Inner Ear Physiology

The oceans are virtually transparent to sound, and opaque to light and radio waves. At a wavelength of 1 m (1,500 Hz), water is nearly 1,000,000 times more transparent to sound than to radio signals (Pilgrim and Lovell, 2002). This fact underlies the intense interest currently being directed toward the acoustical exploration of the ocean. Naturally produced sounds arise from a number of sources, such as breaking waves, heavy rain, volcanic activity, or from marine animals (bio-acoustic sources). Vocalisations such as whale song, along with the grunts and whistles from sonic fish are especially relevant for communication purposes, and during predator prey interactions (Myrberg 1981). There are several types of anthropogenic sources used routinely that produce intense levels of noise, with the most common being the incidental sounds generated by commercial shipping and powered leisure craft, and deliberately produced signals such as the Low Frequency Active Sonar (LFA) used by the military in anti-submarine warfare, or from the airgun arrays used during a seismic survey of the substrate beneath the seafloor by the petroleum industry. These activities can generate noise levels in excess of 253 dB (re 1 µPa at 1 m) (Engås et al., 1996), and are comparable to the noise levels generated by a seafloor volcanic eruption, which can produce a source level of in excess of 255 dB (re 1 µPa) (Northrop, 1974).

Ground dolphin ears and exposed cochlea canal. The ear on the left has been gold coated for an SEM examination

Growing concern by a number of environmental organisations regarding the use of LFA sonar and other intense anthropogenic sounds by the military and oil industry, is stimulating considerable interest in the diagnosis of the existence and extent of hearing loss in marine animals. This interest has prompted a number of studies into the effects of intense noise exposure on the hearing systems of marine mammals (e.g., Costa et al., 2003; Ketten, 1995; Richardson et al., 1995; Todd et al., 1996; Whitlow et al., 1997). High intensity low frequency sounds may be particularly damaging to the vestibular (balance) organs of cetaceans, and may account for the reported disorientation when these animals strand live. Concise morphological and physiological information on the hearing systems is critical to the assessment of the potential effect of anthropogenic noise pollution in the marine environment, being especially relevant where an animal has died as a consequence of intense noise exposure.

Trauma to the auditory system can result in lesions developing along the VIII nerve pathway, or ruptures in the blood vessels surrounding the inner ear. A number of techniques have been developed to study gross physiological damage to the inner ear, though these investigations do not necessarily verify the impairment of hearing and balance. In addition, injuries sustained by the animal may have been caused as it struggles in fishing nets or thrashes about on the shoreline and thus be unrelated to loud noise exposure. If caused by intense noise, signs of trauma (haematoma and nerve lesions) would probably manifest at the highest end of the impact scale, whereas more subtle damage to the ears may only show in the ultrastructure and thus be missed when using conventional examination methodologies. Current literature shows a paucity of information on consistent and meticulous removal of inner ear parts necessary to identify damage to the ultrastructure symptomatic of hearing and balance loss. The usual practice during autopsy is to fix the ear in formalin, though this chemical does not bind the proteins in the ultrastructure and results in the rapid destruction of the cilia, making the sample unusable for SEM microscopy. It is therefore the purpose of this study to attempt the dissection and fixation methodologies relevant to an SEM examination of the mammalian inner ear. However, owing to the scarcity of cetacean ear samples suitable for SEM microscopy, the inner ear of the domestic pig (Sus scrofa) is used to append the dissection and fixation methodologies required to view mammalian ultrastructural hair cells (Link to Study of Mammalian Ultrastructure). The periotic bone containing the inner ear from S. scrofa is dimensionally similar (though slightly thinner) than the cetacean periotic, though it has the considerable advantage of being easy to obtain fresh from commercial sources.

The comparative morphological study of the inner ears from the common dolphin (Delphinus delphis) and the harbour porpoise (Phocoena phocoena) are investigated here, in preparation for a Scanning Electron Microscope study of the cetacean inner ear ultrastructure. The saccule, utricle and semi-circular canals make up the vestibular system, and the scala tympani, scala media and scala vestibuli make up the cochlea (Corti, 1851; Retzius, 1884). Vibrations in the auditory periotic of Odontocetiform animals, caused by sound energy conducted through the mandibular channel oscillate the scala tympani within the inner ear (Whitlow, 1993). It is probable that these oscillations transmit energy to each of the three compartments in the cochlea, through fluid in the scala vestibule, or along the scala media and basilar membrane (the floor of the scala media). The sensory ultrastructure on the organ of Corti in the scala media rests on the basilar membrane and become polarised or hyperpolarised by the oscillating motion of the membrane (Ulfendahl et al., 1996). The hair cells convert the sound energy into bioelectric impulses, which travel via the VIII nerve to nuclei in the auditory pathway during acoustic stimulation (Brill, et al., 1988). To gain familiarity with the odontocetiform ear, samples of tissue were removed (with the authority of the Natural History Museum, London), and examined in respect to the inner ear morphology. The mature harbour porpoise (Phocoena phocoena) (Figure 1) was recovered on the 11^th of February 2005 from Andurn Point, near Plymouth Sound, Ordnance Survey GB grid reading SX495492 (NHM reference SW.2005/30). The tagged carcass was 1.47 meters in length and approximately three years of age (Read, 1999), and was not designated for autopsy due to it being in an advanced state of decay.

Figure 1. Harbour porpoise (Phocoena phocoena) recovered from Andurn Point (click to enlarge)

Figure 2. Common dolphin (Delphinus delphis)recovered from Beacon Point (click to enlarge)

The common dolphin (Delphinus delphis) (Figure 2) was recovered on the 2^nd of February from Beacon Point in Devon (Ordnance Survey GB grid reading SX674406). The tagged carcass was not designated for collection (autopsy), due to its location at the foot of a steep cliff making access difficult. A brief inspection revealed that the animal was a mature male, approximately 2.4 meters in length, and estimated to have been dead for 10 to 12 days; it is highly possible that the animal died as a result of becoming entangled in fishing gear.

The periotic bone containing the inner ears from D. delphis and P. phocoena were separated from the tympanic bone, and washed in 70 % chilled ethanol. Removal of the complete cochlea from the encapsulating periotic bone in P. phocoena required two cuts made using a fine cutting wheel, which was stopped short of penetrating to the inner ear canals by approximately 0.4 mm. The weakness in the bone caused by the hemispherical cut allowed for the two halves of the periotic to be gently separated using minimal leverage, thus exposing the internal structure of the ear. The skeletal remains of the cochlea were not removed from the periotic in D. delphis; instead it was prepared for the EM study whilst still in the encapsulating bone. A cast of the inner ear cavity was then made by injecting Silicone rubber into the cochlea duct and vestibule, and allowed to cure for 24 hours (Figures 1.a through c). The cast was removed by gently separating the three cut sections of the periotic, and by easing the rubberised impression of the ear from the bone segments; a similar procedure has been used successfully by the author on the elasmobranch ear (Lovell, unpublished). The cast was then washed in 100 % ethanol and processed for a low powered Scanning Electron Microscope (SEM) examination of the surface features.

Figure 3. Ventral view of the periotic from P. phocoena showing the position of the two cuts, b. the sections of periotic cut away to free the cochlea, and c. the periotic after the silicone injection moulding procedure. The annotations D. (Dorsal), and A. (Anterior) represent the orientation of the periotic in the skull

An examination of the brain of the recovered dolphin shown in Figure 2, revealed a large VIII auditory nerve, originating at the peripheral end of the nerve in the periotic chamber, and terminating in the medulla. The brain, VIII auditory nerve and auditory periotic bone from the right side of the cranium were removed intact, and the brain weighed using digital precision scales (total weight = 1008 g with the periotic bone absent), then transferred to a large beaker and immersed in 70 % ethanol, prior to fixing in formalin. Figure 4. hyperlinks to a page looking at the physiology of the D. delphis brain. The skull was then photographed in a number of positions and annotated for reference purposes (Figures 5.a through d). The cranium and brain asymmetry in D. delphis examined here, was found to be larger in the right hemisphere (especially evident in the nasal passage).

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Figure 4. (a. b. c. d) Mid-sagittal division of the 1.90 m D. delphis brain into left and right hemispheres

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Fig. 5. a. Dorsal/anterior view of the skull from D. delphis (note there is a slight dominance of the right side). b. ventral view of the skull showing position of the auditory periotic and tympanic bones (appb) and lower mandible (m)

In D. delphis, fibrous tissue surrounds the heavily calcified auditory periotic bone (Figures 6.a to c) and the thin tympanic bone (Figure 6.a), which are connected to the skull by a flexible ligament that effectively isolates the inner ear from the skull. Sound enters the ear most efficiently through the mandibular channel in the lower jawbone, which extends back toward the auditory periotic bone.

Fig. 6.a. The auditory periotic with VIII nerve and the thin tympanic bone from D. delphis, b. dividing the tympanic bone from the auditory periotic bone, and c. cross section through the periotic capsule containing the inner ear, and peripheral VIII nerve fibres

Figure 7 presents a ventral view of a cross section through the periotic bone from D. delphis, showing the lower basal section of the cochlea, and the scala vestibuli and scala tympani (the floor of the scala tympani has been removed). The skeletal remains of the cochlea is visible, and shows detail of the bony spiral lamina, scalar tympani and upper portion of the scalar vestibule and peripheral VIII nerve fossa on the inside edge of the lamina.

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Fig. 7. Cross section through the periotic bone from D. delphis

Figure 8 presents an Electron micrograph of the inner ear cast from D. delphis, reproduced by injecting silicone rubber into the auditory periotic bone surrounding the inner ear. This procedure was necessary as the fine internal structure of the ear had decomposed to an extent where it could not be removed by dissection; rather it had to be washed from the chambers within the periotic using 70 % ethanol. The length of the cochlea from the upper apical tip to the lower basal segment was calculated to be 30.1 mm, with the vestibule etc, making up the remaining 3 mm of the inner ear (total length 33.1 mm).

Figure 8. SEM micrographs of the left inner ear cast from D. delphis (lateral views toward and away from the mid-sagittal plane of the brain)

Morphological Examination of the Auditory System from the Harbour Porpoise Phocoena phocoena

The dissected cochlea from P. phocoena was placed in a watch glass containing 70 % ethanol, and photographed using a digital camera and trinocular microscope (Figure 9). The innervated length of the cochlea was measured on a PC using the analySIS® (Soft Imaging System) program, and found to have a length of 21.8 mm; the total length of the sample was calculated to be 24.8 mm, with the saccule and oval window making up the remaining 3 mm. The investigation of the internal dimensions of the periotic bone was conducted to rule out the possibility that some of the organ of Corti still remained in the periotic, or had decomposed and was no longer visible, thus ensuring an accurate measurement of the organ.

Fig. 9. The organ of corti from P. phocoena. Total innervated cochlea length: 21.8 mm, total sample length: 24.8 mm

The complete cast of the inner ear from P. phocoena in Figures 10 and 11 presents the complete structure viewed laterally, both toward and away from the mid-sagittal plane of the brain.

Figure 10. SEM micrograph of the left inner ear cast from P. phocoena (lateral view away from the mid-sagittal plane of the brain) (Click Thumbnail to enlarge).

Figure 11. SEM micrograph of the left inner ear cast from P. phocoena (lateral view toward the mid-sagittal plane of the brain) (Click Thumbnail to enlarge).

Discussion:

A procedure for the fast removal of the complete cochlea and other end organs of the inner ear undamaged has been demonstrated here, though, as both carcass examined in this study were retrieved in an advanced state of decomposition, SEM examinations of the ultrastructure within the inner ear was not undertaken, as inner ear hair cells are known to deteriorate within a short time after death. The SEM has been used to considerable effect on lower vertebrates such as fish (Platt 1977; Lovell et al., 2005b), and invertebrates (Lovell et al., 2005a) in the examination of the ultrastructure responsible for the mediation of auditory stimuli, though no SEM examinations have been conducted on the inner ear ultrastructure from any of the cetacean species. The relative ease and speed in which the auditory periotic can be dissected from behind the mandible of D. delphis, indicates that it should be possible to remove the complete cetacean inner ear for a Scanning Electron Microscope examination of the hair cells. It is essential that the periotic is rapidly immersed in chilled fixative (2.5% glutaraldehyde in 0.1 M cacodylate buffer with 3.5% sodium chloride), then refrigerated to inhibit sample decomposition (the sample must not be frozen, as ice crystals will destroy the ultrastructure).

The innervated cochlea from P. phocoena was 21.8 mm in length, confirmed by the investigation of the internal dimensions of the encapsulating periotic bone, which had a total length (from the upper apical tip of the cochlea to the oval window) of 24.5 mm in P phocoena, and 33.1 mm in D. delphis. Both the animals investigated in this study were mature individuals (see Read, 1999), thus it is tentatively concluded that the cochlea in P. phocoena is at least 17 mm shorter than reported for T. truncatus (Wever et al., 1971), and 8 mm shorter than D. delphis. All mammalian cochleae appear to function according to the same basic principles; however, the effective frequency range differs between species (Fay, 1988). For example, the range of audible frequencies is about 20 Hz to 16 kHz in the human cochlea and about 100 Hz to 40 kHz in the cat (Felis catus). The length of the basilar membrane from T. truncates is over 38 mm, in the human it is 35 mm, in D. delphis it is 30 mm, whilst in F. cattus it is 25 mm and in P. phocoena it is 22 mm. The fundamental measure of hearing ability for any animal possessing the appropriate receptor mechanism is its audiogram (Myerberg, 1981), which presents the lowest level of sound that the species can hear as a function of frequency. The hearing frequencies or audiograms for a number of odontocetiformes are well characterised, and have been produced using both physiological and behavioural approaches (see Nachtigall et al., 1995; Kastelein, et al., 2003; Sauerland and Dehnhardt, 1998; Gerstein et al., 1999; Kastelein et al., 2002), though an audiogram for D. delphis has as yet to be produced. The bottlenose dolphin (T. truncates) hears frequencies from 100 Hz to 150 kHz (Johnson, 1966; 1967), and the striped dolphin (Stenella coeruleoalba) hears frequencies ranging from around 500 Hz to 150 kHz (Kastelein, 2003; Brill et al., 2001), with both producing broadband clicks for echolocation that range in frequency from 20 Hz to around 200 kHz. P. phocoena hears frequencies between 300 Hz (Kastelein et al., 2002), up to as high as 190 kHz (Bibikov, 1992; Popov, 1986; Kastelein et al., 2002), and utilises a narrow band high frequency sonar of around 120 to 140 kHz (Busnel and Dziedzic, 1966a). It is feasible that this difference in hearing ability between T. truncates and P. phocoena is explained by the larger cochlea in the bottlenose dolphin (Wever et al., 1971; Ketten, 1997). The evidence presented in this study suggests that the audiogram for D. delphis may lie somewhere between the hearing range of T. truncates and P. phocoena; it is therefore concluded that the production of an audiogram for D. delphis (Link to ABR Proposal), is of considerable importance for an accurate assessment of the impact of anthropogenic sounds on the inner ear physiology of this animal. Audiograms produced using the Auditory Brainstem Response (ABR) technique (developed originally for clinical use on humans) are regarded as being the least time consuming and most reliable methodology for acquiring audiological data. The ABR trace is formed by averaging conglomerate responses of peak potentials arising from centres in the auditory pathways from the periphery of the VIII nerve to the midbrain, and detected in the electrophysiological far-field, using two cutanious electrodes (Corwin et al., 1982; Overbeck and Church, 1992).

Acknowledgments

The authors would like to thank Gavin Black of the Devon Biodiversity Records Centre at the Devon Wildlife Trust, for his help in the acquisition of samples.

References

Au, W.W. (1980). Echolocation signals of the Atlantic bottlenose dolphin (Tursips Truncatus) in open waters. Animal sonar systems. pp. 251-282.

Bibikov, N.G. (1992). Auditory brainstem responses in the harbour porpoise (Phocoena phocoena). In: 'Marine Mammal Sensory Systems', 197-211. Thomas, J. et al (eds). Plenum Press, New York.

Brill, R. L., Sevenich, M. L., Sullivan, T. J., Sustmen, J. D., & Witt, R. E. (1988). Behavioral evidence for hearing through the lower jaw by an echolocating dolphin (Tursiops truncates). Mar. Mam. Sci. 4, 223–230.

Brill, R.L., Moore, P.W.B. & Dankiewicz, L.A. (2001). Assessment of dolphin

(Tursiops truncates) auditory sensitivity and hearing loss using jawphones.

JASA, 109(4), 1717-1722.

Busnel, R.G., & Dziedzic, A (1966a). Acoustic signals of the pilot whale Globicephala melaena and of the porpoises Delphinus delphis and Phocoena phocoena. (in K.S. Norris, ed.) Whales, dolphins and porpoises. University of California Press, Berkeley. pp. 607-646

Corti A. (1851). Recherches sur l'organe de l'ouïe des mammifères. Zeitschr wiss Zool. 3:109-169.

Corwin, J.T, Bullock T.H. & Schweitzer J. (1982). The auditory brainstem response in five vertebrate classes. Electroencephalogr. Clin. Neurophysiol. 54: 629-641

Costa, D.P, Crocker, D.E, Gedamke, J, Webb, P.M, Houser, D.S, Blackwell, S.B, Waples, D, Hayes, S.A. & Le Boeuf, B.J. (2003). The effect of a low-frequency sound source (acoustic thermometry of the ocean climate) on the diving behavior of juvenile northern elephant seals, Mirounga angustirostris. Journal of the Acoustical Society of America 113(2):1155-1165.

Cranford, T.W. (2000) In Search of Impulse Sound Sources in Odontocetes. In hearing by Whales and Dolphins (Springer Handbook of Auditory Research series), W.W.L. Au, A.N. Popper and R.R. Fay, Eds. Springer-Verlag, New York, (40 MS pages).

Cranford, T.W., Amundin, M.E. & Norris K.S. (1996) Functional morphology and homology in the odontocete nasal complex: implications for sound generation. Journal of Morphology. 228(3), pp. 223-285.

Engås, A., Løkkeborg, S., Ona, E., & Soldal, A.V. (1996). Effects of seismic shooting on local abundance and catch rates of cod (Gadus Morhua) and haddock (Melanogrammus aeglefinus). Canadian J. of Fisheries and Aquatic Sciences. 53, 2238 - 2249.

Fay R.R. (1988). Hearing in Vertebrates: a Psychophysics Databook. Hill-Fay Associates, Winnetka IL.

Gerstein, E.R., Gerstein, L., Forsythe, S.E, & Blue, J.E. (1999). The underwater audiogram of the West Indian manatee (Trichechus manatus). JASA, 105(6), 3575-3583.

Gray, H. (1918). Anatomy of the Human Body. Lea & Febiger, Philadelphia: Bartleby.com, 2000. www.bartleby.com/107/. [15-02-05].

Johnson, C.S, (1967). Sound detection thresholds in marine mammals. In W.N. Tavolga (ed), Marine bio-acoustics, vol. 2. Pergamon, Oxford, U.K.

Johnson, C.S. (1966). Auditory thresholds of the bottlenosed porpoise (Tursiops truncatus). U.S. Naval Ord. Test Stn., Tech. Oubl., 4178: 1-28.

Kastelein, R.A., Hagedoorn, M., Au, W.W.L. & de Haan, D. (2003). Audiogram of a striped dolphin (Stenella coeruleoalba). JASA, 113(2), 1130-1137.

Kastelein, R.A., Bunskoek, P., Hagedoorn, M., Au, W.L.W. & de Haan, D. (2002). Audiogram of a harbor porpoise (Phocoena phocoena) measured with narrow-band frequency-modulated signals. JASA, 112(1), 334-344.

Kenyon, T.N., Ladich, F. & Yan, H.Y. (1998). A comparative study of the hearing ability in ﬁshes: the auditory brainstem response approach”. J Comp Physiol A 182:307–318.

Ketten, D.R. (1995). Estimates of blast injury and acoustic trauma zones for marine mammals from underwater explosions. In: Sensory Systems of Aquatic Mammals, R. Kastelein, J. Thomas, and P. Nachtigall (eds.), DeSpil Publishers, pp. 391-408

Ketten, D.R. (1997). Structure and Function in Whale Ears, Bioacoustics, vol.8, no. 1, pp. 103-136.

Lovell, J.M, Findlay, M.M, Moate, R.M & Yan H.Y (2005 A). The hearing abilities of the prawn (Palaemon serratus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. Vol 140/1 pp 89-100

Lovell, J.M, Findlay, M.M, Moate, R.M & Pilgrim D.A. (2005 B). The polarization of inner ear ciliary bundles from a scorpaeniform fish. Journal of Fish Biology 66, pp 836–846

Lovell, J.M (unpublished). The ethology and physiology of sonic production and reception in marine animals. BSc honours project (1999), University of Plymouth UK

Myrberg, A.A. (1981). Sound communication and interception in fishes. Hearing and Sound Communication in Fishes. Tavolga WN, Popper AN, and Fay RR, Eds. Springer-Verlag, New York.

Nachtigall, P.E., Au, W.W.L., Pawloski, J.L. & Moore, P.W.B. (1995). Risso’s dolphin (Grampus griseus) hearing thresholds in Kaneohe Bay, Hawaii. In ‘Sensory Systems of Aquatic Mammals’, 49-53. R.A. Kastelein et al (eds). De Spil Publ., Woerden, Netherlands.

Nedwell, J.R., Turnpenny, A.W.H., Lovell, J.M., Langworthy J., Howell, D. & Edwards, B. (2003). The effects of underwater noise from coastal piling on salmon (Salmo salar) and brown trout (Salmo trutta). Subacoustech Report Reference: 576R0113.

Northrup, J. (1974). Detection of low-frequency underwater sounds from a submarine volcano in the western Pacific. J. Acoust. Soc. Am. 56(3), 837-841.

Pilgrim, D.A. & Lovell, J.M., (2002). A review of current publications dealing with the impact of low frequency sounds upon fish. Report to Devon Sea Fishing Association.

Platt, C. (1977). Hair cell distribution and orientation in goldfish otolith organs. Journal of Comparative Neurology. 172, 283-297 pp.

Popov, V.V., Ladygina, T.F. & Supin, A.Ya. (1986). Evoked potentials of the auditory cortex of the porpoise, Phocoena phocoena. J. Comp. Physiol., 158:705-711.

Read, A.J. (1999). Harbour porpoise (Phocoena phocoena). In S.H. Ridgeway & R. Harrison (eds.), Handbook of Marine Mammals, Volume 6: The Second Book of Dolphins and Porpoises. Academic Press. San Diego.

Retzius, G. (1884). Das Gehörorgan der Wirbelthiere. Vols 1 & 2. Stockholm, Sweden: Samson & Wallin

Richardson, W. J., Greene, C. R., Jr., Malme, C. I., & Thomson, D. H. (1995). Effects of Noise on Marine Mammals. Academic, San Diego pp 576.

Sauerland, M. & Dehnhardt, G. (1998). Underwater audiogram of a tucuxi (Sotalia fluviatilis guianensis). JASA, 103(2): 1199-1204.

Todd, S., S. Stevick, J. Lien, F. Marques, & D. Ketten (1996). Behavioural effects of exposure to underwater explosions in humpback whales (Megaptera novaeangliae). Canadian Journal of Zoology, 74(9):1661-1672.

Ulfendahl, M., Khanna, S. M., Fridberger, A., Flock, A., Flock B., & Jager, W. (1996). Mechanical response characteristics of the hearing organ in the low-frequency regions of the cochlea. Journal of Neurophysiology, Vol 76, Issue 6 3850-3862

Wever, E. G., McCormick, J. G., Palin, J. & Ridway, S. H. (1971). The Cochlea of the Dolphin, Tursiops truncatus: Hair Cells and Ganglion Cells. Proceedings of the National Academy of Sciences of the United States of America 68: 2908-2912.

Whitlow, W., Au, W., Nachtigall, P. & Pawloski, J. (1997). Acoutic effects of the ATOC signal (75Hz, 195 dB) on dolphins and whales. Journal of the Acoustical Society of America, 101(5) Pt1

Whitlow, W. (1993). The sonar of dolphins. New York: Springer-Verlag. p. 22-239.

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