Director of the Neuroscience Training Program, Stanford University (2013 - Present)
Director of the Advance Summer Institute, Stanford University (2012 - Present)
Honors & Awards
Excellence in Diversity and Inclusion, Biosciences (2018)
Excellence in Diversity and Inclusion, Stanford Biosciences (2014)
Burt Evans Young Investigator Award, National Organization for Hearing (2002)
Young Investigator Award, Deafness Research Foundation (1999)
Boards, Advisory Committees, Professional Organizations
Board of Scientific Councillors, NIDCD (2014 - 2017)
Postdoc Reviewing Committee, SNI (2017 - Present)
Graduate student Sustainable funding group, Stanford Medical School (2014 - Present)
BDAC committee, Stanford Medical School (2015 - Present)
Nominating Committee, Association for Research in Otolaryngology (2014 - 2015)
Admissions Committee, Neuroscience training program (2006 - Present)
Program Committee, Neuroscience Training Program (2010 - Present)
PhD, Tulane University, Neuroscience (1992)
BA, Case Western Reserve University, Chemistry (1985)
Anthony Ricci, Alan Cheng, Robert Greenhouse. "United States Patent 61/792,256 Aminoglycoside Antibiotics with Reduced Ototoxicity", Leland Stanford Junior University, Mar 15, 2013
Current Research and Scholarly Interests
The auditory system is a remarkable feat of engineering capable of detecting motion at the atomic level and transmitting this information to the brain with precise timing and fidelity. We use advanced electrophysiologic, imaging, molecular and pharmacologic techniques to probe mechanisms of mechanotransduction and synaptic transmission at the auditory periphery. There are several independent lines of research in the laboratory.
Mechanotransduction, the conversion of mechanical stimulation into an electrical signal, is complex and involves a variety of proteins, many of which have not yet been identified. A major goal of the laboratory is to delineate the functional relevance of mechanotransduction and to identify proteins and their function in this process. To date, we have identified and characterized the tuning properties of the sensory hair bundle and mechanotransducer channels, identifying at least two new physiologically relevant contributions of these channels. We have performed the only single channel study of mechanotransducer channels, demonstrating tonotopic variations in the intrinsic channel properties. We have also performed the only kinetic analysis of activation, again demonstrating tonotopic variations in the kinetics of the mechanotransduction channel. In addition, we have pharmacologically characterized and biophysically mapped the transducer channel pore. Recently we have developed a high speed confocal imaging system that will allow us to optically monitor calcium changes associated with mechanotransduction, allowing us to localize the site of mechanotransduction and directly investigate mechanisms of calcium, regulation.
A second major direction of the laboratory is synaptic transmission where we are interested in identifying mechanisms associated with specializing these synapses to graded and tonic release of transmitter at high rates and with high fidelity. We have morphologically and biophysically characterized these synapses, quantifying release properties at different frequency locations. We are one of only a handful of laboratories who have recorded directly from synaptic terminals where we are investigating mechanisms of multivesicular release. Recently we have developed a technique for measuring vesicular fusion during stimulation so that true release parameters can be investigated. We plan to further develop this technique to be used while measuring membrane potential changes.
A third area of interest for our laboratory is the development of the peripheral system. We are particularly interested in identifying mechanisms associated with the establishment of the tonotopic organization of the cochlea. In addition, indentifying factors that control cell differentiation and specialization, those intrinsic and those extrinsic to the cells is a key priority. This work is critical when trying to repair or replace hair cells either via regenerative or stem cell type therapies.
Although fundamentally a basic science laboratory we have strong ties to translational research both directly and through collaborative efforts. Each of our three major research areas have translationally oriented projects associated with them. In addition, we are developing a project to create a nontoxic aminoglycoside based on biophysical data collected while investigating mechanotransduction.
The auditory sensory cell, the hair cell, detects mechanical stimulation at the atomic level and conveys information regarding frequency and intensity to the brain with high fidelity. Our interests are in identifying specializations associated with mechanotransduction and synaptic transmission leading to the amazing sensitivities of the auditory system. We are also interested in the developmental process, particularly in how development gives insight into repair and regenerative mechanisms.
- Responsible Conduct of Neuroscience Research
NEPR 212 (Aut)
Independent Studies (6)
- Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr, Sum)
- Directed Reading in Otolaryngology
OTOHNS 299 (Aut, Win, Spr, Sum)
- Graduate Research
NEPR 399 (Aut, Win, Spr, Sum)
- Graduate Research
OTOHNS 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
OTOHNS 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
OTOHNS 199 (Aut, Win, Spr, Sum)
- Directed Reading in Neurosciences
- Prior Year Courses
TRPV6, TRPM6 and TRPM7 Do Not Contribute to Hair-Cell Mechanotransduction
FRONTIERS IN CELLULAR NEUROSCIENCE
2018; 12: 41
Hair cells of the inner ear transduce mechanical stimuli like sound or head movements into electrical signals, which are propagated to the central nervous system. The hair-cell mechanotransduction channel remains unidentified. We tested whether three transient receptor channel (TRP) family members, TRPV6, TRPM6 and TRPM7, were necessary for transduction. TRPV6 interacted with USH1C (harmonin), a scaffolding protein that participates in transduction. Using a cysteine-substitution knock-in mouse line and methanethiosulfonate (MTS) reagents selective for this allele, we found that inhibition of TRPV6 had no effect on transduction in mouse cochlear hair cells. TRPM6 and TRPM7 each interacted with the tip-link component PCDH15 in cultured eukaryotic cells, which suggested they might be part of the transduction complex. Cochlear hair cell transduction was not affected by manipulations of Mg2+, however, which normally perturbs TRPM6 and TRPM7. To definitively examine the role of these two channels in transduction, we showed that deletion of either or both of their genes selectively in hair cells had no effect on auditory function. We suggest that TRPV6, TRPM6 and TRPM7 are unlikely to be the pore-forming subunit of the hair-cell transduction channel.
View details for DOI 10.3389/fncel.2018.00041
View details for Web of Science ID 000425546400001
View details for PubMedID 29515374
View details for PubMedCentralID PMC5826258
The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse
The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon's contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles.
View details for DOI 10.7554/eLife.30241
View details for Web of Science ID 000423785600001
View details for PubMedID 29328021
View details for PubMedCentralID PMC5794257
Phosphoinositol-4,5-Bisphosphate Regulates Auditory Hair-Cell Mechanotransduction-Channel Pore Properties and Fast Adaptation
JOURNAL OF NEUROSCIENCE
2017; 37 (48): 11632–46
Membrane proteins, such as ion channels, interact dynamically with their lipid environment. Phosphoinositol-4,5-bisphosphate (PIP2) can directly or indirectly modify ion-channel properties. In auditory sensory hair cells of rats (Sprague Dawley) of either sex, PIP2 localizes within stereocilia, near stereocilia tips. Modulating the amount of free PIP2 in inner hair-cell stereocilia resulted in the following: (1) the loss of a fast component of mechanoelectric-transduction current adaptation, (2) an increase in the number of channels open at the hair bundle's resting position, (3) a reduction of single-channel conductance, (4) a change in ion selectivity, and (5) a reduction in calcium pore blocking effects. These changes occur without altering hair-bundle compliance or the number of functional stereocilia within a given hair bundle. Although the specific molecular mechanism for PIP2 action remains to be uncovered, data support a hypothesis for PIP2 directly regulating channel conformation to alter calcium permeation and single-channel conductance.SIGNIFICANCE STATEMENT How forces are relayed to the auditory mechanoelectrical transduction (MET) channel remains unknown. However, researchers have surmised that lipids might be involved. Previous work on bullfrog hair cells showed an effect of phosphoinositol-4,5-bisphosphate (PIP2) depletion on MET current amplitude and adaptation, leading to the postulation of the existence of an underlying myosin-based adaptation mechanism. We find similar results in rat cochlea hair cells but extend these data to include single-channel analysis, hair-bundle mechanics, and channel-permeation properties. These additional data attribute PIP2 effects to actions on MET-channel properties and not motor interactions. Further findings support PIP2's role in modulating a fast, myosin-independent, and Ca2+-independent adaptation process, validating fast adaptation's biological origin. Together this shows PIP2's pivotal role in auditory MET, likely as a direct channel modulator.
View details for DOI 10.1523/JNEUROSCI.1351-17.2017
View details for Web of Science ID 000417372300011
View details for PubMedID 29066559
View details for PubMedCentralID PMC5707765
- Rise time reduction of thermal actuators operated in air and water through optimized pre-shaped open-loop driving JOURNAL OF MICROMECHANICS AND MICROENGINEERING 2017; 27 (4)
Towards the Prevention of Aminoglycoside-Related Hearing Loss
Frontiers in Cellular Neuroscience
2017; 11: 325
Aminoglycosides are potent antibiotics deployed worldwide despite their known side-effect of sensorineural hearing loss. The main etiology of this sensory deficit is death of inner ear sensory hair cells selectively triggered by aminoglycosides. For decades, research has sought to unravel the molecular events mediating sensory cell demise, emphasizing the roles of reactive oxygen species and their potentials as therapeutic targets. Studies in recent years have revealed candidate transport pathways including the mechanotransducer channel for drug entry into sensory cells. Once inside sensory cells, intracellular targets of aminoglycosides, such as the mitochondrial ribosomes, are beginning to be elucidated. Based on these results, less ototoxic aminoglycoside analogs are being generated and may serve as alternate antimicrobial agents. In this article, we review the latest findings on mechanisms of aminoglycoside entry into hair cells, their intracellular actions and potential therapeutic targets for preventing aminoglycoside ototoxicity.
View details for DOI 10.3389/fncel.2017.00325
View details for PubMedCentralID PMC5651232
- Hair Cells and Their Synapses UNDERSTANDING THE COCHLEA 2017; 62: 183–213
Development and localization of reverse-polarity mechanotransducer channels in cochlear hair cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (24): 6767-6772
Cochlear hair cells normally detect positive deflections of their hair bundles, rotating toward their tallest edge, which opens mechanotransducer (MT) channels by increased tension in interciliary tip links. After tip-link destruction, the normal polarity of MT current is replaced by a mechanically sensitive current evoked by negative bundle deflections. The "reverse-polarity" current was investigated in cochlear hair cells after tip-link destruction with BAPTA, in transmembrane channel-like protein isoforms 1/2 (Tmc1:Tmc2) double mutants, and during perinatal development. This current is a natural adjunct of embryonic development, present in all wild-type hair cells but declining after birth with emergence of the normal-polarity current. Evidence indicated the reverse-polarity current seen developmentally was a manifestation of the same ion channel as that evident under abnormal conditions in Tmc mutants or after tip-link destruction. In all cases, sinusoidal fluid-jet stimuli from different orientations suggested the underlying channels were opened not directly by deflections of the hair bundle but by deformation of the apical plasma membrane. Cell-attached patch recording on the hair-cell apical membrane revealed, after BAPTA treatment or during perinatal development, 90-pS stretch-activated cation channels that could be blocked by Ca(2+) and by FM1-43. High-speed Ca(2+) imaging, using swept-field confocal microscopy, showed the Ca(2+) influx through the reverse-polarity channels was not localized to the hair bundle, but distributed across the apical plasma membrane. These reverse-polarity channels, which we propose to be renamed "unconventional" mechanically sensitive channels, have some properties similar to the normal MT channels, but the relationship between the two types is still not well defined.
View details for DOI 10.1073/pnas.1601067113
View details for Web of Science ID 000377948800057
View details for PubMedID 27162344
View details for PubMedCentralID PMC4914197
Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer
JOURNAL OF NEUROSCIENCE
2016; 36 (10): 2945-2956
The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (Popen) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, Popen is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases Popen. Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of Popen to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting Popen. The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates.Classically, changes in extracellular calcium and voltage affect open probability (Popen) through mechanoelectric transduction adaptation, and this mechanism is the only means of controlling the set point of the channel. Here, we further characterize the effects of extracellular calcium and voltage on the channel and for the first time determine that these manipulations occur through a mechanism that is independent of fast adaptation and involves the lipid bilayer. These data additionally demonstrate that effects on Popen are not enough to characterize adaptation and thus may clarify some of the discrepancies within the literature as to mechanisms underlying adaptation.
View details for DOI 10.1523/JNEUROSCI.3011-15.2016
View details for Web of Science ID 000372077000009
View details for PubMedID 26961949
View details for PubMedCentralID PMC4783497
Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells.
Journal of neurophysiology
2016; 115 (1): 226-239
Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking.
View details for DOI 10.1152/jn.00559.2015
View details for PubMedID 26510758
View details for PubMedCentralID PMC4760492
Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses
EUROPEAN JOURNAL OF NEUROSCIENCE
2016; 43 (2): 148-161
Functional maturation of afferent synaptic connections to inner hair cells (IHCs) involves pruning of excess synapses formed during development, as well as the strengthening and survival of the retained synapses. These events take place during the thyroid hormone (TH)-critical period of cochlear development, which is in the perinatal period for mice and in the third trimester for humans. Here, we used the hypothyroid Snell dwarf mouse (Pit1(dw) ) as a model to study the role of TH in afferent type I synaptic refinement and functional maturation. We observed defects in afferent synaptic pruning and delays in calcium channel clustering in the IHCs of Pit1(dw) mice. Nevertheless, calcium currents and capacitance reached near normal levels in Pit1(dw) IHCs by the age of onset of hearing, despite the excess number of retained synapses. We restored normal synaptic pruning in Pit1(dw) IHCs by supplementing with TH from postnatal day (P)3 to P8, establishing this window as being critical for TH action on this process. Afferent terminals of older Pit1(dw) IHCs showed evidence of excitotoxic damage accompanied by a concomitant reduction in the levels of the glial glutamate transporter, GLAST. Our results indicate that a lack of TH during a critical period of inner ear development causes defects in pruning and long-term homeostatic maintenance of afferent synapses.
View details for DOI 10.1111/ejn.13081
View details for Web of Science ID 000368245600003
Glass Probe Stimulation of Hair Cell Stereocilia.
Methods in molecular biology (Clifton, N.J.)
2016; 1427: 487-500
Hair cells are designed to sense mechanical stimuli of sound using their apical stereocilia hair bundles. Mechanical deflection of this hair bundle is converted into an electrical signal through gating of mechano-electric transduction channels. Stiff probe stimulation of hair bundles is an invaluable tool for studying the transduction channel and its associated processes because of the speed and ability to precisely control hair bundle position. Proper construction of these devices is critical to their ultimate performance as is appropriate placement of the probe onto the hair bundle. Here we describe the construction and use of a glass probe coupled to a piezo-electric actuator for stimulating hair bundles, including the basic technique for positioning of the stimulating probe onto the hair bundle. These piezo-electric stimulators can be adapted to other mechanically sensitive systems.
View details for DOI 10.1007/978-1-4939-3615-1_27
View details for PubMedID 27259944
Underestimated Sensitivity of Mammalian Cochlear Hair Cells Due to Splay between Stereociliary Columns
2015; 108 (11): 2633-2647
Current-displacement (I-X) and the force-displacement (F-X) relationships characterize hair-cell mechano-transduction in the inner ear. A common technique for measuring these relationships is to deliver mechanical stimulations to individual hair bundles with microprobes and measure whole cell transduction currents through patch pipette electrodes at the basolateral membrane. The sensitivity of hair-cell mechano-transduction is determined by two fundamental biophysical properties of the mechano-transduction channel, the stiffness of the putative gating spring and the gating swing, which are derived from the I-X and F-X relationships. Although the hair-cell stereocilia in vivo deflect <100 nm even at high sound pressure levels, often it takes >500 nm of stereocilia displacement to saturate hair-cell mechano-transduction in experiments with individual hair cells in vitro. Despite such discrepancy between in vivo and in vitro data, key biophysical properties of hair-cell mechano-transduction to define the transduction sensitivity have been estimated from in vitro experiments. Using three-dimensional finite-element methods, we modeled an inner hair-cell and an outer hair-cell stereocilia bundle and simulated the effect of probe stimulation. Unlike the natural situation where the tectorial membrane stimulates hair-cell stereocilia evenly, probes deflect stereocilia unevenly. Because of uneven stimulation, 1) the operating range (the 10-90% width of the I-X relationship) increases by a factor of 2-8 depending on probe shapes, 2) the I-X relationship changes from a symmetric to an asymmetric function, and 3) the bundle stiffness is underestimated. Our results indicate that the generally accepted assumption of parallel stimulation leads to an overestimation of the gating swing and underestimation of the gating spring stiffness by an order of magnitude.
View details for DOI 10.1016/j.bpj.2015.04.028
View details for Web of Science ID 000355668800005
View details for PubMedID 26039165
Development and Characterization of Chemical Cochleostomy in the Guinea Pig
OTOLARYNGOLOGY-HEAD AND NECK SURGERY
2015; 152 (6): 1113-1118
Creation of an atraumatic, hearing-preservation cochleostomy is integral to the future of minimally invasive inner ear surgery. The goal of this study was to develop and characterize a novel chemical approach to cochleostomy.Prospective animal study.Laboratory.Experimental animal study in which phosphoric acid gel (PAG) was used to decalcify the otic capsule in 25 Hartley guinea pigs. Five animals in each of 5 surgical groups were studied: (1) mechanically opening the auditory bulla alone, (2) PAG thinning of the basal turn otic capsule, leaving endosteum covered by a layer of bone, (3) micro-pick manual cochleostomy, (4) PAG chemical cochleostomy, exposing the endosteum, and (5) combined PAG/micro-pick cochleostomy, with initial chemical thinning and subsequent manual removal of the last osseous layer. Preoperative and postoperative auditory brainstem responses and otoacoustic emissions were obtained at 2, 6, 10, and 16 kHz. Hematoxylin and eosin-stained paraffin sections were compared.Surgical and histologic findings confirmed that application of PAG provided reproducible local bone removal, and cochlear access was enabled. Statistically significant auditory threshold shifts were observed at 10 kHz (P = .048) and 16 kHz (P = .0013) following cochleostomy using PAG alone (group 4) and at 16 kHz using manual cochleostomy (group 3) (P = .028). No statistically significant, postoperative auditory threshold shifts were observed in the other groups, including PAG thinning with manual completion cochleostomy (group 5).Hearing preservation cochleostomy can be performed in an animal model using a novel technique of thinning cochlear bone with PAG and manually completing cochleostomy.
View details for DOI 10.1177/0194599815573703
View details for Web of Science ID 000355692300025
View details for PubMedID 25779472
Activity-dependent regulation of prestin expression in mouse outer hair cells.
Journal of neurophysiology
2015; 113 (10): 3531-3542
Prestin is a membrane protein necessary for outer hair cell (OHC) electromotility and normal hearing. Its regulatory mechanisms are unknown. Several mouse models of hearing loss demonstrate increased prestin, inspiring us to investigate how hearing loss might feedback onto OHCs. To test whether centrally mediated feedback regulates prestin, we developed a novel model of inner hair cell loss. Injection of diphtheria toxin (DT) into adult CBA mice produced significant loss of inner hair cells without affecting OHCs. Thus, DT-injected mice were deaf because they had no afferent auditory input despite OHCs continuing to receive normal auditory mechanical stimulation and having normal function. Patch-clamp experiments demonstrated no change in OHC prestin, indicating that loss of information transfer centrally did not alter prestin expression. To test whether local mechanical feedback regulates prestin, we used Tecta(C1509G) mice, where the tectorial membrane is malformed and only some OHCs are stimulated. OHCs connected to the tectorial membrane had normal prestin levels, whereas OHCs not connected to the tectorial membrane had elevated prestin levels, supporting an activity-dependent model. To test whether the endocochlear potential was necessary for prestin regulation, we studied Tecta(C1509G) mice at different developmental ages. OHCs not connected to the tectorial membrane had lower than normal prestin levels before the onset of the endocochlear potential and higher than normal prestin levels after the onset of the endocochlear potential. Taken together, these data indicate that OHC prestin levels are regulated through local feedback that requires mechanoelectrical transduction currents. This adaptation may serve to compensate for variations in the local mechanical environment.
View details for DOI 10.1152/jn.00869.2014
View details for PubMedID 25810486
View details for PubMedCentralID PMC4461885
Designer aminoglycosides prevent cochlear hair cell loss and hearing loss.
journal of clinical investigation
2015; 125 (2): 583-592
Bacterial infections represent a rapidly growing challenge to human health. Aminoglycosides are widely used broad-spectrum antibiotics, but they inflict permanent hearing loss in up to ~50% of patients by causing selective sensory hair cell loss. Here, we hypothesized that reducing aminoglycoside entry into hair cells via mechanotransducer channels would reduce ototoxicity, and therefore we synthesized 9 aminoglycosides with modifications based on biophysical properties of the hair cell mechanotransducer channel and interactions between aminoglycosides and the bacterial ribosome. Compared with the parent aminoglycoside sisomicin, all 9 derivatives displayed no or reduced ototoxicity, with the lead compound N1MS 17 times less ototoxic and with reduced penetration of hair cell mechanotransducer channels in rat cochlear cultures. Both N1MS and sisomicin suppressed growth of E. coli and K. pneumoniae, with N1MS exhibiting superior activity against extended spectrum β lactamase producers, despite diminished activity against P. aeruginosa and S. aureus. Moreover, systemic sisomicin treatment of mice resulted in 75% to 85% hair cell loss and profound hearing loss, whereas N1MS treatment preserved both hair cells and hearing. Finally, in mice with E. coli-infected bladders, systemic N1MS treatment eliminated bacteria from urinary tract tissues and serially collected urine samples, without compromising auditory and kidney functions. Together, our findings establish N1MS as a nonototoxic aminoglycoside and support targeted modification as a promising approach to generating nonototoxic antibiotics.
View details for DOI 10.1172/JCI77424
View details for PubMedID 25555219
- Cytoarchitecture of the mouse organ of corti from base to apex, determined using in situ two-photon imaging. Journal of the Association for Research in Otolaryngology : JARO 2015; 16 (1): 47-66
Cytoarchitecture of the mouse organ of corti from base to apex, determined using in situ two-photon imaging.
Journal of the Association for Research in Otolaryngology : JARO
2015; 16 (1): 47-66
The cells in the organ of Corti are highly organized, with a precise 3D microstructure hypothesized to be important for cochlear function. Here we provide quantitative data on the mouse organ of Corti cytoarchitecture, as determined using a new technique that combines the imaging capabilities of two-photon microscopy with the autofluorescent cell membranes of the genetically modified mTmG mouse. This combination allowed us to perform in situ imaging on freshly excised tissue, thus minimizing any physical distortions to the tissue that extraction from the cochlea and chemical fixation and staining might have caused. 3D image stacks of the organ of Corti were obtained from base to apex in the cochlear duct, from which 3D lengths and relative angles for inner and outer hair cells, Deiters' cells, phalangeal processes, and inner and outer pillars were measured. In addition, intercellular distances, diameters, and stereocilia shapes were obtained. An important feature of this study is the quantitative reporting of the longitudinal tilts of the outer hair cells towards the base of the cochlea, the tilt of phalangeal processes towards the apex, and Deiters' cells that collectively form a Y-shaped building block that is thought to give rise to the lattice-like organization of the organ of Corti. The variations of this Y-shaped element along the cochlear duct and between the rows of outer hair cells are shown with the third row morphologically different from the other rows, and their potential importance for the cochlear amplifier is discussed.
View details for DOI 10.1007/s10162-014-0497-1
View details for PubMedID 25348579
The how and why of identifying the hair cell mechano-electrical transduction channel.
Pflügers Archiv : European journal of physiology
2015; 467 (1): 73-84
Identification of the auditory hair cell mechano-electrical transduction (hcMET) channel has been a major focus in the hearing research field since the 1980s when direct mechanical gating of a transduction channel was proposed (Corey and Hudspeth J Neurosci 3:962-976, 1983). To this day, the molecular identity of this channel remains controversial. However, many of the hcMET channel's properties have been characterized, including pore properties, calcium-dependent ion permeability, rectification, and single channel conductance. At this point, elucidating the molecular identity of the hcMET channel will provide new tools for understanding the mechanotransduction process. This review discusses the significance of identifying the hcMET channel, the difficulties associated with that task, as well as the establishment of clear criteria for this identification. Finally, we discuss potential candidate channels in light of these criteria.
View details for DOI 10.1007/s00424-014-1606-z
View details for PubMedID 25241775
View details for PubMedCentralID PMC4282604
Role of intracellular calcium stores in hair-cell ribbon synapse.
Frontiers in cellular neuroscience
2014; 8: 162-?
Intracellular calcium stores control many neuronal functions such as excitability, gene expression, synaptic plasticity, and synaptic release. Although the existence of calcium stores along with calcium-induced calcium release (CICR) has been demonstrated in conventional and ribbon synapses, functional significance and the cellular mechanisms underlying this role remains unclear. This review summarizes recent experimental evidence identifying contribution of CICR to synaptic transmission and synaptic plasticity in the CNS, retina and inner ear. In addition, the potential role of CICR in the recruitment of vesicles to releasable pools in hair-cell ribbon synapses will be specifically discussed.
View details for DOI 10.3389/fncel.2014.00162
View details for PubMedID 24971053
Adaptation of Mammalian auditory hair cell mechanotransduction is independent of calcium entry.
2013; 80 (4): 960-972
Adaptation is a hallmark of hair cell mechanotransduction, extending the sensory hair bundle dynamic range while providing mechanical filtering of incoming sound. In hair cells responsive to low frequencies, two distinct adaptation mechanisms exist, a fast component of debatable origin and a slow myosin-based component. It is generally believed that Ca(2+) entry through mechano-electric transducer channels is required for both forms of adaptation. This study investigates the calcium dependence of adaptation in the mammalian auditory system. Recordings from rat cochlear hair cells demonstrate that altering Ca(2+) entry or internal Ca(2+) buffering has little effect on either adaptation kinetics or steady-state adaptation responses. Two additional findings include a voltage-dependent process and an extracellular Ca(2+) binding site, both modulating the resting open probability independent of adaptation. These data suggest that slow motor adaptation is negligible in mammalian auditory cells and that the remaining adaptation process is independent of calcium entry.
View details for DOI 10.1016/j.neuron.2013.08.025
View details for PubMedID 24267652
Response properties from turtle auditory hair cell afferent fibers suggest spike generation is driven by synchronized release both between and within synapses
JOURNAL OF NEUROPHYSIOLOGY
2013; 110 (1): 204-220
Inner ear hair cell afferent fiber synapses are capable of transferring information at high rates, for long periods of time with extraordinary fidelity. As at other sensory synapses, hair cells rely on graded receptor potentials and unique vesicle trafficking and release properties of ribbon synapses to relay intensity information. Postsynaptic recordings from afferent fibers of the turtle auditory papilla identified EPSCs that were fast AMPA receptor-based responses with rapid onset and decay times. EPSCs varied in amplitude by about 15x per fiber, with kinetics that showed a tendency to slow at larger amplitudes. Complex EPSCs, were produced by temporal summation of single events, likely across synapses. Complex EPSCs were more efficient at generating action potentials than were single EPSCs. Potassium-evoked release increased the frequency of EPSCs, in particular complex events, but did not increase EPSC amplitudes. Temporal summation of EPSCs across synapses may underlie action potential generation at these synapses. The broad amplitude histograms were probed for mechanisms of multivesicular release using reduced external Ca(2+) or the introduction of Cd(2+) or Sr(2+) to uncouple release. Results are consistent with broad amplitude histograms being generated by a combination of the variability in synaptic vesicle size and by coordinated release of these vesicles. It is posited that multivesicular release plays less of role in multisynaptic ribbon synapses than in single synaptic afferent fibers.
View details for DOI 10.1152/jn.00121.2013
View details for Web of Science ID 000321197400021
View details for PubMedID 23596330
The elusive hair cell gating spring, a potential role for the lipid membrane.
journal of the Acoustical Society of America
2013; 133 (5): 3509-?
Deflection of auditory hair cell hair bundle results in a nonlinear (i.e., non Hookean) force-displacement relationships whose molecular mechanism remains elusive. A gating spring model posits that mechanosensitive channels are in series with a spring such that channel opening puts the activation gate in series with the spring, thus reducing spring extension until further stimulation is provided. Here we present a theoretical analysis of whether the lipid membrane might be the source of nonlinearity. A hair bundle kinematic model is coupled with a lipid membrane model that includes a diffusible compartment into which the tip-link embeds and a minimally diffusive reservoir pool. Using physiological parameters, this model was capable of reproducing nonlinear force-displacement plots, including a negative stiffness component but required a standing tip-link tension. In addition, this model suggests the mechanotransducer channel is most sensitive to curvature forces that are located within 2 nm of the tip-link. [Work supported in part by Grant Nos. R01-DC07910 and R01-DC03896 from the NIDCD of NIH and by The Timoshenko fund from Mechanical Engineering Department at Stanford University].
View details for DOI 10.1121/1.4806258
View details for PubMedID 23655592
A Novel Ion Channel Formed by Interaction of TRPML3 with TRPV5
2013; 8 (2)
TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.
View details for DOI 10.1371/journal.pone.0058174
View details for Web of Science ID 000315524900242
View details for PubMedID 23469151
Patch-Clamp Recordings from Lateral Line Neuromast Hair Cells of the Living Zebrafish
JOURNAL OF NEUROSCIENCE
2013; 33 (7): 3131-3134
Zebrafish are popular models for biological discovery. For investigators of the auditory and vestibular periphery, manipulations of hair cell and synaptic mechanisms have relied on inferences from extracellular recordings of physiological activity. We now provide data showing that hair cells and supporting cells of the lateral line can be directly patch-clamped, providing the first recordings of ionic channel activity, synaptic vesicle release, and gap junctional coupling in the neuromasts of living fish. Such capabilities will allow more detailed understanding of mechano-sensation of the zebrafish.
View details for DOI 10.1523/JNEUROSCI.4265-12.2013
View details for Web of Science ID 000314887200035
View details for PubMedID 23407967
Integrity and Regeneration of Mechanotransduction Machinery Regulate Aminoglycoside Entry and Sensory Cell Death
2013; 8 (1)
Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23(+/+) and Cdh23(v2J/+) mice, but failed to detect GTTR uptake in Cdh23(v2J/v2J) hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23(v2J/v2J) and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.
View details for DOI 10.1371/journal.pone.0054794
View details for Web of Science ID 000314023600111
View details for PubMedID 23359017
View details for PubMedCentralID PMC3554584
Faster than the Speed of Hearing: Nanomechanical Force Probes Enable the Electromechanical Observation of Cochlear Hair Cells
2012; 12 (12): 6107-6111
Understanding the mechanisms responsible for our sense of hearing requires new tools for unprecedented stimulation and monitoring of sensory cell mechanotransduction at frequencies yet to be explored. We describe nanomechanical force probes designed to evoke mechanotransduction currents at up to 100 kHz in living cells. High-speed force and displacement metrology is enabled by integrating piezoresistive sensors and piezoelectric actuators onto nanoscale cantilevers. The design, fabrication process, actuator performance, and actuator-sensor crosstalk compensation results are presented. We demonstrate the measurement of mammalian cochlear hair cell mechanotransduction with simultaneous patch clamp recordings at unprecedented speeds. The probes can deliver mechanical stimuli with sub-10 μs rise times in water and are compatible with standard upright and inverted microscopes.
View details for DOI 10.1021/nl3036349
View details for Web of Science ID 000312122100012
View details for PubMedID 23181721
Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging
7th Omaha Imaging Symposium
CAMBRIDGE UNIV PRESS. 2012: 753–60
Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality.
View details for DOI 10.1017/S1431927612000542
View details for Web of Science ID 000307171900016
View details for PubMedID 22831554
View details for PubMedCentralID PMC3549604
Permeation properties of the hair cell mechanotransducer channel provide insight into its molecular structure
JOURNAL OF NEUROPHYSIOLOGY
2012; 107 (9): 2408-2420
Mechanoelectric transducer (MET) channels, located near stereocilia tips, are opened by deflecting the hair bundle of sensory hair cells. Defects in this process result in deafness. Despite this critical function, the molecular identity of MET channels remains a mystery. Inherent channel properties, particularly those associated with permeation, provide the backbone for the molecular identification of ion channels. Here, a novel channel rectification mechanism is identified, resulting in a reduced pore size at positive potentials. The apparent difference in pore dimensions results from Ca(2+) binding within the pore, occluding permeation. Driving force for permeation at hyperpolarized potentials is increased because Ca(2+) can more easily be removed from binding within the pore due to the presence of an electronegative external vestibule that dehydrates and concentrates permeating ions. Alterations in Ca(2+) binding may underlie tonotopic and Ca(2+)-dependent variations in channel conductance. This Ca(2+)-dependent rectification provides targets for identifying the molecular components of the MET channel.
View details for DOI 10.1152/jn.01178.2011
View details for Web of Science ID 000303600900010
View details for PubMedID 22323630
Integrating the biophysical and molecular mechanisms of auditory hair cell mechanotransduction
Mechanosensation is a primitive and somewhat ubiquitous sense. At the inner ear, sensory hair cells are refined to enhance sensitivity, dynamic range and frequency selectivity. Thirty years ago, mechanisms of mechanotransduction and adaptation were well accounted for by simple mechanical models that incorporated physiological and morphological properties of hair cells. Molecular and genetic tools, coupled with new optical techniques, are now identifying and localizing specific components of the mechanotransduction machinery. These new findings challenge long-standing theories, and require modification of old and development of new models. Future advances require the integration of molecular and physiological data to causally test these new hypotheses.
View details for DOI 10.1038/ncomms1533
View details for Web of Science ID 000297686500010
View details for PubMedID 22045002
Tracking vesicle fusion from hair cell ribbon synapses using a high frequency, dual sine wave stimulus paradigm.
Communicative & integrative biology
2011; 4 (6): 785-787
Recent experiments describe a technique for tracking membrane capacitance during depolarizations where membrane conductance is varying. This is a major advance over traditional technologies that can only monitor capacitance when conductance is constant because it gives direct information regarding release kinetics from single stimulations. Presented here is additional data supporting the use of this technology with multiple conductances being active including BK-Ca-activated potassium channels, SK Ca-activated potassium conductances and also the rapidly activating sodium conductance. It goes further to illustrate the ability to monitor rapid capacitative changes. And finally, it points out the need to evaluate single step responses because of the use-dependent movement of vesicles.
View details for PubMedID 22446556
Functional Hair Cell Mechanotransducer Channels Are Required for Aminoglycoside Ototoxicity
2011; 6 (7)
Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.
View details for DOI 10.1371/journal.pone.0022347
View details for Web of Science ID 000293175100021
View details for PubMedID 21818312
View details for PubMedCentralID PMC3144223
Calcium-Dependent Synaptic Vesicle Trafficking Underlies Indefatigable Release at the Hair Cell Afferent Fiber Synapse
2011; 70 (2): 326-338
Sensory hair cell ribbon synapses respond to graded stimulation in a linear, indefatigable manner, requiring that vesicle trafficking to synapses be rapid and nonrate-limiting. Real-time monitoring of vesicle fusion identified two release components. The first was saturable with both release rate and magnitude varying linearly with Ca(2+), however the magnitude was too small to account for sustained afferent firing rates. A second superlinear release component required recruitment, in a Ca(2+)-dependent manner, of vesicles not in the immediate vicinity of the synapse. The superlinear component had a constant rate with its onset varying with Ca(2+) load. High-speed Ca(2+) imaging revealed a nonlinear increase in internal Ca(2+) correlating with the superlinear capacitance change, implicating release of stored Ca(2+) in driving vesicle recruitment. These data, supported by a mass action model, suggest sustained release at hair cell afferent fiber synapse is dictated by Ca(2+)-dependent vesicle recruitment from a reserve pool.
View details for DOI 10.1016/j.neuron.2011.01.031
View details for Web of Science ID 000291073700013
View details for PubMedID 21521617
View details for PubMedCentralID PMC3254016
Somatic motility and hair bundle mechanics, are both necessary for cochlear amplification?
2011; 273 (1-2): 109-122
Hearing organs have evolved to detect sounds across several orders of magnitude of both intensity and frequency. Detection limits are at the atomic level despite the energy associated with sound being limited thermodynamically. Several mechanisms have evolved to account for the remarkable frequency selectivity, dynamic range, and sensitivity of these various hearing organs, together termed the active process or cochlear amplifier. Similarities between hearing organs of disparate species provides insight into the factors driving the development of the cochlear amplifier. These properties include: a tonotopic map, the emergence of a two hair cell system, the separation of efferent and afferent innervations, the role of the tectorial membrane, and the shift from intrinsic tuning and amplification to a more end organ driven process. Two major contributors to the active process are hair bundle mechanics and outer hair cell electromotility, the former present in all hair cell organs tested, the latter only present in mammalian cochlear outer hair cells. Both of these processes have advantages and disadvantages, and how these processes interact to generate the active process in the mammalian system is highly disputed. A hypothesis is put forth suggesting that hair bundle mechanics provides amplification and filtering in most hair cells, while in mammalian cochlea, outer hair cell motility provides the amplification on a cycle by cycle basis driven by the hair bundle that provides frequency selectivity (in concert with the tectorial membrane) and compressive nonlinearity. Separating components of the active process may provide additional sites for regulation of this process.
View details for DOI 10.1016/j.heares.2010.03.094
View details for Web of Science ID 000289608100013
View details for PubMedID 20430075
Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection.
International journal of otolaryngology
2011; 2011: 937861-?
Aminoglycosides are commonly prescribed antibiotics with deleterious side effects to the inner ear. Due to their popular application as a result of their potent antimicrobial activities, many efforts have been undertaken to prevent aminoglycoside ototoxicity. Over the years, understanding of the antimicrobial as well as ototoxic mechanisms of aminoglycosides has increased. These mechanisms are reviewed in regard to established and potential future targets of hair cell protection.
View details for DOI 10.1155/2011/937861
View details for PubMedID 22121370
- Elastostatic Analysis of the Membrane Tenting Deformation of Inner-Ear Stereocilia 11th International Workshop on the Mechanics of Hearing AMER INST PHYSICS. 2011
- New Devices for Investigating Hair Cell Mechanical Properties 11th International Workshop on the Mechanics of Hearing AMER INST PHYSICS. 2011
- Exploring the Role of Mechanotransduction Activation and Adaptation Kinetics in Hair Cell Filtering Using a Hodgkin-Huxley Approach 11th International Workshop on the Mechanics of Hearing AMER INST PHYSICS. 2011
- Three-Dimensional Imaging of the Mouse Organ of Corti Cytoarchitecture for Mechanical Modeling 11th International Workshop on the Mechanics of Hearing AMER INST PHYSICS. 2011
Mechanosensitive Hair Cell-like Cells from Embryonic and Induced Pluripotent Stem Cells
2010; 141 (4): 704-716
Mechanosensitive sensory hair cells are the linchpin of our senses of hearing and balance. The inability of the mammalian inner ear to regenerate lost hair cells is the major reason for the permanence of hearing loss and certain balance disorders. Here, we present a stepwise guidance protocol starting with mouse embryonic stem and induced pluripotent stem cells, which were directed toward becoming ectoderm capable of responding to otic-inducing growth factors. The resulting otic progenitor cells were subjected to varying differentiation conditions, one of which promoted the organization of the cells into epithelial clusters displaying hair cell-like cells with stereociliary bundles. Bundle-bearing cells in these clusters responded to mechanical stimulation with currents that were reminiscent of immature hair cell transduction currents.
View details for DOI 10.1016/j.cell.2010.03.035
View details for Web of Science ID 000277623600022
View details for PubMedID 20478259
Rethinking How Hearing Happens
2009; 62 (3): 305-307
Inner ear hair cells convert hair bundle deflection into mechanical force sensed by ion channels via extracellular tip links between adjacent stereocilia. In this Neuron issue, Grillet and colleagues show the protein harmonin mechanically reinforces tip link upper insertion sites. Harmonin loss at this site reduces mechanotransduction kinetics and sensitivity.
View details for DOI 10.1016/j.neuron.2009.04.019
View details for Web of Science ID 000266146100001
View details for PubMedID 19447085
Localization of inner hair cell mechanotransducer channels using high-speed calcium imaging
2009; 12 (5): 553-558
Hair cells detect vibrations of their stereociliary bundle by activation of mechanically sensitive transducer channels. Although evidence suggests the transducer channels are near the stereociliary tops and are opened by force imparted by tip links connecting contiguous stereocilia, the exact channel site remains controversial. We used fast confocal imaging of fluorescence changes reflecting calcium entry during bundle stimulation to localize the channels. Calcium signals were visible in single stereocilia of rat cochlear inner hair cells and were up to tenfold larger and faster in the second and third stereociliary rows than in the tallest first row. The number of functional stereocilia was proportional to transducer current amplitude, indicating that there were about two channels per stereocilium. Comparable results were obtained in outer hair cells. The observations, supported by theoretical simulations, suggest there are no functional mechanically sensitive transducer channels in first row stereocilia and imply the channels are present only at the bottom of the tip links.
View details for DOI 10.1038/nn.2295
View details for Web of Science ID 000265575400011
View details for PubMedID 19330002
LOCALIZING HAIR CELL MECHANOTRANSDUCER CHANNELS USING HIGH SPEED CALCIUM IMAGING
SPRINGER JAPAN KK. 2009: 76–76
View details for Web of Science ID 000271023100369
- Hair bundles teaming up to tune the mammalian cochlea PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2008; 105 (48): 18651-18652
Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer
2008; 455 (7212): 537-U47
Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment. Atonal homologue 1 (encoded by Atoh1, also known as Math1) is a basic helix-loop-helix transcription factor required for hair-cell development, and its misexpression in vitro and in vivo generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory and vestibular dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes and express the ribbon synapse marker carboxy-terminal binding protein 2 (refs 12,13). Moreover, the hair cells are capable of mechanoelectrical transduction and show basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor misexpression produces functional sensory cells in the postnatal mammalian cochlea. We expect that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness.
View details for DOI 10.1038/nature07265
View details for Web of Science ID 000259449600047
View details for PubMedID 18754012
- Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer. Nature 2008; 455: 537-541
Stepwise morphological and functional maturation of mechanotransduction in rat outer hair cells
JOURNAL OF NEUROSCIENCE
2007; 27 (50): 13890-13902
Inner ear mechanosensory hair cells convert mechanical vibrations into electrical signals via the coordinated interaction of multiple proteins precisely positioned within the sensory hair bundle. Present work identifies the time course for the acquisition and maturation of mechanoelectric transduction (MET) in rat cochlea outer hair cells maintained in organotypic cultures. A spatiotemporal developmental progression was observed morphologically and functionally with basal cochlea maturation preceding apical cochlea by 2-3 d in all measured properties. The fraction of mechanosensitive cells increased rapidly, with a midpoint at postnatal day 0 for basal cells, and correlated with myosin IIIa immunoreactivity. MET current magnitude increased over several days. Adaptation lagged the onset of transduction by a day and matured more slowly, overlapping but preceding the rise in myosin Ic immunoreactivity. Less than approximately 25% of myosin Ic expression was required for the mature adaptation response, suggesting multiple roles for this protein in hair bundle function. Directional sensitivity, lacking in immature responses, developed rapidly and correlated with the pruning of radial links and an increase in tenting of stereociliary tips. Morphological and electrophysiological data support a hypothesis in which key elements arrive independently at the site of MET, with a mature response occurring as membrane tension increases, likely by the increased tensioning of the tip link with the onset of adaptation. Organotypic cultures developed normal, tonotopically specific, MET response properties, suggesting that maturation was not influenced significantly by external factors such as innervation, endolymph, normal mechanical stimulation, or an intact organ of Corti.
View details for DOI 10.1523/JNEUROSCI.2159-07.2007
View details for Web of Science ID 000251616000033
View details for PubMedID 18077701
A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (49): 19583-19588
Homozygote varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3 (mucolipin 3), are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. Here we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitint-waddler hair cells as a distinct inwardly rectifying current. We hypothesize that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembrane-spanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels were not affected. We conclude that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. To our knowledge, our study provides the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.
View details for DOI 10.1073/pnas.0709846104
View details for Web of Science ID 000251525800074
View details for PubMedID 18048323
- Hair cell mechanotransduction: The dynamic interplay between structure and function MECHANOSENSITIVE ION CHANNELS, PT B 2007; 59: 339-374
Steady-state adaptation of mechanotransduction modulates the resting potential of auditory hair cells, providing an assay for endolymph [Ca2+]
JOURNAL OF NEUROSCIENCE
2006; 26 (48): 12526-12536
The auditory hair cell resting potential is critical for proper translation of acoustic signals to the CNS, because it determines their filtering properties, their ability to respond to stimuli of both polarities, and, because the hair cell drives afferent firing rates, the resting potential dictates spontaneous transmitter release. In turtle auditory hair cells, the filtering properties are established by the interactions between BK calcium-activated potassium channels and an L-type calcium channel (electrical resonance). However, both theoretical and in vitro recordings indicate that a third conductance is required to set the resting potential to a point on the I(Ca) and I(BK) activation curves in which filtering is optimized like that found in vivo. Present data elucidate a novel mechanism, likely universal among hair cells, in which mechanoelectric transduction (MET) and its calcium-dependent adaptation provide the depolarizing current to establish the hair cell resting potential. First, mechanical block of the MET current hyperpolarized the membrane potential, resulting in broadband asymmetrical resonance. Second, altering steady-state adaptation by altering the [Ca2+] bathing the hair bundle changed the MET current at rest, the magnitude of which resulted in membrane potential changes that encompassed the best resonant voltage. The Ca2+ sensitivity of adaptation allowed for the first physiological estimate of endolymphatic Ca2+ near the MET channel (56 +/- 11 microM), a value similar to bulk endolymph levels. These effects of MET current on resting potential were independently confirmed using a theoretical model of electrical resonance that included the steady-state MET conductance.
View details for DOI 10.1523/JNEUROSCI.3569-06.2006
View details for Web of Science ID 000242387900016
View details for PubMedID 17135414
Mechano-electrical transduction: New insights into old ideas
JOURNAL OF MEMBRANE BIOLOGY
2006; 209 (2-3): 71-88
The gating-spring theory of hair cell mechanotransduction channel activation was first postulated over twenty years ago. The basic tenets of this hypothesis have been reaffirmed in hair cells from both auditory and vestibular systems and across species. In fact, the basic findings have been reproduced in every hair cell type tested. A great deal of information regarding the structural, mechanical, molecular and biophysical properties of the sensory hair bundle and the mechanotransducer channel has accumulated over the past twenty years. The goal of this review is to investigate new data, using the gating spring hypothesis as the framework for discussion. Mechanisms of channel gating are presented in reference to the need for a molecular gating spring or for tethering to the intra- or extracellular compartments. Dynamics of the sensory hair bundle and the presence of motor proteins are discussed in reference to passive contributions of the hair bundle to gating compliance. And finally, the molecular identity of the channel is discussed in reference to known intrinsic properties of the native transducer channel.
View details for DOI 10.1007/s00232-005-0834-8
View details for Web of Science ID 000238293000002
View details for PubMedID 16773495
- The role of mechanoelectric transduction and Journal of Neuroscience 2006; 26: 12526
- Aminoglycoside ototoxicity: permeant drugs cause permanent hair cell loss JOURNAL OF PHYSIOLOGY-LONDON 2005; 567 (2): 359-360
Auditory hair cell-afferent fiber synapses are specialized to operate at their best frequencies
2005; 47 (2): 243-254
Auditory afferent fiber activity is driven by high-fidelity information transfer from the sensory hair cell. Presynaptic specializations, posited to maintain fidelity, are investigated at synapses with characteristic frequencies of 120 Hz and 320 Hz. Morphological data indicate that high-frequency cells have more synapses and higher vesicle density near dense bodies (DBs). Tracking vesicular release via capacitance changes identified three overlapping kinetic components of release corresponding to morphologically identified vesicle pools. High-frequency cells released faster; however, when normalized to release site number, low-frequency cells released faster, likely due to a greater Ca2+ load per synapse. The Ca(2+)-dependence of release was nonsaturating and independent of frequency, suggesting that release, not refilling, was rate limiting. A model of release derived from vesicle equilibration between morphologically defined pools reproduced the capacitance data, supporting a critical role in vesicle trafficking for DBs. The model suggests that presynaptic specializations enable synapses to operate most efficiently at their characteristic frequencies.
View details for DOI 10.1016/j.neuron.2005.06.004
View details for Web of Science ID 000230693900011
View details for PubMedID 16039566
Voltage-clamp errors cause anomalous interaction between independent ion channels
2005; 16 (9): 943-947
In voltage-clamp, uncompensated series resistance results in steady-state voltage errors that scale with the amplitude of the elicited current and are often correctable offline. However, while investigating mechanoelectric transduction currents at hair cells' resting potential, voltage-gated calcium channels and calcium-activated potassium channels (BK) were activated in voltage-clamp by displacing the sensory hair bundle. This resulted from steady-state voltage errors (<1.5 mV) induced by series resistance changing the holding potential. Thus, uncompensated series resistance, interacting with an elicited current, resulted in a voltage error that could induce the erroneous activation of other currents. This error is not correctable offline. Recognizing this type of error is critical when investigating multiple voltage-dependent conductances with steep voltage dependence.
View details for Web of Science ID 000230607600013
View details for PubMedID 15931066
- The transduction Journal of Neursocience 2005; 25: 7831..7839
Probing the pore of the auditory hair cell mechanotransducer channel in turtle
JOURNAL OF PHYSIOLOGY-LONDON
2004; 558 (3): 769-792
Hair cell mechano-electric transducer (MET) channels play a pivotal role in auditory and vestibular signal detection, yet few data exist regarding their molecular nature. Present work characterizes the MET channel pore, a region whose properties are thought to be intrinsically determined. Two approaches were used. First, the channel was probed with antagonists of candidate channel subtypes including: cyclic nucleotide-gated channels, transient receptor potential channels and gap-junctional channels. Eight new antagonists were identified. Most of the effective antagonists had a partially charged amine group predicted to penetrate the channel pore, antagonizing current flow, while the remainder of the molecule prevented further permeation of the compound through the pore. This blocking mechanism was tested using curare to demonstrate the open channel nature of the block and by identifying methylene blue as a permeant channel blocker. The second approach estimated dimensions of the channel pore with simple amine compounds. The narrowest diameter of the pore was calculated as 12.5 +/- 0.8 A and the location of a binding site approximately 45% of the way through the membrane electric field was calculated. Channel length was estimated as approximately 31 A and the width of the pore mouth at < 17 A. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC(50) and minimal diameter. The IC(50) was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore.
View details for DOI 10.1113/jphysiol.2004.061267
View details for Web of Science ID 000223435900005
View details for PubMedID 15181168
Mechano-electrical transduction in the turtle utricle.
Biomedical sciences instrumentation
2004; 40: 441-446
Two classes of mechano-sensory hair cell are present in the vestibular system of mammals, birds and reptiles. Type I hair cells are bottle-shaped and make synaptic contact with afferent calyx terminals. Type II hair cells are cylindrical and contact small bouton afferent terminals. Voltage-dependent basolateral currents have been found to differ between the two cell types and these properties are believed to contribute to the shaping of primary afferent responses. Type I hair cells have a low input resistance, which may be related to the low gain of calyx afferents. In the turtle utricle, type I vestibular hair cells are found only in a narrow band of the sensory epithelium called the striola, whereas type II hair cells are found in both striolar and extrastriolar regions. We have made whole cell patch clamp recordings from type I hair cells, type II hair cells and calyx fibers in order to better understand the processing of vestibular signals. Here we describe responses of hair cells to hair bundle displacement with a stiff glass probe. Mechano-electrical transduction (MET) currents were largest in type I hair cells, where the mean peak amplitude was approximately 500 pA. MET currents in all hair cells showed rapid and slow adaptation.
View details for PubMedID 15133998
Mechano-electrical transduction in the turtle utricle
41st Annual Rocky Mountain Bioengineering Symposium/41st International ISA Biomedical Sciences Instrumentation Symposium
INSTRUMENT SOC AMER. 2004: 441–446
View details for Web of Science ID 000221906600073
Tonotopic variation in the conductance of the hair cell mechanotransducer channel
2003; 40 (5): 983-990
Hair cells in the vertebrate cochlea are arranged tonotopically with their characteristic frequency (CF), the sound frequency to which they are most sensitive, changing systematically with position. Single mechanotransducer channels of hair cells were characterized at different locations in the turtle cochlea. In 2.8 mM external Ca2+, the channel's chord conductance was 118 pS (range 80-163 pS), which nearly doubled (range 149-300 pS) on reducing Ca2+ to 50 microM. In both Ca2+ concentrations, the conductance was positively correlated with hair cell CF. Variation in channel conductance can largely explain the increases in size of the macroscopic transducer current and speed of adaptation with CF. It suggests diversity of transducer channel structure or environment along the cochlea that may be an important element of its tonotopic organization.
View details for Web of Science ID 000187042200014
View details for PubMedID 14659096
Adaptation in auditory hair cells
CURRENT OPINION IN NEUROBIOLOGY
2003; 13 (4): 446-451
The narrow stimulus limits of hair cell transduction, equivalent to a total excursion of about 100nm at the tip of the hair bundle, demand tight regulation of the mechanical input to ensure that the mechanoelectrical transducer (MET) channels operate in their linear range. This control is provided by multiple components of Ca(2+)-dependent adaptation. A slow mechanism limits the mechanical stimulus through the action of one or more unconventional myosins. There is also a fast, sub-millisecond, Ca(2+) regulation of the MET channel, which can generate resonance and confer tuning on transduction. Changing the conductance or kinetics of the MET channels can vary their resonant frequency. The tuning information conveyed in transduction may combine with the somatic motility of outer hair cells to produce an active process that supplies amplification and augments frequency selectivity in the mammalian cochlea.
View details for DOI 10.1016/S0959-4388(03)00094-1
View details for Web of Science ID 000185403000008
View details for PubMedID 12965292
Biophysical and pharmacological characterization of voltage-gated calcium currents in turtle auditory hair cells
JOURNAL OF PHYSIOLOGY-LONDON
2003; 549 (3): 697-717
Hair cell calcium channels regulate membrane excitability and control synaptic transmission. The present investigations focused on determining whether calcium channels vary between hair cells of different characteristic frequencies or if multiple channel types exist within a hair cell, each serving a different function. To this end, turtle auditory hair cells from high- (317 +/- 27 Hz) and low-frequency (115 +/- 6 Hz) positions were voltage clamped using the whole-cell recording technique, and calcium currents were characterized based on activation, inactivation and pharmacological properties. Pharmacological sensitivity to dihydropyridines (nimodipine, Bay K 8644), benzothiazepines (diltiazem) and acetonitrile derivatives (verapamil, D600) and the insensitivity to non-L-type calcium channel antagonists support the conclusion that only L-type calcium channels were present. Fast activation rise times (< 0.5 ms), hyperpolarized half-activation potentials and a relative insensitivity to nimodipine suggest the channels were of the alpha1D (CaV1.3) variety. Although no pharmacological differences were found between calcium currents obtained from high- and low-frequency cells, low-frequency cells activated slightly faster and at hyperpolarized potentials, with half-activating voltages of -43 +/- 1 mV compared to -35 +/- 1 mV. Inactivation was observed in both high- and low-frequency cells. The time course of inactivation required three time constants for a fit. Long depolarizations could result in complete inactivation. The voltage of half-inactivation was -40 +/- 2 mV for high-frequency cells and -46 +/- 2 mV for low-frequency cells. Calcium channel inactivation did not significantly alter hair cell electrical resonant properties elicited from protocols where the membrane potential was hyperpolarized or depolarized prior to characterizing the resonance. A bell-shaped voltage dependence and modest sensitivities to intracellular calcium chelators and external barium ions suggest that inactivation was calcium dependent.
View details for DOI 10.1113/jphysiol.2002.037481
View details for Web of Science ID 000183815200005
View details for PubMedID 12740421
- Tonotopic Variation in the Conductance of Neuron 2003; 40: 1..20
The effects of calcium on mechanotransducer channel kinetics in auditory hair cells
Conference on Biophysics of the Cochlea - Molecules to Models
WORLD SCIENTIFIC PUBL CO PTE LTD. 2003: 65–72
View details for Web of Science ID 000229998300007
Mechanisms of active hair bundle motion in auditory hair cells
JOURNAL OF NEUROSCIENCE
2002; 22 (1): 44-52
Sound stimuli vibrate the hair bundles on auditory hair cells, but the resulting motion attributable to the mechanical stimulus may be modified by forces intrinsic to the bundle, which drive it actively. One category of active hair bundle motion has properties similar to fast adaptation of the mechanotransducer channels and is explicable if gating of the channels contributes significantly to the mechanics of the hair bundle. To explore this mechanism, we measured hair bundle compliance in turtle auditory hair cells under different conditions that alter the activation range of the channel. Force-displacement relationships were nonlinear, possessing a maximum slope compliance when approximately one-half of the transducer channels were open. When the external calcium concentration was reduced from 2.8 to 0.25 mm, the position of maximum compliance was shifted negative, reflecting a comparable shift in the transducer channel activation curve. Assuming that the nonlinearity represents the compliance attributable to channel gating, a single-channel gating force of 0.25 pN was calculated. By comparing bundle displacements with depolarization with and without an attached flexible fiber, the force contributed by each channel was independently estimated as 0.47 pN. These results are consistent with fast active bundle movements resulting from changes in mechanotransducer channel gating. However, several observations revealed additional components of hair bundle motion, with slower kinetics and opposite polarity to the fast movement but also linked to transducer adaptation. This finding argues for multiple mechanisms for controlling hair bundle position in auditory hair cells.
View details for Web of Science ID 000172905700012
View details for PubMedID 11756487
Clues to the cochlear amplifier from the turtle ear
TRENDS IN NEUROSCIENCES
2001; 24 (3): 169-175
Sound stimuli are detected in the cochlea by vibration of hair bundles on sensory hair cells, which activates mechanotransducer ion channels and generates an electrical signal. Remarkably, the process can also work in reverse with additional force being produced by the ion channels as they open and close, evoking active movements of the hair bundle. These movements could supplement the energy of the sound stimuli but to be effective they would need to be very fast. New measurements in the turtle ear have shown that such active bundle movements occur with delays of less than a millisecond, and are triggered by the entry of Ca(2+) into the cell via the mechanotransducer channel. Furthermore, their speed depends on the frequency to which the hair cell is most sensitive, suggesting that such movements could be important in cochlear amplification and frequency discrimination.
View details for Web of Science ID 000168765900012
View details for PubMedID 11182457
Active hair bundle motion linked to fast transducer adaptation in auditory hair cells
JOURNAL OF NEUROSCIENCE
2000; 20 (19): 7131-7142
During transduction in auditory hair cells, hair bundle deflection opens mechanotransducer channels that subsequently reclose or adapt to maintained stimuli, a major component of the adaptation occurring on a submillisecond time scale. Using a photodiode imaging technique, we measured hair bundle motion in voltage-clamped turtle hair cells to search for a mechanical correlate of fast adaptation. Excitatory force steps imposed by a flexible glass fiber attached to the bundle caused an initial movement toward the kinocilium, followed by a fast recoil equivalent to bundle stiffening. The recoil had a time course identical to adaptation of the transducer current, and like adaptation, was most prominent for small stimuli, was slowed by reducing extracellular calcium, and varied with hair cell resonant frequency. In free-standing hair bundles, depolarizations positive to 0 mV evoked an outward current attributable to opening of transducer channels, which was accompanied by a sustained bundle deflection toward the kinocilium. Both processes were sensitive to external calcium concentration and were abolished by blocking the transducer channels with dihydrostreptomycin. The similarity in properties of fast adaptation and the associated bundle motion indicates the operation of a rapid calcium-sensitive force generator linked to the gating of the transducer channels. This force generator may permit stimulus amplification during transduction in auditory hair cells.
View details for Web of Science ID 000089538400005
View details for PubMedID 11007868
Tonotopic variations of calcium signalling in turtle auditory hair cells
JOURNAL OF PHYSIOLOGY-LONDON
2000; 524 (2): 423-436
Turtle cochlear hair cells are electrically tuned by a voltage-dependent Ca2+ current and a Ca2+-dependent K+ current (IBK(Ca)). The effects of intracellular calcium buffering on electrical tuning were studied in hair cells at apical and basal cochlear locations tuned to 100 and 300 Hz, respectively. Increasing the intracellular BAPTA concentration changed the hair cell's resonant frequency little, but optimized tuning at more depolarized membrane potentials due to a positive shift in the half-activation voltage (V ) of the IBK(Ca). The shift in V depended similarly on BAPTA concentration in basal and apical hair cells despite a 2. 4-fold difference in the size of the Ca2+ current at the two positions. The Ca2+ current amplitude increased exponentially with distance along the cochlea. Comparison of V values and tuning properties using different BAPTA concentrations with values measured in perforated-patch recordings gave the endogenous calcium buffer as equivalent to 0.21 mM BAPTA in low-frequency cells, and 0.46 mM BAPTA in high-frequency cells. High conductance Ca2+-activated K+ (BKCa) channels recorded in inside-out membrane patches were 2-fold less Ca2+ sensitive in high-frequency than in low-frequency cells. Confocal Ca2+ imaging using the fluorescent indicator Calcium Green-1 revealed about twice as many hotspots of Ca2+ entry during depolarization in high-frequency compared to low-frequency hair cells. We suggest that each BKCa channel is gated by Ca2+ entry through a few nearby Ca2+ channels, and that Ca2+ and BKCa channels occupy, at constant channel density, a greater fraction of the membrane area in high-frequency cells than in low-frequency cells.
View details for Web of Science ID 000086905000012
View details for PubMedID 10766923
Two components of transducer adaptation in auditory hair cells
JOURNAL OF NEUROPHYSIOLOGY
1999; 82 (5): 2171-2181
Mechanoelectrical transducer currents in turtle auditory hair cells adapted to maintained stimuli via a Ca(2+)-dependent mechanism characterized by two time constants of approximately 1 and 15 ms. The time course of adaptation slowed as the stimulus intensity was raised because of an increased prominence of the second component. The fast component of adaptation had a similar time constant for both positive and negative displacements and was unaffected by the myosin ATPase inhibitors, vanadate and butanedione monoxime. Adaptation was modeled by a scheme in which Ca(2+) ions, entering through open transducer channels, bind at two intracellular sites to trigger independent processes leading to channel closure. It was assumed that the second site activates a modulator with 10-fold slower kinetics than the first site. The model was implemented by computing Ca(2+) diffusion within a single stereocilium, incorporating intracellular calcium buffers and extrusion via a plasma membrane CaATPase. The theoretical results reproduced several features of the experimental responses, including sensitivity to the concentration of external Ca(2+) and intracellular calcium buffer and a dependence on the onset speed of the stimulus. The model also generated damped oscillatory transducer responses at a frequency dependent on the rate constant for the fast adaptive process. The properties of fast adaptation make it unlikely to be mediated by a myosin motor, and we suggest that it may result from Ca(2+) binding to the transducer channel or a nearby cytoskeletal element.
View details for Web of Science ID 000083875700017
View details for PubMedID 10561397
Electrical response properties of avian lagena type II hair cells: a model system for vestibular filtering
AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY
1999; 276 (4): R943-R953
Data presented represent the first electrical recordings from avian lagena type II hair cells. The perforated-patch variant of the whole cell recording technique was used to investigate how the macroscopic currents shaped the voltage response of the hair cells. Voltage-clamp data separated cells into two broad classes on the basis of differences in activation rates, rates and degree of inactivation, and pharmacological sensitivity. Current-clamp recordings revealed low-quality membrane voltage oscillations (Qc < 1) during pulse current injections. Oscillation frequency correlated with activation rate of the macroscopic currents. The quality of membrane oscillations (Qc) varied linearly with frequency for cells with little inactivation. For cells with rapid inactivation, no relationship was found between Qc and frequency. Rapid inactivation may serve to extend the bandwidth of vestibular hair cells. The frequency measured from voltage responses to pulsed currents may reflect the corner frequency of the cell. The filtering properties of avian lagena hair cells are like those found in all other vestibular end organs, suggesting that the electrical membrane properties of these cells are not responsible for specializing them to a particular stimulus modality.
View details for Web of Science ID 000079548600004
View details for PubMedID 10198371
The endogenous calcium buffer and the time course of transducer adaptation in auditory hair cells
JOURNAL OF NEUROSCIENCE
1998; 18 (20): 8261-8277
Mechanoelectrical transducer currents in turtle auditory hair cells adapt to maintained stimuli via a Ca2+-dependent mechanism that is sensitive to the level of internal calcium buffer. We have used the properties of transducer adaptation to compare the effects of exogenous calcium buffers in the patch electrode solution with those of the endogenous buffer assayed with perforated-patch recording. The endogenous buffer of the hair bundle was equivalent to 0.1-0.4 mM BAPTA and, in a majority of cells, supported adaptation in an external Ca2+ concentration of 70 microM similar to that in turtle endolymph. The endogenous buffer had a higher effective concentration, and the adaptation time constant was faster in cells at the high-frequency end than at the low-frequency end of the cochlea. Experiments using buffers with different Ca2+-binding rates or dissociation constants indicated that the speed of adaptation and the resting open probability of the transducer channels could be differentially regulated and imply that the endogenous buffer must be a fast, high-affinity buffer. In some hair cells, the transducer current did not decay exponentially during a sustained stimulus but displayed damped oscillations at a frequency (58-230 Hz) that depended on external Ca2+ concentration. The gradient in adaptation time constant and the tuned transducer current at physiological levels of calcium buffer and external Ca2+ suggest that transducer adaptation may contribute to hair cell frequency selectivity. The results are discussed in terms of feedback regulation of transducer channels mediated by Ca2+ binding at two intracellular sites.
View details for Web of Science ID 000076317600015
View details for PubMedID 9763471
Calcium permeation of the turtle hair cell mechanotransducer channel and its relation to the composition of endolymph
JOURNAL OF PHYSIOLOGY-LONDON
1998; 506 (1): 159-173
1. Recordings of mechanoelectrical transducer currents were combined with calcium imaging of hair bundles in turtle auditory hair cells located near the high-frequency end of the cochlea. The external face of the hair bundles was perfused with a range of Ca2+ concentrations to study the quantitative relationship between Ca2+ influx and transducer adaptation. 2. With Na+ as the monovalent ion, the peak amplitude of the transducer current decreased monotonically as the external [Ca2+] was raised from 25 microns to 20 mm. When Na+ was replaced with the impermeant Tris the transducer current increased with external [Ca2+]. These results indicate that Ca2+ can both permeate and block the transducer channels. The Ca2+ concentration for half-block of the monovalent current was 1 mm. 3. To quantify the Ca2+ influx, the fraction of transducer current carried by Ca2+ was measured using the change in bundle fluorescence in cells loaded with 1 mm Calcium Green-1. The fluorescence change was calibrated by substituting an impermeable monovalent ion to render Ca2+ the sole charge carrier. 4. In the presence of Na+, the fractional Ca2+ current was approximately 10% in 50 microns Ca2+, a concentration similar to that in endolymph, which bathes the hair bundles in vivo. The amount of Ca2+ entering was dependent on the identity of the monovalent ion, and was larger with K+, suggesting that the transducer channel is a multi-ion pore. 5. Over a range of ionic conditions, the rate of transducer adaptation was proportional to Ca2+ influx indicating that adaptation is driven by a rise in intracellular [Ca2+]. 6. Shifts in the current-displacement function along the displacement axis in different external Ca2+ concentrations were predictable from variation in the resting Ca2+ influx. We suggest that changes in the resting open probability of the transducer channels adjust the entry of Ca2+ to keep its concentration constant at an internal site. 7. The results demonstrate that endolymph containing high K+, 50 microns Ca2+ and low Mg2+ concentrations, maximizes the transducer current while still allowing sufficient Ca2+ entry to drive adaptation. The hair cell mechanotransducer channel, in its permeation and block by Ca2+, shows behaviour similar to the voltage-gated Ca2+ channel and the cyclic nucleotide-gated channel.
View details for Web of Science ID 000071648500012
View details for PubMedID 9481679
Vestibular type I and type II hair cells .1. Morphometric identification in the pigeon and gerbil
JOURNAL OF VESTIBULAR RESEARCH-EQUILIBRIUM & ORIENTATION
1997; 7 (5): 393-406
Classically, type I and type II vestibular hair cells have been defined by their afferent innervation patterns. Little quantitative information exists on the intrinsic morphometric differences between hair cell types. Data presented here define a quantitative method for distinguishing hair cell types based on the morphometric properties of the hair cell's neck region. The method is based initially on fixed histological sections, where hair cell types were identified by innervation pattern, type I cells having an afferent calyx. Cells were viewed using light microscopy, images were digitized, and measurements were made of the cell body width, the cuticular plate width, and the neck width. A plot of the ratio of the neck width to cuticular plate width (NPR) versus the ratio of the neck width to the body width (NBR) established four quadrants based on the best separation of type I and type II hair cells. The combination of the two variables made the accuracy of predicting either type I or type II hair cells greater than 90%. Statistical cluster analysis confirmed the quadrant separation. Similar analysis was performed on dissociated hair cells from semicircular canal, utricle, and lagena, giving results statistically similar to those of the fixed tissue. Additional comparisons were made between fixed tissue and isolated hair cells as well as across species (pigeon and gerbil) and between end organs (semicircular canal, utricle, and lagena). In each case, the same morphometric boundaries could be used to establish four quadrants, where quadrant 1 was predominantly type I cells and quadrant 3 was almost exclusively type II hair cells. The quadrant separations were confirmed statistically by cluster analysis. These data demonstrate that there are intrinsic morphometric differences between type I and type II hair cells and that these differences can be maintained when the hair cells are dissociated from their respective epithelia.
View details for Web of Science ID A1997XY11700003
View details for PubMedID 9376913
Vestibular type I and type II hair cells .2. Morphometric comparisons of dissociated pigeon hair cells
JOURNAL OF VESTIBULAR RESEARCH-EQUILIBRIUM & ORIENTATION
1997; 7 (5): 407-420
Morphometric properties of solitary hair cells dissociated from the semicircular canals (SCC), utricles (UTR), and lagenas (LAG) of adult white king pigeons, Columbia livia, were compared. Measurements were made of the cell body, cuticular plate and hair bundle. Cells were divided into two groups: type 1 (group 1) was predominantly type I hair cells, and type 2 (group 3) was primarily type II hair cells. Comparisons are made initially between end organs for each group. A subpopulation of short otolith hair cells was identified. Quantitative comparisons between isolated type 1 and type 2 hair cells demonstrated that type 1 hair cells were more homogeneous both within and between vestibular end organs; while they had shorter, thinner neck regions, narrower apical surfaces, with longer and thinner bodies than did type 2 hair cells. Generally, for both type 1 and type 2 hair cells, two different hair bundle shapes were present, those (unimodal) with a single sharp taper from longest to shortest stereocilia, and those (bimodal) with an initial steep taper followed by a less steep taper. An additional subtype of type 1 hair cells with short hair bundles was identified. SCC hair cells have fewer hair bundles with bimodal tapers across all cell groups when compared to UTR or LAG. All cell subtypes identified for dissociated hair cells were corroborated using histologic sections.
View details for Web of Science ID A1997XY11700004
View details for PubMedID 9376914
The effects of calcium buffering and cyclic AMP on mechanoelectrical transduction in turtle auditory hair cells
JOURNAL OF PHYSIOLOGY-LONDON
1997; 501 (1): 111-124
1. The effects of intracellular Ca2+ buffering on hair cell mechanotransduction were studied in an intact cochlear epithelium where the endolymphatic and perilymphatic surfaces could be separately perfused with different Ca2+ solutions. 2. The speed and extent of transducer adaptation increased as the concentration in the patch electrode of the Ca2+ buffer BAPTA was lowered. In 0.1 mM BAPTA or less, the transducer adapted almost completely, with a mean time constant of 0.8 ms. 3. For a fixed internal BAPTA concentration, the transducer conductance varied with hair cell location, increasing towards the high-frequency end of the cochlea, and the time constant of adaptation decreased proportionally. At a given cochlear location, hair cells with larger transducer conductances displayed faster adaptation. We suggest that transducer adaptation accounts for a variable high-pass filter observed in the acoustic tuning curve. 4. The effects of perfusion of 50 microM Ca2+ endolymph depended on the BAPTA concentration of the electrode: with 3 mM BAPTA, adaptation was abolished, but in most recordings with 0.01 or 0.1 mM BAPTA, rapid adaptation was retained. The current-displacement curve was also shifted less the lower the intracellular BAPTA concentration. Cells in the high-frequency half of the papilla retained adaptation at a higher BAPTA concentration. 5. Treatment with the cAMP agonist, 8-bromo-cAMP, or with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, caused a rightward shift in the current-displacement curve which was independent of the internal BAPTA concentration. 6. We conclude that the free Ca2+ and cyclic nucleotide concentrations of the hair bundle modulate the position of the activation curve of the transducer. The factors which may be important for the correct functioning of adaptation in vivo are discussed.
View details for Web of Science ID A1997XA71400011
View details for PubMedID 9174998
The delayed rectifier, I-KI is the major conductance in type I vestibular hair cells across vestibular end organs
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
1996; 432 (1): 34-42
Hair cells were dissociated from the semicircular canal, utricle, lagena and saccule of white king pigeons. Type I hair cells were identified morphologically based on the ratios of neck width to cuticular plate width (NPR < 0.72) as well as neck width to cell body width (NBR < 0.64). The perforated patch variant of the whole-cell recording technique was used to measure electrical properties from type I hair cells. In voltage-clamp, the membrane properties of all identified type I cells were dominated by a predominantly outward potassium current, previously characterized in semicircular canal as IKI. Zero-current potential, activation, deactivation, slope conductance, pharmacologic and steady-state properties of the complex currents were not statistically different between type I hair cells of different vestibular end organs. The voltage dependence causes a significant proportion of this conductance to be active about the cell's zero-current potential. The first report of the whole-cell activation kinetics of the conductance is presented, showing a voltage dependence that could be best fit by an equation for a single exponential. Results presented here are the first data from pigeon dissociated type I hair cells from utricle, saccule and lagena suggesting that the basolateral conductances of a morphologically identified population of type I hair cells are conserved between functionally different vestibular end organs; the major conductance being a delayed rectifier characterized previously in semicircular canal hair cells as IKI.
View details for Web of Science ID A1996UP58600005
View details for PubMedID 8662265
Electrical filtering in gerbil isolated type I semicircular canal hair cells
JOURNAL OF NEUROPHYSIOLOGY
1996; 75 (5): 2117-2123
1. Membrane potential responses of dissociated gerbil type I semicircular canal hair cells to current injections in whole cell current-clamp have been measured. The input resistance of type I cells was 21.4 +/- 14.3 (SD) M omega, (n = 25). Around the zero-current potential (Vz = -66.6 +/- 9.3 mV, n = 25), pulsed current injections (from approximately -200 to 750 pA) produced only small-amplitude, pulse-like changes in membrane potential. 2. Injecting constant current to hyperpolarize the membrane to around -100 mV resulted in a approximately 10-fold increase in membrane resistance. Current pulses superimposed on this constant hyperpolarization produced larger and more complex membrane potential changes. Depolarizing currents > or = 200 pA caused a rapid transient peak voltage before a plateau. 3. Membrane voltage was able to faithfully follow sine-wave current injections around Vz over the range 1-1,000 Hz with < 25% attenuation at 1 kHz. A previously described K conductance, IKI, which is active at Vz, produces the low input resistance and frequency response. This was confirmed by pharmacologically blocking IKI. This conductance, present in type I cells but not type II hair cells, would appear to confer on type I cells a lower gain, but a much broader bandwidth at Vz, than seen in type II cells.
View details for Web of Science ID A1996UK80900029
View details for PubMedID 8734607
- Filtering properties of vestibular hair cells: An update Conference on New Directions in Vestibular Research NEW YORK ACAD SCIENCES. 1996: 138–149
- A delayed rectifier conductance shapes the voltage response of type I hair cells Conference on New Directions in Vestibular Research NEW YORK ACAD SCIENCES. 1996: 690–692
COMPARATIVE ELECTROPHYSIOLOGICAL PROPERTIES OF GUINEA-PIG (CAVIA-COBAYA) OUTER HAIR-CELLS AND FROG (RANA-PIPIENS) SEMICIRCULAR CANAL HAIR-CELLS
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY
1994; 107 (1): 13-21
View details for Web of Science ID A1994MV32800004
THE INACTIVATING POTASSIUM CURRENTS OF HAIR-CELLS ISOLATED FROM THE CRISTA-AMPULLARIS OF THE FROG
JOURNAL OF NEUROPHYSIOLOGY
1992; 68 (5): 1642-1653
1. A-type outward currents were studied in sensory hair cells isolated from the semicircular canals (SCC) of the leopard frog (Rana pipiens) with whole-cell voltage- and current-clamping techniques. 2. There appear to be two classes of A-type outward-conducting potassium channels based on steady-state, kinetic, pharmacological parameters, and reversal potential. 3. The two classes of A-type currents differ in their steady-state inactivation properties as well as in the kinetics of inactivation. The steady-state inactivation properties are such that a significant portion of the fast channels are available from near the resting potential. 4. The inactivating channels studied do not appear to be calcium dependent. 5. The A-channels in hair cells appear to subserve functions that are analogous to IA functions in neurons, that is, modulating spike latency and Q (the oscillatory damping function). The A-currents appear to temporally limit the hair cell voltage response to a current injection.
View details for Web of Science ID A1992JY68500013
View details for PubMedID 1336044
CYCLIC-AMP MODULATES SENSORY-NEURAL COMMUNICATION AT THE VESTIBULAR END ORGAN
1991; 565 (1): 78-84
Adenosine 3':5'-cyclic phosphate (cAMP) is a second messenger that plays an important role in mediating neuronal interactions in many systems. A possible role for cAMP in sensorineural communication at the vestibular end organ was studied. The putative roles for cAMP action investigated here were: the ability of cAMP to act as the second messenger for the efferent transmitter, acetylcholine, and the possible involvement of cAMP in modulating spontaneous or mechanically-evoked afferent nerve firing. Levels of cAMP were increased pharmacologically with forskolin, 3-isobutyl-1-methyl xanthine (IBMX) and dibutyryl cAMP. Changes in multiunit afferent nerve firing measured from the ampullar nerve of the semicircular canal, and the transepithelial potential measured across the neuroepithelium of the semicircular canal were recorded. At selected doses, all drugs produced a similar increase in spontaneous multiunit afferent nerve firing with a concomitant decrease in the transepithelial potential. Mechanically-evoked hair cell activity and the response to exogenously applied acetylcholine were unaffected by these drugs. We are suggesting that the excitatory aspects of the acetylcholine response are not mediated via a cAMP-dependent mechanism. However, cAMP does play an important role in modulating spontaneous afferent nerve firing in the semicircular canal. The finding that spontaneous afferent nerve firing can be biochemically modulated without altering mechanically-induced afferent firing is novel and deserves further investigation.
View details for Web of Science ID A1991GV92800010
View details for PubMedID 1723024
DIFFERENTIAL MODULATION OF SPONTANEOUS AND EVOKED NEUROTRANSMITTER RELEASE FROM HAIR-CELLS - SOME NOVEL HYPOTHESES
1991; 56 (1-2): 69-78
It has been generally accepted that even in the absence of mechanical stimulation of the transductional elements, a resting depolarizing current exists which is ultimately responsible for the spontaneous release of neurotransmitter. Movement of the transductional elements modulates this resting current and thereby the evoked release of neurotransmitter occurs. Recent data from our laboratory and others have led us to question whether the relationship between spontaneous and evoked neurotransmitter release is as simple as stated. Indeed, a variety of experimental manipulations appear to influence the two modes of release differently. Examination of our results and the results of others has led us to four hypotheses: 1. the two modes of neurotransmitter release are processed differently by the hair cells; 2. cyclic AMP is involved in spontaneous but not evoked neurotransmitter release; 3. there is a positive feedback step involving an excitatory amino acid and its receptor on the hair cell in evoked neurotransmitter release and; 4. different pools of calcium are involved according to the mode of release. Accordingly, there may be several biochemical steps between the transductional movement of the stereocilia at the apex of the hair cells and the ultimate release of the neurotransmitter at the base of these cells. Some of these biochemical steps are different depending on whether the mode of release is spontaneous or evoked. These biochemical steps may amplify or at least interact with the biophysical processes previously described in the hair cells.
View details for Web of Science ID A1991GR40300009
View details for PubMedID 1685158
LACTATE COMPARTMENTATION IN HIPPOCAMPAL SLICES - EVIDENCE FOR A TRANSPORTER
METABOLIC BRAIN DISEASE
1990; 5 (3): 143-154
Lactic acid accumulation has been implicated in the evolution of brain damage after ischemia. Since compartmentation of lactate may play a role in acid-base balance, lactate release from gerbil hippocampal slices was examined during a number of metabolic stresses including elevated [K+]e, ischemia, anoxia, and aglycemia. Slices were preincubated for 1 hr in artificial cerebrospinal fluid (ACSF) equilibrated with 95% O2/5% CO2 (pH 7.4 at 37 degrees C) and then transferred to tubes containing 300 microliters of test medium. The rate of lactate release in control slices was 9.64 nmol/min/mg protein and increased 2.6- and 3.2-fold in the presence of 60 mM potassium and anoxia, whereas the rate of lactate release was decreased by 50 and 25% during ischemia and aglycemia. Lactate release was temperature dependent and was only minimally influenced by removing Ca2+ or by adding 5 mM d-lactate to the ACSF. In contrast, pyruvate inhibited lactate release with an apparent Ki of 2.4 mM. The results suggest that lactate can be released from cells via a saturable and stereospecific lactate transporter with an apparent Km of 10.7 mM and Vmax of 43.7 nmol/mg protein/min. Such a relatively high-capacity transporter system can rapidly equilibrate brain lactate but is probably not involved in regulating intracellular acid-base balance.
View details for Web of Science ID A1990EC44300004
View details for PubMedID 2274000
THE EVOLUTION OF FOCAL ISCHEMIC DAMAGE - A METABOLIC ANALYSIS
METABOLIC BRAIN DISEASE
1990; 5 (1): 33-44
Focal cerebral ischemia in the rat was induced by left middle cerebral artery occlusion. The area of ischemia was determined by infusion of a qualitative perfusion indicator, neutral red. The temporal evolution of alterations in regional energy metabolism was assessed by direct microquantitative histochemical analysis of high-energy phosphates, glucose, glycogen, and lactate content of the tissue. Perfusion analyses demonstrated a perifocal region of diminished, but not absent perfusion up to 6 hr after occlusion. By 24 hr, there was an abrupt demarcation between perfused and nonperfused regions. Profound metabolic alterations were seen as early as 20 min after occlusion. Although there was an area of intermediate metabolic derangement in the more medial portions of the lateral ipsilateral cortex up to 6 hr, by 24 hr there was an abrupt transition from normal to abnormal cortex. No evidence of metabolic recovery was seen in this model of permanent occlusion.
View details for Web of Science ID A1990CX93500004
View details for PubMedID 2336048
IMPAIRMENT OF METABOLIC RECOVERY WITH INCREASING PERIODS OF MIDDLE CEREBRAL-ARTERY OCCLUSION IN RATS
1990; 21 (3): 467-471
We examined the consequences of reflow on metabolic recovery following increasing periods of focal ischemia. The middle cerebral artery of 21 Sprague-Dawley rats was occluded with a snare ligature for 1, 2, or 6 hours followed by 5, 4, or 0 hours of reflow, respectively (seven rats in each group). All animals were injected with neutral red for visual confirmation that the affected regions were reperfused. The brains were frozen in situ, and the concentrations of adenosine triphosphate, phosphocreatine, glycogen, and lactate were determined in those areas corresponding to the normally perfused medial ipsilateral cortex, the perifocal region, and the ischemic focus. Values for the 6 hours' occlusion with no reflow group served as a control to demonstrate restoration of metabolite concentrations. In both groups with reflow, the levels of high-energy phosphates were greater than control, but this effect of reflow was primarily significant for the group with 1 hour's occlusion (p less than 0.05). The levels of glycogen and lactate provided additional evidence that the extent of metabolite restoration was graded; following 2 hours of occlusion, metabolite recovery was compromised (p less than 0.05). Our data strongly support the concept that the window of opportunity for effective treatment of focal ischemia by reperfusion is narrow (of short duration).
View details for Web of Science ID A1990CT20500019
View details for PubMedID 2309272
ROLE FOR GAMMA-AMINOBUTYRIC ACID IN SELECTIVE VULNERABILITY IN GERBILS
1989; 20 (2): 281-287
We tested the efficacy of various putative neuroprotective agents in the gerbil model of delayed neuronal death. The selective loss of anterior CA1 neurons of the hippocampus 4 days after 5 minutes of bilateral ischemia was complete in greater than 90% of the gerbils examined. We tested 11 agents for their ability to protect against neuronal loss. Only those agents that were associated with the GABAergic system exhibited protection and only when administered before the ischemic insult. The possibility that delayed neuronal death is the result of a primary defect in inhibitory neurotransmission is considered.
View details for Web of Science ID A1989T192200018
View details for PubMedID 2919417
EFFECTS OF METABOLIC STRESS ON THE RELEASE OF GLUTAMATE AND GABA FROM HIPPOCAMPAL SLICES
SATELLITE SYMP OF THE 14TH INTERNATIONAL SYMP ON CEREBRAL BLOOD FLOW AND METABOLISM : NEUROTRANSMISSION AND CEREBROVASCULAR FUNCTION
ELSEVIER SCIENCE PUBL B V. 1989: 433–436
View details for Web of Science ID A1989BQ04F00086
A ROLE FOR GAMMA-AMINOBUTYRIC ACID (GABA) IN THE EVOLUTION OF DELAYED NEURONAL DEATH FOLLOWING ISCHEMIA
METABOLIC BRAIN DISEASE
1988; 3 (4): 287-292
A series of putative neuroprotective agents was tested to determine their efficacy in preventing the loss of the CA 1 neurons of the hippocampus at 4 days following 5 min of bilateral ischemia in the gerbil. Agents associated with the GABAergic system were determined to be the most effective, but only when given prior to the ischemic episode, suggesting that there was a gamma-aminobutyric acid (GABA)-related event during ischemia which triggers the delayed neuronal death of these cells. In this report, the unidirectional release of GABA and glutamate from gerbil hippocampal slices was determined under conditions mimicking anoxia and/or ischemia. Pentobarbital, the most effective of the GABAergic agents, had little or no effect on the time-dependent release of glutamate. In contrast, pentobarbital reduced in release of GABA in both anoxia and ischemia, but only after 25 to 30 min of incubation.
View details for Web of Science ID A1988R163400007
View details for PubMedID 2907368