Higher tension levels produced greater increases in responses; at the highest tensions used (about 120 kN m-2) responses were increased by as much as 4.5 dB. The opercularis muscle is therefore specialized for slow but prolonged contractions, and tension is important in its sensory function. A tensed opercularis muscle appears to transmit faithfully motion of the forelimb, produced by vibrations, to the operculum such that the latter moves relative to the inner ear fluids.

Comparative morphology of the amphibian opercularis system: I. General design features and functional interpretation.

The morphology of the opercularis system of anuran and caudate amphibians suggests that it acts to produce motion of the operculum that in turn produces fluid motion within the inner ear. The operculum and opercularis muscle form a lever system, with a narrow connection between the operculum and otic capsule acting as a fulcrum about which the operculum moves in response to forces applied via the muscle. The opercula of many species possess a muscular process on which the muscle inserts, thereby increasing the moment arm through which the muscle acts. The tonicity of the opercularis muscle allows tensile forces produced by substrate vibration or other mechanical energy applied to the forelimb to be effectively transmitted to the operculum; the elasticity of the connective tissue holding the operculum in place should act to return the operculum to its original position. The opercularis systems of frogs and non-plethodontid salamanders are similar structurally and functionally; that of plethodontid salamanders is structurally distinct but also functions as a lever system. Fluid motion produced by opercular motion could stimulate various end organs of the inner ear; the saccule, lagena, and amphibian papilla are in close approximation and wave energy could directly affect their otoconial or tectorial structures. In those anurans with a tympanic ear, the stapedial footplate and operculum articulate, but this articulation allows both to move independently. The stapes-tympanum complex and opercularis system therefore appear to be independent functional systems, and it is unlikely that the opercularis system modulates middle ear responsiveness. The general design of the opercularis system is consistent with a function in reception of substrate vibrations.

Role of the opercularis muscle in seismic sensitivity in the bullfrog Rana catesbeiana.

The inner ear of anuran amphibians appears to be exceptionally sensitive to substrate vibration. The opercularis system, consisting of an opercularis muscle running from the shoulder girdle to a movable, cartilaginous operculum lying next to the inner ear, has been hypothesized to be involved in driving these seismic responses. Removal of the opercularis muscle of adult bullfrogs, Rana catesbeiana, caused clear decreases in microphonic responses of the inner ear to vibrations from 20-250 Hz and 0.05-5.0 cm/sec2 accelerations. Degree of decrease in responsiveness was variable between individuals and between different frequencies of stimulation, ranging up to 90% reduction at certain frequencies and in certain specimens. Decreases were most marked at lower frequencies below about 50 Hz. Additional removal of the levator scapulae superior muscle, which runs alongside the opercularis muscle from the shoulder girdle to ventrolateral portions of the otic capsule, also tended to depress responses, although this effect was substantially less (generally less than 10%) and also less consistent. As the opercularis muscle appears to be derived from the levator scapulae musculature, it is speculated that primitively seismic sensitivity was enhanced by a muscular connection that could transmit motion from the forelimb to the otic region, responsiveness being further enhanced by the subsequent evolution of the specialized opercularis system.

Electromyography of the opercularis muscle of Rana catesbeiana: an amphibian tonic muscle.

The opercularis muscle of Rana catesbeiana originates on the suprascapular cartilage of the shoulder girdle and inserts on the otic opercular element. It is part of the levator scapulae musculature and lies dorsomedial to the levator scapulae superior and inferior muscles. Bipolar electrode recordings from all three muscles show electrical activity linked to cyclical firing of the posterior intermandibularis muscle, an important ventilatory muscle. The opercularis muscle shows low amplitude, erratic signals when animals are submerged. Upon emergence of the snout region, the opercularis muscle shows rhythmic low amplitude activity at twice the rate of buccal pumping. Lung ventilation is synchronized with this rhythm and at ventilation the opercularis muscle shows higher amplitude activity. Upon submergence, opercularis activity again shows low level activity with no rhythmic pattern. Opercularis muscle activity has a major low frequency component (about 30 Hz) that probably corresponds to activity of tonic muscle fibers. Higher frequency signals (about 200-250 Hz) comparable to those of the levator scapulae muscles are also present and probably represent activity of phasic muscle fibers. Activity of the opercularis muscle is correlated with conditions in which aerial respiration is possible, and this pattern of activity supports an opercularis role in aerial hearing and/or detection of substrate vibrations. As far as we know, this is the first report of electromyographic analysis of a vertebrate tonic muscle.

Mechanisms of underwater hearing in larval and adult tiger salamanders Ambystoma tigrinum.

1. A standing wave tube apparatus was used to determine the biophysical basis of underwater hearing in Ambystoma tigrinum. 2. A. tigrinum responds to the pressure component of underwater sound, and the mouth cavity appears responsible for transduction of sound pressure. 3. Near-field displacements produced by pulsations of the air-filled mouth cavity apparently stimulate the inner ear. 4. Salamander head preparations with no air-filled mouth cavity respond to the particle motion component of underwater sound, but only at sound pressure levels 40 dB or more above levels producing clear pressure sensitivity in intact salamanders or head preparations including an air-filled mouth cavity.

Biophysics of underwater hearing in anuran amphibians.

A standing wave tube apparatus was used to determine the biophysical basis of underwater hearing sensitivity in 3 species of Rana and in Xenopus laevis. A speaker inside the base of a vertical, water-filled 3 m steel pipe produced standing waves. Pressure and particle motion were measured with a hydrophone and geophone respectively and were spatially 90 degrees out of phase along the length of the tube. Microphonic responses were recorded from the inner ear of frogs lowered through pressure and particle motion maxima and minima. The air-filled lungs of whole frogs produced distortions of the sound field. Preparations of heads with only an air-filled middle ear produced little distortion and showed clear pressure tracking at sound intensities 10-20 dB above hearing thresholds from 200-3000 Hz. Filling the middle ear with water decreased or abolished microphonic responses. Severing the stapes reduced responses except at certain frequencies below about 1000 Hz which varied with body size and likely represent resonant frequencies of the middle ear cavity. We conclude that the frog species examined respond to underwater sound pressure from about 200-3000 Hz with the middle ear cavity responsible for pressure transduction.