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ABSTRACT: Sensory synapses of the visual and auditory systems must faithfully encode a wide dynamic range of graded signals, and must be capable of sustained transmitter release over long periods of time. Functionally and morphologically, these sensory synapses are unique: their active zones are specialized in several ways for sustained, rapid vesicle exocytosis, but their most striking feature is an organelle called the synaptic ribbon, which is a proteinaceous structure that extends into the cytoplasm at the active zone and tethers a large pool of releasable vesicles. But precisely how does the ribbon function to support tonic release at these synapses? Recent genetic and biophysical advances have begun to open the 'black box' of the synaptic ribbon with some surprising findings and promise to resolve its function in vision and hearing.
Nature Reviews Neuroscience 11/2010; 11(12):812-22. · 26.48 Impact Factor
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ABSTRACT: The mammalian cochlea is innervated by two classes of sensory neurons. Type I neurons make up 90-95% of the cochlear nerve and contact single inner hair cells to provide acoustic analysis as we know it. In contrast, the far less numerous type II neurons arborize extensively among outer hair cells (OHCs) and supporting cells. Their scarcity and smaller calibre axons have made them the subject of much speculation, but little experimental progress for the past 50 years. Here we record from type II fibres near their terminal arbors under OHCs to show that they receive excitatory glutamatergic synaptic input. The type II peripheral arbor conducts action potentials, but the small and infrequent glutamatergic excitation indicates a requirement for strong acoustic stimulation. Furthermore, we show that type II neurons are excited by ATP. Exogenous ATP depolarized type II neurons, both directly and by evoking glutamatergic synaptic input. These results prove that type II neurons function as cochlear afferents, and can be modulated by ATP. The lesser magnitude of synaptic drive dictates a fundamentally different role in auditory signalling from that of type I afferents.
Nature 10/2009; 461(7267):1126-9. · 36.28 Impact Factor
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Julián Taranda,
Jimena A Ballestero,
Hakim Hiel,
Flavio S J de Souza,
Carolina Wedemeyer,
M Eugenia Gómez-Casati,
Marcela Lipovsek,
Douglas E Vetter, Paul A Fuchs,
Eleonora Katz,
A Belén Elgoyhen
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ABSTRACT: Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express alpha10 into adulthood by expressing the alpha10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the alpha10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 alpha10 transgenic mice backcrossed to a Chrna10(-/-) background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the alpha10 transgene restored nAChR function. However, in the alpha10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.
Journal of the Association for Research in Otolaryngology 06/2009; 10(3):397-406. · 2.84 Impact Factor
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ABSTRACT: This review will cover advances in the study of hair cell afferent synaptic function occurring between 2005 and 2008. During this time, capacitance measurements of vesicular fusion have continued to be refined, optical methods have added insights regarding vesicle trafficking, and paired intracellular recordings have established the transfer function of the afferent synapse at high resolution. Further, genes have been identified with forms of deafness known as auditory neuropathy, and their role in afferent signaling explored in mouse models. With these advances, our view of the hair cell afferent synapse has continued to be refined, and surprising properties have been revealed that emphasize the unique role of this structure in neural function.
Current Opinion in Neurobiology 11/2008; 18(4):389-95. · 7.44 Impact Factor
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ABSTRACT: Efferent inhibition of cochlear hair cells is mediated by 'nicotinic' cholinergic receptors functionally coupled to calcium-activated, small conductance (SK2) potassium channels. We recorded from cochlear hair cells in SK2 knockout mice to evaluate further the role of this channel in efferent function. Since cholinergic inhibitory synapses can be found on inner or outer hair cells, depending on developmental age, both cell types were studied. To determine if SK channel activity was indeed eliminated, seconds-long voltage-gated calcium influx was used to activate slowly rising and falling calcium-dependent potassium currents. These were identified as SK currents by their time course, calcium dependence and sensitivity to block by apamin in wild-type IHCs. IHCs from knockout mice had no SK current by these same criteria. Thus, the SK2 gene is solely responsible for encoding the SK channels of inner hair cells. Other aspects of hair cell excitability remained relatively unaffected. Unexpectedly, cholinergic synaptic currents were entirely absent from both inner and outer SK2-knockout hair cells. Further, direct application of ACh caused no change in membrane current, implying absent or otherwise dysfunctional ACh receptors. Immunohistology of whole-mounts using the antibody to the synaptic vesicle protein 2 (SV2) revealed a pronounced reduction of efferent innervation to outer hair cells (OHCs) in the knockout cochleas. Quantitative RT-PCR analysis, however, showed no change in the mRNA levels of alpha9 and alpha10 nicotinic ACh receptor (nAChR) genes. Thus, some aspect of translation or subsequent protein processing leads to non-functional or absent ACh receptors. These results indicate that SK2 channels are required both for expression of functional nAChRs, and for establishment and/or maintenance of efferent terminals in the cochlea.
The Journal of Physiology 10/2008; 586(Pt 22):5471-85. · 4.72 Impact Factor
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ABSTRACT: Modulation of voltage-gated calcium channels was studied in inner hair cells (IHCs) in an ex vivo preparation of the apical turn of the rat organ of Corti. Whole cell voltage clamp in the presence of potassium channel blockers showed inward calcium currents with millisecond activation and deactivation kinetics. When temperature was raised from 22 to 37 degrees C, the calcium currents of immature IHCs [<12 days postnatal (P12)] increased threefold in amplitude, and developed more pronounced inactivation. This was determined to be calcium-dependent inactivation (CDI) on the basis of its reliance on external calcium (substitution with barium), sensitivity to internal calcium-buffering, and voltage dependence (reflecting the calcium driving force). After the onset of hearing at P12, IHC calcium current amplitude and the extent of inactivation were greatly reduced. Although smaller than in prehearing IHCs, CDI remained significant in the mature IHC near the resting membrane potential. CDI in mature IHCs was enhanced by application of the endoplasmic calcium pump blocker, benzo-hydroquinone. Conversely, CDI in immature IHCs was reduced by calmodulin inhibitors. Thus voltage-gated calcium channels in mammalian IHCs are subject to a calmodulin-mediated process of CDI. The extent of CDI depends on the balance of calcium buffering mechanisms and may be regulated by calmodulin-specific processes. CDI provides a means for the rate of spontaneous transmitter release to be adjusted to variations in hair cell resting potential and steady state calcium influx.
Journal of Neurophysiology 05/2008; 99(5):2183-93. · 3.32 Impact Factor
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ABSTRACT: Voltage-gated calcium channels support both spontaneous and sound-evoked neurotransmitter release from ribbon synapses of cochlear hair cells. A variety of regulatory mechanisms must cooperate to ensure the appropriate level of activity in the restricted pool of synaptic calcium channels ( approximately 100) available to each synaptic ribbon. One potential feedback mechanism, calcium-dependent inactivation (CDI) of voltage-gated, L-type calcium channels, can be modulated by calmodulin-like calcium-binding proteins. CDI of voltage-gated calcium current was studied in hair cells of the chicken's basilar papilla (analogous to the mammalian cochlea) after blocking the predominant potassium conductances. For inactivating currents produced by 2.5 s steps to the peak of the current-voltage relation (1 mm EGTA internal calcium buffer), single exponential fits yielded an average decay time constant of 1.92 +/- 0.18 s (mean +/- s.e.m., n = 12) at 20-22 degrees C, while recovery occurred with a half-time of approximately 10 s. Inactivation produced no change in reversal potential, arguing that the observed relaxation did not result from alternative processes such as calcium accumulation or activation of residual potassium currents. Substitution of external calcium with barium greatly reduced inactivation, while inhibition of endoplasmic calcium pumps with t-benzohydroquinone (BHQ) or thapsigargin made inactivation occur faster and to a greater extent. Raising external calcium 10-fold (from 2 to 20 mm) increased peak current 3-fold, but did not alter the extent or time course of CDI. However, increasing levels of internal calcium buffer consistently reduced the rate and extent of inactivation. With 1 mm EGTA buffering and in 2 mm external calcium, the available pool of calcium channels was half-inactivated near the resting membrane potential (-50 mV). CDI may be further regulated by calmodulin-like calcium-binding proteins (CaBPs). mRNAs for several CaBPs are expressed in chicken cochlear tissue, and antibodies to CaBP4 label hair cells, but not supporting cells, equivalent to the pattern seen in mammalian cochlea. Thus, molecular mechanisms that underlie CDI appeared to be conserved across vertebrate species, may provide a means to adjust calcium channel open probability, and could serve to maintain the set-point for spontaneous release from the ribbon synapse.
The Journal of Physiology 10/2007; 583(Pt 3):909-22. · 4.72 Impact Factor
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ABSTRACT: Studies of the electrophysiological response to acetylcholine (ACh) in mammalian outer hair cells (OHCs) are hindered by the presence of a large potassium current, I(K,n), most likely mediated by channels containing the KCNQ4 subunit. Since I(K,n) can be blocked by linopirdine, cholinergic effects might be better revealed in the presence of this compound. The aim of the present work was to study the effects of linopirdine on the ACh-evoked responses through alpha9alpha10-containing native and recombinant nicotinic cholinergic receptors. Responses to ACh were blocked by linopirdine in both OHCs and inner hair cells (IHCs) of rats at postnatal days 21-27 (OHCs) and 9-11 (IHCs). In addition, linopirdine blocked responses of recombinant alpha9alpha10 nicotinic cholinergic receptors (nAChRs) in a concentration-dependent manner with an IC(50) of 5.2 microM. Block by linopirdine was readily reversible, voltage independent, and surmountable at high concentrations of ACh, thus suggestive of a competitive type of interaction with the receptor. The present results contribute to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that linopirdine should be used with caution when analyzing the cholinergic sensitivity of cochlear hair cells.
Journal of the Association for Research in Otolaryngology 10/2004; 5(3):261-9. · 2.84 Impact Factor
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Paul Fuchs
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ABSTRACT: Mechanosensory hair cells of the vertebrate inner ear are so-called 'short' receptors that communicate to the central nervous system by way of chemical synapses with afferent neurons. In turn, hair cells are the targets of olivocochlear fibers that carry efferent inhibitory feedback from the brain. These synaptic activities contribute to, or modulate the hair cell's receptor potentials through the gating of associated ion channels. Thus for example, voltage-gated calcium channels open to trigger vesicle fusion and release of transmitter by entry of extracellular calcium. The inward calcium current also depolarizes the membrane and could lead to generation of 'all-or-none' action potentials. However, regenerative depolarization is prevented in most hair cells by prominent voltage-gated potassium conductances that rapidly repolarize the membrane. The magnitude and speed of these delayed potassium conductances determine the size and shape of the resulting receptor potential, and subsequent transmitter release, produced by sound. Efferent feedback is provided by the release of acetylcholine (ACh) from olivocochlear nerve fibers onto outer hair cells in the mammalian cochlea. The hair cell's ACh receptors are ligand-gated cation channels related to the nicotinic receptors of nerve and muscle. Calcium influx through the ACh receptors activates nearby calcium-gated potassium channels, resulting in hyperpolarization and inhibition of the hair cell. Calcium influx during efferent inhibition is regulated by a 'synaptic cistern' that also may act as a calcium store that is triggered by ACh under some conditions.
Audiology and Neurotology 7(1):40-4. · 2.46 Impact Factor
