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. 2018 Sep 12;9(1):3691.
doi: 10.1038/s41467-018-06033-3.

Neuronal Heterogeneity and Stereotyped Connectivity in the Auditory Afferent System

Affiliations
Free PMC article

Neuronal Heterogeneity and Stereotyped Connectivity in the Auditory Afferent System

Charles Petitpré et al. Nat Commun. .
Free PMC article

Abstract

Spiral ganglion (SG) neurons of the cochlea convey all auditory inputs to the brain, yet the cellular and molecular complexity necessary to decode the various acoustic features in the SG has remained unresolved. Using single-cell RNA sequencing, we identify four types of SG neurons, including three novel subclasses of type I neurons and the type II neurons, and provide a comprehensive genetic framework that define their potential synaptic communication patterns. The connectivity patterns of the three subclasses of type I neurons with inner hair cells and their electrophysiological profiles suggest that they represent the intensity-coding properties of auditory afferents. Moreover, neuron type specification is already established at birth, indicating a neuronal diversification process independent of neuronal activity. Thus, this work provides a transcriptional catalog of neuron types in the cochlea, which serves as a valuable resource for dissecting cell-type-specific functions of dedicated afferents in auditory perception and in hearing disorders.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
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 µm) 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)
Fig. 2
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. eg 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)
Fig. 3
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
Fig. 4
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)
Fig. 5
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)
Fig. 6
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)
Fig. 7
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. bd 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

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