Salicylate-Induced Hearing Loss Trigger Structural Synaptic Modifications in the Ventral Cochlear Nucleus of Rats via Medial Olivocochlear (MOC) Feedback Circuit

Lesion-induced cochlear damage can result in synaptic outgrowth in the ventral cochlear nucleus (VCN). Tinnitus may be associated with the synaptic outgrowth and hyperactivity in the VCN. However, it remains unclear how hearing loss triggers structural synaptic modifications in the VCN of rats subjected to salicylate-induced tinnitus. To address this issue, we evaluated tinnitus-like behavior in rats after salicylate treatment and compared the amplitude of the distortion product evoked otoacoustic emission (DPOAE) and auditory brainstem response (ABR) between control and treated rats. Moreover, we observed the changes in the synaptic ultrastructure and in the expression levels of growth-associated protein (GAP-43), brain-derived neurotrophic factor (BDNF), the microglial marker Iba-1 and glial fibrillary acidic protein (GFAP) in the VCN. After salicylate treatment (300 mg/kg/day for 4 and 8 days), analysis of the gap prepulse inhibition of the acoustic startle showed that the rats were experiencing tinnitus. The changes in the DPOAE and ABR amplitude indicated an improvement in cochlear sensitivity and a reduction in auditory input following salicylate treatment. The treated rats displayed more synaptic vesicles and longer postsynaptic density in the VCN than the control rats. We observed that the GAP-43 expression, predominantly from medial olivocochlear (MOC) neurons, was significantly up-regulated, and that BDNF- and Iba-1-immunoreactive cells were persistently decreased after salicylate administration. Furthermore, GFAP-immunoreactive astrocytes, which is associated with synaptic regrowth, was significantly increased in the treated groups. Our study revealed that reduced auditory nerve activity triggers synaptic outgrowth and hyperactivity in the VCN via a MOC neural feedback circuit. Structural synaptic modifications may be a reflexive process that compensates for the reduced auditory input after salicylate administration. However, massive increases in excitatory synapses in the VCN may represent a detrimental process that causes central hyperactivity, leading to tinnitus.     

Altered Neuronal Intrinsic Properties and Reduced Synaptic Transmission of the Rat's Medial Geniculate Body in Salicylate-Induced Tinnitus

Sodium salicylate (NaSal), an aspirin metabolite, can cause tinnitus in animals and human subjects. To explore neural mechanisms underlying salicylate-induced tinnitus, we examined effects of NaSal on neural activities of the medial geniculate body (MGB), an auditory thalamic nucleus that provides the primary and immediate inputs to the auditory cortex, by using the whole-cell patch-clamp recording technique in MGB slices. Rats treated with NaSal (350 mg/kg) showed tinnitus-like behavior as revealed by the gap prepulse inhibition of acoustic startle (GPIAS) paradigm. NaSal (1.4 mM) decreased the membrane input resistance, hyperpolarized the resting membrane potential, suppressed current-evoked firing, changed the action potential, and depressed rebound depolarization in MGB neurons. NaSal also reduced the excitatory and inhibitory postsynaptic response in the MGB evoked by stimulating the brachium of the inferior colliculus. Our results demonstrate that NaSal alters neuronal intrinsic properties and reduces the synaptic transmission of the MGB, which may cause abnormal thalamic outputs to the auditory cortex and contribute to NaSal-induced tinnitus.     

卡那霉素致聋豚鼠耳蜗电极植入模型的建立及意义

目的:探讨人工耳蜗电极的插入对耳蜗功能的影响,为研究人工耳蜗植入建立相应的动物模型。方法:取听力正常的豚鼠8只,4只注射卡那霉素联合呋塞米致聋,为致聋组;4只仅注射生理盐水,为对照组。对两组动物行听性脑干反应(ABR)及耳声发射(DPOAE)检查后,将耳蜗电极植入左侧耳蜗。结果:致聋组术侧4个频率段ABR阈移随着时间的推移逐渐减小,术后24 h、48 h、72 h时间段比较无显著性差异(P0.05);对照组术侧ABR阈移随着时间的推移逐渐减小,32 kHz频率的三个时间段比较有显著性差异(P0.05),其余3个频率无显著性差异。此外,致聋组与对照组术侧耳ABR阈移比较均无显著性差异(P0.05)。致聋组术前5个频率的DPOAE无法引出,术后DPOAE仍无法引出;对照组术前DPOAE均可引出,术后术侧的DPOAE均无法引出。术后72 h可见电极周围有组织包绕,固定良好,局部未见明显炎症反应。结论:本实验成功建立了卡那霉素致聋的豚鼠耳蜗电极植入模型,可为人工耳蜗植入术后颞骨病理改变的研究提供实验基础。     

Cochlear implants: the view from the brain

The cochlear implant arguably is the most successful neural prosthesis. Studies of the responses of the central auditory system to prosthetic electrical stimulation of the cochlea are revealing the success with which electrical stimulation of a deaf ear can mimic acoustic stimulation of a normal-hearing ear. Understanding of the physiology of central auditory structures can lead to improved restoration of hearing with cochlear implants. In turn, the cochlear implant can be exploited as an experimental tool for examining central hearing mechanisms isolated from the effects of cochlear mechanics and transduction.     

Local NMDA Receptor Blockade Attenuates Chronic Tinnitus and Associated Brain Activity in an Animal Model

Chronic tinnitus has no broadly effective treatment. Identification of specific markers for tinnitus should facilitate the development of effective therapeutics. Recently it was shown that glutamatergic blockade in the cerebellar paraflocculus, using an antagonist cocktail was successful in reducing chronic tinnitus. The present experiment examined the effect of selective N-methyl d-aspartate (NMDA) receptor blockade on tinnitus and associated spontaneous brain activity in a rat model. The NMDA antagonist, D(−)-2-amino-5-phosphonopentanoic acid (D-AP5) (0.5 mM), was continuously infused for 2 weeks directly to the ipsilateral paraflocculus of rats with tinnitus induced months prior by unilateral noise exposure. Treated rats were compared to untreated normal controls without tinnitus, and to untreated positive controls with tinnitus. D-AP5 significantly decreased tinnitus within three days of beginning treatment, and continued to significantly reduce tinnitus throughout the course of treatment and for 23 days thereafter, at which time testing was halted. At the conclusion of psychophysical testing, neural activity was assessed using manganese enhanced magnetic resonance imaging (MEMRI). In agreement with previous research, untreated animals with chronic tinnitus showed significantly elevated bilateral activity in their paraflocculus and brainstem cochlear nuclei, but not in mid or forebrain structures. In contrast, D-AP5-treated-tinnitus animals showed significantly less bilateral parafloccular and dorsal cochlear nucleus activity, as well as significantly less contralateral ventral cochlear nucleus activity. It was concluded that NMDA-mediated glutamatergic transmission in the paraflocculus appears to be a necessary component of chronic noise-induced tinnitus in a rat model. Additionally, it was confirmed that in this model, elevated spontaneous activity in the cerebellar paraflocculus and auditory brainstem is associated with tinnitus.     

Dysregulation of limbic and auditory networks in tinnitus

Tinnitus is a common disorder characterized by ringing in the ear in the absence of sound. Converging evidence suggests that tinnitus pathophysiology involves damage to peripheral and/or central auditory pathways. However, whether auditory system dysfunction is sufficient to explain chronic tinnitus is unclear, especially in light of evidence implicating other networks, including the limbic system. Using functional magnetic resonance imaging and voxel-based morphometry, we assessed tinnitus-related functional and anatomical anomalies in auditory and limbic networks. Moderate hyperactivity was present in the primary and posterior auditory cortices of tinnitus patients. However, the nucleus accumbens exhibited the greatest degree of hyperactivity, specifically to sounds frequency-matched to patients' tinnitus. Complementary structural differences were identified in ventromedial prefrontal cortex, another limbic structure heavily connected to the nucleus accumbens. Furthermore, tinnitus-related anomalies were intercorrelated in the two limbic regions and between limbic and primary auditory areas, indicating the importance of auditory-limbic interactions in tinnitus.     

Expression of Immediate-Early Genes in the Inferior Colliculus and Auditory Cortex in Salicylate-Induced Tinnitus in Rat

Tinnitus could be associated with neuronal hyperactivity in the auditory center. As a neuronal activity marker, immediate-early gene (IEG) expression is considered part of a general neuronal response to natural stimuli. Some IEGs, especially the activity-dependent cytoskeletal protein (Arc) and the early growth response gene-1 (Egr-1), appear to be highly correlated with sensory-evoked neuronal activity. We hypothesize, therefore, an increase of Arc and Egr-1 will be observed in a tinnitus model. In our study, we used the gap prepulse inhibition of acoustic startle (GPIAS) paradigm to confirm that salicylate induces tinnitus-like behavior in rats. However, expression of the Arc gene and Egr-1 gene were decreased in the inferior colliculus (IC) and auditory cortex (AC), in contradiction of our hypothesis. Expression of N-methyl D-aspartate receptor subunit 2B (NR2B) was increased and all of these changes returned to normal 14 days after treatment with salicylate ceased. These data revealed long-time administration of salicylate induced tinnitus markedly but reversibly and caused neural plasticity changes in the IC and the AC. Decreased expression of Arc and Egr-1 might be involved with instability of synaptic plasticity in tinnitus.Key words: salicylate, inferior colliculus, auditory cortex, activity-dependent cytoskeletal protein, early growth response gene-1     

Muscarinic Receptors in the Cochlear Nucleus and Auditory Nerve of the Guinea Pig

The specific-binding properties of l-[3H]quinuclidinyl benzilate, a muscarinic acetylcholine-receptor antagonist, were investigated in synaptic and other membrane preparations of the guinea pig cochlear nucleus and auditory nerve. Binding parameters for all experiments were consistent with a single binding site with a Hill coefficient of 1.0. The binding of the ligand was specific and of high affinity, with values of KD in the range of 30-80 pM. Bmax was 0.352 +/- 0.023 pmol/mg protein for the dorsal cochlear nucleus and 0.215 +/- 0.011 pmol/mg protein for the ventral cochlear nucleus. The dorsal cochlear nucleus/ventral cochlear nucleus ratio for density of muscarinic receptors (1.6/1.0) was maintained across two different buffer systems, which varied with respect to the inclusion of proteolysis inhibitors. The results for auditory nerve indicated a level of binding much below that of the cochlear nucleus, with Bmax = 0.052 +/- 0.011 pmol/mg protein. The results of specific-binding experiments for l-[3H]quinuclidinyl benzilate support a role for acetylcholine as a neurotransmitter in the cochlear nucleus. The greater density of muscarinic receptors in the dorsal cochlear nucleus may indicate greater cholinergic activity in the dorsal relative to the ventral cochlear nucleus.     

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser. Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible. The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.     

Coding of communication calls in the subcortical and cortical structures of the auditory system

The processing of species-specific communication signals in the auditory system represents an important aspect of animal behavior and is crucial for its social interactions, reproduction, and survival. In this article the neuronal mechanisms underlying the processing of communication signals in the higher centers of the auditory system--inferior colliculus (IC), medial geniculate body (MGB) and auditory cortex (AC)--are reviewed, with particular attention to the guinea pig. The selectivity of neuronal responses for individual calls in these auditory centers in the guinea pig is usually low--most neurons respond to calls as well as to artificial sounds; the coding of complex sounds in the central auditory nuclei is apparently based on the representation of temporal and spectral features of acoustical stimuli in neural networks. Neuronal response patterns in the IC reliably match the sound envelope for calls characterized by one or more short impulses, but do not exactly fit the envelope for long calls. Also, the main spectral peaks are represented by neuronal firing rates in the IC. In comparison to the IC, response patterns in the MGB and AC demonstrate a less precise representation of the sound envelope, especially in the case of longer calls. The spectral representation is worse in the case of low-frequency calls, but not in the case of broad-band calls. The emotional content of the call may influence neuronal responses in the auditory pathway, which can be demonstrated by stimulation with time-reversed calls or by measurements performed under different levels of anesthesia. The investigation of the principles of the neural coding of species-specific vocalizations offers some keys for understanding the neural mechanisms underlying human speech perception.     

Functional correlates of characteristic frequency in single cochlear nerve fibers of the Mongolian gerbil

Summary Single-unit recordings obtained from the auditory nerve of the Mongolian gerbil, Meriones unguiculatus, revealed functional differences in the response properties of neurons tuned to low and high frequencies. The distribution of neural thresholds displayed a distinct rise for auditory nerve fibers with characteristic frequencies] (CFs) between 3–5 kHz. This frequency band also marked abrupt changes in both the distribution of spontaneous discharge rates and the shape of the neural tuning curve. For neurons of all CFs, spontaneous firing rates were inversely related to neural threshold but unrelated to sharpness of neural tuning. The range of CF thresholds encountered, even when data from many animals were combined, rarely exceeded 20 dB, suggesting that cochlear nerve responses obtained from this species display little inter-animal variability. These results are compared with similar data from other species and discussed in terms of recent studies on sound communication and cochlear anatomy in gerbils.Abbreviations CF characteristic frequency - SR spontaneous discharge rate     

Uptake and Release of D-Aspartate in the Guinea Pig Cochlear Nucleus

Abstract: This study attempted to determine if l -glutamate (L-Glu) and/or l -aspartate (L-Asp) might be the transmitters of neurons that provide synaptic endings to the cochlear nucleus of the medulla. The uptake and release of D-[³H]aspartate (D-Asp), a putative marker for l -Glu and l -Asp, were measured in the guinea pig cochlear nucleus before and after destruction of the cochlear afferents by cochlear ablation. The cochlear nucleus was dissected into the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) cochlear nuclei. Subdivisions from unlesioned animals took up D-Asp, achieving concentrations in the tissues that were 13–20 times that in the medium. Subsequently, electrical stimulation evoked a Ca²⁺-dependent release of part of the D-Asp from each subdivision. Disarticulation of the middle ear ossicles, which attenuates acoustic stimulation, produced a modest inhibition of D-Asp release in each subdivision, but did not alter the uptake of D-Asp. Cochlear ablation strongly depressed both the uptake and the release of D-Asp in each subdivision, presumably as a result of destruction of the cochlear nerve endings in the cochlear nucleus. Nevertheless, after lesions, there was a preservation of the uptake and release of D-Asp in the DCN relative to the AVCN and PVCN. These residual activities in the DCN may be mediated by the axonal endings of the granule cells of the cochlear nucleus. The present findings support the hypothesis that the granule cells of the cochlear nucleus, as well as the cochlear nerve fibers, use l -Glu and/or l -Asp as transmitters.     

Both Central and Peripheral Auditory Systems Are Involved in Salicylate-Induced Tinnitus in Rats: A Behavioral Study

Objective

This study was designed to establish a low dose salicylate-induced tinnitus rat model and to investigate whether central or peripheral auditory system is involved in tinnitus.

Methods

Lick suppression ratio (R), lick count and lick latency of conditioned rats in salicylate group (120 mg/kg, intraperitoneally) and saline group were first compared. Bilateral auditory nerves were ablated in unconditioned rats and lick count and lick latency were compared before and after ablation. The ablation was then performed in conditioned rats and lick count and lick latency were compared between salicylate group and saline group and between ablated and unablated salicylate groups.

Results

Both the R value and the lick count in salicylate group were significantly higher than those in saline group and lick latency in salicylate group was significantly shorter than that in saline group. No significant changes were observed in lick count and lick latency before and after ablation. After ablation, lick count and lick latency in salicylate group were significantly higher and shorter respectively than those in saline group, but they were significantly lower and longer respectively than those in unablated salicylate group.

Conclusion

A low dose of salicylate (120 mg/kg) can induce tinnitus in rats and both central and peripheral auditory systems participate in the generation of salicylate-induced tinnitus.     

Modulation of activity in starling cochlear ganglion units by middle-ear muscle contractions,perilymph movements and lagena stimuli

In the present study three groups of cochlear ganglion neurons were detected which differed in respect to their tone-evoked and spontaneous activity: auditory units which showed an irregular spontaneous discharge, non-auditory neurones with regular activity and such with an irregular spontaneous discharge pattern. Electrically-elicited contractions of the middle-ear muscle influenced the tone-evoked and/or the spontaneous activity of the auditory and the non-auditory neurones with irregular spontaneous discharge but not, however, the regularly firing units. Similar results were obtained with imposed perilymph movements in the cochlea (evoked via the vestibular system. Fractions of all three groups of cochlear ganglion neurones were responsive to direct deformations of the membraneous lagena. Several (auditory and non-auditory) units with irregular discharge were excited during a basilar membrane displacement towards scala vestibuli whereas a basilar membrane motion towards scala tympani resulted in a decrease of the discharge rate. A few units showed a different reaction. The results provide evidence that the neurones with periodic spontaneous discharge innervate the lagena and that this sense organ has no auditory significance in birds. The peripheral origin of the 'non-auditory' neurones with irregular spontaneous activity remains undecided and might be the macula lagenae or the apical portion of the basilar papilla.     

Single unit activity in the guinea-pig cochlear nucleus during sleep and wakefulness.

The effects of waking and sleep on the response properties of auditory units in the ventral cochlear nucleus (CN) were explored by using extracellular recordings in chronic guinea-pigs. Significant increases and decreases in firing rate were detected in two neuronal groups, a) the "sound-responding" and b) the "spontaneous" (units that do not show responses to any acoustic stimuli controlled by the experimenter). The "spontaneous" may be considered as belonging to the auditory system because the corresponding units showed a suppression of their discharge when the receptor was destroyed. The auditory CN units were characterized by their PSTH in response to tones at their characteristic frequency and also by the changes in firing rate and probability of discharge evaluated during periods of waking, slow wave and paradoxical sleep. The CNS performs functions dependent on sensory inputs during wakefulness and sleep phases. By studying the auditory input at the level of the ventral CN with constant sound stimuli, it was shown that, in addition to the firing rate shifts, some units presented changes in the temporal probability of discharge, implying central actions on the corresponding neurons. The mean latency of the responses, however, did not show significant changes throughout the sleep-waking cycle. The auditory efferent pathways are postulated to modulate the auditory input at CN level during different animal states. The probability of firing and the changes in the temporal pattern, as shown by the PSTH, are thus dependent on both the auditory input and the functional brain state related to the sleep-waking cycle.     

The Spatial Origins of Cochlear Amplification Assessed by Stimulus-Frequency Otoacoustic Emissions

Cochlear amplification of basilar membrane traveling waves is thought to occur between a tone’s characteristic frequency (CF) place and within one octave basal of the CF. Evidence for this view comes only from the cochlear base. Stimulus-frequency otoacoustic emissions (SFOAEs) provide a noninvasive alternative to direct measurements of cochlear motion that can be measured across a wide range of CF regions. Coherent reflection theory indicates that SFOAEs arise mostly from the peak region of the traveling wave, but several studies using far-basal suppressor tones claimed that SFOAE components originate many octaves basal of CF. We measured SFOAEs while perfusing guinea pig cochleas from apex to base with salicylate or KCl solutions that reduced outer-hair-cell function and SFOAE amplification. Solution effects on inner hair cells reduced auditory nerve compound action potentials (CAPs) and provided reference times for when solutions reached the SFOAE-frequency CF region. As solution flowed from apex to base, SFOAE reductions generally occurred later than CAP reductions and showed that the effects of cochlear amplification usually peaked ∼1/2 octave basal of the CF region. For tones ≥2 kHz, cochlear amplification typically extended ∼1.5 octaves basal of CF, and the data are consistent with coherent reflection theory. SFOAE amplification did not extend to the basal end of the cochlea, even though reticular lamina motion is amplified in this region, which indicates that reticular lamina motion is not directly coupled to basilar membrane traveling waves. Previous reports of SFOAE-frequency residuals produced by suppressor frequencies far above the SFOAE frequency are most likely due to additional sources created by the suppressor. For some tones <2 kHz, SFOAE amplification extended two octaves apical of CF, which highlights that different vibratory motions produce SFOAEs and CAPs, and that the amplification region depends on the cochlear mode of motion considered. The concept that there is a single “cochlear amplification region” needs to be revised.     

Dynamic characteristics of the auditory cortex of guinea pigs observed with multichannel optical recording

The spatiotemporal characteristics of neural activity in the guinea pig auditory cortex are investigated to determine their importance in neural processing and coding of the complex sounds. A multi-channel optical recording system has been developed for observing the cortical field of the mammalian brain in vivo. Using the voltage-sensitive dye: RH795, optical imaging was used to visualize neural activity in the guinea pig auditory cortex. Experimental results reveal a boomerang-shaped pattern of movement of activated neural cell regions for the evoked response to click as complex sounds. Parallel and sequential neural processing structure was observed. Although the exact frequency selectivities of single cells and tonotopical organization observed using microelectrode were not visible, the similar feature to the microelectrode evidences was imaged by extracting the strongly response field from the optical data.     

Adaptation of Cochlear Amplification to Low Endocochlear Potential

Endocochlear potential (EP) is essential for cochlear amplification by providing the voltage source needed to drive outer hair cell (OHC) transducer current, which leads to OHC electromechanical force. An early study using furosemide to reversibly reduce EP showed that distortion product otoacoustic emissions (DPOAEs) recovered before EP. This indicated that cochlear amplification may be able to adjust to a new, lower EP. To investigate the mechanism of this adjustment, the extracellular OHC voltage, which we term local cochlear microphonic (LCM), was measured simultaneously with DPOAE and EP while using intraperitoneal (IP) and intravenous injection of furosemide to reversibly reduce EP. With IP injection, the DPOAEs recovered fully, whereas the EP was reduced, but LCM showed a similar time course as EP. The DPOAEs failed to accurately report the variation of cochlear amplification. With intravenous injection, for which both reduction and recovery of EP are known to occur relatively quickly compared to IP, the cochlear amplification observed in LCM could attain nearly full or even full recovery with reduced EP. This showed the cochlea has an ability to adjust to diminished operating condition. Furthermore, the cochlear amplifier and EP recovered with different time courses: cochlear amplification just started to recover after the EP was nearly fully recovered and stabilized. Using a Boltzmann model and the second harmonic of the LCM to estimate the mechanoelectric transducer channel operating point, we found that the recovery of cochlear amplification occurred with recentering of the operating point.     

Noise-Induced Inner Hair Cell Ribbon Loss Disturbs Central Arc Mobilization: A Novel Molecular Paradigm for Understanding Tinnitus

Increasing evidence shows that hearing loss is a risk factor for tinnitus and hyperacusis. Although both often coincide, a causal relationship between tinnitus and hyperacusis has not been shown. Currently, tinnitus and hyperacusis are assumed to be caused by elevated responsiveness in subcortical circuits. We examined both the impact of different degrees of cochlear damage and the influence of stress priming on tinnitus induction. We used (1) a behavioral animal model for tinnitus designed to minimize stress, (2) ribbon synapses in inner hair cells (IHCs) as a measure for deafferentation, (3) the integrity of auditory brainstem responses (ABR) to detect differences in stimulus-evoked neuronal activity, (4) the expression of the activity-regulated cytoskeletal protein, Arc, to identify long-lasting changes in network activity within the basolateral amygdala (BLA), hippocampal CA1, and auditory cortex (AC), and (5) stress priming to investigate the influence of corticosteroid on trauma-induced brain responses. We observed that IHC ribbon loss (deafferentation) leads to tinnitus when ABR functions remain reduced and Arc is not mobilized in the hippocampal CA1 and AC. If, however, ABR waves are functionally restored and Arc is mobilized, tinnitus does not occur. Both central response patterns were found to be independent of a profound threshold loss and could be shifted by the corticosterone level at the time of trauma. We, therefore, discuss the findings in the context of a history of stress that can trigger either an adaptive or nonadaptive brain response following injury.     

Basilar membrane responses to two-tone and broadband stimuli.

The responses to sound of mammalian cochlear neurons exhibit many nonlinearities, some of which (such as two-tone rate suppression and intermodulation distortion) are highly frequency specific, being strongly tuned to the characteristic frequency (CF) of the neuron. With the goal of establishing the cochlear origin of these auditory-nerve nonlinearities, mechanical responses to clicks and to pairs of tones were studied in relatively healthy chinchilla cochleae at a basal site of the basilar membrane with CF of 8-10 kHz. Responses were also obtained in cochleae in which hair cell receptor potentials were reduced by systemic furosemide injection. Vibrations were recorded using either the M?ssbauer technique or laser Doppler-shift velocimetry. Responses to tone pairs contained intermodulation distortion products whose magnitudes as a function of stimulus frequency and intensity were comparable to those of distortion products in cochlear afferent responses. Responses to CF tones could be selectively suppressed by tones with frequency either higher or lower than CF; in most respects, mechanical two-tone suppression resembled rate suppression in cochlear afferents. Responses to clicks displayed a CF-specific compressive nonlinearity, similar to that present in responses to single tones, which could be profoundly and selectively reduced by furosemide. The present findings firmly support the hypothesis that all CF-specific nonlinearities present in the auditory nerve originate in analogous phenomena of basilar membrane vibration. However, because of their lability, it is almost certain that the mechanical nonlinearities themselves originate in outer hair cells.