The Cl--channel TMEM16A is involved in the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks in mice
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Evaluation Summary:
This paper addresses the very extremely interesting question of how spontaneous activity in the cochlea prior to hearing onset impacts the development of auditory circuits in the brainstem. The study has many strengths, including the use of complementary in vitro and in vivo recording techniques to characterize both peripheral and central defects resulting from conditional deletion of the gene for the chloride channel TMEM16A. The reviewers identified some concerns over the interpretation of the data and felt that the results could be discussed more in the context of other work, which might require some additional experiments.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Before hearing onset (postnatal day 12 in mice), inner hair cells (IHCs) spontaneously fire action potentials, thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISCs) of Kölliker’s organ through the activity of the Ca 2+ -activated Cl - -channel TMEM16A (ANO1). Here, we show that conditional deletion of Ano1 ( Tmem16a ) in mice disrupts Ca 2+ waves within Kölliker’s organ, reduces the burst-firing activity and the frequency selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the functional refinement of MNTB projections to the lateral superior olive. These results reveal the importance of the activity of Kölliker’s organ for the refinement of central auditory connectivity. In addition, our study suggests the involvement of TMEM16A in the propagation of Ca 2+ waves, which may also apply to other tissues expressing TMEM16A.
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Author Response
Reviewer #3 (Public Review):
Strengths of the paper include the use of complementary techniques to characterize both peripheral and central defects in TMEM16A knockout mice. They use calcium imaging in excised cochlear preparations to demonstrate the altered patterns of calcium waves in TMEM16A knockout mice, which are far smaller and faster than in control animals, and less frequent. Tests of auditory function (ABRs) in normal animals demonstrate overall normal hearing thresholds and auditory nerve function. They use in vivo recordings from medial nucleus of the trapezoid body (MNTB) neurons identified by their characteristic pre-spike to show altered patterns of spiking in developing MNTB neurons, with a smaller coefficient of variation that indicates less bursting activity. In hearing MNTB neurons, TMEM16A mutants …
Author Response
Reviewer #3 (Public Review):
Strengths of the paper include the use of complementary techniques to characterize both peripheral and central defects in TMEM16A knockout mice. They use calcium imaging in excised cochlear preparations to demonstrate the altered patterns of calcium waves in TMEM16A knockout mice, which are far smaller and faster than in control animals, and less frequent. Tests of auditory function (ABRs) in normal animals demonstrate overall normal hearing thresholds and auditory nerve function. They use in vivo recordings from medial nucleus of the trapezoid body (MNTB) neurons identified by their characteristic pre-spike to show altered patterns of spiking in developing MNTB neurons, with a smaller coefficient of variation that indicates less bursting activity. In hearing MNTB neurons, TMEM16A mutants have a higher sound threshold, and slightly wider tuning curves. They then use in vitro recordings from neurons of the lateral superior olive (LSO) which receive tonotopically refined inhibitory synaptic inputs from MNTB neurons to compute horizontal sound localization paired with glutamate uncaging activation of pre-synaptic MNTB neurons to demonstrate that single LSO neurons receive inputs from a larger region of the MNTB in TMEM16A mutants compared to control animals. This further indicates aberrant refinement of tonotopically distributed neurons in the ascending auditory system.
There are a few weaknesses in interpretation of the data. There is an unsupported claim that TMEM16A is upstream of ATP release from inner supporting cells, the opposite of which has been proposed by other groups.
We would like to thank the reviewer for the appreciation of our work and the comments that helped us to further improve our manuscript.
We agree with the reviewer that we have no clear evidence that TMEM16A is upstream of ATP release. Our data and the data from the literature stipulate the idea that TMEM16A may amplify ATP release from ISCs via connexin hemichannels either due to changes in cell volume or changes in membrane potential and thus promotes the propagation of Ca2+ waves. Since many epithelia, which express TMEM16A, exhibit ATP-dependent Ca2+ waves, this scenario may apply more generally. We changed the discussion in our revised manuscript accordingly.
The authors did not adequately discuss whether TMEM16A may influence other parts of the sound localization circuitry besides cochlear supporting cells.
As we show in the supplementary Figure 4, TMEM16A is not expressed in auditory pathways of the brainstem. To be more explicit, as requested by the reviewer, we modified the discussion section: “Because neurons of the auditory pathway do not express TMEM16A, less bursting activity and impaired tonotopic refinement of auditory projections in cKO mice likely stem from less synchronized prehearing IHC acitivity: In the absence of TMEM16A mediated Cl- efflux, no simultaneous K+ release will be triggered, and hence, the firing patterns of neighboring IHCs will not be synchronized.”
The alterations in frequency selectivity of MNTB neurons in TMEM16A mutants seems too subtle to account for the broader deficits in frequency-specific inputs to LSO neurons, which could be discussed.
The alterations in frequency selectivity, though significant, indeed seem insufficient to account for the differences observed in the LSO. However, the LSO receives projections from both ears, and there are likely some changes at the level of the cochlear nucleus (CN) as well. It is possible that the deficits in frequency-specific inputs to LSO neurons converge from changes in both the CN and the MNTB.
Finally, the authors did not adequately discuss how their present work fits in with previous work using extremely similar techniques in a different knockout animal that also implicated patterns of spontaneous activity in the cochlea in tonotopic refinement of ascending auditory projections (Clause et al 2014). The authors should compare the patterns of aberrant bursting measured in MNTB neurons in TMEM16A mutants to those measured in alpha9 AChR knockout animals used in the Clause et al 2014 paper. Then, they should clearly state what their work adds to the existing literature, namely that while other papers have linked the bursting patterns of the ascending auditory system with tonotopic refinement of MNTB projections to the LSO, this work more clearly links an earlier step in the process, the spreading calcium waves in developing supporting cells, with refinement of ascending circuits.
Thank you for your suggestions. The discussion was revised and following lines were added: “While otoferlin KO mice have almost no discernable bursting activity, TMEM16A cKO mice showed a drastic reduction of the number of bursts. In contrast, the number of bursts was not changed in α9 KO mice. Firing rates within bursts were 7080% higher in α9 KO compared to WT mice but did not differ between TMEM16A cKO and WT mice. Overall firing rates, however, do not differ between both KO models. As a consequence, we infer that the overall firing rate and firing rates within bursts, as well as the number of bursts, do not influence the physiological refinement of the MNTB-LSO pathway. Notably, the duration of bursts was markedly reduced in α9 KO and TMEM16A cKO mice (50% in α9 KO, and 56% in TMEM16A cKO mice). Average ISIs, however, were significantly longer in TMEM16A cKO and shorter in α9 KO mice. Thus, it seems that both subtle and drastic changes in the temporal pattern and/or the duration of bursts can lead to a severe disruption of tonotopic maps.
Although recent publications already established a causal connection between the bursting patterns of the ascending auditory system and the tonotopic refinement of MNTB projections to the LSO (Clause et al. and Müller et al.), our work links the TMEM16A-dependent amplification of ATP release in developing supporting cells with the refinement of ascending circuits.”
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Evaluation Summary:
This paper addresses the very extremely interesting question of how spontaneous activity in the cochlea prior to hearing onset impacts the development of auditory circuits in the brainstem. The study has many strengths, including the use of complementary in vitro and in vivo recording techniques to characterize both peripheral and central defects resulting from conditional deletion of the gene for the chloride channel TMEM16A. The reviewers identified some concerns over the interpretation of the data and felt that the results could be discussed more in the context of other work, which might require some additional experiments.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The …
Evaluation Summary:
This paper addresses the very extremely interesting question of how spontaneous activity in the cochlea prior to hearing onset impacts the development of auditory circuits in the brainstem. The study has many strengths, including the use of complementary in vitro and in vivo recording techniques to characterize both peripheral and central defects resulting from conditional deletion of the gene for the chloride channel TMEM16A. The reviewers identified some concerns over the interpretation of the data and felt that the results could be discussed more in the context of other work, which might require some additional experiments.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study addresses how spontaneous activity in the developing cochlea impacts the development of auditory response in the MTNB of the brain stem. To this end the authors make a targeted KO mouse in which the calcium activated chloride channel is knocked out in the developing cochlea. They showed that the targeted KO greatly reduces spontaneous activity, recapitulating what was show from Wang et al in the whole animal KO of the same channel. They show this reduction of activity is observed in the ex vivo cochlea leads to a change in the bursting activity in the MNTB in vivo, though units recorded exhibited many different firing patterns. They conclude that the change in the organization of the bursting activity leads to a reduction in refinement of spiral ganglion neurons to their target in the MTNB based …
Reviewer #1 (Public Review):
This study addresses how spontaneous activity in the developing cochlea impacts the development of auditory response in the MTNB of the brain stem. To this end the authors make a targeted KO mouse in which the calcium activated chloride channel is knocked out in the developing cochlea. They showed that the targeted KO greatly reduces spontaneous activity, recapitulating what was show from Wang et al in the whole animal KO of the same channel. They show this reduction of activity is observed in the ex vivo cochlea leads to a change in the bursting activity in the MNTB in vivo, though units recorded exhibited many different firing patterns. They conclude that the change in the organization of the bursting activity leads to a reduction in refinement of spiral ganglion neurons to their target in the MTNB based on two measurements, 1) a reduction in frequency selectivity of MNTB neurons and 2) an expansion In the number and spatial distribution of glutamatergic inputs from MNTB to LSO neurons.
The manuscript has many strengths - it is addressing an extremely interesting question as to how disrupting activity in the cochlea prior to hearing impacts the developing auditory circuits in the brain stem. Moreover, the targeted knockout approach removed difficulty associated with expression of this channel throughout the brain. However, the approaches used by authors are limited in their interpretation and therefore it is difficult to assign the changes they observed to a lack of refinement.
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Reviewer #2 (Public Review):
In this study, Maul and colleagues examined the impact of the spontaneous activity within the developing cochlea on the refinement of the medial nucleus of the trapezoid body (MNTB) and lateral superior olive (LSO) connectivity. Using the TMEM16A knock-out mouse, in which the spontaneous activity leading to the excitement of the cochlear sensory cells is strongly diminished, the authors demonstrate the frequency-coding within the medial nucleus of the trapezoid body (MNTB) is degraded in the TMEM16A KO mouse. Then, they show an increase in the MNTB- LSO projections. Interestingly, they find-out that both high- and low-frequency regions in the MNTB project onto LSO neurons suggesting a failure in the tonotopic refinement. Thus, the authors conclude that the spontaneous activity within the immature cochlea is …
Reviewer #2 (Public Review):
In this study, Maul and colleagues examined the impact of the spontaneous activity within the developing cochlea on the refinement of the medial nucleus of the trapezoid body (MNTB) and lateral superior olive (LSO) connectivity. Using the TMEM16A knock-out mouse, in which the spontaneous activity leading to the excitement of the cochlear sensory cells is strongly diminished, the authors demonstrate the frequency-coding within the medial nucleus of the trapezoid body (MNTB) is degraded in the TMEM16A KO mouse. Then, they show an increase in the MNTB- LSO projections. Interestingly, they find-out that both high- and low-frequency regions in the MNTB project onto LSO neurons suggesting a failure in the tonotopic refinement. Thus, the authors conclude that the spontaneous activity within the immature cochlea is essential for a proper maturation within the higher auditory centers.
Although it was known that the deletion of TMEM16A led to a reduced activity of the cochlear epithelium before the onset of hearing, the authors took care to repeat part of these experiments. Next, the analysis of the receptive field is pretty straightforward to appreciate the frequency-selectivity coding of the MNTB neurons. Finally, the functional mapping of the MNTB-LSO projections is very elegant. In addition, the authors examined the functional state of the cochlea of the TMEM16A KO mouse as well as the TMEM16A expression along the ascending auditory pathway to link the central auditory neurons maturation to the sole defect in the developing cochlea.
Thus, this study is an important contribution to the field on the input from periphery in the neural circuitry establishment and function.
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Reviewer #3 (Public Review):
Strengths of the paper include the use of complementary techniques to characterize both peripheral and central defects in TMEM16A knockout mice. They use calcium imaging in excised cochlear preparations to demonstrate the altered patterns of calcium waves in TMEM16A knockout mice, which are far smaller and faster than in control animals, and less frequent. Tests of auditory function (ABRs) in normal animals demonstrate overall normal hearing thresholds and auditory nerve function. They use in vivo recordings from medial nucleus of the trapezoid body (MNTB) neurons identified by their characteristic pre-spike to show altered patterns of spiking in developing MNTB neurons, with a smaller coefficient of variation that indicates less bursting activity. In hearing MNTB neurons, TMEM16A mutants have a higher sound …
Reviewer #3 (Public Review):
Strengths of the paper include the use of complementary techniques to characterize both peripheral and central defects in TMEM16A knockout mice. They use calcium imaging in excised cochlear preparations to demonstrate the altered patterns of calcium waves in TMEM16A knockout mice, which are far smaller and faster than in control animals, and less frequent. Tests of auditory function (ABRs) in normal animals demonstrate overall normal hearing thresholds and auditory nerve function. They use in vivo recordings from medial nucleus of the trapezoid body (MNTB) neurons identified by their characteristic pre-spike to show altered patterns of spiking in developing MNTB neurons, with a smaller coefficient of variation that indicates less bursting activity. In hearing MNTB neurons, TMEM16A mutants have a higher sound threshold, and slightly wider tuning curves. They then use in vitro recordings from neurons of the lateral superior olive (LSO) which receive tonotopically refined inhibitory synaptic inputs from MNTB neurons to compute horizontal sound localization paired with glutamate uncaging activation of pre-synaptic MNTB neurons to demonstrate that single LSO neurons receive inputs from a larger region of the MNTB in TMEM16A mutants compared to control animals. This further indicates aberrant refinement of tonotopically distributed neurons in the ascending auditory system.
There are a few weaknesses in interpretation of the data. There is an unsupported claim that TMEM16A is upstream of ATP release from inner supporting cells, the opposite of which has been proposed by other groups. The authors did not adequately discuss whether TMEM16A may influence other parts of the sound localization circuitry besides cochlear supporting cells. The alterations in frequency selectivity of MNTB neurons in TMEM16A mutants seems too subtle to account for the broader deficits in frequency-specific inputs to LSO neurons, which could be discussed. Finally, the authors did not adequately discuss how their present work fits in with previous work using extremely similar techniques in a different knockout animal that also implicated patterns of spontaneous activity in the cochlea in tonotopic refinement of ascending auditory projections (Clause et al 2014). The authors should compare the patterns of aberrant bursting measured in MNTB neurons in TMEM16A mutants to those measured in alpha9 AChR knockout animals used in the Clause et al 2014 paper. Then, they should clearly state what their work adds to the existing literature, namely that while other papers have linked the bursting patterns of the ascending auditory system with tonotopic refinement of MNTB projections to the LSO, this work more clearly links an earlier step in the process, the spreading calcium waves in developing supporting cells, with refinement of ascending circuits.
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