“…The presence of the inward Ca 2+ current (inset in Figure 4E) was used to confirm hair cell-identity in tomt nl16 mutants. The peak of the Ca 2+ current at –31 mV was 9.2 ± 2.4 pA ( n = 8), which was similar to that previously reported (Olt et al, 2016). 10.7554/eLife.28474.006Figure 4.Tomt-deficient hair cells have no mechanotransduction (MET) current.( A , B ) Examples of K + currents recorded from lateral line hair cells in wild-type sibling (A) and tomt nl16 mutant (B) zebrafish.…”
Section: Resultssupporting
confidence: 91%
“…To confirm whether Tomt-deficient hair cells have a specific defect in mechanotransduction, we performed electrophysiological recordings from lateral-line hair cells in wild-type and mercury mutants. Hair cells from the lateral line organ of wild-type and tomt nl16 mutant zebrafish (3.0–5.2 dpf) showed a similar complement of K + currents (Figure 4A,B), in agreement with that previously described for wild-type hair cells (Olt et al, 2016, 2014). The size of the peak K + current measured at 0 mV was found to be similar between wild-type (261 ± 26 pA, n = 4) and mutant hair cells (352 ± 43 pA, n = 3) (Figure 4C).…”
Transmembrane O-methyltransferase (TOMT/LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.DOI:
http://dx.doi.org/10.7554/eLife.28474.001
“…The presence of the inward Ca 2+ current (inset in Figure 4E) was used to confirm hair cell-identity in tomt nl16 mutants. The peak of the Ca 2+ current at –31 mV was 9.2 ± 2.4 pA ( n = 8), which was similar to that previously reported (Olt et al, 2016). 10.7554/eLife.28474.006Figure 4.Tomt-deficient hair cells have no mechanotransduction (MET) current.( A , B ) Examples of K + currents recorded from lateral line hair cells in wild-type sibling (A) and tomt nl16 mutant (B) zebrafish.…”
Section: Resultssupporting
confidence: 91%
“…To confirm whether Tomt-deficient hair cells have a specific defect in mechanotransduction, we performed electrophysiological recordings from lateral-line hair cells in wild-type and mercury mutants. Hair cells from the lateral line organ of wild-type and tomt nl16 mutant zebrafish (3.0–5.2 dpf) showed a similar complement of K + currents (Figure 4A,B), in agreement with that previously described for wild-type hair cells (Olt et al, 2016, 2014). The size of the peak K + current measured at 0 mV was found to be similar between wild-type (261 ± 26 pA, n = 4) and mutant hair cells (352 ± 43 pA, n = 3) (Figure 4C).…”
Transmembrane O-methyltransferase (TOMT/LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.DOI:
http://dx.doi.org/10.7554/eLife.28474.001
“…In addition, at rest, we were unable to detect any difference in cell membrane capacitance between genotypes (WT: 2.8 ± 0.3 pF, n = 25; ribeye b-EGFP : 3.3 ± 0.6 pF, n = 31, p = 0.491), which suggests that the overall size of the hair cells is similar. Furthermore, there were no differences in the complement of K + currents, which included I K,Ca and I A as previously described, between WT and ribeye b-EGFP hair cells (data not shown) (Olt et al, 2014, 2016b). …”
Section: Resultssupporting
confidence: 66%
“…Our recording setup for action currents has been described in detail previously (Trapani and Nicolson, 2010; Olt et al, 2016b). For all experiments, recordings were performed in normal extracellular solution (see above) on afferent neurons innervating zebrafish primary neuromasts (L1-L4).…”
Section: Methodsmentioning
confidence: 99%
“…Whole-cell patch clamp experiments were performed from hair cells of the zebrafish primary neuromasts (L1-L4) as previously described (Olt et al, 2014, 2016b). The zebrafish were placed in a microscope chamber, with continuous perfusion via a peristaltic pump in the following extracellular solution in m m : 135 NaCl, 1.3 CaCl 2 , 5.8 KCl, 0.9 MgCl 2 , 0.7 NaH 2 PO 4 , 5.6 d -glucose, 10 HEPES-NaOH.…”
In sensory hair cells of auditory and vestibular organs, the ribbon synapse is required for the precise encoding of a wide range of complex stimuli. Hair cells have a unique presynaptic structure, the synaptic ribbon, which organizes both synaptic vesicles and calcium channels at the active zone. Previous work has shown that hair-cell ribbon size is correlated with differences in postsynaptic activity. However, additional variability in postsynapse size presents a challenge to determining the specific role of ribbon size in sensory encoding. To selectively assess the impact of ribbon size on synapse function, we examined hair cells in transgenic zebrafish that have enlarged ribbons, without postsynaptic alterations. Morphologically, we found that enlarged ribbons had more associated vesicles and reduced presynaptic calcium-channel clustering. Functionally, hair cells with enlarged ribbons had larger global and ribbon-localized calcium currents. Afferent neuron recordings revealed that hair cells with enlarged ribbons resulted in reduced spontaneous spike rates. Additionally, despite larger presynaptic calcium signals, we observed fewer evoked spikes with longer latencies from stimulus onset. Together, our work indicates that hair-cell ribbon size influences the spontaneous spiking and the precise encoding of stimulus onset in afferent neurons.SIGNIFICANCE STATEMENT Numerous studies support that hair-cell ribbon size corresponds with functional sensitivity differences in afferent neurons and, in the case of inner hair cells of the cochlea, vulnerability to damage from noise trauma. Yet it is unclear whether ribbon size directly influences sensory encoding. Our study reveals that ribbon enlargement results in increased ribbon-localized calcium signals, yet reduces afferent spontaneous activity and disrupts the timing of stimulus onset, a distinct aspect of auditory and vestibular encoding. These observations suggest that varying ribbon size alone can influence sensory encoding, and give further insight into how hair cells transduce signals that cover a wide dynamic range of stimuli.
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