An analysis of cochlear response harmonics: Contribution of neural excitation.

In this report an analysis of cochlear response harmonics is developed to derive a mathematical function to estimate the gross mechanics involved in the in vivo transfer of acoustic sound into neural excitation (f(Tr)). In a simulation it is shown that the harmonic distortion from a nonlinear system can be used to estimate the nonlinearity, supporting the next phase of the experiment: Applying the harmonic analysis to physiologic measurements to derive estimates of the unknown, in vivo f(Tr). From gerbil ears, estimates of f(Tr) were derived from cochlear response measurements made with an electrode at the round window niche from 85 Hz tone bursts. Estimates of f(Tr) before and after inducing auditory neuropathy-loss of auditory nerve responses with preserved hair cell responses from neurotoxic treatment with ouabain-showed that the neural excitation from low-frequency tones contributes to the magnitude of f(Tr) but not the sigmoidal, saturating, nonlinear morphology.

Temporal expression of cytokines and signal transducer and activator of transcription factor 3 activation after neonatal hypoxia/ischemia in mice.

Hypoxia/ischemia (HI) is a prevalent reason for neonatal brain injury with inflammation being an inevitable phenomenon following such injury; but there is a scarcity of data regarding the signaling pathway involved and the effector molecules. The signal transducer and activator of transcription factor 3 (STAT3) is known to modulate injury following imbalance between pro- and anti-inflammatory cytokines in peripheral and central nervous system injury making it a potential molecule for study. The current study investigates the temporal expression of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, IL-1ra, IL-4, IL-10, IL-13 and phosphorylated STAT3 (pSTAT3) after carotid occlusion and hypoxia (8% O2, 55 min) in postnatal day 7 C57BL/6 mice from 3 h to 21 days after hypoxia. Protein array illustrated notable changes in cytokines expressed in both hemispheres in a time-dependent manner. The major pro-inflammatory cytokines showing immediate changes between ipsi- and contralateral hemispheres were IL-6 and IL-1ß. The anti-inflammatory cytokines IL-4 and IL-13 demonstrated a delayed augmentation with no prominent differences between hemispheres, while IL-1ra showed two distinct peaks of expression spread over time. We also illustrate for the first time the spatiotemporal activation of pSTAT3 (Y705 phosphorylation) after a neonatal HI in mice brain. The main regions expressing pSTAT3 were the hippocampus and the corpus callosum. pSTAT3+ cells were mostly a subpopulation of activated astrocytes (GFAP+) and microglia/macrophages (F4/80+) seen only in the ipsilateral hemisphere at most time points studied (till 7 days after hypoxia). The highest expression of pSTAT3+ cells was observed to be around 24-48 h, where the presence of pSTAT3+ astrocytes and pSTAT3+ microglia/macrophages was seen by confocal micrographs. In conclusion, our study highlights a synchronized expression of some pro- and anti-inflammatory cytokines, especially in the long term not previously defined. It also points towards a significant role of STAT3 signaling following micro- and astrogliosis in the pathophysiology of neonatal HI-related brain injury. In the study, a shift from pro-inflammatory to anti-inflammatory cytokine profile was also noted as the injury progressed. We suggest that while designing efficient neuroprotective therapies using inflammatory molecules, the time of intervention and balance between the pro- and anti-inflammatory cytokines must be considered.

Neuroprotective and neurodegenerative effects of the chronic expression of tumor necrosis factor α in the nigrostriatal dopaminergic circuit of adult mice.

Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine, has been implicated in both neuronal death and survival in Parkinson's disease (PD). The substantia nigra (SN), a CNS region affected in PD, is particularly susceptible to inflammatory insults and possesses the highest density of microglial cells, but the effects of inflammation and in particular TNF-α on neuronal survival in this region remains controversial. Using adenoviral vectors, the CRE/loxP system and hypomorphic mice, we achieved chronic expression of two levels of TNF-α in the SN of adult mice. Chronic low expression of TNF-α levels reduced the nigrostriatal neurodegeneration mediated by intrastriatal 6-hydroxydopamine administration. Protective effects of low TNF-α level could be mediated by TNF-R1, GDNF, and IGF-1 in the SN and SOD activity in the striatum (ST). On the contrary, chronic expression of high levels of TNF-α induced progressive neuronal loss (63% at 20 days and 75% at 100 days). This effect was accompanied by gliosis and an inflammatory infiltrate composed almost exclusively by monocytes/macrophages. The finding that chronic high TNF-α had a slow and progressive neurodegenerative effect in the SN provides an animal model of PD mediated by the chronic expression of a single cytokine. In addition, it supports the view that cytokines are not detrimental or beneficial by themselves, i.e., their level and time of expression among other factors can determine its final effect on CNS damage or protection. These data support the view that new anti-parkinsonian treatments based on anti-inflammatory therapies should consider these dual effects of cytokines on their design.

The influence of noise exposure on the parameters of a convolution model of the compound action potential.

The influence of noise exposure on the parameters of a convolution model of the compound action potential (CAP) was examined. CAPs were recorded in normal-hearing gerbils and in gerbils exposed to a 117 dB SPL 8 kHz band of noise for various durations. The CAPs were fitted with an analytic CAP to obtain the parameters representing the number of nerve fibers (N), the probability density function [P(t)] from a population of nerve fibers, and the single-unit waveform [U(t)]. The results showed that the analytic CAP fitted the physiologic CAPs well with correlations of approximately 0.90. A subsequent analysis using hierarchical linear modeling quantified the change in the parameters as a function of both signal level and hearing threshold. The results showed that noise exposure caused some of the parameter-level functions to simply shift along the signal level axis in proportion to the amount of hearing loss, whereas others shifted along the signal level axis and steepened. Significant changes occurred in the U(t) parameters, but they were not related to hearing threshold. These results suggest that noise exposure alters the physiology underlying the CAP, some of which can be explained by a simple lack of gain, whereas others may not.

Distinguishing cochlear pathophysiology in 4-aminopyridine and furosemide treated ears using a nonlinear systems identification technique.

To test the adequacy of physiologic indices derived from a third-order polynomial model quantifying cochlear mechano-electric transduction (MET), 24 Mongolian gerbils were exposed to either 250-mM glucose (control), 150-mM 4-aminopyridine (4-AP), or 30-mM furosemide solutions applied to the round window (RW) membrane. The cochlear microphonic (CM) was recorded from the RW in response to 68- and 88-dB SPL Gaussian noise. A nonlinear systems identification technique (NLID) provided the frequency-domain parameters and physiologic indices of the polynomial model of MET. The control group showed no change in both compound action potential (CAP) thresholds and CM. Exposure to 4-AP and furosemide resulted in a similar elevation in CAP thresholds and a reduction in CM. However, the polynomial model of MET showed different changes. The operating point, slope, and symmetry of the MET function, the polynomial model parameters, and related nonlinear coherences differed between the experimental groups. It is concluded that the NLID technique is sensitive and specific to alterations in the cochlear physiology.

Characterizing non-linearity in the cochlear microphonic using the instantaneous frequency.

In this paper, we examine the non-linearity of mechano-electric transduction in the cochlea by computing the instantaneous frequency (IF) of the cochlear microphonic (CM) in response to sinusoidal stimuli. In contrast to a linear system which yields a constant IF when driven with a sinusoid, the IF of the CM varied during one period of oscillation. This variation was not symmetric, but differed for positive and negative slopes of the CM. Administration of tetrodotoxin to eliminate neural activity indicated that the variation of the IF was not due to neural contamination. Moreover, comparing the IF of the stimulus to that of the CM indicated that the IF was not due to non-linearity in the acoustic signal. Signal frequency, signal level and acoustic trauma altered the IF. A cochlear model of the CM was developed to determine the influence of the saturation of hair-cell receptor currents and vector summation on the IF. Results indicated that these factors could not fully account for the variation in the IF. We conclude that the variation in IF within one period of cochlear partition vibration indicates that the mechanical and/or electrical oscillations which produce the CM differ from those of a linear system.

Mathematical decomposition of the round window potential.

We present an algorithm called the median transform which can be used to decompose the round window auditory potential into AC and DC components. The first of these is identified with the cochlear microphonic, and the second with the combined summating and compound action potentials. Elsewhere in this volume, the algorithm is employed as an intermediate step in obtaining the instantaneous frequency of the CM. Since the algorithm is easily implemented and operates entirely in the time domain, it may prove useful to clinicians as well as researchers.

Insights into linear and nonlinear cochlear transduction: application of a new system-identification procedure on transient-evoked otoacoustic emissions data.

Transient-evoked otoacoustic emissions (TEOAE) were used to characterize linear and nonlinear cochlear transduction using a new system-identification procedure. In this technique, a computational model of the system is first developed. From the measured stimulus and response records, spectral-density functions and multiple coherence functions are calculated. The coherence functions allow the characterization of linear/nonlinear processes as a function of frequency. Summations of linear and nonlinear coherences provide a goodness-of-fit of the chosen model. Finite impulse response pulses with a bandwidth of 1-8 kHz were used to evoke otoacoustic emissions. Eleven adults with normal hearing served as subjects. Third- and fifth-order polynomial models were used to model the data, and the results indicate that the fifth-order model is a better fit to the TEOAE data. The results of this study suggest that this system-identification procedure can be successfully applied to model cochlear transduction using a broadband stimulus. Most importantly, coherence functions provide useful insights into linear and nonlinear cochlear processes and have the potential to be developed as a clinical measure for monitoring changes in cochlear status.

Effects of in-situ calibration of click stimuli on the auditory brainstem response.

We studied the effects on the auditory brainstem response (ABR) of applying a computerized calibration procedure for click stimuli that corrects for individual transducer characteristics and ear canal acoustics. The "calibrated" signal at the eardrum possesses a nearly flat spectrum from 500 to 10,000 Hz. ABRs were recorded from normal-hearing subjects using both calibrated and uncalibrated clicks. The preponderance of energy for the latter stimulus was between 1000 and 4000 Hz. When compared to the responses evoked by the uncalibrated signal, ABRs to calibrated clicks displayed shorter component latencies, increased component amplitudes, and a more sensitive wave V relative to behavioral threshold.

Differentiation of cochlear pathophysiology in ears damaged by salicylate or a pure tone using a nonlinear systems identification technique.

Mongolian gerbils were exposed to either alpha-ketoglutarate, salicylate, or an 8-kHz pure tone. Cochlear microphonic (CM) was recorded from the round window in response to 68 and 88 dB SPL Gaussian noise. A nonlinear systems identification technique provided the frequency-domain parameters of a third-order polynomial model characterizing cochlear mechano-electric transduction (MET). A series of physiologic indices were derived from further exploration of the model. Exposure to the 8-kHz pure tone and round window application of salicylate resulted in different changes in the polynomial parameters and physiologic indices even though the threshold shifts were similar. A general reduction of CM magnitude was found after the tone exposure, and an increase at low-mid frequencies was demonstrated in the salicylate group especially at the lower signal level. The slope of the MET curve was reduced by the acoustic overstimulation. The root or the operating point of the MET was shifted in opposite directions after the two treatments. Sound-pressure levels that saturate MET expanded in the tone exposure group and narrowed in the salicylate group. The signal level also had effects on these indices.

Application of a stimulus spectral calibration routine to click evoked otoacoustic emissions.

This study examined the influence of a calibrated transient stimulus on click evoked otoacoustic emissions (CEOAEs). The calibration procedure produced a spectrally uniform stimulus (1-8 kHz) at the plane of the ear probe that was very similar among individuals. However, the calibrated signal reduced the overall level and repeatability of the CEOAE, probably due to the minimization of the energy peak at 2 kHz, which is enhanced in the uncalibrated signal. The amplitude of CEOAEs obtained with the calibrated signal was less variable among individuals compared to CEOAEs obtained with the uncalibrated signal.

Characterizing cochlear mechano-electric transduction in ears damaged with pure tones.

Cochlear microphonics were recorded in response to Gaussian noise from the round window of Mongolian gerbils. A nonlinear systems identification procedure provided the frequency-domain parameters of a third-order polynomial equation describing cochlear mechano-electric transduction (MET). Exposure to an 8 kHz pure tone at 100 dB SPL for 20 min reduced the magnitude of the linear, quadratic, and cubic terms significantly. Animals exposed to a 1- or 4-kHz pure tone showed changes in the quadratic term. Differentiation of the polynomial equation and algebraic manipulations of the coefficients provided physiologic indices of MET. The sensitivity, saturation voltages, and sound pressures required to saturate MET were altered in animals exposed to an 8-kHz pure tone. Limited changes occurred in animals exposed to a 1- or 4-kHz pure tone.

Characterizing cochlear mechano-electric transduction using a nonlinear systems identification procedure.

A nonlinear systems identification technique [J. S. Bendat, Nonlinear System Analysis and Identification from Random Data (Wiley, New York, 1990)] provided the frequency-domain parameters of a third-order system polynomial equation describing cochlear mechano-electric transduction (MET) in Mongolian gerbils. The magnitude of the linear system term was the largest followed by the cubic and quadratic terms. The phase of the linear and cubic system terms differed by approximately 180 deg and the phase of the quadratic term lay between. Between-animal and within-animal variability was smallest for the linear and cubic terms, and largest for the quadratic term. Summing linear and nonlinear coherence functions revealed that the third-order system polynomial equation characterized approximately 83% of MET for low frequencies and 92% for high frequencies. Animals exposed to a 4-kHz pure tone at 100 dB SPL for 20 min showed changes in the magnitude and phase of the parameters of the third-order polynomial equation. An increase in linear coherence and a decrease in nonlinear coherence occurred at frequencies centered at the exposure frequency. Below the exposure frequency, linear coherence decreased and nonlinear coherence increased. Summation of the coherence functions showed that the third-order polynomial equation characterized MET better after exposure to the pure tone.

An in-situ calibration procedure for click stimuli.

The use of insert earphones for delivering acoustic signals to subjects and patients has grown in popularity in auditory research and clinical audiology. In conjunction with transducer characteristics, the insertion of an earphone into the ear canal modifies ear canal acoustics and results in an acoustic signal in the ear canal unlike the original signal delivered to the earphone. The modified ear canal signal will differ within and between individuals depending on the depth of earphone insertion and ear canal geometry. We describe a new procedure for correcting earphone and ear canal acoustics. When applied to the calibration of click stimuli, the resultant ear canal signal in each individual is an impulse with a spectrum almost identical to the rectangular pulse delivered to the transducer.

Comparison of the envelope following response in the Mongolian gerbil using two-tone and sinusoidally amplitude-modulated tones.

Two-tone (TT) and sinusoidally amplitude-modulated (SAM) signals, although differing in spectra, are both periodic; the period corresponds to the difference between the two frequencies (f2,1 = f2-f1) in the former and to the frequency of the modulation tone (fmod) in the latter. Here the results of a study comparing the steady-state electrophysiologic responses to TT and SAM stimuli recorded from Nembutal-anesthetized Mongolian gerbils are reported. In the first experiment a modulation rate transfer function (MRTF) was obtained for each stimulus type by setting the SAM carrier frequency (fc) and f1 of the TT signal at the same frequency while fmod and f2,1 were covaried. MRTFs were obtained for f1s and fcs of 1, 3, and 5 kHz, with envelopes which varied between 50 and 500 Hz in 50-Hz increments. Stimuli were presented at 75 dB peak sound-pressure level (pSPL). Responses to the two stimulus types yielded MRTFs which were very similar and generally low pass in shape. In the second experiment responses to the TT and SAM signals were recorded in the presence of a continuous interfering tone of 85-dB pSPL which was varied between 650 Hz and 3 kHz. In these experiments a maximum reduction in the response to the TT and SAM signals, measured at f2,1 and fmod as well as at fc and f1, occurred within a narrow frequency band above the frequency of the probe carrier and a broader region of reduced response extending to higher frequencies. This reduction in response was asymmetrical, spreading more to high than to low frequencies. The similarity of both MRTFs and interference response patterns supports the view that the envelope following responses to TT and SAM stimuli are manifestations of the same nonlinear phenomena.

Characterization of cochlear outer hair cell turgor.

The cochlear outer hair cell (OHC) is a cylindrical cell with structural features suggestive of a hydraulic skeleton, i.e., an elastic shell with a positive internal pressure. This study characterizes the role of the OHC elevated cytoplasmic pressure in maintaining the cell shape. Intracellular pressure of OHCs from guinea pig is estimated by measuring changes in cell morphology in response to increasing or decreasing osmolarity. Cells collapse when subjected to a continuous increase in osmolarity. Collapse occurs at an average of 8 mosM above the standard medium, suggesting that normal cells have an effective intracellular pressure of 128 mmHg. Fewer cells collapse when exposed to slow rates of osmolarity increase than cells exposed to fast rates of osmolarity increase, although the final change in osmolarity in the perfusion chamber is similar. Furthermore, cells undergo a slow, spontaneous increase in volume on exposure to either no osmolarity change or slow rates of osmolarity increase, suggesting that the cell's internal osmolarity increases in vitro. After volume reduction or elevation, cells do not return to their initial volume.

In vitro organotin administration alters guinea pig cochlear outer hair cell shape and viability.

Trimethyltin (TMT) and triethyltin (TET) disrupt auditory function at doses far below those shown to be neurotoxic. In vivo studies suggest that the initial effect of TMT on hearing occurs at the inner hair cell/spiral ganglion cell synapse, while later, the outer hair cell (OHC) undergoes structural and functional damage. TET produces acute effects upon afferent neurotransmission similar to those observed following TMT, but TET's effects on OHC structure and function have not been examined. OHCs are motile elements within the cochlea, believed to modulate the sensitivity and tuning within the inner ear. Changes in OHC length may alter hearing function, and length changes have been reported following exposure to various ototoxic agents in vitro. In the present study, 77 OHCs from 45 pigmented male guinea pigs were isolated in primary culture and exposed for 90 min to concentrations between 30 microM and 1.0 mM of TMT or TET and then to bathing medium for 30 min to remove the toxicant. Significant shortening of the OHC cell body occurred at all doses to both organotins, with a mean reduction in length of 15.1 and 20.2% for 1.0 mM TMT and TET, respectively, at the end of testing; control cells were only 3.4% shorter at the end of 90 min of perfusion with bathing medium. The effect of organotin exposure on OHC volume was not consistently related to either TMT or TET concentration or altered cell length. In addition, disruption of the plasma membrane characterized by bleb formation, the forceful ejection of cytoplasm, or bursting was seen in 80% of cells exposed to 1.0 mM TET, although not TMT; lower concentrations of both organotins disrupted the cell membrane in 10-30% of cells. Membrane rupture was not reliably associated with either increased cell volume or decreased length, implicating a weakening of the plasma membrane or cortical lattice as the basis for this effect. Consistent with the irreversible structural weakening of the lateral wall, resorption of organotin-induced cytoplasmic blebs was never evidenced. Qualitatively, subcellular elements in the central core of many organotin-treated OHCs appeared pathological. These changes are similar to histopathological changes observed following in vivo organotin administration and may represent one target of acute alkyltin ototoxicity.

Auditory distortion products measured with averaged auditory evoked potentials.

The purpose of this investigation was to describe the properties of averaged auditory evoked potential distortion products (AEP-DPs) in guinea pigs. This study provided a step toward developing a clinical index of nonlinear processing of auditory signals and supplied a baseline for studies evaluating the effect of cochlear damage on AEP-DPs. The amplitude of the AEP-DPs was evaluated as a function of f2/f1 ratio (1.12-1.52) and primary frequency (500 Hz-2000 Hz). The amplitude of the AEP cubic difference tone (AEP-CDT) increased with increasing f2/f1 ratio for the 500-Hz f1 primary and remained constant for the 800-Hz and 1700-Hz f1 primaries. The AEP-CDT generated by the 1100-Hz and 1400-Hz f1 primaries was maximum for the middle f2/f1 ratios (1.22, 1.32, and 1.42). The AEP-CDT could not be distinguished from the noise floor for the 2000-Hz f1 primary. The AEP difference tone (AEP-DT) was larger and more frequently identified than the AEP-CDT. The amplitude of the AEP-DT decreased with an increase in f2/f1 ratio. The decrease was more pronounced for low-frequency f1 primaries than for high-frequency f1 primaries.

Electrophysiological evidence of nonlinear distortion products to two-tone stimuli.

Spectral analysis of auditory-evoked potential recordings from ten normal-hearing subjects to two-tone signals revealed energy at difference tone (DT = f2-f1) and cubic difference (CDT = 2f1-f2) frequencies that was not present in the acoustic signal. Control experiments and calibrations provided substantial evidence supportive of the biological nature of these auditory nonlinearities, suggesting that they are not the result of electromagnetic, acoustic, or analytic artifact. Amplitudes of DT- and CDT-evoked responses were evaluated for rarefaction and condensation signals with f1 = 510 and 800 Hz across frequency ratios (f2/f1) of 1.16, 1.26, 1.36, and 1.46. Additionally, time-domain summation and subtraction of separately collected evoked responses to rarefaction and condensation signals were performed to demonstrate that these electrophysiological DT and CDT responses reflect their expected quadratic and cubic nature. Suggestions for development of clinical applications of assessing auditory nonlinearities using this methodology are provided.

Auditory nonlinearities measured with auditory-evoked potentials.

This article describes the use of auditory-evoked potentials (AEPs) as a tool to assess nonlinear processes in the auditory system. Two-tone signals were used as stimuli to obtain AEPs in both animal and human subjects. Frequency analysis of the physiologic waveforms revealed frequencies in the evoked potential that were not present in the acoustic signal. The largest distortion product in the evoked potential corresponded to the difference between the two primary frequencies (f2-f1). This distortion product was present in all subjects tested. Other distortion products at frequencies defined by n(f2-f1), where n less than 5, were also present in some individuals. These frequencies represent distortion components generated from an even-order nonlinear system. Extensive acoustic and electric calibration procedures provided substantial evidence that the distortion products recorded in the AEP were biologic in origin and not the result of acoustic or recording artifact.