Auditory performance in bald eagles and red-tailed hawks: a comparative study of hearing in diurnal raptors.

Collision with wind turbines is a conservation concern for eagles with population abundance implications. The development of acoustic alerting technologies to deter eagles from entering hazardous air spaces is a potentially significant mitigation strategy to diminish associated morbidity and mortality risks. As a prelude to the engineering of deterrence technologies, auditory function was assessed in bald eagles (Haliaeetus leucocephalus), as well as in red-tailed hawks (Buteo jamaicensis). Auditory brainstem responses (ABRs) to a comprehensive battery of clicks and tone bursts varying in level and frequency were acquired to evaluate response thresholds, as well as suprathreshold response characteristics of wave I of the ABR, which represents the compound potential of the VIII cranial nerve. Sensitivity curves exhibited an asymmetric convex shape similar to those of other avian species, response latencies decreased exponentially with increasing stimulus level and response amplitudes grew with level in an orderly manner. Both species were responsive to a frequency band at least four octaves wide, with a most sensitive frequency of 2 kHz, and a high-frequency limit of approximately 5.7 kHz in bald eagles and 8 kHz in red-tailed hawks. Findings reported here provide a framework within which acoustic alerting signals might be developed.

Input-output curves of low and high spontaneous rate auditory nerve fibers are exponential near threshold.

Input-output (IO) properties of cochlear transduction are frequently determined by analyzing the average discharge rates of auditory nerve fibers (ANFs) in response to relatively long tonal stimulation. The ANFs in cats have spontaneous discharge rates (SRs) that are bimodally distributed, peaking at low (<0.5 spikes/s) and high (∼60 spikes/s) rates, and rate-level characteristics differ depending upon SR. In an effort to assess the instantaneous IO properties of ANFs having different SRs, static IO-curves were constructed from period histograms based on phase-locking of spikes to the stimulus waveform. These curves provide information unavailable in conventional average rate-level curves. We find that all IO curves follow an exponential trajectory. It is argued that the exponential behavior represents the transduction in the IHC and that the difference among ANFs having different SRs is predominantly a difference in gain attributed most likely to synaptic drive. © 2018 The authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A mouse model of miR-96, miR-182 and miR-183 misexpression implicates miRNAs in cochlear cell fate and homeostasis.

Germline mutations in Mir96, one of three co-expressed polycistronic miRNA genes (Mir96, Mir182, Mir183), cause hereditary hearing loss in humans and mice. Transgenic FVB/NCrl- Tg(GFAP-Mir183,Mir96,Mir182)MDW1 mice (Tg1MDW), which overexpress this neurosensory-specific miRNA cluster in the inner ear, were developed as a model system to identify, in the aggregate, target genes and biologic processes regulated by the miR-183 cluster. Histological assessments demonstrate Tg1MDW/1MDW homozygotes have a modest increase in cochlear inner hair cells (IHCs). Affymetrix mRNA microarray data analysis revealed that downregulated genes in P5 Tg1MDW/1MDW cochlea are statistically enriched for evolutionarily conserved predicted miR-96, miR-182 or miR-183 target sites. ABR and DPOAE tests from 18 days to 3 months of age revealed that Tg1MDW/1MDW homozygotes develop progressive neurosensory hearing loss that correlates with histologic assessments showing massive losses of both IHCs and outer hair cells (OHCs). This mammalian miRNA misexpression model demonstrates a potency and specificity of cochlear homeostasis for one of the dozens of endogenously co-expressed, evolutionally conserved, small non-protein coding miRNA families. It should be a valuable tool to predict and elucidate miRNA-regulated genes and integrated functional gene expression networks that significantly influence neurosensory cell differentiation, maturation and homeostasis.

Identifying microRNAs involved in degeneration of the organ of corti during age-related hearing loss.

MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA targets, are important regulators of cellular senescence and aging. We questioned which miRNAs are involved in age-related degeneration of the organ of Corti (OC), the auditory sensory epithelium that transduces mechanical stimuli to electrical activity in the inner ear. Degeneration of the OC is generally accepted as the main cause of age-related hearing loss (ARHL), a progressive loss of hearing in individuals as they grow older. To determine which miRNAs are involved in the onset and progression of ARHL, miRNA gene expression in the OC of two mouse strains, C57BL/6J and CBA/J, was compared at three different ages using GeneChip miRNA microarray and was validated by real-time PCR. We showed that 111 and 71 miRNAs exhibited differential expression in the C57 and CBA mice, respectively, and that downregulated miRNAs substantially outnumbered upregulated miRNAs during aging. miRNAs that had approximately 2-fold upregulation included members of miR-29 family and miR-34 family, which are known regulators of pro-apoptotic pathways. In contrast, miRNAs that were downregulated by about 2-fold were members of the miR-181 family and miR-183 family, which are known to be important for proliferation and differentiation, respectively. The shift of miRNA expression favoring apoptosis occurred earlier than detectable hearing threshold elevation and hair cell loss. Our study suggests that changes in miRNA expression precede morphological and functional changes, and that upregulation of pro-apoptotic miRNAs and downregulation of miRNAs promoting proliferation and differentiation are both involved in age-related degeneration of the OC.

Discrimination of individual tigers (Panthera tigris) from long distance roars.

This paper investigates the extent of tiger (Panthera tigris) vocal individuality through both qualitative and quantitative approaches using long distance roars from six individual tigers at Omaha's Henry Doorly Zoo in Omaha, NE. The framework for comparison across individuals includes statistical and discriminant function analysis across whole vocalization measures and statistical pattern classification using a hidden Markov model (HMM) with frame-based spectral features comprised of Greenwood frequency cepstral coefficients. Individual discrimination accuracy is evaluated as a function of spectral model complexity, represented by the number of mixtures in the underlying Gaussian mixture model (GMM), and temporal model complexity, represented by the number of sequential states in the HMM. Results indicate that the temporal pattern of the vocalization is the most significant factor in accurate discrimination. Overall baseline discrimination accuracy for this data set is about 70% using high level features without complex spectral or temporal models. Accuracy increases to about 80% when more complex spectral models (multiple mixture GMMs) are incorporated, and increases to a final accuracy of 90% when more detailed temporal models (10-state HMMs) are used. Classification accuracy is stable across a relatively wide range of configurations in terms of spectral and temporal model resolution.

Probing cochlear tuning and tonotopy in the tiger using otoacoustic emissions.

Otoacoustic emissions (sound emitted from the ear) allow cochlear function to be probed noninvasively. The emissions evoked by pure tones, known as stimulus-frequency emissions (SFOAEs), have been shown to provide reliable estimates of peripheral frequency tuning in a variety of mammalian and non-mammalian species. Here, we apply the same methodology to explore peripheral auditory function in the largest member of the cat family, the tiger (Panthera tigris). We measured SFOAEs in 9 unique ears of 5 anesthetized tigers. The tigers, housed at the Henry Doorly Zoo (Omaha, NE), were of both sexes and ranged in age from 3 to 10 years. SFOAE phase-gradient delays are significantly longer in tigers--by approximately a factor of two above 2 kHz and even more at lower frequencies--than in domestic cats (Felis catus), a species commonly used in auditory studies. Based on correlations between tuning and delay established in other species, our results imply that cochlear tuning in the tiger is significantly sharper than in domestic cat and appears comparable to that of humans. Furthermore, the SFOAE data indicate that tigers have a larger tonotopic mapping constant (mm/octave) than domestic cats. A larger mapping constant in tiger is consistent both with auditory brainstem response thresholds (that suggest a lower upper frequency limit of hearing for the tiger than domestic cat) and with measurements of basilar-membrane length (about 1.5 times longer in the tiger than domestic cat).

Adapted to roar: functional morphology of tiger and lion vocal folds.

Vocal production requires active control of the respiratory system, larynx and vocal tract. Vocal sounds in mammals are produced by flow-induced vocal fold oscillation, which requires vocal fold tissue that can sustain the mechanical stress during phonation. Our understanding of the relationship between morphology and vocal function of vocal folds is very limited. Here we tested the hypothesis that vocal fold morphology and viscoelastic properties allow a prediction of fundamental frequency range of sounds that can be produced, and minimal lung pressure necessary to initiate phonation. We tested the hypothesis in lions and tigers who are well-known for producing low frequency and very loud roaring sounds that expose vocal folds to large stresses. In histological sections, we found that the Panthera vocal fold lamina propria consists of a lateral region with adipocytes embedded in a network of collagen and elastin fibers and hyaluronan. There is also a medial region that contains only fibrous proteins and hyaluronan but no fat cells. Young's moduli range between 10 and 2000 kPa for strains up to 60%. Shear moduli ranged between 0.1 and 2 kPa and differed between layers. Biomechanical and morphological data were used to make predictions of fundamental frequency and subglottal pressure ranges. Such predictions agreed well with measurements from natural phonation and phonation of excised larynges, respectively. We assume that fat shapes Panthera vocal folds into an advantageous geometry for phonation and it protects vocal folds. Its primary function is probably not to increase vocal fold mass as suggested previously. The large square-shaped Panthera vocal fold eases phonation onset and thereby extends the dynamic range of the voice.

Mature mice lacking Rbl2/p130 gene have supernumerary inner ear hair cells and supporting cells.

Adult mammalian auditory hair cells (HCs) and their associated supporting cells (SCs) do not proliferate, and HC death leads to irreversible neurosensory hearing loss and balance impairment. In nonmammalian vertebrates, loss of HCs induces mitotic proliferation of adjacent nonsensory SCs and/or direct SC transdifferentiation to generate replacement cells. This results in the structural and functional recovery of the nonmammalian sensory systems. Potential replacement of mammalian auditory HCs, either by transplanting cells or by transforming existing cells through molecular therapy, has long been proposed. However, HC replacement strategies with clear therapeutic potential remain elusive. The retinoblastoma (pRB) family of cell cycle regulators, Rb1, Rbl1 (p107), and Rbl2 (p130), regulate the G(1)- to S-phase transition in proliferating cells. In the inner ear, the biochemical and molecular pathways involving pRBs, particularly p107 and p130, are relatively unexplored and their therapeutic suitability is yet to be determined. In this study, we analyzed the cochleae of adult p130 knock-out (p130(-/-)) mice and showed that lack of the p130 gene results in extra rows of HCs and SCs in the more apical regions of the cochlea. No evidence of transdifferentiation of these supernumerary SCs into HCs was observed in the p130(-/-) mouse. Nevertheless, unscheduled proliferation of SCs in the adult p130(-/-) cochlea coupled to downregulation of bona fide cell cycle inhibitors provides a mechanistic basis for the role of p130 as a regulator of SC and HC mitotic quiescence in the more apical regions of the cochlea. Interestingly, p130(-/-) mice exhibited nearly normal peripheral auditory sensitivity.

Vocal power and pressure-flow relationships in excised tiger larynges.

Despite the functional importance of loud, low-pitched vocalizations in big cats of the genus Panthera, little is known about the physics and physiology of the mechanisms producing such calls. We investigated laryngeal sound production in the laboratory using an excised-larynx setup combined with sound-level measurements and pressure-flow instrumentation. The larynges of five tigers (three Siberian or Amur, one generic non-pedigreed tiger with Bengal ancestry and one Sumatran), which had died of natural causes, were provided by Omaha's Henry Doorly Zoo over a five-year period. Anatomical investigation indicated the presence of both a rigid cartilaginous plate in the arytenoid portion of the glottis, and a vocal fold fused with a ventricular fold. Both of these features have been confusingly termed 'vocal pads' in the previous literature. We successfully induced phonation in all of these larynges. Our results showed that aerodynamic power in the glottis was of the order of 1.0 W for all specimens, acoustic power radiated (without a vocal tract) was of the order of 0.1 mW, and fundamental frequency ranged between 20 and 100 Hz when a lung pressure in the range of 0-2.0 kPa was applied. The mean glottal airflow increased to the order of 1.0 l s(-1) per 1.0 kPa of pressure, which is predictable from scaling human and canine larynges by glottal length and vibrational amplitude. Phonation threshold pressure was remarkably low, on the order of 0.3 kPa, which is lower than for human and canine larynges phonated without a vocal tract. Our results indicate that a vocal fold length approximately three times greater than that of humans is predictive of the low fundamental frequency, and the extraordinarily flat and broad medial surface of the vocal folds is predictive of the low phonation threshold pressure.

Gamma-actin is required for cytoskeletal maintenance but not development.

Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.

The influence of thyroid hormone deficiency on the development of cochlear nonlinearities.

It is well known that failure to treat severe congenital hypothyroidism leads to profound auditory disability, and it has been suggested that an intracochlear defect, or defects, associated with the condition diminishes the efficacy of an active, physiologically vulnerable nonlinear transduction process commonly referred to as cochlear amplification. We address this question directly by tracking the development of threshold-frequency (tuning) curves and two-tone suppression in hypothyroid, Tshr mutant mice born to hypothyroid dams and comparing those findings with findings observed in euthyroid mice. Like sharp tuning, two-tone suppression is a product of transduction nonlinearity and is a useful indicator of the functional status of cochlear amplification. In contrast to euthyroid mice that acquire sharp tuning, normal two-tone suppression, and adultlike sensitivity by the end of the third postnatal week, as shown in earlier studies, hypothyroid mice remained grossly insensitive to sound throughout life. In addition, tuning was generally broad in hypothyroid mice, tuning curve "tips" were frequently missing, and two-tone suppression was rarely observed. However, unlike tip thresholds, tuning curve "tail" thresholds, a feature that reflects the functional status of passive elements of transduction, acquired normal values over a roughly 2-month postnatal time period. These observations collectively suggest that active transduction micromechanics, at least in the frequency region studied here, are profoundly affected by thyroid hormone and support speculation that abnormal outer hair cell function may be the cause of the primary, enduring peripheral auditory defect associated with profound, congenital hypothyroidism in the Tshr mutant mouse.

Development of cochlear amplification, frequency tuning, and two-tone suppression in the mouse.

It is generally believed that the micromechanics of active cochlear transduction mature later than passive elements among altricial mammals. One consequence of this developmental order is the loss of transduction linearity, because an active, physiologically vulnerable process is superimposed on the passive elements of transduction. A triad of sensory advantage is gained as a consequence of acquiring active mechanics; sensitivity and frequency selectivity (frequency tuning) are enhanced and dynamic operating range increases. Evidence supporting this view is provided in this study by tracking the development of tuning curves in BALB/c mice. Active transduction, commonly known as cochlear amplification, enhances sensitivity in a narrow frequency band associated with the "tip" of the tuning curve. Passive aspects of transduction were assessed by considering the thresholds of responses elicited from the tuning curve "tail," a frequency region that lies below the active transduction zone. The magnitude of cochlear amplification was considered by computing tuning curve tip-to-tail ratios, a commonly used index of active transduction gain. Tuning curve tip thresholds, frequency selectivity and tip-to-tail ratios, all indices of the functional status of active biomechanics, matured between 2 and 7 days after tail thresholds achieved adultlike values. Additionally, two-tone suppression, another product of active cochlear transduction, was first observed in association with the earliest appearance of tuning curve tips and matured along an equivalent time course. These findings support a traditional view of development in which the maturation of passive transduction precedes the maturation of active mechanics in the most sensitive region of the mouse cochlea.

A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.

Thyroid hormone receptors TRalpha1 and TRbeta differentially regulate gene expression of Kcnq4 and prestin during final differentiation of outer hair cells.

Thyroid hormone (TH or T3) and TH-receptor beta (TRbeta) have been reported to be relevant for cochlear development and hearing function. Mutations in the TRbeta gene result in deafness associated with resistance to TH syndrome. The effect of TRalpha1 on neither hearing function nor cochlear T3 target genes has been described to date. It is also uncertain whether TRalpha1 and TRbeta can act simultaneously on different target genes within a single cell. We focused on two concomitantly expressed outer hair cell genes, the potassium channel Kcnq4 and the motor protein prestin Slc26a5. In outer hair cells, TH enhanced the expression of the prestin gene through TRbeta. Simultaneously Kcnq4 expression was activated in the same cells by derepression of TRalpha1 aporeceptors mediated by an identified THresponse element, which modulates KCNQ4 promoter activity. We show that T3 target genes can differ in their sensitivity to TH receptors having the ligand either bound (holoreceptors) or not bound (aporeceptors) within single cells, and suggest a role for TRalpha1 in final cell differentiation.

Consequences of combined maternal, fetal and persistent postnatal hypothyroidism on the development of auditory function in Tshrhyt mutant mice.

Tshrhyt/hyt mutant mice express a point mutation in the gene encoding the thyrotropin receptor, and affected animals are congenitally hypothyroid and profoundly deaf as a consequence when the condition is untreated. In this investigation, a previously unrecognized developmental stage was identified in the hypothyroid, mutant progeny of hypothyroid dams by tracking developmental changes in the auditory brainstem response (ABR). ABR thresholds develop rapidly in normal, euthyroid animals, decreasing as much as 80 dB between P12 (postnatal day 12) and P15, with mature sensitivity being gradually acquired by P18. In contrast, Tshrhyt/hyt mutant mice remained profoundly deaf on P24 and although thresholds improved by approximately 30 dB by P60, residual frequency-dependent deficits of 20-70 dB were observed in animals exhibiting end-stage disease. The rate of threshold improvement in mutant mice was approximately ten times slower than in normal mice. While ABR wave latencies and interpeak intervals decreased early in postnatal life, values decreased over a delayed and protracted time period, reaching adult values well after those of controls attained maturity. As with normal mice, slopes of wave I latency-intensity curves were significantly steeper in immature animals than those observed in adults and decreased during development, but failed to achieve normal adult values and remained significantly steeper than those for controls. Findings reported here suggest that passive aspects of electromechanical transduction achieve maturity in Tshrhyt/hyt progeny of Tshrhyt/hyt mice and that development, limited as it may be, occurs most prominently in the basal half of the cochlea.

The very large G-protein-coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles.

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Frequency- and level-dependent changes in auditory brainstem responses (ABRS) in developing mice.

The development of the auditory brainstem response was studied to quantitatively assess its dependence on stimulus frequency and level. Responses were not observed to stimuli > or =16 kHz on P12, however, the full range of responsive frequencies included in the study was observed by P14. Response thresholds were high on P12, exceeding 100 dB SPL for all stimuli tested. The rate of threshold development increased progressively for stimulus frequencies between -2 and 10 kHz, with the most rapid changes occurring at frequencies >10 kHz. Adultlike thresholds were observed by P18. Response latencies and interpeak intervals matured rapidly over the course of the second and third postnatal weeks and did not achieve adultlike characteristics until after P18. Latencies of higher-order peaks were progressively and sequentially delayed relative to wave I. Wave I amplitudes developed nonmonotonically, growing during the first 24 days and stabilizing at adult values by approximately P36. Slopes of wave I amplitude-and latency-level curves were significantly steeper than those of adults during the neonatal period and the outcome of input-output analyses, as well as frequency-specific maturational profiles, support developmental models in which function initially matures in the mid-frequency range and proceeds, simultaneously, in both apical and basal directions.

Local community vs. national industry : The TMI and Santa Barbara protests compared

Comparing two examples of local protest-one a failure and the other a success. This paper shows the importance of the interaction between local groups and national social movements in predicting the outcomes of struggles pitting communities against established industries. A critical difference between the unsuccessful attempt of Santa Barbara activists to prevent the resumption of oil drilling after the 1969 spill, on the one hand, and the successful efforts of TMI residents to block the restart of the reactor adjacent to the one damaged in the 1979 nuclear accident, on the other, is the maturity of the national environmental movement. Focusing primarily on the relatively successful TMI protest, the paper emphasizes the importance of the national antinuclear movement's resources in forcing resourseful actors (individual and organizational) into adversarial positions vis-à-vis the offending utility (AU)