Experience-dependent flexibility in a molecularly diverse central-to-peripheral auditory feedback system
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This important paper provides detailed cellular and molecular characterization of the olivocochlear efferents that project to the inner ear. These specialized motoneurons are the only source of feedback from the brain to the ear and have been difficult to access. This study convincingly categorizes the efferents, using single nucleus RNA-sequencing and 3D reconstructions of individual fibers and their pre-synaptic contacts onto target neurons in the cochlea.
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Abstract
Brainstem olivocochlear neurons (OCNs) modulate the earliest stages of auditory processing through feedback projections to the cochlea and have been shown to influence hearing and protect the ear from sound-induced damage. Here, we used single-nucleus sequencing, anatomical reconstructions, and electrophysiology to characterize murine OCNs during postnatal development, in mature animals, and after sound exposure. We identified markers for known medial (MOC) and lateral (LOC) OCN subtypes, and show that they express distinct cohorts of physiologically relevant genes that change over development. In addition, we discovered a neuropeptide-enriched LOC subtype that produces Neuropeptide Y along with other neurotransmitters. Throughout the cochlea, both LOC subtypes extend arborizations over wide frequency domains. Moreover, LOC neuropeptide expression is strongly upregulated days after acoustic trauma, potentially providing a sustained protective signal to the cochlea. OCNs are therefore poised to have diffuse, dynamic effects on early auditory processing over timescales ranging from milliseconds to days.
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Author Response
Reviewer #2 (Public Review):
The molecular characteristics of OCNs in normal or ototoxic conditions are poorly understood before. The strength of this study is that it provides the first single-cell RNA-seq database of OCNs as well as surrounding facial branchial motor neurons. By thoroughly analyzing the database, they found high heterogeneities within OCN populations and identified distinct markers that are enriched in different OCN subtypes. Furthermore, a few previously unknown neuropeptides are revealed, including Npy which is more enriched in the LOC-2 located on the medial side. They also found that neuropeptide expression levels and distributions are subjected to hearing experience and noise exposure. On the other hand, the weakness of the study is that the numbers of single-cell RNA-seq are not sufficient, …
Author Response
Reviewer #2 (Public Review):
The molecular characteristics of OCNs in normal or ototoxic conditions are poorly understood before. The strength of this study is that it provides the first single-cell RNA-seq database of OCNs as well as surrounding facial branchial motor neurons. By thoroughly analyzing the database, they found high heterogeneities within OCN populations and identified distinct markers that are enriched in different OCN subtypes. Furthermore, a few previously unknown neuropeptides are revealed, including Npy which is more enriched in the LOC-2 located on the medial side. They also found that neuropeptide expression levels and distributions are subjected to hearing experience and noise exposure. On the other hand, the weakness of the study is that the numbers of single-cell RNA-seq are not sufficient, and may underscore the MOC heterogeneity (Figure 3A). Moreover, the physiological functions of the LOC-2 are not revealed in this study, and no specific markers in one OCN subtype are identified that can predict the morphological or projecting axon features. Those might be addressed in the following studies.
We agree that this study does not allow us to make conclusions about MOC heterogeneity or LOC2 functions. These are certainly interesting avenues to pursue in the future.
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eLife assessment
This important paper provides detailed cellular and molecular characterization of the olivocochlear efferents that project to the inner ear. These specialized motoneurons are the only source of feedback from the brain to the ear and have been difficult to access. This study convincingly categorizes the efferents, using single nucleus RNA-sequencing and 3D reconstructions of individual fibers and their pre-synaptic contacts onto target neurons in the cochlea.
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Reviewer #1 (Public Review):
This study comprehensively categorizes the olivocochlear efferents, using single nucleus RNA-sequencing and 3D reconstructions of individual fibers and their pre-synaptic contacts onto target neurons in the cochlea.
The major strengths of the methods and results are the gene expression studies, which reveal 5 clusters of olivocochlear neurons. Traditionally, efferents have been divided into two groups, medial olivocochlear neurons that terminate on outer hair cells, and the lateral olivocochlear neurons, that terminate on spiral ganglion neurons postsynaptic to the auditory hair cells. The analyses here revealed 3 main clusters, one large cluster of medial olivocochlear neurons, and two clusters of lateral olivocochlear neurons.
In a second major strength, the study shows changing patterns of physiologically …
Reviewer #1 (Public Review):
This study comprehensively categorizes the olivocochlear efferents, using single nucleus RNA-sequencing and 3D reconstructions of individual fibers and their pre-synaptic contacts onto target neurons in the cochlea.
The major strengths of the methods and results are the gene expression studies, which reveal 5 clusters of olivocochlear neurons. Traditionally, efferents have been divided into two groups, medial olivocochlear neurons that terminate on outer hair cells, and the lateral olivocochlear neurons, that terminate on spiral ganglion neurons postsynaptic to the auditory hair cells. The analyses here revealed 3 main clusters, one large cluster of medial olivocochlear neurons, and two clusters of lateral olivocochlear neurons.
In a second major strength, the study shows changing patterns of physiologically relevant gene expression over development. The authors further showed changes in the neuropeptide expression in the lateral olivocochlear neurons days after acoustic trauma.
There are no significant weaknesses, barring the issue of a gap between gene and protein expression. This is mitigated by a close match with previous protein expression studies.
Thus, the authors have achieved their aims to characterize the phenotypes and arborization patterns of the cochlear efferents. They have confirmed and enlarged upon what has previously known about this important population.
Since these neurons have been difficult to characterize, and are important for auditory function, particularly in noise, the likely impact of the work on the field is high.
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Reviewer #2 (Public Review):
The molecular characteristics of OCNs in normal or ototoxic conditions are poorly understood before. The strength of this study is that it provides the first single-cell RNA-seq database of OCNs as well as surrounding facial branchial motor neurons. By thoroughly analyzing the database, they found high heterogeneities within OCN populations and identified distinct markers that are enriched in different OCN subtypes. Furthermore, a few previously unknown neuropeptides are revealed, including Npy which is more enriched in the LOC-2 located on the medial side. They also found that neuropeptide expression levels and distributions are subjected to hearing experience and noise exposure. On the other hand, the weakness of the study is that the numbers of single-cell RNA-seq are not sufficient, and may underscore …
Reviewer #2 (Public Review):
The molecular characteristics of OCNs in normal or ototoxic conditions are poorly understood before. The strength of this study is that it provides the first single-cell RNA-seq database of OCNs as well as surrounding facial branchial motor neurons. By thoroughly analyzing the database, they found high heterogeneities within OCN populations and identified distinct markers that are enriched in different OCN subtypes. Furthermore, a few previously unknown neuropeptides are revealed, including Npy which is more enriched in the LOC-2 located on the medial side. They also found that neuropeptide expression levels and distributions are subjected to hearing experience and noise exposure. On the other hand, the weakness of the study is that the numbers of single-cell RNA-seq are not sufficient, and may underscore the MOC heterogeneity (Figure 3A). Moreover, the physiological functions of the LOC-2 are not revealed in this study, and no specific markers in one OCN subtype are identified that can predict the morphological or projecting axon features. Those might be addressed in the following studies.
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Reviewer #3 (Public Review):
The research question is highly relevant as far too little is known about the efferent olivocochlear system, and the methods are state-of-the-art. This is high-quality work both for molecular analysis as well as for LOC physiology. The study is well-designed and executed, the manuscript is elegantly prepared. The high-quality gene expression data from a region of the ventral brainstem at 3 different postnatal time points (P1, P5, P26-28) is impactful in terms of development, heterogeneity, and physiological relevance of OCNs. I expect the data of this study to become instrumental for future functional studies on the lateral efferent olivocochlear system.
One issue inherent to transcriptomics studies is the challenge of linking RNA levels to protein levels for functional interpretation. I would ask the …
Reviewer #3 (Public Review):
The research question is highly relevant as far too little is known about the efferent olivocochlear system, and the methods are state-of-the-art. This is high-quality work both for molecular analysis as well as for LOC physiology. The study is well-designed and executed, the manuscript is elegantly prepared. The high-quality gene expression data from a region of the ventral brainstem at 3 different postnatal time points (P1, P5, P26-28) is impactful in terms of development, heterogeneity, and physiological relevance of OCNs. I expect the data of this study to become instrumental for future functional studies on the lateral efferent olivocochlear system.
One issue inherent to transcriptomics studies is the challenge of linking RNA levels to protein levels for functional interpretation. I would ask the authors to acknowledge this and (still more) carefully draw conclusions. For example, name the differentiation of LOC from MOC based on collagen (Col4a4) expression or Gad2 vs. Htr2c for differentiating OCNs from FMNs. Moreover, the lack of physiological differences in soma recordings would seem to suggest a rather homogeneous phenotype but certainly does not exclude the postulated different presynaptic functions of LOC2 and LOC1 neurons.
I am worried that the NPY-based identity in the sparse labeling experiment meant to selectively report LOC2 might not be such a safe approach. This is even more concerning considering that the NPY identity of presynaptic terminals varies within a given axon. Therefore, wonder why the authors did not perform more immunohistochemical labeling of LOC2 and LOC1 markers in the cochlea. Also, it would be great to see how LOC subtype specification changes in genetically deaf and noise-deafened mice.
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