Outer hair cell driven reticular lamina mechanical distortion in living cochleae.

Cochlear distortions afford researchers and clinicians a glimpse into the conditions and properties of inner ear signal processing mechanisms. Until recently, our examination of these distortions has been limited to measuring the vibration of the basilar membrane or recording acoustic distortion output in the ear canal. Despite its importance, the generation mechanism of cochlear distortion remains a substantial task to understand. The ability to measure the vibration of the reticular lamina in rodent models is a recent experimental advance. Surprising mechanical properties have been revealed. These properties merit both discussion in context with our current understanding of distortion, and appraisal of the significance of new interpretations of cochlear mechanics. This review focusses on some of the recent data from our research groups and discusses the implications of these data on our understanding of vocalization processing in the periphery, and their influence upon future experimental directions. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.

Maternity records in Edinburgh and Aberdeen in 1936: a comparison.

Historians have long used maternity records to understand the evolution of maternity services. More recently, epidemiologists have become interested in obstetric hospital records as a source of data (e.g. birth weight, social class), to study the influence of early life on future health and disease: life course epidemiology. Edinburgh and Aberdeen are unusual in holding detailed records from several maternity institutions. The records of 1936 are of particular interest because all children born in this year and at school in Scotland at age 11 sat a cognitive ability test, the Scottish Mental Survey 1947. This study aims to describe the maternity services in Edinburgh and Aberdeen in 1936, between the First and Second World Wars. Understanding the richness of data in birth records, the manner in which they were recorded, and the context of the institutions in their community is essential for interpreting life course epidemiology studies.

Functional expression of P2X4 receptor in capillary endothelial cells of the cochlear spiral ligament and its role in regulating the capillary diameter.

The cochlear lateral wall generates the endocochlear potential (EP), which creates a driving force for the hair cell transduction current and is essential for normal hearing. Blood flow at the cochlear lateral wall is critically important for maintaining the EP. The vulnerability of the EP to hypoxia suggests that the blood flow in the cochlear lateral wall is dynamically and precisely regulated to meet the changing metabolic needs of the cochlear lateral wall. It has been reported that ATP, an important extracellular signaling molecule, plays an essential role in regulating cochlear blood flow. However, the cellular mechanism underlying ATP-induced regional blood flow changes has not been investigated. In the current study, we demonstrate that 1) the P2X4 receptor is expressed in endothelial cells (ECs) of spiral ligament (SL) capillaries. 2) ATP elicits a characteristic current through P2X4 on ECs in a dose-dependent manner (EC(50) = 0.16 mM). The ATP current has a reversal potential at ∼0 mV; is inhibited by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD), LaCl(3), pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), and extracellular acidosis; and is less sensitive to α,ß-methyleneadenosine 5'-triphosphate (α,ß-MeATP) and 2'- and 3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP). 3) ATP elicits a transient increase of intracellular Ca(2+) in ECs. 4) In accordance with the above in vitro findings, perilymphatic ATP (1 mM) caused dilation in SL capillaries in vivo by 11.5%. N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nonselective inhibitor of nitric oxide synthase, or 5-BDBD, the specific P2X4 inhibitor, significantly blocked the dilation. These findings support our hypothesis that extracellular ATP regulates cochlear lateral blood flow through P2X4 activation in ECs.

Effect of capsaicin on potassium conductance and electromotility of the guinea pig outer hair cell.

Capsaicin, the classic activator of TRPV-1 channels in primary sensory neurons, evokes nociception. Interestingly, auditory reception is also modulated by this chemical, possibly by direct actions on outer hair cells (OHCs). Surprisingly, we find two novel actions of capsaicin unrelated to TRPV-1 channels, which likely contribute to its auditory effects in vivo. First, capsaicin is a potent blocker of OHC K conductances (I(K) and I(K,n)). Second, capsaicin substantially alters OHC nonlinear capacitance, the signature of electromotility - a basis of cochlear amplification. These new findings of capsaicin have ramifications for our understanding of the pharmacological properties of OHC I(K), I(K,n) and electromotility and for interpretation of capsaicin pharmacological actions.

Effect of salicylate on KCNQ4 of the guinea pig outer hair cell.

Salicylate causes a moderate hearing loss and tinnitus in humans at high-dose levels. Salicylate-induced hearing loss has been attributed to impaired sound amplification by outer hair cells (OHCs) through its direct action on the OHC motility sensor and/or motor. However, there is a disparity of salicylate concentrations between the clinical and animal studies, i.e., extremely high extracellular concentrations of salicylate (from 1 to 10 mM) is required to produce a significant reduction of electromotility in animal studies. Such concentrations are above the clinical/physiological range for humans. Here, we showed that clinical/physiological concentration range of salicylate caused concentration-dependent and reversible reductions in I(K,n) (KCNQ4) and subsequent depolarization of OHCs. Salicylate reduced the maximal tail current of the activation curve of I(K,n) without altering the voltage-sensitivity (V(half)). The salicylate-induced reduction of I(K,n) was almost completely blocked by linopirdine (0.1 mM) and BaCl2 (10 mM). Consistent with the finding in OHCs, salicylate significantly reduced KCNQ4-mediated current expressed in Chinese hamster ovarian (CHO) cells by comparable amplitude to OHCs without significantly shifting V(half). Nonstationary fluctuation analysis shows that salicylate significantly reduced the estimated single-channel current amplitude and numbers. Intracellular Ca²+ elevation resulting from cytoplasmic acidosis also contributes to the current reduction of I(K,n) (KCNQ4) of OHCs. These results indicate a different model for the salicylate-induced hearing loss through the reduction of KCNQ4 and subsequent depolarization of OHCs, which reduces the driving force for transduction current and electromotility. The major mechanism underlying the reduction of I(K,n) (KCNQ4) is the direct blocking action of salicylate on KCNQ4.

The influence of loud sound stress on expression of osmotic stress protein 94 in the murine inner ear.

Osmotic stress protein 94 (OSP94), a member of the heat shock protein 110/SSE subfamily, is expressed in certain organs such as the kidney, testis, and brain where it can act as a molecular chaperon. In general, its alteration in expression is in response to hyper-ionic and osmotic stress as well as heat shock stress. Since many cells in the inner ear are involved in active ion transportation and are constantly exposed to two ionic different environments, we hypothesize that OSP94 may be expressed in the inner ear and its expression may be influenced by loud sound stress (LSS). With immunohistochemistry combined with confocal microscopy, immunoblotting, and reverse transcription polymerase chain reaction techniques, we found that OSP94 was widely expressed in various cells in the murine cochlea including the stria vascularis, the organ of Corti, the interdental cells, spiral ganglion cells, the spiral ligament, and Reissner's membrane. Under the unstressed condition, the transcription and protein level of OSP94 expression in the inner ear was quantitatively similar to that of the kidney. Furthermore, its expression in the inner ear by LSS from broadband noise at 117 dB/SPL was upregulated, but remained unchanged in the kidney. In particular, the upregulation of OSP94 in the cochlear lateral wall tissue was slowly elicited in a LSS time-dependent manner compared with the response of two other HSPs; HSP25 and HSP70 are considered to play a cytoprotective role under stressful conditions. Our results show that OSP94 is expressed in the inner ear and indicate this may be necessary for cells in a special ionic and osmotic environment such as endo-perilymphatic ion compartments. The organ-specific upregulation of OSP94 by acoustic overstimulation reveals that OSP94 in the murine inner ear is potentially important for cellular functional adaptation to LSS.

Proteomic analysis of scleroderma lesional skin reveals activated wound healing phenotype of epidermal cell layer.

OBJECTIVE: To identify using proteomic analysis, proteins of altered abundance in the skin of patients with SSc. METHODS: 4 mm excision biopsies were obtained from the forearm involved skin of 12 diffuse SSc patients and 12 healthy controls. Two-dimensional gel electrophoresis was used to separate and define proteins in normal and SSc skin biopsy material. Proteins of altered abundance in the disease were formally identified by mass spectroscopy. Abnormalities of the epidermis were confirmed by immunohistochemistry. RESULTS: Proteomic analysis revealed altered abundance of proteins involved in extracellular matrix production, myofibroblast contractility, energy metabolism and response to oxidative stress. In addition, proteins specific to the epidermis and involved in epidermal cell differentiation were altered in abundance in the disease. SSc epidermis is thickened, has an expanded nucleated cell layer, and exhibits abnormal persistence of basal marker keratin 14, delayed expression of maturation markers keratin 1/10 and the induction of keratins 6 and 16, normally absent from interfollicular skin and induced following epidermal injury. These changes closely resemble the activated phenotype seen during wound healing. CONCLUSIONS: Consistent with previous models of SSc pathogenesis these data are showing increased contractility, increased extracellular matrix and response to oxidative stress in the involved skin of recent onset SSc patients. In addition, we show that SSc epidermis has an activated, wound healing phenotype. These findings are important because epidermal cells activated by injury induce and regulate local fibroblasts during wound repair.

Co-localization of the vanilloid capsaicin receptor and substance P in sensory nerve fibers innervating cochlear and vertebro-basilar arteries.

Evidence suggests that capsaicin-sensitive substance P (SP)-containing trigeminal ganglion neurons innervate the spiral modiolar artery (SMA), radiating arterioles, and the stria vascularis of the cochlea. Antidromic electrical or chemical stimulation of trigeminal sensory nerves results in neurogenic plasma extravasation in inner ear tissues. The primary aim of this study was to reveal the possible morphological basis of cochlear vascular changes mediated by capsaicin-sensitive sensory nerves. Therefore, the distribution of SP and capsaicin receptor (transient receptor potential vanilloid type 1-TRPV1) was investigated by double immunolabeling to demonstrate the anatomical relationships between the cochlear and vertebro-basilar blood vessels and the trigeminal sensory fiber system. Extensive TRPV1 and SP expression and co-localization were observed in axons within the adventitial layer of the basilar artery, the anterior inferior cerebellar artery, the SMA, and the radiating arterioles of the cochlea. There appears to be a functional relationship between the trigeminal ganglion and the cochlear blood vessels since electrical stimulation of the trigeminal ganglion induced significant plasma extravasation from the SMA and the radiating arterioles. The findings suggest that stimulation of paravascular afferent nerves may result in permeability changes in the basilar and cochlear vascular bed and may contribute to the mechanisms of vertebro-basilar type of headache through the release of SP and stimulation of TPVR1, respectively. We propose that vertigo, tinnitus, and hearing deficits associated with migraine may arise from perturbations of capsaicin-sensitive trigeminal sensory ganglion neurons projecting to the cochlea.

Expression of Trk A receptors in the mammalian inner ear.

Tyrosine kinase receptors, including Trk A, Trk B and Trk C, participate in many different biological processes that are regulated by neurotrophic factors. Nerve growth factor (NGF)-triggered Trk A signaling is involved in growth, survival and differentiation of neurons in the central nervous system and in neural crest-derived cells. Trk A, Trk B and Trk C expression has been reported in the rat ventral cochlear nucleus. In the present study, we explored the immunocytochemical distribution of Trk A in the rodent inner ear. Rat and mouse cochleae were immunolabeled with a rabbit anti-Trk A polyclonal antibody (Chemicon) that has no reported cross-reactivity with Trk B and Trk C. In embryonic day 16 mice, no Trk A immunolabeling could be detected in the developing neuroepithelium. At postnatal day 6, weak Trk A labeling could be observed in both inner and outer hair cells. At postnatal day 12, enhanced punctate Trk A immunoexpression was present in hair cells. In adult mice and rats, intense Trk A labeling was observed in outer and inner hair cell bodies, in supporting cell bodies throughout the cochlea, and in spiral ganglion neurons. Trk A was not observed in stria vascularis, hair cell stereocilia, nor in the Trk B- and Trk C-rich cerebellum. This distribution pattern of Trk A suggests that its ligand, NGF, exerts significant trophic effects in the rodent inner ear.

Spontaneous basilar membrane oscillation and otoacoustic emission at 15 kHz in a guinea pig.

A spontaneous otoacoustic emission (SOAE) measured in the ear canal of a guinea pig was found to have a counterpart in spontaneous mechanical vibration of the basilar membrane (BM). A spontaneous 15-kHz BM velocity signal was measured from the 18-kHz tonotopic location and had a level close to that evoked by a 14-kHz, 15-dB SPL tone given to the ear. Lower-frequency pure-tone acoustic excitation was found to reduce the spontaneous BM oscillation (SBMO) while higher-frequency sound could entrain the SBMO. Octave-band noise centered near the emission frequency showed an increased narrow-band response in that frequency range. Applied pulses of current enhanced or suppressed the oscillation, depending on polarity of the current. The compound action potential (CAP) audiogram demonstrated a frequency-specific loss at 8 and 12 kHz in this animal. We conclude that a relatively high-frequency spontaneous oscillation of 15 kHz originated near the 15-kHz tonotopic place and appeared at the measured BM location as a mechanical oscillation. The oscillation gave rise to a SOAE in the ear canal. Electric current can modulate level and frequency of the otoacoustic emission in a pattern similar to that for the observed mechanical oscillation of the BM.

Two resting potential levels regulated by the inward-rectifier potassium channel in the guinea-pig spiral modiolar artery.

1. Intracellular in vitro recordings were made from 771 cells from the spiral modiolar artery (SMA). The initial resting potentials (RPs) displayed a bimodal distribution that was well modelled as a mixture of two Gaussian distributions. About half of the cells had an average RP of -74 mV, and were termed high-RP cells, whereas the other half had an average RP around -41 mV, and were termed low-RP cells. Preparations that were incubated for longer than 24 h contained significantly more high-RP cells than those incubated for less than 8 h. 2. When labelled with the fluorescent dye propidium iodide, 68 and 36 cells were identified as smooth muscle cells (SMC) and endothelial cells (EC), respectively. The RP and input resistance were not significantly different between these two types of cell. Dye coupling was observed only in ECs. Dual cell recordings with 0.2-1.0 mm separation demonstrated the simultaneous existence of high- and low-RP cells and a heterogeneous low-strength electrical coupling. 3. The high-RP cells were depolarized by ACh and by high extracellular potassium concentration (high K(+)). The low-RP cells were usually hyperpolarized by moderately high K(+) (7.5-20 mM) and by ACh. The high K(+)-induced hyperpolarization was suppressed by barium (Ba(2+), 10-50 microM). The putative gap junction blocker 18 beta-glycyrrhetinic acid suppressed the ACh-induced responses in SMCs, but not in ECs. 4. Low-RP cells could rapidly shift the membrane potential to a permanent high-RP state spontaneously or, more often, after a brief application of hyperpolarizing agents including high K(+), ACh, nitric oxide and pinacidil. Once shifted to a high-RP state, the responses of these cells to high K(+) and ACh became similar to those of the original high-RP cells. 5. High-RP cells occasionally shifted their potentials to a low-RP state either spontaneously or after a brief application of 10-50 microM Ba(2+) or 100 microM ouabain. Once shifted to the low-RP state, the response of these cells to high K(+) and ACh became a hyperpolarization. The shift between high- and low-RP states was largely mimicked by wash-in and wash-out of low concentrations of Ba(2+). The shift often showed a regenerative process as a fast phase in its middle course. 6. It is concluded that the cochlear SMA in vitro is composed of poorly and heterogeneously coupled SMCs and ECs, simultaneously resting in one of two distinct states, one a high-RP state and the other a low-RP state. The two RP states are exchangeable mainly due to all-or-none-like conductance changes of the inward-rectifier K(+) channel.

Noise-induced hearing loss: the effect of melanin in the stria vascularis.

Conflicting investigations regarding the potential protective effect of melanin against noise-induced sensorineural hearing loss have suggested that eumelanin and pheomelanin may have differing effects within the stria vascularis. Three strains of C57BL/6J mice, (+/+, a/a) wild-types (dark coats/black eyes), (c2j/c2j, a/a), albinos (white coats/pink eyes), and (+/+, Ay/Ay) yellow mice (yellow coats/black eyes), were subjected to five consecutive days of broad band noise exposure at 112 dB(A) SPL for 3 h/day. Cochlear function was evaluated with auditory brainstem response audiometry to pure tones immediately pre-exposure, 5-6 h postexposure, and 14 days post-exposure. No significant difference in the degree of sensorineural hearing loss induced in the three strains of mice was identified. The eumelanin and pheomelanin content of each stria vascularis and amount of protein per stria for both mouse and guinea pig (2/NCR) were determined via high performance liquid chromatography. No pheomelanin was found in the stria of yellow mice, suggesting that coat color is not an accurate predictor of strial melanin content. The melanin content per mg of strial protein was higher in mice than in guinea pigs. A species-specific difference in melanin content does not explain the absence of a protective effect in mice.

Quinine-induced alterations of electrically evoked otoacoustic emissions and cochlear potentials in guinea pigs.

Quinine is a well-known ototoxic drug which may affect portions of the auditory system with different biochemical effects, causing reversible hearing loss and tinnitus. Recent investigations indicate that quinine at high concentrations can act directly on cochlear outer hair cells to affect their motility and the mechanical response of the basilar membrane. This study aimed to investigate the effect of quinine on the electromotility of outer hair cells in vivo by means of measuring the electrically evoked otoacoustic emissions (EEOAEs), and the relationship between EEOAE and hearing sensitivity alterations in guinea pigs. Quinine was infused into the scala tympani with concentrations between 0.05 and 5 mM. An alternating current (35 microA RMS) swept from 400 Hz to 40 kHz was applied to the round window to evoke the EEOAE. The compound action potential (CAP), cochlear microphonic (CM) and summating potential (SP) were also measured. Results show that quinine affects the EEOAE in a dose-dependent manner and that its effects are reversible. Two aspects of the EEOAE were affected by quinine, depending on concentration: (1) the 'fine structure' only for concentrations below 0.1 mM and (2) the overall amplitude and the 'fine structure' for concentrations above 0.1 mM. At 5 mM the fine structure was completely absent and the mean amplitude of the EEOAE greatly decreased. Multiple component analysis shows the short delay component of the EEOAE is related to the mean value of the amplitude spectrum while the long delay component is related to the fine structure. The alterations of the EEOAE are roughly comparable to that of the cochlear potentials. A 'threshold concentration' for quinine's effects was found at 25 microM. CAP was significantly affected at 25 microM while EEOAE, CM and SP were not. Enhancement of the EEOAE amplitude was noticed in five out of 20 animals in the current study. The enhancement appears only related to the EEOAE mean level or short delay component. The results suggest that quinine can affect in vivo electromotility of outer hair cells at low concentration and therefore change the cochlear amplifier performance via an effect on electro-mechanical transduction. Its effects on the cochlear spiral ganglion neurons and/or their presynaptic process are also suggested, and these are speculated to be the primary sites for quinine's effects on the auditory system.

Co-existence of tyrosine hydroxylase and calcitonin gene-related peptide in cochlear spiral modiolar artery of guinea pigs.

The distribution of tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP) on the cochlear spiral modiolar artery (SMA) was investigated in the guinea pig. The SMA was dissected from the modiolus so that the entire length of the vessel and many of its branches could be observed. Immunohistochemical labeling and double immunofluorescence were employed to localize each compound and to determine whether the TH and CGRP co-exist in neurons of the SMA. Microscopic examination of whole vessel preparations revealed numerous TH- and CGRP-positive neural networks innervating the SMA and its branches. The labeled neurons showed distinct arborization, varicosities and overlap, and were of different diameters. Confocal immunofluorescence microscopy of double-labeled TH and CGRP neurons showed that a number of the TH- and CGRP-positive neurons were co-labeled. Thus, TH and CGRP partially co-exist within the neuronal innervation of SMA. These findings support a hypothesis that specific neuropeptide and adrenergic neurons regulate cochlear blood flow.

Capsaicin stimulation of the cochlea and electric stimulation of the trigeminal ganglion mediate vascular permeability in cochlear and vertebro-basilar arteries: a potential cause of inner ear dysfunction in headache.

Trigeminal neurogenic inflammation is one explanation for the development of vascular migraine. The triggers for this inflammation and pain are not well understood, but are probably vasoactive components acting on the blood vessel wall. Migraine-related inner ear symptoms like phonophobia, tinnitus, fluctuation in hearing perception and increased noise sensitivity provide indirect evidence that cochlear blood vessels are also affected by basilar artery migraine. The purpose of this investigation was to determine if a functional connection exists between the cochlea and the basilar artery. Neuronally mediated permeability changes in the cochlea and basilar artery were measured by colloidal silver and Evans Blue extravasation, following orthodromic and antidromic stimulation of the trigeminal ganglion innervating the cochlea. Capsaicin and electrical stimulation induced both dose- and time-dependent plasma extravasation of colloidal silver and Evans Blue from the basilar artery and anterior inferior cerebellar artery. Both orthodromic and antidromic activation of trigeminal sensory fibers also induced cochlear vascular permeability changes and significant quantitative differences between the treated and control groups in spectrophotometric assays. These results characterize a vasoactive connection between the cochlea and vertebro-basilar system through the trigeminal sensory neurons. We propose that vertigo, tinnitus and hearing deficits associated with basilar migraine could arise by excitation of the trigeminal nerve fibers in the cochlea, resulting in local plasma extravasation. In addition, cochlear "dysfunction" may also trigger basilar and cluster headache by afferent input to the trigeminal system.

Recording depth of the heterodyne laser interferometer for cochlear vibration measurement.

Measurement of the cochlear partition vibration as a function of the optical-axis (z-axis) position in the gerbil cochlea showed that the velocity distributes over a range of more than 300 microm, which is larger than the thickness of the cochlear partition. This finding suggests that the recording depth (RD) of the heterodyne interferometer probably is not as small as reported in the literature. In the current experiment, the RD of the heterodyne laser interferometer was studied by measuring the velocity of a vibrating mirror as a function of the z-axis position. Results demonstrate that the optical sectioning characteristic, measured by the intensity of the reflected laser beam as a function of the z-axis position, is not able to correctly estimate the RD of the heterodyne interferometer: the RD is much larger than optical sectioning, indicating a poor spatial resolution along the z axis.

Electrically evoked otoacoustic emissions from apical and basal perilymphatic electrode positions in the guinea pig cochlea.

Stimulation of the cochlea with sinusoidal current results in the production of an otoacoustic emission at the primary frequency of the stimulus current. In this study we test the hypothesis that the wide frequency response from round window (RW) stimulation is due to the involvement of a relatively large spatial segment of the organ of Corti. Tonotopically organized group delays would be evident from perilymphatic electrode locations that restrict the spatial extent of hair cell stimulation. Monopolar and bipolar-paired stimulus electrodes were placed in perilymphatic areas of the first or third cochlear turns and the electrically evoked otoacoustic emissions (EEOAE) produced by these electrodes were compared to that from the RW monopolar electrode in the anesthetized guinea pig. Current stimuli of 35 microA RMS were swept across the frequency range between 60 Hz and 100 kHz. The EEOAE was measured using a microphone coupled to the ear canal. It was found that the bandwidth of EEOAEs from RW stimulation extended to at least 40 kHz and was a relatively insensitive to electrode location on the RW. The group delay of the EEOAE from stimulation at the RW membrane (corrected to stapes motion) was about 53 micros. First and third turn stimulations from electrode placements in perilymph near the bony wall of cochlea yielded narrower band EEOAE magnitude spectra but which had the same short group delays as for RW stimulation. A confined current (from a bipolar electrode pair) applied close to the basilar membrane (BM) in the first turn produced the narrowest frequency-band magnitude emissions and a mean corrected group delay of 176 micros for a location approximately 3 mm from the high frequency end of the BM (corresponding to about the 18 kHz best frequency location). Bipolar electrodes in the third turn scala tympani produced low pass EEOAE magnitude functions with corrected group delays ranging between approximately 0.3 and 1 ms. The average phase slopes did not change with altered cochlear sensitivity and postmortem. These data indicate that the EEOAE from RW stimulation is the summed response from a wide-tonotopic distribution of outer hair cells. A preliminary model study indicates that short time delayed emissions are the result of a large spatial distribution of current applied to perilymphatic locations possibly giving rise to "wave-fixed" emissions.

Nitric oxide distribution and production in the guinea pig cochlea.

Production sites and distribution of nitric oxide (NO) were detected in cochlear lateral wall tissue, the organ of Corti and in isolated outer hair cells (OHCs) from the guinea pig using the fluorescent dye, 4,5-diaminofluorescein diacetate. Fluorescent signal, indicating the presence of NO, was found in the afferent nerves and their putative endings near inner hair cells (IHCs) and putative efferent nerve endings near OHCs, the IHCs and OHCs, the endothelial cells of blood vessels of the spiral ligament, the stria vascularis, and the spiral blood vessels of the basilar membrane. An increased NO signal was observed following exposure to the substrate for NO, L-arginine, while exposure to NO synthase inhibitors resulted in a decrease in NO signal. Observation of OHCs at the subcellular level revealed differentially strong fluorescent signals at the locations of cuticular plate, the subcuticular plate region, the infranuclear region, and the region adjacent to the lateral wall. The findings indicate the presence of NO in the cochlea and suggest that NO may play an important role in both regulating vascular tone and mediating neurotransmission in guinea pig cochlea.

Basilar membrane vibration in the basal turn of the sensitive gerbil cochlea.

The basal membrane (BM) velocity responses to pure tones were measured using a newly developed laser interferometer microscope that does not require placing a reflecting object on the BM. It was demonstrated that the instrument is able to measure sub-nanometer vibration from the cochlear partition in the basal turn of the gerbil. The overall shape of the amplitude spectra shows typical tuning features. The 'best' frequencies (BFs) for the BM locations studied were between 14 kHz and 27 kHz, depending on the longitudinal position. For a given BM location, tuning sharpness was input level dependent, indicated by the Q(10dB), which varied from approximately 3 at low stimulus levels to near 1.5 at high input levels. At frequencies below BF, parallel amplitude/frequency curves across stimulus levels indicate a linear growth function. However, at frequencies near BF, the velocity increased linearly at low levels (<40 dB SPL) and became compressed between 40 and 50 dB SPL. Although the velocity gain for the frequency range below BF was a function of frequency, for a given frequency the gains were approximately constant across different levels. At frequencies near BF, the velocity gain at low sound pressure level was greater than that at a high sound pressure level, indicating a nonlinear negative relationship to stimulus level. The data also showed that the BF shifts toward the low frequencies with stimulus intensity increase. The phase spectra showed two important features: (1) at frequencies about half octave below the BF, phase slope is very small, indicating an extremely short delay; (2) the greatest phase lag occurs at frequencies near the BF, indicating a significant delay near this frequency range.

Adhesion-dependent tyrosine phosphorylation of (beta)-dystroglycan regulates its interaction with utrophin.

Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated (beta)-dystroglycan and utrophin from cell extracts. Western blotting of (&bgr;)-dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and (beta)-dystroglycan by co-immunoprecipitation with endogenous (beta)-dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous (beta)-dystroglycan was tyrosine phosphorylated, (beta)-dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide 'SPOTs' assays confirmed that tyrosine phosphorylation of (beta)-dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y(892) in the (beta)-dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the (beta)-dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.