[International expert consensus on gene therapy for hereditary hearing loss: based on clinical trials].
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Journal Article↗
Biomedical subjects
Tobias Moser
Publications and source records attributed to Tobias Moser.
Measurements of membrane patch capacitance using a software-based lock-in system.
On-cell patch-clamp capacitance measurements can resolve the fusion of individual vesicles to a membrane patch and the accompanying dilation of the fusion pore. So far, these measurements have used a patch-clamp amplifier in combination with a hardware lock-in amplifier. Usually, solely the capacitance and conductance outputs of hardware lock-in amplifiers were recorded, which needed to be filtered rather heavily to suppress spectral components at the stimulus frequency. Therefore, the temporal resolution was limited, and information carried in the patch current was not utilized. In this paper, we describe an alternative and more versatile approach for measuring patch capacitance and conductance, using a digitally controlled patch-clamp amplifier. The software lock-in system showed better bandwidth and identical signal-to-noise performance needing less instrumentation. High temporal resolution measurements on patches of chromaffin cells showed that vesicle fission can be completed in only tens of microseconds. Capacitance calculation based on the patch current allows for straightforward offline phase correction. Moreover, the close inspection of direct current for the first time revealed small current changes accompanying the fusion and fission of large secretory vesicles, promising new insights into the vesicles' membrane properties. A practical guide to high-resolution on-cell patch-clamp capacitance measurements using the software lock-in is provided.
Algorithms↗
Intracavity generation of radially polarized CO2 laser beams based on a simple binary dielectric diffraction grating.
We present experimental results on the intracavity generation of radially polarized light by incorporation of a polarization-selective mirror in a CO(2)-laser resonator. The selectivity is achieved with a simple binary dielectric diffraction grating etched in the backsurface of the mirror substrate. Very high polarization selectivity was achieved, and good agreement of simulation and experimental results is shown. The overall radial polarization purity of the generated laser beam was found to be higher than 90%.
Journal Article↗
Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse.
The auditory inner hair cell (IHC) ribbon synapse operates with an exceptional temporal precision and maintains a high level of neurotransmitter release. However, the molecular mechanisms underlying IHC synaptic exocytosis are largely unknown. We studied otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness. We show that otoferlin expression in the hair cells correlates with afferent synaptogenesis and find that otoferlin localizes to ribbon-associated synaptic vesicles. Otoferlin binds Ca(2+) and displays Ca(2+)-dependent interactions with the SNARE proteins syntaxin1 and SNAP25. Otoferlin deficient mice (Otof(-/-)) are profoundly deaf. Exocytosis in Otof(-/-) IHCs is almost completely abolished, despite normal ribbon synapse morphogenesis and Ca(2+) current. Thus, otoferlin is essential for a late step of synaptic vesicle exocytosis and may act as the major Ca(2+) sensor triggering membrane fusion at the IHC ribbon synapse.
Animals↗
Hair cell ribbon synapses.
Hearing and balance rely on the faithful synaptic coding of mechanical input by the auditory and vestibular hair cells of the inner ear. Mechanical deflection of their stereocilia causes the opening of mechanosensitive channels, resulting in hair cell depolarization, which controls the release of glutamate at ribbon-type synapses. Hair cells have a compact shape with strong polarity. Mechanoelectrical transduction and active membrane turnover associated with stereociliar renewal dominate the apical compartment. Transmitter release occurs at several active zones along the basolateral membrane. The astonishing capability of the hair cell ribbon synapse for temporally precise and reliable sensory coding has been the subject of intense investigation over the past few years. This research has been facilitated by the excellent experimental accessibility of the hair cell. For the same reason, the hair cell serves as an important model for studying presynaptic Ca(2+) signaling and stimulus-secretion coupling. In addition to common principles, hair cell synapses differ in their anatomical and functional properties among species, among the auditory and vestibular organs, and among hair cell positions within the organ. Here, we briefly review synaptic morphology and connectivity and then focus on stimulus-secretion coupling at hair cell synapses.
Animals↗
Mechanisms underlying the temporal precision of sound coding at the inner hair cell ribbon synapse.
Our auditory system is capable of perceiving the azimuthal location of a low frequency sound source with a precision of a few degrees. This requires the auditory system to detect time differences in sound arrival between the two ears down to tens of microseconds. The detection of these interaural time differences relies on network computation by auditory brainstem neurons sharpening the temporal precision of the afferent signals. Nevertheless, the system requires the hair cell synapse to encode sound with the highest possible temporal acuity. In mammals, each auditory nerve fibre receives input from only one inner hair cell (IHC) synapse. Hence, this single synapse determines the temporal precision of the fibre. As if this was not enough of a challenge, the auditory system is also capable of maintaining such high temporal fidelity with acoustic signals that vary greatly in their intensity. Recent research has started to uncover the cellular basis of sound coding. Functional and structural descriptions of synaptic vesicle pools and estimates for the number of Ca(2+) channels at the ribbon synapse have been obtained, as have insights into how the receptor potential couples to the release of synaptic vesicles. Here, we review current concepts about the mechanisms that control the timing of transmitter release in inner hair cells of the cochlea.
Afferent Pathways↗
Expression pattern and functional characterization of connexin29 in transgenic mice.
Using newly generated transgenic mice in which the coding region of the connexin29 (Cx29) gene was replaced by the lacZ reporter gene, we confirmed previous immunochemical results that Cx29 is expressed in Schwann cells, oligodendrocytes and Bergmann glia cells. In addition, we detected lacZ/Cx29 in Schwann cells of the sciatic nerve and in particular of the spiral ganglion in the inner ear, as well as at low abundance in the stria vascularis. Furthermore, we found lacZ/Cx29 expression in nonmyelinating Schwann cells of the adrenal gland, in chondrocytes of intervertebral discs and the epiphysis of developing bones. Electron microscopic analyses of myelin sheaths in the central and peripheral nervous system of Cx29-deficient mice detected no abnormalities. The nerve conduction in the sciatic nerve of adult Cx29-deficient mice and the auditory brain stem response as well as visually evoked potentials in 4- to 10-week-old Cx29-deficient mice were not different from wild-type littermate controls. Thus, in contrast to connexin32 and connexin47, which are also expressed in myelinating cells, Cx29 does not contribute to the function of myelin in adult mice.
Adrenal Medulla↗
CSPalpha-deficiency causes massive and rapid photoreceptor degeneration.
Cysteine string protein (CSP) alpha is an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40-type cochaperones. Previous studies showed that deletion of CSPalpha in mice leads to massive lethal neurodegeneration but did not clarify how the neurodegeneration affects specific subpopulations of neurons. Here, we analyzed the effects of the CSPalpha deficiency on tonically active ribbon synapses of the retina and the inner ear. We show that CSPalpha-deficient photoreceptor terminals undergo dramatic and rapidly progressive neurodegeneration that starts before eye opening and initially does not affect other retinal synapses. These changes are associated with progressive blindness. In contrast, ribbon synapses of auditory hair cells did not exhibit presynaptic impairments in CSPalpha-deficient mice. Hair cells, but not photoreceptor cells or central neurons, express CSPbeta, thereby accounting for the lack of a hair-cell phenotype in CSPalpha knockout mice. Our data demonstrate that tonically active ribbon synapses in retina are particularly sensitive to the deletion of CSPalpha and that expression of at least one CSP isoform is essential to protect such tonically active synapses from neurodegeneration.
Animals↗
Mice with altered KCNQ4 K+ channels implicate sensory outer hair cells in human progressive deafness.
KCNQ4 is an M-type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4-/- mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the I(K,n) current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near-normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.
Animals↗
[Hearing loss -- can heat shock protein 70 be cited as prognostic marker?].
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Administration, Oral↗
Few CaV1.3 channels regulate the exocytosis of a synaptic vesicle at the hair cell ribbon synapse.
Hearing relies on faithful sound coding at hair cell ribbon synapses, which use Ca2+-triggered glutamate release to signal with submillisecond precision. Here, we investigated stimulus-secretion coupling at mammalian inner hair cell (IHC) synapses to explore the mechanisms underlying this high temporal fidelity. Using nonstationary fluctuation analysis on Ca2+ tail currents, we estimate that IHCs contain approximately 1700 Ca2+ channels, mainly of CaV1.3 type. We show by immunohistochemistry that the CaV1.3 Ca2+ channels are localized preferentially at the ribbon-type active zones of IHCs. We argue that each active zone holds approximately 80 Ca2+ channels, of which probably <10 open simultaneously during physiological stimulation. We then manipulated the Ca2+ current by primarily changing single-channel current or open-channel number. Effects on exocytosis of the readily releasable vesicle pool (RRP) were monitored by membrane capacitance recordings. Consistent with the high intrinsic Ca2+ cooperativity of exocytosis, RRP exocytosis changed nonlinearly with the Ca2+ current when varying the single-channel current. In contrast, the apparent Ca2+ cooperativity of RRP exocytosis was close to unity when primarily manipulating the number of open channels. Our findings suggest a Ca2+ channel-release site coupling in which few nearby CaV1.3 channels impose high nanodomain [Ca2+] on release sites in IHCs during physiological stimulation. We postulate that the IHC ribbon synapse uses this Ca2+ nanodomain control of exocytosis to signal with high temporal precision already at low sound intensities.
Animals↗
Hair cell synaptic ribbons are essential for synchronous auditory signalling.
Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). At present, the function of presynaptic ribbons at these synapses is still largely unknown. Here we show that anchoring of IHC ribbons is impaired in mouse mutants for the presynaptic scaffolding protein Bassoon. The lack of active-zone-anchored synaptic ribbons reduced the presynaptic readily releasable vesicle pool, and impaired synchronous auditory signalling as revealed by recordings of exocytic IHC capacitance changes and sound-evoked activation of spiral ganglion neurons. Both exocytosis of the hair cell releasable vesicle pool and the number of synchronously activated spiral ganglion neurons co-varied with the number of anchored ribbons during development. Interestingly, ribbon-deficient IHCs were still capable of sustained exocytosis with normal Ca2+-dependence. Endocytic membrane retrieval was intact, but an accumulation of tubular and cisternal membrane profiles was observed in ribbon-deficient IHCs. We conclude that ribbon-dependent synchronous release of multiple vesicles at the hair cell afferent synapse is essential for normal hearing.
Action Potentials↗
BetaIVSigma1 spectrin stabilizes the nodes of Ranvier and axon initial segments.
Saltatory electric conduction requires clustered voltage-gated sodium channels (VGSCs) at axon initial segments (AIS) and nodes of Ranvier (NR). A dense membrane undercoat is present at these sites, which is thought to be key for the focal accumulation of channels. Here, we prove that betaIVSigma1 spectrin, the only betaIV spectrin with an actin-binding domain, is an essential component of this coat. Specifically, betaIVSigma1 coexists with betaIVSigma6 at both AIS and NR, being the predominant spectrin at AIS. Removal of betaIVSigma1 alone causes the disappearance of the nodal coat, an increased diameter of the NR, and the presence of dilations filled with organelles. Moreover, in myelinated cochlear afferent fibers, VGSC and ankyrin G clusters appear fragmented. These ultrastructural changes can explain the motor and auditory neuropathies present in betaIVSigma1 -/- mice and point to the betaIVSigma1 spectrin isoform as a master-stabilizing factor of AIS/NR membranes.
Animals↗
CaV1.3 channels are essential for development and presynaptic activity of cochlear inner hair cells.
Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, afferent synaptic transmission is triggered by spontaneous Ca2+ spikes of IHCs, which are under efferent cholinergic control. Around the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired, and Ca2+ spikes as well as the cholinergic innervation are lost. Here, we performed patch-clamp measurements in IHCs of mice lacking the CaV1.3 channel (CaV1.3-/-) to investigate the role of this prevailing voltage-gated Ca2+ channel in IHC development and synaptic function. The small Ca2+ current remaining in IHCs from 3-week-old CaV1.3-/- mice was mainly mediated by L-type Ca2+ channels, because it was sensitive to dihydropyridines but resistant to inhibitors of non-L-type Ca2+ channels such as omega-conotoxins GVIA and MVIIC and SNX-482. Depolarization induced only marginal exocytosis in CaV1.3-/- IHC, which was solely mediated by L-type Ca2+ channels, whereas robust exocytic responses were elicited by photolysis of caged Ca2+. Secretion triggered by short depolarizations was reduced proportionally to the Ca2+ current, suggesting that the coupling of the remaining channels to exocytosis was unchanged. CaV1.3-/- IHCs lacked the Ca2+ action potentials and displayed a complex developmental failure. Most strikingly, we observed a continued presence of efferent cholinergic synaptic transmission and a lack of functional large-conductance Ca2+-activated K+ channels up to 4 weeks after birth. We conclude that CaV1.3 channels are essential for normal hair cell development and synaptic transmission.
Aging↗
LIMP-2/LGP85 deficiency causes ureteric pelvic junction obstruction, deafness and peripheral neuropathy in mice.
In previous overexpression studies we revealed a role for the lysosomal membrane protein LIMP-2/LGP85 in lysosomal biogenesis. LIMP-2-deficient mice show an increased postnatal mortality which is associated with a development of a uni- or bilateral hydronephrosis caused by an obstruction of the ureteropelvic junction. An accumulation of lysosomes in epithelial cells of the ureter adjacent to the ureteral lumen and a disturbed apical expression of uroplakin was observed, suggesting an impairment of membrane transport processes. Serious hearing impairment in LIMP-2-deficient animals was indicated by deficits in acoustic startle responses, in brainstem evoked auditory potentials and a reduced endochondral potential. LIMP-2-deficient mice suffer from a massive decline of spiral ganglia in the cochlea concomitant with that of the inner and outer hair cells. These pathological changes begin at the age of 3 months and are probably secondary to a degeneration of the stria vascularis. LIMP-2-deficient mice are also characterized by a peripheral demyelinating neuropathy. Demyelinization was found to be associated with a massive loss of peripheral myelin proteins and an increased activity and expression of lysosomal proteins highlighting a hitherto unknown role of the lysosomal compartment in the development of this myelination disorder. The phenotype of LIMP-2-deficient mice stimulates the search for mutations in human disorders associated with degeneration of the stria vascularis and/or demyelinization of peripheral nerves.
Animals↗
The afferent synapse of cochlear hair cells.
Mechanosensory hair cells of the cochlea must serve as both transducers and presynaptic terminals, precisely releasing neurotransmitter to encode acoustic signals for the postsynaptic afferent neuron. Remarkably, each inner hair cell serves as the sole input for 10-30 individual afferent neurons, which requires extraordinary precision and reliability from the synaptic ribbons that marshal vesicular release onto each afferent. Recent studies of hair cell membrane capacitance and postsynaptic currents suggest that the synaptic ribbon may operate by simultaneous multi-vesicular release. This mechanism could serve to ensure the accurate timing of transmission, and further challenges our understanding of this synaptic nano-machine.
Afferent Pathways↗
Calcium regulates exocytosis at the level of single vesicles.
Ca2+ microdomains that form during the opening of voltage-gated Ca2+ channels have been implicated in regulating the kinetics of hormone and transmitter release. Direct assessment of the interaction between a single Ca2+ microdomain and a single secretory vesicle has been impossible because of technical limitations. Using evanescent field imaging of near-membrane micromolar Ca2+ concentration ([Ca2+]) and fluorescently labeled vesicles, we have observed exocytosis of individual chromaffin dense-core vesicles that was triggered by Ca2+ microdomains. Ca2+ microdomains selectively triggered the release of vesicles that were docked within 300 nm. Not all vesicles exposed to a Ca2+ microdomain were released, indicating that some vesicles are docked but are not ready for release. In addition to its established role as a trigger for release, elevated near-membrane [Ca2+] reduced the distance between docked vesicles and Ca2+ entry sites. Our results suggest a new mechanism for stimulation-dependent facilitation of exocytosis, whereby vesicles are moved closer to Ca2+ entry sites, thereby increasing a Ca2+ microdomain's efficacy to trigger vesicle fusion.
Action Potentials↗