Abstract
Inner hair cell ribbon synapses indefatigably transmit acoustic information. The proteins mediating their fast vesicle replenishment (hundreds of vesicles per s) are unknown. We found that an aspartate to glycine substitution in the C2F domain of the synaptic vesicle protein otoferlin impaired hearing by reducing vesicle replenishment in the pachanga mouse model of human deafness DFNB9. In vitro estimates of vesicle docking, the readily releasable vesicle pool (RRP), Ca2+ signaling and vesicle fusion were normal. Moreover, we observed postsynaptic excitatory currents of variable size and spike generation. However, mutant active zones replenished vesicles at lower rates than wild-type ones and sound-evoked spiking in auditory neurons was sparse and only partially improved during longer interstimulus intervals. We conclude that replenishment does not match the release of vesicles at mutant active zones in vivo and a sufficient standing RRP therefore cannot be maintained. We propose that otoferlin is involved in replenishing synaptic vesicles.
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References
Kiang, N.Y.-S., Watanabe, T., Thomas, E.C. & Clark, L.F. Discharge Pattern of Single Fibers in the Cat's Auditory Nerve (MIT Press, Cambridge, Massachusetts, 1965).
Geisler, C.D. From Sound to Synapse (Oxford University Press, New York, 1998).
Parsons, T.D., Lenzi, D., Almers, W. & Roberts, W.M. Calcium-triggered exocytosis and endocytosis in an isolated presynaptic cell: capacitance measurements in saccular hair cells. Neuron 13, 875–883 (1994).
Moser, T. & Beutner, D. Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse. Proc. Natl. Acad. Sci. USA 97, 883–888 (2000).
Schnee, M.E., Lawton, D.M., Furness, D.N., Benke, T.A. & Ricci, A.J. Auditory hair cell-afferent fiber synapses are specialized to operate at their best frequencies. Neuron 47, 243–254 (2005).
Griesinger, C.B., Richards, C.D. & Ashmore, J.F. Fast vesicle replenishment allows indefatigable signaling at the first auditory synapse. Nature 435, 212–215 (2005).
Keen, E.C. & Hudspeth, A.J. Transfer characteristics of the hair cell's afferent synapse. Proc. Natl. Acad. Sci. USA 103, 5537–5542 (2006).
Goutman, J.D. & Glowatzki, E. Time course and calcium dependence of transmitter release at a single ribbon synapse. Proc. Natl. Acad. Sci. USA 104, 16341–16346 (2007).
von Gersdorff, H. & Matthews, G. Depletion and replenishment of vesicle pools at a ribbon-type synaptic terminal. J. Neurosci. 17, 1919–1927 (1997).
Gomis, A., Burrone, J. & Lagnado, L. Two actions of calcium regulate the supply of releasable vesicles at the ribbon synapse of retinal bipolar cells. J. Neurosci. 19, 6309–6317 (1999).
Zenisek, D., Steyer, J.A. & Almers, W. Transport, capture and exocytosis of single synaptic vesicles at active zones. Nature 406, 849–854 (2000).
Thoreson, W.B., Rabl, K., Townes-Anderson, E. & Heidelberger, R. A highly Ca2+-sensitive pool of vesicles contributes to linearity at the rod photoreceptor ribbon synapse. Neuron 42, 595–605 (2004).
Rabl, K., Cadetti, L. & Thoreson, W.B. Kinetics of exocytosis is faster in cones than in rods. J. Neurosci. 25, 4633–4640 (2005).
Singer, J.H. & Diamond, J.S. Vesicle depletion and synaptic depression at a mammalian ribbon synapse. J. Neurophysiol. 95, 3191–3198 (2006).
Jackman, S.L. et al. Role of the synaptic ribbon in transmitting the cone light response. Nat. Neurosci. 12, 303–310 (2009).
Hosoi, N., Sakaba, T. & Neher, E. Quantitative analysis of calcium-dependent vesicle recruitment and its functional role at the calyx of Held synapse. J. Neurosci. 27, 14286–14298 (2007).
Saviane, C. & Silver, R.A. Fast vesicle reloading and a large pool sustain high bandwidth transmission at a central synapse. Nature 439, 983–987 (2006).
Khimich, D. et al. Hair cell synaptic ribbons are essential for synchronous auditory signaling. Nature 434, 889–894 (2005).
Moser, T., Neef, A. & Khimich, D. Mechanisms underlying the temporal precision of sound coding at the inner hair cell ribbon synapse. J. Physiol. (Lond.) 576, 55–62 (2006).
Wittig, J.H. Jr. & Parsons, T.D. Synaptic ribbon enables temporal precision of hair cell afferent synapse by increasing the number of readily releasable vesicles: a modeling study. J. Neurophysiol. 100, 1724–1739 (2008).
Eisen, M.D., Spassova, M. & Parsons, T.D. Large releasable pool of synaptic vesicles in chick cochlear hair cells. J. Neurophysiol. 91, 2422–2428 (2004).
Johnson, S.L., Marcotti, W. & Kros, C.J. Increase in efficiency and reduction in Ca2+ dependence of exocytosis during development of mouse inner hair cells. J. Physiol. (Lond.) 563, 177–191 (2005).
Roux, I. et al. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell 127, 277–289 (2006).
Roux, I. et al. Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses. Hum. Mol. Genet. 18, 4615–4628 (2009).
Heidrych, P. et al. Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell. Hum. Mol. Genet. 18, 2779–2790 (2009).
Schwander, M. et al. A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function. J. Neurosci. 27, 2163–2175 (2007).
Brandt, A., Khimich, D. & Moser, T. Few CaV1.3 channels regulate the exocytosis of a synaptic vesicle at the hair cell ribbon synapse. J. Neurosci. 25, 11577–11585 (2005).
Frank, T., Khimich, D., Neef, A. & Moser, T. Mechanisms contributing to synaptic Ca2+ signals and their heterogeneity in hair cells. Proc. Natl. Acad. Sci. USA 106, 4483–4488 (2009).
Spassova, M.A. et al. Evidence that rapid vesicle replenishment of the synaptic ribbon mediates recovery from short-term adaptation at the hair cell afferent synapse. J. Assoc. Res. Otolaryngol. 5, 376–390 (2004).
Meyer, A.C. et al. Tuning of synapse number, structure and function in the cochlea. Nat. Neurosci. 12, 444–453 (2009).
Li, G.L., Keen, E., Andor-Ardo, D., Hudspeth, A.J. & von Gersdorff, H. The unitary event underlying multiquantal EPSCs at a hair cell's ribbon synapse. J. Neurosci. 29, 7558–7568 (2009).
Glowatzki, E. & Fuchs, P.A. Transmitter release at the hair cell ribbon synapse. Nat. Neurosci. 5, 147–154 (2002).
Grant, L., Yi, E. & Glowatzki, E. Two modes of release shape the postsynaptic response at the inner hair cell ribbon synapse. J. Neurosci. 30, 4210–4220 (2010).
Taberner, A.M. & Liberman, M.C. Response properties of single auditory nerve fibers in the mouse. J. Neurophysiol. 93, 557–569 (2005).
Strenzke, N. et al. Complexin-I is required for high-fidelity transmission at the endbulb of held auditory synapse. J. Neurosci. 29, 7991–8004 (2009).
Starr, A., Michalewski, H.J., Feng, G. & Moser, T. Perspectives on auditory neuropathy: disorders of inner hair cell, auditory nerve and their synapse. in The Senses: a Comprehensive Reference (eds. Dallos, P. & Oertel, D.) 397–412 (Elsevier, Amsterdam, 2008).
Obholzer, N. et al. Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells. J. Neurosci. 28, 2110–2118 (2008).
Seal, R.P. et al. Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3. Neuron 57, 263–275 (2008).
Ruel, J. et al. Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice. Am. J. Hum. Genet. 83, 278–292 (2008).
Hosoi, N., Holt, M. & Sakaba, T. Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse. Neuron 63, 216–229 (2009).
Wadel, K., Neher, E. & Sakaba, T. The coupling between synaptic vesicles and Ca2+ channels determines fast neurotransmitter release. Neuron 53, 563–575 (2007).
Wölfel, M., Lou, X. & Schneggenburger, R. A mechanism intrinsic to the vesicle fusion machinery determines fast and slow transmitter release at a large CNS synapse. J. Neurosci. 27, 3198–3210 (2007).
Neef, J. et al. The Ca2+ channel subunit beta2 regulates Ca2+ channel abundance and function in inner hair cells and is required for hearing. J. Neurosci. 29, 10730–10740 (2009).
de Wit, H. et al. Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes. Cell 138, 935–946 (2009).
Sterling, P. & Matthews, G. Structure and function of ribbon synapses. Trends Neurosci. 28, 20–29 (2005).
Dulon, D., Safieddine, S., Jones, S.M. & Petit, C. Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses. J. Neurosci. 29, 10474–10487 (2009).
Neef, A. et al. Probing the mechanism of exocytosis at the hair cell ribbon synapse. J. Neurosci. 27, 12933–12944 (2007).
Beutner, D., Voets, T., Neher, E. & Moser, T. Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse. Neuron 29, 681–690 (2001).
Goodman, M.B. & Lockery, S.R. Pressure polishing: a method for re-shaping patch pipettes during fire polishing. J. Neurosci. Methods 100, 13–15 (2000).
McCue, M.P. & Guinan, J. Jr. Acoustically responsive fibers in the vestibular nerve of the cat. J. Neurosci. 14, 6058–6070 (1994).
Acknowledgements
We would like to thank E. Glowatzki and D. Khimich for teaching us the postsynaptic patch clamp, K. Tittman, D. Fasshauer and R. Jahn for advice and support for protein biochemistry, C.P. Richter and M.A. Cheatham for advice on the electrocochleography, A. Leonov and C. Griesinger for the DM-nitrophen, P. Jonas for the parvalbumin taqman probe, members of the InnerEarLab for discussion, M. Rutherford, J. Singer, E. Neher, J. Siegel, P. Heil, T. Sakaba, R. Nouvian, A. Lysakowski and A. Lee for comments on the manuscript and C. Rüdiger, S. Blume, N. Dankenbrink-Werder and M. Köppler for expert technical assistance. This work was supported by a fellowship of the Alexander von Humboldt Foundation to T.P., a fellowship of the Boehringer Ingelheim Fonds to K.R., a fellowship of MED-EL company to H.T., grants from the German Research Foundation (Center for Molecular Physiology of the Brain, T.M. and N.B.; Fellowship to N.S.), the European Commission (Eurohear, T.M.), the Max-Planck-Society (Tandemproject, T.M. and N.B.), the German Ministry for Education and Science via the Bernstein Focus Neurotechnology Goettingen (grant no. 01GQ0810 to T.M.), the State of Lower Saxony ('VW-Vorab' to T.M. and Christoph Matthias), and the US National Institutes of Health (DC007704, U.M.).
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The study was designed by T.M., T.P., U.M., N.S., E.R. and N.B. T.P. carried out the IHC patch clamp and flash photolysis, extracellular postsynaptic recordings and immunohistochemistry and contributed to the electron microscopy. L.L. performed in vivo single-unit recordings, electrocochleography and auditory brainstem responses. K.R. carried out real-time PCR, protein purification, circular dichroism spectroscopy, fluorimetry and the floatation assay. H.T. performed postsynaptic recordings. M.S. carried out the ENU screen and initial auditory testing. D.R. performed electron microscopy. T.F. carried out Ca2+ imaging. N.S. performed in vivo physiology. E.R. generated the knockout mice. J.S.B. and L.M.T. started the ENU screen. T.M. and T.P. prepared the manuscript.
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Supplementary Text and Figures
Supplementary Figs. 1–4 and Tables 1–3 (PDF 6382 kb)
Supplementary Movie 1
Tomogram of the ribbon in OtofPga/Pga IHC fixed in stimulatory condition from Figure 3d. Gold particles are visible on both surfaces of the sections. (MOV 3212 kb)
Supplementary Movie 2
Model of the same ribbon synapse in OtofPga/Pga IHC (as in Supplementary Movie 1). (PDF 737 kb)
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Pangršič, T., Lasarow, L., Reuter, K. et al. Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells. Nat Neurosci 13, 869–876 (2010). https://doi.org/10.1038/nn.2578
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DOI: https://doi.org/10.1038/nn.2578
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