Synaptic contributions to cochlear outer hair cell Ca 2+ homeostasis
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Abstract
For normal cochlear function, outer hair cells (OHCs) require a precise regulation of intracellular Ca 2+ levels. Influx of Ca 2+ occurs both at the stereocillia tips and through the basolateral membrane. In this latter compartment, two different origins for Ca 2+ influx have been poorly explored: voltage-gated Ca 2+ channels (VGCC) at synapses with type II afferent neurons, and α9α10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in rodent OHCs, we report that these two Ca 2+ entry sites are closely positioned, but present different regulation mechanisms. Ca 2+ spread from MOC synapses is contained by cisternal Ca 2+ -ATPases. Considered a weak drive for transmitter release, we unexpectedly found that VGCC Ca 2+ signals are comparable in size to those elicited by α9α10 and can be potentiated by ryanodine receptors. Finally, we showed that sorcin, a highly expressed gene product in OHCs with reported Ca 2+ control function in cardiomy-ocytes, regulates basal Ca 2+ levels and MOC synaptic activity in OHCs.
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###Reviewer #3:
The focus of this manuscript from Moglie et al. is to investigate calcium entry in post-hearing OHCs via the activation of either voltage-gated calcium channels or the MOC efferent fibers. Based on the literature reported, very little is known about how OHCs handle increases in cellular calcium, although oncomodulin is believed to be the major calcium buffer in these cells. Therefore, this work attempts to address this gap in our knowledge by using a combination of calcium imaging and electrophysiology. From the results presented, the authors conclude that the large calcium signals generated by the opening of calcium channels appear to be modulated by ryanodine receptors. In addition, the opening of nicotinic receptors, caused by ACh released from active efferent fibers produced calcium transients that were contained by …
###Reviewer #3:
The focus of this manuscript from Moglie et al. is to investigate calcium entry in post-hearing OHCs via the activation of either voltage-gated calcium channels or the MOC efferent fibers. Based on the literature reported, very little is known about how OHCs handle increases in cellular calcium, although oncomodulin is believed to be the major calcium buffer in these cells. Therefore, this work attempts to address this gap in our knowledge by using a combination of calcium imaging and electrophysiology. From the results presented, the authors conclude that the large calcium signals generated by the opening of calcium channels appear to be modulated by ryanodine receptors. In addition, the opening of nicotinic receptors, caused by ACh released from active efferent fibers produced calcium transients that were contained by cisternal calcium-ATPases. The authors have also provided results that sorcin, a calcium binding protein involved in controlling calcium in myocytes, appears to control basal calcium levels and MOC synaptic activity in OHCs. The topic of the study is very interesting but unfortunately there are several major shortcomings in the design and execution of the work that drastically lower its impact. Moreover, the work appears to be designed and written for a specialized "auditory" audience.
The main issue of the paper is that the imaging data is used as the primary means of quantifying calcium changes under different experimental conditions, including the measurements of basal calcium level. However, all experiments were performed with a non-ratiometric calcium dye, making most of the conclusions and assumptions extremely difficult to interpret.
Another problem is that the authors make very specific conclusions regarding the mechanisms involved in calcium handling in OHCs, which are used to explain/understand how OHCs operate in vivo. However, experiments were done using whole-cell patch clamp, which is far from physiological, using unphysiological voltages (-100 mV) and at room temperature. The authors should provide evidence that the mechanisms proposed using the above experimental conditions are physiologically relevant.
Figures 1 and 2 describe the same aspect, and should be combined. Also, it is not clear why 1 mM Ach was used for the experiments. How do the authors know that this is a physiologically saturating concentration?
Figure 3G-H highlights another major issue with the method used. The similarity of the calcium change between the different stimulus durations could just be due to dye saturation, which is in fact suggested by the initially flat response in panel G despite the reduction in current. This finding should be corroborated by evidence indicating that the calcium dye is not saturated under their experimental conditions.
Figure 4 describes an even more problematic result. Here calcium changes are reported as DF instead of DF/F0, which is highly inappropriate as it makes comparing different recordings extremely unreliable (F0 can vary significantly between experiments, see Figure 4F). Similarly, DF measurements are done in other experiments (e.g. Figure 5D), in which data for the control condition comes from a different cell. As mentioned above, this problem could be avoided by using a ratiometric dye (e.g., fura-2 or furaptra see Beutner-Moser 2000?).
Figure 5B. It is surprising to see that a similar variance in baseline calcium level to that reported in Figure 4E (again using non-ratiometric measurements), is now just significant and used to support one of the main conclusions of the paper. Considering that the method used does not provide quantifiable baseline calcium levels, how are the authors able to exclude bias in their measurements due to experimental variability? What is the biological replica needed to validate their statistics based on the mean +/- sem? Also, the fact that adding sorcin "increases" the resting calcium level does not prove that it has a role in OHC function; it only shows that sorcin affects calcium levels, which is not surprising since it is a calcium binding protein.
Figure 7D is a bit puzzling to me but I may have missed some underlying reason from published work. Why do Ryn concentrations that are known to either facilitate or block the receptors cause the same change in calcium level?
The method section should contain a statistical statement. It should also explain the reason for using non-parametric analysis for the statistical comparisons. Also, most of the methods are only briefly described; although the authors have probably published these methods before, the method section should be more self-explanatory e.g. exactly how was the photobleaching correction performed?
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###Reviewer #2:
In this study, the group of Juan Goutman investigated Ca2+ signaling in immature cochlear outer hair cells (OHCs). The work focuses on the basolateral compartment analyzing Ca2+ signals mediated by afferent ribbon-type active zones and by efferent synapses. Ca2+ influx at the ribbon-type active zones is substantial, which is in keeping with the large ribbons found in OHCs. The authors show that it can be potentiated by ryanodine which indicates an interesting interplay between voltage-gated Ca2+ influx and ryanodine receptor mediated Ca2+ release from internal stores. Finally, adding recombinant sorcin, a Ca2+ binding protein prominently expressed in cardiomyocytes to the patch-pipette modulated the basal [Ca2+]i and efferent Ca2+ signalling in OHCs. The authors provide characterization of efferent and afferent Ca2+ …
###Reviewer #2:
In this study, the group of Juan Goutman investigated Ca2+ signaling in immature cochlear outer hair cells (OHCs). The work focuses on the basolateral compartment analyzing Ca2+ signals mediated by afferent ribbon-type active zones and by efferent synapses. Ca2+ influx at the ribbon-type active zones is substantial, which is in keeping with the large ribbons found in OHCs. The authors show that it can be potentiated by ryanodine which indicates an interesting interplay between voltage-gated Ca2+ influx and ryanodine receptor mediated Ca2+ release from internal stores. Finally, adding recombinant sorcin, a Ca2+ binding protein prominently expressed in cardiomyocytes to the patch-pipette modulated the basal [Ca2+]i and efferent Ca2+ signalling in OHCs. The authors provide characterization of efferent and afferent Ca2+ signals. However, there are a number of issues which are discussed below:
Novelty:
The approach taken, and some of the conclusions, is similar to what the group presented for immature inner hair cells, that also feature afferent and efferent synapses in close proximity and with functional interaction. This is absolutely reasonable to do but presents an extension of the same concept to a related cell type.
Relevance for understanding OHC function in the mature cochlea:
The authors have performed experiments on organs of Corti from mice at postnatal days 12-14. This is around the onset of hearing in mice and represents a time window during which substantial changes have been shown to occur. Figures 4 and 5 of Hackney et al., JN2005 show that the cytosolic abundance Ca2+ binding proteins parvalbumin a, parvalbumin ß, and calretinin changes dramatically around this stage of development. Hence, the presented data should not be taken to conclude on the situation in the mature cochlea.
Statistical data basis/sample size:
Analyzing highly variable Ca2+ signals in hair cells poses the challenge of capturing the underlying distribution by sufficient sample size. Several experiments in the present study fall short in acquiring such sample size.
Role of sorcin:
I highly recommend the authors to provide their own sorcin immunohistochemistry. Perfusion of the cytosol with recombinant Ca2+ binding proteins is expected to affect Ca2+ signalling (reducing amplitude and spread) and in a way similar to the addition of synthetic Ca2+ chelators. With 3 µM of recombinant protein, it seems difficult to achieve a sizable effect (even when considering fully functional multiple EF-hands. In the present study, a non-significant trend towards a reduced amplitude of afferent Ca2+ signals was observed during whole-cell patch clamp with sorcin (molar concentration should be provided). The relevance of sorcin function for OHC function remains to be studied by deleting sorcin expression in OHCs and performing comparative perforated-patch recordings from sorcin-deficient mice or siRNA knock-down.
Specific comments:
Mention species in title and/or abstract
What is meant by "we found that VGCC Ca2+ signals are larger than expected" please disambiguate or remove?
Also consider replacing "VGCC Ca2+ signals" by afferent or presynaptic Ca2+ signals, as the proposed CICR contribution indicates a more complex origin of Ca2+ contributing to these signals.
Line 56: "we found that Ca2+ signals from VGCC are unexpectedly large," see my comment above
Line 57 and throughout: consider clarifying that you refer to signal amplitude not spatial extent of the signal (perhaps replace size by dF/F0 or amplitude)
Line 61: "control Ca2+-based excitation-contraction coupling in cardiomyocytes"?
Line 62: "among the most differentially expressed genes in OHCs" this statement is not useful without mentioning the cells used for the comparison
Line 64: "Thus, the present results shed light into Ca2+ homeostasis in the hair cells involved in sound amplification at the cochlea, and unveil a role for the novel protein sorcin."
I don't think so, please see major concerns.
Line 70 and following: I think this first section is mainly confirmatory (work by the Mammano lab and others) and hence might better serve as supplementary information. Please add whether the data points in C-E correspond to cells and single trials or represent average responses of each OHC.
Line 88: So, do you assume that the hotspot corresponds to a single efferent OHC synapse being activated?
Line 97:Was this averaging including the failures? If not the example shown in Fig. 2B does not really seem representative? Consider adding a note relating the dF/F0 for ACh and efferent transmission: 2 orders of magnitude difference. Also please reflect on finding failing Ca2+ signals despite successful IPSC.
Legend to fig. 2 should mention the imaging approach used here. Please add whether the data points in C-E correspond to cells and singe trials or represent average responses of each OHC. "during double-pulse"
Line 102: Consider to move this explanation up to where you introduce the experiment.
Line 117: A methods section detailing the statistical analysis is missing completely. How was the use of a non-parametric test (Friedman's test) justified: i.e. how was normality tested?
Line 122: "localized Ca2+ rise with a measurable spread which accounted for 31 {plus minus} 5 % of the area corresponding to the imaged OHC area." How was "measurable spread" defined?
Line 128: The maximal Ca2+ signal with 80 Hz stimulation of efferent synapses is still an order of magnitude lower than that found with ACh. The authors suggest that the Ca2+ rise is limited by SERCA pumps, but do they assume, indeed, that this clearance mechanism is not at work during ACh application?
Line 141: How sure can the authors be that this cytosolic Ca2+ rise does not result from a store-depletion related Ca2+ entry?
Line 155: I recommend keeping the order from 20-80 Hz as above and below to make reading easier.
Line 178: How confident can we be that the recombinant sorcin was Ca2+ free, in other words, could the elevated basal Ca2+ simply reflect preloading of sorcin?
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###Reviewer #1:
This manuscript describes convincing measurements of cytoplasmic Ca2+ signals attributable to voltage gated Ca2+ channels and efferent nAChR channels. These channel coexist on the basolateral surface of OHCs and may, with the MET channels, contribute to OHC Ca2+ homeostasis. The main conclusions are that the two channel types are differentially modulated, ryanodine receptor action potentiating VGCC but not efferents, which disagrees with previous claims (Lioudyno et al 2004); and efferent responses were reduced by sorcin, a Ca2+-binding protein recently localized to OHCs, and known inhibitor of ryanodine receptors. Neither the concentration nor exact mechanism of sorcin's action was determined.
Specific comments:
In the fluorescent images in the various figures, the image orientation is unclear- is it a radial or a …
###Reviewer #1:
This manuscript describes convincing measurements of cytoplasmic Ca2+ signals attributable to voltage gated Ca2+ channels and efferent nAChR channels. These channel coexist on the basolateral surface of OHCs and may, with the MET channels, contribute to OHC Ca2+ homeostasis. The main conclusions are that the two channel types are differentially modulated, ryanodine receptor action potentiating VGCC but not efferents, which disagrees with previous claims (Lioudyno et al 2004); and efferent responses were reduced by sorcin, a Ca2+-binding protein recently localized to OHCs, and known inhibitor of ryanodine receptors. Neither the concentration nor exact mechanism of sorcin's action was determined.
Specific comments:
In the fluorescent images in the various figures, the image orientation is unclear- is it a radial or a transverse view? It would help if in some figures, a representation of an OHC, either as a Nomarski image or as a drawing, can accompany the fluorescent image.
L126. in describing Ca2+ spread, especially with long stimulation, there is concern that the high affinity dye Fluo4 will saturate. This should be discussed. I would not have used this dye - preferred a lower-affinity dye such as Fluo5.
Fig. 3F and L:124. Express the spread in absolute units rather than percent of OHC diameter. I assume the conclusion (not stated) is that the Ca2+ rise is not confined to the sub-cisternal space but spreads throughout the cell. Why does it not activate release of the afferent neurotransmitter? A point not mentioned is that the efferent SK2 and BK channels are distributed along the lateral membrane.
Fig. 6F. Ideally the spread of Ca2+ signals at the peak should be presented as (overlapping) Gaussians for the two sources. The significance of the 3.7 um separation (L319) between the sources needs some context.
L169. State explicitly that the ryanodine results disagree with (Lioudyno et al 2004).
L177. Refer to Corey's Shield database (Scheffer et al 2015) who first reported the presence of sorcin mRNA in OHCs.
L178. A concern is over the physiological significance of the sorcin effects. Sorcin is a Ca2+ binding protein that if present at high concentrations could supplement oncomodulin in addition to inhibiting RyRs. Can the authors determine the sorcin concentration in OHC cytoplasm? In addition it seems strange that the reported effect to sorcin is to inhibit RyRs so limiting the temporal spread of the CICR, but the present results suggest Can the authors clarify these problems.
L199-204. The authors could have resolved whether sorcin affected SK2 channels by (briefly) switching to -40 holding potential where the nAChR and SK2 currents would be of opposite polarity.
L350 omit 'novel' Sorcin is not a novel protein having been described in the 1990's
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
There was a consensus that the scope of the work is of general interest for the hearing field. However, three major critiques were raised:
A high-affinity Ca2+ indicator was used, raising the possibility that the fluorescence signals might be saturated in some experiments (Fig.3G-F; Fig. 4G-H; Fig.5D) and thus confounding the conclusions that can be made from the observation of unchanged Ca2+ signals. In particular, could saturation explain why ryonidine has no effect on the Ca2+ influx from efferent …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
There was a consensus that the scope of the work is of general interest for the hearing field. However, three major critiques were raised:
A high-affinity Ca2+ indicator was used, raising the possibility that the fluorescence signals might be saturated in some experiments (Fig.3G-F; Fig. 4G-H; Fig.5D) and thus confounding the conclusions that can be made from the observation of unchanged Ca2+ signals. In particular, could saturation explain why ryonidine has no effect on the Ca2+ influx from efferent synapses, an observation that contradicts published observations by Lioudino et al 2014?
The variability of the data is large and the results are often on the verge of statistical significance, which calls for special care in the statistical methods used to evaluate the effects reported here and ensure that the sample size is large enough to reach a reliable conclusion.
The experiments with sorcin appear preliminary. In particular it is worrisome that sorcin may change the Ca2+ concentration only because it is a Ca2+-binding protein.
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