Effects of Age on the Distortion Product Otoacoustic Emission Growth Functions

Hearing Research. Jan, 2002  |  Pubmed ID: 11788199

Age-related hearing loss (presbycusis) is thought to result from age-related degeneration (aging) of the cochlea plus the cumulative effects of extrinsic damage (noise and other ototoxic agents) and intrinsic disorders (e.g. systemic diseases). Previous studies have implicated dysfunction of the hair cells (sensory presbycusis) as the principal mechanism of age-related hearing loss. However, recent evidence from quiet-reared gerbils suggests that cochlear aging results primarily from atrophy of the stria vascularis, which is associated with diminished endocochlear potential (EP), spiral ganglion atrophy, and a relatively flat audiometric loss, termed metabolic presbycusis. Because it is not currently possible to measure EP directly in the clinical setting, we wondered if cochlear metabolic dysfunction might be evidenced indirectly from existing clinical tests, specifically, the input-output (IO) growth function of the distortion product (DP) otoacoustic emissions in relation to behavioral hearing threshold levels (HTL). We anticipated finding discordance between the IO functions and HTL with either a greater decline with age in HTL than in IO functions if an age-related metabolic dysfunction of the cochlea was operant, or a greater loss of IO function than HTL if outer hair cell dysfunction was the dominant pathology. To address this supposition we analyzed existing auditory data from a large cohort of adults to determine the change with age in three aspects of the DP IO function: area under the curve, threshold, and slope. The analyses demonstrated a greater effect of age on HTL than on the DP IO measures. This effect supports the hypothesis that strial dysfunction is a substantive factor in cochlear aging. The etiology and mechanisms for this dysfunction are conjectural at present.

Choosing Axonal Real Estate: Location, Location, Location

The Journal of Comparative Neurology. Jun, 2002  |  Pubmed ID: 12012372

Bcl-2 Overexpression Eliminates Deprivation-induced Cell Death of Brainstem Auditory Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2002  |  Pubmed ID: 12040073

Deprivation of afferent input in young animals results in transneuronal degeneration of postsynaptic sensory neurons in a variety of species and sensory pathways. Transneuronal degeneration is generally not seen in adult animals. The cellular and molecular basis for this dramatic developmental change in susceptibility is not understood. One possibility is that genes involved in the apoptotic process are involved in determining cell death or survival after afferent deprivation. To further investigate this possibility, we performed unilateral cochlear ablation on wild-type and bcl-2-overexpressing mice at a variety of ages. In postnatal day 5 (P5) or P8 wild-type mice, cochlea removal resulted in a 54% or 31% neuronal loss in the anteroventral cochlear nucleus (AVCN), respectively. When the same manipulation is performed on a P30 mouse, no loss of AVCN neurons occurs. This confirmed a rather abrupt change in the sensitivity to disruption of afferent input, a critical period. However, in littermates expressing bcl-2 under a neuron-specific enolase promoter, no significant loss of AVCN neurons was observed at any age after unilateral cochlear ablation. Furthermore, wild-type mice demonstrate rapid expression of activated caspase-3 in AVCN neurons within hours of deafferentation, whereas bcl-2-overexpressing mice do not. This suggests that bcl-2 can influence cell survival after removal of afferent input during the critical period and is consistent with the hypothesis that caspase-3 is one effector of cell death under these circumstances. These data are the first to indicate that known apoptotic mediators can play a role in central neuronal plasticity in models of afferent deprivation.

Auditory System Development: Primary Auditory Neurons and Their Targets

Annual Review of Neuroscience. 2002  |  Pubmed ID: 12052904

The neurons of the cochlear ganglion transmit acoustic information between the inner ear and the brain. These placodally derived neurons must produce a topographically precise pattern of connections in both the inner ear and the brain. In this review, we consider the current state of knowledge concerning the development of these neurons, their peripheral and central connections, and their influences on peripheral and central target cells. Relatively little is known about the cellular and molecular regulation of migration or the establishment of precise topographic connection to the hair cells or cochlear nucleus (CN) neurons. Studies of mice with neurotrophin deletions are beginning to yield increasing understanding of variations in ganglion cell survival and resulting innervation patterns, however. Finally, existing evidence suggests that while ganglion cells have little influence on the differentiation of their hair cell targets, quite the opposite is true in the brain. Ganglion cell innervation and synaptic activity are essential for normal development of neurons in the cochlear nucleus.

Vocal Memory and Learning in Adult Bengalese Finches with Regenerated Hair Cells

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2002  |  Pubmed ID: 12196601

Critical learning periods are common in vertebrate development. In many birds, song learning is limited by a critical period; juveniles copy songs from adult birds by forming memories of those songs during a restricted developmental period and then using auditory feedback to practice their own vocalizations. Adult songs are stable over time regardless of exposure to other birds, but auditory feedback is required for the maintenance of stable adult song. A technique was developed to reversibly deafen Bengalese Finches by destruction and regeneration of inner ear auditory hair cells. With this approach, we asked two questions about the plasticity of song information stored in the adult brain. First, do adult birds store memories or "templates" of their songs that exist independent of auditory reinforcement? Such memories could be used to control vocal output by acting as fixed models of song to which ongoing vocalizations are matched. Second, can adult song learning, which does not normally occur in this species, be induced by removing and then restoring hearing? Studying changes in adult song behavior during hair cell loss and regeneration revealed two findings: (1) adult birds store memories or templates of their songs that exist independent of auditory input and can be used to restore normal vocal behavior when hearing is restored; (2) under experimental circumstances, adult birds can be induced to acquire song material from other birds. Results suggest that, in Bengalese Finches, the degree of behavioral and neural plasticity in juvenile and adult birds may be less distinct that previously thought.

Expression of EphB Receptors and EphrinB Ligands in the Developing Chick Auditory Brainstem

The Journal of Comparative Neurology. Oct, 2002  |  Pubmed ID: 12205709

Nucleus magnocellularis (NM) in the avian auditory brainstem receives auditory input from nerve the VIIIth and projects bilaterally to nucleus laminaris (NL). This projection preserves binaural segregation in that ipsilateral NM projects to dorsal dendrites of NL and contralateral NM projects to ventral dendrites of NL. We have begun to examine the molecular signals that influence segregation of inputs onto discrete regions of NL cells. We previously showed that the Eph receptor, EphA4, is expressed selectively in the dorsal NL neuropil from embryonic day (E) 9 to E11, when NM axons grow into the NL neuropil. This asymmetric distribution suggests that EphA4 acts as a guidance molecule during binaural segregation. We report here on the developmental changes in the expression of two other Eph receptors, EphB2 and EphB5, and two ligands, ephrin-B1 and ephrin-B2, in the chick auditory brainstem. These proteins are expressed in the auditory nuclei during the maturation of the NM-NL projection. EphB2, EphB5, and ephrin-B1 are expressed in dorsal and ventral NL neuropil and at the midline of the brainstem at E10-E12. At this age, ephrin-B2, a ligand for EphB receptors and for EphA4, is expressed in NL cell bodies and NM-NL axons. The expression of these proteins diminishs in the posthatch ages examined. These results suggest that several members of the Eph family are involved in maturation of the nuclei and their projections. Moreover, ephrin-B2 in growing axons may interact with the asymmetrically expressed EphA4 during the establishment of binaural segregation.

Caspase Activation in Hair Cells of the Mouse Utricle Exposed to Neomycin

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2002  |  Pubmed ID: 12351727

Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating neomycin-induced hair cell death in this preparation.

Hair Cell Death in the Avian Basilar Papilla: Characterization of the in Vitro Model and Caspase Activation

Journal of the Association for Research in Otolaryngology : JARO. Mar, 2003  |  Pubmed ID: 12417974

Caspases are a family of proteases that have been implicated as key mediators of cell death. Although nonspecific inhibition of caspase activation has been reported to prevent mammalian sensory hair cell death, the exact roles of individual caspases during hair cell death are unclear. In other systems, the activation of initiator caspases, such as caspase-8 and caspase-9, can lead to the activation of the effector caspase-3. We have begun to systematically characterize hair cell death in an in vitro system by examining the activation of these specific caspases in degenerating hair cells after acutely damaging the whole avian basilar papilla with gentamicin. Basilar papillae (BP) displayed a dose-dependent hair cell loss after a 24-h treatment with gentamicin at concentrations of 0.1, 0.5, and 2.0 mM. When treated with 0.5 mM gentamicin for 6, 12, or 24 h, hair cells first began to degenerate in the basal third of the BP and damage progressed apically. Supplementation of z-VAD-fmk, a general caspase inhibitor, provided short-term protection against gentamicin-induced hair cell death. Treatment with gentamicin for 6 or 12 h promoted the expression of active caspase-3 and active caspase-9 in many hair cells along the BP as shown by immunohistochemistry. At these time-points, specific fluorescent-labeled peptide substrates detected more active caspase-3, caspase-8, and caspase-9 in gentamicin-treated hair cells relative to controls. Our data indicate that auditory hair cells degenerate as a result of gentamicin exposure in a caspase-dependent manner. Specifically, the upstream caspases, caspase-8 and caspase-9, and the downstream caspase-3 are activated in aminoglycoside-damaged hair cells.

Hair Cell Regeneration: Winging Our Way Towards a Sound Future

Current Opinion in Neurobiology. Feb, 2003  |  Pubmed ID: 12593990

The discovery of hair cell regeneration in the inner ear of birds provides new optimism that there may be a treatment for hearing and balance disorders. In this review we describe the process of hair cell regeneration in birds; including restoration of function, recovery of perception and what is currently known about molecular events, such as growth factors and signalling systems. We examine some of the key recent findings in both birds and mammals.

Ultrastructural Analysis of [3H]thymidine-labeled Cells in the Rat Utricular Macula

The Journal of Comparative Neurology. Aug, 2003  |  Pubmed ID: 12815755

Ototoxic drugs stimulate cell proliferation in adult rat vestibular sensory epithelia, as does the infusion of transforming growth factor alpha (TGFalpha) plus insulin. We sought to determine whether new hair cells can be regenerated by means of a mitotic pathway. Previously, studies have shown that the nuclei of some newly generated cells are located in the lumenal half of the sensory epithelium, suggesting that some may be newly generated sensory hair cells. The aim of this study was to examine the ultrastructural characteristics of newly proliferated cells after TGFalpha stimulation and/or aminoglycoside damage in the utricular sensory epithelium of the adult rat. The cell proliferation marker tritiated-thymidine was infused, with or without TGFalpha plus insulin, into the inner ears of normal or aminoglycoside-damaged rats for 3 or 7 days by means of osmotic pumps. Autoradiographic techniques and light microscopy were used to identify cells synthesizing DNA. Sections with labeled cells were re-embedded, processed for transmission electron microscopy, and the ultrastructural characteristics of the labeled cells were examined. The following five classes of tritiated-thymidine labeled cells were identified in the sensory epithelium: (1) labeled cells with synaptic specializations that appeared to be newly generated hair cells, (2) labeled supporting cells, (3) labeled leukocytes, (4) labeled cells that we have classified as "active cells" in that they are relatively nondescript but contain massive numbers of polyribosomes, and (5) labeled degenerating hair cells. These findings suggest that new hair cells can be generated in situ by means of a mitotic mechanism in the vestibular sensory epithelium of adult mammals.

Neomycin-induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio Rerio)

Journal of the Association for Research in Otolaryngology : JARO. Jun, 2003  |  Pubmed ID: 12943374

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 microM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24-48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Timing and Topography of Nucleus Magnocellularis Innervation by the Cochlear Ganglion

The Journal of Comparative Neurology. Nov, 2003  |  Pubmed ID: 14566951

This series of experiments examined the arrival and organization of cochlear nerve axons in the primary auditory brainstem nucleus, nucleus magnocellularis (NM), of the chick. DiI and DiD were injected into the cochlear nerve, cochlear ganglion, and basilar papilla (i.e., avian cochlea) in fixed tissue and labeled axons were studied in NM and its vicinity. Cochlear nerve axons first penetrate NM between stages 29 (E6) and 36 (E10). Axons penetrate NM in a middle-to-posterior-to-anterior developmental sequence; the anterior, high-frequency region of NM receives axons last. When cochlear nerve axons arrive in the NM, they are already organized in a topographic map related to the position of their cell bodies along the basilar papilla, foreshadowing the tonotopic mapping observed between NM and the basilar papilla later in development. Evidence of a topographic map was also observed in the other primary auditory brainstem nucleus, nucleus angularis. These results indicate that topographic mapping of position (and ultimately characteristic frequency) between the basilar papilla and NM is established as cochlear nerve axons arrive in the NM prior to the onset of synaptic activity. .

Developmental Differences in Susceptibility to Neomycin-induced Hair Cell Death in the Lateral Line Neuromasts of Zebrafish (Danio Rerio)

Hearing Research. Dec, 2003  |  Pubmed ID: 14644458

Mechanosensory hair cells of the inner ear are susceptible to death when exposed to a variety of drugs including aminoglycoside antibiotics. During avian and mammalian development, there is a period of relative insensitivity to aminoglycoside-induced hair cell death. This study was designed to test the hypothesis that zebrafish (Danio rerio) have developmental differences in sensitivity to aminoglycoside-induced hair cell death in the lateral line neuromasts. Larval zebrafish of various ages were exposed to several concentrations of neomycin, and their hair cells were examined using the potentiometric vital dye, DASPEI. Results indicate that zebrafish larvae aged 4 days post-fertilization are relatively insensitive to aminoglycoside-induced hair cell death compared to older fish. Thus zebrafish hair cells show developmental differences in sensitivity to aminoglycoside-induced death similar to those reported for inner ear hair cells of birds and mammals.

Electron Microscopy of Degenerative Changes in the Chick Basilar Papilla After Gentamicin Exposure

The Journal of Comparative Neurology. Mar, 2004  |  Pubmed ID: 14750159

We present a sequential study of the substructural alterations in the chick basilar papilla at the earliest signs of hair cell degeneration. Three-day posthatch chicks received a single injection of gentamicin (300 mg/kg) and were killed at 6, 8, 12, 15, 18, 21, and 24 hours after the injection. The basilar papillae were studied by conventional transmission electron microscopy. Examination was limited to the basal region, where all hair cells are eliminated by this treatment. As early as 8 hours and clearly by 12 hours, altered fine structure was seen in hair cells. Changes included rounding and swelling of the hair cells, condensation of nuclear chromatin, dissolution of ribosomes, dilatation of the mitochondria, and accumulation of inclusion bodies and lysosomes. By 15-18 hours, lysosomes increased and became denser, afferent terminals appeared swollen, and the first cell extrusion was seen. Efferents were unaffected, and supporting cells, though having inclusion bodies now, retained normal intercellular junctions. By 21-24 hours, large regions of complete hair cell loss were composed of expanded supporting cell processes with normal-appearing intercellular junctions and portions of extruded hair cells, partially attached to the supporting cell surface. These observations demonstrate that auditory hair cells undergo a rapid and controlled process of hair cell extrusion that allows preservation of the reticular lamina and minimal contamination of surrounding structures by intracytoplasmic contents of the damaged hair cells.

Activity-dependent Regulation of the Potassium Channel Subunits Kv1.1 and Kv3.1

The Journal of Comparative Neurology. Feb, 2004  |  Pubmed ID: 14755528

Afferent activity, especially in young animals, can have profound influences on postsynaptic neuronal structure, function and metabolic processes. Most studies evaluating activity regulation of cellular components have examined the expression of ubiquitous cellular proteins as opposed to molecules that are specialized in the neurons of interest. Here we consider the regulation of two proteins (voltage-gated potassium channel subunits Kv1.1 and Kv3.1) that auditory brainstem neurons in birds and mammals express at uniquely high levels. Unilateral removal of the avian cochlea leads to rapid and dramatic reduction in the expression of both proteins in the nucleus magnocellularis (NM; a division of the avian cochlear nucleus) neurons as detected by immunocytochemistry. Uniform downregulation of Kv1.1 was reliable by 3 hours after cochlea removal, was sustained through 96 hours, and returned to control levels in the surviving neurons by 2 weeks. The activity-dependent changes in Kv3.1 appear to be bimodal and are more transient, being observed at 3 hours after cochlea removal and recovering to control levels within 24 hours. We also explored the functional properties of Kv1.1 in NM neurons deprived of auditory input for 24 hours by whole-cell recordings. Low-threshold potassium currents in deprived NM neurons were not significantly different from control neurons in their amplitude or sensitivity to dendrotoxin-I, a selective K+ channel antagonist. We conclude that the highly specialized abundant expression of Kv1.1 and 3.1 channel subunits is not permanently regulated by synaptic activity and that changes in overall protein levels do not predict membrane pools.

A New Method for Imaging and 3D Reconstruction of Mammalian Cochlea by Fluorescent Confocal Microscopy

Brain Research. Mar, 2004  |  Pubmed ID: 15053969

Traditional methods for anatomical and morphometric studies of cochlear tissues have relied upon either microdissection of the organ of Corti or the generation of serial sections of the cochlea. Such methods are time-consuming, disruptive to three-dimensional relationships and often restrict sampling to very limited numbers of cells. We have found that cells and tissue components of the cochlear duct may be labelled by fluorescent markers within intact cochleae, which are then embedded in epoxy resin for subsequent viewing by fluorescent microscopy methods. This approach allows imaging through thick optical volumes with preservation of three-dimensional relationships. Unlike sectioned tissue, alignment of the sample relative to the focal axis may be easily corrected by re-orientation of the optical volume with common image processing software. Fluorescently labelled cochleae embedded in epoxy can be viewed by most fluorescent microscopy methods including laser scanning confocal microscopy, multi-photon confocal microscopy and widefield epi-fluorescence microscopy with deconvolution. Furthermore, semi-thin sections made from these preparations are compatible with traditional histological stains, as well as allowing brightly labelled epi-fluorescent images.

EphA4 Signaling Promotes Axon Segregation in the Developing Auditory System

Developmental Biology. May, 2004  |  Pubmed ID: 15081355

Precision of synaptic connections within neural circuits is essential for the accurate processing of sensory information. Specificity is exemplified at cellular and subcellular levels in the chick auditory brainstem, where nucleus magnocellularis (NM) neurons project bilaterally to nucleus laminaris (NL). Dorsal dendrites of NL neurons receive input from ipsilateral, but not contralateral, branches of NM axons whereas ventral dendrites are innervated by contralateral NM axons. This organization is analogous to that of the mammalian medial superior olive (MSO) and represents an important component of the circuitry underlying sound localization. However, the molecular mechanisms that establish segregated inputs to individual regions of NL neurons have not been identified. During synapse formation in NL, the EphA4 receptor is expressed in dorsal, but not ventral NL, neuropil, suggesting a potential role in targeting synapses to appropriate termination zones. Here, we directly tested this role by ectopically expressing EphA4 and disrupting EphA4 signaling using in ovo electroporation. We found that both misexpression of EphA4 and disruption of EphA4 signaling resulted in an increase in the number of NM axons that grow aberrantly across NL cell bodies into inappropriate regions of NL neuropil. EphA4 signaling is thus essential for targeting axons to distinct subsets of dendrites. Moreover, loss of EphA4 function resulted in morphological abnormalities of NL suggestive of errors in cell migration. These results suggest that EphA4 has multiple roles in the formation of auditory brainstem nuclei and their projections.

Round Window Gentamicin Application: an Inner Ear Hair Cell Damage Protocol for the Mouse

Hearing Research. Jun, 2004  |  Pubmed ID: 15157964

It is important to develop an inner ear damage protocol for mice that avoids systemic toxicity and produces damage in a relatively rapid fashion, allowing for study of early cellular and molecular mechanisms responsible for hair cell death and those that underlie the lack of hair cell regeneration in mammals. Ideally, this damage protocol would reliably produce both partial and complete lesions of the sensory epithelium. We present a method for in vivo induction of hair cell damage in the mouse via placement of gentamicin-soaked Gelfoam in the round window niche of the inner ear, an adaptation of a method developed to study hair cell regeneration in chicks. A total of 82 subjects underwent the procedure. Variable doses of gentamicin were used (25, 50, 100 and 200 microg). Saline-soaked Gelfoam, sham-operations and the contralateral, non-operated cochlea were used as controls. Survival periods were 1, 3 and 14 days. Damage was assessed on scanning electron microscopy. We found that this method produces relatively rapid hair cell damage that varies with dose and can extend the entire length of the sensory epithelium. In addition, this protocol produces no systemic toxicity and preserves the contralateral ear as a control.

Tonotopic Gradients of Eph Family Proteins in the Chick Nucleus Laminaris During Synaptogenesis

Journal of Neurobiology. Jul, 2004  |  Pubmed ID: 15188270

Topographically precise projections are established early in neural development. One such topographically organized network is the auditory brainstem. In the chick, the auditory nerve transmits auditory information from the cochlea to nucleus magnocellularis (NM). NM in turn innervates nucleus laminaris (NL) bilaterally. These projections preserve the tonotopy established at the level of the cochlea. We have begun to examine the expression of Eph family proteins during the formation of these connections. Optical density measurements were used to describe gradients of Eph proteins along the tonotopic axis of NL in the neuropil, the somata, and the NM axons innervating NL at embryonic day 10, when synaptic connections from NM to NL are established. At E10-11, NL dorsal neuropil expresses EphA4 at a higher concentration in regions encoding high frequency sounds, decreasing in concentration monotonically toward the low frequency (caudolateral) end. In the somata, both EphA4 and ephrin-B2 are concentrated at the high frequency end of the nucleus. These tonotopic gradients disappear between E13 and E15, and expression of these molecules is completely downregulated by hatching. The E10-11 patterns run counter to an apparent gradient in dendrite density, as indicated by microtubule associated protein 2 (MAP2) immunolabeling. Finally, ephrin-B2 is also expressed in a gradient in tissue ventral to the NL neuropil. Our findings thus suggest a possible conserved mechanism for establishing topographic projections in diverse sensory systems. These results of this study provide a basis for the functional examination of the role of Eph proteins in the formation of tonotopic maps in the brainstem.

Overexpression of Bcl-2 Prevents Neomycin-induced Hair Cell Death and Caspase-9 Activation in the Adult Mouse Utricle in Vitro

Journal of Neurobiology. Jul, 2004  |  Pubmed ID: 15188275

Mechanosensory hair cells of the inner ear are especially sensitive to death induced by exposure to aminoglycoside antibiotics. This aminoglycoside-induced hair cell death involves activation of an intrinsic program of cellular suicide. Aminoglycoside-induced hair cell death can be prevented by broad-spectrum inhibition of caspases, a family of proteases that mediate apoptotic and programmed cell death in a wide variety of systems. More specifically, aminoglycoside-induced hair cell death requires activation of caspase-9. Caspase-9 activation requires release of mitochondrial cytochrome c into the cytoplasm, indicating that aminoglycoside-induced hair cell death is mediated by the mitochondrial (or "intrinsic") cell death pathway. The Bcl-2 family of pro-apoptotic and anti-apoptotic proteins are important upstream regulators of the mitochondrial apoptotic pathway. Bcl-2 is an anti-apoptotic protein that localizes to the mitochondria and promotes cell survival by preventing cytochrome c release. Here we have utilized transgenic mice that overexpress Bcl-2 to examine the role of Bcl-2 in neomycin-induced hair cell death. Overexpression of Bcl-2 significantly increased hair cell survival following neomycin exposure in organotypic cultures of the adult mouse utricle. Furthermore, Bcl-2 overexpression prevented neomycin-induced activation of caspase-9 in hair cells. These results suggest that the expression level of Bcl-2 has important effects on the pathway(s) important for the regulation of aminoglycoside-induced hair cell death.

Activation of Metabotropic Glutamate Receptors Inhibits High-voltage-gated Calcium Channel Currents of Chicken Nucleus Magnocellularis Neurons

Journal of Neurophysiology. Mar, 2005  |  Pubmed ID: 15371493

Using whole cell patch-clamp recordings, we pharmacologically characterized the voltage-gated Ca2+ channel (VGCC) currents of chicken nucleus magnocellularis (NM) neurons using barium as the charge carrier. NM neurons possessed both low- and high-voltage-activated Ca2+ channel currents (HVA I(Ba2+)). The N-type channel blocker (omega-conotoxin-GVIA) inhibited more than half of the total HVA I(Ba2+), whereas blockers of L- and P/Q-type channels each inhibited a small fraction of the current. Metabotropic glutamate receptor (mGluR)-mediated modulation of the HVA I(Ba2+) was examined by bath application of glutamate (100 microM), which inhibited the HVA I(Ba2+) by an average of 16%. The inhibitory effect was dose dependent and was partially blocked by omega-conotoxin-GVIA, indicating that mGluRs modulate N and other type HVA I(Ba2+). The nonspecific mGluR agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarbosylic acid (1S,3R-ACPD), mimicked the inhibitory effect of glutamate on HVA I(Ba2+). Group I-III mGluR agonists showed inhibition of the HVA current with the most potent being the group III agonist L(+)-2-amino-4-phosphonobutyric acid. 1S,3R-ACPD (200 microM) had no effect on K+ or Na+ currents. The firing properties of NM neurons were also not altered by 1S,3R-ACPD. We propose that the inhibition of VGCC currents by mGluRs limits depolarization-induced Ca2+ entry into these highly active NM neurons and regulates their Ca2+ homeostasis.

GABA(B) Receptor Activation Modulates GABA(A) Receptor-mediated Inhibition in Chicken Nucleus Magnocellularis Neurons

Journal of Neurophysiology. Mar, 2005  |  Pubmed ID: 15483063

Neurons of nucleus magnocellularis (NM), a division of avian cochlear nucleus that performs precise temporal encoding, receive glutamatergic excitatory input solely from the eighth nerve and GABAergic inhibitory input primarily from the ipsilateral superior olivary nucleus. GABA activates both ligand-gated Cl- channels [GABA(A) receptors (GABA(A)Rs)] and G protein-coupled receptors (GABA(B) receptors). The net effect of GABA(A)R-mediated input to NM is inhibitory, although depolarizing. Several studies have shown that this shunting, inhibitory GABAergic input can evoke action potentials in postsynaptic NM neurons, which could interfere with their temporal encoding. While this GABA-mediated firing is limited by a low-voltage-activated K+ conductance, we have found evidence for a second mechanism. We investigated modulation of GABA(A)R-mediated responses by GABA(B)Rs using whole cell recording techniques. Bath-applied baclofen, a GABA(B)R agonist, produced dose-dependent suppression of evoked inhibitory postsynaptic currents (eIPSCs). This suppression was blocked by CGP52432, a potent and selective GABA(B)R antagonist. Baclofen reduced the frequency but not the amplitude of miniature IPSCs (mIPSCs) and did not affect postsynaptic currents elicited by puff application of a specific GABA(A)R agonist muscimol, suggesting a presynaptic mechanism for the GABA(B)R-mediated modulation. Firing of NM neurons by synaptic stimulation of GABAergic inputs to NM was eliminated by baclofen. However, endogenous GABA(B)R activity in the presynaptic inhibitory terminals was not observed. We propose that presynaptic GABA(B)Rs function as autoreceptors, regulating synaptic strength of GABA(A)R-mediated inhibition, and prevent NM neurons from generating firing during activation of the inhibitory inputs.

Avian Superior Olivary Nucleus Provides Divergent Inhibitory Input to Parallel Auditory Pathways

The Journal of Comparative Neurology. Jan, 2005  |  Pubmed ID: 15558730

The avian auditory brainstem displays parallel processing, a fundamental feature of vertebrate sensory systems. Nuclei specialized for temporal processing are largely separate from those processing other aspects of sound. One possible exception to this parallel organization is the inhibitory input provided by the superior olivary nucleus (SON) to nucleus angularis (NA), nucleus magnocellularis (NM), and nucleus laminaris (NL) and contralateral SON (SONc). We sought to determine whether single SON neurons project to multiple targets or separate neuronal populations project independently to individual target nuclei. We introduced two different fluorescent tracer molecules into pairs of target nuclei and quantified the extent to which retrogradely labeled SON neurons were double labeled. A large proportion of double-labeled SON somata were observed in all cases in which injections were made into any pair of ipsilateral targets (NA and NM, NA and NL, or NM and NL), suggesting that many individual SON neurons project to multiple targets. In contrast, when injections involved the SONc and any or all of the ipsilateral targets, double labeling was rare, suggesting that contralateral and ipsilateral targets are innervated by distinct populations of SON neurons arising largely from regionally segregated areas of SON. Therefore, at the earliest stages of auditory processing, there is interaction between pathways specialized to process temporal cues and those that process other acoustic features. We present a conceptual model that incorporates these results and suggest that SON circuitry, in part, functions to offset interaural intensity differences in interaural time difference processing.

Expression of GABA(B) Receptor in the Avian Auditory Brainstem: Ontogeny, Afferent Deprivation, and Ultrastructure

The Journal of Comparative Neurology. Aug, 2005  |  Pubmed ID: 15977167

Nucleus magnocellularis (NM), nucleus angularis (NA), and nucleus laminaris (NL), second- and third-order auditory neurons in the avian brainstem, receive GABAergic input primarily from the superior olivary nucleus (SON). Previous studies have demonstrated that both GABA(A) and GABA(B) receptors (GABA(B)Rs) influence physiological properties of NM neurons. We characterized the distribution of GABA(B)R expression in these nuclei during development and after deafferentation of the excitatory auditory nerve (nVIII) inputs. We used a polyclonal antibody raised against rat GABA(B)Rs in the auditory brainstem during developmental periods that are thought to precede and include synaptogenesis of GABAergic inputs. As early as embryonic day (E)14, dense labeling is observed in NA, NM, NL, and SON. At earlier ages immunoreactivity is present in somas as diffuse staining with few puncta. By E21, when the structure and function of the auditory nuclei are known to be mature, GABA(B) immunoreactivity is characterized by dense punctate labeling in NM, NL, and a subset of NA neurons, but label is sparse in the SON. Removal of the cochlea and nVIII neurons in posthatch chicks resulted in only a small decrease in immunoreactivity after survival times of 14 or 28 days, suggesting that a major proportion of GABA(B)Rs may be expressed postsynaptically or on GABAergic terminals. We confirmed this interpretation with immunogold TEM, where expression at postsynaptic membrane sites is clearly observed. The characterization of GABA(B)R distribution enriches our understanding of the full complement of inhibitory influences on central auditory processing in this well-studied neuronal circuit.

Gene Expression Differences over a Critical Period of Afferent-dependent Neuron Survival in the Mouse Auditory Brainstem

The Journal of Comparative Neurology. Dec, 2005  |  Pubmed ID: 16261529

Deprivation of auditory nerve input in young mice results in dramatic neuron death in the anteroventral cochlear nucleus (CN), while the same manipulation performed in older mice does not result in significant neuronal loss. The molecular basis underlying this critical period of susceptibility to loss of afferent input remains largely unknown. One possibility is that developmental differences in baseline mRNA expression of specific genes could predispose CN neurons to either death or survival after deafferentation. We used two microarray platforms to identify differentially expressed genes in the CN during and after this critical period. Results across platforms were also compared to each other. Three ages were examined; during the critical period (postnatal day (P)7), at the closing of the critical period (P14), and 1 week after the critical period (P21). Of all the genes surveyed (22,690 or 15,247), 1,082 were identified as significantly changed in expression during the critical period relative to after. Real-time reverse transcription polymerase chain reaction and immunohistochemistry confirmed and extended the microarray results for a subset of these genes. Further analysis of genes related to apoptotic pathways showed 6 out of 7 differentially expressed known pro-apoptotic genes had higher expression during the critical period. In contrast, 9 out of 11 differentially expressed known pro-survival genes increased after the critical period when CN neurons survive deprivation. This finding supports the concept that multiple neuroprotective mechanisms increase and pro-apoptotic factors decrease over development to protect mature neurons from stressful insults, making them less dependent on afferent input for survival.

Mechanisms of Hair Cell Death and Protection

Current Opinion in Otolaryngology & Head and Neck Surgery. Dec, 2005  |  Pubmed ID: 16282762

Sensory hair cells are mechanotransducers of the inner ear that are essential for hearing and balance. Hair cell death commonly occurs following acoustic trauma or exposure to ototoxins, such as the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Loss of these inner ear sensory cells can lead to permanent sensorineural hearing loss, balance disturbance, or both. Currently, the only effective clinical intervention is prevention from exposure to known ototoxic insults. To help improve therapeutic strategies, a better understanding of the molecular mechanisms underlying hair cell degeneration is required. Current knowledge of these cell death mechanisms and potential therapeutic targets are discussed in this review.

The Level and Integrity of Synaptic Input Regulates Dendrite Structure

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2006  |  Pubmed ID: 16452677

How localized synaptic input regulates dendritic branch structure is not well understood. For these experiments, we used single-cell electroporation, live cell imaging, in vitro deafferentation, pharmacology, and electrophysiological stimulation to study how local alterations in synaptic input affect dendritic branch structure in nucleus laminaris (NL). We found that interrupting or modulating synaptic input to distinct sets of NL dendrites can regulate their structure on a very short timescale. Specifically, eliminating synaptic input by deafferenting only one set of the bitufted NL dendrites caused a selective reduction in the total dendritic branch length of the deafferented dendrites but relatively few changes in the normally innervated dendrites on the same cell. An analysis of individual dendritic branch changes demonstrated that both control and deafferented NL dendrites exhibit branch extension and retraction. However, the presence of intact synaptic inputs balanced these changes, maintaining the total dendritic branch length of control dendrites. When glutamate receptor signaling was blocked (DNQX and AP-5), NL neurons exhibited significant dendrite retraction, demonstrating that NL dendrite maintenance depends in part on presynaptic glutamatergic input. Electrophysiological experiments further confirmed that modulating the level of synaptic input regulates NL dendrite structure. Differential stimulation of the two sets of dendrites resulted in a selective reduction in the total dendritic branch length of the unstimulated dendrites and a selective increase in the total dendritic branch length of the stimulated dorsal dendrites. These results suggest that balanced activation of the two sets of NL dendrites is required to maintain the relative amount of dendritic surface area allotted to each input.

Lateral Line Hair Cell Maturation is a Determinant of Aminoglycoside Susceptibility in Zebrafish (Danio Rerio)

Hearing Research. Mar, 2006  |  Pubmed ID: 16459035

Developmental differences in hair cell susceptibility to aminoglycoside-induced cell death has been observed in multiple species. Increased sensitivity to aminoglycosides has been temporally correlated with the onset of mechanotransduction-dependent activity. We have used in vivo fluorescent vital dye markers to further investigate the determinants of aminoglycoside induced hair cell death in the lateral line of zebrafish (Danio rerio). Labeling hair cells of the lateral line in vivo with the dyes FM 1-43, To-Pro-3, and Yo-Pro-1 served as reliable indicators of hair cell viability. Results indicate that hair cell maturation is a determinant of developmental differences in susceptibility. The age dependent differences in susceptibility to aminoglycosides are independent of the onset of mechanotransduction-dependent activity as measured by FM 1-43 uptake and independent of hair cell ability to take up fluorescently conjugated aminoglycosides.

Formation of the Avian Nucleus Magnocellularis from the Auditory Anlage

The Journal of Comparative Neurology. Oct, 2006  |  Pubmed ID: 16874806

In the avian auditory system, the neural network for computing the localization of sound in space begins with bilateral innervation of nucleus laminaris (NL) by nucleus magnocellularis (NM) neurons. We used antibodies against the neural specific markers Hu C/D, neurofilament, and SV2 together with retrograde fluorescent dextran labeling from the contralateral hindbrain to identify NM neurons within the anlage and follow their development. NM neurons could be identified by retrograde labeling as early as embryonic day (E) 6. While the auditory anlage organized itself into NM and NL in a rostral-to-caudal fashion between E6 and E8, labeled NM neurons were visible throughout the extent of the anlage at E6. By observing the pattern of neuronal rearrangements together with the pattern of contralaterally projecting NM fibers, we could identify NL in the ventral anlage. Ipsilateral NM fibers contacted the developing NL at E8, well after NM collaterals had projected contralaterally. Furthermore, the formation of ipsilateral connections between NM and NL neurons appeared to coincide with the arrival of VIIIth nerve fibers in NM. By E10, immunoreactivity for SV2 was heavily concentrated in the dorsal and ventral neuropils of NL. Thus, extensive pathfinding and morphological rearrangement of central auditory nuclei occurs well before the arrival of cochlear afferents. Our results suggest that NM neurons may play a central role in formation of tonotopic connections in the auditory system.

Afferent Regulation of Neuron Number in the Cochlear Nucleus: Cellular and Molecular Analyses of a Critical Period

Hearing Research. Jun-Jul, 2006  |  Pubmed ID: 16874907

The neurons of the cochlear nucleus are dependent on input from the auditory nerve for survival during a critical period of development in a variety of vertebrate species. The molecules that underlie this age-dependent vulnerability to deafferentation are for the most part unknown, although recent studies have begun to yield interesting candidate genes. Here, we review the studies that originally described the presence of afferent dependent neuron survival in the cochlear nucleus and the age-dependency of this effect, as well as more recent work that seeks to understand the mechanisms underlying the neuron loss that occurs and the basis of this critical period. While much of the past work on cochlear nucleus neuronal susceptibility has been conducted looking at one or two genes at a time, recent advances in genomics make it possible to screen tens of thousands of genes while looking for candidate genes that are determinants of the critical period response to afferent deprivation.

JNK Signaling in Neomycin-induced Vestibular Hair Cell Death

Hearing Research. Nov, 2006  |  Pubmed ID: 17005344

Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to neomycin. CEP-11004 inhibited neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of neomycin. However, the results were not uniform across the dose-response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither Chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in neomycin-induced vestibular hair cell death and caspase-9 activation.

Development of Spontaneous Miniature EPSCs in Mouse AVCN Neurons During a Critical Period of Afferent-dependent Neuron Survival

Journal of Neurophysiology. Jan, 2007  |  Pubmed ID: 17079338

During a critical period prior to hearing onset, cochlea ablation leads to massive neuronal death in the mouse anteroventral cochlear nucleus (AVCN), where cell survival is believed to depend on glutamatergic input. We investigated the development of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in AVCN neurons using whole cell patch-clamp techniques during [postnatal day 7 (P7)] and after (P14, P21) this critical period. We also examined the effects of unilateral cochlea ablation on mEPSC development. The two main AVCN neuron types, bushy and stellate cells, were distinguished electrophysiologically. Bushy cell mEPSCs became more frequent and faster between P7 and P14/P21 but with little change in amplitude. Dendritic filtering of mEPSCs was not detected as indicated by the lack of correlation between 10 and 90% rise times and decay time constants. Seven days after cochlea ablation at P7 or P14, mEPSCs in surviving bushy cells were similar to controls, except that rise and decay times were positively correlated (R = 0.31 and 0.14 for surgery at P7 and P14, respectively). Consistent with this evidence for a shift of synaptic activity from the somata to the dendrites, SV2 staining (a synaptic vesicle marker) forms a ring around somata of control but not experimental bushy cells. In contrast, mEPSCs of stellate cells showed few significant changes over these ages with or without cochlea ablation. Taken together, mEPSCs in mouse AVCN bushy cells show dramatic developmental changes across this critical period, and cochlea ablation may lead to the emergence of excitatory synaptic inputs impinging on bushy cell dendrites.

A Developmental Switch to GABAergic Inhibition Dependent on Increases in Kv1-type K+ Currents

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2007  |  Pubmed ID: 17314306

Mature nucleus magnocellularis (NM) neurons, the avian homolog of bushy cells of the mammalian anteroventral cochlear nucleus, maintain high [Cl-]i and depolarize in response to GABA. Depolarizing GABAergic postsynaptic potentials (GPSPs) activate both the synaptic conductance and large outward currents, which, when coupled together, inhibit spikes via shunting and spike threshold accommodation. We studied the maturation of the synaptic and voltage-dependent components of inhibition in embryonic NM neurons using whole-cell and gramicidin-perforated patch-clamp techniques to measure Cl- reversal potential, GABAergic synaptic responses, and voltage-dependent outward currents. We found that GABA enhanced excitability in immature NM neurons, undergoing a switch to inhibitory between embryonic day 14 (E14) and E18. Low-voltage-activated Kv1-type (dendrotoxin-I sensitive) K+ currents increased in amplitude between E14 and E18, whereas Cl- reversal potential and synaptic conductances remained relatively stable during this period. GABA was rendered inhibitory because of this increase in low-voltage activated outward currents. GPSPs summed with other inputs to increase spike probability at E14. GPSPs shunted spikes at E18, but blocking Kv1 channels transformed this inhibition to excitation, similar to E14 neurons. Subthreshold depolarizing current steps, designed to activate outward currents similar to depolarizing GPSPs, enhanced excitability at E14 but inhibited spiking in E18 neurons. Blocking Kv1 channels reversed this effect, rendering current steps excitatory. We present the novel finding that the developmental transition of GABAergic processing from increasing neuronal excitability to inhibiting spiking can depend on changes in the expression of voltage-gated channels rather than on a change in Cl- reversal potential.

Ultrastructural Analysis of Aminoglycoside-induced Hair Cell Death in the Zebrafish Lateral Line Reveals an Early Mitochondrial Response

The Journal of Comparative Neurology. Jun, 2007  |  Pubmed ID: 17394157

Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycoside neomycin. Similarly, ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of neomycin exposure. Animals exposed to a low, 25-microM concentration of neomycin exhibited hair cells with swollen mitochondria, but little other damage. Animals treated with higher concentrations of neomycin (50-200 microM) had more severe and heterogeneous cellular changes, as well as fewer hair cells. Both necrotic-like and apoptotic-like cellular damage were observed. Quantitation of the types of alterations observed indicated that mitochondrial defects appear earlier and more predominantly than other structural alterations. In vivo monitoring demonstrated that mitochondrial potential decreased following neomycin treatment. These results indicate that perturbation of the mitochondrion is an early, central event in aminoglycoside-induced damage.

Auditory Feedback and Song Production Do Not Regulate Seasonal Growth of Song Control Circuits in Adult White-crowned Sparrows

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2007  |  Pubmed ID: 17581968

An important area of research in neuroscience is understanding what properties of brain structure and function are stimulated by sensory experience and behavioral performance. We tested the roles of experience and behavior in seasonal plasticity of the neural circuits that regulate learned song behavior in adult songbirds. Neurons in these circuits receive auditory input and show selective auditory responses to conspecific song. We asked whether auditory input or song production contribute to seasonal growth of telencephalic song nuclei. Adult male Gambel's white-crowned sparrows were surgically deafened, which eliminates auditory input and greatly reduces song production. These birds were then exposed to photoperiod and hormonal conditions that regulate the growth of song nuclei. We measured the volumes of the nuclei HVC, robust nucleus of arcopallium (RA), and area X at 7 and 30 d after exposure to long days plus testosterone in deafened and normally hearing birds. We also assessed song production and examined protein kinase C (PKC) expression because previous research reported that immunostaining for PKC increases transiently after deafening. Deafening did not delay or block the growth of the song nuclei to their full breeding-condition size. PKC activity in RA was not altered by deafening in the sparrows. Song continued to be well structured for up to 10 months after deafening, but song production decreased almost eightfold. These results suggest that neither auditory input nor high rates of song production are necessary for seasonal growth of the adult song control system in this species.

Cisplatin-induced Hair Cell Loss in Zebrafish (Danio Rerio) Lateral Line

Hearing Research. Nov, 2007  |  Pubmed ID: 17709218

We have used time-lapse imaging to study cisplatin-induced hair cell death in lateral line neuromasts of zebrafish larvae in vivo. We found that cisplatin-induced hair cell death occurred much more slowly than had been shown to occur in aminoglycoside-induced hair cell death. By prelabeling hair cells with FM1-43FX, and assessing hair cell damage, it was established that cisplatin causes hair cell loss in the lateral line in a dose-dependent fashion. The kinetics of hair cell loss during exposure to different concentrations of cisplatin was also assessed and it was found that the onset of hair cell loss correlated with the accumulated dose of cisplatin. These data demonstrate the feasibility and repeatability of cisplatin damage protocols in the zebrafish lateral line and set the stage for future evaluations of modulation of cisplatin-induced hair cell death.

Notch Signaling Regulates the Extent of Hair Cell Regeneration in the Zebrafish Lateral Line

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2008  |  Pubmed ID: 18305259

Mechanosensory hair cells within the zebrafish lateral line spontaneously regenerate after aminoglycoside-induced death. Exposure of 5-d-old larvae to 400 microM neomycin for 1 h results in death of almost all lateral line hair cells. Regeneration of new hair cells is observed by 24 h after neomycin treatment with nearly complete replacement by 72 h. Using bromodeoxyuridine incorporation, we show that the majority of new hair cells are generated from a transient increase in support cell proliferation that occurs between 12 and 21 h after neomycin damage. Additional observations reveal two distinct subsets of proliferating support cells within the neuromasts that differ in position, morphology, and temporal pattern of proliferation in response to neomycin exposure. We hypothesize that proliferative hair cell progenitors are located centrally within the neuromasts, whereas peripheral support cells may have a separate function. Expression of Notch signaling pathway members notch3, deltaA, and atoh1a transcripts are all upregulated within the first 24 h after neomycin treatment, during the time of maximum proliferation of support cells and hair cell progenitor formation. Treatment with a gamma-secretase inhibitor results in excess regenerated hair cells by 48 h after neomycin-induced death but has no effect without previous damage. Excess hair cells result from increased support cell proliferation. These results suggest a model where Notch signaling limits the number of hair cells produced during regeneration by regulating support cell proliferation.

Using the Zebrafish Lateral Line to Screen for Ototoxicity

Journal of the Association for Research in Otolaryngology : JARO. Jun, 2008  |  Pubmed ID: 18408970

The zebrafish is a valuable model for studying hair cell development, structure, genetics, and behavior. Zebrafish and other aquatic vertebrates have hair cells on their body surface organized into a sensory system called the lateral line. These hair cells are highly accessible and easily visualized using fluorescent dyes. Morphological and functional similarities to mammalian hair cells of the inner ear make the zebrafish a powerful preparation for studying hair cell toxicity. The ototoxic potential of drugs has historically been uncovered by anecdotal reports that have led to more formal investigation. Currently, no standard screen for ototoxicity exists in drug development. Thus, for the vast majority of Food and Drug Association (FDA)-approved drugs, the ototoxic potential remains unknown. In this study, we used 5-day-old zebrafish larvae to screen a library of 1,040 FDA-approved drugs and bioactives (NINDS Custom Collection II) for ototoxic effects in hair cells of the lateral line. Hair cell nuclei were selectively labeled using a fluorescent vital dye. For the initial screen, fish were exposed to drugs from the library at a 100-muM concentration for 1 h in 96-well tissue culture plates. Hair cell viability was assessed in vivo using fluorescence microscopy. One thousand forty drugs were rapidly screened for ototoxic effects. Seven known ototoxic drugs included in the library, including neomycin and cisplatin, were positively identified using these methods, as proof of concept. Fourteen compounds without previously known ototoxicity were discovered to be selectively toxic to hair cells. Dose-response curves for all 21 ototoxic compounds were determined by quantifying hair cell survival as a function of drug concentration. Dose-response relationships in the mammalian inner ear for two of the compounds without known ototoxicity, pentamidine isethionate and propantheline bromide, were then examined using in vitro preparations of the adult mouse utricle. Significant dose-dependent hair cell loss in the mouse utricle was demonstrated for both compounds. This study represents an important step in validating the use of the zebrafish lateral line as a screening tool for the identification of potentially ototoxic drugs.

Identification of Genetic and Chemical Modulators of Zebrafish Mechanosensory Hair Cell Death

PLoS Genetics. Feb, 2008  |  Pubmed ID: 18454195

Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.

Three-dimensional Imaging of the Intact Mouse Cochlea by Fluorescent Laser Scanning Confocal Microscopy

Hearing Research. Sep, 2008  |  Pubmed ID: 18573326

The complex anatomy of the mammalian cochlea is most readily understood by representation in three-dimensions. However, the cochlea is often sectioned to minimize the effects of its anatomic complexity and optical properties on image acquisition by light microscopy. We have found that optical aberrations present in the decalcified cochlea can be greatly reduced by dehydration through graded ethanols followed by clearing with a mixture of five parts methyl salicylate and three parts benzyl benzoate (MSBB). Clearing the cochlea with MSBB enables acquisition of high-resolution images with multiple fluorescent labels, through the full volume of the cochlea by laser scanning confocal microscopy. The resulting images are readily applicable to three-dimensional morphometric analysis and volumetric visualizations. This method promises to be particularly useful for three-dimensional characterization of anatomy, innervation and expression of genes or proteins in the many new animal models of hearing and balance generated by genetic manipulation. Furthermore, the MSBB is compatible with most non-protein fluorophores used for histological labeling, and may be removed with traditional transitional solvents to allow subsequent epoxy embedding for sectioning.

Afferent Deprivation Elicits a Transcriptional Response Associated with Neuronal Survival After a Critical Period in the Mouse Cochlear Nucleus

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2008  |  Pubmed ID: 18945907

The mechanisms underlying enhanced plasticity of synaptic connections and susceptibilities to manipulations of afferent activity in developing sensory systems are not well understood. One example is the rapid and dramatic neuron death that occurs after removal of afferent input to the cochlear nucleus (CN) of young mammals and birds. The molecular basis of this critical period of neuronal vulnerability and the transition to survival independent of afferent input remains to be defined. Here we used microarray analyses, real-time reverse transcription PCR, and immunohistochemistry of the mouse CN to show that deafferentation results in strikingly different sets of regulated genes in vulnerable [postnatal day (P)7] and invulnerable (P21) CN. An unexpectedly large set of immune-related genes was induced by afferent deprivation after the critical period, which corresponded with glial proliferation over the same time frame. Apoptotic gene expression was not highly regulated in the vulnerable CN after afferent deprivation but, surprisingly, did increase after deafferentation at P21, when all neurons ultimately survive. Pharmacological activity blockade in the eighth nerve mimicked afferent deprivation for only a subset of the afferent deprivation regulated genes, indicating the presence of an additional factor not dependent on action potential-mediated signaling that is also responsible for transcriptional changes. Overall, our results suggest that the cell death machinery during this critical period is mainly constitutive, whereas after the critical period neuronal survival could be actively promoted by both constitutive and induced gene expression.

CC2D2A is Mutated in Joubert Syndrome and Interacts with the Ciliopathy-associated Basal Body Protein CEP290

American Journal of Human Genetics. Nov, 2008  |  Pubmed ID: 18950740

Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.

Identification of FDA-approved Drugs and Bioactives That Protect Hair Cells in the Zebrafish (Danio Rerio) Lateral Line and Mouse (Mus Musculus) Utricle

Journal of the Association for Research in Otolaryngology : JARO. Jun, 2009  |  Pubmed ID: 19241104

The hair cells of the larval zebrafish lateral line provide a useful preparation in which to study hair cell death and to screen for genes and small molecules that modulate hair cell toxicity. We recently reported preliminary results from screening a small-molecule library for compounds that inhibit aminoglycoside-induced hair cell death. To potentially reduce the time required for development of drugs and drug combinations that can be clinically useful, we screened a library of 1,040 FDA-approved drugs and bioactive compounds (NINDS Custom Collection II). Seven compounds that protect against neomycin-induced hair cell death were identified. Four of the seven drugs inhibited aminoglycoside uptake, based on Texas-Red-conjugated gentamicin uptake. The activities of two of the remaining three drugs were evaluated using an in vitro adult mouse utricle preparation. One drug, 9-amino-1,2,3,4-tetrahydroacridine (tacrine) demonstrated conserved protective effects in the mouse utricle. These results demonstrate that the zebrafish lateral line can be used to screen successfully for drugs within a library of FDA-approved drugs and bioactives that inhibit hair cell death in the mammalian inner ear and identify tacrine as a promising protective drug for future studies.

Response of Mechanosensory Hair Cells of the Zebrafish Lateral Line to Aminoglycosides Reveals Distinct Cell Death Pathways

Hearing Research. Jul, 2009  |  Pubmed ID: 19285126

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 min, regardless of concentration or exposure time. Gentamicin-induced loss of half of the hair cells within 90 min and substantial additional loss, which was prolonged and cumulative over exposure times up to at least 24h. 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.

Extracellular Divalent Cations Modulate Aminoglycoside-induced Hair Cell Death in the Zebrafish Lateral Line

Hearing Research. Jul, 2009  |  Pubmed ID: 19285547

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 h) 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.

Cisplatin-induced Hair Cell Death Requires STAT1 and is Attenuated by Epigallocatechin Gallate

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2009  |  Pubmed ID: 19321781

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 after exposure of utricles to cisplatin. STAT1 phosphorylation peaked after 4 h of cisplatin exposure and returned to control levels by 8 h 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 h postexposure. 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-gamma, which also causes STAT1 activation, also induced hair cell death in wild-type 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.

Compartment-specific Regulation of Plasma Membrane Calcium ATPase Type 2 in the Chick Auditory Brainstem

The Journal of Comparative Neurology. Jun, 2009  |  Pubmed ID: 19365819

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 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 after 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 with the other neuropil that continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for 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.

Effects of Restricted Basilar Papillar Lesions and Hair Cell Regeneration on Auditory Forebrain Frequency Organization in Adult European Starlings

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. May, 2009  |  Pubmed ID: 19474314

The frequency organization of neurons in the forebrain Field L complex (FLC) of adult starlings was investigated to determine the effects of hair cell (HC) destruction in the basal portion of the basilar papilla (BP) and of subsequent HC regeneration. Conventional microelectrode mapping techniques were used in normal starlings and in lesioned starlings either 2 d or 6-10 weeks after aminoglycoside treatment. Histological examination of the BP and recordings of auditory brainstem evoked responses confirmed massive loss of HCs in the basal portion of the BP and hearing losses at frequencies >2 kHz in starlings tested 2 d after aminoglycoside treatment. In these birds, all neurons in the region of the FLC in which characteristic frequencies (CFs) normally increase from 2 to 6 kHz had CF in the range of 2-4 kHz. The significantly elevated thresholds of responses in this region of altered tonotopic organization indicated that they were the residue of prelesion responses and did not reflect CNS plasticity. In the long-term recovery birds, there was histological evidence of substantial HC regeneration. The tonotopic organization of the high-frequency region of the FLC did not differ from that in normal starlings, but the mean threshold at CF in this frequency range was intermediate between the values in the normal and lesioned short-recovery groups. The recovery of normal tonotopicity indicates considerable stability of the topography of neuronal connections in the avian auditory system, but the residual loss of sensitivity suggests deficiencies in high-frequency HC function.

A Simple Method for Multiday Imaging of Slice Cultures

Microscopy Research and Technique. Jan, 2010  |  Pubmed ID: 19565635

The organotypic slice culture (Stoppini et al. A simple method for organotypic cultures of nervous tissue. 1991;37:173-182) 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. Single-cell electroporation for gene transfer in vivo. 2001;29:583-591). 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 during the course of multiple days and over many continuous hours, without noticeable damage to the tissue or photobleaching. Our method uses 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.

Mechanisms for Adjusting Interaural Time Differences to Achieve Binaural Coincidence Detection

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2010  |  Pubmed ID: 20053889

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.

Deafness and Retinal Degeneration in a Novel USH1C Knock-in Mouse Model

Developmental Neurobiology. Mar, 2010  |  Pubmed ID: 20095043

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 1 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.

Drug Screening for Hearing Loss: Using the Zebrafish Lateral Line to Screen for Drugs That Prevent and Cause Hearing Loss

Drug Discovery Today. Apr, 2010  |  Pubmed ID: 20096805

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 and 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. The 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.

Chemical Screening for Hair Cell Loss and Protection in the Zebrafish Lateral Line

Zebrafish. Mar, 2010  |  Pubmed ID: 20192852

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.

Development of Glutamatergic Synaptic Transmission in Binaural Auditory Neurons

Journal of Neurophysiology. Sep, 2010  |  Pubmed ID: 20668278

Glutamatergic synaptic transmission is essential for binaural auditory processing in birds and mammals. Using whole cell voltage clamp recordings, we characterized the development of synaptic ionotropic glutamate receptor (iGluR) function from auditory neurons in the chick nucleus laminaris (NL), the first nucleus responsible for binaural processing. We show that synaptic transmission is mediated by AMPA- and N-methyl-d-aspartate (NMDA)-type glutamate receptors (AMPA-R and NMDA-R, respectively) when hearing is first emerging and dendritic morphology is being established across different sound frequency regions. Puff application of glutamate agonists at embryonic day 9 (E9) revealed that both iGluRs are functionally present prior to synapse formation (E10). Between E11 and E19, the amplitude of isolated AMPA-R currents from high-frequency (HF) neurons increased 14-fold. A significant increase in the frequency of spontaneous events is also observed. Additionally, AMPA-R currents become faster and more rectifying, suggesting developmental changes in subunit composition. These developmental changes were similar in all tonotopic regions examined. However, mid- and low-frequency neurons exhibit fewer spontaneous events and evoked AMPA-R currents are smaller, slower, and less rectifying than currents from age-matched HF neurons. The amplitude of isolated NMDA-R currents from HF neurons also increased, reaching a peak at E17 and declining sharply by E19, a trend consistent across tonotopic regions. With age, NMDA-R kinetics become significantly faster, indicating a developmental switch in receptor subunit composition. Dramatic increases in the amplitude and speed of glutamatergic synaptic transmission occurs in NL during embryonic development. These changes are first seen in HF neurons suggesting regulation by peripheral inputs and may be necessary to enhance coincidence detection of binaural auditory information.

Dynamic Spike Thresholds During Synaptic Integration Preserve and Enhance Temporal Response Properties in the Avian Cochlear Nucleus

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2010  |  Pubmed ID: 20826669

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 electrophysiology 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.

GABAergic Inhibition Sharpens the Frequency Tuning and Enhances Phase Locking in Chicken Nucleus Magnocellularis Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2010  |  Pubmed ID: 20826670

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 to 4 weeks posthatch chickens show that firing rates of auditory nerve fibers 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.

Topography and Morphology of the Inhibitory Projection from Superior Olivary Nucleus to Nucleus Laminaris in Chickens (Gallus Gallus)

The Journal of Comparative Neurology. Feb, 2011  |  Pubmed ID: 21165979

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 between 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.

Relative Input Strength Rapidly Regulates Dendritic Structure of Chick Auditory Brainstem Neurons

The Journal of Comparative Neurology. Oct, 2011  |  Pubmed ID: 21500196

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 dendrites receive 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, whereas the stimulated dendrites exhibited a tendency to grow. In this system, balanced input leads to balanced changes in the two sets of dendrites, but 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.

Inhibition in the Balance: Binaurally Coupled Inhibitory Feedback in Sound Localization Circuitry

Journal of Neurophysiology. Jul, 2011  |  Pubmed ID: 21525367

Interaural time differences (ITDs) are the primary cue animals, including humans, use to localize low-frequency sounds. In vertebrate auditory systems, dedicated ITD processing neural circuitry performs an exacting task, the discrimination of microsecond differences in stimulus arrival time at the two ears by coincidence-detecting neurons. These neurons modulate responses over their entire dynamic range to sounds differing in ITD by mere hundreds of microseconds. The well-understood function of this circuitry in birds has provided a fruitful system to investigate how inhibition contributes to neural computation at the synaptic, cellular, and systems level. Our recent studies in the chicken have made significant progress in bringing together many of these findings to provide a cohesive picture of inhibitory function.

Tonotopic Organization of the Superior Olivary Nucleus in the Chicken Auditory Brainstem

The Journal of Comparative Neurology. Nov, 2011  |  Pubmed ID: 22102107

Topographic maps are salient features of neuronal organization in sensory systems. Inhibitory components of neuronal circuitry are often embedded within this organization making them difficult to isolate experimentally. The auditory system provides opportunities to study the topographic organization of inhibitory long-range projection nuclei, such as the superior olivary nucleus (SON). We analyzed the topographic organization of response features of neurons in SON of chickens. Quantitative methods were developed to assess and communicate this organization. These analyses led to three main conclusions: (1) sound frequency is linearly arranged from dorsal (low frequencies) to ventral (high frequencies) in SON; (2) this tonotopic organization is less precise than the organization of the excitatory nuclei in the chicken auditory brainstem; and (3) neurons with different response patterns to pure tone stimuli are interspersed throughout SON and show similar tonotopic organizations. This work provides a predictive model to determine the optimal stimulus frequency for a neuron from its spatial location in SON. J. Comp. Neurol., 2011. © 2011 Wiley-Liss, Inc.

Screening for Chemicals That Affect Hair Cell Death and Survival in the Zebrafish Lateral Line

Hearing Research. Jan, 2012  |  Pubmed ID: 22310494

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.