Gentamicin Blocks Both Fast and Slow Effects of Olivocochlear Activation in Anesthetized Guinea Pigs

Naohiro Yoshida,1,2,4 M. Charles Liberman,1,2,3 M. Christian Brown,1,2 and William F. Sewell1,2,3

 1Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary;  2Department of Otology and Laryngology, Harvard Medical School, Boston 02114;  3Program in Neurosciences, Harvard Medical School, Boston, Massachusetts 02115; and  4Department of Otolaryngology, Tohoku University, Graduate School of Medicine, Sendai 980-8574, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

Yoshida, Naohiro, M. Charles Liberman, M. Christian Brown, and William F. Sewell. Gentamicin Blocks Both Fast and Slow Effects of Olivocochlear Activation in Anesthetized Guinea Pigs. J. Neurophysiol. 82: 3168-3174, 1999. The medial olivocochlear (MOC) efferent system, which innervates cochlear outer hair cells, suppresses cochlear responses. MOC-mediated suppression includes both slow and fast components, with time courses differing by three orders of magnitude. Pharmacological studies in anesthetized guinea pigs suggest that both slow and fast effects on cochlear responses require an initial acetylcholine activation of alpha -9 nicotinic receptors on outer hair cells and that slow effects require additional intracellular events downstream from those mediating fast effects. Gentamicin, an aminoglycoside antibiotic, has been reported to block fast effects of sound-evoked OC activation following intramuscular injection in unanesthetized guinea pigs, without changing slow effects. In the present study, we show that electrically evoked fast and slow effects in the anesthetized guinea pig are both blocked by either intramuscular or intracochlear gentamicin, with similar time courses and/or dose-response curves. We suggest that sound-evoked slow effects in unanesthetized animals are fundamentally different from electrically evoked slow effects in anesthetized animals, and that the former may arise from effects of the lateral OC system.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In the mammalian cochlea, efferent fibers originate in the olivary complex of the brain stem and synapse on either the outer hair cells (OHC) via the medial olivocochlear (MOC) bundle or afferent nerve fibers in the inner hair cell (IHC) area via the lateral olivocochlear (LOC) bundle. The principal neurotransmitter released by both MOC and LOC fibers is acetylcholine (ACh). The peripheral effects of electrically activating the OC bundle (which contains both MOC and LOC fibers) have been well characterized, and there is convincing evidence that this OC-mediated suppression of cochlear responses is effected largely (if not exclusively) by the MOC innervation of OHCs (recently reviewed by Guinan 1996). The MOC neurons respond to sound and thus are part of a sound-evoked reflex to the auditory periphery, but the responses and function of the LOC neurons are unknown.

Suppression of auditory responses by MOC efferent fibers is mediated by the release of ACh from MOC nerve terminals onto OHCs. The cholinergic receptor on the OHC is an alpha -9 nicotinic receptor (Elgoyhen et al. 1994), which, when activated, is permeable to Ca2+ (Blanchet et al. 1996; Erostegui et al. 1994; Evans 1996; Fuchs and Murrow 1992; Housley and Ashmore 1991). Ca2+ entering via the receptor interacts with Ca2+ activated K+ (KCa) channels to increase outward K+ current, which hyperpolarizes the OHC. This hyperpolarization is thought to decrease the amplification of basilar membrane motion normally mediated by OHC electromotility, and hence decrease the stimulation of IHCs. IHCs, in turn, encode auditory information via afferent neurotransmitter release onto auditory nerve fibers (reviewed by Sewell 1996).

Early studies of the time course of OC effects showed that electrically evoked OC activity can suppress sound-evoked discharge of auditory nerve fibers with an onset time constant of ~100 ms (Wiederhold and Kiang 1970). In addition to this classical "fast effect" of the OC system, OC activation also evokes a slower suppression of cochlear responses, which builds up and recovers with a time constant of >= 30 s. Pharmacological analysis suggests that both fast and slow effects require ACh release from MOC terminals and activation of alpha -9 nicotinic receptors on OHCs (Sridhar et al. 1995). Whereas fast effects are well explained by opening of KCa channels near the MOC synapse (Fuchs and Murrow 1992), further pharmacological evidence suggested that slow effects require different cellular mechanisms that may involve Ca2+-induced Ca2+ release (Sridhar et al. 1997). Circumstantial evidence suggests that slow rather than fast effects of OC activation are responsible for the protective effects of OC stimulation in acoustic trauma (Reiter and Liberman 1995).

Aminoglycoside antibiotics have long been known to block the effects of ACh and efferent stimulation on the cochlea at doses that do not affect hearing sensitivity (Daigneault et al. 1970; Smith et al.1994), presumably by blocking cholinergic transmission between the MOC terminals and OHCs. Such a block would be expected to reduce both the fast and slow OC effects (Sridhar et al. 1995). Recently, however, Lima da Costa et al. (1997) reported that intramuscular injections of gentamicin (an aminoglycoside antibiotic) could differentially block fast and slow effects of sound-evoked OC activation. At gentamicin dosages of 150 mg/kg, fast effects were completely blocked with little change in slow effects, whereas higher doses were required to block both fast and slow effects. This interpretation, if correct, has strong implications for understanding the slow effect. The interpretation of these results, however, is complicated by methodological differences between studies. In the study by Sridhar et al., OC fibers were activated electrically (because sound-evoked OC activity can be depressed by anesthesia), and cochlear responses to clicks or tone pips were examined for suppression. In the study by Lima da Costa et al., the OC activity was evoked by contralateral acoustic noise, and the cochlear response metric was the biological noise level at the round window (RW noise, or ensemble background activity) in the absence of experimentally applied ipsilateral sound. Thus it is not clear whether the fast and slow effects seen by Lima da Costa et al. (1997) are the same fast and slow effects seen by Sridhar et al. (1995).

The aim of the present study was to evaluate the effects of gentamicin on fast and slow effects of electrically evoked OC activation. Cochlear responses as well as RW noise were monitored in anesthetized guinea pigs. If gentamicin differentially modulated the electrically evoked fast and slow effects, the drug could be useful in probing the molecular mechanisms that differentiate the effects. In fact, no differential effects were seen, raising interesting questions as to the possible origin of a sound-evoked slow effect that may be demonstrable only in unanesthetized animals.


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Albino guinea pigs of either sex weighing between 350 and 500 g were anesthetized with urethan (1.5 g/kg ip), droperidol (10 mg/kg im), and fentanyl (0.2 mg/kg im). The animals received boosters of urethan (<FR><NU>1</NU><DE>3</DE></FR> original dose) every 8 h and boosters of droperidol and fentanyl (<FR><NU>1</NU><DE>3</DE></FR> original dose) every 2 h. Albino animals of the type used in the present study show a genetic anomaly in orientation of the first-row stereocilia in one-fourth of animals, consistent with an autosomal recessive mutation: when present, this anomaly is associated with a <10-dB elevation cochlear thresholds (Yoshida and Liberman 1999). A similar anomaly has been noted in both pigmented and albino guinea pigs from Europe (Comis et al. 1989; Furness et al. 1990); thus it is not yet clear whether any source of animals is free of the problem.

Animals were tracheostomized and connected to a respirator. The temperature within the experimental chamber was maintained at 32-33°C, and the animal's rectal temperature was maintained between 36 and 39°C. The pinnae were removed, and one cochlea was exposed by a dorsolateral approach. Calibrated acoustic stimuli were delivered with a 1-in. condenser microphone housed in a brass coupler that sealed tightly around the cartilaginous portion of the external ear (Kiang et al. 1965). The care and use of animals in this study was approved by the Animal Care Committee of Massachusetts Eye and Ear Infirmary.

Cochlear potentials

Cochlear potentials were measured with a silver wire placed near the round window, referenced to the neck muscles. Signals were amplified 10,000 times by an AC-coupled amplifier (pass band 300-3,000 Hz). The resulting signal was digitized with 30-µs sampling via a 12-bit A/D converter (A2000; National Instruments), and the digital waveforms were averaged from groups of eight responses to acoustic stimuli presented at 1/s. Acoustic stimuli for measurement of compound action potential (CAP) of the auditory nerve were either 1) 100-µs clicks or 2) 5-ms tone pips with 0.5 ms rise-fall times and cos2 shaping. For cochlear microphonic (CM) measurement, only clicks were used. For measurement of RW noise, 115 ms of waveform from the electrode was digitized, beginning 7.5 ms after each click or tone pip. A fast Fourier transform (FFT) was performed on the waveform, and the resultant magnitude spectra were averaged over eight consecutive stimulus cycles (with an overall repetition rate of 1/s). The average power was computed from the average FFT over the frequency range 600-1,200 Hz.

Olivocochlear bundle (OCB) stimulation

A posterior craniotomy was performed, and a portion of the cerebellum was aspirated to expose the floor of the IVth ventricle. The OCB was stimulated electrically with electrodes placed along the midline, where the OCB runs close to the surface of the brain stem (White and Warr 1983). The stimulator consisted of a linear rake of six fine, silver wires (0.4 mm spacing). After placement of the rake, the optimum electrode pair for eliciting OC activity was determined. Shocks were monophasic pulses of 150 µs duration, delivered at 300/s in trains of 300 ms duration. To eliminate possible middle-ear muscle contractions, paralysis was induced with d-tubocurarine (d-TC; 0.8 mg/kg im) and maintained with boosters (<FR><NU>1</NU><DE>3</DE></FR> original dose) every 2 h. Although d-TC blocks the effects of OCB stimulation (for review, Eybalin 1993), it does not cross the blood-cochlear barrier when administered systemically (Guth et al. 1976). Nevertheless, we were careful to match d-TC dosing across all animals in the study. Before injection of paralytic, the shock level required to elicit facial twitches was noted; after paralysis, shock levels were set 5-10 dB above the facial twitch threshold.

Drug delivery

For intramuscular delivery, gentamicin (Fujisawa) at a dose of 150 mg/kg or 0.9% NaCl was injected into the leg muscle in a volume of 1.2-1.5 ml. For intracochlear perfusion, gentamicin was dissolved in artificial perilymph solution (in mM: 120 NaCl, 3.5 KCl, 1.5 CaCl2, 5.5 glucose, and 20 HEPES, titrated with NaOH to pH 7.5; total Na+, 130 mM). To perfuse, an inlet hole was drilled in scala tympani just apical to the round window with a 0.2-mm, hand-held pivot drill, and a perfusion pipette (31 gauge, stainless steel cannula) was inserted. An outlet hole over the cochlear apex was created with a fine pick. Artificial perilymph was warmed to near body temperature as it passed through the heated room and perfused continuously with a peristaltic pump at 4 µl/min. Because of a variable amount of leakage between pipette tip and hole, the actual perfusion rate was less than the flow rate. During perfusion, the bulla drained through a ventrally placed hole, and the middle ear cavity was drained with a gauze wick. Only animals in which threshold to a 14-kHz tone remained within 10 dB of the predrilling value are included in this report. Perilymph containing gentamicin was loaded into a parallel loop such that drug delivery was accomplished by turning a valve, eliminating any mechanical artifact associated with drug application. The delay between valve opening and drug arrival at the ear was estimated to be 10 min by monitoring (in separate experiments) the CAP decrement following perfusion with high-K+ perilymph.


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Measurement and definition of fast and slow effects

Our paradigm allows simultaneous quantification of fast and slow effects of OC stimulation. One run of the paradigm (Fig. 1) begins with a set of measurements of CAP amplitude (gray boxes in Fig. 1A, gray circles in Fig. 1B), 10 of which are averaged to form a "baseline" amplitude. To measure the fast effect, a with-shocks trial is presented in which each click is immediately preceded by an OCB shock train (open boxes in Fig. 1A, open circles in Fig. 1B). Fast effect is defined as the percent decrease between baseline CAP amplitude (dashed line) and the suppressed CAP amplitude from the first with-shocks trial. The average fast effect suppression in this study was 51%. The slow effect is evoked by interleaving a series of five no-shocks trials (black boxes in Fig. 1A and black circles in Fig. 1B) among the five with-shocks trials. The CAP amplitude in these interleaved no-shocks trials gradually declines to a minimum (usually achieved by the 4th or 5th trial pair). Slow effect is defined as the average percent decrease between baseline CAP and all five interleaved no-shocks trials. Continuous CAP monitoring after the five paired trials clearly documents the slow recovery from suppressive effects of OC stimulation. The average slow effect suppression in this study was 20%.



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Fig. 1. Fast and slow olivocochlear bundle (OCB) effects on compound action potential (CAP) amplitude were measured with an alternating shock burst paradigm. Illustrated is the experimental paradigm (A) and representative data from one run (B). Each point in B represents the averaged peak-to-peak amplitude of the CAP in response to 8 acoustic stimuli (either clicks or tone pips) presented once per second. The acoustic stimuli were presented with (open circles) or without (filled circles) a shock burst (300/s for 300 ms) preceding the acoustic stimulus by 10 µs. The fast effect of OCB stimulation is the decrease in CAP magnitude with the 1st set of shocks, relative to a baseline of an average of 10 no-shock trials (dashed line). The slow effect is the steady decrease in the response to acoustic stimuli with repeated bursts of shocks (black circles) compared with CAP amplitude measured before the onset of the repeated bursts of shocks (gray circles). The slow effect is defined as the difference between the baseline CAP and the CAP amplitudes measured during all 5 no-shock trials of one run. Intervals between runs were ~10 min. In the case illustrated in B, the acoustic stimulus was a click 30 dB above the visual detection threshold. OCB shock levels were 10 dB above the facial twitch threshold.

Intramuscular gentamicin blocks both fast and slow effects

To assess the degree of blockade of fast and slow effects by intramuscularly injected gentamicin, we monitored CAP continuously for 5-9 h, performing an OCB run (5 paired with-shocks/no-shocks trials) approximately every 10 min. Saline injections were also performed in other animals. Results obtained from three gentamicin animals and three saline animals are summarized in Fig. 2. In each experiment, after monitoring baseline CAP and OC effects for ~30 min, gentamicin or NaCl (0.9%) was injected intramuscularly (time 0 on Fig. 2).



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Fig. 2. Intramuscular injections of gentamicin blocks both fast and slow effects without significantly affecting baseline CAP responses. Data from 6 different animals are shown, including 3 in which gentamicin (150 mg/kg) was injected at time 0 (filled symbols), and 3 animals in which saline (0.9% NaCl) was injected instead (open symbols). Fast and slow effects of MOC stimulation were monitored as illustrated in Fig. 1, for 4-10 h after injection in each case, with OCB shock trials performed every 10 min and baseline CAP amplitude measured continuously in the intervening periods. Acoustic stimuli were, with one exception, 14 kHz tone pips at 25 dB above CAP threshold. The exception (NAO16) used a click 20 dB above visual detection threshold. Baseline CAP amplitude (A) was measured at least 9 min after any OCB shock trial, and is normalized to the first CAP measures obtained before any OCB shock trials. Fast and slow effects magnitudes (B and C) are computed as described in Fig. 1, and all values are normalized to "control" values measured on the first run of the experiment.

In all three gentamicin cases, both fast and slow effects were completely eliminated within ~3 h postinjection. In no case did either fast or slow effects return. In both control and gentamicin experiments, the baseline CAP amplitude either remained unchanged or declined slightly during the 8-h experiment. In saline injections, OC effects showed no systematic or consistent changes with time. The average effects of saline versus gentamicin administration, as measured 4 h after the injection, are listed in Table 1 (percentage change in amplitude from preinjection values, mean ± SE). To better compare the time courses for gentamicin blockade of fast versus slow effects, the relationship between the fast and slow effect magnitudes at each time point is plotted in Fig. 3, combining data from all three intramuscular gentamicin experiments. In general, suppression of the slow effect paralleled that of the fast effect. If anything, slow effect blockade was slightly larger at each time point than fast effect blockade.


                              
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Table 1. Percentage change in amplitude by intramuscular injections of gentamicin



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Fig. 3. Slow and fast effects of MOC stimulation are similarly affected by intramuscular gentamicin (150 mg/kg). Each point plots the fast and slow effect magnitudes extracted from a single OCB shock trial. Data are replotted from the same 3 experimental animals of Fig. 2, with the same symbol conventions.

Intracochlear gentamicin perfusion also blocks fast and slow effects

To allow more rapid delivery and clearance of drug, as well as more precise control over intracochlear drug concentration, we delivered gentamicin directly via cochlear perfusion at concentrations of 1, 10, or 100 µg/ml dissolved in artificial perilymph. Gentamicin is a mixture of three related aminoglycosides with similar molecular weights; the estimated molarity of the three doses is 2, 20, and 200 µM. Intracochlear gentamicin reversibly and completely blocked both fast and slow effects of OC stimulation, in parallel and symmetrical fashion. Results from one experiment with gentamicin perfusion at 100 µg/ml are illustrated in Fig. 4. Within 20 min after perfusion onset, both fast and slow effects were almost completely blocked, and both completely recovered within 30 min after washing with artificial perilymph. Baseline CAP (to 14 kHz tone-pip at 50 dB SPL) was little affected throughout the perfusion, demonstrating that this dose of gentamicin did not change the general cochlear condition.



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Fig. 4. Intracochlear perfusion of gentamicin (100 µg/ml) blocks both slow and fast effects of OCB shocks on the CAP. A: CAP amplitudes during a 1.5-h period in which 10 OCB shock runs were presented: symbol conventions are as described in Fig. 1. As shown in B, the intracochlear perfusion of gentamicin was initiated immediately after the 3rd OCB shock run. B also shows the baseline CAP and the magnitudes of the fast and slow effects, extracted from the data shown in A and normalized to the magnitudes seen for the 1st shock trial. The box indicates the time during which drug was perfused. All values are normalized to "control" values measured on the 1st run of the experiment. Acoustic stimuli were 14-kHz tone-pip presented at 25 dB above CAP threshold. Shocks were delivered 5 dB above facial twitch threshold.

Dose-response curves constructed with data from 14 experiments (Fig. 5) at the three gentamicin concentrations suggest that fast and slow effect are equally susceptible to gentamicin blockade: the EC50 for gentamicin's block of fast and slow effects were 30.5 and 20.3 µg/ml, respectively. In this regard, gentamicin is not a particularly potent blocker. These EC50s are equivalent to ~40-60 µM, which can be compared with EC50 (in this preparation) of 1 µM for strychnine and 10 µM for atropine (Sridhar et al. 1995).



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Fig. 5. Dose-response relationships for effects of intracochlear gentamicin on fast and slow effects of OCB stimulation are very similar. Data are taken from 16 perfusions performed in 10 different animals. As in previous figures, the magnitude of the slow and fast effects in each case is computed as defined in Fig. 1 and normalized in each case to the initial values seen for the 1st OCB shock trial in that case. Sigmoidal functions are fit to the 2 sets of data by the following equation: y(D) = ymax[1/1 + (EC50/D)n], where y(D) is the percentage suppression of OCB effects and y max is the maximal block, 100.

To better localize the site of action of gentamicin blockade, we simultaneously monitored OC effects on CAPs and CM during gentamicin perfusion. CM is generated largely by the OHCs (Dallos and Cheatham 1976) and is normally enhanced, rather than suppressed, by OC stimulation (Fex 1959; Konishi and Slepian 1971) presumably because the OC-mediated conductance increase of the OHC membrane increases total transepithelial current in response to sound (Geisler 1974). Fast and slow effects of OC stimulation are demonstrable in CM as well as CAP (Sridhar et al. 1995), and both effects on CM were reversibly reduced by gentamicin, similar to the effect on the CAP (Fig. 6). Thus it would appear that gentamicin is blocking OC effects at the level of the OHCs.



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Fig. 6. Intracochlear perfusion of gentamicin (100 µg/ml) blocks OCB effects on both cochlear microphonic (CM) and CAP. The magnitudes of the slow and fast effects on CAP and CM amplitude were separately computed for each trial and normalized with respect to the effect magnitude seen on the 1st trial. The box indicates the time during which drug was perfused. All values are normalized to "control" values measured on the 1st run of the experiment. Acoustic stimuli were clicks presented at 30 dB above visual detection threshold. Shocks were delivered 8 dB above facial twitch threshold.

To more directly compare our results to those obtained with gentamicin injection in the unanesthetized guinea pig (Lima da Costa et al. 1997), we measured RW noise and looked for fast and slow effects in a total of eight animals. RW noise is measured in the absence of applied sound, and the component centered at ~800 Hz is thought to reflect background spike activity in a population of auditory nerve fibers innervating the basal turn of the cochlea (Dolan et al. 1990). In seven of eight animals, we were unable to demonstrate any convincing OC effects using this metric, either fast or slow. In one animal, which also showed the highest levels of RW noise of the group, we saw hints of fast and slow effects of OCB stimulation. In this case, gentamicin at 100 µg/ml appeared to block both fast and slow effects of OCB stimulation in reversible fashion.


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Gentamicin mechanisms of action

Aminoglycoside antibiotics such as gentamicin are well known for their ototoxic effects (reviewed in Garetz and Schacht 1996; Hawkins 1976). When delivered daily at doses of 300-400 mg/kg for several days, permanent loss of inner and outer hair cells can result, especially in the basal half of the cochlea (Kiang et al. 1976). In the present experiment, gentamicin was injected only once, at 150 mg/kg. This dose is unlikely to produce toxicity over the time scale of these experiments, as evidenced by the stability of baseline CAP measurements (Fig. 2), sensitive indicators of OHC function.

Other observations bolster the conclusion that gentamicin blockade of OC effects is unrelated to its ototoxicity. Chronic deefferentation does not lead to hair cell loss (Walsh et al. 1998), thus blockade of ACh release, per se, should not be ototoxic. Genetic knockout of the alpha -9 receptor in mice (Vetter et al. 1999) does not lead to hair cell loss or dysfunction (other than loss of MOC effects), thus loss of receptor function, per se, also cannot readily explain gentamicin's ototoxic effects.

The most likely site of gentamicin's OC blockade is at the cholinergic synapse on OHCs. Aminoglycoside antibiotics have long been known to block cholinergic transmission at the neuromuscular junction (Timmerman et al. 1959), and gentamicin is one of the more potent aminoglycosides in this regard (Paradelis et al. 1980). Cholinergic transmission in our preparation begins when electrical shocks generate action potentials in MOC fibers, which, on reaching their cochlear terminals, activate voltage-dependent Ca2+ channels to evoke ACh release. ACh diffuses across the synaptic cleft to the OHC, where it binds to alpha -9 receptors. Activation of this receptor allows Na+ and Ca2+ to enter the OHC, thereby activating KCa channels [suggested by Fuchs and Murrow (1992) and Housley and Ashmore (1991) and demonstrated by Blanchet et al. (1996) and Evans (1996)]. Because gentamicin blocks both fast and slow OC effects, action on a common process is most likely. The action could be either 1) a presynaptic blockade of ACh release (Fiekers 1983a) or 2) a postsynaptic action on the cholinergic receptor (Fiekers 1983b). As for possible presynaptic effects, voltage-dependent Ca2+ entry is essential for ACh release from MOC fibers, and aminoglycosides block voltage-dependent Ca2+ channels in nerve terminals (Parsons et al. 1992; Redman and Silinsky 1994), including MOC terminals on OHCs (Takeuchi and Wangemann 1993). Although doses of gentamicin that blocked MOC effects did not reduce CAP (Fig. 2), which also requires voltage-dependent transmitter release (from IHCs rather than MOC terminals), Ca2+ channels in IHCs could be less sensitive to aminoglycosides than those in the MOC terminals (for example, see Nakagawa et al. 1992). Also arguing in favor of a postsynaptic effect of gentamicin on the alpha -9 receptor is the finding that aminoglycosides block the cochlear effects of intra-arterially administered ACh (Daigneault et al. 1970).

Lima da Costa et al. (1997) suggest that gentamicin may inhibit KCa channels in OHCs, either directly, or by blocking Ca2+ entry. Blockade of Ca2+ entry through the cholinergic receptor is a possible mechanism for gentamicin's action, but we would expect it to block both fast and slow effects (as we have observed), rather than cause selective blockade of fast effects (as reported by Lima da Costa et al. 1997). Blockade of Ca2+ entry by aminoglycosides in isolated OHCs associated with K+-induced depolarization has been demonstrated (Dulon et al. 1989; Nakagawa et al. 1992), but this seems an unlikely mechanism for OC stimulation, which would be expected to hyperpolarize, not to depolarize, the OHC.

Gentamicin action on fast versus slow OC effects

Lima da Costa et al. (1997) reported that OC activity evoked by contralateral sound could suppress RW noise of awake or lightly sedated guinea pigs. This suppression had both fast and slow components with time courses and magnitudes remarkably similar to the OC effects on CAP and CM described in our laboratory (Sridhar et al. 1995, 1997). The slow effects in both laboratories built up over a period of seconds and decayed with a time constant of 30-40 s. The magnitude of Lima da Costa's fast effects were 41% compared with our 51% suppression, and Lima da Costa's slow effects were 15.6% compared with our 20% suppression. Lima da Costa et al. (1997) further reported that an intramuscular gentamicin dose of 150 mg/kg selectively blocked the fast effect in their experimental paradigm, while leaving the slow effect intact. By contrast, we found, for both intramuscular and intracochlear gentamicin, and at a variety of dosages, that fast and slow effects of electrically evoked OC activity in anesthetized guinea pigs were always blocked to a similar extent and with a similar onset and recovery of blockade.

Although the conclusions of the two studies appear to be contradictory, three major differences in methodology could contribute to the discrepancy: 1) use of RW noise versus sound-evoked CAP or CM as a metric of cochlear function; 2) use of OC activity evoked by contralateral sounds versus electric shock at the floor of the IVth ventricle; and 3) use of awake or lightly sedated versus deeply anesthetized animals. To this list, the difference in animal pigmentation should be added for completeness; however, it is difficult to assess the likelihood that gentamicin effects on the OC system would be fundamentally different in pigmented versus albino animals.

The difference is probably not attributable to use of RW noise versus CAP. We observed slow and fast effects on RW noise in only one animal; nevertheless, in that one case, gentamicin at 100 µg/ml blocked both the fast and slow OC effects. An incomplete understanding of the origin of the noise measure itself complicates the interpretation of these results. RW noise, measured in the absence of externally applied sound, shows a spectral peak near 800 Hz. Neural blockade with kainic acid suggests that the primary source of this peak is neural action potentials (Dolan et al. 1990); this finding fits with the known spectrum of single-fiber contributions to the RW potential (Kiang et al. 1976). RW noise can be suppressed by high-frequency tones, suggesting that it originates in the basal turn (Dolan et al. 1990). However, these masking data do not fit with the notion that RW noise represents spontaneous activity in auditory nerve fibers, because spontaneous activity is not usually suppressed by external stimuli (Kiang et al. 1965).

It is unlikely that the difference in the two studies can be attributed to the means of activation of OC fibers. Peripheral effects of sound-evoked versus shock-evoked OC activity appear fundamentally similar, at least in anesthetized animals (Warren and Liberman 1989; Wiederhold and Kiang 1970). Most convincing is the similarity in OC-blockade pharmacology observed using contralateral-noise suppression of distortion-product emissions (Kujawa et al. 1993, 1994) versus shock-evoked suppression of the CAP (Sridhar et al. 1995). The larger magnitude of shock-evoked effects matches the observation that sound-evoked rates in MOC fibers (<100 spikes/s) are significantly lower than the shock rates typically used (300/s) (Liberman 1988).

A final possibility is that the lack of anesthesia reveals another type of slow effect, which is fundamentally different from that seen in anesthetized animals. In particular, it is possible that contralateral sound in unanesthetized animals either increases or decreases activation of LOC fibers contacting auditory nerve terminals in the IHC area. Although peripheral effects of the LOC system are poorly understood, LOC terminals on afferent dendrites are well positioned to affect background discharge rate in auditory nerve fibers, and, therefore possibly to affect the RW noise. Putative sound-evoked activation in this system might conceivably be abolished by anesthesia, if LOC fibers are more sensitive to anesthetic effects. A difference in the gentamicin susceptibility of a putative LOC slow effect could arise for several reasons: 1) relatively poorer access of drug to the inner versus the outer hair cell area (given the larger extracellular spaces in the latter), 2) presence of different cholinergic receptors on afferent fibers, which do not express alpha -9 mRNA (Elgoyhen et al. 1994), or 3) action of other neurotransmitters, e.g., GABA, known to be present in the LOC system (Vetter et al. 1991). Whatever causes the difference, these gentamicin-resistant slow effects in unanesthetized animals appear to represent an opportunity to study the LOC system and its functions.


    ACKNOWLEDGMENTS

This work was supported by National Institute on Deafness and Other Communication Disorders Grant PO1 DC-0119.


    FOOTNOTES

Address for reprint requests: W. F. Sewell, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 May 1999; accepted in final form 13 August 1999.


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