Author response: Experience-dependent flexibility in a molecularly diverse central-to-peripheral auditory feedback system
Year: 2023
Type: peer-review
Abstract: Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation This paper provides a 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 comprehensively 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. https://doi.org/10.7554/eLife.83855.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Just as our pupils dilate or shrink depending on the amount of light available to our eyes, our ears adjust their sensitivity based on the sound environment we encounter. Evidence suggests that a group of cells known as olivocochlear neurons (OCNs for short) may be involved in this process. These cells are located in the brainstem but project into the cochlea, the inner ear structure that converts sound waves into the electrical impulses relayed to the brain. OCNs may mediate how sounds are detected and encoded "at the source." Historically, OCNs have been divided into two groups (medial or lateral OCNs) based on different morphologies and roles in hearing. For instance, medial OCNs are thought to protect our ears against loud sounds by sending molecular signals to the inner ear cells that amplify certain auditory signals. However, it remains difficult to disentangle the precise function of the different types of OCNs, in part because scientists still lack markers that would allow them to distinguish between medial and lateral cells simply based on genetic activity. Frank et al. aimed to eliminate this bottleneck by identifying which genes were switched on and to what degree in individual mouse medial and lateral OCNs; this was done throughout development and after exposure to loud noises. The experiments uncovered a range of genetic markers for medial and lateral OCNs, showing that these cells switch on different sets of genes relevant to their role over development. This gene expression data also revealed that two distinct groups of lateral OCNs exist, one of which is characterised by the production of large amounts of neuropeptides, a type of chemical messenger that can modulate neural circuit activity. Further work in both developing and adult mice showed that this production is shaped by the activity of the cells, with the neuropeptide levels increasing when the animals are exposed to damaging levels of noise. This change lasts for several days, suggesting that such an experience can have long-lasting effects on how the brain provides feedback to the ear. Overall, the results by Frank et al. will help to better identify and characterize the different types of OCNs and the role that they have in hearing. By uncovering the chemical messengers that mediate the response to loud noises, this research may contribute to a better understanding of how to prevent or reduce hearing loss. Introduction All sensory systems are modulated by feedback circuits that dynamically tune sensory information in response to both external experience and internal state. In the sense of vision, for instance, central efferent pathways mediate the reflexive restriction of the pupil in response to bright light and also modulate pupil diameter with changes in arousal state (McGinley et al., 2015). Analogously, a small group of several hundred olivocochlear neurons (OCNs) in the superior olivary complex (SOC) extend projections all the way to the peripheral hearing organ, the cochlea, enabling direct regulation of initial detection and encoding of auditory stimuli (Rasmussen, 1946; Rasmussen, 1953; Figure 1A and B). By integrating information from both ascending auditory pathways and descending central circuits, OCNs serve as a conduit for an animal's experiences and needs to modulate sound information at the earliest stages of sensory processing. Figure 1 with 2 supplements see all Download asset Open asset Identification of OCNs by single-nucleus sequencing of brainstem neurons. (A, B) Schematic of medial (MOC) and lateral (LOC) olivocochlear neuron (OCN) subtypes housed in the brainstem (A) and their projections into the cochlea, shown in cross-section (A) and top-down (B). D, dorsal; L, lateral; LSO, lateral superior olive; VNTB, ventral nucleus of the trapezoid body; SOC, superior olivary complex; IHC, inner hair cell; OHC, outer hair cell; SGN, spiral ganglion neuron; IGSB, intraganglionic spiral bundle; ISB, inner spiral bundle; TSB, tunnel spiral bundle. (C) Schematic of single-nucleus collection protocol. The ventral brainstem of ChatCre; Rosa26Sun1-GFP mice was dissected, including the SOC, facial motor nucleus (VII), and trigeminal motor nucleus (V), followed by collection and sequencing of individual GFP+ nuclei. A, anterior; P, posterior. (D) UMAP plot summarizing sequencing data of 45,828 single nuclei from 61 animals across three ages. The data includes 56 clusters, including MOCs, LOCs, and several different motor neuron types, as well as a few non-neuronal populations. (E–H) Feature plots denoting normalized expression levels of selected genetic markers. For visualization purposes, high expression on the color scale is capped at the 95th percentile of gene expression. The majority of clusters express the neuronal marker Snap25 (E). The main non-neuronal population is labeled by Mog, an oligodendrocyte marker (F). Motor neuron clusters express the motor neuron determinant Isl1 (G). Among these brainstem motor neurons, OCNs are identified by expression of the transcription factor Gata3 (H). (I, J) Differential expression of known markers Ucn (I) and Atp1a3 (J) denotes LOCs and MOCs, respectively. (K–M) Cadps2 is a novel marker for OCNs that is expressed by both MOCs and LOCs but not FMNs. (K) Violin plots summarizing transcriptional expression of Cadps2 in mature LOCs, MOCs, and FMNs. (L, M) Fluorescent in situ hybridization (FISH) verifies that Cadps2 is detected in both MOCs and LOCs (L) but not in FMNs (M). Representative images are shown from a total of three animals of either sex, P28. The impact of OCNs on cochlear function is poorly understood, as few markers for these cells have been identified and the population is difficult to target either genetically or physiologically. OCNs express a variety of neurotransmitters (Eybalin, 1993; Kitcher et al., 2022; Sewell, 2011) and can even change their neurotransmitter profiles in response to intense sounds (Niu and Canlon, 2002; Wu et al., 2020), making it difficult to identify and catalog cell types. Developmentally and evolutionarily, OCNs are closely related to the facial branchial motor neurons (FMNs; Frank and Goodrich, 2018; Fritzsch and Elliott, 2017). Both populations derive from common motor neuron progenitors and share expression of motor neuron markers such as Islet-1 (Frank and Goodrich, 2018; Fritzsch and Elliott, 2017), although OCNs are not considered motor neurons. Expression of the zinc-finger transcription factor GATA3 seems to be crucial for specifying an OCN fate, but little else is known about how OCNs and FMNs diverge, or how OCN subtypes differentiate from one another and acquire their specialized attributes (Pata et al., 1999; Karis et al., 2001). Thus, identifying markers that can distinguish OCN subtypes from one another and from neighboring FMNs is critical for understanding the anatomical and molecular logic of OCN connectivity. OCNs fall into two major groups: medial olivocochlear neurons (MOCs), which sit in the ventral nucleus of the trapezoid body (VNTB), and lateral olivocochlear neurons (LOCs), which are intermingled with afferent principal neurons in the lateral superior olive (LSO; Figure 1A). Both MOCs and LOCs extend peripheral axons that spiral along the length of the cochlea, often spanning large frequency domains (Brown, 2011; Figure 1B). MOCs terminate mainly on outer hair cells (OHCs), a group of mechanosensory cells that influence cochlear responses to auditory stimuli through rapid, sound-induced cell-body movements (van der Heijden and Vavakou, 2022; Ashmore, 2019). It is widely accepted that MOCs affect cochlear gain through inhibitory, cholinergic synapses that reduce OHC motility (Mountain, 1980; Siegel and Kim, 1982; Blanchet et al., 1996; Dallos et al., 1997; Fuchs and Lauer, 2019), much as visual efferent pathways control gain by altering pupil diameter. As well as protecting cochlear circuitry from the impact of acoustic trauma (Reiter and Liberman, 1995; Taranda et al., 2009; Maison et al., 2013; Boero et al., 2018; Fuente, 2015; Rajan, 1995; Tong et al., 2013), MOCs are thought to contribute to the detection of signals in noise (Guinan, 2018; Kawase et al., 1993; Winslow and Sachs, 1987) and to tune sensory responses based on states of attention (Delano et al., 2007; Terreros et al., 2016; Oatman, 1976; Oatman, 1971). Consistent with this range of effects on the perception of sound, electrophysiological characterization suggests that MOCs are controlled both by inputs from the cochlear nucleus and by descending inputs from multiple sources (Romero and Trussell, 2022). In contrast, we know little about the LOCs, as they are difficult to access for recording, stimulation, or surgical ablation. Anatomically, LOCs terminate on the peripheral processes of Type I spiral ganglion neurons (SGNs), primary sensory neurons that carry perceptual auditory information from mechanosensory inner hair cells (IHCs) into the brain. Functionally, the nature of communication between LOCs and SGNs remains opaque: although LOCs express acetylcholine, a variety of other signaling molecules have also been reported in these cells, including GABA, CGRP, dopamine, enkephalin, dynorphin, and urocortin (Ucn) (Eybalin, 1993; Kitcher et al., 2022; Sewell, 2011; Safieddine and Eybalin, 1992; Safieddine et al., 1997; Kaiser et al., 2011; Takeda et al., 1987; Adams et al., 1987). However, definitive evidence for many of these transmitters is lacking. Moreover, indirect activation of the auditory efferent system can elicit both excitatory and inhibitory effects on SGNs, hinting at the possibility of multiple LOC subtypes that can direct distinct effects on their downstream targets (Groff and Liberman, 2003). No specific role in hearing has been definitively linked to LOCs, although they have been implicated in sound localization (Darrow et al., 2006a; Larsen and Liberman, 2010; Irving et al., 2011) and appear to protect the ear from noise damage (Fuente, 2015; Darrow et al., 2007; Kujawa and Liberman, 1997). The mechanisms underlying this protective effect remain unclear. One promising model holds that dopamine—and potentially other neurotransmitters—released from LOCs inhibits SGN firing, thereby dampening excitotoxicity (Wu et al., 2020; Ruel et al., 2001). However, this model has yet to be tested directly, and other, as yet unidentified, pathways may contribute a protective role as well. Here, we took a multi-pronged approach to investigate key molecular, anatomical, and physiological features of the auditory efferent system. We found many novel transcriptional features of MOCs and LOCs in both developing and mature OCNs, including cell-type specific markers, genes that could confer distinct physiological properties, and developmental gene-expression changes that highlight pathways involved in OCN maturation. In addition, we found that LOCs cluster into two molecularly and anatomically distinct subtypes based on expression of neuropeptides. However, regardless of the amount of neuropeptide Y in their pre-synaptic puncta, LOCs elaborate axon arborizations that vary extensively and terminate on multiple SGN subtypes. Further, LOCs change their neuropeptide expression profiles upon the onset of hearing and after acoustic trauma, indicating that variability in peptide expression may serve as a way to modulate sensory circuits based on prior experience. Results To assess transcriptional variability within OCNs, we sequenced cell nuclei from individual cholinergic neurons, which were labeled using ChatCre to drive expression of a nuclear-localized GFP (Rosa26Sun1-GFP) (Rossi et al., 2011; Mo et al., 2015). We enriched for OCNs and the closely related FMNs by dissecting a region of the ventral brainstem that includes the SOC as well as facial and trigeminal motor neurons. We then used fluorescence-activated cell-sorting (FACS) to isolate dissociated GFP+ cholinergic nuclei and employed the 10x Genomics platform to encapsulate nuclei and generate barcoded single-nucleus libraries (Figure 1C). To examine the maturation of these cell types and identify markers that are expressed consistently across postnatal development, nuclei were collected at two pre-hearing timepoints, postnatal day (P)1 (n=13 animals) and P5 (n=16 animals), the latter of which is an important time of synapse refinement in auditory circuits (Frank and Goodrich, 2018; Yu and Goodrich, 2014). We also collected nuclei at P26–P28 (n=32 animals), when auditory circuitry is grossly mature. After filtering out low-quality cells and infrequently expressed genes (see Materials and methods), our dataset includes 45,828 nuclei: 16,753 cells from P1 animals; 15,542 from P5 animals; and 13,533 cells from P26–P28 animals. Unsupervised, graph-based clustering analysis identified 56 clusters (Figure 1D). These clusters primarily consist of neurons, as indicated by the expression of neuronal markers like Snap25 (Figure 1E). Populations of oligodendrocyte precursors, Mog-positive oligodendrocytes (Figure 1F), and astrocytes were also identified based on expression of well-established markers (Zhang, 2001). Each neuronal cluster includes cells from all three timepoints (Figure 1—figure supplement 1A). Non-neuronal cells primarily originate from the adult dataset, consistent with prior reports showing an increase in myelination of auditory brainstem neurons and elevated expression of oligodendrocyte markers over the first few postnatal weeks (Long et al., 2018). Although cell types vary somewhat in the number of genes and unique molecular identifiers (UMI) detected, the structure of the data is not driven by these technical variables (Figure 1—figure supplement 1B–D). Cell types were also similar in the fraction of genes mapping to the mitochondrial genome, suggesting that clusters are not affected by differences in cell health (Ilicic et al., 2016; Luecken and Theis, 2019; Figure 1—figure supplement 1E). The neuronal clusters include several motor neuron clusters, as defined by co-expression of Isl1 (Figure 1G), Tbx20, and Phox2b. Among these, two OCN clusters were identified based on expression of Gata3, which is expressed in OCNs but not in canonical motor neuron populations (Figure 1H; Karis et al., 2001). LOC and MOC clusters were identified by expression of the LOC-specific peptide Urocortin (Ucn) (Kaiser et al., 2011; Figure 1I) and the Na,K-ATPase Atp1a3, which is expressed in MOCs but not LOCs in mature rats (McLean et al., 2009; Figure 1J). Gene-expression profiles that distinguish LOCs and MOCs from FMNs and each other LOCs and MOCs arise from a common progenitor pool with and develop alongside FMNs, raising the question of how OCNs deviate from a typical motor neuron fate to play such an unorthodox role in peripheral sensory modulation (Frank and Goodrich, 2018; Fritzsch and Elliott, 2017). To learn more about the transcriptional origins of their unique developmental and mature properties, we compared all OCNs to a cluster of FMNs, which was identified based on co-expression of Etv1 and Epha7 (Figure 1—figure supplement 2A, B; Tenney et al., 2019). Our analysis revealed substantial transcriptional differences between OCNs and FMNs that could contribute to their distinct functions and anatomy (Figure 1—figure supplement 2C). For instance, OCNs show selective expression of Cadps2, which encodes the calcium-dependent activator protein for secretion 2 (CADPS2, also known as CAPS-2; Figure 1K). CADPS2 is a member of the Munc13 and CAPS family of priming proteins (Imig et al., 2014; Jockusch et al., 2007; Nestvogel et al., 2020) and has been linked to exocytosis of dense-core vesicles (Tandon et al., 1998; Speidel et al., 2003; Grishanin et al., 2004; Renden et al., 2001), consistent with neuropeptide release from OCNs (Eybalin, 1993; Kitcher et al., 2022). Fluorescent in situ hybridization (FISH) in P27–P28 mice confirmed that Cadps2 is expressed in both MOCs and LOCs but is not detectable in FMNs (Figure 1L and M; n=3 animals). This finding validates our OCN and FMN clusters and establishes a new OCN-specific marker. Despite their early shared origins, FMNs and OCNs express unique combinations of genes encoding transcription factors, adhesion molecules, and other receptors needed for proper neuronal migration, targeting, and synaptic specificity. These differences are particularly prominent at P1 and P5, while OCN axons are still growing to their final targets in the inner ear and before many developmentally salient molecules become downregulated (Figure 1—figure supplement 2D, E). Cell-type-specific transcription factor genes include Pbx3 in FMNs and Sall3 in OCNs (Figure 1—figure supplement 2G). Genes for many guidance and adhesion molecules are also differentially expressed, including Kirrel3 in OCNs and Pcdh15 in FMNs (Figure 1—figure supplement 2H). Consistent with their integration into fundamentally different circuitry, OCNs and FMNs also differ in the expression of molecules relevant to mature function (Figure 1—figure supplement 2F, I). In particular, OCNs express Gad2, a gene involved in FMNs express the gene (Figure 1—figure supplement Although OCNs share several that distinguish them from FMNs, OCNs are with LOCs and MOCs distinct roles in auditory circuitry (Guinan, 2018). To few markers for either population have been For instance, although Atp1a3 is enriched in MOCs in it is expressed in both LOCs and MOCs developmentally (Figure supplement To this we identified several genes that are enriched in either the MOC or LOC cluster across all three timepoints and their expression in mature animals by (Figure Figure supplement We found that is expressed in all LOCs in mature animals, is expressed in all MOCs (Figure These the of MOC and LOC clusters and new markers to distinguish MOC and LOC neurons across development. Figure 2 with 1 supplement see all Download asset Open asset MOCs and LOCs distinct gene-expression (A) summarizing expression of the genes with the change for LOCs and For visualization purposes, expression levels were capped at and Fluorescent in situ hybridization in ChatCre; animals D, validates novel markers with enriched expression in sequencing data from LOCs or MOCs, shown in plots E). The gene is expressed by cholinergic neurons in the (C) but not in the shown also as images with each cholinergic In contrast, the gene is expressed in MOCs but is from LOCs (F). Representative images are shown from three animals of either sex, P28. plots indicating differentially expressed genes between mature MOCs and LOCs that transcription guidance and adhesion molecules and receptors and are for all genes with an change 1 and Violin plots denoting normalized expression levels of genes for neurotransmitter Both MOCs and LOCs generate to receptors for including and In several MOCs and LOCs express genes for different or of receptors (L, MOCs and LOCs also differ in their expression of genes that more specialized including the and of for the transcription factor and several neurotransmitter and genes expression changes across postnatal development and with for multiple In mature animals, LOCs and MOCs are into distinct circuits, from multiple brain and to neurotransmitters and (Brown, 2011; and Trussell, 2022). our data to many transcription and adhesion molecules that could direct their unique developmental including to different nuclei in the SOC and guidance of axons to different target cells in the cochlea (Figure I). In addition, genes for many physiologically relevant receptors and are differentially expressed (Figure including receptors and for neurotransmitters such as acetylcholine, and (Figure Differential expression analysis between and mature timepoints identified including several that neurotransmitter receptors and (Figure Figure supplement a that mature physiological of LOCs et al., 2022). We also identified expression of transcription factor like that may these developmental changes (Figure anatomically subtype of LOCs is defined by neuropeptide expression One in understanding LOC function has been the of within this population (Brown, 2011; and Trussell, 2022). we mature OCNs, on differences This analysis revealed OCN clusters (Figure the number of genes detected and similar all clusters (Figure supplement on expression of MOC and LOC markers, we identified a large cluster of MOCs (Figure and two main clusters of LOCs cells, Figure This analysis also revealed two clusters with only cells One of these clusters expressed MOC markers, including the other to consist of cells as these cells not express either MOC or LOC markers. We identified only a small number of differentially expressed genes in the and clusters (Figure supplement to cell We were therefore to definitively identify either cluster and not them Figure with 1 supplement see all Download asset Open asset LOC neurons include a summarizing results of OCNs from ChatCre; Rosa26Sun1-GFP animals OCNs into different groups Feature plots normalized expression levels of the MOC marker and the LOC marker For the of expression was capped at the 95th (D) plot summarizing differences in gene expression between and Violin plots expression of genes encoding adhesion molecules transcription and that differ OCN subtypes. Representative images and of and Ucn on showing expression in the medial of the animals of either sex, Representative of on ChatCre; genetic of LOCs in the medial of the MOCs in the are labeled in this mouse animals of either sex, of a ChatCre; cochlea to that was for to and each show genetically labeled LOCs in the the at three different frequency animals of either sex, of the two LOC subtypes revealed that are by of genes for the and Ucn (Figure 3D and and also differ in expression of the genes for the transcription factor and for guidance and adhesion molecules, such as and (Figure suggesting that these clusters distinct cell types the pool of cells in different Consistent with this are to the medial of the animals of either sex, as revealed by for and Ucn in from an mouse (Figure expression is although all adult LOCs express of CGRP, as reported (Brown, 2011; Wu et al., 2018; Maison et al., not express both and Ucn (Figure seems to be a particularly marker for as Ucn was not expressed in cells and few cells expressed only an genetic approach in which ChatCre expression of the the of LOC restriction to the medial of the (Figure MOCs in the Figure or the lateral of the animals of either sex, Thus, we that a distinct cell type that is poised to release multiple in the cochlea. have indicated that LOCs an of signaling molecules, including acetylcholine, GABA, CGRP, and (Eybalin, 1993; Safieddine and Eybalin, confirmed expression of Gad2, and across OCNs (Figure 1—figure supplement 2F, Figure supplement 1G), we to for were detected in either LOC However, we were to expression of either or in LOC subtype in either our sequencing data or (Figure supplement The lack of that LOC subtype produces although we out the of a population or that expression is OCNs the cochlea that the frequency of LOC axons is in a with medial neurons to of the cochlea and lateral neurons to (Brown, 2011; et al., 1987; et al., These projections are consistent with the of principal neurons, which are from along a et al., The of to the medial therefore the possibility of effects in the cochlea. However, although the of labeled by ChatCre; from to LOC axons the of the length (Figure to OCN axons can extensively along
Source: eLife
Language: English
Subfield: Sensory Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4367333938