Hearing Research, 61 (1992) 106-116 © 1992 Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/$05.00
106
HEARES 01772
Intracochlear application of acetylcholine alters sound-induced mechanical events within the cochlear partition * S h a r o n G. K u j a w a l, T h e o d o r e J. G l a t t k e 1, M a u r e e n F a l l o n 2 a n d R i c h a r d P. B o b b i n 2 I Department of Speech and Hearing Sciences, University of Arizona, Tucson, Arizona, USA and z Kresge Hearing Research Laboratory of the South, Department of Otorhinolaryngology and Biocommunication, Louisiana State Unh'ersi~. Medical Center, New Orleans, Louisiana, USA (Received 4 November 1991; Revision received 18 March 1992; Accepted 26 March 1992)
Activation of olivocochlear (PC) efferent fibers has been suggested to alter micromechanical events occurring within the cochlear partition, possibly through an effect of the efferent neurotransmitter (acetylcholine; ACh) on outer hair cells (OHCs). Based on the widely-accepted assumption that otoacoustic emissions reflect OHC activity, we investigated the in vivo influence of ACh on OHCs by studying alterations in emission amplitude with local ACh application. Distortion product otoacoustic emissions (DPOAEs) were measured in anesthetized guinea pigs before, during, and after intracochlear application of ACh (250 /zM) with the cholinesterase inhibitor, eserine (20 /zM). Perfusion of ACh/eserine was associated with a desensitizing reduction in D P O A E amplitude of approximately 4.4 dB. This reduction was intensity-dependent, with greater and more consistent reductions observed for DPOAEs elicited by low- than by moderate-intensity primaries. The response reduction was not seen during consecutive ACh perfusions performed without an intervening artificial perilymph wash, and was effectively blocked in the presence of pharmacologic antagonists of P C efferent activity (curare, 50 ~M, strychnine, 50 izM). Finally, a similar alteration in DPOAE amplitude was never seen during perfusion of the control (artificial perilymph) solution alone. It is argued that these results support the hypothesis that P C efferent activation can alter sound-induced cochlear mechanical events. Efferents; Acetylcholine; Acoustic distortion products; Cochlear mechanics; Olivocochlear neurons; Outer hair cells
Introduction
Our understanding of the mechanical function of the cochl~.a has been revised substantially in the past decade. I iae traditional view of the cochlea as a passive organ that transduces mechanical displacements into neural activity has been altered by two important discoveries: the finding that outer hair cells (OHCs) have motile capabilities (Brownell et al., 1985) and the demonstration that the cochlea can produce acoustic energy detected as otoacoustic emissions (OAEs) (Kemp, 1978). Both of these phenomena have been taken as support for active, physiologically-vulnerable processes within the cochlea that amplify and sharpen the cochlear mechanical response to sound stimulation. Recent investigations have suggested that these processes are under the influence of the olivocochlear (PC) efferent neurons. Specifically, it has been argued that the medial olivocochlear (MOC) efferents, through
Correspondence to: Richard P. Bobbin, Kresge Hearing Research Laboratory, LSU .~]edical Center, 2020 Gravier Street, Suite A, New Orleans, LA 70112, USA. Fax: (504) 568-4460. * A preliminary report was presented at the Midwinter Research Meeting of the Association far Research in Otolaryngology in February, 1991.
numerous and direct connections with OHCs, can alter the local cochlear mechanical response to sound stimulation and provide a 'gain control' (Guinan and Gifford, 1988) of the cochlear amplifier. Newer models of cochlear function describe an active cochlea - one capable of executing modifications to its response patterns to increase or decrease afferent outflow from the cochlea. Although a contribution to these processes by the OHCs is widely accepted, the role of the PC efferents in modulating cochlear mechanical events has been more difficult to define. Investigations undertaken to study the efferent influence at the level of the cochlea traditionally have recorded changes in gross sound-induced electrical responses from the cochlea and the eighth nerve during electrical activation of P C efferent fibers (see Wiederhold, 1986 for review). Such studies have revealed an inhibitory role for the PC efferents. The precise mechanism of this inhibitory influence is unknown; however, it has been suggested to involve a mechanical component at the OHCs (Dallos, 1985). A more direct reflection of sound-induced cochlear mechanical events can be obtained through the study of OAEs. OAEs are widely regarded t o reflect the integrity of cochlear mechanical processes in general, and OHC function in particular (e.g., Kim, 1986). Distort.;on product otoacoustic emissions (DPOAEs) rep-
107
resent one class of evoked emissions. These distortions are frequency-specific signals produced by the normal nonlinear mechanical response of the cochlea. Through their association with OHCs, DPOAEs have been linked to the active processes responsible for the cochlea's sensitivity and frequency selectivity (Kim, 1980). At low levels of stimulation, these emissions may provide a direct and sensitive reflection of OHC function (Brown et al., 1989). Therefore, DPOAEs are particularly appropriate tools to study the cochlear mechanical consequences of efferent activation. Employing DPOAEs, an efferent effect on cochlear mechanics has been demonstrated previously by Mountain (1980) an~d Siegel and Kim (1982). In these investigations, DPOAE amplitudes (f2-fl; 2f~-f2) were altered during electrical stimulation of OC efferent fibers at the floor of the fourth ventricle. The proposed efferent effects or, distortion products were not straighfforJcard, however, and inconsistencies between the two investigations remain unresolved. Acoustic stimulation of the contralateral ear has also been employed as a strategy to study auditory efferent effects on cochlear mechanics. Distortion product amplitudes are reduced in awake humans (Brown and Norton, 1990; Moulin and Collet, 1992) and anesthetized guinea pigs (Puel and Rebillard, 1990) during contralatcral sound stimulation. Contralateral sound suppression is suggested to be mediated primarily by uncrossed MOC efferents, and these fibers comprise only about one third of the MOC projections to each cochlea (Guinan et al., 1983). Investigations employing contraiateral sound, then, would be expected to provide information about only a portion of the OC efferents to OHCs. The nature of the OC efferent influence on cochlear mechanical events thus remains unclear, and further study is needed. Acetylcholine (ACh) is the major neurotransmitter released onto OHCs during MOC efferent stimulation (see Bledsoe et al., 1988 for review). We attempted to mimic MOC efferent activation by utilizing intracochlear ACh application. The influence of this manipulation on sound-induced cochlear mechanical events was evaluated by monitoring 2fl-f2 DPOAE amplitude.
Method
Subjects Experiments were performed on 10 pigmented guinea pigs (Carla cobaya) of either sex weighing between 250 and 350 g. Animals were treated in accordance with Federal, state, and institutional guidelines and the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1985). All
animals included in the study demonstrated auditory nerve compound action potential (CAP) thresholds consistent with laboratory norms (Bobbin, unpublished), and all had normal distortion product amplitude characteristics (Brown, 1987; Brown and Gaskill, 1990).
Stimulus generation and response recording Compound action potential Acoustic stimuli used to obtain CAP thresholds were 4, 8, 10, 12, 16, and 20 kHz tone bursts (0.25 ms rise/fall, 10 ms duration, 200 ms interstimulus interval). These signals were produced by an external signal generator (Wavetek 148A), transduced by a speaker with an extended high frequency response (to 25 kHz), and delivered to the right ear of each animal under computer control with intensity incremented in 6 dB steps. Electrophysiologic activity was detected by a flame¢dLI|~U~ silver electrode (insulated, except for the tip) b~U~.~ placed on the round window membrane. Electrical signals were amplified (Grass P15; gain = 1000), averaged (over 20 trials) and stored on disk. For measurement purposes, stored voltages were filtered (KrohnHite 3202R; 24 dB/octave; low pass = 2 kHz) and displayed on an oscilloscope. Threshold was defined as the lowest stimulus intensity at which the following two criteria could be met: a) an averaged NI-P i amplitude of > 5/zV was obtained and b) the magnitude of this peak to peak amplitude increased with the next stimulus increment.
Distortion products The 2fl-f2 DPOAE at 8 kHz was elicited by equalintensity 'primary' stimuli at 10 kHz (ft) and 12 kHz (f2), yielding an fz/fi ratio of 1.2. This ratio was associated with among the largest 2 f l - f 2 DPOAE amplitudes evaluated in this laboratory (Littman, 1990), a finding in good agreement with the outcomes of other investigations of 2fl-f2 DPOAE characteristics in guinea pigs (Brown, 1987; Brown and Gaskill, 1990). Pure tone signals were generated by oscillators (Hewlett Packard 200CD; 200ABR), and were routed through attenuators (Hewlett Packard 350D) to Etymotic Research ER-2 receivers. The outputs from these receivers were delivered to an acoustic probe assembly which contained a low-noise microphone system (Etymotic Research, ER-10) and which was coupled to the right ear of each animal by a hollow ear bar. Responses to this bitonal stimulation were detected by the probe microphone system, the output of which was directed via a microphone preamplifier (Etymotic Research, ER-1072) to a Hewlett Packard 3561A signal analyzer. The ear canal soundfield containing the DPOAE was sampled, digitized, and submitted to Fast
108
fl =f2=60 dB SPL
2fl-f2
;ENTER 9000 Hz
RMS 16
SPAN 6250 H
Fig. 1. DPOAE response to primary pure tone stimulation at 60 dB SPL. Shown are the primary tones (f~ = I0 kHz; f2 = 12 kHz) and the resulting 2 f l - f 2 P P O A E at 8 kHz as recorded in the open bulla condition. The 3 f l - 2 f 2 DPOAE at 6 kHz is also present in this trace.
Fourier Transform (FFT) analysis, and the resulting spectrum (averaged over 16 samples) was displayed on the analyzer using a 6250 Hz window (see Fig. 1). When displayed in this manner, the spectrum average contained the two primary stimuli and the 2f~-f2 DPOAE. A distortion product corresponding to the frequency 3f~-2f2 was occasionally present within this display window as well, but was not monitored in the present investigation. Distortion product amplitude, defined as the spectral peak corresponding to the 2fl-f2 frequency (8 kHz) was recorded manually from such FFT spectra. The noise floor associated with this instrumentation was approximately - 1 2 dB SPL at points 150 Hz above and below the 2fl-f2 DPOAE frequency (2 kHz window; BW = 7.5 Hz), and averaged approximately - 2 dB SPL when a 6250 Hz window (BW = 23.4 Hz) was employed. When evaluated in a passive cavity of appropriate volume, harmonic and intermodulation distortion components were more than 75 dB below the level of the primaries. Stimulus intensity and frequency calibrations of the sound system/acoustic probe-ear bar assembly were performed daily according to the procedures described by Littman (1990).
Experimental procedures Surgical procedures and associated pre-perfusion measurements All procedures were conducted in an acousticallyand electrically-shielded room. Animals were anesthetized with pentobarbital sodium (Nembutal, 30 mg/kg, i.p.) and chlorprothixene (Taractan, 30 mg/kg, i.p.) and were tracheotomized and artificially respi-
rated. ECG and rectal temperature were monitored throughout each experiment and temperature was maintained at 38°+ I°C by a heating pad. Additional pentobarbital (30 mg/kg, i.p.) was administered as required to maintain an adequate depth of anesthesia. Cartilaginous ear canals were exposed and removed to allow optimum placement of the ear bars used to secure the animal in the headholder. The right auditory bulla was exposed using a ventrolateral approach and a small opening was made in the dorsolateral bulla just sufficient to gain access to the round window. The round window electrode was then positioned for baseline recordings of CAP thresholds. Baseline measures of the 2fl-f2 DPOAE (five consecutive trials of 16 spectra each) were also obtained at this time for both 40 and 60 dB SPL primaries. Finally, DPOAE amplitude growth functions, obtained by increasing primary stimulus levels from 25 to 75 dB SPL in 10 dB steps, were recorded for later comparison. Following these measurements, the bulla was opened adequately to permit access to the cochlea for perfusion experiments. The periosteum covering the basal coil of the cochlea was removed, znd two small holes (approximately 100/~m each) were bored into the basal turn with a pick: a hole in scala tympani for the introduction of perfusates and an effluent hole in scala vestibuli to allow fluid escape. Tendons of the middle ear muscles were sectioned. The 2f~-f2 DPOAE response to 40 and 60 dB SPL primaries was monitored at intervals throughout the various surgical procedures, including immediately before and after opening the bulla for round window recordings, and again after each of the following: the wider opening of the bulla for perfusion experiments, sectioning of the tendons of middle ear muscles, and the boring of perfusion holes. CAP thresholds and DPOAE growth functions were also obtained following this last procedure to ensure that the animal's auditory sensitivity had not been compromised by the surgical exposure and preparation of the cochlea for perfusion. For inclusion in this investigation, CAP thresholds had to be consistent with normative laboratory data obtained using the same instrumentation, round window recording site and N~ amplitude criterion (Bobbin, unpublished), and the 2f~-f2 distortion product elicited by primaries at 40 dB SPL was required to exceed the surrounding noise floor (+ 150 Hz) by at least 5 dB following the completion of all surgical procedures necessary to prepare the cochlea for perfusion. Finally, animals with spontaneous otoacoustic emissions (two in this series) were excluded.
Cochlear perfusion experiments Perfusion experiments were undertaken following completion of the post-surgical measures described
109
above. The vehicle (artificial perilymph) employed in this investigation had a composition of (in raM): NaCl, 137; KCI, 5; CaC! 2, 2; NaH~PO 4, 1; MgCI2, 1; glucose, ll; NaHCO 3, 12, with a resulting pH range of 7.3-7.4 when brought into solution in deionized and filtered water. All perfusion solutions were freshly prepared. Experimental drugs used were: aeetylcholine (ace~lcholine chloride, Sigma No. 420-150), eserine (physostigmine hemisulfate, Sigma No. E8625), curare (dtubocurarine, Sigma No. T-2379), strychnine (strychnine sulfate, Sigma No. S-7001), and salicylate (sodium salicylate, Sigma No. 3007). These agents were dissolved in the artificial perilymph at desired concentrations and pH-adjusted as needed. Perfusates were introduced into scala tympani at room temperature through z pipette coupled to a syringe pump. The rate of infusion was slow, approximately 2.5 #l/min, to minimize displacement of cochlear tissues. Effluent was absorbed within the bulla using small cotton wicks. All perfusions were 10 min in duration. An approximately 15 min interval elapsed between perfusions, during which time the perfusion pipette was withdrawn from the hole in scala tympani, thoroughly rinsed, filled with the next perfusion solution, and repositioned for the start of the next perfusion. Beginning 1 min after the start of each perfusion, and continuing through the following 4 rain, the averaged amplitudes of the 2fl-f2 DPOAEs elicited by 40 dB SPL primaries were recorded at 10 second intervals. Thereafter, distortion product responses to both 40 and 60 dB SPL primaries were recorded every 30 seconds to the end of the perfusion. Monitoring at 30 second intervals continued for an additional 3 min following withdrawal of the perfusion pipette. DPOAE growth functions (25-75 dB SPL) were again obtained immediately following this monitoring. In three additional animals, DPOAEs recorded during the entire 10 rain of each perfusion were eiici*ed by primaries at 60 dB SPL. A standard perfusion protocol was employed. In all animals (N = 10), the first two perfusions were of artificial perilymph alone. This was accomplished so that a stable post-perfusion 2fl-f2 DPOAE baseline could be obtained for later comparison. These perfusions were followed by a series of perfusions with ACh (250 /zM) together with the cholinesterase inhibitor, eserine (20/zM). Each animal received at least three ACh/eserine perfusions. Those animals ( N = 7) demonstrating a transient reduction in DPOAE amplitude of 3 dB or greater that occurred during the first 5 rain of ACh/eserine perfusion were classified as having a response to perfusion of this drug combination. Responding animals also received 'consecutive' ACh/eserine peffusions (a second ACh/eserine perfusion which was not separated from the first by an artificial perilymph wash). A subgroup (N = 5) of these responders was tested with an ACh/eserine solution to
which known pharmacologic blockers of auditory efferent activity (curare, 50 tzM; strychnine, 50 p.M) were added. We employed both of these agents in this initial study because of suggestions that ACh may act at separate 'strychnine-preferring' and 'curare-preferring' sites (Norris et al., 1988). Before that perfusate was tested, however, a solution containing only the antagonists in artificial perilymph was perfused to ensure that there was no DPOAE response to the presence of these substances in the cochlear perilymph. Animals ( N - - 3 ) that did not demonstrate this DPOAE alteration to ACh/eserine perfusion each received a total of three ACh/eserine perfusions separated by appropriate artificial perilymph washes. Consecutive A C h / eserine perfusions and treatments with pharmacologic blockers were not accomplished for these animals. As a final treatment, all animals received a perfusion of salicylate (2.5 raM) as an internal control, undertaken to establish the continued responsiveness of the preparation (as determined by changes in 2 f r f 2 DPOAE amplitude) to intracochlear application of a drug with known DPOAE effects (Kujawa et al., 1991). Perfusions (2-3) with artificial perilymph alone were employed to wash this dru~ from cochlear perilymph. Repeated measures analysis of variance (ANOVA) and appropriate post-hoe tests (Tukey) were performed to quantify the effects of these perfusion solutions on DPOAE amplitude.
Results
Pre-perfusion measures Compound action potential CAP thresholds (4-20 kHz) obtained following completion of all surgical procedures required to prepare the cochlea for perfusion were not significantly different from baseline values (P > 0.01). Further, both sets of thresholds conformed to laboratory norms for round window CAP sensitivity and thus met the stated selection criteria.
Distortion products For inclusion in this investigation, guinea pigs were required to exhibit a pre-perfusion 2 f t - f 2 DPOAE at 8 kHz which exceeded its surrounding noise floor by at least 5 dB when elicited by primaries at 40 dB SPL. The original intent was to obtain initial recordings of DPOAE amplitude following administration of the anesthetic agents, but before surgical exposure of the cochlea was undertaken. When distortion product measurements were attempted prior to placement of a hole in the buila wall to gain round window access, however, distortion product responses to 40 dB SPL primaries were often unrecordable, or were of an am-
110
plitude smaller than the stated criterion. Over the course of these experiments, DPOAEs recorded at this time in response to either 40 or 60 dB SPL primaries were consistently of lower amplitude than those recorded following the initial opening of the bulla. Although venting the bulla can be expected to change the resonance characteristics of that space (e.g., Guinan and Peake, 1967; Ruggero et al., 1990), it is speculated that abnormal pressure in the bulla/middle ear space associated with the anesthetized state of these animals compromised reverse transmission characteristics of the middle ear and, as a result, DPOAE amplitudes were reduced. Consistent with this interpretation is the finding that DPOAEs of appropriate amplitude would immediately emerge from the noise floor following introduction of a small hole in the bulla wall. This hole could be sealed with bone wax and distortion product amplitude would gradually (over the course of several minutes) fall again toward the noise floor - with immediate return of distortion product amplitude again effected by removing the bone wax plug. Similar gradual changes in N t sensitivity associated with prolonged bulla closure in anesthetized cats were previously reported by Guinan and Peake (1967). Thus, the initial DPOAE amplitude recordings that were employed for the purposes of subject selection were those obtained in conjunction with the initial CAP recordings - that is to say, at the time of the round window exposure. Distortion product amplitudes associated with both 40 and 60 dB 5PL primaries were similarly obtained following the final surgical exposure and preparation of the cochlea for perfusion. For a given primary level, these values (40 dB SPL primaries: 9.54 -+ 0.969 dB 8PL; 60 dB SPL primaries: 25.72 + 0.580 dB SPL) were not significantly different ( P > 0.05) from values recorded at the time of the round window exposure (40 dB SPL primaries: 9.61 + 0.957 dB SPL; 66 dB SPL
._1 12. tO nn -0 ~-L~ rn
6O 50 40 3O
J O_
2O
0.05) from those recorded prior to the start of the perfusion experiments. Finally, repeated perfusions of artificial perilymph alone in three additional animals revealed the 2f~-f2 DPOAE to remaia stable within _+ 1.5 dB of pre-peffusion levels over the course of eight successive 10-rain perfusions. For each animal, mean 2fl-fz DPOAE amplitude (5 trials of 16 spectra each) as recorded following the second artificial perilymph perfusion was employed as the distortion product baseline to which all subsequent responses were compared.
Experimental drug perfusions
I 45
I 55
I 65
I 75
PRIMARY LEVEL (dB SPL) Fig. 2. 2ft-f2 DPOAE growth function (pre-perfusion). Shown are DPOAE amplitude means (_+ S.E.) as a function of primary stimulus level across the range 35 to 75 dB SPL.
ACh/eserine perfusions separated by artificial perilymph washes were associated with a transient reduction in the amplitude of the 2f~-f2 DPOAE elicited by 40 dB SPL primaries in 21 perfusions (100%) in 7 animals. The magnitude Of this reduction averaged 4.4 _+0.416 dB (mean _S.E.). In the remaining three animals, perfusion of this drug combination was not associated with a consistent alteration in DPOAE amplitude. Fig. 3 displays 2fc-f2 DPOAE amplitude as recorded during and for 3 min following perfusion of artificial perilymph alone and artificial perilymph containing ACh/eserine for one representative animal.
111 15
10 Q_ U3
5
m v hJ
0
t-_J 13
106
HEARES 01772
Intracochlear application of acetylcholine alters sound-induced mechanical events within the cochlear partition * S h a r o n G. K u j a w a l, T h e o d o r e J. G l a t t k e 1, M a u r e e n F a l l o n 2 a n d R i c h a r d P. B o b b i n 2 I Department of Speech and Hearing Sciences, University of Arizona, Tucson, Arizona, USA and z Kresge Hearing Research Laboratory of the South, Department of Otorhinolaryngology and Biocommunication, Louisiana State Unh'ersi~. Medical Center, New Orleans, Louisiana, USA (Received 4 November 1991; Revision received 18 March 1992; Accepted 26 March 1992)
Activation of olivocochlear (PC) efferent fibers has been suggested to alter micromechanical events occurring within the cochlear partition, possibly through an effect of the efferent neurotransmitter (acetylcholine; ACh) on outer hair cells (OHCs). Based on the widely-accepted assumption that otoacoustic emissions reflect OHC activity, we investigated the in vivo influence of ACh on OHCs by studying alterations in emission amplitude with local ACh application. Distortion product otoacoustic emissions (DPOAEs) were measured in anesthetized guinea pigs before, during, and after intracochlear application of ACh (250 /zM) with the cholinesterase inhibitor, eserine (20 /zM). Perfusion of ACh/eserine was associated with a desensitizing reduction in D P O A E amplitude of approximately 4.4 dB. This reduction was intensity-dependent, with greater and more consistent reductions observed for DPOAEs elicited by low- than by moderate-intensity primaries. The response reduction was not seen during consecutive ACh perfusions performed without an intervening artificial perilymph wash, and was effectively blocked in the presence of pharmacologic antagonists of P C efferent activity (curare, 50 ~M, strychnine, 50 izM). Finally, a similar alteration in DPOAE amplitude was never seen during perfusion of the control (artificial perilymph) solution alone. It is argued that these results support the hypothesis that P C efferent activation can alter sound-induced cochlear mechanical events. Efferents; Acetylcholine; Acoustic distortion products; Cochlear mechanics; Olivocochlear neurons; Outer hair cells
Introduction
Our understanding of the mechanical function of the cochl~.a has been revised substantially in the past decade. I iae traditional view of the cochlea as a passive organ that transduces mechanical displacements into neural activity has been altered by two important discoveries: the finding that outer hair cells (OHCs) have motile capabilities (Brownell et al., 1985) and the demonstration that the cochlea can produce acoustic energy detected as otoacoustic emissions (OAEs) (Kemp, 1978). Both of these phenomena have been taken as support for active, physiologically-vulnerable processes within the cochlea that amplify and sharpen the cochlear mechanical response to sound stimulation. Recent investigations have suggested that these processes are under the influence of the olivocochlear (PC) efferent neurons. Specifically, it has been argued that the medial olivocochlear (MOC) efferents, through
Correspondence to: Richard P. Bobbin, Kresge Hearing Research Laboratory, LSU .~]edical Center, 2020 Gravier Street, Suite A, New Orleans, LA 70112, USA. Fax: (504) 568-4460. * A preliminary report was presented at the Midwinter Research Meeting of the Association far Research in Otolaryngology in February, 1991.
numerous and direct connections with OHCs, can alter the local cochlear mechanical response to sound stimulation and provide a 'gain control' (Guinan and Gifford, 1988) of the cochlear amplifier. Newer models of cochlear function describe an active cochlea - one capable of executing modifications to its response patterns to increase or decrease afferent outflow from the cochlea. Although a contribution to these processes by the OHCs is widely accepted, the role of the PC efferents in modulating cochlear mechanical events has been more difficult to define. Investigations undertaken to study the efferent influence at the level of the cochlea traditionally have recorded changes in gross sound-induced electrical responses from the cochlea and the eighth nerve during electrical activation of P C efferent fibers (see Wiederhold, 1986 for review). Such studies have revealed an inhibitory role for the PC efferents. The precise mechanism of this inhibitory influence is unknown; however, it has been suggested to involve a mechanical component at the OHCs (Dallos, 1985). A more direct reflection of sound-induced cochlear mechanical events can be obtained through the study of OAEs. OAEs are widely regarded t o reflect the integrity of cochlear mechanical processes in general, and OHC function in particular (e.g., Kim, 1986). Distort.;on product otoacoustic emissions (DPOAEs) rep-
107
resent one class of evoked emissions. These distortions are frequency-specific signals produced by the normal nonlinear mechanical response of the cochlea. Through their association with OHCs, DPOAEs have been linked to the active processes responsible for the cochlea's sensitivity and frequency selectivity (Kim, 1980). At low levels of stimulation, these emissions may provide a direct and sensitive reflection of OHC function (Brown et al., 1989). Therefore, DPOAEs are particularly appropriate tools to study the cochlear mechanical consequences of efferent activation. Employing DPOAEs, an efferent effect on cochlear mechanics has been demonstrated previously by Mountain (1980) an~d Siegel and Kim (1982). In these investigations, DPOAE amplitudes (f2-fl; 2f~-f2) were altered during electrical stimulation of OC efferent fibers at the floor of the fourth ventricle. The proposed efferent effects or, distortion products were not straighfforJcard, however, and inconsistencies between the two investigations remain unresolved. Acoustic stimulation of the contralateral ear has also been employed as a strategy to study auditory efferent effects on cochlear mechanics. Distortion product amplitudes are reduced in awake humans (Brown and Norton, 1990; Moulin and Collet, 1992) and anesthetized guinea pigs (Puel and Rebillard, 1990) during contralatcral sound stimulation. Contralateral sound suppression is suggested to be mediated primarily by uncrossed MOC efferents, and these fibers comprise only about one third of the MOC projections to each cochlea (Guinan et al., 1983). Investigations employing contraiateral sound, then, would be expected to provide information about only a portion of the OC efferents to OHCs. The nature of the OC efferent influence on cochlear mechanical events thus remains unclear, and further study is needed. Acetylcholine (ACh) is the major neurotransmitter released onto OHCs during MOC efferent stimulation (see Bledsoe et al., 1988 for review). We attempted to mimic MOC efferent activation by utilizing intracochlear ACh application. The influence of this manipulation on sound-induced cochlear mechanical events was evaluated by monitoring 2fl-f2 DPOAE amplitude.
Method
Subjects Experiments were performed on 10 pigmented guinea pigs (Carla cobaya) of either sex weighing between 250 and 350 g. Animals were treated in accordance with Federal, state, and institutional guidelines and the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1985). All
animals included in the study demonstrated auditory nerve compound action potential (CAP) thresholds consistent with laboratory norms (Bobbin, unpublished), and all had normal distortion product amplitude characteristics (Brown, 1987; Brown and Gaskill, 1990).
Stimulus generation and response recording Compound action potential Acoustic stimuli used to obtain CAP thresholds were 4, 8, 10, 12, 16, and 20 kHz tone bursts (0.25 ms rise/fall, 10 ms duration, 200 ms interstimulus interval). These signals were produced by an external signal generator (Wavetek 148A), transduced by a speaker with an extended high frequency response (to 25 kHz), and delivered to the right ear of each animal under computer control with intensity incremented in 6 dB steps. Electrophysiologic activity was detected by a flame¢dLI|~U~ silver electrode (insulated, except for the tip) b~U~.~ placed on the round window membrane. Electrical signals were amplified (Grass P15; gain = 1000), averaged (over 20 trials) and stored on disk. For measurement purposes, stored voltages were filtered (KrohnHite 3202R; 24 dB/octave; low pass = 2 kHz) and displayed on an oscilloscope. Threshold was defined as the lowest stimulus intensity at which the following two criteria could be met: a) an averaged NI-P i amplitude of > 5/zV was obtained and b) the magnitude of this peak to peak amplitude increased with the next stimulus increment.
Distortion products The 2fl-f2 DPOAE at 8 kHz was elicited by equalintensity 'primary' stimuli at 10 kHz (ft) and 12 kHz (f2), yielding an fz/fi ratio of 1.2. This ratio was associated with among the largest 2 f l - f 2 DPOAE amplitudes evaluated in this laboratory (Littman, 1990), a finding in good agreement with the outcomes of other investigations of 2fl-f2 DPOAE characteristics in guinea pigs (Brown, 1987; Brown and Gaskill, 1990). Pure tone signals were generated by oscillators (Hewlett Packard 200CD; 200ABR), and were routed through attenuators (Hewlett Packard 350D) to Etymotic Research ER-2 receivers. The outputs from these receivers were delivered to an acoustic probe assembly which contained a low-noise microphone system (Etymotic Research, ER-10) and which was coupled to the right ear of each animal by a hollow ear bar. Responses to this bitonal stimulation were detected by the probe microphone system, the output of which was directed via a microphone preamplifier (Etymotic Research, ER-1072) to a Hewlett Packard 3561A signal analyzer. The ear canal soundfield containing the DPOAE was sampled, digitized, and submitted to Fast
108
fl =f2=60 dB SPL
2fl-f2
;ENTER 9000 Hz
RMS 16
SPAN 6250 H
Fig. 1. DPOAE response to primary pure tone stimulation at 60 dB SPL. Shown are the primary tones (f~ = I0 kHz; f2 = 12 kHz) and the resulting 2 f l - f 2 P P O A E at 8 kHz as recorded in the open bulla condition. The 3 f l - 2 f 2 DPOAE at 6 kHz is also present in this trace.
Fourier Transform (FFT) analysis, and the resulting spectrum (averaged over 16 samples) was displayed on the analyzer using a 6250 Hz window (see Fig. 1). When displayed in this manner, the spectrum average contained the two primary stimuli and the 2f~-f2 DPOAE. A distortion product corresponding to the frequency 3f~-2f2 was occasionally present within this display window as well, but was not monitored in the present investigation. Distortion product amplitude, defined as the spectral peak corresponding to the 2fl-f2 frequency (8 kHz) was recorded manually from such FFT spectra. The noise floor associated with this instrumentation was approximately - 1 2 dB SPL at points 150 Hz above and below the 2fl-f2 DPOAE frequency (2 kHz window; BW = 7.5 Hz), and averaged approximately - 2 dB SPL when a 6250 Hz window (BW = 23.4 Hz) was employed. When evaluated in a passive cavity of appropriate volume, harmonic and intermodulation distortion components were more than 75 dB below the level of the primaries. Stimulus intensity and frequency calibrations of the sound system/acoustic probe-ear bar assembly were performed daily according to the procedures described by Littman (1990).
Experimental procedures Surgical procedures and associated pre-perfusion measurements All procedures were conducted in an acousticallyand electrically-shielded room. Animals were anesthetized with pentobarbital sodium (Nembutal, 30 mg/kg, i.p.) and chlorprothixene (Taractan, 30 mg/kg, i.p.) and were tracheotomized and artificially respi-
rated. ECG and rectal temperature were monitored throughout each experiment and temperature was maintained at 38°+ I°C by a heating pad. Additional pentobarbital (30 mg/kg, i.p.) was administered as required to maintain an adequate depth of anesthesia. Cartilaginous ear canals were exposed and removed to allow optimum placement of the ear bars used to secure the animal in the headholder. The right auditory bulla was exposed using a ventrolateral approach and a small opening was made in the dorsolateral bulla just sufficient to gain access to the round window. The round window electrode was then positioned for baseline recordings of CAP thresholds. Baseline measures of the 2fl-f2 DPOAE (five consecutive trials of 16 spectra each) were also obtained at this time for both 40 and 60 dB SPL primaries. Finally, DPOAE amplitude growth functions, obtained by increasing primary stimulus levels from 25 to 75 dB SPL in 10 dB steps, were recorded for later comparison. Following these measurements, the bulla was opened adequately to permit access to the cochlea for perfusion experiments. The periosteum covering the basal coil of the cochlea was removed, znd two small holes (approximately 100/~m each) were bored into the basal turn with a pick: a hole in scala tympani for the introduction of perfusates and an effluent hole in scala vestibuli to allow fluid escape. Tendons of the middle ear muscles were sectioned. The 2f~-f2 DPOAE response to 40 and 60 dB SPL primaries was monitored at intervals throughout the various surgical procedures, including immediately before and after opening the bulla for round window recordings, and again after each of the following: the wider opening of the bulla for perfusion experiments, sectioning of the tendons of middle ear muscles, and the boring of perfusion holes. CAP thresholds and DPOAE growth functions were also obtained following this last procedure to ensure that the animal's auditory sensitivity had not been compromised by the surgical exposure and preparation of the cochlea for perfusion. For inclusion in this investigation, CAP thresholds had to be consistent with normative laboratory data obtained using the same instrumentation, round window recording site and N~ amplitude criterion (Bobbin, unpublished), and the 2f~-f2 distortion product elicited by primaries at 40 dB SPL was required to exceed the surrounding noise floor (+ 150 Hz) by at least 5 dB following the completion of all surgical procedures necessary to prepare the cochlea for perfusion. Finally, animals with spontaneous otoacoustic emissions (two in this series) were excluded.
Cochlear perfusion experiments Perfusion experiments were undertaken following completion of the post-surgical measures described
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above. The vehicle (artificial perilymph) employed in this investigation had a composition of (in raM): NaCl, 137; KCI, 5; CaC! 2, 2; NaH~PO 4, 1; MgCI2, 1; glucose, ll; NaHCO 3, 12, with a resulting pH range of 7.3-7.4 when brought into solution in deionized and filtered water. All perfusion solutions were freshly prepared. Experimental drugs used were: aeetylcholine (ace~lcholine chloride, Sigma No. 420-150), eserine (physostigmine hemisulfate, Sigma No. E8625), curare (dtubocurarine, Sigma No. T-2379), strychnine (strychnine sulfate, Sigma No. S-7001), and salicylate (sodium salicylate, Sigma No. 3007). These agents were dissolved in the artificial perilymph at desired concentrations and pH-adjusted as needed. Perfusates were introduced into scala tympani at room temperature through z pipette coupled to a syringe pump. The rate of infusion was slow, approximately 2.5 #l/min, to minimize displacement of cochlear tissues. Effluent was absorbed within the bulla using small cotton wicks. All perfusions were 10 min in duration. An approximately 15 min interval elapsed between perfusions, during which time the perfusion pipette was withdrawn from the hole in scala tympani, thoroughly rinsed, filled with the next perfusion solution, and repositioned for the start of the next perfusion. Beginning 1 min after the start of each perfusion, and continuing through the following 4 rain, the averaged amplitudes of the 2fl-f2 DPOAEs elicited by 40 dB SPL primaries were recorded at 10 second intervals. Thereafter, distortion product responses to both 40 and 60 dB SPL primaries were recorded every 30 seconds to the end of the perfusion. Monitoring at 30 second intervals continued for an additional 3 min following withdrawal of the perfusion pipette. DPOAE growth functions (25-75 dB SPL) were again obtained immediately following this monitoring. In three additional animals, DPOAEs recorded during the entire 10 rain of each perfusion were eiici*ed by primaries at 60 dB SPL. A standard perfusion protocol was employed. In all animals (N = 10), the first two perfusions were of artificial perilymph alone. This was accomplished so that a stable post-perfusion 2fl-f2 DPOAE baseline could be obtained for later comparison. These perfusions were followed by a series of perfusions with ACh (250 /zM) together with the cholinesterase inhibitor, eserine (20/zM). Each animal received at least three ACh/eserine perfusions. Those animals ( N = 7) demonstrating a transient reduction in DPOAE amplitude of 3 dB or greater that occurred during the first 5 rain of ACh/eserine perfusion were classified as having a response to perfusion of this drug combination. Responding animals also received 'consecutive' ACh/eserine peffusions (a second ACh/eserine perfusion which was not separated from the first by an artificial perilymph wash). A subgroup (N = 5) of these responders was tested with an ACh/eserine solution to
which known pharmacologic blockers of auditory efferent activity (curare, 50 tzM; strychnine, 50 p.M) were added. We employed both of these agents in this initial study because of suggestions that ACh may act at separate 'strychnine-preferring' and 'curare-preferring' sites (Norris et al., 1988). Before that perfusate was tested, however, a solution containing only the antagonists in artificial perilymph was perfused to ensure that there was no DPOAE response to the presence of these substances in the cochlear perilymph. Animals ( N - - 3 ) that did not demonstrate this DPOAE alteration to ACh/eserine perfusion each received a total of three ACh/eserine perfusions separated by appropriate artificial perilymph washes. Consecutive A C h / eserine perfusions and treatments with pharmacologic blockers were not accomplished for these animals. As a final treatment, all animals received a perfusion of salicylate (2.5 raM) as an internal control, undertaken to establish the continued responsiveness of the preparation (as determined by changes in 2 f r f 2 DPOAE amplitude) to intracochlear application of a drug with known DPOAE effects (Kujawa et al., 1991). Perfusions (2-3) with artificial perilymph alone were employed to wash this dru~ from cochlear perilymph. Repeated measures analysis of variance (ANOVA) and appropriate post-hoe tests (Tukey) were performed to quantify the effects of these perfusion solutions on DPOAE amplitude.
Results
Pre-perfusion measures Compound action potential CAP thresholds (4-20 kHz) obtained following completion of all surgical procedures required to prepare the cochlea for perfusion were not significantly different from baseline values (P > 0.01). Further, both sets of thresholds conformed to laboratory norms for round window CAP sensitivity and thus met the stated selection criteria.
Distortion products For inclusion in this investigation, guinea pigs were required to exhibit a pre-perfusion 2 f t - f 2 DPOAE at 8 kHz which exceeded its surrounding noise floor by at least 5 dB when elicited by primaries at 40 dB SPL. The original intent was to obtain initial recordings of DPOAE amplitude following administration of the anesthetic agents, but before surgical exposure of the cochlea was undertaken. When distortion product measurements were attempted prior to placement of a hole in the buila wall to gain round window access, however, distortion product responses to 40 dB SPL primaries were often unrecordable, or were of an am-
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plitude smaller than the stated criterion. Over the course of these experiments, DPOAEs recorded at this time in response to either 40 or 60 dB SPL primaries were consistently of lower amplitude than those recorded following the initial opening of the bulla. Although venting the bulla can be expected to change the resonance characteristics of that space (e.g., Guinan and Peake, 1967; Ruggero et al., 1990), it is speculated that abnormal pressure in the bulla/middle ear space associated with the anesthetized state of these animals compromised reverse transmission characteristics of the middle ear and, as a result, DPOAE amplitudes were reduced. Consistent with this interpretation is the finding that DPOAEs of appropriate amplitude would immediately emerge from the noise floor following introduction of a small hole in the bulla wall. This hole could be sealed with bone wax and distortion product amplitude would gradually (over the course of several minutes) fall again toward the noise floor - with immediate return of distortion product amplitude again effected by removing the bone wax plug. Similar gradual changes in N t sensitivity associated with prolonged bulla closure in anesthetized cats were previously reported by Guinan and Peake (1967). Thus, the initial DPOAE amplitude recordings that were employed for the purposes of subject selection were those obtained in conjunction with the initial CAP recordings - that is to say, at the time of the round window exposure. Distortion product amplitudes associated with both 40 and 60 dB 5PL primaries were similarly obtained following the final surgical exposure and preparation of the cochlea for perfusion. For a given primary level, these values (40 dB SPL primaries: 9.54 -+ 0.969 dB 8PL; 60 dB SPL primaries: 25.72 + 0.580 dB SPL) were not significantly different ( P > 0.05) from values recorded at the time of the round window exposure (40 dB SPL primaries: 9.61 + 0.957 dB SPL; 66 dB SPL
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0.05) from those recorded prior to the start of the perfusion experiments. Finally, repeated perfusions of artificial perilymph alone in three additional animals revealed the 2f~-f2 DPOAE to remaia stable within _+ 1.5 dB of pre-peffusion levels over the course of eight successive 10-rain perfusions. For each animal, mean 2fl-fz DPOAE amplitude (5 trials of 16 spectra each) as recorded following the second artificial perilymph perfusion was employed as the distortion product baseline to which all subsequent responses were compared.
Experimental drug perfusions
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PRIMARY LEVEL (dB SPL) Fig. 2. 2ft-f2 DPOAE growth function (pre-perfusion). Shown are DPOAE amplitude means (_+ S.E.) as a function of primary stimulus level across the range 35 to 75 dB SPL.
ACh/eserine perfusions separated by artificial perilymph washes were associated with a transient reduction in the amplitude of the 2f~-f2 DPOAE elicited by 40 dB SPL primaries in 21 perfusions (100%) in 7 animals. The magnitude Of this reduction averaged 4.4 _+0.416 dB (mean _S.E.). In the remaining three animals, perfusion of this drug combination was not associated with a consistent alteration in DPOAE amplitude. Fig. 3 displays 2fc-f2 DPOAE amplitude as recorded during and for 3 min following perfusion of artificial perilymph alone and artificial perilymph containing ACh/eserine for one representative animal.
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