David F. Dolan

Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.

Laser doppler velocimetry of basilar membrane vibration

A method is described for the measurement of basilar membrane (BM) vibration velocimeter (LDV). The instrumentation was coupled to a compound microscope which served to visualize reflective glass microbeads placed on the BM. The laser beam of the LDV was focused in the microscope object plane and positioned over the reflective bead. We show examples of frequency tuning curves and displacement input/output intensity functions obtained with the technique.

Masked cochlear whole‐nerve response intensity functions altered by electrical stimulation of the crossed olivocochlear bundle

Cochlear whole-nerve response (CAP) intensity functions were recorded from the guinea pig round window. The intensity functions were obtained in the presence of masking noise, with electrical stimulation of the crossed olivocochlear bundle (COCB), and the combination of masking noise and COCB stimulation. Electrical stimulation of the COCB produced the expected reduction of CAP magnitude for low- to moderate-intensity tone bursts. Masking noise produced reductions in CAP magnitude over the whole signal intensity range used in this study. The combination of electrical stimulation of the COCB with the masking noise produced CAP magnitude changes graded between reduction and enhancement dependent on signal and masker levels. In general, at low signal levels, the masked CAP magnitude is reduced compared to the masked alone condition. At high signal levels, the masked CAP is increased in magnitude. These results confirm and extend the earlier observations of Nieder and Nieder [Exp. Neurol. 28, 174-188 (1970); also, Nature 227, 184-185 (1970)].

Glial cell line-derived neurotrophic factor has a dose dependent influence on noise-induced hearing loss in the guinea pig cochlea

We examined the effectiveness of glial cell line-derived neurotrophic factor (GDNF) to attenuate cochlear damage from intense noise stress. Subjects were exposed to 115 dB SPL one octave band noise centered at 4 kHz for 5 h. They received artificial perilymph with or without GDNF into the left scala tympani at 0.5 microliter/h from 4 days before noise exposure through 8 days following noise exposure. Different concentrations of GDNF (1 ng/ml, 10 ng/ml, 100 ng/ml, and 1 microgram/ml) were applied chronically directly into the guinea pig cochlea via a microcannula and osmotic pump. Noise-induced hearing loss was assessed with pure tone auditory brainstem responses (at 2, 4, 8 and 20 kHz), measured prior to surgery, 1 day before noise exposure, and 7 days following noise exposure. Subjects were killed on day 8 following exposure for histological preparation and quantitative assessment of hair cell (HC) damage. A dose-dependent protective effect of GDNF on both sensory cell preservation and hearing function was found in the treated ears. At 1 ng/ml, GDNF showed no significant protection; at 10 ng/ml, GDNF showed significant HC protection; and at 100ng/ml, it was greater and bilateral. At 1 microgram/ml, GDNF appeared to have a toxic effect under noise stress in some cochleae. These findings indicate that GDNF at certain concentrations can effectively protect the inner ear from noise-induced hearing loss.

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.

Asynchronous neural activity recorded from the round window.

Voltage recorded from an electrode on the round window (RW) of guinea pig has characteristics that reflect the activity of auditory-nerve fibers in the absence of acoustic stimulation. Fast Fourier transformation (FFT) of the noise recorded from the RW electrode shows a broad spectral peak from 0.8-1.0 kHz. The magnitude of the biological noise is increased by high-frequency, bandlimited acoustic noise stimulation. Pure tones can suppress or enhance the spectral components around 0.8-1.0 kHz depending on frequency and intensity. Kainic acid applied to the intact RW membrane eliminates the biological noise (and the evoked cochlear whole-nerve responses) without alteration of the cochlear microphonic or the summating potential. The spectral characteristics of the biological noise seem to be related to the elemental waveform contributed by the individual auditory-nerve fibers to the voltage recorded at the RW electrode [Kiang et al., Electrocochleography, edited by R. J. Ruben, C. Elbering, and G. Solomon (University Park, Baltimore, 1976)].

Age-related auditory pathology in the CBA/J mouse

Commercially obtained aged male CBA/J mice presented a complex pattern of hearing loss and morphological changes. A significant threshold shift in auditory brainstem responses (ABR) occurred at 3 months of age at 4 kHz without apparent loss of hair cells, rising slowly at later ages accompanied by loss of apical hair cells. A delayed high-frequency deficit started at 24 kHz around the age of 12 months. At 20-26 months, threshold shifts at 12 and 24 kHz and the accompanying hair cell loss at the base of the cochlea were highly variable with some animals appearing almost normal and others showing large deficits. Spiral ganglion cells degenerated by 18 months in all regions of the cochlea, with cell density reduced by approximately 25%. There was no degeneration of the stria vascularis and the endocochlear potential remained stable from 3 to 25 months of age regardless of whether the animals had normal or highly elevated ABR thresholds. The slow high-frequency hearing loss combined with a modest reduction of ganglion cell density and an unchanged endocochlear potential suggest sensorineural presbycusis. The superimposed early hearing loss at low frequencies, which is not seen in animals bred in-house, may complicate the use of these animals as a presbycusis model.

c- fos mRNA Induction in Acute and Chronic Audiogenic Stress: Possible Role of the Orbitofrontal Cortex in Habituation

To study putative brain circuits involved in habituation to stress, rats were exposed daily (30 min for 15 days) to an environment in the presence (Chronic) or absence (Acute) of loud noise (105 dB sound pressure level--SPL A Scale). Behavioral and endocrine measures of stress were taken throughout this habituation period, and both measures displayed strong habituation in the Chronic group. All rats were killed immediately after the day 16 exposure, constituting an acute stressor for the Acute group, and regional brain activity was assessed using c- fos mRNA induction with in situ hybridization. Hearing damage could not easily explain these results because additional rats exposed to a similar stress protocol exhibited no changes in auditory brainstem evoked potentials. c- fos mRNA induction in the central auditory system was similar between the Acute and Chronic groups, particularly at lower auditory processing levels, also arguing against a simple reduction in auditory processing in the chronically stressed rats. However, c- fos mRNA expression was reduced in chronically, as compared to acutely, stressed rats in several regions previously implicated in audiogenic stress (lateral septum, bed nucleus of the stria terminalis, some preoptic areas, and the paraventricular hypothalamic nucleus). Interestingly, the orbitofrontal cortex was the only region displaying higher c- fos mRNA induction in the chronically as compared to acutely stressed rats. This region has connections to several stress-responsive areas and may thus be a critical region actively inhibiting stress.

Frequency-dependent enhancement of basilar membrane velocity during olivocochlear bundle stimulation

Basilar membrane (BM) velocity responses were measured in the presence of olivocochlear bundle (OCB) stimulation. Frequency threshold tuning curves (FTCs) were derived from tone-evoked input-output (I/O) functions. Efferent nerve activation produced decreases in velocity amplitude for frequencies around best frequency (BF) at low stimulus levels with little or no effect for stimuli well below the BF. A level-dependent efferent reduction/enhancement of BM velocity was found for certain stimulus frequencies above the BF. Efferent activation either had no effect or caused small reductions in the velocity response produced by low level sound, whereas, at higher stimulus levels, efferent activation increased the velocity response. The derived FTCs, therefore, showed criterion-dependent changes with efferent activation. For low BM criterion velocities, FTCs showed the classic desensitization of the tip region without a shift of BF. Some BM velocity criterion values showed FTCs with an expanded high-frequency response area, also without a shift of BF. The results suggest that the effect of OCB activation changes the gain of the voltage-dependent outer hair cell motility such that BM velocity response near BF is decreased while increasing the response for tones well above BF.

Two-tone suppression of inner hair cell and basilar membrane responses in the guinea pig.

Recordings of receptor potentials from inner hair cells (IHCs) and the basilar membrane (BM) motion were made in pigmented guinea pigs. The acoustic stimuli were single tones near best frequency (BF) and two-tone complexes. Single tone input/output (I/O) functions had a saturating growth for the magnitude and their phase shifts were strongly dependent on the tone frequency relative to BF. For IHCs, a BF tone stimulus produced no phase shift in the ac receptor potential response. Phase lag or lead occurred for tones below or above BF, respectively. BM velocity I/O functions were not as compressively saturating as IHC ac I/O curves. BM phase shifts (in relation to BF) were similar to those of the IHCs. Two-tone suppression was observed in both IHC and BM response measures. Suppressor tones on the low-frequency side of BF produced complex suppression results, which were inconsistent with a simple attenuation model for suppression. The growth of suppression was faster than the attenuation from equivalent level reductions of the probe tone, and phase shifts were phase lead. Depending upon experimental conditions, phase change with suppression may be in the opposite direction from phase change observed from pure attenuation of the probe tone. High-frequency suppressors (relative to BF) are consistent with an attenuation model of suppression for the IHCs of the current study. High side suppression of basilar membrane velocity, however, differed from the IHCs in a systematic way. The phase change caused by suppression of BM velocity was always smaller than that of an equivalent reduction in the probe tone level.