Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells

We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2kbtsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the “T” antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of a9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation

FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel

Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4°C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca2+ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca2+-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca2+ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca2+ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca2+ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca2+, intracellular Ca2+ buffering, and membrane potential, by their common effect on intracellular free Ca2+. Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3⇓–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca2+ entry through the channel itself (6⇓⇓⇓⇓⇓⇓–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14⇓–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca2+, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca2+ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca2+-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca2+ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20⇓⇓–23) compared with the piezo-driven glass rod (19, 24)

Tmc1 point mutation affects Ca2+ sensitivity and block by dihydrostreptomycin of the mechanoelectrical transducer current of mouse outer hair cells

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1Bth/Bth) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca2+ permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1Bth/Bth OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca2+ dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1Bth/Bth OHCs, whereas raising the intracellular concentration of the Ca2+ chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca2+ permeability of the mutated MET channel indirectly causing the Ca2+ sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca2+ influx as a compensatory mechanism

Shifting visual perspective during memory retrieval reduces the accuracy of subsequent memories

Memories for events can be retrieved from visual perspectives that were never experienced, reflecting the dynamic and reconstructive nature of memories. Characteristics of memories can be altered when shifting from an own eyes perspective, the way most events are initially experienced, to an observer perspective, in which one sees oneself in the memory. Moreover, recent evidence has linked these retrieval-related effects of visual perspective to subsequent changes in memories. Here we examine how shifting visual perspective influences the accuracy of subsequent memories for complex events encoded in the lab. Participants performed a series of mini-events that were experienced from their own eyes, and were later asked to retrieve memories for these events while maintaining the own eyes perspective or shifting to an alternative observer perspective. We then examined how shifting perspective during retrieval modified memories by influencing the accuracy of recall on a final memory test. Across two experiments, we found that shifting visual perspective reduced the accuracy of subsequent memories and that reductions in vividness when shifting visual perspective during retrieval predicted these changes in the accuracy of memories. Our findings suggest that shifting from an own eyes to an observer perspective influences the accuracy of long-term memories

In vivo physiological recording from the lateral line of juvenile zebrafish

Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post-fertilization, dpf, at 28.5°C). In larval zebrafish, the functional properties of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l(-1) was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l(-1) did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS-222, which reduces the size of basolateral membrane currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish. This article is protected by copyright. All rights reserved

Identification of ion-channel modulators that protect against aminoglycoside-induced hair cell death

Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red–conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem

Effect of cell sample size in atomic force microscopy nanoindentation

Single-cell technologies are powerful tools to evaluate cell characteristics. In particular, Atomic Force Microscopy (AFM) nanoindentation experiments have been widely used to study single cell mechanical properties. One important aspect related to single cell techniques is the need for sufficient statistical power to obtain reliable results. This aspect is often overlooked in AFM experiments were sample sizes are arbitrarily set. The aim of the present work was to propose a tool for sample size estimation in the context of AFM nanoindentation experiments of single cell. To this aim, a retrospective approach was used by acquiring a large dataset of experimental measurements on four bone cell types and by building saturation curves for increasing sample sizes with a bootstrap resampling method. It was observed that the coefficient of variation (CV%) decayed with a function of the form y = axb with similar parameters for all samples tested and that sample sizes of 21 and 83 cells were needed for the specific cells and protocol employed if setting a maximum threshold on CV% of 10% or 5%, respectively. The developed tool is made available as an open-source repository and guidelines are provided for its use for AFM nanoindentation experimental design

Exocytosis in mouse vestibular Type II hair cells shows a high‐order Ca2+ dependence that is independent of synaptotagmin‐4

Mature hair cells transduce information over a wide range of stimulus intensities and frequencies for prolonged periods of time. The efficiency of such a demanding task is reflected in the characteristics of exocytosis at their specialized presynaptic ribbons. Ribbons are electron‐dense structures able to tether a large number of releasable vesicles allowing them to maintain high rates of vesicle release. Calcium entry through rapidly activating, non‐inactivating CaV1.3 (L‐type) Ca2+ channels in response to cell depolarization causes a local increase in Ca2+ at the ribbon synapses, which is detected by the exocytotic Ca2+ sensors. The Ca2+ dependence of vesicle exocytosis at mammalian vestibular hair cell (VHC) ribbon synapses is believed to be linear, similar to that observed in mature cochlear inner hair cells (IHCs). The linear relation has been shown to correlate with the presence of the Ca2+ sensor synaptotagmin‐4 (Syt‐4). Therefore, we studied the exocytotic Ca2+ dependence, and the release kinetics of different vesicle pool populations, in Type II VHCs of control and Syt‐4 knockout mice using patch‐clamp capacitance measurements, under physiological recording conditions. We found that exocytosis in mature control and knockout Type II VHCs displayed a high‐order dependence on Ca2+ entry, rather than the linear relation previously observed. Consistent with this finding, the Ca2+ dependence and release kinetics of the ready releasable pool (RRP) of vesicles were not affected by an absence of Syt‐4. However, we did find that Syt‐4 could play a role in regulating the release of the secondary releasable pool (SRP) in these cells. Our findings show that the coupling between Ca2+ influx and neurotransmitter release at mature Type II VHC ribbon synapses is faithfully described by a nonlinear relation that is likely to be more appropriate for the accurate encoding of low‐frequency vestibular information, consistent with that observed at low‐frequency mammalian auditory receptors