Afferent input regulates neuronal dendritic patterning locally and globally through distinct mechanisms. To begin to understand these mechanisms, we differentially manipulate afferent input in vivo and assess effects on dendritic patterning of individual neurons in chicken nucleus laminaris (NL). Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Blocking action potentials from one ear, by either cochlea removal or temporary treatment with tetrodotoxin (TTX), leads to rapid and significant retraction of affected NL dendrites (dorsal ipsilaterally and ventral contralaterally) within 8h as compared to the other dendrites of the same neurons. The degree of retraction is comparable to that induced by direct deafferentation resulting from transection of NM axons. Importantly, when inner ear activity is allowed to recover from TTX treatments, retracted NL dendrites regrow to their normal length within 48h. The retraction and growth involve elimination of terminal branches and addition of new branches. Examination of changes in NL dendrites at 96h following unilateral cochlear removal, a manipulation that induces cell loss in NM and persistent blockage of afferent excitatory action potentials, reveals a significant correlation between cell death in the ipsilateral NM and the degree of dendritic retraction in NL. These results demonstrate that presynaptic action potentials rapidly and reversibly regulate dendritic patterning of postsynaptic neurons in a compartment specific manner, while long-term dendritic maintenance may be regulated in a way that is correlated with the presence of silent presynaptic appositions.
Competition between presynaptic inputs has been suggested to shape dendritic form. This hypothesis can be directly tested on bitufted, auditory neurons in chicken nucleus laminaris (NL). Each NL neuron contains two relatively symmetrical dendritic arbors; the dorsal dendrite receives excitatory glutamatergic input from the ipsilateral ear and the ventral dendrites receive corresponding input from the contralateral ear. To assess the effect of relative synaptic strength on NL dendrites, we used single cell electroporation, electrophysiology and live, two-photon laser scanning microscopy to manipulate both the amount and the balance of synaptic input to the two matching sets of dendrites. With simultaneous activation, both sets of dendrites changed together, either growing or retracting over the imaging period. In contrast, stimulation of only one set of dendrites (either dorsal or ventral) resulted in the unstimulated dendrites losing total dendritic branch length, while the stimulated dendrites exhibited a tendency to grow. In this system, balanced input leads to balanced changes in the two sets of dendrites while imbalanced input results in differential changes. Time-lapse imaging revealed that NL dendrites respond to differential stimulation by first decreasing the size of their unstimulated dendrites, and then increasing the size of their stimulated dendrites. This result suggests that the relative activity of presynaptic neurons dynamically controls dendritic structure in NL, and that dendritic real estate can rapidly be shifted from inactive inputs to active inputs.
dendrites; activity; competition; auditory; time-lapse imaging
The external location of the zebrafish lateral line makes it a powerful model for studying mechanosensory hair cell regeneration. We have developed a chemical screen to identify FDA-approved drugs and biologically active compounds that modulate hair cell regeneration in zebrafish. Of the 1,680 compounds evaluated, we identified 2 enhancers and 6 inhibitors of regeneration. The two enhancers, dexamethasone and prednisolone, are synthetic glucocorticoids that potentiated hair cell numbers during regeneration and also induced hair cell addition in the absence of damage. BrdU analysis confirmed that the extra hair cells arose from mitotic activity. We found that dexamethasone and prednisolone, like other glucocorticoids, suppress zebrafish caudal fin regeneration, indicating that hair cell regeneration occurs by a distinctly different process. Further analyses of the regeneration inhibitors revealed that two of the six, flubendazole and topotecan, significantly suppress hair cell regeneration by preventing proliferation of hair cell precursors. Flubendazole halted support cell division in M-phase, possibly by interfering with normal microtubule activity. Topotecan, a topoisomerase inhibitor, killed both hair cells and proliferating hair cell precursors. A third inhibitor, fulvestrant, moderately delays hair cell regeneration by reducing support cell proliferation. Our observation that hair cells do not regenerate when support cell proliferation is impeded confirms previous observations that cell division is the primary route for hair cell regeneration after neomycin treatment in zebrafish.
Neurons of the cochlear nuclei are anatomically and physiologically specialized to optimally encode temporal and spectral information about sound stimuli, in part for binaural auditory processing. The avian cochlear nucleus magnocellularis (NM) integrates excitatory eighth nerve inputs and depolarizing GABAergic inhibition such that temporal fidelity is enhanced across the synapse. The biophysical mechanisms of this depolarizing inhibition, and its role in temporal processing, are not fully understood. We used whole-cell electro-physiology and computational modeling to examine how subthreshold excitatory inputs are integrated and how depolarizing IPSPs affect spike thresholds and synaptic integration by chick NM neurons. We found that both depolarizing inhibition and subthreshold excitatory inputs cause voltage threshold accommodation, nonlinear temporal summation, and shunting. Inhibition caused such large changes in threshold that subthreshold excitatory inputs were followed by a refractory period. We hypothesize that these large shifts in threshold eliminate spikes to asynchronous inputs, providing a mechanism for the enhanced temporal fidelity seen across the eighth nerve/cochlear nucleus synapse. Thus, depolarizing inhibition and threshold shifting hone the temporal response properties of this system so as to enhance the temporal fidelity that is essential for auditory perception.
Auditory experience during development is necessary for normal language acquisition in humans. Although songbirds, some cetaceans, and maybe bats may also be vocal learners, vocal learning has yet to be well established for a laboratory mammal. Mice are potentially an excellent model organism for studying mechanisms underlying vocal communication. Mice vocalize in different social contexts, yet whether they learn their vocalizations remains unresolved. To address this question, we compared ultrasonic courtship vocalizations emitted by chronically deaf and normal hearing adult male mice. We deafened CBA/CaJ male mice, engineered to express diphtheria toxin (DT) receptors in hair cells, by systemic injection of DT at postnatal day 2 (P2). By P9, almost all inner hair cells were absent and by P16 all inner and outer hair cells were absent in DTR mice. These mice did not show any auditory brainstem responses as adults. Wild-type littermates, also treated with DT at P2, had normal hair cells and normal auditory brainstem responses. We compared the temporal structure of vocalization bouts, the types of vocalizations, the patterns of syllables, and the acoustic features of each syllable type emitted by hearing and deaf males in the presence of a female. We found that almost all of the vocalization features we examined were similar in hearing and deaf animals. These findings indicate that mice do not need auditory experience during development to produce normal ultrasonic vocalizations in adulthood. We conclude that mouse courtship vocalizations are not acquired through auditory feedback-dependent learning.
Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.
The organotypic slice culture (Stoppini et al., 1991) has become the method of choice to answer a variety of questions in neuroscience. For many experiments however, it would be beneficial to image or manipulate a slice culture repeatedly, for example over the course of many days.
We prepared organotypic slice cultures of the auditory brainstem of P3 and P4 mice and kept them in vitro for up to 4 weeks. Single cells in the auditory brainstem were transfected with plasmids expressing fluorescent proteins by way of electroporation (Haas et al., 2001). The culture was then placed in a chamber perfused with oxygenated ACSF and the labeled cell imaged with an inverted wide-field microscope repeatedly for multiple days, recording several time-points per day, before returning the slice to the incubator.
We describe a simple method to image a slice culture preparation over to the course of multiple days and over many continuous hours, without noticeable damage to the tissue or photobleaching. Our method employs a simple, inexpensive custom-built insulator constructed around the microscope to maintain controlled temperature, and uses a perfusion chamber as used for in vitro slice recordings.
slice culture; single-cell gene electroporation; multi-day imaging
Understanding binaural perception requires detailed analyses of the neural circuitry responsible for the computation of interaural time differences (ITDs). In the avian brainstem, this circuit consists of internal axonal delay lines innervating an array of coincidence detector neurons that encode external ITDs. Nucleus magnocellularis (NM) neurons project to the dorsal dendritic field of the ipsilateral nucleus laminaris (NL) and to the ventral field of the contralateral NL. Contralateral-projecting axons form a delay line system along a band of NL neurons. Binaural acoustic signals in the form of phase-locked action potentials from NM cells arrive at NL and establish a topographic map of sound source location along the azimuth. These pathways are assumed to represent a circuit similar to the Jeffress model of sound localization, establishing a place code along an isofrequency contour of NL. Three-dimensional measurements of axon lengths reveal major discrepancies with the current model; the temporal offset based on conduction length alone makes encoding of physiological ITDs impossible. However, axon diameter and distances between Nodes of Ranvier also influence signal propagation times along an axon. Our measurements of these parameters reveal that diameter and internode distance can compensate for the temporal offset inferred from axon lengths alone. Together with other recent studies these unexpected results should inspire new thinking on the cellular biology, evolution and plasticity of the circuitry underlying low frequency sound localization in both birds and mammals.
Sound; Localization; Auditory; Brainstem; Axon; Conduction; Velocity
Calcium signaling plays a role in synaptic regulation of dendritic structure, usually on the time scale of hours or days. Here we use immunocytochemistry to examine changes in expression of the plasma membrane calcium ATPase type 2 (PMCA2), a high-affinity calcium efflux protein, in the chick nucleus laminaris (NL) following manipulations of synaptic inputs. Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Deprivation of the contralateral projection from NM to NL leads to rapid retraction of ventral, but not the dorsal, dendrites of NL neurons. Immunocytochemistry revealed symmetric distribution of PMCA2 in two neuropil regions of normally innervated NL. Electron microscopy confirmed that PMCA2 localizes in both NM terminals and NL dendrites. As early as 30 minutes following transection of the contralateral projection from NM to NL or unilateral cochlea removal, significant decreases in PMCA2 immunoreactivity were seen in the deprived neuropil of NL compared to the other neuropil which continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for the microtubule-associated protein 2, which affects cytoskeleton stabilization. These results suggest that PMCA2 is regulated independently in ventral and dorsal NL dendrites and/or their inputs from NM in a way that is correlated with presynaptic activity. This provides a potential mechanism by which deprivation can change calcium transport that, in turn, may be important for rapid, compartment-specific dendritic remodeling.
calcium homeostasis; afferent regulation; dendritic remodeling; activity-dependency; nucleus laminaris
Sex steroids modulate vertebrate sensory processing, but the impact of circulating hormone levels on forebrain function remains unclear. We tested the hypothesis that circulating sex steroids modulate single-unit responses in the avian telencephalic auditory nucleus, field L. We mimicked breeding or non-breeding conditions by manipulating plasma 17β-estradiol levels in wild-caught female Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii). Extracellular responses of single neurons to tones and conspecific songs presented over a range of intensities revealed that estradiol selectively enhanced auditory function in cells that exhibited monotonic rate-level functions to pure tones. In these cells, estradiol treatment increased spontaneous and maximum evoked firing rates, increased pure tone response strengths and sensitivity, and expanded the range of intensities over which conspecific song stimuli elicited significant responses. Estradiol did not significantly alter the sensitivity or dynamic ranges of cells that exhibited non-monotonic rate-level functions. Notably, there was a robust correlation between plasma estradiol concentrations in individual birds and physiological response properties in monotonic, but not non-monotonic neurons. These findings demonstrate that functionally distinct classes of anatomically overlapping forebrain neurons are differentially regulated by sex steroid hormones in a dose-dependent manner.
Cisplatin is a chemotherapy drug that frequently causes auditory impairment due to the death of mechanosensory hair cells. Cisplatin ototoxicity may result from oxidative stress, DNA damage, and inflammatory cytokines. The transcription factor STAT1, an important mediator of cell death, can regulate all of these processes in other cell types. We used cultured utricles from mature Swiss Webster mice to investigate the role of STAT1 in cisplatin-induced hair cell death. We show that STAT1 phosphorylation is an early event in both hair cells and support cells following exposure of utricles to cisplatin. STAT1 phosphorylation peaked after 4 hours of cisplatin exposure and returned to control levels by 8 hours of exposure. The STAT1 inhibitor epigallocatechin gallate (EGCG) attenuated STAT1 phosphorylation in cisplatin-treated utricles and resulted in concentration-dependent increases in hair cell survival at 24 hours post-exposure. Furthermore, we show that utricular hair cells from STAT1-deficient mice are resistant to cisplatin toxicity. EGCG failed to provide additional protection from cisplatin in STAT1-deficient mice, further supporting the hypothesis that the protective effects of EGCG are due to its inhibition of STAT1. Treatment with IFN-γ, which also causes STAT1 activation, also induced hair cell death in wildtype but not STAT1-deficient mice. These results show that STAT1 is required for maximal cisplatin-induced hair cell death in the mouse utricle and suggest that treatment with EGCG may be a useful strategy for prevention of cisplatin ototoxicity.
vestibular; inflammation; stress; transcription factor; cell fate; hair cells; toxicity
The zebrafish lateral line is an efficient model system for the evaluation of chemicals that protect and damage hair cells. Located on the surface of the body, lateral line hair cells are accessible for manipulation and visualization. The zebrafish lateral line system allows rapid screens of large chemical libraries, as well as subsequent thorough evaluation of interesting compounds. In this review, we focus on the results of our previous screens and the evolving methodology of our screens for chemicals that protect hair cells, and chemicals that damage hair cells using the zebrafish lateral line.
zebrafish; lateral line; hair cells; chemical screen
Differential innervation of segregated dendritic domains in the chick nucleus laminaris (NL), composed of third-order auditory neurons, provides a unique model to study synaptic regulation of dendritic structure. Altering the synaptic input to one dendritic domain affects the structure and length of the manipulated dendrites while leaving the other set of unmanipulated dendrites largely unchanged. Little is known about the effects of neuronal input on the cytoskeletal structure of NL dendrites and whether changes in the cytoskeleton are responsible for dendritic remodeling following manipulations of synaptic inputs. In this study, we investigate changes in the immunoreactivity of high-molecular weight microtubule associated protein 2 (MAP2) in NL dendrites following two different manipulations of their afferent input. Unilateral cochlea removal eliminates excitatory synaptic input to the ventral dendrites of the contralateral NL and the dorsal dendrites of the ipsilateral NL. This manipulation produced a dramatic decrease in MAP2 immunoreactivity in the deafferented dendrites. This decrease was detected as early as three hours following the surgery, well before any degeneration of afferent axons. A similar decrease in MAP2 immunoreactivity in deafferented NL dendrites was detected following a midline transection that silences the excitatory synaptic input to the ventral dendrites on both sides of the brain. These changes were most distinct in the caudal portion of the nucleus where individual deafferented dendritic branches contained less immunoreactivity than intact dendrites. Our results suggest that the cytoskeletal protein MAP2, which is distributed in dendrites, perikarya, and postsynaptic densities, may play a role in deafferentation-induced dendritic remodeling.
deafferentation; dendritic plasticity; afferent regulation; cytoskeleton
We developed a transgenic mouse to permit conditional and selective ablation of hair cells in the adult mouse utricle by inserting the human diphtheria toxin receptor (DTR) gene into the Pou4f3 gene, which encodes a hair cell-specific transcription factor. In adult wild-type mice, administration of diphtheria toxin (DT) caused no significant hair cell loss. In adult Pou4f3
+/DTR mice, DT treatment reduced hair cell numbers to 6% of normal by 14 days post-DT. Remaining hair cells were located primarily in the lateral extrastriola. Over time, hair cell numbers increased in these regions, reaching 17% of untreated Pou4f3
+/DTR mice by 60 days post-DT. Replacement hair cells were morphologically distinct, with multiple cytoplasmic processes, and displayed evidence for active mechanotransduction channels and synapses characteristic of type II hair cells. Three lines of evidence suggest replacement hair cells were derived via direct (nonmitotic) transdifferentiation of supporting cells: new hair cells did not incorporate BrdU, supporting cells upregulated the pro-hair cell gene Atoh1, and supporting cell numbers decreased over time. This study introduces a new method for efficient conditional hair cell ablation in adult mouse utricles and demonstrates that hair cells are spontaneously regenerated in vivo in regions where there may be ongoing hair cell turnover.
The chick embryo (Gallus domesticus) is one of the most important model systems in vertebrate developmental biology. The development and function of its auditory brainstem circuitry is exceptionally well studied. These circuits represent an excellent system for genetic manipulation to investigate mechanisms controlling neural circuit formation, synaptogenesis, neuronal polarity and dendritic arborization. The present study investigates the auditory nucleus, nucleus magnocellularis (NM). The neurotrophin receptor TrkB regulates dendritic structure in CNS neurons. TrkB is expressed at NM neurons at E7–E8 when these neurons have dendritic arbors. Downregulation of TrkB occurs after E8 followed by retraction of dendrites and by E18 most NM cells are adendritic. Is cessation of TrkB expression in NM necessary for dendritic retraction? To answer this question we combined focal in ovo electroporation with transposon mediated gene transfer to obtain stable expression of Doxycycline (Dox) regulated transgenes, specifically TrkB coexpressed with EGFP in a temporally controlled manner. Electroporation was performed at E2 and Dox added onto the chorioallointoic membrane from E7.5 to E16. Expression of EGFP had no effect on development of the embryo, or cell morphology and organization of auditory brainstem nuclei. NM cells expressing EGFP and TrkB at E17–E18 had dendrites and biophysical properties uncharacteristic for normal NM cells, indicating that cessation of TrkB expression is essential for dendrite retraction and functional maturation of these neurons. These studies indicate that expression of transposon based plasmids is an effective method to genetically manipulate events in mid to late embryonic brain development in chick.
The relationships between protein synthesis and neuronal survival are poorly understood. In chicken nucleus magnocellularis (NM), significant alterations in overall protein synthesis precede neuronal death induced by deprivation of excitatory afferent activity. Previously we demonstrated an initial reduction in the overall rate of protein synthesis in all deprived NM neurons, followed by quick recovery (starting at 6h) in some, but not all, neurons. Neurons with recovered protein synthesis ultimately survive while others become “ghost” cells (no detectable Nissl substance) at 12–24h and die within 48h. To explore the mechanisms underlying this differential influence of afferent input on protein synthesis and cell survival, the current study investigates the involvement of eukaryotic translation elongation factor 2 (eEF2), the phosphorylation of which reduces overall protein synthesis. Using immunocytochemistry for either total or phosphorylated eEF2 (p-eEF2), we find significant reductions in the level of phosphorylated, but not total, eEF2 in NM neurons as early as 0.5h to 1h following cochlea removal. Unexpectedly, neurons with low levels of p-eEF2 show reduced protein synthesis at 6h indicated by a marker for active ribosomes. At 12h, all “ghost” cells exhibited little or no p-eEF2 staining although not every neuron with a comparable low level of p-eEF2 was a “ghost” cell. These observations demonstrate that a reduced level of p-eEF2 is not responsible for immediate responses (including reduced overall protein synthesis) of a neuron to compromised afferent input, but may impair the neuron’s ability to initiate recovery signaling for survival and make the neuron more vulnerable to death.
apoptosis; cell death; protein synthesis; cochlea removal; afferent deprivation; nucleus magnocellularis
Spectral counting methods provide an easy means of identifying proteins with differing abundances between complex mixtures using shotgun proteomics data. The crux spectral-counts command, implemented as part of the Crux software toolkit, implements four previously reported spectral counting methods, the spectral index (SIN), the exponentially modified protein abundance index (emPAI), the normalized spectral abundance factor (NSAF), and the distributed normalized spectral abundance factor (dNSAF).
We compared the reproducibility and the linearity relative to each protein’s abundance of the four spectral counting metrics. Our analysis suggests that NSAF yields the most reproducible counts across technical and biological replicates, and both SIN and NSAF achieve the best linearity.
With the crux spectral-counts command, Crux provides open-source modular methods to analyze mass spectrometry data for identifying and now quantifying peptides and proteins. The C++ source code, compiled binaries, spectra and sequence databases are available at
GABAergic modulation of activity in avian cochlear nucleus neurons has been studied extensively in vitro. However, how this modulation actually influences processing in vivo is not known. We investigated responses of chicken nucleus magnocellularis (NM) neurons to sound while pharmacologically manipulating the inhibitory input from the superior olivary nucleus (SON). SON receives excitatory inputs from nucleus angularis (NA) and nucleus laminaris (NL), and provides GABAergic inputs to NM, NA, NL, and putatively to the contralateral SON. Results from single unit extracellular recordings from 2–4 wks posthatch chickens show that firing rates of auditory nerve fibers (ANFs) increased monotonically with sound intensity, while that of NM neurons saturated or even decreased at moderate or loud sound levels. Blocking GABAergic input with local application of TTX into the SON induced an increase in firing rate of ipsilateral NM while that of the contralateral NM decreased at high sound levels. Moreover, local application of bicuculline to NM also increased the firing rate of NM neurons at high sound levels, reduced phase-locking, and broadened the frequency tuning properties of NM neurons. Following application of DNQX, clear evidence of inhibition was observed. Furthermore, the inhibition was tuned to a broader frequency range than the excitatory response areas. We conclude that GABAergic inhibition from SON has at least three physiological influences on the activity of NM neurons: it regulates the firing activity of NM units in a sound-level dependent manner; it improves phase selectivity; and it sharpens frequency tuning of NM neuronal responses.
Superior olivary nucleus; Cochlear nucleus; Bicuculline; GABA; Auditory; In vivo
The avian nucleus laminaris (NL) is involved in computation of interaural time differences (ITDs) that encode the azimuthal position of a sound source. Neurons in NL are bipolar, with dorsal and ventral dendritic arbors receiving input from separate ears. NL neurons act as coincidence detectors that respond maximally when input from each ear arrives at the two dendritic arbors simultaneously. Computational and physiological studies demonstrated that the sensitivity of NL neurons to coincident inputs is modulated by an inhibitory feedback circuit via the superior olivary nucleus (SON). To understand the mechanism of this modulation, the topography of the projection from SON to NL was mapped, and the morphology of the axon terminals of SON neurons in NL was examined in chickens (Gallus gallus). In vivo injection of AlexaFluor 568 dextran amine into SON demonstrated a coarse topographic projection from SON to NL. Retrogradely labeled neurons in NL were located within the zone of anterogradely labeled terminals, suggesting a reciprocal projection from SON to NL. In vivo extracellular physiological recording further demonstrated that this topography is consistent with tonotopic maps in SON and NL. In addition, three-dimensional reconstruction of single SON axon branches within NL revealed that individual SON neurons innervate a large area of NL and terminate on both dorsal and ventral dendritic arbors of NL neurons. The organization of the projection from SON to NL supports its proposed functions of controlling the overall activity level of NL and enhancing the specificity of frequency mapping and ITD detection.
auditory brainstem; axonal projection; γ-aminobutyric acid (GABA); interaural time difference (ITD); tonotopic organization
The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration.
We report a series of experiments investigating the kinetics of hair cell loss in lateral line neuromasts of zebrafish larvae following exposure to aminoglycoside antibiotics. Comparisons of the rate of hair cell loss and the differential effects of acute versus chronic exposure to gentamicin and neomycin revealed markedly different results. Neomycin induced rapid and dramatic concentration-dependent hair cell loss that is essentially complete within 90 minutes, regardless of concentration or exposure time. Gentamicin induced loss of half of the hair cells within 90 minutes and substantial additional loss, which was prolonged and cumulative over exposure times up to at least 24 hr. Small molecules and genetic mutations that inhibit neomycin-induced hair cell loss were ineffective against prolonged gentamicin exposure supporting the hypothesis that these two drugs are revealing at least two cellular pathways. The mechanosensory channel blocker amiloride blocked both neomycin and gentamicin-induced hair cell death acutely and chronically indicating that these aminoglycosides share a common entry route. Further tests with additional aminoglycosides revealed a spectrum of differential responses to acute and chronic exposure. The distinctions between the times of action of these aminoglycosides indicate that these drugs induce multiple cell death pathways.
ototoxicity; aminoglycoside; hair cell; mechanosensory; lateral line; zebrafish; hair cell
Song in oscine birds is a learned behavior that plays important roles in breeding. Pronounced seasonal differences in song behavior, and in the morphology and physiology of the neural circuit underlying song production are well documented in many songbird species. Androgenic and estrogenic hormones largely mediate these seasonal changes. While much work has focused on the hormonal mechanisms underlying seasonal plasticity in songbird vocal production, relatively less work has investigated seasonal and hormonal effects on songbird auditory processing, particularly at a peripheral level. We addressed this issue in Gambel’s white-crowned sparrow (Zonotrichia leucophrys gambelii), a highly seasonal breeder. Photoperiod and hormone levels were manipulated in the laboratory to simulate natural breeding and non-breeding conditions. Peripheral auditory function was assessed by measuring the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs) of males and females in both conditions. Birds exposed to breeding-like conditions demonstrated elevated thresholds and prolonged peak latencies compared with birds housed under non-breeding-like conditions. There were no changes in DPOAEs, however, which indicates that the seasonal differences in ABRs do not arise from changes in hair cell function. These results suggest that seasons and hormones impact auditory processing as well as vocal production in wild songbirds.
songbird; hormone; season; auditory; ABR
Several animal models have been used for the study of mechanosensory hair cells and hearing loss. Because of the difficulty of tissue acquisition and large animal size, these traditional models are impractical for high-throughput screening. The zebrafish has emerged as a powerful animal model for screening drugs that cause or prevent hair cell death. The unique characteristics of the zebrafish enable rapid in vivo imaging of hair cells and hair cell death. We have used this model to screen for and identify multiple drugs that protect hair cells from aminoglycoside-induced death. Identification of multiple drugs and drug-like compounds that inhibit multiple hair cell death pathways might enable the development of protective cocktails to achieve complete hair cell protection.
Usher syndrome is the leading cause of combined deaf-blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock-in mice. Absent auditory-evoked brainstem responses indicated that the mutant mice are deaf at one month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock-in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies.
Usher syndrome; deafness; retinal degeneration; mouse model
In humans, most hearing loss results from death of hair cells, the mechanosensory receptors of the inner ear. Two goals of current hearing research are to protect hair cells from degeneration and to regenerate new hair cells, replacing those that are lost due to aging, disease, or environmental challenges. One limitation of research in the auditory field has been the relative inaccessibility of the mechanosensory systems in the inner ear. Zebrafish possess hair cells in both their inner ear and their lateral line system that are morphologically and functionally similar to human hair cells. The external location of the mechanosensory hair cells in the lateral line and the ease of in vivo labeling and imaging make the zebrafish lateral line a unique system for the study of hair cell toxicity, protection, and regeneration. This review focuses on the lateral line system as a model for understanding loss and protection of mechanosensory hair cells. We discuss chemical screens to identify compounds that induce hair cell loss and others that protect hair cells from known toxins and the potential application of these screens to human medicine.