Alligator Lizard Research Articles

Components of cochlear electric responses in the alligator lizard.

Electric responses to clicks and tones were recorded at the round windows of anesthetized alligator lizards before and after tetrodotoxin (TTX) was added to scala tympani. By combining click responses obtained in the presence and absence of TTX and at high and low click repetition rates, we trisected click responses into three components: (1) a rate-insensitive, TTX-insensitive component (that we identify as the cochlear microphonic potential or CM); (2) a rate-sensitive, TTX-sensitive component (that we identify as the neural component); (3) a rate-sensitive, TTX-resistant component (which has not been identified previously and which we call component X). Component X is generated in the inner ear and has a latency between that of the CM and the neural component. Several possible origins for component X are discussed of which the most likely is that component X represents the compound post-synaptic potential of the nerve terminals. Measurements of responses to tones in the presence and absence of TTX demonstrate that the contribution of the neural component to the response is appreciable below 1.5 kHz and negligible above this frequency.

Frequency selectivity of hair cells and nerve fibres in the alligator lizard cochlea.

Receptor potentials of hair cells and spike discharges of cochlear nerve fibres were recorded with micropipettes from the free-standing region of the basilar papilla of anaesthetized alligator lizards in response to tones. In this region the hair-cell stereocilia are free-standing, i.e. they protrude directly into endolymph and are not in contact with a tectorial membrane. The frequency selectivity of hair-cell responses was measured by means of isovoltage contours of the d.c. (V0) and fundamental-a.c. (V1) component of the receptor potential, i.e. iso-V0 and iso-V1 contours. The frequency selectivity of the nerve-fibre discharge was measured by iso-rate (iso-V0) contours. Iso-V0, iso-V1 and iso-V0 contours are basically V-shaped with a characteristic frequency (c.f.) defined as the frequency at which minimum sound pressure (Pmin) is required to evoke the criterion value of the response. Receptor potential iso-V0 contours and neural iso-V0 contours have similar slopes: the mean slopes of the low-frequency sides (dB/decade) are -43.0 and -44.3; the slopes of the high-frequency sides are 85.0 and 80.2. The band widths of iso-V0 and iso-V0 contours away from c.f. are similar (mean values of Q30dB are 0.40 and 0.53, respectively). The band widths of iso-V0 contours near c.f. are narrower than those of iso-V0 contours (mean values of Q10dB are 2.34 and 1.20, respectively). However, the shapes of the contours near c.f. depend on the iso-response criteria, and we have not determined whether or not iso-V0 and iso-V0 contours are similar near c.f. The shapes of iso-V1 contours differ from those of iso-V0 and iso-V0 contours. Nerve fibre c.f.s are tonotopically organized in the nerve, with lowest c.f.s recorded from fibres innervating the border of free-standing and tectorial regions, a region in which hair-cell stereocilia are longest, and the highest c.f.s recorded from fibres innervating the end of the free-standing region in which hair-cell stereocilia are shortest. The c.f. of nerve-fibre response (and by implication hair-cell response) is, therefore, correlated with the height of the stereociliary tuft. The shapes of iso-V0 contours vary systematically with c.f. and, therefore, tonotopically with nerve position.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanical tuning of free-standing stereociliary bundles and frequency analysis in the alligator lizard cochlea.

Excised cochleae of alligator lizards were prepared to permit microscopic observations of motion of hair-cell free-standing stereociliary bundles and of the underlying basilar papilla during acoustic stimulation, using stroboscopic illumination. In response to tones of frequency from 0.2 to 5 kHz, the papilla rocks about an axis parallel to its length, displacing stereociliary bundles in the morphologically predicted direction of hair cell sensitivity. The papilla moves in phase along its entire length; for frequencies above about 3 kHz, the amplitude of motion of the most basal region is several times larger than that of the rest of the papilla. Over the basal two-thirds of the organ, stereociliary bundles stand freely in endolymph. In this region, maximum bundle height gradually decreases from about 30 to 12 micron; phase vs. frequency characteristics of bundle displacement with respect to the underlying papilla are those of nearly critically damped mechanical resonators. Resonant frequencies measured along the papilla vary inversely with a power (between 3/2 and 2) of bundle height and are close in value to auditory nerve fiber CFs measured in vivo at corresponding locations across the nerve. We suggest that length-dependent mechanical tuning of stereociliary bundles determines neural frequency selectivity and tonotopic organization in this part of the organ.

The ultrastructure of germinal beds in the ovary of Gerrhonotus coeruleus (Reptilia: Anguidae).

A study of ovarian structure in adult Alligator Lizards (Gerrhonotus coeruleus) was conducted by light microscopy and transmission electron microscopy. Particular attention was directed to characterizing the ultrastructure of germ-line cells, prior to follicle formation. General ovarian structure in this lizard is similar to that of other lizards. The paired organs are hollow, thin-walled sacs containing follicles in roughly 3 to 4 size classes. Ovarian germinal tissue consists of oogonia (diploid cells which divide mitotically) and oocytes (meiotic cells), intermixed with ovarian surface epithelial cells. Germ cells reside in two dorsal patches of epithelium per ovary (germinal beds), as is common in lizards. Oogonia in interphase show a highly dispersed chromatin pattern. Within oogonia cytoplasm, Golgi complexes are scarce, rough endoplasmic reticulum is absent, and lipid droplets are rare. Ribosomes are scattered in small clusters. Small, round vesicles are common in all oogonia; glycogen-like granules are present in some. Mitochondria form a juxta-nuclear mass within which groups of several mitochondria surround a dense granule. 'Nuage' granules also are found unassociated with mitochondria. Oocytes are present in stages of meiotic prophase up to diplotene. Synaptinemal complexes are seen in several (pachytene) cells. The cytoplasm of oocytes differs from that of oogonia in that mitochondria do not form groups, and nuage and glycogen are absent, whereas small round vesicles and large irregular vesicles are common. The ultrastructural similarities in germ cells of a reptile as compared to those of other vertebrates strengthens the notion that germ-line cells possess (or lack) qualities related to the undifferentiated state of these cells.

Author index

No adequate theory for the signal-transmission properties of the peripheral auditory system exists for any vertebrate ear. Because the mammalian ear seems to pose conceptual and technical problems that complicate the development of an adequate theory, it is worthwhile to investigate simpler ears. The ear of the alligator lizard is simpler than mammalian ears in several respects: the motion of the basilar membrane is approximately independent of longitudinal position and is approximately linearly related to the sound pressure at the tympanic membrane; in a large region of the cochlea the hair cells have free-standing stereocilia that are not in contact with a tectorial membrane; the receptor potential of these hair cells is related to the sound pressure at the tympanic membrane in a relatively simple manner; the cochlear-nerve fiber responses from this region do not exhibit two-tone rate suppression. Also, the relative accessibility of this ear has enabled measurement of several response variables: tympanic-membrane volume velocity, extracolumella velocity, basilar-membrane velocity, hair-cell stereociliary displacement, hair-cell receptor potentials, and cochlearnerve-fiber discharges. A model is developed to represent these results in terms of underlying anatomical structures and physiological mechanisms. The model consists of four cascaded signal-processing stages: 1.(1) a macromechanical stage that relates the sound pressure at the tympanic membrane to the velocity of the basilar membrane by an acoustico-mechanical network model of the middle and inner ear and which acts as a bandpass, linear, time-invariant filter;2.(2) a micromechanical stage that relates the velocity of the basilar membrane to the angular displacement of the hair-cell stereocilia by a hydrodynamic system that acts as a tonotopically-organized, bandpass, linear, time-invariant filter;3.(3) a mechanoelectric transduction stage that relates the angular displacement of the stereocilia to a change in membrane resistance through a set of angular-displacement-controlled membrane ionic channels and which behaves as a memoryless nonlinear function;4.(4) an electric stage that relates the change in resistance to the receptor potential by an electric network model of a hair cell that behaves approximately as a lowpass filter. This model predicts most — but not all — of the measured signal-transmission properties of this ear.

Whole‐nerve response to tone bursts in the alligator lizard

The peripheral auditory system of the alligator lizard (Gerrhonotus multicarinatus) has been studied extensively; however, limited data are available for the whole‐nerve (AP) response. Could the AP serve as a convenient monitor of the condition of the ear during experimentation? AP responses to tone burst acoustic stimuli of various frequencies were recorded from the alligator lizard and three measures using the AP response were obtained. (1) Averaged suprathreshold AP responses were related to primary auditory nerve fiber activity. (2) AP thresholds were compared to single‐unit thresholds. (3) AP excitatory tuning curves were compared to single‐unit tuning curves. The good agreement between AP responses and single‐unit data indicate that the AP is a useful tool for measuring the physiology of the peripheral auditory system in the alligator lizard.

Cochlear ganglion cells in the alligator lizard.

Two types of ganglion cells are present in the cochlear ganglion of the alligator lizard. Myelinated cells are the predominant type but a small population of unmyelinated cells is also present. Ganglion cells are reconstructed and morphometrically analyzed from serial light and electron micrographs. The results are interpreted to indicate that the unmyelinated ganglion cells are degenerating neurons. No normal population of unmyelinated nerve fibers was found. The anatomy of the cochlear nerve of the lizard is described and compared with the mammalian auditory nerve.

Receptor potentials of lizard hair cells with free-standing stereocilia: responses to acoustic clicks.

Receptor potentials of single hair cells in the free-standing region of the basilar papilla of the anaesthetized alligator lizard were measured intracellularly with micropipettes. Stimuli were primarily acoustic pulses (clicks) delivered to the tympanic membrane. The receptor potential was independent of click repetition rate for the range 10-150 clicks/s. This property is presumed to be the basis of the rate independence of the extracellular cochlear microphonic potential. The receptor potential wave-form consisted of a fast oscillatory component (or oscillation) superimposed on a usually positive (depolarizing) slow component. Reversal of the stimulus polarity resulted in a reversal of the polarity of the oscillations; the polarity of the slow component remained unchanged. The relative magnitudes of the two components depended on click level. At the higher click levels the magnitudes of the slow and oscillatory components were comparable. The relation of the receptor potential to the stimulus was non-linear; the peak-to-peak magnitude of the receptor potential increased less than proportionately with increasing sound-pressure level, and reversal of the stimulus polarity did not result in a reversal of the receptor potential. The receptor-potential magnitude for high-level clicks ranged from 1-13 mV peak-to-peak with an average value of 3.5 mV. At the lower click levels the magnitude of the slow component was much smaller than that of the oscillatory component. The relation of the receptor potential to the acoustic stimulus approached that of a linear system, the magnitude of the receptor potential became approximately proportional to the sound-pressure level, and reversal of the stimulus polarity resulted in approximate reversal of the receptor potential. For low-level stimuli the frequency of the oscillations of the receptor potential in response to clicks was approximately equal to the frequency of maximal a.c. response to tones. Apparently, both phenomena reflect the frequency selectivity of the processes generating the receptor potential. The frequency of oscillations in the click response varied from one cell to another (range of 1.0-2.2 kHz in this study). The results are qualitatively consistent with a model (Weiss, Mulroy & Altmann, 1974) that contains a linear, band-pass filter followed by a rectifier followed by a low-pass filter.

Some current concepts of cochlear mechanics.

The development of the current concepts is reviewed in historical perspective. Helmholtz's hypothesis of basilar-membrane resonance was partially confirmed and partially defeated by Békésy's experiments on models and postmortem cochlear preparations. He discovered that sound was propagated in the cochlea in the form of traveling waves which reached a flat maximum at a frequency-dependent location. Mathematical theory explained this type of sound propagation as a special case of surface waves. Johnstone and his coworkers discovered that the maximum of cochlear vibration in living animals was much sharper than postmortem, and more recently Khanna and Johnstone independently determined the maximum to be nearly as sharp as the tuning curves of the inner hair cells and the auditory-nerve fibers. These findings, together with the work at the Massachusetts Institute of Technology on alligator lizards, have led to new concepts of cochlear mechanics which include hypothesized micromechanical processes in the organ of Corti. These concepts deal not only with the sharpness of basilar-membrane tuning but also with the details of the basilar-membrane amplitude and phase characteristics, as well as with the hair cell and neural tuning curves and response phases. They suggest that some sharpening of the tuning curves occurs between the basilar membrane and hair-cell responses. Such sharpening has been demonstrated in lizards, but in the mammalian ear, the relation is less clear.

Supporting-cell and extracellular responses to acoustic clicks in the free-standing region of the alligator lizard cochlea.

1. Acoustic clocks were delivered to the tympanic membrane of anesthetized alligator lizards, and electric responses were measured within the free-standing region of the cochlea using glass micropipettes. Responses were recorded intracellularly in supporting cells and extracellularly in the receptor organ and in the scalae. Gross responses were also recorded with wire electrodes in scala tympani. 2. Intra- and extracellular responses contain two components: (1) an early (with latent period less than 0.15 ms after the onset of the click), rate-independent component presumed to originate in the receptor cells, which we call the 'receptor component'; and (2) a later (with latent period from 2 to 5 ms after the onset of the click), rate-dependent component presumed to originate in the primary neurons, which we call the 'neural component'. 3. The receptor component consists of a positive, slow, polarity-independent potential which is superimposed on a small, oscillatory, polarity-dependent potential. The average magnitude of the receptor component in supporting cells (0.72 mV for -20 dB clicks) is about 10 times that in the extracellular spaces and about 1/5 of that recorded in receptor cells. This component depends nonlinearly on the sound stimulus in the -20 to -55 dB range of click levels. 4. The average magnitude of the neural component in supporting cells (0.3 mV for clicks at -20 dB and 10 clicks/s) is about 5-10 times larger than that in the extracellular spaces. 5. The receptor and neural components have different distributions within the cochlea. The slow potential of the receptor component has positive polarity within the receptor organ and in scala tympani, and negative polarity in scala media. In contrast, the neural component has approximately the same biphasic (negative then positive) waveform in all extracellular compartments where it was detected. However, the neural component has a larger magnitude and an inverted (positive then negative) waveform in supporting cells. The neural component has not been detected in receptor cells.

Bidirectional transduction in vertebrate hair cells: a mechanism for coupling mechanical and electrical processes.

It has been proposed that the sharp frequency selectivity exhibited by cochlear hair cells and neurons of alligator lizards results from a mechanical resonance of the stereociliary-tectorial structures of hair cells. In contrast, in the red-eared turtle this selectivity has been attributed to an electrical resonance mechanism located in the hair-cell membrane. In this report a new mechanism is proposed, one which is consistent with observations in the lizard and turtle preparations and in which hair-cell resonances result from coupling of mechanical properties of hair-cell stereociliary-tectorial structures with electrical properties of the hair-cell membrane through a receptor membrane process that has bidirectional, mechanoelectric and electromechanical, transduction properties. This same mechanism could also be the physical basis for diverse phenomena observed in mammals, including changes in mechanical properties of the ear in response to electrical stimulation in the cochlea and central nervous system, and the presence of sustained, narrow-band, acoustic emissions in the ear canals of humans.

Changes in the organization of actin filaments in the stereocilia of noise-damaged lizard cochleae.

Alligator lizards exposed to 105 dB broadband noise for 24 h showed a 33 dB loss in hearing which was almost completely recovered 11 days after removal from the noise. Two lesions were found in the actin filament organization which could affect the rigidity of the stereocilia and thus could account for this hearing loss. These lesions preferentially affect the tallest stereocilia. The more common one is a depolymerization of the actin filaments at the base of the stereocilium where it makes contact with the cuticular plate. This results in a displacement and detachment of the stereocilium from its rootlet, thereby affecting the orientation of the tallest stereocilium. The other lesion involves a loss in crossbridges between adjacent actin filaments in the stereocilium. We demonstrate that such a loss will dramatically affect the rigidity of the stereocilia.

Actin in cochlear hair cells-implications for stereocilia movement.

The distribution and polarity of actin in chinchilla cochlear hair cells was determined by decoration of actin filaments with myosin subfragment S1. Actin is present in the stereocilia above the cuticular plate and there are actin filaments randomly oriented within the cuticular plate. These results in chinchilla cochlear hair cells agree with the findings in the frog, guinea pig, and alligator lizard inner ears. In the cross-striated region at the base of the cuticular plate of inner hair cells there are actin filaments which label with S1. These decorated filaments appear closely associated with the dense bands but the thin filaments perpendicular to the dense bands appear unlabeled. The presence of actin filaments in this cross-striated region at the base of the cuticular plate supports a mechanism that has been suggested for actin-myosin mediated movement of stereocilia [10], which would cause the stereocilia of inner hair cells to fan out into the endolymph.

Mechanical and electrical mechanisms in the ear: Alligator lizard tales

The ear of the alligator lizard is similar in structure and function to mammalian ears, but somewhat simpler and more accessible to in vivo experimental studies. Our focus has been on signal transmission through the middle ear and the portion of the inner ear having hair cells with free-standing stereocilia. We have measured two-tone distortion products in the sound pressure and the input admittance at the tympanic membrane; velocity of the extrastapes, stapes, and basilar membrane; intracellular receptor potential of hair cells; and spike discharges of cochlear nerve fibers. Many features of these measurements have been represented by a model of the underlying mechanical and electrical mechanisms. Conclusions suggested by the experimental and theoretical work include: (1) Nonlinearities detected in the external and middle ear responses appear to be independent of mechano-electric transduction in the cochlea. (2) The nonlinearities observed in the receptor potential at low-stimulus levels are associated with hair-cell processes. (3) The frequency selectivity of the receptor potential is determined not only by the middle-ear—basilar-membrane mechanical system but also by stereociliary-tuft resonances whose frequencies depend on tuft morphology. [Supported by NIH and the Whitaker Health Sciences Fund.]

Middle-ear mechanics: Comparison of cat and alligator lizard

Although the middle ears of cat and alligator lizard have similar components [tympanic membrane (TM), middle-ear cavities, and ossicular system], there are clear differences such as the number of ossicles and the structure of the TM. Mechanical responses of these two types of middle ear were investigated with measurements of the input admittance at the TM with the cavities open (Yd). Comparisons are made over the 10 Hz–15 kHz range. For cat Yd is compliancelike below 1 kHz and approximately resistive at higher frequencies. In lizard Yd is also compliance dominated below 1 kHz, and resistive near 2 kHz, but is masslike above 3 kHz. Admittance was also measured with the cochlear load removed and interrupted or fixed ossicular systems. Preliminary conclusions are: (1) In cat and the cochlear load has a large effect on Yd, while in lizard Yd is relatively independent of the cochlea. (2) In the lizard Yd is primarily determined by the TM. (3) The ossicular systems, by themselves, contribute little to Yd in either species. [Work supported by NIH and the Whitaker Health Sciences Fund.]

Introcellular responses of hair cells in the alligator lizard: Dependence on tone frequency and level

Receptor potentials of hair cells in the basal region of the basilar papilla of anesthetized alligator lizards were recorded with micropipettes. The steady‐state tone response consists of a dc component and ac components at multiples of the tone frequency. The isopotential contour (“tuning curve”) of the dc or the fundamental ac component has a sharply tuned “tip” on a more broadly tuned region. The dependence of receptor potential components on sound‐pressure level (“level functions”) was measured at different tone frequencies. These functions saturate at high sound‐pressure levels; the maximum value of the dc component is independent of frequency, while the maximum value of the fundamental ac component decreases with increasing tone frequency. Shapes of level functions at tone frequencies in the tuning curve “tip” differ from those at other frequencies, suggesting the presence of a frequency dependent non‐linearity in hair cell transduction. (Research supported by NIH.)

The organization of actin filaments in the stereocilia of cochlear hair cells.

Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin filaments with > 3,000 filaments near the tip and only 18-29 filaments at the base where the bundle enters into the cuticular plate; there the filaments splay out as if on the surface of a cone, forming the rootlet. Decoration of the hair cells with subfragment 1 of myosin reveals that all the filaments in the stereocilia, including those that extend into the cuticular plate forming the rootlet, have unidirectional polarity, with the arrowheads pointing towards the cell center. The rest of the cuticular plate is composed of actin filaments that show random polarity, and numerous fine, 30 A filaments that connect the rootlet filaments to each other, to the cuticular plate, and to the membrane. A careful examination of the packing of the actin filaments in the stereocilia by thin sectin and by optical diffraction reveals that the filaments are packed in a paracrystalline array with the crossover points of all the actin helices in hear-perfect register. In transverse sections, the actin filaments are not hexagonally packed but, rather, are arranged in scalloped rows that present a festooned profile. We demonstrated that this profile is a product of the crossbridges by examining serial sections, sections of different thicknesses, and the same stereocilium at two different cutting angles. The filament packing is not altered by fixation in different media, removal of the limiting membrane by detergent extraction, or incubation of extracted hair cells in EGTA, EDTA, and Ca++ and ATP. From our results, we conclude that the stereocilia of the ear, unlike the brush border of intestinal epithelial cells, are not designed to shorten, nor do the filaments appear to slide past one another. In fact, the stereocilium is like a large, rigid structure designed to move as a lever.

Micromechanics in the theory of cochlear mechanics

The consensus of empirical data suggests that the shear motion between the tectorial membrane and the reticular lamina, which is believed to control the stimulation of the hair cells, is more sharply tuned than the transversal vibration of the basilar membrane. Interaction between the length of basilar-membrane waves and the longitudinal coupling within the tectorial membrane may contribute to the required sharpening. However, such a mechanism is unlikely to be sufficient, acting alone. Simple calculations indicate that damping of the tectorial membrane coupled viscoelastically to the organ of Corti is low enough to allow the system to exhibit local radial responses above about 150 Hz. Some effects of such resonances are illustrated by means of simple mechanical models. In particular, it is demonstrated that radial vibration of the tectorial membrane nearly at right angles to the transversal vibration of the basilar membrane constitutes an inherently nonlinear system. The radial vibration can produce strong loading of the basilar membrane, affecting its tuning and introducing nonlinearities. Nonlinear longitudinal coupling within the tectorial membrane, if present, can produce quadratic and cubic distortion products and strong two-tone suppression by second tones at frequencies above and below the resonance frequency. Such a coupling is suggested by the finding that, in the alligator lizard, the part of auditory papilla endowed with a tectorial membrane produces both effects, but the part not so endowed does not.

Relations between frequency selectivity and two-tone rate suppression in lizard cochlear-nerve fibers

Cochlear-nerve fibers innervating the apicial region of the alligator lizard basilar papilla show sharp frequency selectivity in response to single tones (measured with the frequency threshold contour, or FTC), and the phenomenon of two-tone rate suppression (TTRS) in response to two simultaneously presented tones (measured with the iso-TTRS contour, or ITC). The gross shapes of the FTCs, as characterized by the slopes of the sides and Q 10dB, vary systematically with the fiber's characteristic frequency (CF). ‘Fine-structural’ features are also found: below CF, notches (frequency regions of relatively high threshold) occur in the FTC at frequencies related to CF. Above CF, a break frequency, which varies with CF, divides the FTC into segments of different slope. Features of the ITC also vary with CF. The detailed shapes of the FTCs and ITCs are related: lobes of the ITC interdigitate with notches in the FTC; the side of the FTC with steepest slope is closely associated with the side of the ITC with steepest slope. The close relation that is observed between sharp frequency selectivity and TTRS suggests that both phenomena arise from a common cochlear mechanism.

Basilar membrane motion in the alligator lizard: Its relation to tonotopic organization and frequency selectivity

The configuration of the inner ear in the alligator lizard allows (from a single approach) electrical and mechanical probing of the entire sensory organ. Studies of nerve fiber responses [Weiss, Mulroy, Turner, and Pike, Brain Res. 115, 71–90 (1977)] and intracellular responses in the receptor organ [Weiss, Mulroy, and Altmann, J. Acoust. Soc. Am. 55, 606–619 (1974)] have demonstrated that responses can be placed in two categories based on their frequency selectivity [viz., a high characteristic frequency (CF) group associated with the basal end of the organ and a low CF group associated with the apical end]. These two regions of the cochlea differ in morphology of the cilia and tectorial membrane [Mulroy, Brain Behav. Evol. 10, 69–87 (1974)]. Measurements of velocity using the Mossbauer method indicate that the motion of the basilar membrane is virtually the same in both regions. Thus, tonotopic organization and frequency selectivity are not determined by the basilar membrane motion. This finding suggests that other mechanisms, perhaps involving the cilia and tectorial membrane, provide spatially varying frequency selectivity in this species. Similar mechanisms may play a role in mammalian cochleas, where the basilar membrane apparently provides tonotopic organization and at least some of the frequency selectivity. [Supported by NIH.]