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1.  Extracellular divalent cations modulate aminoglycoside-induced hair cell death in the zebrafish lateral line 
Hearing research  2009;253(1-2):42-51.
Aminoglycoside antibiotics cause death of sensory hair cells. Research over the past decade has identified several key players in the intracellular cascade. However, the role of the extracellular environment in aminoglycoside ototoxicity has received comparatively little attention. The present study uses the zebrafish lateral line to demonstrate that extracellular calcium and magnesium ions modulate hair cell death from neomycin and gentamicin in vivo, with high levels of either divalent cation providing significant protection. Imaging experiments with fluorescently tagged gentamicin show that drug uptake is reduced under high calcium conditions. Treating fish with the hair cell transduction blocker amiloride also reduces aminoglycoside uptake, preventing the toxicity, and experiments with variable calcium and amiloride concentrations suggest complementary effects between the two protectants. Elevated magnesium, in contrast, does not appear to significantly attenuate drug uptake, suggesting that the two divalent cations may protect hair cells from aminoglycoside damage through different mechanisms. These results provide additional evidence for calcium- and transduction-dependent aminoglycoside uptake. Divalent cations provided differential protection from neomycin and gentamicin, with high cation concentrations almost completely protecting hair cells from neomycin and acute gentamicin toxicity, but offering reduced protection from continuous (6 hr) gentamicin exposure. These experiments lend further support to the hypothesis that aminoglycoside toxicity occurs via multiple pathways in a both a drug and time course-specific manner.
doi:10.1016/j.heares.2009.03.004
PMCID: PMC5139914  PMID: 19285547
Hair cell; Ototoxicity; Aminoglycoside; Lateral line; Calcium; Magnesium
2.  Mitochondrial calcium uptake underlies ROS generation during aminoglycoside-induced hair cell death 
The Journal of Clinical Investigation  null;126(9):3556-3566.
Exposure to aminoglycoside antibiotics can lead to the generation of toxic levels of reactive oxygen species (ROS) within mechanosensory hair cells of the inner ear that have been implicated in hearing and balance disorders. Better understanding of the origin of aminoglycoside-induced ROS could focus the development of therapies aimed at preventing this event. In this work, we used the zebrafish lateral line system to monitor the dynamic behavior of mitochondrial and cytoplasmic oxidation occurring within the same dying hair cell following exposure to aminoglycosides. The increased oxidation observed in both mitochondria and cytoplasm of dying hair cells was highly correlated with mitochondrial calcium uptake. Application of the mitochondrial uniporter inhibitor Ru360 reduced mitochondrial and cytoplasmic oxidation, suggesting that mitochondrial calcium drives ROS generation during aminoglycoside-induced hair cell death. Furthermore, targeting mitochondria with free radical scavengers conferred superior protection against aminoglycoside exposure compared with identical, untargeted scavengers. Our findings suggest that targeted therapies aimed at preventing mitochondrial oxidation have therapeutic potential to ameliorate the toxic effects of aminoglycoside exposure.
doi:10.1172/JCI84939
PMCID: PMC5004972  PMID: 27500493
3.  Selective Deletion of Cochlear Hair Cells Causes Rapid Age-Dependent Changes in Spiral Ganglion and Cochlear Nucleus Neurons 
The Journal of Neuroscience  2015;35(20):7878-7891.
During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival.
doi:10.1523/JNEUROSCI.2179-14.2015
PMCID: PMC4438131  PMID: 25995473
cochlea; cochlear nucleus; critical period; diphtheria toxin receptor knock-in; neuronal death; spiral ganglion
4.  A Simple Method for Multi-Day Imaging of Slice Cultures 
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.
doi:10.1002/jemt.20750
PMCID: PMC2797827  PMID: 19565635
slice culture; single-cell gene electroporation; multi-day imaging
5.  ER–Mitochondrial Calcium Flow Underlies Vulnerability of Mechanosensory Hair Cells to Damage 
The Journal of Neuroscience  2014;34(29):9703-9719.
Mechanosensory hair cells are vulnerable to environmental insult, resulting in hearing and balance disorders. We demonstrate that directional compartmental flow of intracellular Ca2+ underlies death in zebrafish lateral line hair cells after exposure to aminoglycoside antibiotics, a well characterized hair cell toxin. Ca2+ is mobilized from the ER and transferred to mitochondria via IP3 channels with little cytoplasmic leakage. Pharmacological agents that shunt ER-derived Ca2+ directly to cytoplasm mitigate toxicity, indicating that high cytoplasmic Ca2+ levels alone are not cytotoxic. Inhibition of the mitochondrial transition pore sensitizes hair cells to the toxic effects of aminoglycosides, contrasting with current models of excitotoxicity. Hair cells display efficient ER–mitochondrial Ca2+ flow, suggesting that tight coupling of these organelles drives mitochondrial activity under physiological conditions at the cost of increased susceptibility to toxins.
doi:10.1523/JNEUROSCI.0281-14.2014
PMCID: PMC4099547  PMID: 25031409
lateral line; zebrafish
6.  Using the zebrafish lateral line to uncover novel mechanisms of action and prevention in drug-induced hair cell death 
The majority of hearing loss and balance disorders are caused by the permanent loss of mechanosensory hair cells of the inner ear. Identification of genes and compounds that modulate susceptibility to hair cell death is frequently confounded by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for genetic and chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of mechanosensory hair cells within the lateral line sensory organ, uniquely position it as an ideal model organism for the study of hair cell toxicity. We have used this model to screen for genes and compounds that affect hair cell survival during ototoxin exposure and have identified agents that would not be expected to play a role in this process based on a priori knowledge of their function. The identification of such agents yields better understanding of hair cell death and holds promise to stem hearing loss and balance disorders in the human population.
doi:10.3389/fncel.2015.00046
PMCID: PMC4332341  PMID: 25741241
aminoglycoside; chemical genetics; cisplatin; hair cell; lateral line; mechanotransduction; ototoxicity; zebrafish
7.  Cilia-Associated Genes Play Differing Roles in Aminoglycoside-Induced Hair Cell Death in Zebrafish 
G3: Genes|Genomes|Genetics  2016;6(7):2225-2235.
Hair cells possess a single primary cilium, called the kinocilium, early in development. While the kinocilium is lost in auditory hair cells of most species it is maintained in vestibular hair cells. It has generally been believed that the primary role of the kinocilium and cilia-associated genes in hair cells is in the establishment of the polarity of actin-based stereocilia, the hair cell mechanotransduction apparatus. Through genetic screening and testing of candidate genes in zebrafish (Danio rerio) we have found that mutations in multiple cilia genes implicated in intraflagellar transport (dync2h1, wdr35, ift88, and traf3ip), and the ciliary transition zone (cc2d2a, mks1, and cep290) lead to resistance to aminoglycoside-induced hair cell death. These genes appear to have differing roles in hair cells, as mutations in intraflagellar transport genes, but not transition zone genes, lead to defects in kinocilia formation and processes dependent upon hair cell mechanotransduction activity. These mutants highlight a novel role of cilia-associated genes in hair cells, and provide powerful tools for further study.
doi:10.1534/g3.116.030080
PMCID: PMC4938675  PMID: 27207957
aminoglycosides; cilia; hair cells; intraflagellar transport; transition zone
8.  Fractalkine Signaling Regulates Macrophage Recruitment into the Cochlea and Promotes the Survival of Spiral Ganglion Neurons after Selective Hair Cell Lesion 
The Journal of Neuroscience  2015;35(45):15050-15061.
Macrophages are recruited into the cochlea in response to injury caused by acoustic trauma or ototoxicity, but the nature of the interaction between macrophages and the sensory structures of the inner ear remains unclear. The present study examined the role of fractalkine signaling in regulating the injury-evoked behavior of macrophages following the selective ablation of cochlear hair cells. We used a novel transgenic mouse model in which the human diphtheria toxin receptor (huDTR) is selectively expressed under the control of Pou4f3, a hair cell-specific transcription factor. Administration of diphtheria toxin (DT) to these mice resulted in nearly complete ablation of cochlear hair cells, with no evident pathology among supporting cells, spiral ganglion neurons, or cells of the cochlear lateral wall. Hair cell death led to an increase in macrophages associated with the sensory epithelium of the cochlea. Their numbers peaked at 14 days after DT and then declined at later survival times. Increased macrophages were also observed within the spiral ganglion, but their numbers remained elevated for (at least) 56 d after DT. To investigate the role of fractalkine signaling in macrophage recruitment, we crossed huDTR mice to a mouse line that lacks expression of the fractalkine receptor (CX3CR1). Disruption of fractalkine signaling reduced macrophage recruitment into both the sensory epithelium and spiral ganglion and also resulted in diminished survival of spiral ganglion neurons after hair cell death. Our results suggest a fractalkine-mediated interaction between macrophages and the neurons of the cochlea.
SIGNIFICANCE STATEMENT It is known that damage to the inner ear leads to recruitment of inflammatory cells (macrophages), but the chemical signals that initiate this recruitment and the functions of macrophages in the damaged ear are unclear. Here we show that fractalkine signaling regulates macrophage recruitment into the cochlea and also promotes the survival of cochlear afferents after selective hair cell lesion. Because these afferent neurons carry sound information from the cochlea to the auditory brainstem, their survival is a key determinant of the success of cochlear prosthetics. Our data suggest that fractalkine signaling in the cochlea is neuroprotective, and reveal a previously uncharacterized interaction between cells of the cochlea and the innate immune system.
doi:10.1523/JNEUROSCI.2325-15.2015
PMCID: PMC4642237  PMID: 26558776
cochlea; fractalkine; hair cells; macrophages; neuroprotection; spiral ganglion neurons
9.  A Brief History of Hair Cell Regeneration Research and Speculations on the Future 
Hearing research  2013;297:42-51.
Millions of people worldwide suffer from hearing and balance disorders caused by loss of the sensory hair cells that convert sound vibrations and head movements into electrical signals that are conveyed to the brain. In mammals, the great majority of hair cells are produced during embryogenesis. Hair cells that are lost after birth are virtually irreplaceable, leading to permanent disability. Other vertebrates, such as fish and amphibians produce hair cells throughout life. However, hair cell replacement after damage to the mature inner ear was either not investigated or assumed to be impossible until studies in the late 1980s proved this to be false. Adult birds were shown to regenerate lost hair cells in the auditory sensory epithelium after noise- and ototoxic drug-induced damage. Since then, the field of hair cell regeneration has continued to investigate the capacity of the auditory and vestibular epithelia in vertebrates (fishes, birds, reptiles, and mammals) to regenerate hair cells and to recover function, the molecular mechanisms governing these regenerative capabilities, and the prospect of designing biologically-based treatments for hearing loss and balance disorders. Here, we review the major findings of the field during the past 25 years and speculate how future inner ear repair may one day be achieved.
doi:10.1016/j.heares.2012.12.014
PMCID: PMC3657556  PMID: 23321648
hair cell regeneration; historical review; proliferation; transdifferentiation; sensory epithelia; hair cells; supporting cells
10.  Transgenic Quail as a Model for Research in the Avian Nervous System – A Comparative Study of the Auditory Brainstem 
Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proven problematic. As a result, experiments aimed at genetic manipulations on birds remained difficult for this popular research tool. Recently, lentiviral methods have enabled production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal-specificity and stable expression of eGFP across generations (termed here as GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of gene manipulation, we compared the development, organization, structure and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) to that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM) and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.
doi:10.1002/cne.23187
PMCID: PMC3488602  PMID: 22806400
transgenic quail; auditory brainstem
11.  Identification of small molecule inhibitors of cisplatin-induced hair cell death: Results of a 10,000 compound screen in the zebrafish lateral line 
Hypothesis
The zebrafish lateral line can be used to identify small molecules that protect against cisplatin-induced hair cell death.
Background
Cisplatin is a commonly used chemotherapeutic which causes hearing loss by damaging hair cells of the inner ear. There are currently no FDA-approved pharmacologic strategies for preventing this side effect. The zebrafish lateral line has been used successfully in the past to study hair cell death and protection.
Methods
In this study we used the zebrafish lateral line to screen a library of 10,000 small molecules for protection against cisplatin-induced hair cell death. Dose-response relationships for identified protectants were determined by quantifying hair cell protection. The effect of each protectant on cisplatin uptake was quantified.
Results
From this screen we identified two compounds exhibiting dose-dependent protection: Cisplatin Hair Cell Protectant 1 and 2 (CHCP1 and 2). CHCP1 reduced uptake of a fluorescent cisplatin analog suggesting its protective effects may be due to decreased cisplatin uptake. CHCP2 did not affect uptake, which suggests an intracellular mechanism of action. Evaluation of analogs of CHCP2 revealed three additional compounds that significantly reduced cisplatin-induced hair cell death, though none exceed the effectiveness or potency of the parent compound.
Conclusion
The zebrafish lateral line was used to identify two small molecules that protected against cisplatin-induced hair cell death.
doi:10.1097/MAO.0000000000000487
PMCID: PMC4332566  PMID: 25687728
12.  Disruption of intracellular calcium regulation is integral to aminoglycoside-induced hair cell death 
Intracellular Ca2+ is a key regulator of life or death decisions in cultured neurons and sensory cells. The role of Ca2+ in these processes is less clear in vivo, as the location of these cells often impedes visualization of intracellular Ca2+ dynamics. We generated transgenic zebrafish lines that express the genetically encoded Ca2+ indicator GCaMP in mechanosensory hair cells of the lateral line. These lines allow us to monitor intracellular Ca2+ dynamics in real time during aminoglycoside-induced hair cell death. Following exposure of live larvae to aminoglycosides, dying hair cells undergo a transient increase in intracellular Ca2+ that occurs shortly after mitochondrial membrane potential collapse. Inhibition of intracellular Ca2+ elevation through either caged chelators or pharmacological inhibitors of Ca2+ effectors mitigates toxic effects of aminoglycoside exposure. Conversely, artificial elevation of intracellular Ca2+ by caged Ca2+ release agents sensitizes hair cells to the toxic effects of aminoglycosides. These data suggest that alterations in intracellular Ca2+ homeostasis play an essential role in aminoglycoside-induced hair cell death, and indicate several potential therapeutic targets to stem ototoxicity.
doi:10.1523/JNEUROSCI.4559-12.2013
PMCID: PMC3703319  PMID: 23616556
13.  Estradiol selectively enhances auditory function in avian forebrain neurons 
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.
doi:10.1523/JNEUROSCI.3938-12.2012
PMCID: PMC3532894  PMID: 23223283
14.  Glial Cell Contributions to Auditory Brainstem Development 
Glial cells, previously thought to have generally supporting roles in the central nervous system, are emerging as essential contributors to multiple aspects of neuronal circuit function and development. This review focuses on the contributions of glial cells to the development of auditory pathways in the brainstem. These pathways display specialized synapses and an unusually high degree of precision in circuitry that enables sound source localization. The development of these pathways thus requires highly coordinated molecular and cellular mechanisms. Several classes of glial cells, including astrocytes, oligodendrocytes and microglia, have now been explored in these circuits in both avian and mammalian brainstems. Distinct populations of astrocytes are found over the course of auditory brainstem maturation. Early appearing astrocytes are associated with spatial compartments in the avian auditory brainstem. Factors from late appearing astrocytes promote synaptogenesis and dendritic maturation, and astrocytes remain integral parts of specialized auditory synapses. Oligodendrocytes play a unique role in both birds and mammals in highly regulated myelination essential for proper timing to decipher interaural cues. Microglia arise early in brainstem development and may contribute to maturation of auditory pathways. Together these studies demonstrate the importance of non-neuronal cells in the assembly of specialized auditory brainstem circuits.
doi:10.3389/fncir.2016.00083
PMCID: PMC5073128  PMID: 27818624
nucleus laminaris; nucleus magnocellularis; medial nucleus of the trapezoid body; astrocyte; oligodendrocyte; delay line; microglia; calyx of Held
15.  In vivo Reversible Regulation of Dendritic Patterning by Afferent Input in Bipolar Auditory Neurons 
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.
doi:10.1523/JNEUROSCI.1737-12.2012
PMCID: PMC3435435  PMID: 22895732
16.  RELATIVE INPUT STRENGTH RAPIDLY REGULATES DENDRITIC STRUCTURE of CHICK auditory brainstem NEURONS 
The Journal of comparative neurology  2011;519(14):2838-2851.
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.
doi:10.1002/cne.22656
PMCID: PMC3196591  PMID: 21500196
dendrites; activity; competition; auditory; time-lapse imaging
17.  Identification of Modulators of Hair Cell Regeneratin in the Zebrafish Lateral Line 
The Journal of Neuroscience  2012;32(10):3516-3528.
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.
doi:10.1523/JNEUROSCI.3905-11.2012
PMCID: PMC3318954  PMID: 22399774
18.  Dynamic Spike Thresholds during Synaptic Integration Preserve and Enhance Temporal Response Properties in the Avian Cochlear Nucleus 
The Journal of Neuroscience  2010;30(36):12063-12074.
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.
doi:10.1523/JNEUROSCI.1840-10.2010
PMCID: PMC3390778  PMID: 20826669
19.  Topography and Morphology of the Inhibitory Projection From Superior Olivary Nucleus to Nucleus Laminaris in Chickens (Gallus gallus) 
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.
doi:10.1002/cne.22523
PMCID: PMC3299086  PMID: 21165979
auditory brainstem; axonal projection; γ-aminobutyric acid (GABA); interaural time difference (ITD); tonotopic organization
20.  Response of mechanosensory hair cells of the zebrafish lateral line to aminoglycosides reveals distinct cell death pathways 
Hearing research  2009;253(1-2):32-41.
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.
doi:10.1016/j.heares.2009.03.001
PMCID: PMC3167481  PMID: 19285126
ototoxicity; aminoglycoside; hair cell; mechanosensory; lateral line; zebrafish; hair cell
21.  Peripheral auditory processing changes seasonally in Gambel’s white-crowned sparrow 
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.
doi:10.1007/s00359-010-0545-1
PMCID: PMC2909381  PMID: 20563817
songbird; hormone; season; auditory; ABR
22.  Seasonal Plasticity of Precise Spike Timing in the Avian Auditory System 
The Journal of Neuroscience  2015;35(8):3431-3445.
Vertebrate audition is a dynamic process, capable of exhibiting both short- and long-term adaptations to varying listening conditions. Precise spike timing has long been known to play an important role in auditory encoding, but its role in sensory plasticity remains largely unexplored. We addressed this issue in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii), a songbird that shows pronounced seasonal fluctuations in circulating levels of sex-steroid hormones, which are known to be potent neuromodulators of auditory function. We recorded extracellular single-unit activity in the auditory forebrain of males and females under different breeding conditions and used a computational approach to explore two potential strategies for the neural discrimination of sound level: one based on spike counts and one based on spike timing reliability. We report that breeding condition has robust sex-specific effects on spike timing. Specifically, in females, breeding condition increases the proportion of cells that rely solely on spike timing information and increases the temporal resolution required for optimal intensity encoding. Furthermore, in a functionally distinct subset of cells that are particularly well suited for amplitude encoding, female breeding condition enhances spike timing-based discrimination accuracy. No effects of breeding condition were observed in males. Our results suggest that high-resolution temporal discharge patterns may provide a plastic neural substrate for sensory coding.
doi:10.1523/JNEUROSCI.3407-14.2015
PMCID: PMC4339354  PMID: 25716843
auditory; hormone; neural coding; plasticity; seasonal; spike timing
23.  Mechanisms for Adjusting Interaural Time Differences to Achieve Binaural Coincidence Detection 
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.
doi:10.1523/JNEUROSCI.3464-09.2010
PMCID: PMC2822993  PMID: 20053889
Sound; Localization; Auditory; Brainstem; Axon; Conduction; Velocity
24.  Compartment-Specific Regulation of Plasma Membrane Calcium ATPase Type 2 in the Chick Auditory Brainstem 
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.
doi:10.1002/cne.22045
PMCID: PMC2702515  PMID: 19365819
calcium homeostasis; afferent regulation; dendritic remodeling; activity-dependency; nucleus laminaris
25.  Cisplatin-Induced Hair Cell Death Requires STAT1 and is Attenuated by Epigallocatechin Gallate 
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.
doi:10.1523/JNEUROSCI.5842-08.2009
PMCID: PMC2707781  PMID: 19321781
vestibular; inflammation; stress; transcription factor; cell fate; hair cells; toxicity

Results 1-25 (63)