Hair Cell Mechanotransduction Regulates Spontaneous Activity and Spiral Ganglion Subtype Specification in the Auditory System

Abstract

Type I spiral ganglion neurons (SGNs) transmit sound information from cochlear hair cells to the CNS. Using transcriptome analysis of thousands of single neurons, we demonstrate that murine type I SGNs consist of subclasses that are defined by the expression of subsets of transcription factors, cell adhesion molecules, ion channels, and neurotransmitter receptors. Subtype specification is initiated prior to the onset of hearing during the time period when auditory circuits mature. Gene mutations linked to deafness that disrupt hair cell mechanotransduction or glutamatergic signaling perturb the firing behavior of SGNs prior to hearing onset and disrupt SGN subtype specification. We thus conclude that an intact hair cell mechanotransduction machinery is critical during the pre-hearing period to regulate the firing behavior of SGNs and their segregation into subtypes. Because deafness is frequently caused by defects in hair cells, our findings have significant ramifications for the etiology of hearing loss and its treatment.

Keywords: hair cell; mechanotransduction; neuronal diversity; neuronal subtypes; single-cell RNA sequencing; spiral ganglion neuron; spontaneous activity.

Fig. 1.. Identification of subtype of type I SGNs by scRNA-seq.

(A) Diagram on the left: cochlear cross-section; inner hair cells, IHCs; outer hair cells, OHCs. Right: top view onto the sensory epithelium showing IHCs, OHCs and SGNs. Efferent neurons are shown in pink but are not studied here. (B) tSNE plot from the sensory compartment of the mouse inner ear at 9 weeks of age. Putative Type I SGNs (Tubb3⁺) in red; putative type II SGNs (Prph⁺) in grey. (C) Violin plots showing select genes that are differentially expressed between final sets of type I and type II SGNs (see Fig. S5A-B and Methods for SGNs refinement); y-axis is a log scale. (D) tSNE plot of type I SGNs showing three subtypes (IA, IB, IC). (E) Heat maps showing standardized expression of the top 10 differentially expressed genes (p<0.01 from pairwise comparisons, highest average log-fold change). Expression is averaged across all cells from an individual mouse for each cluster. Rows are genes, columns are averages for each mouse, grouped by cluster. (F) Violin plots of select genes from (E) within subtypes. Y-axis in C and F, log-normalized transcript counts. See also Figure S1-S7, Table S1-S9, and the STAR Methods.

Fig. 2.. Validation of molecular subtypes of SGNs.

(A) P28 sections stained with antibodies to TuJ1 (red) and NGFR (green) to identify type I and type II SGNs. (B) P28 cochlear whole mounts stained for CALB2 to label IHCs and for NGFR (green). Note labeling of type II nerve fibers (arrows) within the OHC region. (C) P28 sections stained with the indicated antibodies to distinguish type IA, IB, and IC SGNs (arrowheads: cells only expressing TuJ1; arrows: cells co-expressing TuJ1 with CALB2, CALB1 or POU4F1). (D) Percentage of type I SGNs expressing CALB2, CALB1 and POU4F1 at P28 (serial sections from three mice; values are mean ± SEM). (E) P28 sections stained for CALB2, CALB1 and POU4F1. In bottom right panel, segregation of markers as determined by RNAscope. (F) Quantification of the percentage of type I SGNs co-expressing markers (serial sections from three mice; values are mean ± SEM). (G) RNAscope analysis of Lypd1 expression. Left: overview of the cochlea; right: spiral ganglion. Scale bars: 20μm.

Fig. 3.. Molecular characterization of type I SGN subtypes and innervation patterns.

(A) Dot plots showing differentially expressed genes (log fold-change >0.1 and p<0.01 from pairwise comparisons) in type I SGN subtypes grouped by functional category; color scale: average expression of all single cells in each cluster; dot size: percentage of single cells with detectable expression (>1 transcript). (B) Violin plots of select genes. (C) Violin plots of fraction of total transcripts in single cells from the 13 protein-coding genes in the mitochondrial genome and 1,092 cellular genes associated with mitochondrial function (Calvo et al., 2016). All pairwise comparisons are statistically significant (p<0.0001, nested model ANOVA). (D) Violin plots of neurofilament gene expression. Lines in C and D indicate the peak density of cells in the violin plots. The dotted line is an extension of the line from type IA neurons to facilitate comparison. Y-axis in B and D, log-normalized transcript counts. Y-axis in C, fraction of total transcripts in each single cell. The genes shown are differentially expressed between subtypes (log fold-change >0.1; p<0.01 from pairwise comparisons). (E) 3D reconstructions of an IHC and innervating nerve endings at P28. Upper panels: Cochlear whole mounts stained for TuJ1 (red; arrows) to identify type I SGNs projections and for CALB2 (green; arrowheads) to identify IHCs and type IA SGN projections. Lower panels: Whole mounts co-stained for CALB1 (green; arrows) and CALB2 (red; arrowheads). (F) 3D renderings (10 mice; 5–15 hair cells per sample) to quantify innervation patterns. We divided the base of IHCs into modiolar, middle and pillar side according to the TuJ1 innervation area and quantified the number of cells where CALB2⁺ nerve fibers contacted IHCs. Values are mean ± SEM. Scale bar: 8μm.

Fig. 4.. Temporal specification of SGN subtypes.

(A) Diagram showing sequential steps during the maturation of the peripheral auditory sense organ. (B) Histological section through the spiral ganglion were stained with the indicated antibodies (large arrows: single-positive cells; small arrows: double-positive cells). (C) Quantification of the % of cells expressing different molecular markers at the indicated time points (serial sections from three mice at each time point; values are mean ± SEM). Scale bars: 20μm.

Fig. 5.. Defects in hair cell mechanotransduction affects SGN subtype specification.

(A) Hair cell diagram. Inset: enlargement of the tip-link region indicating molecules of the mechanotransduction complex. (B) Sections through the spiral ganglion of control wild-type mice and Tmie-ko mice at P28 stained with the indicated antibodies. (C) Numbers of CALB2⁺, CALB1⁺ and POU4F1⁺ SGNs in wild-type, Tmie-ko, Lhfpl5-ko and Pcdh15-av3j mice at P28. (D) SGN numbers at P28. (E) Numbers of CALB2⁺, CALB1⁺ and POU4F1⁺ SGNs at P28. Total numbers of double positive cells were divided by numbers of cell expressing a single marker. (F) Histological sections of wild-type and Tmie-ko mice at P28 stained with the indicated antibodies (arrows: cells co-expressing TuJ1 and NGFR). (G) Numbers of NGFR⁺ and NGFR⁺/TuJ1⁺ SGNs at P28. (H) Numbers of CALB2⁺, CALB1⁺, POU4F1⁺ and NGFR⁺ SGNs in mice of the indicated genotype at P0 and P14. For all experiments, serial sections from three animals of each genotype were analyzed. Values are mean ± SEM; two tailed unpaired t-test; *** p<0.001; ** p<0.01; * p<0.05. Scale bars: 20μm.

Fig. 6.. Defects in glutamatergic signaling by IHCs affects SGN subtype specification.

(A) Diagram of an IHC with its innervating type I SGNs. Inset: enlargement of a ribbon synapse between IHCs and SGNs. Glutamate receptors (GluR) localizes to nerve terminals and VGLUT3 to synaptic vesicles. (B) Section through the spiral ganglion of control wild-type mice and Vlgut3-ko mice at P28 stained with the indicated antibodies. (C) Numbers of CALB2⁺, CALB1⁺ and POU4F1⁺ SGNs in wild-type and mutants at P28. (D) SGN numbers at P28. (E) Numbers of CALB2⁺, CALB1⁺ and POU4F1⁺ SGNs at P28. Total numbers of double positive cells were divided by numbers of cell expressing a single marker. (F) Histological sections of wild-type and Tmie-ko mice at P28 stained with the indicated antibodies (G) Numbers of Ngfr⁺ and Ngfr⁺/TuJ1⁺ SGNs at P28. (H) Numbers of CALB2⁺, CALB1⁺, POU4F1⁺ and Ngfr⁺ SGNs in mice of the indicated genotype at P0 and P14. For all experiments, serial sections from three animals of each genotype were analyzed. Values are mean ± SEM; two tailed unpaired t-test; *** p<0.001; ** p<0.01; * p<0.05.. Scale bars: 20μm.

Figure 7.. SGNs in Tmie-ko and Vglut3-ko mice exhibit altered burst firing before hearing onset.

(A) Diagram showing the juxtacellular recording configuration used to assess SGNs. ISC: inner supporting cell. (B, C) Spontaneous action potentials from SGNs in control (Tmie^+/+ or ^+/− littermates) and Tmie-ko mice. Green bars indicate discrete action potential bursts. (D, E) Raster plots indicating the average firing rate of SGNs (bin: 1 second) in control and Tmie-ko mice. (F) Quantification of average action potential (AP) frequency, frequency and duration of spontaneous bursts, and number of action potentials per burst from control and Tmie-ko mice. (G) Average log-binned interspike interval (ISI) histograms from control and Tmie-ko mice. (H) Quantification of average action potential frequency, frequency and duration of spontaneous bursts, and number of action potentials per burst from cochleae treated with d-tubocurarine (d-TC; 50 μM). (I, J) Spontaneous action potentials recorded from SGNs of control (Vglut3^+/+ or ^+/− littermates) and Vglut3-ko mice. Green bars indicate discrete action potential bursts. (K, L) Raster plots indicating average SGN firing rates (bin: 1 second) in control and Vglut3-ko mice. (M) Quantification of average action potential frequency, frequency and duration of spontaneous bursts, and number of action potentials per burst in cochleae from control and Vglut3-ko mice. All values are mean ± SEM. Statistical significance calculated with two-tailed paired t-test, Bonferroni correction applied; ** p<0.01, * p<0.05, ns: not significant. (M) Average log-binned interspike interval (ISI) histograms from control and Vglut3-ko mice.