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
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ABSTRACT |
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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 -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.
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INTRODUCTION |
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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 -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
-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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METHODS |
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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 ( original dose) every 8 h and boosters of
droperidol and fentanyl (
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 (
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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RESULTS |
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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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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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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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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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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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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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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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DISCUSSION |
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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
-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
-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
-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 -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.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute on Deafness and Other Communication Disorders Grant PO1 DC-0119.
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FOOTNOTES |
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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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REFERENCES |
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