functional aspects of anuran middle ear structures · lack a middle ear cavity and tympanum, the...

J. Exp. Biol. (1974). 61, 71-93 7 1" " " 14 figures

mnt ted in Great Britain

FUNCTIONAL ASPECTS OF ANURAN MIDDLE EARSTRUCTURES

BY R. ERIC LOMBARD*

Department of Anatomy, University of Chicago, Chicago, Illinois, 60637

AND IAN R. STRAUGHAN*

Department of Biology, University of Southern California,Los Angeles, California, 90007

(Received 19 November 1973)

SUMMARY

1. The opercular complex of amphibians functions to enhance perceptionof airborne environmental sounds below 1 KHz.

2. The columella and tympanum of frogs function in the perception ofacoustic information above 1 KHz.

3. The opercular complex and amphibian papilla comprise the generalhearing mechanism in amphibians.

4. The tympanum, columella and basilar papilla, present in totality onlyin frogs, are concerned with reproductive communication.

5. Interaction of the two systems in frogs provides a mechanism forenhancing input signal to noise ratio during chorusing.

6. Experimental determinations of acoustic perception in amphibians mustbe controlled for anaesthesia and forelimb disposition. The presence ofanaesthesia or unnatural limb position can affect the animal's ability to per-ceive low frequency sounds by preventing normal opercularis function.

INTRODUCTION

Seasonally, frogs are among the most soniferous of vertebrates. Biologically,vocalization is a major feature of the anuran adaptive radiation, for it is primarilyby vocal means that the diverse species maintain their reproductive isolation. Thus,frogs would be expected to have a well developed sense of hearing, capable of dis-criminating between the various calls important to their behaviour. Early studies(van Bergeijk, 1957; Geisler, van Bergeijk & Frishkopf, 1964) examined the ear ofthe frog as if its function were analogous to that of mammals: to provide discrimina-tion of all environmental sounds within a given sensitivity of frequency and intensity.The resultant model, though theoretically feasible, has not been substantiated andremains unsupported.

In 1959, Lettvuve* al. demonstrated that the eye of a frog did not provide a com-plete visual image of its environment but processed at the receptor (the retina) onlyevents significant to the immediate well-being of the animal. This teleologicalapproach, i.e. investigating how biologically significant stimuli would be processed,

• The authors names are in alphabetical order. The sequence does not imply seniority.

R. E. LOMBARD AND I. R. STRAUGHAN

I mm

Ex col

Fig. i. Dorsal graphic reconstruction from serial sections of the right middle ear elements ofH. regilla. Anterior is to the right and the scalar bar (i mm) lies in the mid-sagittal line. Thecontour interval is 60 ft, and bone is stippled. The pars superior of the otic labyrinth is out-lined for orientation. Abbreviations: Col, columella; Col fp, columellar footplate; Ex col,extracolumella; Op, operculum; Op m, opercular muscle; ss, suprascapula; Tym, tympanum.

was then adopted in studies of anuran ear function, primarily in the bullfrog, Ranacatesbeiana (see Frishkopf, Capranica & Goldstein, 1968, for a review of these studies).These investigations produced evidence that there are two classes of fibres in theauditory nerve of the bullfrog: (1) 'complex' units which respond to vibrations andlow frequencies and are inhibited by the presence of certain other frequencies, and(2)' simple' units which are spontaneously active, respond to higher frequencies, andcannot be inhibited. On anatomical evidence, the complex units have been associatedwith the amphibian papilla and the simple units with the basilar pap ^ ^

Anuran middle ear 73

ldstein, 1963; Sachs, 1964; Frishkopf & Geisler, 1966). In R. cateibeiana, the bestfrequency response characteristics of these two groups of units were shown to cor-respond to the frequency bandwidths of maximum energy in the so-called matingcall. This was interpreted to indicate that detection of significant sound was accom-plished by the simultaneous activation of the two groups of units, i.e. the reason thatfrogs have two papillae is to process significant sounds at the receptor (analogous tothe visual processing in the retina) (Capranica, 1965). To date there has been noinvestigation of how the frequency response characteristics of each of these sensorypapillae are obtained.

The presence of two papillae is unique to amphibians. The basilar papilla and itsassociated accessory structures have been regarded as the precursors of the amniotecochlea, although the structure is simple in amphibians. The amphibian papilla isfound only in amphibians and is generally larger and more complex than the basilarpapilla (Paterson, i960; Geisler et al. 1964). Further this is the only papilla in manysalamanders (Lombard, 1971).

A second unique feature of the auditory system of most frogs and salamanders isthe presence of two ossicles in the fenestra ovalis. One of these is the proximal ex-pansion or foot plate of the columella and the other, lying more posteriorly, thecartilaginous or bony operculum. The columellar foot plate is connected to thetympanic membrane in frogs by the columella proper and its distal cartilaginous pro-jection, the extracolumella (Col fp, Tymp, Col, Ex col, Fig. 1). In salamanders, whichlack a middle ear cavity and tympanum, the columella attaches to the palatoquadrateof the suspensorium or squamosal bone. The columella, with its foot plate and extra-columella, is a derivative of the hyomandibular of fish and is homologous to thecolumella or stapes of amniotes. The operculum provides origin for a muscle whichproceeds postero-dorsally to insert on the ventral face of the suprascapula (Op, Op m,ss, Fig. 1). This muscle is evident in all frogs and salamanders which have a terrestrialphase in their life-history. The operculum, a de novo structure in amphibians, isthought to be a freed portion of the otic capsular wall (Kingsbury & Reed, 1909), andhas no homologue in other vertebrate groups.

While the function of the columella and tympanum are fairly obvious, the functionalsignificance of the operculum - opercularis muscle complex is more obscure. Twogeneral functions have been proposed: (1) that the complex functions as a link in anacoustical transmission route for perception of acoustic disturbances in the substratevia the forelimbs (Kingsbury & Reed, 1909), and (2) that the complex functions in themaintenance of balance via the vestibular system by monitoring body position (Ecke,1934). The former proposal has been widely accepted with little criticism and noevidence for many years and is presented as fact in many texts (Romer, 1970; Gobi &Goin, 1971; Porter, 1972). The later hypothesis, though less widely accepted, hasreceived some supportive evidence (Baker, 1969). In this paper we report on the roleof the opercularis muscle and other middle ear structures associated with it.

MATERIALS

Specimens of the following species were used: Hyla regiUa, 6; H. verstcolor, 2;[. cinerea, 3, and Smilisca baudinii, 2, of the family Hylidae; and Leptodactylus

74 R. E. LOMBARD AND I. R. STRAUGHAN

melanonotus, i, of the family Leptodactylidae. The H. regilla were collected frd^Catalina Island and San Dimas Canyon, Los Angeles Co., California. The H. versi-color specimens were taken from east Texas populations, the H. cinerea from thevicinity of Tampa, Florida, and the Smilisca from the Yucatan Peninsula of Mexico.The Leptodactylus was collected in Baja California, Mexico. Accurate field data for allanimals have been recorded in personal files.

METHODSSurgical procedures

All animals were anaesthetized for surgery by immersion in an 0*5 % solution ofurethane. After surgery, full recovery from anaesthesia was generally allowed beforephysiological recording was attempted. For single unit experiments the eighth nervewas exposed at its entry into the brain case by dissecting away a small region of theoral epithelium and connective tissue and a portion of the braincase floor from theroof of the mouth. The nerve sheath was then dissected off. For recording from themidbrain, a triangle of skin, with the apex midway between the eyes and the baseslightly behind the suprascapula was folded back over the body, thus exposing theroof of the brain case. By drilling out the bone with a dental drill or dissecting offthe cartilage, the dorsal surfaces of the optic tecta were exposed. The dura was splitmedially and pulled off to both sides. Circulation to the brain could be monitored inthe medial cerebral vein, which was always left intact. In animals used repeatedly, theflap of skin was returned to the normal position between experiments. Post-operativefrogs showed no discernible impediments.

Experimental surgery was performed simply by: (1) severing the opercularis muscleat its attachment to the suprascapula, (2) severing the nerves to the muscle peripheralto branches to other structures, or (3) dissecting out the tympanum from the sur-rounding tympanic annulus. Three additional experimental manipulations, not in-volving surgery were performed during threshold/frequency determinations: (1)adpression of the forelimbs, (2) keeping the animal under anaesthesia, and (3) isolatingthe animal from the substrate. Anaesthesia was maintained by covering the animal inan absorbent tissue soaked in 0-5 % urethane. Isolation from the substrate wasaccomplished by placing the animal on a 5 cm thick cotton pad. This experimentalseries was designed to counterfit possible traumatic effects resulting from surgery inthe experimental procedures. It also enabled us to record the overall response of thereceptor while making changes in the peripheral structures without having to repeatthe procedure many times and sum the responses from many experiments. In addition,advantage could be taken of the apparent total crossing of eighth nerve projections tothe tori. This enabled unilateral surgery so that the animal could provide its owncontrol under experimental conditions.

Care was taken at all times to ensure that the animal had fully recovered fromsurgical anaesthesia. Any degree of anaesthesia during threshold-frequency determina-tions often resulted in loss of the low frequency sensitive region. Two control curvesdetermined under anaesthesia are shown in Figs. 7 and 8. In both the low frequencysensitive region is absent or poorly developed.

For all midbrain recordings the unanaesthetized animals were restrained only bya pin through the nasal region. By keeping the animals moist and running the ^ i

Anuran middle ear m

Fig. 2. Schematic diagram of the experimental chamber used for all experiments. For descrip-tion see materials and methods. Abbreviations: Elec, micromanipulator; Mic, microphone;Pit, paraffin platform: Sp, speaker.

ments in the dark, little problem was encountered with movement. Finally, theposition of the forelimbs was maintained in a normal position at all times except undercertain experimental conditions. Random or uncontrolled placement of the forelimbsoften resulted in the loss of the low frequency sensitive region as shown in Fig. 14.For the single unit recordings where the animal was on its back, the forelimbs weremanipulated until a strong low frequency response was achieved. They were thenheld in position with pins. In this experiment the animal was restrained by a wettissue blanket as well as pins.

Experimental set-up

All physiological procedures were conducted in an acoustically, vibrationally, andelectrically isolated chamber measuring 1-25 X075 XO75 m internally (Fig. 2). Thewalls were laminated wood and acoustic tile and lined with fibre wool insulation. Overthe operating frequency and intensity ranges used no difficulty with echo interferenceor standing waves occurred. A loud speaker (J. B. L. LE8T) flat (± 1-5 dB) over theoperating range 1-5 KHz was centrally mounted in one end. A heavy metal stand(1, Fig. 2) isolated from the floor of the chamber by a metal-rubber-metal vibrationabsorbing sandwich (2, Fig. 2) was fixed in the middle of the chamber. Fixed to thisstand was a dissecting microscope, a micromanipulator (Narashigi M3) (Elec, Fig. 2)and monitoring microphone (B and K 4131) (Mic, Fig. 2). The microscope was fitted

Wkh an ocular grid which was used to centre the frog in a constant position relative


Waveformgenerator

Audiokeyer

Audioamplifier

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Audiomonitor

Indifferent electrode

Microclectrode PreamplifierOperational

amplifier

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Fig. 3. Block schematic of experimental equipment used. For explanation see materials andmethods. Abbreviations: C.A.T., computer of average transients.

to the speaker and to position the microelectrode in the midbrain. With the electrodeholder kept at a constant angle, we were able to obtain accurate and repeatableelectrode placement. A rotatable paraffin platform attached magnetically to the standenabled orientation of the experimental animal (Pit, Fig. 2). The microphone wasplaced as close to the animal as possible and equidistant from the speaker (about60 cm).

A block diagram of the experimental apparatus is shown in Fig. 3. Sound signalswere produced by gating a pure tone from a sine wave generator (Hewlett Packard 5D6)through an audio keyer. The duration and repetition rate of sound pulses could bevaried but in all experiments a pulse of 20 msec duration repeated every 0-5 sec wasused. Rise and fall times varied with frequency to produce a smooth transient free of'popping'. The signal was fed through the audio amplifier of a Crown 8000 seriestape recorder to the loud speaker and to one channel of the master oscilloscope(Tektronix 5O2d) for continuous monitoring.

The microelectrodes were insulated tungsten with a 1-3 /i diameter tip for recordingcompound action potentials in the midbrain and with a 0-5 /i tip for recording singleunits extracellularly from the eighth nerve. The electrode was followed by a pre-amplifier (Bioelectric BFI) and a differential amplifier (Tektronix 122) which thenfed into the master oscilloscope. The signal could be monitored by audition througha speaker and further analysed by a computer of average transients (C. A. T. Instru-ment Co.).

The monitory microphone was connected to a precision sound level meter (B andK 2003) and a band pass filter (B and K 1613) for recording sound intensity levels atthe animal's ear during experiments. The sound chamber was maintained at a constanttemperature (19 °C) during the experiments.


Experimented procedure

Experimental studies of amphibian middle ear components are complicated by themajor difficulty in maintaining the mechanical auditory system intact while recordingneurophysiological responses in the auditory pathway. The eighth nerve is notaccessible dorsally in most frogs without surgical damage to part of the otic capsule.Normally, to avoid this problem, a ventral approach to the auditory nerve is employed.However, this involves positioning the animal on its back and exposing the nervethrough the roof of the mouth. Since the opercularis complex can be affected by therelative disposition of the skull and shoulder girdle (see below), such an unnaturalposture is better avoided in functional studies.

In examining the neural response to acoustic stimuli, three major techniques havebeen applied in frogs: (i) recording single units from the midbrain (Potter, 1965),(2) recording single units from the eighth nerve (Frishkopf & Goldstein, 1963), andmonitoring midbrain slow wave evoked responses (Loftus-Hills & Johnstone, 1969).Our technique is a combination of those of Potter and Loftus-Hills & Johnstone. Tomeasure overall response, we used the compounded potentials at a group of units ratherthan single units or the slow wave summation. Potter found that units in the torusproduced simple responses to sound stimuli. The midbrain is readily accessible dorsallyafter removing part of the calvarium without any damage to the functional componentsof the middle or inner ear. The animal can then be positioned to reflect the normaldisposition of the middle ear components. To determine the overall response of theentire system, summation of responses from many single units is required. We foundthat in the torus we could record either single units (as extracellular action potentials)with fine electrodes (0̂ 5 ft) or multiunit extracellular responses with coarser electrodes.Even with the coarse electrodes, at low stimulus intensities the number of units ina multiple recording was reduced. Near threshold, such recording became indis-tinguishable from single unit recording made with fine electrodes. Potter (1965) foundthat single units vary considerably in threshold, latency and peak potential such thatspecific units could be sorted out from a multiple recording. Since we were primarilyinterested in overall response, the multiplicity of units was always maximized for eachelectrode placement.

To see if this measure was comparable to single unit recordings in more peripherallevels of the pathway, we conducted a series of experiments to determine 'bestfrequencies' obtained by responses in the brain and compared these to 'best fre-quencies' obtained from single units in the eighth nerve using H. cinerea. The experi-mental procedures for both determinations were the same except for the orientationof the animal (on his back for single unit recording) and size of electrode. H. cinereais a much larger and more robust animal than our principal experimental speciesH. regilla and is capable of enduring a longer series of experimentation. The results(Fig. 5) show that our measure of 'best' frequency is indeed equivalent to that ob-tained from summation of single unit responses in the auditory nerve and has theadvantage that the full range of the audiogram can be used to detect changes insensitivity due to experimental manipulations.

After surgery and gross electrode placement, a search stimulus of either normalpulses of an appropriate frequency (if the response characteristics were already


Fig. 4. Representative near threshold response from the torus semicircularis of S. baudinii(upper trace) to a 20 msec signal of o-8 KHz at 50 dB, re 00002 dynes/cm1 (lower trace).

known) or of pulses of white noise was presented at relatively high intensity (70-80 dBre 0-0002 dyne3/cma). For midbrain recording the visual cortex and ventricular spacewere then penetrated to gain the auditory region of the torus semicircularis. When astrong compound action potential (or an extracellular action potential of a single unitin the nerve) was encountered, the search stimulus was switched to the experimentalseries which ran from 1 to 5 KHz in c-1 KHz intervals. At each frequency the soundintensity was raised until the response was evident, then gradually lowered until itdisappeared. The sound intensity as measured in decibels (re SPL 0-0002 dynes/cms)by the sound level meter at this point was recorded as threshold for each frequency.Disappearance of the response was judged by visual and auditory means. To removesubjective bias, replications were always made with alternate observers. This pro-cedure was tested against the more time-consuming, but potentially more accuratemethod of Loftus-Hills & Johnstone, i.e. reducing the sound intensity 2 dB at a timeand averaging 50 responses at each intensity level with the computer of averagetransients. With a good signal to noise ratio, we found this procedure to be unnecessaryfor accuracy or repeatability, and it was used only in rare cases when clear signalswere not available.

The response signal (Fig. 4) was essentially a temporal summation of the midbrainsingle units described by Potter. The initiation of the response had an average latencyof the order of 14 msec. Depending on the intensity of the stimulus and degree ofelectrode isolation, the length of the response was from 1-2 msec up to 40 msec. Themaximum amplitude of response exceeded 500 /tV and at threshold was aboutBackground noise levels were usually about 35 /iV.


^Statistical treatment was performed after converting dB values to dynes/cm1. Themeans were then converted back to dB values for graphic display.

RESULTS

The threshold/frequency curves determined show, in control and experimentalsituations, consistent major features in the animals examined. Though some variationin shape and absolute threshold is evident when comparing details even in the curvesfrom animals of the same species (for example, H. regilla, Figs. 6, 9, 13, 14), we do notintend to examine them. Instead, focus will be maintained on the general aspects ofcurve shape, both inter- and intraspecifically. No differences in ability to hear wereevident in the data from right to left ears in a given animal. This general finding isshown in Fig. 5. In all subsequent graphs the data from both ears have been lumped.No differences in ability to hear at any frequency were evident in situations where theanimal was isolated from the substrate (right ear determination in Fig. 5). Thisexperiment was performed with the other species used in a random manner. At notime were any differences in perceptive ability noticed. Data from these determinationswere integrated with that from substrate contact determinations for presentation.

Control threshold/frequency curves

In control situations two broad regions of maximum sensitivity are seen, onecentred below 1 KHz and the other centred higher, around 2-3 KHz (solid lines,Figs. 5-14). Exceptions to this pattern can be found in Figs. 7 and 8, where the lowersensitivity region is missing. These discrepancies will be discussed in a later section.A general feature of these two 'peak' regions of sensitivity is a relatively lowerthreshold in the lower frequency region. This is seen in Figs. 5, 6, 9, 10, 11 and 12.This feature is not evident in Figs. 7 and 8 due to the absence of a low frequencyregion. In Fig. 13 the minimal threshold is lower in the high frequency region, andin Fig. 14 the thresholds are equivalent. It is evident from the stippled range limitsabout the solid lines that a fair degree of consistency was possible from one determina-tion to the next. For this reason only the curves determined by the mean response ata given frequency will be discussed.

H. cinerea, Figs. 5, 8 and 11. In H. cinerea the low frequency sensitive region liesbetween 0-3 and i*6 KHz (Figs. 5 and 11). The higher frequency sensitivity region liesbetween 2̂ 3 and 3-5 KHz. There is a broad region of lessened sensitivity between i-6and 2*3 KHz. From the regions of ' peak' sensitivity the animal's ability to hear fallsrapidly at both the high and low ends of the spectrum. In Fig. 8, this general patternis confused by the absence of a broad low frequency sensitive region.

H. regilla, Figs. 6, 9, 13 and 14. In H. regilla the low frequency sensitive region liesbetween o-i and i-o KHz. The higher frequency sensitive region lies between i-o andabout 3-3 KHz. The higher frequency sensitive region can be further subdivided intotwo subordinate sensitivity regions. The lower of these lies between about 1-5 and2-2 KHz and the higher between about 2-4 and 3-0 KHz. From the regions of 'peak'sensitivity the animal's ability to hear falls rapidly at both the high and low ends ofthe spectrum.

{. versicolor, Fig. 7. The curve presented for H. versicolor shows a higher frequency


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Fig. 5. Comparison of threshold/frequency data obtained from the tori semicirculares ofH. cinerea (upper curves) with data gained from single unit recordings (lower histogram)from the eighth nerve of the same animal. Circles (•), right torus; stars (•), left torus;single units recorded in the right nerve. In the histogram, the height of each bar indicates thenumber of single units found that were maximally responsive to frequencies within the o-1 KHzbar interval (dB re 0-0002 dynes/cm*).

sensitive region between i*o and 3-4 KHz. Sensitivity appears at its maximum be-tween i*6 and 2*1 KHz. A lower frequency sensitive area is not apparent in this curve.In other H. versicolor studied but not presented here, a lower frequency sensitiveregion occurred between o-i and 0-9 KHz. In these cases the animal's ability to hearfalls off rapidly at both the high and low end of the spectrum. This same sensitivity'fall-off' is evident at the high frequency region of Fig. 7.

L. melanonotus, Fig. 12. In L. melanonotus the low frequency sensitive region liesbetween o-i and 1-5 KHz. The higher frequency sensitive region lies between o-i and1 *5 KHz. The higher frequency sensitive region lies between i*8 and 3*4 KHz. In thehigh region, sensitivity appears at a maximum between 2-6 and 3-4 KHz. Though thesensitivity ' fall-off' from the low frequency region toward lower frequencies is rapid,that of the high frequency region toward higher frequencies is less pronounced.Indeed, some minor areas of relatively greater sensitivity centred at 3-7 and 4-3 KHzare evident.

<S. baudinii, Fig. 10. In S. baudinii the low frequency sensitive region lies betweeno-i and I-1 KHz. The higher frequency sensitive region lies between i-6 and 2-7 KHz,with maximum sensitivity at 2-1 KHz. There is a region of lessened sensitivity betwl


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Fig. 6. Effect of bilateral removal of the tympana in H. regilla. The solid line represents theaverage of three threshold/frequency determinations with the tympani intact. The range at anyfrequency is represented by stippling. The dashed line represents the average of threethreshold/frequency determinations with the tympani removed. The range at any frequencyis represented by vertical lines. Each of the six curve* summarized was done with a differentelectrode placement: three in the right torus and three in the left. The lower curve summarizesthe loss in sensitivity (-dB, re ocooa dynes/cm1).

i*i and i-6 KHz. From the region of 'peak' sensitivity the animal's ability to hearfalls off rapidly toward lower frequencies but more gradually toward higher fre-quencies.

Experimental threshold/frequency curvesSingle units

Fig. 5 shows a comparison between threshold/frequency data obtained from thetori semicirculares and single unit summations from the right eighth nerve of thesame animal. There is good correspondence between the number of single units foundmaximally responsive to a given frequency, and the sensitivity of the animal, deter-mined at the midbrain, to the same frequency. Where the eighth nerve evidenced agreater number of single units responsive to a given frequency, the midbrain showedmaximum sensitivity. Where the eighth nerve produced fewer or no single units, themidbrain indicates a lesser sensitivity. Regions of maximal sensitivity by eithermethod occur at 0-5, i-o and 3-O-3-4 KHz.


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Fig. 7. Effect of bilateral removal of the tympana in H. versicolor. The solid line representsthe average of two threshold/frequency determinations with the tympani intact. The range atany frequency is represented by stippling. The dashed line represents the average of threethreshold/frequency determinations with the tympana removed. The range at any onefrequency is represented by vertical lines. Each of the five curves summarized was determinedwith a different electrode placement: two in the right torus and three in the left. All curves weredetermined under anaesthesia. The lower curve summarizes the loss in sensitivity (- dB,re o-ooo2 dynes/cm1).

TympanumThreshold/frequency curves before and after bilateral removal of the tympana are

shown in Fig. 6 for H. regilla and Fig. j for H. versicolor. Threshold/frequency curvesfrom a H. cinerea with a small, malformed left tympanum are shown in Fig. 8. In allthree cases the maximal reduction in sensitivity occurs at higher frequencies, generallyabove i KHz. Though sensitivity loss does occur below i KHz, it is irregular, in-consistent and less pronounced.

Comparison of the control and experimental extremes at a given frequency belowi KHz in Fig. 6 (vertical bars: experimentals; stippled envelope: controls) shows afair degree of overlap. This is also the case in H. cinerea (Fig. 8) where there is verylittle difference in the curves below i KHz. In H. versicolor (Fig. 7), the loss appearsto increase irregularly from about 0-2 KHz but does not reach a consistently highlevel until 1 KHz. In all three animals the loss in sensitivity is fairly constant once amaximum level is achieved. Comparison of the control and experimental curves 'ma given animal shows that once a maximum differential occurs the two curves are™

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Fig. 8. Effect of a naturally malformed, small left tympanum in H. cmerea. The solid line depictsthe threshold/frequency data obtained from the left torus. The dashed line shows the averagethreshold/frequency data obtained from two locations in the right torus. The range at anyfrequency is represented by the vertical lines. All curves were determined under anaesthesia.The lower curve summarizes the loss in sensitivity (- dB, re 0-0002 dynes/cm*).

the same shape, i.e. both contain the same 'peaks' and 'valleys', even in detail. Ingeneral the pattern shows three phases: (1) an irregular small loss in sensitivity below1 KHz, (2) a transitional zone between 1 and 2 KHz where sensitivity loss increasesfairly regularly, and (3) a zone > 2 KHz where the loss is maximal and consistent.

Opercularis muscle

Comparative threshold/frequency curves, before and after experimental inter-ference with normal opercularis function, are shown in Figs. 9-14. In all cases,maximum loss of sensitivity occurs below 1 KHz after ablating normal muscle action(dashed lines). Loss of sensitivity above 1 KHz is small and irregular, if present, andnot significant. In general, the sensitivity loss is maximum with experimental manipula-tion in the region of 'peak' sensitivity under non-experimental conditions. Thesensitivity loss also generally falls off rapidly on either side of the maximum loss. InS. baudinii (Fig. 10) and H. cinerea (Fig. 11), however, the loss in sensitivity decreasesmuch less precipitously from the maximum towards the higher frequency side,

(a) Muscle transection. Figs. 9-12 show the results of surgically cutting the oper-aris muscle. In H. regiUa (Fig. 9) the operation was bilateral and in S. baudinii,

and L. melanonotus (Figs. 10-12) the operation was unilateral. In all cases6-2


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s'•!., ' i

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Fig. 9. Effects of bilaterally severing the operculares muscles in H. regilla. The solid linerepresents the average of five threshold/frequency determinations with the muscle* intact. Therange at any frequency is represented by stippling. TTxe dashed line represents the average oftwo threshold/frequency determinations with the muscles severed. The range at any frequencyis represented by vertical lines. Each of the seven curves summarized was done with a differentelectrode placement: three in the right torus and four in the left. The lower curve summarizesthe loss in sensitivity (- dB, re 00002 dynes/cm1).

the sensitivity loss was at a maximum below 1 KHz (dashed lines). Also, maximumloss occurs in the region of optimum low frequency sensitivity. Loss above 1-5 KHzis minimal and not significant. Between 1 KHz and (1) 1-5 KHz in H. regiUa (Fig. 9)and L. melanonotus (Fig. 12) and (2) 2 KHz in S. baudimi (Fig. 10) and H. drierea(Fig. 11), there is a zone of decreasing sensitivity loss.

In those cases where the muscle section was unilateral, only recordings from thecontralateral torus semicircularis indicated a sensitivity loss (dashed lines, Figs, io,11 and 12). Recordings from the ipsilateral torus in each case indicated no deviationfrom normal (dotted lines Figs. 10 and 11). The mean ipsilateral response is seen tolie almost entirely within the envelope of extremes of non-experimental conditions atall frequencies. Ipsilateral recordings for H. regiUa (Fig. 9) are not shown because theanimal expired during that phase of the experiment.

(b) Nerve transection. The effect of bilateral surgical section of the opercularis nervesupply is shown in Fig. 13. Normal disposition of the operculares muscles was notaltered during surgery. The sensitivity loss is at a maximum below 1 KHz (dashedline). The maximum loss also occurs in the region of optimum low frequency :tivity. Loss of sensitivity above 1 KHz is irregular and insignificant. Above 1

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Fig. io. Effect of unilateral (left) section of the opercularis muscle in S. baudhm. The solid linerepresents the average of three threshold/frequency determinations with the muscles intact.The range at any frequency is represented by stippling. Each of the curves summarized wasdone with a different electrode placement: two in the left torus and one in the right. Thedashed line represents the threshold/frequency determination found in the right torus aftersection of the left opercularis muscle. The dotted line represents the threshold/frequencydetermination found in the left torus after section of the left opercularis muscle. The lowercurve summarizes the loss in sensitivity (- dB, re 0-0002 dynes/cm1).

the experimental threshold/frequency curve lies almost entirely within the envelopeof extremes found under non-experimental conditions. The region of maximum lossconforms to that found with experimental section of the opercularis muscle (Fig. 9),and positional alteration (Fig. 14) in the same species.

(c) Postural modification. The effect of postural modification (bilateral adpressionof the forelimbs) on the threshold/frequency curve of H. regilla is shown by the dashedline in Fig. 14. The sensitivity loss is at a maximum below 1 KHz and occurs in theregion of optimum low frequency sensitivity under non-experimental conditions.Sensitivity loss above 1 KHz is very irregular and difficult to generalize. In mostinstances the experimental curve of means lies within the range found under normalconditions. Reversal of the postural modification eradicates the sensitivity loss (dottedline). The threshold/frequency curve after regaining normal posture is, with a coupleof minor exceptions, completely coincident with the range of pre-postural changecurves. The region of maximum loss under experimental conditions conforms to that

1 using other methods of interfering with normal opercularis function in the same3 (Figs. 9 and 13).


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H. cinerea

I

kHz

-10

- 2 0 « -

FIR- I I . Effect of unilateral (right) section of the opercularis muscle in H. cinerea. The solidline represents the average of three threshold/frequency determinations with the musclesintact. The range at any frequency is represented by stippling. Each of the curves summarizedwas done with a different electrode placement: two in the right torus and one in the left. Thedashed line represents the threshold/frequency determination found in the left torus aftersection of the right opercularis muscle. The dotted line represents the threshold/frequencyfound in the right torus after section of the right opercularis muscle. The lower curve sum-marizes the loss in sensitivity (-dB, re 00002 dynes/cm1).

(d) Anaesthesia. The effects of anaesthesia on the threshold/frequency curves ofH. versicolor and H. cinerea are shown in Figs. 7 and 8. In neither case is a low fre-quency sensitive region clearly discernible. A low frequency sensitive region is presentin unanaesthetized animals of the same species (H. cinerea, Figs. 5 and 11, H. versi-color not shown).

DISCUSSION

Functional and evolutionary interpretations

Controls in all species examined showed a clear-cut division of the auditory responseinto two basic components as had been found previously (Capranica, 1965; Potter,1965; Loftus-Hills & Johnstone, 1969). There is a low frequency zone below 1 KHzthat has been associated with the amphibian papilla, and a high frequency zone, above1 KHz that has been associated with the basilar papilla. That is, low frequency soundsand vibrations pass through one receptor and high frequencies through another. Ourresults show that the middle ear components also function differently in respect tQeach of these 'channels'.

Anuran middle ear

80 r-

70

60

50

40

30

-10

-20 L

- L. melenenotus

. I

Fig. i a. Effect of unilateral (right) section of the opercularis muscle in L. melanonotut. The solidline represents the average of two threshold/frequency determinations in the left torus with themuscle intact. Each determination was done with a different electrode placement and the rangeis indicated by stippling. The dashed line represents threshold/frequency data obtained fromthe left torus after section of the right opercularis muscle. The lower curve summarizes theloss in sensitivity (- dB, re 0-0002 dynes/cm1).

Interference with the tympanum - columella system does not appreciably affectsensitivity (as measured in the auditory centre of the brain) to low frequency but doessignificantly lower the sensitivity to high frequencies. Conversely, there is a markeddrop in sensitivity to low frequencies when normal functioning of the opercularissystem is prevented, but response to frequencies above 1 KHz is hardly affected.Capranica (personal communication) has reported that sensitivity (recorded as micro-phonics in the basilar papilla) is unaffected by severing the opercularis muscle, con-firming that the high frequency 'channel' is not dependent on the opercularis system.A property that might be expected of these systems is that they should operate atpeak efficiency at a particular frequency. This frequency would depend on their forcetransfer efficiency and time constant characteristics as determined by physicaldimensions. This would provide some degree of frequency tuning that is character-istic of anuran ears. Our experiments show, however, a consistent loss of sensitivityin the higher frequency part of the audiogram with tympanum - columella complexinterference. If there was tuning, removal of the system would be maximally effectivea^he tuned frequency and lower at frequencies away from this point. In the low^(uency portion, affected by manipulations of the opercularis system, the best


80 r-

70

60

50

30

-10

- 2 0 1 -

Fig. 13. Effect of bilateral section of the nerves to the opercularia muscles in H. regilla. Thesolid line repreients the average of three threshold/frequency determinations with the nervesintact. The range at any frequency is represented by stippling. Two curves were determinedfrom the right torus and one from the left. The dashed line represents the average of twothreshold/frequency determinations with the nerves to the opercularis muscles severed. Therange is represented by vertical lines. The lower curve summarizes the loss in sensitivity(-dB, re 00002 dynes/cm1).

frequency of hearing is maximally affected and the degree of change falls off sharplyat the lowest frequencies tested. In fact, the very low frequencies were almost un-affected, and as far as we could tell without quantitative measure, neither was theresponse to vibrations. If this can be verified quantitatively it will likely show that theopercular system is not necessary for vibration detection (as suggested by Smith,1968). Rather, the complex functions only to provide a low frequency airborne soundchannel with peak efficiency between about 0-3 KHz for large frogs such as R. cates-beiana and 0-7 KHz for small species such as H. regilla.

The direct connexion between an intact opercularis system and normal response inthe low frequency-vibration zone would appear to support the hypothesis of Kings-burg & Reed (1909): that the opercularis muscle provides a mechanical link betweenshoulder girdle and inner ear for the transmission of low frequency vibrations fromthe substrate by way of the forelimbs. However, animals isolated from the substrateby a non-transmitting pad showed no alteration in their response at any frequency.Also, when making single unit recordings from the auditory nerve; i.e. when the limbswere not in contact with the substrate, sensitivity to low frequencies was the same 2&that for normal postures.

Anuran middle ear

ov

70

60

50

40

30

0

10

on

_ H. regilla

v f m\ / ^

i

\ /WVi i . - 1

/ ' ^ " JcHz

V,,,,-.Mf,,

,1

hfW

i i

/"\3

Af

i

4i |

5

Fig. 14. Effect of bilateral adpression of the forelimbs in H. regilla. The eolid line represents theaverage of four threshold/frequency determinations with the animal in a normal position(humeri at right angles to the body and forearms at right angles to the humeri). The range atany frequency is represented by stippling. Two curves were determined in the right torus andtwo in the left. The dashed line represents threshold/frequency data obtained from the lefttorus with the forelimbs adpressed. The dotted line represents threshold data obtained fromthe left torus with the forelimbs returned to a normal position. The loss in sensitivity issummarized in the lower curve (- dB, re 00002 dynes/cm1).

If the opercularis complex is not a direct transmission line for sounds then it mustfunction in some other fashion to enhance the reception of low frequencies at the innerear. Fig. 1 shows a reconstructed opercularis complex and columella demonstratingthe interlocking nature of the operculum and the columellar foot plate. Tension inthe muscle would pull the operculum into locked position with the columellar footplate producing a coupled plate occupying the entire oval window. The fact that re-moval of the tympanum and the distal portion of the columellar shaft does not severelyreduce sensitivity to low frequencies indicates that low frequencies impinge on theoval window with about equal force whether or not there is direct coupling to thetympanum. Under this condition, the efficiency of force transfer of the sound pressureto fluid displacement in the inner ear appears directly proportional to the surface areaof the plate in the oval window. In addition, the mass of the plate also increases withcoupling, thus producing an increase in inertia of the now compound plate. Finally,the tension of the opercularis muscle would conceivably enhance this inciease inuiertia by 'stiffening' the middle ear complex. Since displacement acceleration is slowWt low frequencies the compromise of increased efficiency of force transfer for slower


reaction time can be met. As the frequency increases so does displacement accelerationand at some point loss of responsiveness due to inertia outweighs the gain due tohigh force transfer efficiency. Also, as the frequency increases the sound pressure atthe oval window decreases if no mechanical link to the outside is provided, as indicatedby high frequency loss on removal of the typanum. Therefore if the oval window platewere a single unit (of area equivalent to the two elements) it could not serve both lowand high frequency transmission with equal efficiency.

By uncoupling the two elements (as represented in our experiments by severing themuscle or otherwise preventing its normal state of tension), the columellar foot plateis free to move independently of the operculum. The mass of the moving plate isdecreased, thus reducing inertia and allowing responsiveness to greater displacementacceleration (i.e. higher frequencies). Also, the system gains mechanical advantage ofthe order of the ratio of the tympanum area to the area of the columellar foot plate,which is of little consequence when the area of the inner plate is not small relative totympanic area (as when the two elements are coupled). It may be, thus, that the ap-parent near equality of force transfer at the oval window and without this mechanicallink is due to the fact that when the oval window elements are coupled there islittle mechanical advantage derived from the link. Thus the middle ear components(tympanum-columella system and the opercularis complex) provide alternativemechanical efficiencies to the transmission at low or high frequency sounds, eitherbeing brought into play simply by a change in tension in the opercularis muscle.Reception at low frequencies is enhanced by coupling the operculum and columellarfoot plate into a single oval window plate of large surface area which has high forcetransfer efficiency but slow reaction time. Removal of opercularis muscle tensionuncouples the two oval window elements, allowing the efficient transmission at highfrequencies through the tympanum-columella system, which has a faster reactiontime. The structures involved are so small in the animals themselves that empiricaltests of the relationships between force transfer efficiency and area of the oval windowplate, frequency, inertia, and mechanical advantage cannot easily be made directlyon the animal. We are at present engaged in both comparative studies and experi-ments with dynamic models to more directly deal with this problem.

Our functional interpretations can be tested indirectly by comparison of the relativedevelopment and distribution of the various functional components in different groupsof amphibians that inhabit different major adaptive zones. The role of the highfrequency tympanum-columella-basilar papilla system in anuran communication hasbeen well documented (see Straughan, 1973). The role of low frequency opercularcomplex-amphibian papilla is not so clearly defined. Because of its response tovibratory stimuli and its presence in terrestrial salamanders (sensu lato), which ingeneral do not communicate acoustically, its primary function is likely monitoringenvironmental sounds in terrestrial situations. A best low frequency response(o-2-o-5 KHz) is common to both frogs and salamanders (Lombard, in preparation)and corresponds to the frequency bandwidth of potentially important ambientsounds (Hardy, 1956). In salamanders this response cannot be associated withvocalizations because they are largely mute (Maslin, 1950). In an evolutionarysense, we believe that in terrestrial situations the primary function of the amphibiauditory system is the detection of general environmental sounds and


communication has been a later derivation in the Anura. This interpretationis supported by the ontogeny and relative distribution of this system in the amphibia.Temporally, the appearance of a complete opercular complex is essentially a meta-morphic event. Thus, no amphibian larvae possess this system. This supports thenotion of Kingsbury & Reed (1909) that the opercular complex is an adaptation forterrestrial perception. In addition, all paedogenetic urodeles and some totally aquaticmembers of derived families which have no terrestrial phase fail to develop theopercular complex (Kingsbury & Reed, 1909; Dunn, 1941; Monath, 1965). Amongthe Anura all members of the Liopelmatidae and Pipidae (Wagner, 1934; Stephenson,1951) and some aglossids (de Villiers, 1932; Sedra & Michael, 1959) lack an opercularcomplex. All of these animals are primarily adapted to an aquatic existence. There areapparent exceptions to these generalities. Many frogs and salamanders which spenda great deal of time in aquatic situations such as Rana and Ambystoma retain an oper-cular complex. Where this is true, however, the animal also spends a fair amount oftime on land where an opercular complex would be adaptive.

In anurans which have secondarily lost their high frequency communication channel,as in some species at high elevations, many aquatic (McDiarmid, 1971), and someburrowing forms (Emerson, 1970), the corresponding functional components - thetympanum and columellar shaft-become reduced or lost. In two such species examined,Bufo periglenes and Kalula pulchra, the low frequency threshold was equivalent toother 'vocal' species of the same size, but high frequency response was completelylacking (Straughan and Lombard, unpublished data).

A parallel situation exists in the Caudata where terrestrial acoustic communicationhas never been demonstrated. Completely terrestrial adults are not universal in thisorder but at least two independent terrestrial lines have evolved (Wake, 1966). Thepresence of an apparatus sensitive to environmental sound stimuli in air would be anadaptive advantage to these groups. Since they have independently arisen fromdifferent primarily aquatic ancestors (which would have no need for such a device)each line has developed an opercular complex, similar in basic form but differing indetail. There has thus been a convergence towards the development of the samefunctional system. The major difference seen involves the derivation of the opercularismuscle. In the Plethodontidae the opercularis is a derivative of the cucullaris. In otherterrestrial forms the muscle is a derivative of the levator scapulae (as it is in theAnura) (Dunn, 1941; Monath, 1965). This strongly supports the notion of theadaptive importance of this system to terrestrial amphibians. In salamanders, wherethere has been no development of an alternate sound transmission channel (such asa typanum), the system is open to evolutionary modification resulting in a singleuncoupled plate suitable for low frequency transmission only. On the evidence avail-able (Monath, 1965) it would appear that such an evolutionary modification has beenthe rule in terrestrial salamanders.

Finally, presumptive interactions of the two perception systems as they exist infrogs needs comment. During reproductive chorusing an ability to inactivate the lowfrequency system would confer a distinct advantage. With a decrease in sensitivity toambient environmental sound, the ability to perceive specific reproductive information

ould be relatively enhanced. That is, the total system would gain a more favourableut signal to noise ratio. Our experiments show that a 20-30 dB loss in sensitivity


results at the optimum low frequency when the opercular complex is not functioninpThis represents a considerable drop in sensitivity. The higher frequency region, whichis matched to preponderance of energy in the call, is not affected by inactivation ofthe opercular complex. The animal thus has a system favourable to 'focusing' onspecies specific information during reproductive interactions.

The bulk of this research was supported by grant 71-2112 from the AFOSRadministered by the University of Southern California. Terminal stages of the workwere supported by grant-SOi-RR-05367-11 from the USPHS administered by theUniversity of Chicago. The technical help of L. G. Bishop, D. R. Dvorak, L. Masuokaand C. Hillary, the University of Southern California, is gratefully acknowledged.The efforts of R. McDiarmid and R. Harris in sending specimens of H. versicolor,H. cinerea, S. baudimi and L. melanonotus to us are gratefully acknowledged. Theillustrations were prepared for publication by M. Oster, Department of Anatomy,University of Chicago. Special thanks are due D. B. Wake and J. A. Hopson forcritical reading of the manuscript.

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VAN BERGEJJK, W. A. (1957)- Observations on models of the basilar papilla of the frog's ear. J. Acoust.Soc. Amer. 39, 1159-63.

CAPBANICA, R R (1965). The evoked vocal response of the bull-frog. A study of communication bysound. Research Monog. No. 33, MIT Press.

DUNN, E. R. (1941). The 'opercularis' muscle of salamanders. J. Morph. 69, 307-16.ECKB, H. (1934). Anatomische und histologische Untersuchungen am Labyrinth der Erdkrote (Bufo

vulgaris, Laur). Z. Morph. Okol. Tiere. 39, 79-113.EMERSON, S. B. (1970). The fossorial frog adaptive zone: a study of convergence and parallelism in

the anura. Dissertation, University of Southern California.FRISHKOPF, L. S., CAPRANICA, R. R. & GOLDSTEIN, M. H., Jr. (1968). Neural coding in the bullfrogs

auditory system - a teleological approach. Proc. IEEE 56, 969-̂ 80.FRISHKOPF, L. S. & GBISLER, C. D. (1966). Peripheral origin of auditory responses recorded from the

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eighth nerve of the bullfrog. J. acoust. Soc. Am. 35, 1219-28.GBISLER, C. D., VAN BERGEIJK, A. A. & FRISHKOPF, L. S. (1964). The inner ear of the bullfrog. J. Morph.

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tells the frogs brain. Proc. IRE 47, 1941-51.LOMBARD, R. E. (1971). A comparative morphological analysis of the salamander inner ear. Disserta-

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Lond, 34, 509-46-PORTER, W. J. (1972). Herpetology. W. B. Saunders.POTTER, H. D. (1965). Patterns of acoustically evoked discharges of neurons in the mesencephlon j f

the bullfrog. J. Neurophysiol. a8, 1155-84.

Anuran middle ear 93R, A. S. (1970). The Vertebrate Body. 4th «L W. B. Saunders Co.

SACHS, M. B. (1964). Responses to acoustic stimuli from single units in the eighth nerve of the greenfrog. J. acoust. Soc. Am. 36, 1956-8.

SEDRA, S. N. & MICHAKL, M. I. (1959). The ontogenesis of the sound conducting apparatus of theEgyptian toad Bvfo regularis Reuss, with a review of this apparatus in salentia. J. Morph. 104,359-73-

SMITH, J. J. B. (1968). Hearing in terrestrial urodeles: a vibration sensitive mechanism in the ear.J. exp. Biol. 48, 191-205.

STBPHBNSON, N. G. (1951). On the development of the chondrocranium and visceral arches oiLiopdmaarcheyi. Trans. Zoo!. Soc. Land. 37, 303-53.

STRAUOHAN, I. R. (1973). Evolution of anuran mating calls: bioacoustical aspects. In EvolutionaryBiology of the Anurant (ed. J. A. Vial). University of Missouri Press.

DE VILLIERS, C. G. S. (1932). Uber das Gehorskelett der aglossen Anuren. Anat. Ana. 74, 33-55.WAGNER, D. S. (1934). The structure of the inner ear in relation to the reduction of the middle ear in

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functional aspects of anuran middle ear structures · lack a middle ear cavity and tympanum, the...

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