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Effects of electrical stimulation of the inferior colliculus on 2f1−f2 distortion product otoacoustic emissions in anesthetized guinea pigs
Hearing Research 170 (2002) 116^126 www.elsevier.com/locate/heares
E¡ects of electrical stimulation of the inferior colliculus on 2f1 3f2 distortion product otoacoustic emissions in anesthetized guinea pigs J. Popelar , J. Mazelova¤, J. Syka Institute of Experimental Medicine, Academy of Sciences, V|¤denska¤ 1083, 142 20 Prague 4, Czech Republic Received 20 November 2001; accepted 4 April 2002
Abstract The effects of electrical stimulation of the inferior colliculus (IC) on the activation of olivocochlear nerve fibers were investigated in guinea pigs in which the 2f1 3f2 distortion product otoacoustic emissions (DPOAE) were recorded. Animals were anesthetized with ketamine (33 mg/kg) and xylazine (6.6 mg/kg). Bipolar electrical stimulation of the IC by a train of pulses with currents less than the threshold for evoking muscle twitches resulted in a small depression of the DPOAE amplitude by 0.1^2 dB. The maximal effect was observed when the stimulating electrodes were located in the rostro-medial or ventral parts of the IC. The suppression of electrically evoked DPOAE was similar to the DPOAE suppression produced by acoustical stimulation of the contralateral ear by a broad-band noise. Suppression of DPOAE amplitude in response to both acoustical and electrical stimulation was abolished 1^2 h after a single intramuscular injection of gentamicin (210^250 mg/kg). The results indicate that electrical stimulation of the IC can activate the efferent system and produce DPOAE changes by similar mechanisms as does acoustical stimulation of the contralateral ear. 9 2002 Elsevier Science B.V. All rights reserved.
1. Introduction The inferior colliculus (IC) represents an important relay nucleus for the a¡erent auditory pathway, receiving inputs from the cochlear nucleus and the superior olivary complex and projecting to the auditory cortex through the medial geniculate body. The IC also represents an important part of the e¡erent, descending auditory system. The IC receives many axons of pyramidal cells of layer V of the auditory cortex, which terminate mainly in the dorsal and external cortices of the IC (Diamond et al., 1969; Andersen et al., 1980; Druga and Syka, 1984; Faye-Lund, 1985; Coleman and Clerici, 1987; Druga et al., 1997; Winer et al., 1998). Addi-
* Corresponding author. Tel.: +420 (2) 4106 2689; Fax: +420 (2) 4106 2787. E-mail address: [email protected] (J. Popelar). Abbreviations: BBN, broad-band noise; CAP, compound action potential of the auditory nerve; CyOx, cytochrome oxidase; DPOAE, 2f1 3f2 distortion product otoacoustic emissions; ECIC, external cortex of the inferior colliculus; IC, inferior colliculus; LOC, lateral olivocochlear system; MOC, medial olivocochlear system; NADPHd, nicotinamide adenine dinucleotide phosphate-diaphorase
tional parts of the e¡erent pathway are represented by ¢bers projecting from the IC to the nuclei of the lateral lemniscus, superior olivary complex (Syka et al., 1988a; Scho¢eld and Cant, 1999) and cochlear nuclei (for review see Syka et al., 1988b; Spangler and Warr, 1991). Some target regions within the superior olivary complex, mainly the rostral periolivary region and the ventral nucleus of the trapezoid body, contain olivocochlear neurons, which innervate sensory hair cells within the cochlea. Two principal olivocochlear pathways have been described that di¡er anatomically and physiologically ^ the medial (MOC) and lateral (LOC) olivocochlear systems (White and Warr, 1983). The LOC neurons closely surround the lateral superior olive (LSO) ; their axons are mostly unmyelinated and synapse on ipsilateral a¡erent neuron terminals at the inner hair cells. The MOC neurons tend to surround the medial, ventral and rostral parts of the medial superior olive; their ¢bers are mostly myelinated, and two-thirds of them project to outer hair cell bodies in the contralateral cochlea. Limited data are available on the function of the e¡erent auditory system. It is thought that the descending projections are involved in selective attention, fre-
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quency selectivity, intensity coding, sound localization, adjustment of dynamic range, speech discrimination and the detection of tones in noise (Scharf et al., 1987; Puel et al., 1988; Micheyl and Collet, 1996; Giraud et al., 1997). In addition to these functions, the e¡erent olivocochlear bundle is thought to protect the cochlea from overexposure to noise. However, the mechanisms of these processes are not completely known. Previous studies have demonstrated that activation of the olivocochlear e¡erent system (by contralateral acoustic stimulation or by direct electrical stimulation of the olivocochlear nerve ¢bers or contralateral round window) alters the amplitude of most cochlear potentials (Fex, 1959; Gi¡ord and Guinan, 1987; Liberman, 1989; Popelar et al., 1996, 1999) and otoacoustic emissions (Puel and Rebillard, 1990; Veuillet et al., 1991; Berlin et al., 1993; Hood et al., 1996). A few studies have reported on the e¡ects of IC stimulation on the compound action potential of the auditory nerve (CAP) in guinea pigs (Mulders and Robertson, 2000) or distortion product otoacoustic emissions (DPOAE) in rats (Scates et al., 1999). Rajan (1990) demonstrated a reduction of the temporary threshold shifts in guinea pigs in which the IC was electrically stimulated during noise overexposure. On the other hand, he failed to ¢nd any direct e¡ect of IC stimulation on the CAP or DPOAE. The aim of our work was to investigate the e¡ects of electrical stimulation of di¡erent parts of the IC on DPOAE recorded in anesthetized guinea pigs. DPOAE suppression elicited by IC electrical stimulation was compared with DPOAE suppression produced by acoustic stimulation of the contralateral ear. The mechanisms of DPOAE suppression elicited by electrical stimulation were studied by using a single intramuscular injection of gentamicin, which was shown previously to eliminate the e¡ect of contralateral acoustic stimulation (by blocking the Ca2þ entry necessary to elicit the acetylcholine response in outer hair cells). The results contribute to understanding the function of descending pathways in the central auditory system.
2. Materials and methods Experiments were performed on 20 pigmented guinea pigs weighing 250^300 g. Animals were anesthetized with an intramuscular injection of 33 mg/kg ketamine (Narkamon 5%, Spofa) and 6.6 mg/kg xylazine (Sedazine, Phoenix Scienti¢que). All recordings were conducted in a sound-attenuated and anechoic chamber. 2.1. Surgery The skin and underlying muscles covering the skull
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were retracted to expose the dorsal cranium between points bregma and lambda. A small hole was made by a trephine on the right side of the skull above the IC. The animal’s head was rigidly held in a stereotaxic apparatus by a special holder, ¢xed to the skull by two screws and secured by acrylic resin. This type of ¢xation enabled the animal’s head to be free for electrode penetration, acoustic stimulation and otoacoustic emission recording. A rectal temperature of 37^38‡C was automatically maintained by a DC-powered electric heating pad. 2.2. Electrical stimulation of the IC Two metal electrodes (insulated nichrome wire, diameter 0.002 inch) with a tip separation of 1 mm, glued on a rigid wire as a carrier, were used for electrical stimulation. Electrodes were inserted into the ipsilateral IC through the overlying cortex in a dorsoventral direction using an electrically driven micromanipulator. The IC was stimulated in a bipolar mode with a continuous train of monophasic or biphasic rectangular pulses (pulse duration 0.3 ms, pulse frequency 100^300 Hz, current up to 1.5 mA), generated by an optically coupled constant current source. During penetration of the IC, electrode movement was discontinued every 0.3^0.5 mm. At these positions of the electrode tip, the electrical current was continuously increased until the ¢rst detectable muscle twitches were observed (pinna or eye movement, small contractions of facial musculature, limb £exion). Then, the stimulation current was set approximately 10% lower, and the e¡ect of electrical stimulation on the DPOAE amplitude was tested. 2.3. Recording the IC evoked responses To con¢rm the correct electrode position within the IC, the IC evoked responses were recorded during electrode penetration. Bipolar clicks (100 Ws duration) were generated by SigGen TDT (Tucker-Davis Technologies) software and processed by a TDT D/A converter and programmable attenuator. Stimuli were delivered to the contralateral ear by a piezoelectric stimulator coupled to the outer ear canal via a 7 cm long silastic tube (outer diameter 5 mm, inner diameter 3 mm). The end of the silastic tube was ¢xed in the entrance to the outer ear canal by acrylic glue. The stimulating sound system was calibrated in an arti¢cial ear (adapted for the impedance of the guinea pig ear canal) with a Bruel and Kjaer (4134) 1/2 in microphone and in a group of anesthetized guinea pigs (Popelar et al., 1996). The signal from the electrodes (one stimulating electrode against a reference electrode in the neck muscles) was ampli¢ed by a DAM di¡erential ampli¢er
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(gain 60 dB, ¢lters 10 Hz^1 kHz), processed with a TDT A/D converter, and the responses were averaged using BioSig software. 2.4. DPOAE recordings DPOAE were recorded in guinea pigs using an ILO 96 analyzer (Otodynamics) and ILO H-probes. Cubic 2f1 3f2 DPOAE were recorded using two primary tones, f1 and f2 (ratio f2 /f1 = 1.22), presented with two phones, either with equilevel stimuli (50/50 or 60/60 dB SPL) or with an L2 level that was 5^10 dB lower than L1 (60/50, 55/45 or 55/50 dB SPL). At the beginning of the experiment, a DP-gram (the function of DPOAE level on increasing stimulus frequency, four points per octave) was recorded, followed by DP-gram measurement during simultaneous contralateral acoustic stimulation with broad-band noise (BBN). The recording series was ended by a second control DPOAE recording. According to the shape of the DP-gram, two frequencies (f1 and f2 ) were chosen for recording of the 2f1 3f2 DPOAE using the ILO software Spectral history. This software continuously monitors and displays every 1.4 s the 2f1 3f2 DPOAE amplitude (16 DPOAE subaverages) to stimuli of ¢xed frequency and intensity. Recording sessions in the course of single electrode penetration usually consisted of DPOAE recordings during IC electrical stimulation at di¡erent electrode positions alternated with 20 s periods of control DPOAE recordings. 2.5. DPOAE suppression produced by acoustic stimulation of the contralateral ear The suppression of DPOAE was tested by stimulating the contralateral ear with continuous BBN at 51^71 dB SPL, generated with a Hewlett Packard (33120A) waveform generator and ampli¢ed and shaped with an electronic switch. BBN was presented to the animal from a piezoelectric stimulator coupled to the outer ear canal via a 7 cm long silastic tube. It was con¢rmed in previous experiments that a BBN intensity of 51^71 dB SPL was 40^60 dB above the hearing threshold, which was low enough to prevent contralateral crosstalk (Popelar et al., 1994). 2.6. Functional ablation of the olivocochlear e¡erent system by gentamicin Previous studies demonstrated that a single gentamicin injection (150^250 mg/kg) reversibly and temporarily eliminates the e¡ect of contralateral acoustic stimulation (Smith et al., 1994; Popelar et al., 1996; Yoshida et al., 1999). In order to test the hypothesis that the mechanisms of DPOAE suppression produced
by contralateral acoustic stimulation and electrical stimulation of the IC were similar, a gentamicin injection was used in two guinea pigs. When IC electrodes were located in a position where a pronounced e¡ect of electrical stimulation on DPOAE was found, the animals were injected intramuscularly with gentamicin at a dose of 210 or 250 mg/kg, and the DPOAE suppression in response to acoustic and electrical stimulation was tested in 15 min periods. 2.7. Histological control At the end of the experiments, the stimulation electrodes were left in the deepest position of the last penetration within the IC and carefully cut just above the surface of the brain. The guinea pig was sacri¢ced with an overdose of 1^2 ml of pentobarbital (Spofa, 50 mg/ ml) and perfused intracardially with 10% formaldehyde. The brain with the cut electrode was removed from the skull and post¢xed in the same solution for several days. Before sectioning, the electrode was carefully removed from the brain. Subsequently, the brain was sectioned in the frontal plane on a freezing MICROM K400 microtome (slice thickness 40 Wm) and stained with cresyl violet. The whole extent of the electrode track was clearly discernible in several serial sections. The coordinates of the electrode track were determined according to an atlas of the guinea pig brain (Luparello, 1967), and the location of individual stimulation sites within the IC was assessed on the basis of the depth coordinates indicated on the electronically driven microdrive. The boundaries of the IC subnuclei were determined in previous experiments (unpublished data) on the basis of Nissl staining, the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and cytochrome oxidase (CyOx) positivity. For NADPH-d staining the method of Scherer-Singler et al. (1983) was used, and for CyOx histochemistry the Wong-Riley (1979) method was used. Whereas CyOx staining was most pronounced in the central nucleus of the IC, intensely stained NADPH-d-positive neurons and moderately positive neuropil were localized only in the dorsal cortex of the IC and in the external cortex of the IC (ECIC). On the basis of the above-described histochemical staining, the boundaries of the IC subnuclei were determined in eight serial IC frontal sections (the distance between individual sections was 200^250 Wm), and the schema was used for the localization of electrode penetrations in the IC. The care and use of animals reported on in this study were approved by the Ethics Committee of the Institute of Experimental Medicine of the Academy of Sciences of the Czech Republic and followed the guidelines of the Declaration of Helsinki.
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Fig. 1. An example of 2f1 3f2 DP-grams elicited with f1 and f2 primaries of intensities L1 = 55 dB SPL and L2 = 50 dB SPL, recorded in one guinea pig (MIC 12) under control conditions (solid line) and during contralateral acoustic stimulation with BBN (intensity 71 dB SPL, dashed line).
3. Results 3.1. DPOAE recording Experiments were performed in 20 healthy guinea pigs weighing 250^350 g. DPOAE were regularly recorded in all tested animals. Fig. 1 shows an example of 2f1 3f2 DPOAE (DP-gram) elicited by two primaries, f1 and f2 , with stimulus intensities L1 = 55 dB SPL and L2 = 50 dB SPL recorded in one guinea pig (MIC 12). The DPOAE curve exceeds the noise £oor by 6^32 dB in the frequency range from 1 to 6.3 kHz. The interrupted line in Fig. 1 represents the DP-gram recorded during acoustic stimulation of the contralateral ear. The DPOAE amplitudes are suppressed by 0.5^2.2 dB in the frequency range 1^5.6 kHz, and the DP-gram is shifted to lower values. On the basis of the shape of DP-grams observed in individual animals, a pair of primaries with ¢xed parameters were chosen for continuous DPOAE recording using the Spectral history software. Since DPOAE suppression by contralateral sound was elicited almost throughout the whole frequency range, the DPOAE suppression produced by electrical stimulation of the IC was tested in a broad range of frequencies. 3.2. E¡ect of IC electrical stimulation The e¡ect of contralateral acoustic and IC electrical
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stimulation was tested using continuous recording of DPOAE to ¢xed stimulus frequency and intensity (Spectral history ILO 96 software). The e¡ect of electrical stimulation of the IC on the DPOAE amplitude was investigated at di¡erent positions of the stimulating electrode in the IC. At each position the electrical current was increased from zero to the level at which the ¢rst facial muscle or pinna twitches appeared. Then the stimulation current was decreased by approximately 10%, and DPOAE were recorded for at least 20^30 s. Periods of DPOAE recordings with IC electrical stimulation were alternated with control recordings without stimulation. At individual IC electrode positions, evoked responses to click stimulation were recorded as well. As a rule, it was possible to stimulate the dorsal parts of the IC (up to a depth of 2 mm) using an electrical current reaching 1^1.5 mA without any sign of muscle twitches or DPOAE suppression. The deeper the electrodes were inserted into the IC, the lower the observed electric current threshold for evoking muscle twitches. In the ventral layers of the IC the muscle reactions occurred even with an electrical current lower than 100 WA. In some electrode positions electrical stimulation produced the suppression of DPOAE. Fig. 2 shows an example of a single electrode penetration through the IC and the resulting e¡ect of electrical stimulation on the DPOAE in animal MIC 202. Open circles in the histological section (Fig. 2a) represent positions with no e¡ect on DPOAE, whereas in positions marked by black circles the suppressive e¡ect was maximal and in positions marked by gray circles the suppression of DPOAE reached approximately half of the maximal value. DPOAE amplitude changes over time (f1 /f2 = 2002/2417 kHz, L1 /L2 = 60/60 dB SPL) for individual IC locations are demonstrated in Fig. 2b. The duration of electrical stimulation (20 s) is shown in the bottom part of the ¢gure. In each panel, the ¢rst three and the last three data points represent control DPOAE values preceding and following the electrical stimulation. This ¢gure documents that pronounced DPOAE suppression in response to IC electrical stimulation was seen only in two electrode positions which were 0.3 mm apart. Switching the electrical stimulation on produced a fast decrease of 1.4^1.6 dB in DPOAE amplitude, and this level was maintained with small variations of Q 0.2^0.4 dB during the whole period of electrical stimulation. Switching the electrical stimulation o¡ caused a fast recovery of DPOAE amplitude to prestimulation levels. In neighboring positions, a weak e¡ect was obtained at depths of both 2.4 and 3.2 mm. Repeated stimulation in individual electrode positions usually resulted in reproducible e¡ects. In this animal, the diameter of the area of the IC where the DPOAE suppression was observed was 0.8 mm.
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Fig. 2. (a) A Nissl-stained IC section with schematic reconstruction of an electrode penetration into the IC and positions of the electrode tip in the IC during electrical stimulation. (b) Recordings of the DPOAE amplitude (f1 /f2 = 2002/2417 Hz, 60/60 dB SPL) during electrical stimulation at individual IC positions (marked by arrows). Empty circles, no e¡ect; gray circles, weak e¡ect; black circles, pronounced e¡ect of IC electrical stimulation. Electrical stimulation current varied between 0.4 WA in the most dorsal position and 0.1 WA in the most ventral position.
In some guinea pigs DPOAE suppression was tested using di¡erent values of the stimulation current. Fig. 3 demonstrates an example of such recordings. When the IC was stimulated with electrical pulses of 60 WA or less, no DPOAE suppression was observed (not presented in Fig. 3). Electrical stimulation with 70 WA (Fig. 3b) produced a DPOAE suppression of 1.5 dB, which is comparable with the DPOAE suppression produced by acoustic stimulation of the contralateral ear (Fig. 3a). When the IC was stimulated with a current of electrical pulses of more than 75 WA (Fig. 3e), the course of DPOAE amplitude changes was di¡erent. Such stimulation produced a large and fast drop of DPOAE amplitude (approximately 5 dB) followed by slow exponential recovery. However, during stimulation with such an intensity, pinna and facial muscle twitches were already visible. In addition, the Spectral history software of ILO 96, used for DPOAE acquisition, continuously monitors the sound pressure level in the ear
canal of both primary tones. The sound pressure level of both primaries was usually stable during the whole recording session. In the case of a large DPOAE amplitude decrease during excessive IC electrical stimulation, a small drop in the sound pressure level (by several tenths of dB) was simultaneously detected in either the f1 or f2 intensity level. A short-lived decrease of 0.2 dB in f1 intensity during IC electrical stimulation with a 75 WA pulse train is demonstrated in Fig. 3c, whereas the f2 intensity remains stable (Fig. 3d). The amount of maximal DPOAE suppression in response to IC electrical stimulation roughly corresponded to the DPOAE suppression produced by contralateral acoustic stimulation with BBN. This is documented in an example of acoustically evoked suppression (Fig. 4a) and suppression of DPOAE elicited in the same animal by electrical stimulation of the IC (Fig. 4b). Panels c and d in Fig. 4 summarize the averaged results obtained from 28 DPOAE recordings dur-
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Fig. 5. A schematic representation of IC electrode penetrations in all guinea pigs, illustrating the e⁄ciency of electrical stimulation in individual positions in which the e¡ect of electrical stimulation on DPOAE was tested. Fig. 3. Examples of DPOAE suppression produced in guinea pig MIC 3 by contralateral acoustic stimulation (a) and by IC electrical stimulation with currents of 70 WA (b) and 75 WA (e). Panels c and d demonstrate f1 and f2 intensity levels measured in the outer ear canal during IC electrical stimulation with a current of 75 WA.
ing BBN stimulation and 35 recordings during IC electrical stimulation (in IC positions where electrical stimulation produced pronounced DPOAE suppression). The average values of DPOAE suppression produced by both types of stimulation are almost identical. In individual animals, only a few IC positions were e¡ective in producing DPOAE suppression, whereas many electrode penetrations were ine¡ective. A schema summarizing the e¡ectiveness of all electrode positions in four representative sections of the IC (intersection distance 400^500 Wm) is shown in Fig. 5. The places where IC stimulation produced a suppressive e¡ect on DPOAE were situated at the rostral pole of the IC (rostral ECIC), in the deep layers of the ventral part of the IC (ventral and lateral ECIC) and the ventral part of the central nucleus of the IC. 3.3. Functional ablation of the olivocochlear e¡erent system by gentamicin
Fig. 4. Examples of DPOAE suppression produced by acoustic stimulation of the contralateral ear by BBN (intensity 61 dB SPL) (a) and by electrical stimulation of the IC in the electrode position with maximal e¡ect on DPOAE amplitude (b). (c) Average values of DPOAE amplitudes obtained from 28 DPOAE recordings during BBN stimulation and 35 recordings during successful IC electrical stimulations in individual animals (d). Bars represent Q S.E.M.
In order to investigate in more detail the mechanisms of DPOAE suppression produced by contralateral acoustic stimulation and electrical stimulation of the IC, gentamicin was injected in two guinea pigs to temporarily inactivate the e¡erent system. When IC-stimulating electrodes were placed in a position where electrical stimulation produced a pronounced DPOAE suppression, the animals were injected intramuscularly with gentamicin at a dose of 210 or 250 mg/kg. Then, the DPOAE suppression in response to acoustic and electrical stimulation was measured for several hours in 15 min periods after gentamicin injection. The data obtained in both experiments are demonstrated in Fig. 6. In guinea pig MIC 206 (Fig. 6a), the extent of
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Fig. 6. Elimination of DPOAE amplitude suppression after intramuscular gentamicin injection tested in guinea pigs MIC 206 (a) and MIC 3 (b).
DPOAE suppression produced by contralateral acoustic stimulation and IC electrical stimulation before gentamicin injection was 1 dB. One hour after gentamicin injection, DPOAE suppression, produced by both kinds of suppressors, decreased to approximately 0.3 dB. The experiment was stopped 1 h after gentamicin injection (before reaching complete elimination of e¡erent system function) for technical reasons. In the second experiment (guinea pig MIC 3, Fig. 6b), complete elimination of DPOAE suppression was reached 2 h after gentamicin injection (250 mg/kg). However, the ILO measuring probe in the outer ear canal moved slightly during the recording session (after injecting a supplementary dose of anesthesia), and the DPOAE value after re¢xing the probe was higher than the original one. Despite technical problems, both experiments demonstrated that gentamicin injection did not in£uence the basic DPOAE level (guinea pig MIC 206) and that 2 h after gentamicin injection the function of the olivocochlear bundle is fully eliminated (guinea pig MIC 3). These experiments demonstrated that DPOAE suppression produced by
acoustic and electrical stimulation has a similar mechanism of origin.
4. Discussion Results of the present paper demonstrate that electrical stimulation of some electrode positions in the IC suppresses the amplitude of DPOAE recorded in the ipsilateral ear. Even though this suppression amounted to less than 2 dB, this value was consistent during repeated stimulation and was comparable with DPOAE suppression produced by acoustic stimulation of the contralateral ear. 4.1. Value of DPOAE suppression The values of DPOAE suppression found in this study were relatively small, but they were comparable with data obtained by other authors using contralateral acoustic stimulation or electrical stimulation of the
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round window or electrical stimulation of the e¡erent nerve ¢bers. A suppression of 1^5 dB (which is equivalent to a 20^40% response reduction) was reported for the CAP (Rajan and Johnstone, 1983; Liberman, 1989; Smith et al., 1994; Popelar et al., 2001) or otoacoustic emissions (Mountain, 1980; Siegel and Kim, 1982; Puel and Rebillard, 1990; Popelar et al., 1999) in cats or guinea pigs. Mulders and Robertson (2000) studied the e¡ect of IC electric stimulation on cochlear output in paralyzed guinea pigs and reported a decrease in CAP amplitude equivalent to a 3^6 dB change in acoustic input in the frequency range 6^10 kHz, whereas CAP amplitudes evoked by stimulus frequencies above 14 kHz were often increased during IC stimulation. A frequency of 8 kHz for evoking the CAP has been shown previously to be an optimal stimulus frequency for demonstrating maximal CAP suppression by contralateral acoustic stimulation (Liberman, 1989; Smith et al., 1994), whereas CAP amplitudes evoked by 16 kHz tone pips were even slightly increased during contralateral acoustic stimulation (Popelar et al., 2001). In the present experiments the DPOAE were evoked by conventional primary tones used by ILO 96 equipment, i.e. f2 = 1^6.3 kHz, and DPOAE amplitudes were suppressed by contralateral acoustic stimulation in the frequency range 1^4 kHz. This is the reason why DPOAE for testing the e¡ect of IC electrical stimulation were evoked by f1 and f2 primary tones in the frequency range 1^4 kHz. It should be emphasized that the present experiments were performed in ketamine^xylazine anesthetized guinea pigs. Some authors have reported that the suppressive e¡ect of contralateral acoustic stimulation is smaller under anesthesia (Lima da Costa et al., 1997; Guitton and Avan, 2001). Previously, we have found comparable values of suppression of click-evoked transient otoacoustic emissions produced by contralateral acoustic stimulation (1.04 Q 0.48 dB) or electrical stimulation at the round window (0.94 Q 0.53 dB) in awake or slightly sedated guinea pigs (Popelar et al., 1999). Relatively small values of DPOAE suppression resulted from the use of a low level of contralateral BBN stimulation (51^71 dB SPL) to prevent acoustic crosstalk.
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Scates et al. (1999) demonstrated that electrical stimulation of the IC in anesthetized rats produces a large (7^25 dB) initial DPOAE suppression followed by a sustained smaller (3^15) suppression. Mulders and Robertson (2000) repeated such experiments and con¢rmed the results. However, in paralyzed rats, the suppression of DPOAE amplitudes for individual f2 frequencies fell to 1^2 dB. Careful observation of nonparalyzed rats during IC electrical stimulation revealed muscle activity (limb movement, slight tympanic membrane movement). When the stimulating current was dropped below the muscle twitch threshold, the DPOAE suppressive e¡ect disappeared. To avoid the confounding e¡ect of an activation of middle ear muscles, Mulders and Robertson (2000) studied the effect of electrical stimulation of the IC on cochlear responses in paralyzed guinea pigs. They found a 3^6 dB change in CAP amplitude evoked by acoustic stimuli in the frequency range 6^10 kHz. In our experiments, DPOAE amplitude suppression to electrical stimulation of the IC was measured in anesthetized guinea pigs. However, the eye and head musculature were controlled microscopically during IC stimulation, and the stimulating current was set at least 10% below the threshold for pinna and muscle twitches. In such experiments, the degree of DPOAE suppression was similar to that observed with acoustic stimulation of the contralateral ear. In cases of DPOAE recording during small eye or muscle twitches (an example of which is shown in Fig. 3), the magnitude and time pattern of DPOAE suppression were signi¢cantly di¡erent. Muscle activity was usually accompanied by a small shift of several tenths of dB in either f1 or f2 , which were monitored during DPOAE recording by Spectral history of ILO 96 software. During IC stimulation that did not evoke any eye or muscle twitches, the f1 and f2 stimulus pressure levels were stable. We believe that changes in sound pressure levels in the outer ear canal re£ect changes in middle ear impedance, and thus the stable stimulus pressure level indicates no change of middle ear muscle contraction. 4.3. IC positions e¡ective in activating the e¡erent system
4.2. The e¡ect of middle ear muscles The possible in£uence of the middle ear muscle re£ex on the magnitude of suppression of otoacoustic emissions is frequently discussed in many studies. It has been shown previously that electrical stimulation of the tectum in cats elicits muscle contractions (eye, head and pinna movement, facial muscle twitches, limb £exion) (Syka and Straschill, 1970; Syka and Radil-Weiss, 1971). These motor pathways are thought to be involved in acoustico-motor orienting responses.
The electrode positions throughout the whole IC were systematically tested with the aim of ¢nding those electrode positions the electrical stimulation of which can evoke the activation of the e¡erent system and thus DPOAE amplitude suppression. In accordance with other published data (Scates et al., 1999; Mulders and Robertson, 2000), the suppressive e¡ect was not seen in dorsal cortex of the IC, but was evident after a minimum penetration of 2 mm into the IC (except the very rostral part of the IC). This ¢nding corresponds to
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the fact that the dorsal cortex of the IC is a target of descending corticotectal ¢bers (Druga et al., 1997; Winer et al., 1998), but neurons of this part of the IC do not send descending ¢bers to the superior olivary complex or cochlear nucleus. However, it was not possible to elicit a suppression of DPOAE amplitudes from many electrode positions in the IC. In some animals, it was possible to evoke DPOAE suppression from only one electrode penetration out of several penetrations tested. Electrode positions with maximal suppressive e¡ects (i.e. rostro-medial, the ventral and lateral external cortices of the IC and the ventral part of the central nucleus of the IC) corresponded to anatomically veri¢ed origins of descending pathways from the IC (Syka et al., 1988a; Scho¢eld and Cant, 1999). The relatively low number of e⁄cient electrode positions within the IC that evoked DPOAE suppression could be explained in two ways: (i) electrical stimulation of only a few places in the IC could activate the IC descending ¢bers, or (ii) the threshold of the appearance of the ¢rst eye and muscle twitches was lower than the threshold for evoking DPOAE suppression. Another critical factor could also be injury of the IC by penetrating stimulation electrodes. In previous experiments we recorded DPOAE in awake guinea pigs and electrically stimulated the IC from implanted electrodes (unpublished data). Even though we stimulated similar IC regions as in the present acute experiments, or implanted multiple electrodes to stimulate more IC regions or a larger IC area, we never observed any e¡ect on CAP or otoacoustic emission amplitudes. We suppose that the descending colliculo-olivary system is very sensitive to any damaging manipulations and that the implantation of electrodes into the IC can seriously a¡ect its function. These unsuccessful results in experiments with implanted IC electrodes did not allow us to proceed in developing an animal model of the protective e¡ect of chronic electrical stimulation of the IC. Several recent papers, however, demonstrated a modulatory role of the auditory cortex on cochlear activity realized through either the descending corticotectal or direct cortico-olivary ¢bers (Xiao and Suga, 2002; Khalfa et al., 2001). Further research will be necessary to distinguish the role of individual cortical and subcortical structures in the e¡erent control of auditory signal processing in the cochlea. 4.4. The e¡ect of gentamicin injection In order to test the hypothesis that the mechanism of DPOAE suppression produced by contralateral acoustic stimulation and electrical stimulation of the IC is similar, two guinea pigs were treated with a single intramuscular injection of 210^250 mg/kg of gentamicin, and DPOAE suppression by both types of stimulation was
tested at postinjection periods. In agreement with other studies (Smith et al., 1994; Popelar et al., 1996; Avan et al., 1996; Lima da Costa et al., 1997; Yoshida et al., 1999), suppression of DPOAE was partly or fully eliminated 1^2 h after gentamicin injection. The above-mentioned authors also demonstrated that a single gentamicin injection does not signi¢cantly a¡ect the CAP audiogram, CAP input/output function or DPOAE amplitudes. Elimination of e¡erent olivocochlear system function by gentamicin is thought to be produced by the inactivation of acetylcholine receptors on outer hair cells (by blocking the Ca2þ entry necessary to elicit the acetylcholine response ^ Dulon et al., 1989; Nakagawa et al., 1992). Since gentamicin injection eliminated DPOAE suppression produced by both kinds of stimuli, we conclude that DPOAE suppression produced by acoustic and electrical stimulation is caused by the activation of the e¡erent olivocochlear system. 4.5. Conclusions The results of the present paper demonstrate that ¢bers descending from the IC are functionally connected to e¡erent ¢bers of the olivocochlear bundle and that electrical stimulation of the IC can suppress the amplitude of DPOAE recorded in the ipsilateral ear. We hypothesize that the descending system from the IC may control and modulate the micromechanics of the cochlea through the olivocochlear e¡erent bundle and thus contribute to the adjustment and improvement of some parameters of auditory information processing in the cochlea, such as selective attention, frequency selectivity, intensity coding, sound localization, speech discrimination or detection of tones in noise. However, the existence of low thresholds of middle ear muscle contractions to IC electrical stimulation preclude the use of electrical stimulation of the IC for chronic activation of the e¡erent auditory system.
Acknowledgements The authors wish to thank Rastislav Druga for his assistance in the evaluation of histological data. The study was supported by Grants AV OZ 5039906, the Grant Agency of the Czech Ministry of Health NK/ 6454-3 and the Grant Agency of the Czech Republic 309/01/1063. References Andersen, R.A., Snyder, R.L., Merzenich, M.M., 1980. The topographic organization of corticocollicular projections from physiologically identi¢ed loci in the A I, A II and anterior auditory cortical ¢elds of the cat. J. Comp. Neurol. 191, 479^494.
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J. Popelar et al. / Hearing Research 170 (2002) 116^126 Avan, P., Erre, J.P., Lima da Costa, D., Aran, J.M., Popelar, J., 1996. The e¡erent-mediated suppression of of otoacoustic emissions in awake guinea pigs and its reversible blockade by gentamicin. Exp. Brain Res. 109, 9^16. Berlin, C.I., Hood, L.J., Wen, H., Szabo, P., Cecola, R.P., Rigby, P., Jackson, D.F., 1993. Contralateral suppression of non-linear clickevoked otoacoustic emissions. Hear. Res. 71, 1^11. Coleman, J.R., Clerici, W.J., 1987. Sources of projections to subdivisions of the inferior colliculus in the rat. J. Comp. Neurol. 262, 215^226. Diamond, I.T., Jones, E.G., Powel, T.P.S., 1969. The projection of the auditory cortex upon the diencephalon and brain stem in the cat. Brain Res. 15, 305^340. Druga, R., Syka, J., 1984. Ascending and descending projections to the inferior colliculus in the rat. Physiol. Bohemoslov. 33, 31^42. Druga, R., Syka, J., Rajkowska, G., 1997. Prijections of auditory cortex onto the inferior colliculus in the rat. Physiol. Res. 46, 215^222. Dulon, D., Zajic, G., Aran, J.M., Schacht, J., 1989. Aminoglycoside antibiotics impair calcium entry but not viability and motility in isolated cochlear outer hair cells. J. Neurosci. Res. 24, 338^346. Faye-Lund, H., 1985. The neocortical projection to the inferior colliculus in the albino rat. Anat. Embryol. 173, 53^70. Fex, J., 1959. Augmentation of cochlear microphonic by stimulation of e¡erent ¢bers to the cochlea. Acta Otolaryngol. 50, 540^541. Gi¡ord, M.L., Guinan, J.J.Jr., 1987. E¡ects of electrical stimulation of medial olivocochlear neurons on ipsilateral and contralateral cochlear responses. Hear. Res. 29, 179^194. Giraud, A.L., Garnier, S., Micheyl, C., Lina, G., Chery-Croze, S., 1997. Auditory e¡erents involved in speech-in-noise intelligibility. NeuroReport 8, 1779^1783. Guitton, M., Avan, P., 2001. Medial olivocochlear e¡erents in awake guinea pigs (Abstract). 24th Midwinter Res. Meeting, Assoc. Res. Otolaryngol., No. 24. Hood, L.J., Berlin, C.I., Hurley, A., Cecola, R.P., Bell, B., 1996. Contralateral suppression of transient-evoked otoacoustic emissions in humans: Intensity e¡ects. Hear. Res. 37, 29^46. Khalfa, S., Bougeard, R., Morand, N., Veuillet, E., Isnard, J., Guenot, M., Ryvlin, P., Fischer, C., Collet, L., 2001. Evidence of peripheral auditory activity modulation by the auditory cortex in humans. Neuroscience 104, 347^358. Liberman, M.C., 1989. Rapid assessment of sound-evoked olivocochlear feedback: suppression of compound action potentials by contralateral sound. Hear. Res. 38, 47^56. Lima da Costa, D., Erre, J.P., Charlet de Sauvage, R., Popelar, J., Aran, J.M., 1997. Bioelectrical cochlear noise and its contralateral suppression: relation to background activity of the eighth nerve and e¡ects of sedation and anesthesia. Exp. Brain Res. 116, 259^ 269. Luparello, T.J., 1967. Stereotaxic Atlas of the Forebrain of the Guinea Pig. Karger, Basel. Micheyl, C., Collet, L., 1996. Involvement of the olivocochlear bundle in the detection of tones in noise. J. Acoust. Soc. Am. 99, 1604^ 1610. Mountain, D.C., 1980. Changes in endolymphatic potential and crossed olivocochlear bundle stimulation alter cochlear mechanics. Science 210, 71^72. Mulders, W.H., Robertson, D., 2000. E¡ects on cochlear responses of activation of descending pathways from the inferior colliculus. Hear. Res. 149, 11^23. Nakagawa, T., Kakehata, S., Akaike, N., Komune, S., Takasaka, T., Uemura, T., 1992. E¡ects of Ca2þ antagonists and aminoglykoside antibiotics on Ca2þ current isolated outer hair cells of guinea pig cochlea. Brain Res. 580, 345^347.
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Popelar, J., Erre, J.P., Aran, J.M., Cazals, Y., 1994. Plastic changes in ipsi-contralateral di¡erences of auditory cortex and inferior colliculus evoked potentials after injury to one ear in the adult guinea pig. Hear. Res. 72, 125^134. Popelar, J., Lima da Costa, D., Erre, J.P., Avan, P., Aran, J.M., 1996. Contralateral suppression of the ensemble background activity of the auditory nerve in awake guinea pigs: E¡ects of gentamicin. Audit. Neurosci. 3, 425^433. Popelar, J., Valvoda, J., Syka, J., 1999. Acoustically and electrically evoked contralateral suppression of otoacoustic emissions in guinea pigs. Hear. Res. 135, 61^70. Popelar, J., Erre, J.P., Syka, J., Aran, J.M., 2001. E¡ects of contralateral acoustical stimulation on three measures of cochlear function in the guinea pig. Hear. Res. 152, 128^138. Puel, J.L., Rebillard, G., 1990. E¡ect of contralateral sound stimulation on the distortion product 2F1-F2: evidence that the medial e¡erent system is involved. J. Acoust. Soc. Am. 87, 1630^1635. Puel, J.L., Bon¢ls, P., Pujol, R., 1988. Selective attention modi¢es the active micromechanical properties of the cochlea. Brain Res. 447, 380^383. Rajan, R., 1990. Electrical stimulation of the inferior colliculus at low rates protects the cochlea from auditory desensitization. Brain Res. 506, 192^204. Rajan, R., Johnstone, B.M., 1983. E¡erent e¡ects elicited by electrical stimulation at the round window of the guinea pig. Hear. Res. 12, 405^417. Scates, K.W., Woods, C.I., Azeredo, W.J., 1999. Inferior colliculus stimulation and changes in 2f1 3f2 distortion product otoacoustic emissions in the rat. Hear. Res. 128, 51^60. Scharf, B., Quigley, S., Aoki, C., Peachey, N., Reever, A., 1987. Focused auditory attention and frequency selectivity. Percept. Psychophys. 42, 215^223. Scherer-Singler, U., Vincent, S.R., Kimura, H., McGeer, E.G., 1983. Demonstration of a unique population of neurons with NADPHdiaphorase histochemistry. J. Neurosci. Methods 9, 229^234. Scho¢eld, B.R., Cant, N.B., 1999. Descending auditory pathways: projections from the inferior colliculus contact superior olivary cells that project bilaterally to the cochlear nuclei. J. Comp. Neurol. 409, 210^223. Siegel, J.H., Kim, D.O., 1982. E¡erent neural control of cochlear mechanics? Olivocochlear bundle stimulation a¡ects cochlear biomechanical nonlinearity. Hear. Res. 6, 171^182. Smith, D.W., Erre, J.P., Aran, J.M., 1994. Rapid, reversible elimination of medial olivocochlear e¡erent function following single injections of gentamicin in the guinea pig. Brain Res. 652, 243^ 248. Spangler, K.M., Warr, W.B., 1991. The descending auditory system. In: Altschuler, R.A., Bobbin, R.P., Clopton, B.M., Ho¡man, D.W. (Eds.), Neurobiology of Hearing: The Central Auditory System. Raven Press, New York, pp. 27^45. Syka, J., Radil-Weiss, T., 1971. Activation of superior colliculus neurons and motor responses after electrical stimulation of the inferior colliculus. Exp. Neurol. 28, 384^392. Syka, J., Straschill, M., 1970. Electrical stimulation of the tectum in freely moving cats. Brain Res. 28, 567^572. Syka, J., Robertson, D., Johnstone, B.M., 1988a. E¡erent descending projections from the inferior colliculus in guinea pig. In: Syka, J., Masterton, R.B. (Eds.), Auditory Pathway. Plenum Press, New York, pp. 299^303. Syka, J., Popelar, J., Druga R., Vlkova, A., 1988b. Descending central auditory pathway ^ structure and function. In: Syka, J., Masterton, R.B. (Eds.), Auditory Pathway. Plenum Press, New York, pp. 279^292. Veuillet, E., Collet, L., Duclaux, R., 1991. E¡ect of contralateral acoustic stimulation on active cochlear micromechanical proper-
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ties in human subjects: dependence on stimulus variables. J. Neurophysiol. 65, 724^735. White, J.S., Warr, W.B., 1983. The dual origins of the olivocochlear bundle in the albino rat. J. Comp. Neurol. 219, 203^214. Winer, J.A., Larue, D.T., Diehl, J.J., Hefti, B.J., 1998. Auditory cortical projections to the cat inferior colliculus. J. Comp. Neurol. 400, 147^174. Wong-Riley, M., 1979. Changes in the visual system of monocularly
sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. 171, 11^28. Xiao, Z., Suga, N., 2002. Modulation of cochlear hair cells by the auditory cortex in the mustached bat. Nature Neurosci. 5, 57^63. Yoshida, N., Liberman, M.C., Brown, M.C., Sewell, W.F., 1999. Gentamicin blocks both fast and slow e¡ects of olivocochlear activation in anesthetized guinea pigs. J. Neurophysiol. 82, 3168^ 3174.
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E¡ects of electrical stimulation of the inferior colliculus on 2f1 3f2 distortion product otoacoustic emissions in anesthetized guinea pigs J. Popelar , J. Mazelova¤, J. Syka Institute of Experimental Medicine, Academy of Sciences, V|¤denska¤ 1083, 142 20 Prague 4, Czech Republic Received 20 November 2001; accepted 4 April 2002
Abstract The effects of electrical stimulation of the inferior colliculus (IC) on the activation of olivocochlear nerve fibers were investigated in guinea pigs in which the 2f1 3f2 distortion product otoacoustic emissions (DPOAE) were recorded. Animals were anesthetized with ketamine (33 mg/kg) and xylazine (6.6 mg/kg). Bipolar electrical stimulation of the IC by a train of pulses with currents less than the threshold for evoking muscle twitches resulted in a small depression of the DPOAE amplitude by 0.1^2 dB. The maximal effect was observed when the stimulating electrodes were located in the rostro-medial or ventral parts of the IC. The suppression of electrically evoked DPOAE was similar to the DPOAE suppression produced by acoustical stimulation of the contralateral ear by a broad-band noise. Suppression of DPOAE amplitude in response to both acoustical and electrical stimulation was abolished 1^2 h after a single intramuscular injection of gentamicin (210^250 mg/kg). The results indicate that electrical stimulation of the IC can activate the efferent system and produce DPOAE changes by similar mechanisms as does acoustical stimulation of the contralateral ear. 9 2002 Elsevier Science B.V. All rights reserved.
1. Introduction The inferior colliculus (IC) represents an important relay nucleus for the a¡erent auditory pathway, receiving inputs from the cochlear nucleus and the superior olivary complex and projecting to the auditory cortex through the medial geniculate body. The IC also represents an important part of the e¡erent, descending auditory system. The IC receives many axons of pyramidal cells of layer V of the auditory cortex, which terminate mainly in the dorsal and external cortices of the IC (Diamond et al., 1969; Andersen et al., 1980; Druga and Syka, 1984; Faye-Lund, 1985; Coleman and Clerici, 1987; Druga et al., 1997; Winer et al., 1998). Addi-
* Corresponding author. Tel.: +420 (2) 4106 2689; Fax: +420 (2) 4106 2787. E-mail address: [email protected] (J. Popelar). Abbreviations: BBN, broad-band noise; CAP, compound action potential of the auditory nerve; CyOx, cytochrome oxidase; DPOAE, 2f1 3f2 distortion product otoacoustic emissions; ECIC, external cortex of the inferior colliculus; IC, inferior colliculus; LOC, lateral olivocochlear system; MOC, medial olivocochlear system; NADPHd, nicotinamide adenine dinucleotide phosphate-diaphorase
tional parts of the e¡erent pathway are represented by ¢bers projecting from the IC to the nuclei of the lateral lemniscus, superior olivary complex (Syka et al., 1988a; Scho¢eld and Cant, 1999) and cochlear nuclei (for review see Syka et al., 1988b; Spangler and Warr, 1991). Some target regions within the superior olivary complex, mainly the rostral periolivary region and the ventral nucleus of the trapezoid body, contain olivocochlear neurons, which innervate sensory hair cells within the cochlea. Two principal olivocochlear pathways have been described that di¡er anatomically and physiologically ^ the medial (MOC) and lateral (LOC) olivocochlear systems (White and Warr, 1983). The LOC neurons closely surround the lateral superior olive (LSO) ; their axons are mostly unmyelinated and synapse on ipsilateral a¡erent neuron terminals at the inner hair cells. The MOC neurons tend to surround the medial, ventral and rostral parts of the medial superior olive; their ¢bers are mostly myelinated, and two-thirds of them project to outer hair cell bodies in the contralateral cochlea. Limited data are available on the function of the e¡erent auditory system. It is thought that the descending projections are involved in selective attention, fre-
0378-5955 / 02 / $ ^ see front matter 9 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 2 ) 0 0 3 9 7 - 0
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quency selectivity, intensity coding, sound localization, adjustment of dynamic range, speech discrimination and the detection of tones in noise (Scharf et al., 1987; Puel et al., 1988; Micheyl and Collet, 1996; Giraud et al., 1997). In addition to these functions, the e¡erent olivocochlear bundle is thought to protect the cochlea from overexposure to noise. However, the mechanisms of these processes are not completely known. Previous studies have demonstrated that activation of the olivocochlear e¡erent system (by contralateral acoustic stimulation or by direct electrical stimulation of the olivocochlear nerve ¢bers or contralateral round window) alters the amplitude of most cochlear potentials (Fex, 1959; Gi¡ord and Guinan, 1987; Liberman, 1989; Popelar et al., 1996, 1999) and otoacoustic emissions (Puel and Rebillard, 1990; Veuillet et al., 1991; Berlin et al., 1993; Hood et al., 1996). A few studies have reported on the e¡ects of IC stimulation on the compound action potential of the auditory nerve (CAP) in guinea pigs (Mulders and Robertson, 2000) or distortion product otoacoustic emissions (DPOAE) in rats (Scates et al., 1999). Rajan (1990) demonstrated a reduction of the temporary threshold shifts in guinea pigs in which the IC was electrically stimulated during noise overexposure. On the other hand, he failed to ¢nd any direct e¡ect of IC stimulation on the CAP or DPOAE. The aim of our work was to investigate the e¡ects of electrical stimulation of di¡erent parts of the IC on DPOAE recorded in anesthetized guinea pigs. DPOAE suppression elicited by IC electrical stimulation was compared with DPOAE suppression produced by acoustic stimulation of the contralateral ear. The mechanisms of DPOAE suppression elicited by electrical stimulation were studied by using a single intramuscular injection of gentamicin, which was shown previously to eliminate the e¡ect of contralateral acoustic stimulation (by blocking the Ca2þ entry necessary to elicit the acetylcholine response in outer hair cells). The results contribute to understanding the function of descending pathways in the central auditory system.
2. Materials and methods Experiments were performed on 20 pigmented guinea pigs weighing 250^300 g. Animals were anesthetized with an intramuscular injection of 33 mg/kg ketamine (Narkamon 5%, Spofa) and 6.6 mg/kg xylazine (Sedazine, Phoenix Scienti¢que). All recordings were conducted in a sound-attenuated and anechoic chamber. 2.1. Surgery The skin and underlying muscles covering the skull
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were retracted to expose the dorsal cranium between points bregma and lambda. A small hole was made by a trephine on the right side of the skull above the IC. The animal’s head was rigidly held in a stereotaxic apparatus by a special holder, ¢xed to the skull by two screws and secured by acrylic resin. This type of ¢xation enabled the animal’s head to be free for electrode penetration, acoustic stimulation and otoacoustic emission recording. A rectal temperature of 37^38‡C was automatically maintained by a DC-powered electric heating pad. 2.2. Electrical stimulation of the IC Two metal electrodes (insulated nichrome wire, diameter 0.002 inch) with a tip separation of 1 mm, glued on a rigid wire as a carrier, were used for electrical stimulation. Electrodes were inserted into the ipsilateral IC through the overlying cortex in a dorsoventral direction using an electrically driven micromanipulator. The IC was stimulated in a bipolar mode with a continuous train of monophasic or biphasic rectangular pulses (pulse duration 0.3 ms, pulse frequency 100^300 Hz, current up to 1.5 mA), generated by an optically coupled constant current source. During penetration of the IC, electrode movement was discontinued every 0.3^0.5 mm. At these positions of the electrode tip, the electrical current was continuously increased until the ¢rst detectable muscle twitches were observed (pinna or eye movement, small contractions of facial musculature, limb £exion). Then, the stimulation current was set approximately 10% lower, and the e¡ect of electrical stimulation on the DPOAE amplitude was tested. 2.3. Recording the IC evoked responses To con¢rm the correct electrode position within the IC, the IC evoked responses were recorded during electrode penetration. Bipolar clicks (100 Ws duration) were generated by SigGen TDT (Tucker-Davis Technologies) software and processed by a TDT D/A converter and programmable attenuator. Stimuli were delivered to the contralateral ear by a piezoelectric stimulator coupled to the outer ear canal via a 7 cm long silastic tube (outer diameter 5 mm, inner diameter 3 mm). The end of the silastic tube was ¢xed in the entrance to the outer ear canal by acrylic glue. The stimulating sound system was calibrated in an arti¢cial ear (adapted for the impedance of the guinea pig ear canal) with a Bruel and Kjaer (4134) 1/2 in microphone and in a group of anesthetized guinea pigs (Popelar et al., 1996). The signal from the electrodes (one stimulating electrode against a reference electrode in the neck muscles) was ampli¢ed by a DAM di¡erential ampli¢er
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(gain 60 dB, ¢lters 10 Hz^1 kHz), processed with a TDT A/D converter, and the responses were averaged using BioSig software. 2.4. DPOAE recordings DPOAE were recorded in guinea pigs using an ILO 96 analyzer (Otodynamics) and ILO H-probes. Cubic 2f1 3f2 DPOAE were recorded using two primary tones, f1 and f2 (ratio f2 /f1 = 1.22), presented with two phones, either with equilevel stimuli (50/50 or 60/60 dB SPL) or with an L2 level that was 5^10 dB lower than L1 (60/50, 55/45 or 55/50 dB SPL). At the beginning of the experiment, a DP-gram (the function of DPOAE level on increasing stimulus frequency, four points per octave) was recorded, followed by DP-gram measurement during simultaneous contralateral acoustic stimulation with broad-band noise (BBN). The recording series was ended by a second control DPOAE recording. According to the shape of the DP-gram, two frequencies (f1 and f2 ) were chosen for recording of the 2f1 3f2 DPOAE using the ILO software Spectral history. This software continuously monitors and displays every 1.4 s the 2f1 3f2 DPOAE amplitude (16 DPOAE subaverages) to stimuli of ¢xed frequency and intensity. Recording sessions in the course of single electrode penetration usually consisted of DPOAE recordings during IC electrical stimulation at di¡erent electrode positions alternated with 20 s periods of control DPOAE recordings. 2.5. DPOAE suppression produced by acoustic stimulation of the contralateral ear The suppression of DPOAE was tested by stimulating the contralateral ear with continuous BBN at 51^71 dB SPL, generated with a Hewlett Packard (33120A) waveform generator and ampli¢ed and shaped with an electronic switch. BBN was presented to the animal from a piezoelectric stimulator coupled to the outer ear canal via a 7 cm long silastic tube. It was con¢rmed in previous experiments that a BBN intensity of 51^71 dB SPL was 40^60 dB above the hearing threshold, which was low enough to prevent contralateral crosstalk (Popelar et al., 1994). 2.6. Functional ablation of the olivocochlear e¡erent system by gentamicin Previous studies demonstrated that a single gentamicin injection (150^250 mg/kg) reversibly and temporarily eliminates the e¡ect of contralateral acoustic stimulation (Smith et al., 1994; Popelar et al., 1996; Yoshida et al., 1999). In order to test the hypothesis that the mechanisms of DPOAE suppression produced
by contralateral acoustic stimulation and electrical stimulation of the IC were similar, a gentamicin injection was used in two guinea pigs. When IC electrodes were located in a position where a pronounced e¡ect of electrical stimulation on DPOAE was found, the animals were injected intramuscularly with gentamicin at a dose of 210 or 250 mg/kg, and the DPOAE suppression in response to acoustic and electrical stimulation was tested in 15 min periods. 2.7. Histological control At the end of the experiments, the stimulation electrodes were left in the deepest position of the last penetration within the IC and carefully cut just above the surface of the brain. The guinea pig was sacri¢ced with an overdose of 1^2 ml of pentobarbital (Spofa, 50 mg/ ml) and perfused intracardially with 10% formaldehyde. The brain with the cut electrode was removed from the skull and post¢xed in the same solution for several days. Before sectioning, the electrode was carefully removed from the brain. Subsequently, the brain was sectioned in the frontal plane on a freezing MICROM K400 microtome (slice thickness 40 Wm) and stained with cresyl violet. The whole extent of the electrode track was clearly discernible in several serial sections. The coordinates of the electrode track were determined according to an atlas of the guinea pig brain (Luparello, 1967), and the location of individual stimulation sites within the IC was assessed on the basis of the depth coordinates indicated on the electronically driven microdrive. The boundaries of the IC subnuclei were determined in previous experiments (unpublished data) on the basis of Nissl staining, the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and cytochrome oxidase (CyOx) positivity. For NADPH-d staining the method of Scherer-Singler et al. (1983) was used, and for CyOx histochemistry the Wong-Riley (1979) method was used. Whereas CyOx staining was most pronounced in the central nucleus of the IC, intensely stained NADPH-d-positive neurons and moderately positive neuropil were localized only in the dorsal cortex of the IC and in the external cortex of the IC (ECIC). On the basis of the above-described histochemical staining, the boundaries of the IC subnuclei were determined in eight serial IC frontal sections (the distance between individual sections was 200^250 Wm), and the schema was used for the localization of electrode penetrations in the IC. The care and use of animals reported on in this study were approved by the Ethics Committee of the Institute of Experimental Medicine of the Academy of Sciences of the Czech Republic and followed the guidelines of the Declaration of Helsinki.
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Fig. 1. An example of 2f1 3f2 DP-grams elicited with f1 and f2 primaries of intensities L1 = 55 dB SPL and L2 = 50 dB SPL, recorded in one guinea pig (MIC 12) under control conditions (solid line) and during contralateral acoustic stimulation with BBN (intensity 71 dB SPL, dashed line).
3. Results 3.1. DPOAE recording Experiments were performed in 20 healthy guinea pigs weighing 250^350 g. DPOAE were regularly recorded in all tested animals. Fig. 1 shows an example of 2f1 3f2 DPOAE (DP-gram) elicited by two primaries, f1 and f2 , with stimulus intensities L1 = 55 dB SPL and L2 = 50 dB SPL recorded in one guinea pig (MIC 12). The DPOAE curve exceeds the noise £oor by 6^32 dB in the frequency range from 1 to 6.3 kHz. The interrupted line in Fig. 1 represents the DP-gram recorded during acoustic stimulation of the contralateral ear. The DPOAE amplitudes are suppressed by 0.5^2.2 dB in the frequency range 1^5.6 kHz, and the DP-gram is shifted to lower values. On the basis of the shape of DP-grams observed in individual animals, a pair of primaries with ¢xed parameters were chosen for continuous DPOAE recording using the Spectral history software. Since DPOAE suppression by contralateral sound was elicited almost throughout the whole frequency range, the DPOAE suppression produced by electrical stimulation of the IC was tested in a broad range of frequencies. 3.2. E¡ect of IC electrical stimulation The e¡ect of contralateral acoustic and IC electrical
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stimulation was tested using continuous recording of DPOAE to ¢xed stimulus frequency and intensity (Spectral history ILO 96 software). The e¡ect of electrical stimulation of the IC on the DPOAE amplitude was investigated at di¡erent positions of the stimulating electrode in the IC. At each position the electrical current was increased from zero to the level at which the ¢rst facial muscle or pinna twitches appeared. Then the stimulation current was decreased by approximately 10%, and DPOAE were recorded for at least 20^30 s. Periods of DPOAE recordings with IC electrical stimulation were alternated with control recordings without stimulation. At individual IC electrode positions, evoked responses to click stimulation were recorded as well. As a rule, it was possible to stimulate the dorsal parts of the IC (up to a depth of 2 mm) using an electrical current reaching 1^1.5 mA without any sign of muscle twitches or DPOAE suppression. The deeper the electrodes were inserted into the IC, the lower the observed electric current threshold for evoking muscle twitches. In the ventral layers of the IC the muscle reactions occurred even with an electrical current lower than 100 WA. In some electrode positions electrical stimulation produced the suppression of DPOAE. Fig. 2 shows an example of a single electrode penetration through the IC and the resulting e¡ect of electrical stimulation on the DPOAE in animal MIC 202. Open circles in the histological section (Fig. 2a) represent positions with no e¡ect on DPOAE, whereas in positions marked by black circles the suppressive e¡ect was maximal and in positions marked by gray circles the suppression of DPOAE reached approximately half of the maximal value. DPOAE amplitude changes over time (f1 /f2 = 2002/2417 kHz, L1 /L2 = 60/60 dB SPL) for individual IC locations are demonstrated in Fig. 2b. The duration of electrical stimulation (20 s) is shown in the bottom part of the ¢gure. In each panel, the ¢rst three and the last three data points represent control DPOAE values preceding and following the electrical stimulation. This ¢gure documents that pronounced DPOAE suppression in response to IC electrical stimulation was seen only in two electrode positions which were 0.3 mm apart. Switching the electrical stimulation on produced a fast decrease of 1.4^1.6 dB in DPOAE amplitude, and this level was maintained with small variations of Q 0.2^0.4 dB during the whole period of electrical stimulation. Switching the electrical stimulation o¡ caused a fast recovery of DPOAE amplitude to prestimulation levels. In neighboring positions, a weak e¡ect was obtained at depths of both 2.4 and 3.2 mm. Repeated stimulation in individual electrode positions usually resulted in reproducible e¡ects. In this animal, the diameter of the area of the IC where the DPOAE suppression was observed was 0.8 mm.
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Fig. 2. (a) A Nissl-stained IC section with schematic reconstruction of an electrode penetration into the IC and positions of the electrode tip in the IC during electrical stimulation. (b) Recordings of the DPOAE amplitude (f1 /f2 = 2002/2417 Hz, 60/60 dB SPL) during electrical stimulation at individual IC positions (marked by arrows). Empty circles, no e¡ect; gray circles, weak e¡ect; black circles, pronounced e¡ect of IC electrical stimulation. Electrical stimulation current varied between 0.4 WA in the most dorsal position and 0.1 WA in the most ventral position.
In some guinea pigs DPOAE suppression was tested using di¡erent values of the stimulation current. Fig. 3 demonstrates an example of such recordings. When the IC was stimulated with electrical pulses of 60 WA or less, no DPOAE suppression was observed (not presented in Fig. 3). Electrical stimulation with 70 WA (Fig. 3b) produced a DPOAE suppression of 1.5 dB, which is comparable with the DPOAE suppression produced by acoustic stimulation of the contralateral ear (Fig. 3a). When the IC was stimulated with a current of electrical pulses of more than 75 WA (Fig. 3e), the course of DPOAE amplitude changes was di¡erent. Such stimulation produced a large and fast drop of DPOAE amplitude (approximately 5 dB) followed by slow exponential recovery. However, during stimulation with such an intensity, pinna and facial muscle twitches were already visible. In addition, the Spectral history software of ILO 96, used for DPOAE acquisition, continuously monitors the sound pressure level in the ear
canal of both primary tones. The sound pressure level of both primaries was usually stable during the whole recording session. In the case of a large DPOAE amplitude decrease during excessive IC electrical stimulation, a small drop in the sound pressure level (by several tenths of dB) was simultaneously detected in either the f1 or f2 intensity level. A short-lived decrease of 0.2 dB in f1 intensity during IC electrical stimulation with a 75 WA pulse train is demonstrated in Fig. 3c, whereas the f2 intensity remains stable (Fig. 3d). The amount of maximal DPOAE suppression in response to IC electrical stimulation roughly corresponded to the DPOAE suppression produced by contralateral acoustic stimulation with BBN. This is documented in an example of acoustically evoked suppression (Fig. 4a) and suppression of DPOAE elicited in the same animal by electrical stimulation of the IC (Fig. 4b). Panels c and d in Fig. 4 summarize the averaged results obtained from 28 DPOAE recordings dur-
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Fig. 5. A schematic representation of IC electrode penetrations in all guinea pigs, illustrating the e⁄ciency of electrical stimulation in individual positions in which the e¡ect of electrical stimulation on DPOAE was tested. Fig. 3. Examples of DPOAE suppression produced in guinea pig MIC 3 by contralateral acoustic stimulation (a) and by IC electrical stimulation with currents of 70 WA (b) and 75 WA (e). Panels c and d demonstrate f1 and f2 intensity levels measured in the outer ear canal during IC electrical stimulation with a current of 75 WA.
ing BBN stimulation and 35 recordings during IC electrical stimulation (in IC positions where electrical stimulation produced pronounced DPOAE suppression). The average values of DPOAE suppression produced by both types of stimulation are almost identical. In individual animals, only a few IC positions were e¡ective in producing DPOAE suppression, whereas many electrode penetrations were ine¡ective. A schema summarizing the e¡ectiveness of all electrode positions in four representative sections of the IC (intersection distance 400^500 Wm) is shown in Fig. 5. The places where IC stimulation produced a suppressive e¡ect on DPOAE were situated at the rostral pole of the IC (rostral ECIC), in the deep layers of the ventral part of the IC (ventral and lateral ECIC) and the ventral part of the central nucleus of the IC. 3.3. Functional ablation of the olivocochlear e¡erent system by gentamicin
Fig. 4. Examples of DPOAE suppression produced by acoustic stimulation of the contralateral ear by BBN (intensity 61 dB SPL) (a) and by electrical stimulation of the IC in the electrode position with maximal e¡ect on DPOAE amplitude (b). (c) Average values of DPOAE amplitudes obtained from 28 DPOAE recordings during BBN stimulation and 35 recordings during successful IC electrical stimulations in individual animals (d). Bars represent Q S.E.M.
In order to investigate in more detail the mechanisms of DPOAE suppression produced by contralateral acoustic stimulation and electrical stimulation of the IC, gentamicin was injected in two guinea pigs to temporarily inactivate the e¡erent system. When IC-stimulating electrodes were placed in a position where electrical stimulation produced a pronounced DPOAE suppression, the animals were injected intramuscularly with gentamicin at a dose of 210 or 250 mg/kg. Then, the DPOAE suppression in response to acoustic and electrical stimulation was measured for several hours in 15 min periods after gentamicin injection. The data obtained in both experiments are demonstrated in Fig. 6. In guinea pig MIC 206 (Fig. 6a), the extent of
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Fig. 6. Elimination of DPOAE amplitude suppression after intramuscular gentamicin injection tested in guinea pigs MIC 206 (a) and MIC 3 (b).
DPOAE suppression produced by contralateral acoustic stimulation and IC electrical stimulation before gentamicin injection was 1 dB. One hour after gentamicin injection, DPOAE suppression, produced by both kinds of suppressors, decreased to approximately 0.3 dB. The experiment was stopped 1 h after gentamicin injection (before reaching complete elimination of e¡erent system function) for technical reasons. In the second experiment (guinea pig MIC 3, Fig. 6b), complete elimination of DPOAE suppression was reached 2 h after gentamicin injection (250 mg/kg). However, the ILO measuring probe in the outer ear canal moved slightly during the recording session (after injecting a supplementary dose of anesthesia), and the DPOAE value after re¢xing the probe was higher than the original one. Despite technical problems, both experiments demonstrated that gentamicin injection did not in£uence the basic DPOAE level (guinea pig MIC 206) and that 2 h after gentamicin injection the function of the olivocochlear bundle is fully eliminated (guinea pig MIC 3). These experiments demonstrated that DPOAE suppression produced by
acoustic and electrical stimulation has a similar mechanism of origin.
4. Discussion Results of the present paper demonstrate that electrical stimulation of some electrode positions in the IC suppresses the amplitude of DPOAE recorded in the ipsilateral ear. Even though this suppression amounted to less than 2 dB, this value was consistent during repeated stimulation and was comparable with DPOAE suppression produced by acoustic stimulation of the contralateral ear. 4.1. Value of DPOAE suppression The values of DPOAE suppression found in this study were relatively small, but they were comparable with data obtained by other authors using contralateral acoustic stimulation or electrical stimulation of the
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round window or electrical stimulation of the e¡erent nerve ¢bers. A suppression of 1^5 dB (which is equivalent to a 20^40% response reduction) was reported for the CAP (Rajan and Johnstone, 1983; Liberman, 1989; Smith et al., 1994; Popelar et al., 2001) or otoacoustic emissions (Mountain, 1980; Siegel and Kim, 1982; Puel and Rebillard, 1990; Popelar et al., 1999) in cats or guinea pigs. Mulders and Robertson (2000) studied the e¡ect of IC electric stimulation on cochlear output in paralyzed guinea pigs and reported a decrease in CAP amplitude equivalent to a 3^6 dB change in acoustic input in the frequency range 6^10 kHz, whereas CAP amplitudes evoked by stimulus frequencies above 14 kHz were often increased during IC stimulation. A frequency of 8 kHz for evoking the CAP has been shown previously to be an optimal stimulus frequency for demonstrating maximal CAP suppression by contralateral acoustic stimulation (Liberman, 1989; Smith et al., 1994), whereas CAP amplitudes evoked by 16 kHz tone pips were even slightly increased during contralateral acoustic stimulation (Popelar et al., 2001). In the present experiments the DPOAE were evoked by conventional primary tones used by ILO 96 equipment, i.e. f2 = 1^6.3 kHz, and DPOAE amplitudes were suppressed by contralateral acoustic stimulation in the frequency range 1^4 kHz. This is the reason why DPOAE for testing the e¡ect of IC electrical stimulation were evoked by f1 and f2 primary tones in the frequency range 1^4 kHz. It should be emphasized that the present experiments were performed in ketamine^xylazine anesthetized guinea pigs. Some authors have reported that the suppressive e¡ect of contralateral acoustic stimulation is smaller under anesthesia (Lima da Costa et al., 1997; Guitton and Avan, 2001). Previously, we have found comparable values of suppression of click-evoked transient otoacoustic emissions produced by contralateral acoustic stimulation (1.04 Q 0.48 dB) or electrical stimulation at the round window (0.94 Q 0.53 dB) in awake or slightly sedated guinea pigs (Popelar et al., 1999). Relatively small values of DPOAE suppression resulted from the use of a low level of contralateral BBN stimulation (51^71 dB SPL) to prevent acoustic crosstalk.
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Scates et al. (1999) demonstrated that electrical stimulation of the IC in anesthetized rats produces a large (7^25 dB) initial DPOAE suppression followed by a sustained smaller (3^15) suppression. Mulders and Robertson (2000) repeated such experiments and con¢rmed the results. However, in paralyzed rats, the suppression of DPOAE amplitudes for individual f2 frequencies fell to 1^2 dB. Careful observation of nonparalyzed rats during IC electrical stimulation revealed muscle activity (limb movement, slight tympanic membrane movement). When the stimulating current was dropped below the muscle twitch threshold, the DPOAE suppressive e¡ect disappeared. To avoid the confounding e¡ect of an activation of middle ear muscles, Mulders and Robertson (2000) studied the effect of electrical stimulation of the IC on cochlear responses in paralyzed guinea pigs. They found a 3^6 dB change in CAP amplitude evoked by acoustic stimuli in the frequency range 6^10 kHz. In our experiments, DPOAE amplitude suppression to electrical stimulation of the IC was measured in anesthetized guinea pigs. However, the eye and head musculature were controlled microscopically during IC stimulation, and the stimulating current was set at least 10% below the threshold for pinna and muscle twitches. In such experiments, the degree of DPOAE suppression was similar to that observed with acoustic stimulation of the contralateral ear. In cases of DPOAE recording during small eye or muscle twitches (an example of which is shown in Fig. 3), the magnitude and time pattern of DPOAE suppression were signi¢cantly di¡erent. Muscle activity was usually accompanied by a small shift of several tenths of dB in either f1 or f2 , which were monitored during DPOAE recording by Spectral history of ILO 96 software. During IC stimulation that did not evoke any eye or muscle twitches, the f1 and f2 stimulus pressure levels were stable. We believe that changes in sound pressure levels in the outer ear canal re£ect changes in middle ear impedance, and thus the stable stimulus pressure level indicates no change of middle ear muscle contraction. 4.3. IC positions e¡ective in activating the e¡erent system
4.2. The e¡ect of middle ear muscles The possible in£uence of the middle ear muscle re£ex on the magnitude of suppression of otoacoustic emissions is frequently discussed in many studies. It has been shown previously that electrical stimulation of the tectum in cats elicits muscle contractions (eye, head and pinna movement, facial muscle twitches, limb £exion) (Syka and Straschill, 1970; Syka and Radil-Weiss, 1971). These motor pathways are thought to be involved in acoustico-motor orienting responses.
The electrode positions throughout the whole IC were systematically tested with the aim of ¢nding those electrode positions the electrical stimulation of which can evoke the activation of the e¡erent system and thus DPOAE amplitude suppression. In accordance with other published data (Scates et al., 1999; Mulders and Robertson, 2000), the suppressive e¡ect was not seen in dorsal cortex of the IC, but was evident after a minimum penetration of 2 mm into the IC (except the very rostral part of the IC). This ¢nding corresponds to
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the fact that the dorsal cortex of the IC is a target of descending corticotectal ¢bers (Druga et al., 1997; Winer et al., 1998), but neurons of this part of the IC do not send descending ¢bers to the superior olivary complex or cochlear nucleus. However, it was not possible to elicit a suppression of DPOAE amplitudes from many electrode positions in the IC. In some animals, it was possible to evoke DPOAE suppression from only one electrode penetration out of several penetrations tested. Electrode positions with maximal suppressive e¡ects (i.e. rostro-medial, the ventral and lateral external cortices of the IC and the ventral part of the central nucleus of the IC) corresponded to anatomically veri¢ed origins of descending pathways from the IC (Syka et al., 1988a; Scho¢eld and Cant, 1999). The relatively low number of e⁄cient electrode positions within the IC that evoked DPOAE suppression could be explained in two ways: (i) electrical stimulation of only a few places in the IC could activate the IC descending ¢bers, or (ii) the threshold of the appearance of the ¢rst eye and muscle twitches was lower than the threshold for evoking DPOAE suppression. Another critical factor could also be injury of the IC by penetrating stimulation electrodes. In previous experiments we recorded DPOAE in awake guinea pigs and electrically stimulated the IC from implanted electrodes (unpublished data). Even though we stimulated similar IC regions as in the present acute experiments, or implanted multiple electrodes to stimulate more IC regions or a larger IC area, we never observed any e¡ect on CAP or otoacoustic emission amplitudes. We suppose that the descending colliculo-olivary system is very sensitive to any damaging manipulations and that the implantation of electrodes into the IC can seriously a¡ect its function. These unsuccessful results in experiments with implanted IC electrodes did not allow us to proceed in developing an animal model of the protective e¡ect of chronic electrical stimulation of the IC. Several recent papers, however, demonstrated a modulatory role of the auditory cortex on cochlear activity realized through either the descending corticotectal or direct cortico-olivary ¢bers (Xiao and Suga, 2002; Khalfa et al., 2001). Further research will be necessary to distinguish the role of individual cortical and subcortical structures in the e¡erent control of auditory signal processing in the cochlea. 4.4. The e¡ect of gentamicin injection In order to test the hypothesis that the mechanism of DPOAE suppression produced by contralateral acoustic stimulation and electrical stimulation of the IC is similar, two guinea pigs were treated with a single intramuscular injection of 210^250 mg/kg of gentamicin, and DPOAE suppression by both types of stimulation was
tested at postinjection periods. In agreement with other studies (Smith et al., 1994; Popelar et al., 1996; Avan et al., 1996; Lima da Costa et al., 1997; Yoshida et al., 1999), suppression of DPOAE was partly or fully eliminated 1^2 h after gentamicin injection. The above-mentioned authors also demonstrated that a single gentamicin injection does not signi¢cantly a¡ect the CAP audiogram, CAP input/output function or DPOAE amplitudes. Elimination of e¡erent olivocochlear system function by gentamicin is thought to be produced by the inactivation of acetylcholine receptors on outer hair cells (by blocking the Ca2þ entry necessary to elicit the acetylcholine response ^ Dulon et al., 1989; Nakagawa et al., 1992). Since gentamicin injection eliminated DPOAE suppression produced by both kinds of stimuli, we conclude that DPOAE suppression produced by acoustic and electrical stimulation is caused by the activation of the e¡erent olivocochlear system. 4.5. Conclusions The results of the present paper demonstrate that ¢bers descending from the IC are functionally connected to e¡erent ¢bers of the olivocochlear bundle and that electrical stimulation of the IC can suppress the amplitude of DPOAE recorded in the ipsilateral ear. We hypothesize that the descending system from the IC may control and modulate the micromechanics of the cochlea through the olivocochlear e¡erent bundle and thus contribute to the adjustment and improvement of some parameters of auditory information processing in the cochlea, such as selective attention, frequency selectivity, intensity coding, sound localization, speech discrimination or detection of tones in noise. However, the existence of low thresholds of middle ear muscle contractions to IC electrical stimulation preclude the use of electrical stimulation of the IC for chronic activation of the e¡erent auditory system.
Acknowledgements The authors wish to thank Rastislav Druga for his assistance in the evaluation of histological data. The study was supported by Grants AV OZ 5039906, the Grant Agency of the Czech Ministry of Health NK/ 6454-3 and the Grant Agency of the Czech Republic 309/01/1063. References Andersen, R.A., Snyder, R.L., Merzenich, M.M., 1980. The topographic organization of corticocollicular projections from physiologically identi¢ed loci in the A I, A II and anterior auditory cortical ¢elds of the cat. J. Comp. Neurol. 191, 479^494.
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