Учебники / Hair Cell Regeneration, Repair, and Protection Salvi 2008

deteriorates as frequency increases, and between 3.0 and 4.0 kHz is lost in the noise. Figure 3.7C shows the VS in a sample of units just removed from the 0.9-kHz, 120-dB sound exposure, and these results reveal that VS in exposed and control units are the same. This observation is interesting because these exposed units exhibited the same changes in CF threshold, tuning sharpness, spontaneous activity, and rate-level activity shown in Figures 3.3, 3.4, and 3.5. The fact that synchronization was unaffected, even though overlain on substantial changes in discharge activity, suggests that the hair cell membrane and synaptic properties that determine the degree of synchronization were unaffected by the exposure.

2.3.6 Adaptation

Adaptation in the cochlear nerve response represents a progressive decline in discharge activity with the passage of stimulus time. Adaptation is identified in poststimulus time histograms (PSTH), which describe the accumulation of spike discharges in successive time intervals (bins) that occur with repeated tone-burst presentations. The PSTH exhibits several stages of adaptation; a phasic portion at stimulus onset where the number of discharges per bin are high, followed by a decline in activity to a tonic level where the number of discharges per bin are lower and relatively constant. The decline in discharge activity is orderly, and characterized by fitting the histogram data to an exponential decay function. Figure 3.8A shows a PST histogram for a tone burst with discharge activity collected at a 0.1-ms resolution. Figure 3.8B reduces the data to 1.0-ms bins, and fits the results with an exponential decay (solid line), revealing a time constant of 16.8 ms. Adaptation in chick occurs in three different time compartments called rapid, short term, and long term with time constants of approximately 2–3 ms, 15–17 ms, and 20–40 s. An example of long-term adaptation appears in Figure 3.8C, where discharge rates to a continuous tone were measured in 2-s intervals, every 4 s, for 43 min. The time constant was 30.9 s, but what is remarkable about this curve is that tonic activity (approximately 65 S/s) was maintained for the next 2530 s.

The presynaptic membrane of the hair cell appears to control adaptation through the number and release kinetics of neurotransmitter vesicles (Spassova et al. 2004; Crumling and Saunders 2006). One possible scenario is that vesicles lying beneath the dense body (the synaptic ribbon in chick) in immediate proximity to the hair cell plasma membrane, undergo exocytosis first (this is the immediately releasable pool). This is then followed by the exocytosis of those vesicles tethered to the dense body (the readily releasable pool). Finally, a steady level of vesicle mobilization, probably from the hair cell cytoplasm, is transported to the dense body and hence the release sites (this is the mobilizable pool). The three stages may correspond to the initial burst of activity, followed by the rapid decay and then tonic levels of activity, as vesicles in the various releasable pools are exhausted. Mobilization of cytoplasmic vesicles to the release site permits sustained discharge activity over long time intervals.

94 J.C. Saunders and R.J. Salvi

Figure 3.8. (A) Peristimulus time histogram plotted at a resolution of 0.1 ms. (B) Activity is binned to a resolution of 1.0 ms and fitted with a single component exponential decay function. The waveform of the non-phase-locked tone-burst stimulus is seen below. (C) Adaptation in a unit recorded with a 4-s resolution over a 2550-s interval. The value ofindicates the time constant. The upper and lower sections of (D) show PST histograms of neural activity to tone burst stimuli, averaged across a sample of units (14–30 units per curve) with approximately the same indicated CF. The data were obtained from agematched 0-day exposed and control animals. The early phasic component of the PSTH is elevated in the exposed adaptation curves while adapted rates exhibit the same discharge rate in both groups. (Data from Crumling 2006, with permission.)

Figure 3.8D shows changes in adaptation for units measured shortly after removal from the 120-dB, 0.9-kHz pure-tone exposure. The curves in each panel depict an average PST histogram (14–30 units per curve) in control or exposed units, all with approximately the same CF. Exposed units with CFs at or above the exposure frequency show an increase in the phasic discharge rate, but the same level of activity for the tonic region of the curve, compared to the control PSTH. The time constant of these exposed and control PSTHs were much the same (Crumling and Saunders 2006). These results indicated that the kinetics of discharge decay were unaffected by the exposure (as revealed by the similar time constants); however, the phasic portion of the response was increased in exposed units. Adaptation functions for control and exposed units with CFs below the exposure frequency (0.9 kHz) were nearly identical.

The mechanisms controlling vesicle release rate were unaffected by the exposure; however, the number of vesicles released (and the volume of neuro-

transmitter available to influence the postsynaptic membrane) was larger, but only for the vesicle pools associated with the initial level of exocitic activity. How this can be is not clear at present because the dense body normally has a 60% vesicle packing density. It could be that the exposure causes more vesicles to cluster at the membrane surface, filling the remaining 40% of the dense body, or the dense body gets larger in exposed hair cells. Contributions from efferent feedback or changes in the postsynaptic membrane have yet to be defined, but they could also play a role in this observation. It is interesting to speculate further that the enhanced phasic response is some form of compensatory mechanism by the hair cell to maintain a measure of discharge sensitivity in the face of destructive overstimulation.

2.3.7 Spatial Tuning Curves

The issue of the incompletely healed tectorial membrane and its effect on the chick peripheral auditory system has been noted above and revisited by examining spatial excitation for CFs distributed across the basilar papilla surface (Lifshitz et al. 2004). Spectral response tuning curves were examined in agematched control chicks, and in chicks exposed to intense sound, but allowed to recover for 12 days. The discharge activity at criterion frequencies and intensities were noted for each unit and then plotted against the CF of the unit. When the CF of the unit was the same as the criterion frequency, unit discharge rate was expectedly high. As units were examined with CFs above or below the criterion frequency, activity diminished. Finally, when the unit CF was sufficiently distant from the criterion frequency, only spontaneous activity was observed. These spatial tuning curves were constructed for a number of criterion frequencies, and the excitation patterns provided a proxy of the mechanical activity on the papilla surface. Spatial tuning curves for control chicks at 10 criterion frequencies appear in Figure 3.9 at one criterion intensity (40 dB SPL). Using an equation that described the distribution of unit CFs along the tonotopic axis of the basilar papilla (Chen et al. 1994), the spatial tuning curve data were plotted against axes for the percent distance and tonotopic frequency from the apical end of the papilla.

Figure 3.9B illustrates the excitation patterns in chicks exposed to a 120-dB SPL, 48-hour, 0.9-kHz pure tone, and then allowed 12 days to recover. Above the spatial tuning curves is a cartoon of the papilla depicting the location and extent of the partially healed, patch lesion. The accuracy of the equation transforming unit CF to papilla location is seen in the fact that the spatial tuning curve in Figure 3.9B with a criterion frequency of 0.95 kHz is centered almost in the middle of the patch created by a 0.9-kHz exposure tone.

The spatial tuning curves with criterion frequencies within the patch lesion are the same as those in the age-matched control group in Figure 3.9A. Thus, the damage to the tectroial membrane does not appear to play a role in the processes that determine the spatial tuning of units across the sensory surface of the basilar papilla (Lifshitz et al. 2004).

2.4 Changes in Cochlear Nerve Activity

After Ototoxic Trauma

2.4.1 Ototoxic Damage and Recovery to the Papilla

The application of ototoxic agents such as gentamicin or kanamycin is potentially lethal due to nephrotoxic side effects, and animal survival depends on the number of daily injections and drug dose level. These treatments are very destructive to the receptor cells and cause the loss of all neural and abneural hair cells beginning in the high-frequency region of the papilla. As dosing progresses, the lesion extends into the low frequencies, but even with long treatment regimens, rarely goes beyond the basal 50% of the papilla. Despite the hair cell losses, the injury to the overall papilla is less traumatic than with acoustic overstimulation. The tectorial membrane, for example, appears normal, although it is disconnected from the reticular surface of the papilla.

Single, high-dose injections have been used to more precisely time the course of injury and recovery, and these also cause hair cell loss to the approximate basal half of the papilla. A refinement in procedure applies gentamicin-loaded collagen sponges directly to the round window membrane. Nephrotoxicity is greatly reduced and complete loss of all hair cells now occurs over as much as 70% of the papilla (Husmann et al. 1998; Müller and Smolders 1998, 1999).

The time of emergence of new hair cells is difficult to determine with multiple injections, but with a single-dose treatment new hair cells are evident after 3–5 days in young chicks. In general, within 4 weeks posttreatment the sensory epithelium is completely repopulated with new hair cells, although hair bundle orientation may remain abnormal. Also, when a local application of gentamicin is used, it results in fewer regenerated hair cells than found in the original population. Reinnervation of the regenerated hair cells also occurs. Full details of the development and recovery of ototoxic damage to the papilla can be found in Cotanche (1999).

2.4.2 Functional Changes

Figure 3.10A illustrates auditory brainstem response (ABR) threshold shifts in the adult pigeon after application of a gentamicin sponge to the round window (Müller and Smolders 1999). The parameter is recovery duration. At 5 days after the sponge application, the threshold shift is between 63 and 69 dB and between 1.41 and 5.66 kHz. Over the next 37 days considerable recovery occured, but after 70 days there was little further improvement. Similar ABR results have been reported in the chick after aminoglycoside injections that began just after hatching. Thresholds recovered with time, but after durations as long as 14–20 weeks a high-frequency (>1 5 kHz) permanent shift of 20–30 dB was noted (Tucci and Rubel 1990; Girod et al. 1991; Duckert and Rubel 1993).

Cochlear nerve unit activity has been reported after either systemic kanamycin injections in the adult chicken or localized gentamicin treatment in the pigeon (Salvi et al. 1994; Müller and Smolders 1998). The results of both studies were largely

consistent with each other. Shortly after the end of treatment, lower frequency CFs showed abnormal thresholds, tuning curves, lower spontaneous activity, and changes in the proportion of units showing preferred intervals of spontaneous activity. It was difficult if not impossible to record units with higher frequency CFs because the basal region of the papilla was completely stripped of hair cells. After long recovery intervals (approximately 10–20 weeks), activity in low-frequency units exhibited complete recovery. Units with higher CFs could be identified as the basal papilla hair cells regenerated and matured; however, they often failed to exhibit normal levels of activity. These effects are illustrated in Figure 3.10B by plotting only the cochlear nerve units that showed the most sensitive CF thresholds in control animals and in gentamicin-treated pigeons (Müller and Smolders 1998). The units in the treated animals were recorded 14 weeks after the aminoglycoside application. The “best” CF thresholds are approximately the same in both groups up to about 0.25 kHz, and then differ by increasing amounts as CF rises. The differences are startling given that hair cell complements in the high-frequency region of the papilla have returned to nearly normal levels. Measures of Q10dB, spontaneous activity, and the dynamic range of rate-intensity functions were also diminished in units with CFs above 0.25 kHz.

2.4.3 Differences Between Acoustic and Ototoxic Trauma

The effects of acoustic and ototoxic trauma to the papilla offer interesting and contrasting models of damage. Acoustic lesions are often confined to the abneural hair cells at a restricted location on the sensory surface that corresponds to the location stimulated by the exposure frequency. Overstimulation produces lesions that result in hair cell loss, cellular disruption of the sensory epithelium, and severe damage to the tectorial membrane. The latter damage does not appear to recover (Cotanche 1999). An ototoxic lesion destroys all neural and abneural hair cells over a large area of the sensory sheet, extending perhaps from the mid-papilla to the basal end. With ototoxic damage, the tectorial membrane remains intact, though disconnected from the reticular papilla surface in the area where hair cells disappear. Hair cell regeneration and neuronal reinnervation occur after acoustic and aminoglycoside trauma. The patterns of functional recovery accompanying these lesions differ with nearly complete return of function seen after acoustic damage and only partial functional recovery after ototoxic damage. These differences offer important comparative possibilities for understanding the consequences of papilla repair. Unfortunately, the utility of these comparisons has yet to be fully exploited.

3. Otoacoustic Emissions in Normal and Damaged

Bird Ears

3.1 Acoustically Evoked Otoacoustic Emissions

As noted in the preceding text, single-neuron activity that describes ratelevel functions and two-tone rate suppression reflect nonlinear activity within

100 J.C. Saunders and R.J. Salvi

the cochlea. Another peripheral auditory response originating from nonlinear cochlear activity is the otoacoustic emission. These emissions have served as an indicator of active cochlear processes in both mammals and birds (Manley 2001).

Mammalian otoacoustic emissions are closely linked to somatic motility of OHCs (Brownell 1990). This motility is an active process driven by the motor protein prestin, which is heavily expressed along the lateral wall of OHCs (Dallos and Fakler 2002; Zheng et al. 2002). A sound-evoked change in the OHC membrane potential elicits a contractile response that causes feedback of mechanical energy into the cochlear partition. This in turn enhances the sensitivity and frequency selectivity of the inner ear, and as a by product gives, rise to otoacoustic emissions. These emissions are propagated out of the cochlea via the middle ear, and can be identified as acoustic signals in the ear canal. Support for this mechanism of producing emissions is found in lesion studies, which show that selective destruction of OHCs abolishes the response (Brown et al. 1989; Hofstetter et al. 1997; Wang et al. 1997).

Synchronously evoked otoacoustic emissions were first detected from the ear of the starling (Sternus vulgaris; Manley et al. 1987), and sound-evoked distortion product otoacoustic emissions (DPOAEs) have been observed in the chicken (Norton and Rubel 1990; Burkard et al. 1996). The DPOAE consists of acoustic components not identified in the stimuli used to generate them (Probst et al. 1991). The cubic difference tone is considered the most prominent DPOAE. It is defined mathematically as 2f1 − f2, where f1 and f2 are the frequencies of two equally intense primary stimulus tones that share the relation f1 < f2.

The cubic difference tone component (which we now refer to, by convention, as the DPOAE) was examined in adult chickens after intense sound exposures (120 dB SPL) at either 1.5 or 0.53 kHz (Froymovich et al. 1995; Trautwein et al. 1996). Shortly after removal from the exposure, DPOAE amplitude exhibited a substantial reduction at or above the exposure frequency. After 8–16 weeks of recovery, DPOAE frequencies at or near the exposure tone showed little recovery whereas the frequencies lying further away from the exposure returned to normal levels. The failure of the DPOAE to exhibit recovery was attributed to the persistent tectorial membrane lesion, and suggested that this membrane was somehow involved in the production of the emission signal.

Changes in the DPOAE have also been examined in young chicks after intense tone exposure (Ipakchi et al. 2005), and Figures 3.11A and B show DP-grams and response-level functions from two groups of control animals (3 and 15 days old). The DP-grams (Fig. 3.11A) were obtained by averaging the DPOAE levels at each test frequency between 0.2 and 2.54 kHz across primary levels between 75 and 90 dB SPL. The emission level declines at a rate of approximately 2 dB/octave as frequency increases. The response-level functions (Fig. 3.11B) averaged emission levels across frequency (0.4–2.54 kHz), at each primary tone level, from 60 to 100 dB SPL. This input/output curve shows systematic increases in the DPOAE response as the primary tone levels increase. A slight but reliable difference between the two age groups is also seen.