scRNAseq identifies new neuron types in the cochlea

The majority of SG neurons are type I neurons; they are myelinated, contact the inner hair cells (IHCs) and are the principal carrier of the auditory signal. The other minority population (5%) of neurons are called type II neurons; they are unmyelinated and innervate the outer hair cells (OHCs), which modulate the output of the organ of Corti1. The few in vitro studies on their function indicate they could report cochlear trauma10,11.

To identify neuron types in adult SG neurons, a total of 487 tdTomato positive (TOM+) cells from PVCre;R26TOM cochlea of postnatal stage 17 (P17), P21, and P33 were processed for single-cell transcriptome analysis (Fig. 1a–c). Note that all SG neurons express parvalbumin (PV) and are TOM+ after recombination (Fig. 1a). The cell expression data were clustered using R package SEURAT and visualized using bi-dimensional t-distributed stochastic neighbor embedding (t-SNE), showing four distinct neuronal types (Fig. 1d–e). The type II neurons represented the smallest cluster and were identified by their expression of the known markers Prph12 (peripherin) and Th13 (Fig. 1e–f). We confirmed the type I identity of the three other clusters (thereafter named Ia, Ib, and Ic types) by their high expression of the transcription factor Prox114 and lack of Prph (Fig. 1e, Supplementary Fig. 1a). Importantly, our initial clustering showed that these neuron type identities were conserved at P17, P21, and P33 (Supplementary Fig. 1b).

Fig. 1 Identification and validation of four neuronal types in adult SG. a Genetic tracing of SG neurons (β3Tub+/RFP+) on P21 sections from PVcre;R26TOM mice. b Sketch depicting dissection of SG from the organ of Corti and their dissociation. c Fluorescence-side scatter plot of dissociated single cells showing isolation of Tom+ SG neurons through FAC sorting. d Heat map showing single-cell expression of the top 20 differentially expressed genes in the four types of SG neurons, from combined data of P17, P21, and P33 neurons (SGNs, SG neurons). Dendritic tree shows the similarity between neuronal types. e tSNE plot showing four distinct types of SG neurons. f Violin plots showing the expression of marker genes in log-transformed scale among the four different populations of SG neurons. g In vivo validation of the identified SG neuron types by immunohistochemical and fluorescent in situ hybridization using identified marker genes in P21 cochlea. Type II neurons were identified by peripherin (Peri), Plk5, TH, and Cacna1g specifically. Ia neurons were identified by Calb1, Pou4f1, Runx1, and calretinin (CR). Ib neurons were identified by Lypd1, Runx1, and Pou4f1 and Ic neurons, by Rxrg, Pcdh20, and CR expression. Note that co-localization on sections could never be observed for markers expressed in different populations of neurons in the scRNAseq data. h Schematic representation of neuronal types with their key markers and their average soma size (in µm2) at P21. i Proportion of SG neurons types along the tonotopic gradient (from base to apex) quantified by Runx1, Peri, and CR expression at P21 (n = 3 animals; Data are represented as mean ± SEM). Scale bars: 20 μm (a,g) Full size image

We next explored this data set to identify novel markers of the four types of SG neurons (Fig. 1f, Supplementary Fig 1c; Supplementary Data 1, 2). All type I neurons (Ia, Ib, and Ic) were characterized by the expression of Slc17a6 (VGlut1), Trpm2, Epha4, and abundant levels of Prox1, and the type II population, by its expression of Prph, Npy (neuropeptide Y), Th, Piezo2, and high levels of Cux2. The different subclasses of type I neurons could be further distinguished based on unique and combinatorial molecular profiles. Type Ia expressed Runx1, Ttn (Titin), Calb1 and 2 (calretinin, CR), and low levels of Pou4f1 (Brn3a). Type Ib was characterized by the expression of Grm8 (mGluR8), Kcnc2 (K v 3.2), Lypd1, Runx1, and abundant levels of Pou4f1. Finally, type Ic expressed Trim54 (MuRF3), Rxrg and high levels of Calb2. We next confirmed in vivo the existence of the four types of SG neurons by validating the expression of genes enriched in specific cell types using combinatorial labeling with new markers and transgenic mouse lines (Fig. 1g). In addition, we analyzed the soma size of each neuronal type (Fig. 1h) and calculated the percentage of each type along the baso-apical axis of the cochlea, which physiologically reflects the tonotopic gradient necessary for encoding frequency specificity within the auditory nerve8,15. Using different combinations of newly identified specific markers, the proportion of the four neuron types was found to be relatively constant throughout the length of the cochlea: 7% for type II, 26% for type Ia, 24% for type Ib, and 43% for type Ic (Fig. 1i, Supplementary Fig. 1d-f). Also, we could not observe any specific spatial patterning within the SG of any of the type I neurons subclasses (Supplementary Fig. 1f-g).

Together, these results demonstrate the existence and validate markers of four types of SG neurons. We investigate below the transcriptional basis that contributes to define their distinct neuronal identity.

Comparative analysis of SG neurons

To get insights into the major differences between the four types of SG neurons, we first conducted gene set enrichment analysis (GSEA) of the two most distinct populations, i.e., type I and type II neurons. While highly enriched gene ontology (GO) terms were associated with neuronal cell functions, such as “neurotransmission”, “ion transport”, and “axogenesis”, the most enriched category in the type I group was “metabolism” (Fig. 2a, c, d), likely reflecting the large energy demand of the myelinated type I neurons to ensure their high sensitivity and temporal fidelity16,17. Also, this analysis identified an enrichment of genes among the unmyelinated type II neurons involved in “response to stress” and “pain” mechanisms (Fig. 2b), which is consistent with their activation by cochlear damage11.

Fig. 2 Comparative analysis of SG neurons transcriptomes. a, b Gene set enrichment analysis of types I (a) and type II neurons (b) visualized by network. Each node represents a GO term, edges are drawn when there are shared genes between two GO terms. c Gene ontology analysis of the type I and type II group. The graph shows most significant terms reflecting neuronal features. d Heatmaps showing expression of genes associated with energy metabolism in each subclass of SG neurons. e–g Differential expression of transcription factors (e), cell-adhesion molecules including Cadherin, Semaphorin, and Ephrin family (f) and of cytoskeleton-related genes among the four subclasses of SG neurons (g) (see also Supplementary Fig. 2) Full size image

To further complete the characterization of the four types of SG neurons, we carried out a comparative analysis of their differential expression of transcription factors, adhesion, and cytoskeleton molecules. While numerous genes were specifically expressed in type II neurons, a strict ON/OFF division amongst the three subclasses of type I neurons was rarely observed (Fig. 2e–g, Supplementary Fig. 2), likely reflecting the importance of a combinatorial basis of the code specifying a cell type.

Neurotransmission-related machinery in SG neurons

We next analyzed in more detail gene families implicated in generic neuronal transmission, including voltage-gated ion channels (VGICs), synaptic vesicle complex, neurotransmitter (NT) receptors and transporters, and calcium-binding proteins, as they all participate in the classification of neuron types by regulating their electrophysiological profile (Fig. 3a–c). SG neurons respond mainly to glutamate released by HCs16,18. Interestingly, the relative expression of ionotropic glutamate receptors (iGluR)—AMPA (Gria), Kainate (Grik), and N-methyl-d-aspartate (NMDA) (Grin) receptors—and of metabotropic glutamate receptors (mGluRs, Grm) was particularly heterogeneous between the type I and type II neurons. Only type I neurons were found to express mGluRs (Grm7 in all three type I, and Grm8 only in type Ib) which are mostly pre-synaptic and decrease neurotransmitter release at the central synapse19 (Fig. 3a–f, Supplementary Figs. 3 and 4a,b). Also, while type I neurons exhibited a homogeneous pattern of iGluR expression, Gria3 and Grin1 were expressed at much lower levels in type II neurons than in all other types, whereas type II neurons instead specifically expressed Grik3, Grin2c, and 3a. Grik4 and 5 were detected in all subtypes, albeit at very low levels. These results suggest that type II neurons use both AMPA and Kainate receptors, while type I neurons rely mainly on AMPA receptors, as previously shown16,18. Moreover, the distinct composition of NMDARs subunits suggests cell-type-dependent activation of different sets of NMDARs-interacting molecules20. Thus, together with the morphological differences of the OHC-type II afferent synapse, the different expression of iGluR by type II neurons could participate in vivo in establishing their distinct synaptic responses; the frequency of synaptic events and the size of synaptic potentials are considerably smaller in type II afferent dendrites compared to type I afferents10,21.

Fig. 3 Input–output communication transcriptional signature of SG neurons. a Differential expression of voltage-gated ion channels family among SG neurons. b, c Differential expression of neurotransmission systems including neurotransmitter (NT) receptors, peptides-related molecules, NT transporters, and synaptic vesicles (b) and of calcium-binding protein (c) among SG neurons (see also Supplementary Fig. 3). d Sketch of the SG neuron (SGN) synaptic connection with HC peripherally and with the cochlear nuclei (CN) in the brainstem. Arrows shows direction of the signal transmission. e, f Schematic representation of the transcriptional portrait of the post-synaptic (e) and pre-synaptic (f) sites of SG neuron types, based on differentially expressed gene shown in a, b Full size image

The electrophysiological properties of neurons are shaped by the expression of several families of VGICs, which include the sodium (Na v ), potassium (K v ), calcium (Ca v ) channels, and the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels22. VGICs exhibited extensive differential expression profiles amongst the different types of SG neurons, sharply distinguishing type II from type I neurons (Fig. 3a–f). Although Ca v , Na v , and HCN channels families showed modest differences amongst the different subclasses of type I neurons, K v channels differed substantially in these neuron types. Activation of K v channels shapes the pattern of neuronal firing and excitability and drives the rate of adaptation to sustained stimuli23. Their differential expression could participate in the expected distinct physiological characteristics of the different subclasses of type I neurons5,8,24 (Fig. 3d–f and Supplementary Fig. 4a and b).

The excitability of auditory afferents is also regulated by the lateral and medial olivocochlear (LOC and MOC) efferents which innervate respectively the type I and type II peripheral endings underneath the HCs and modulate their sensitivity1,2,25. Note that the MOC efferents make also direct synaptic contacts with OHCs. The MOC system is cholinergic and GABAergic while the LOC system can be divided into at least cholinergic and dopaminergic components and also contain serotonin, GABA and many neuropeptides, including enkephalins, dynorphins, and calcitonin gene-related peptide (CGRP)25,26. Surprisingly, the receptors for these peptides were absent from all SG neurons, as were dopamine receptors, with the exception of Drd5 that was expressed in type II neurons, and Drd1a in type Ib neurons, albeit both at low levels (Supplementary Fig. 4c). The ionotropic GABA A receptor subunit Gabrb3 and metabotropic receptor Gabbr1/2 (GABA B1/2 ) were uniformly expressed amongst SG neurons. In contrast, nicotinic acetylcholine receptors (nAChRs, which are ionotropic) were differentially expressed: Chrnb2 in all SG neurons, Chrna7 in type II and Chrna4 in type Ic, all at low levels in general. Interestingly, only the type II neurons expressed serotonin receptors (mostly Htr2c and 7) (Fig. 3b; Supplementary Fig. 4c). Thus, with regards to the very low abundance of receptors for acetylcholine (Ach), dopamine, and opioids, these results suggest that the prevalent negative feedback control of SG afferents activity is GABAergic, through both ionotropic and metabotropic receptors. In parallel, a serotonergic positive feedback could also modulate the type II neurons (Fig. 3d–f).

One of the ways by which the strength of activation of a synapse can be modulated is by regulating neurochemical delivery by pre-synaptic terminal through actions on exocytosis27. During this process, the cooperation between Synaptotagmins (Syt), complexins (Cplx), N-ethylmaleimide sensitive factor (Nsf), the SNAREs proteins synaptobrevin 1 (Vamp1), syntaxin (Stx), and SNAP-25 (Snap25), as well as Munc18-1 (Stxbp1) and Rab3a (Rab3a) is essential for synaptic targeting and membrane fusion27. Their enrichment within the type I neurons group supports a fast exocytosis at the auditory nerve endings (Fig. 3b, Supplementary Fig. 4c). In line with this, the enrichment of Slc17a7 (VGLUT1) in type I afferents enables efficient recovery of synaptic vesicles during prolonged stimulation28 (Fig. 3b, Supplementary Fig. 4c). In addition, by regulating calcium availability, which triggers the fusion step27, the differential expression of calcium-binding proteins could also participate in the distinct firing dynamics of SG neurons29 (Fig. 3c).

Altogether, our data provide core molecular features of SG neuron types whose differential expression may underlie their input–output communication properties.

Electrophysiological profiles of type I neurons

We next asked whether the three distinct subclasses of type I SG neurons also exhibit unique electrophysiological properties. Whole-cell recordings were made from isolated SG neurons after dissociation to capture their key electrophysiological properties at the soma level. Analysis of current-clamp recordings revealed a high degree of diversity amongst the 133 recorded neurons in terms of action potential firing patterns and intrinsic passive and active properties. Two main groups could be easily distinguished based on their accommodation rate to step current injections: unitary spike accommodating/phasic cells (UA, n = 107 cells, 80% of neurons) and multiple spikes accommodating cells (MA, n = 26 cells, 20% of neurons, also characterized by a sustained firing over 200 ms long current steps) (Supplementary Fig. 5a), as shown at earlier stages24. UA cells were not able to fire more than a single action potential in response to a prolonged depolarizing step, while MA fired multiple action potentials with frequencies that increased by incrementing current intensity (Supplementary Fig. 5a). Post hoc immunostaining on 50 cells revealed that all Ia and Ic neurons (28 TOM+/CR+ neurons in a PVCre;R26TOM context) corresponded to UA type (Fig. 4a, b, e–g, Supplementary Fig. 5b), while the Ib population (22 TOM+/CR− neurons) was equally comprising either UA (11 cells) or MA type (11 cells) (Fig. 4c, d, e–g) raising the possibility that the Ib type could be further subdivided. Moreover, neurons with an MA profile showed high variability in the number of spikes they fired with prolonged depolarization current (Fig. 4h).

Fig. 4 Electrophysiological characterization of SG neurons types. a, c Immunohistochemistry of RFP+ neurons after culture and patch-clamp recordings from SG neurons from P21, PVCre;R26TOM mice illustrating Ia/Ic types (CR+) and Ib type (CR−). b, d Correspondence of SG neuron types and their firing patterns, illustrating that all Ia/Ic neurons (n = 28) are unitary spike accommodating (UA) and 50% of Ib neurons (n = 11) are UA while the other 50% (n = 11) are multiple spikes accommodating (MA). e Representative whole-cell current-clamp recordings from UA and MA neurons from P21 SGNs. f Different accommodation rates and action potential firing patterns of representative UA (left) and MA (right) neurons in response to suprathreshold step current injections. g Graphs of the current–voltage relationship illustrating the state and peak IV responses for UA (left) and MA (right) types. Note steeper slope for peak voltage in MA cells than UA suggesting stronger rectification. h Plots of inter-spike interval (ISI) vs action potential (AP) max of the stained SG neurons, illustrating the diversity of type Ib neurons. i Schematic representation of measured action potential parameters (fAHP—fast after-hyperpolarization). Bottom, AP shape of UA (blue) and MA (gray) neurons showing different AP threshold and rheobase values, along with different AP kinetics (latency, duration, and fAHP). j Comparison of basic electrophysiological parameters highlighted in (h) between Ia/Ic, Ib UA, and Ib MA SG neurons. Data are represented as mean ± SEM (**P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; t-test between Ia/c UA and Ib MA population.) Scale bars: 20 μm (a,c) Full size image

To further describe the two SG neuron populations, we performed a more detailed analysis of their action potential properties. Only the Ib neurons with a MA profile differed significantly in most analyzed parameters (Fig. 4i–j, Supplementary Fig. 5c). In general, UA cells exhibited higher depolarization threshold compared to MA cells, their resting membrane potential was more hyperpolarized and they required stronger current injections to discharge action potentials. MA cells fired in response to smaller current injections, responded slowly and with a longer latency and had wider action potentials. Also, a slow after-hyperpolarization (AHP) was more prominent among MA cells. Other distinctive features of MA cells were their pronounced rectification of the current–voltage (I–V) relationship, larger Ih-mediated sag, shorter membrane time constant, and a high variability in their maximal frequency discharge, reaching up to 106 Hz (mean = 51 Hz).

To correlate the physiological data of SG neurons to their molecular profile, we found that Kcnc2 (K v 3.2) exhibited a contrasted expression profile in type Ib neurons, identifying two populations, which reinforces the possibility of a subdivision of type Ib in two subtypes (Supplementary Fig. 5d). One did not express Kcnc2 while the other expressed variable levels of Kcnc2. K v 3.2 has been shown to play an important role for sustaining repetitive firing23,30. Thus, these two populations could refer to the UA and MA types of Ib neurons, respectively. In line with this, type Ia and Ic neurons, which are UA type, did not express Kcnc2 (Supplementary Fig. 5d). To the best of our analysis, Kcnc2 was the only candidate gene in our neurotransmission-related gene data set to show this contrasted expression in type Ib neurons. In view of the importance of K v 3 channels in the regulation of the firing properties of neurons31, it will be interesting to assess in vitro and in vivo the role of K v 3.2 in the physiology of type Ib neurons and in hearing.

Although our data obtained on dissociated neurons do not reflect the actual biophysical properties of the post-synaptic SG neurons during synaptic transmission of inputs from IHCs, they do demonstrate a high heterogeneity of electrophysiological properties among SG neuron types. These neuron types could conceivably correspond to the auditory units with distinct thresholds to sound stimulation in vivo5,8.

Spatial segregation of type I afferent terminals

Multiple type I afferents (10–20 in mice) receive input from one IHC32—in comparison, a single type II neuron receives input from a dozen of outer HCs (OHCs)1. Remarkably, the position of these type I afferent terminals around the IHC circumference determines the fibers’ threshold sensitivity8,33,34. Fibers with high thresholds (HT afferents) to acoustic stimuli have been shown to contact the modiolar side (facing the modiolus) of the IHC while those with lower acoustic thresholds (LT afferents), their opposite, pillar side (facing the pillar cells)8,9,34,35 (see scheme in Fig. 5a). Differences in threshold sensitivity are important for expanding the dynamic range of the cochlea, and would provide a means for discriminating sounds in a background noise5,34. They also strongly suggest the existence of distinct ascending circuits and, consistent with this, specialized neuron types. To test whether the molecular differences we identified between SG neuron types could underlie the cellular basis of this functional diversity, we first made use of the high expression of Pou4f1 (Brn3a) in type Ib neurons to selectively trace their nerve endings below IHCs using Brn3aCreERT2;R26TOM mice and limiting tamoxifen exposure (a single injection at P21) (Fig. 5b). Indeed, a single injection of tamoxifen in P21 Brn3aCreERT2;R26TOM mice lead to a sparse labeling of mostly type Ib neurons at P30 (94% of RFP+ type I neurons are Ib neurons, with 25% efficiency within the Ib population). Combined with CR immunostaining to label the projections of type Ia and Ic neurons, this sparse labeling strategy revealed on cross-sections and in cochlea whole mount that TOM+ Ib fibers consistently innervated the modiolar side of IHC, while CR+ Ia and Ic fibers, the pillar side (Fig. 5c, Supplementary Fig. 6a-c). Importantly, both CR and TOM positive fibers did not co-localize with synaptotagmin positive pre-synaptic nerve endings underneath the IHCs, excluding the possibility that these fibers were efferents (Supplementary Fig. 6b-c-e). A quantitative analysis of CR+ (only in Ia/Ic neurons) vs PV+ (in all neurons) fibers underneath the IHCs in cochlea whole mount of WT mice further confirmed the specific projection of Ib neurons to the modiolar and of the Ia/Ic neurons to the pillar (Ia/Ic) side of the IHCs (Fig. 5d–f, Supplementary Fig. 6d-e). Strikingly, the segregation of type I afferent projections was already defined in the osseus spiral lamina region, where peripheral axons of all SG neurons merge and project towards the sensory epithelium (Fig. 5g). Thus, Ib fibers were systematically positioned on the scala vestibuli (SV) side of the nerve bundle, while the Ia and Ic fibers, on the scala tympani (ST) side. Altogether, these data demonstrate that genetically distinct subclasses of type I SG neurons are associated with specific peripheral projection profiles with IHCs that correlate well with the distinction between LT and HT afferents8,34 (Fig. 5h).

Fig. 5 Innervation pattern of IHCs by type I neurons. a Sketch representing the afferent innervation of the mature organ of Corti, and illustrating the spatial segregation of the peripheral projections and synaptic contacts of high threshold (HT) and low threshold (LT) SG neuron fibers with IHCs. HT fibers innervate the modiolar side while LT fibers, the opposite, pillar side of IHCs. b Genetic labeling of Ib neurons using Brn3aCreERT2;R26TOM, injected with tamoxifen at P21 and analyzed on cross-section at P30. About 96% of RFP+ cells were Lypd1+ and were CR−, confirming their Ib identity (n = 3 animals). c In Brn3aCreERT2;R26TOM mice, RFP+ Ib fibers innervate the modiolar side, while CR+ Ia and Ic fibers, the pillar side of IHCs. In the merged panel for the CR (Ia/Ic fibers) and Myo6 (IHC) staining, the IHC is shadowed to better visualize the innervation. d Schematic of the position of sections shown in e and f. e Whole mount staining of P21 cochlea, using CR and PV immunostaining in WT mice. The images show the presence of CR+ fibers on the pillar side (PS, section #1) and their absence on the modiolar side (MS, section #4) of IHCs, while PV+ afferents are observed on either side (Aff: afferents). f Quantification of the distribution of CR+ afferent fibers at different section levels of the IHCs (from the modiolar side to the pillar side) by measuring the area of the CR+ fibers within the area of PV+ fibers at different levels of the IHC innervation, as shown in d and e (n = 4 animals). Note that no CR+ fibers were observed outside the PV+ fibers area. g In Brn3aCreERT2;R26TOM mice (see b), the peripheral projections of Ib (RFP+) and of Ia/Ic (CR+) neurons within the osseus lamina are segregated and occupy the scala vestibuli (SV) and scala tympani (ST) sides, respectively. h Schematic summary of the IHC innervation by type I afferents. Data are presented as mean ± SEM. Scale bars: 20 μm (b,c); 10 μm (e,g) Full size image

Neuronal diversity in the cochlea is established at birth

In mice, pups are born deaf and become responsive to sound stimulation after P1036. Before the onset of hearing, neuronal activity in the SG is triggered by IHCs spontaneous activity37, which begins at about P4 and affects the maturation of the IHC-afferent synapses and certainly of different other cell types in the cochlea37,38,39. We therefore investigated the timing of establishment of SG neurons diversity, with respect to this critical maturation period. Unbiased clustering of 478 SG neurons from P3 cochlea mice revealed again four distinct types of SG neurons, in proportion similar (P > 0.05) to those observed in adult mice (Fig. 6a, Supplementary Fig. 7a-b). As in adult SG, the expression of transcription factors such as Pou4f1 and Runx1 was specific to the Ia and Ib types. The type Ic and II neurons were characterized respectively by a specific, transient expression of Cxcl14 and Etv4 (PEA3) (Fig. 6b, c; Supplementary Fig. 7c), distinguishing the two types from adult stages. Interestingly, both Epha4 and Prph, described previously as early postnatal type I and type II neuron markers respectively13,40, were more widely expressed, with EphA4 found in all neuron types, and Prph, in both type II and Ic neurons (Fig. 6b, Supplementary Fig. 7c-f; Supplementary Data 3, 4). Moreover, the four neuron types could be already identified at P0 (Supplementary Fig. 7g), suggesting that the molecular pathways that specify the generation of the four SG neuron types precede and are independent of the postnatal maturation of the organ of Corti and of spontaneous or stimulus-driven activity.

Fig. 6 SG neuron types in new born mice and comparative analysis of their transcriptome with adult SG neurons. a t-SNE of SG neurons showing four different clusters at P3. b Violin plots showing the expression of marker genes in log-transformed scale. c In vivo validation of the identified neuron types by immunohistochemical and fluorescent in situ hybridization using identified marker genes in P3 cochlea. Type II SG neurons were identified by Peripherin (Peri), Plk5, Etv4 (Etv4GFP transgenic mouse), TH, and Gabrg2. Ia neurons were identified by Pou4f1, Runx1, and CR. Ib neurons were identified by Runx1 and Pou4f1 and Ic neurons, by Rxrg, Pcdh20, and CR expression. Lypd1 and Calb1 expression could not distinguish type Ia from Ib neurons at this stage. Note that co-localization on sections could never be observed for markers expressed in different populations of neurons in the scNRAseq data. d Gene set enrichment analysis of P3 type I and type II SG neurons visualized by network. e Correlation analysis of SG neuron types from adult and P3 stages, using average expression of all differentially expressed genes as input. f, g Visualization of SG neuron types from adult and P3 stages using tSNE, revealing the conserved subclass identities between the two samples. h Volcano plots of gene expression differences between adult and P3 SG neuron types for type Ia (top panel) and type II (bottom panel). Genes differentially expressed in adult or P3 are marked by red or blue dots respectively. Scale bar: 20 μm (c) Full size image

Through GSEA of P3 SG neuron types, we observed that “neurogenesis” and “morphogenesis/neuronal projection” were among GO terms with the highest representation in type II neurons (Fig. 6d). By contrast, the most enriched term in type I neurons concerned genes associated with metabolism, as was described earlier for the adult type I group (Fig. 6d). In line with this, a correlation analysis of adult and P3 SG neuron types revealed high similarity between the two stages (Fig. 6e–g). This was particularly visible for gene families related to neurotransmission (Fig. 6h, Supplementary Fig. 8), in which only a few number of genes showed different expression between P3 and adult stages. These results strengthen the idea that functional diversification of SG neuron types displayed prior to experience includes mature and distinct synaptic communication signatures that persist in adult neurons.

Cell-to-cell communication machinery in P3 SG neurons

We next compared transcriptional differences amongst neonatal SG neuron types, focusing on gene sets commonly involved in neurodevelopmental processes, such as “axon guidance”, “adhesion”, and “signaling molecules”. These most likely participate to the establishment of their innervation pattern and to the specificity of their synaptic connections with HCs (Fig. 7a), efferents and central targets. As expected, the different types of SG neurons exhibited various combinations of gene expression (Fig. 7b–d, Supplementary Fig. 9). For instance, several genes of the Eph-Ephrin families (Ephb1/3, Epha8, Efna1/5, Efnb1) were particularly enriched in type II neurons, likely participating in the intricate growth of their projections within the OHCs area (Fig. 7a, b). Interestingly, several members of the cadherin, proto-cadherin and contactin cell surface molecules, which are critical in the control of synaptic partner specificity, displayed very distinct expression profiles across the four populations of neurons41 (Fig. 7b). In the cadherin family for instance, Cdh7 and 8 were enriched in type Ib, Cdh9 in types Ia and Ic and Cdh4 and 13, in type II neurons. For those categories of molecules, their relative levels of expression in different neighboring cells can generate discrete, local synaptic connection patterns. Thus, their interactions between synaptic partners could participate in the correct matching of each afferent types with particular cellular domains of HCs and efferent endings during development42,43,44.

Fig. 7 Functional signature of neonatal SG neuron types. a Schematic illustration of the mature connection pattern of auditory afferents with HCs. A single type II afferent travels through the OHCs area and receives synaptic inputs from several OHCs. A single-IHC makes synaptic contact with several type I neurons. Ia and one Ic afferent contact the pillar side, and Ib afferent contact the modiolar side of IHC. b–d Differential expression of adhesion-related genes (RTP, receptor tyrosine phosphatase), of guidance molecules and of genes linked to key signaling pathways including Bmp signaling, Wnt signaling, and growth factors among SG neurons at P3 (see also Supplementary Fig. 9). e Schematic illustration of the Bmp signaling using gene expression data from P3 SG neuron types. Note in red the type II enriched expression of genes coding for inhibitory proteins of the Bmp signaling Full size image

We also found that whereas all SG neurons expressed the machinery necessary for the activation of the TGFB signaling, many inhibitory modulators of this pathway (Smad6, Smad7, Nog, Nbl1, Smad9, Smurf2) were particularly enriched in the type II neurons, arguing for a specific role of this signaling only in type I neurons (Fig. 7d, e). More generally, each of the major signaling pathways manifested differential expression amongst SG neuron types, highlighting their potential contribution to the characteristic connection patterns and physiological properties of each cell type.

Altogether, our results provide an extensive set of differentially expressed genes among neonatal SG neurons that will help future investigations of the molecular programs that control the diversification and connection patterns of SG neuron types.