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

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2.2 Ototoxic Drug Administration

Previous work has shown that considerable variation exists in the response of the auditory system to acoustic overexposure and that structures other than hair cells are damaged if the exposure is intense or prolonged enough (reviewed in Cotanche 1999). Moreover, the apparently paradoxical finding that a considerable amount of functional recovery occurs before hair cell regeneration is complete further complicates interpretation. In part for this reason, more recent studies of behavioral recovery after hair cell regeneration have used ototoxic drug administration to cause hair cell loss. Typically in birds, hair cells in the basal end of the papilla are damaged and are lost first, with the loss proceeding toward the apical region with increased dose and time (Cotanche 1999). Studies of recovery from insult with ototoxic drugs have the advantage of being able to attribute the recovery of hearing more completely to the regeneration of new auditory hair cells with less of a confounding influence of surviving hair cells or mechanical damage to other structures such as the basilar papillae or tectorial membrane.

In budgerigars given 100 mg/kg or 200 mg/kg of kanamycin (KM) for 10 days, threshold shifts of about 20–60 dB occurred for frequencies of 2.0 kHz and above, depending on the dosage of KM (Hashino and Sokabe 1989). Threshold shifts reached maximal values 3–5 days after the end of the KM treatment. Slightly larger threshold shifts occurred at 0.5 kHz, reaching nearly 80 dB at day 3 for the higher dose condition. Recovery of thresholds reached asymptotic levels around 15 days posttreatment. Residual hearing losses of less than 20 dB occurred for frequencies of 2.0 kHz and above in birds given the higher dosage, but returned to normal levels in birds given the lower dosage. Permanent threshold shifts of about 10 dB and 40 dB at 0.5 kHz occurred in birds given the lower and higher KM dosage, respectively. Because aminoglycosides induce hair cell loss primarily in the basal region of the basilar papilla (Hashino et al. 1992; Dooling et al. 1997), a permanent low-frequency hearing loss would not be expected so this pattern of low-frequency hearing loss remains a curious one.

Other studies have reported returns to near-normal absolute sensitivity for pure tones after ototoxic drug-induced hair cell loss and regeneration in European starlings (Sturnus vulgaris; Marean et al. 1993) and budgerigars (Dooling et al. 1997). Experiments on budgerigars show that by about the fifth day of injections, absolute thresholds began to increase dramatically, and the threshold shift at 2.86 kHz by the time injections were completed (10 days) was greater than 60 dB. Immediately after KM treatment, the greatest shift occurred for frequencies above 2.0 kHz (Dooling et al. 1997). Recovery of hearing began immediately after cessation of injections. By 8 weeks postinjection, absolute thresholds improved to within 5–15 dB of normal for frequencies below 2.0 kHz and to within 20–30 dB of normal above 2.0 kHz. Recovery of threshold eventually reached an asymptote with a permanent threshold shift on average of 23 dB.

In a more recent study on budgerigars, Dooling et al. (2006) measured thresholds at six frequencies and found them to be differentially affected by the

KM injections. Initial threshold shift was greater at high frequencies (50–60 dB above 2 kHz) than it was at low frequencies (10–30 dB below 2 kHz). Thresholds gradually recovered to within about 15–25 dB of preinjection thresholds by 8 weeks after injections, with the most rapid recovery occurring at the lowest frequencies. Absolute thresholds for all frequencies reached asymptote by 8 weeks after cessation of KM injections with a return to within 5–15 dB of normal for frequencies below 2 kHz and within 20–30 dB of normal above 2 kHz (Dooling et al. 2006).

Marean and colleagues (1995) compared behavioral detection thresholds from starlings before, during, and after 11 days of subcutaneous injections of KM. The starlings were given 100 mg/kg injections for 2 days and then 200 mg/kg injections for 9 days. The birds showed large threshold shifts initially at high frequencies and then at progressively lower frequencies. These shifts were in excess of 60 dB at 4–7 kHz but none of the birds showed any change in auditory thresholds for frequencies below 3 kHz. The recovery of hearing began within a few days of the end of the KM injections, and lasted for approximately 50 days. After a second course of KM injections, the starlings showed less of a threshold shift than after the first injections. Subsequent studies showed that the smaller threshold shift after the second course of injections was likely due to metabolic changes sustained after the first course of antibiotics and not to a resistance to antibiotic damage by regenerated hair cells (Marean et al. 1995). The time course of recovery and the amount of permanent threshold shift in starlings, compared to budgerigars, suggests that they may be less sensitive to damage from aminoglycoside antibiotics, similar to Bengalese finches (Lonchura domestica; Marean et al. 1993, 1998; Woolley et al. 2001).

As is the case with acoustic overexposure, there are a number of physiological estimates of threshold recovery after ototoxic drug administration that in general parallel behavioral findings. Tucci and Rubel (1990) used frequencyspecific auditory evoked potentials and tested 16to 20-week-old chicks (Gallus gallus domesticus) after gentamicin injections. EPs were increased immediately after injections ceased, and continued to increase for up to 5 weeks afterward. This is in contrast to behavioral thresholds that generally show a decrease in thresholds following termination of injections. They suggested that the increase in EP threshold during the early stage of recovery was due to a lack of neural synchrony. Partial recovery of thresholds was seen by 16–20 weeks, predominantly at low and mid frequencies. Residual hearing loss was greatest at the highest frequencies. Interestingly, recovery of thresholds lagged hair cell regeneration by 14–18 weeks. Administration of KM also results in temporarily increased CAP and EP thresholds, primarily at higher frequencies in chickens and quail (Chen et al. 1993; Lou et al. 1994; Trautwein et al. 1998). Some residual loss persists at the highest frequencies tested, however.

In a more recent study, Woolley et al. (2001) measured hair cell regeneration and the recovery of auditory thresholds in Bengalese finches using the aminoglycoside amikacin (alternating doses of 150 mg/kg and 300 mg/kg daily for 7 days). ABR thresholds were measured for frequencies ranging from 0.25

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to 6 kHz. Heavy hair cell losses on the basal end of the basilar papilla correlated with hearing losses above 2 kHz. Recovery of auditory thresholds began at about 1 week after treatments and ceased approximately 4 weeks later. Thresholds at high frequencies remained elevated at 8–12 weeks. One conclusion is that the temporal course of hearing recovery is more similar among closely related species because the pattern in finches is more similar to that of the starling (Marean et al. 1993) than that of either the budgerigar (Dooling et al. 1997) or the chick (Tucci and Rubel 1990).

In aggregate, these comparative results are similar in some respects to the comparative results on recovery of hearing after noise exposures, where canaries and zebra finches were similar to each other but different from budgerigars and quail (Ryals et al. 1999b). It is also important to realize the difficulties inherent in comparisons between behavioral and EP measures of hearing. Hearing is a behavior whereas EPs are a measure of neural synchrony due to stimulus onset, and usually correlate well with stimulus intensity. However, the minimum stimulus intensity required to produce a measurable evoked potential is typically 10–30 dB higher than behavioral detection thresholds. Thus, behavioral measures of hearing remain the gold standard for determining the effect of ototoxicity or acoustic overstimulation on hearing.

Figure 4.2 shows behavioral audiograms from several species of birds measured behaviorally (A-budgerigars and B-starlings) compared with ABR (C-pigeons and D-Bengalese finches), and CAP (E-chickens) threshold curves. These functions show the preinjection thresholds for each species, along with data from several time periods after the ototoxic drug administration. In general, the findings are quite similar across species. Hearing losses are greatest at the high frequencies, minimal at the lowest frequencies, and recovery generally proceeds from the lowest frequencies first to the higher frequencies at later time periods.

These findings from several species of birds show that hair cell damage from ototoxic drug administration proceeds from the base to the apex of the basilar papilla, that hearing loss is greatest at high frequencies, and that functional recovery of hearing follows the hair cell replacement that one might expect from a place representation of frequency on the basilar membrane. The only exception from this general pattern is an unusual report that budgerigars experienced a low-frequency hearing loss to KM-induced hair cell destruction (Hashino and Sokabe 1989). The exact reasons for the anomalous results are still unknown.

An unusual bird, the Belgian Waterslager (BW) canary, also shows an interesting response to hair cell loss and regeneration. These birds have been bred for a distinctive low-frequency song and have an inherited auditory pathology resulting in, on average, 30% fewer hair cells on their basilar papilla, a 12% reduction in the number of auditory nerve fibers, and abnormal cochlear nuclei cell volumes compared to non-BW canaries (Gleich et al. 1994, 2001; Kubke et al. 2002). Hair cells on the abneural edge of the papilla, which are primarily innervated by efferent fibers, appear to be most affected, and this pathology develops after hatching and extends the length of the papilla (Gleich et al. 1994;

3. Effects of Hair Cell Loss and Regeneration on Auditory Discrimination

Not surprisingly, almost all of the work on the effect of hair cell loss and regeneration on hearing has focused on the audiogram. However, a subject’s ability to discriminate complex features of sounds after hair cell regeneration may be equally or more important than simply assessing threshold. Several recent studies have addressed this issue.

3.1 Discrimination Tests: Changes in Spectral, Intensity,

and Temporal Resolution

Behavioral studies of auditory masking and discrimination have now been conducted on several species of birds including starlings, budgerigars, and chickens (Saunders and Salvi 1995; Dooling et al. 1997; Marean et al. 1998).

3.1.1 Spectral and Intensive Measures

Saunders and Salvi (1995) examined pure tone masking patterns in chickens using a tone-on-tone paradigm before and after exposure to a 525-Hz pure tone at 120 dB SPL for 48 hours. Two months after the exposure, masking patterns were reassessed in these birds and the postexposure masking patterns were virtually identical to the preexposure masking patterns. Frequency selectivity has also been shown to decrease in birds immediately after aminoglycoside treatment. Critical ratios, a crude estimate of auditory filter bandwidth, have been shown to increase immediately after KM treatment in budgerigars (Hashino and Sokabe 1989), indicating a broadening of the auditory filters. Marean et al. (1998) measured auditory filter widths in starlings using notched noise maskers before, during, and after KM injections. Auditory filters at 5 kHz were significantly wider after hair cell regeneration while spectral resolution was virtually unchanged at the other frequencies. In budgerigars, intensity (IDLs) and frequency difference limens (FDLs) were measured before, during, and after 10 days of KM administration for pure tones at 1.0 and 2.86 kHz. In spite of mild but permanently elevated absolute thresholds 4 weeks after KM treatment, IDLs were not significantly affected by the KM injections and FDLs were only slightly elevated at both frequencies as shown in Figure 4.4 (Dooling et al. 2006).

3.1.2 Temporal Measures

Marean et al. (1998) also measured minimum temporal resolution in starlings over the time course of hair cell loss and regeneration using temporal modulation transfer functions (TMTFs). TMTFs were mostly unaffected after KM administration. Some decrease in temporal resolution was evident for band-limited noise centered at 5.0 kHz, but these observed effects may have been due to poor

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Figure 4.4. (A) FDLs and (B) IDLs for 1.0- and 2.86-kHz pure tones before and at three time periods after injections for three subjects. Error bars represent standard errors. (Replotted from Dooling et al. 2006.)

audibility of the signal as a result of threshold shifts, rather than to disruption of temporal resolution per se.

Maximum temporal integration (increasing threshold with decreasing stimulus duration) in birds, as in most vertebrates, is typically around 200–300 ms (Dooling et al. 2000). Saunders et al. (1995) showed that maximum temporal integration decreases immediately after acoustic trauma in chickens; it then returned to normal approximately 10–20 days after exposure. The slopes of threshold-duration functions decreased immediately after exposure, but recovered to near-normal levels after hair cell regeneration. Figure 4.5 shows the maximum temporal integration functions for the chicken at 1 kHz after acoustic overexposure to a 525-Hz pure tone (Saunders and Salvi, 1993; Saunders et al. 1995).

3.2 Discrimination Tests: Vocalizations

Hearing in humans and animals is typically assessed with simple sounds. But we know from work with both normal hearing and hearing impaired listeners that auditory tests with pure tones are less than perfect predictors of how well a human listener can detect, discriminate, or understand speech. Recent tests on budgerigars recovering from KM treatment were designed with parallels to human speech perception in mind. Budgerigars have been particularly useful

Figure 4.5. (A) Maximum temporal integration functions at 1 kHz for chickens from Saunders et al. (1995) and Saunders and Salvi (1993) for two subjects. Thresholds are shown relative to the subjects’ thresholds for the 512-ms stimulus condition.

avian models for behavioral studies of auditory perception as well as complex vocal production. These birds learn new vocalizations throughout life, especially in response to changes in their social milieu (Dooling 1986; Dooling et al. 1987; Farabaugh et al. 1994; Hile et al. 2000) and they require hearing and auditory feedback for the maintenance of their adult vocal repertoire. Deafening, by cochlear removal during development or in adulthood, results in an impoverished vocal repertoire (Dooling et al. 1987; Heaton et al. 1999). Moreover, budgerigars trained to produce particular vocalizations under controlled experimental conditions show strong evidence of the Lombard effect (increases in vocal intensity when placed in a noisy environment), suggesting a real time monitoring of vocal output (Manabe et al. 1998).

In a series of tests on the abilities of budgerigars to discriminate among complex vocal signals before and after KM treatment, birds were tested on their ability to discriminate between pairs of calls in a test set of five different speciesspecific budgerigar contact calls and their synthetic analogs for a total of 10 calls in the test set (see Fig. 4.6A). Each bird was tested on the entire 10-call set both before and approximately every 4–6 weeks after injections up to about 24 weeks after cessation of KM injections. Discriminations between contact call types were relatively easy while discrimination between a natural contact call and its synthetic analog was difficult (Dooling et al. 2006).

The relatively easy discriminations between contact call types as measured by percent correct response returned to pretreatment levels by only 4 weeks after cessation of KM injections while the more difficult discriminations between a

natural call and its synthetic analog showed prolonged disruption (Fig. 4.6B). Additional analyses of response latencies also show that the birds’ perceptual space for contact calls was still somewhat distorted four weeks into recovery. Some calls that were perceptually distinct before KM administration were being perceived as similar many weeks into the recovery process (Dooling et al. 2006). These data on the budgerigar suggest a more complicated picture of hearing recovery in these birds when the task involves the discrimination of complex vocalizations.

3.3 Identification Tests: Recognition of Contact Calls

The previous results focused on the recovery in the detection and discrimination of simple and complex sounds after hair cell regeneration. However, a higher order task that needs to be addressed after hair cell regeneration is the ability to understand what is being said. This reminds us that it is one thing to hear speech, and even to discriminate among speech sounds but it is quite another thing to understand what is being said. A recent experiment addressed the effect of hair cell loss and regeneration on this broader question by testing whether budgerigars recognized contact calls that were previously familiar. This task is inherently more difficult than the discrimination task described in the preceding text in that the birds had to remember from trial to trial which call was the “go” call and which call was the “no go” call.

Figure 4.7A shows the average percent correct for the six budgerigars on the classification task before, during, and after 10 days of KM injections. Before injections of KM, the birds’ performance was well above the 85% criterion level. However, performance fell to chance levels (50%) after several days of KM injections and remained there when assessed in a single 100-trial test session 24 and 38 days later. At 38 days (4 weeks after cessation of KM injections), the birds were retrained and tested daily on the same task in 100-trial daily sessions. The birds returned to preinjection performance levels in 4 days.

Response latencies for “go” stimuli remained below 1 s and relatively unchanged throughout the experiment, attesting to the birds’ excellent health and behavioral responsiveness (Fig. 4.7B). Response latencies to the “no-go” stimuli averaged near 5 s before and during the first few days of treatment with KM. But, as testing continued and the birds began to respond by pecking the report key to both “go” and “no-go” stimuli, response latencies to the “no-go” approached the levels recorded to the “go” stimuli.

The six KM-treated birds tested after 4 weeks of recovery required an average of more than four 100-trial sessions to reach criterion instead of the average of less than two 100-trial sessions to reach criterion after a 4-week pause in testing but with no KM treatment. The results of important control experiments necessary for interpretation are summarized in Figure 4.7C. The first two control conditions are for four birds that were not injected with KM but were nonetheless given either a 2-week or a 4-week pause in testing. When testing resumed, the number of trials to reach criterion was much less than the time required to learn a