Cochlear Efferent Innervation Is Sparse in Humans and Decreases with Age

Abstract

The mammalian cochlea is innervated by two cholinergic feedback systems called the medial olivocochlear (MOC) and lateral olivocochlear (LOC) pathways, which send control signals from the brainstem back to the outer hair cells and auditory-nerve fibers, respectively. Despite countless studies of the cochlear projections of these efferent fibers in animal models, comparable data for humans are almost completely lacking. Here, we immunostained the cochlear sensory epithelium from 23 normal-aging humans (14 males and 9 females), 0-86 years of age, with cholinergic markers to quantify the normal density of MOC and LOC projections, and the degree of age-related degeneration. In younger ears, the MOC density peaks in mid-cochlear regions and falls off both apically and basally, whereas the LOC innervation peaks near the apex. In older ears, MOC density decreases dramatically, whereas the LOC density does not. The loss of MOC feedback may contribute to the age-related decrease in word recognition in noise; however, even at its peak, the MOC density is lower than in other mammals, suggesting the MOC pathway is less important for human hearing.SIGNIFICANCE STATEMENT The cochlear epithelium and its sensory innervation are modulated by the olivocochlear (OC) efferent pathway. Although the medial OC (MOC) reflex has been extensively studied in humans, via contralateral sound suppression, the cochlear projections of these cholinergic neurons have not been described in humans. Here, we use immunostaining to quantify the MOC projections to outer hair cells and lateral OC (LOC) projections to the inner hair cell area in humans 0-89 years of age. We show age-related loss of MOC, but not LOC, innervation, which likely contributes to hearing impairments, and a relative paucity of MOC terminals at all ages, which may account for the relative weakness of the human MOC reflex and the difficulty in demonstrating a robust functional role in human experiments.

Keywords: aging; efferent; hearing.

Figure 1.

Schematic illustration of the organ of Corti showing the afferent and efferent innervation of the IHCs and OHCs, including both the lateral and medial divisions of the OC feedback system. Efferents in the IHC area spiral in two bundles, the inner spiral bundle (ISB) and the tunnel spiral bundle (TSB), while afferents underneath the OHCs spiral in the outer spiral bundles (OSBs).

Figure 2.

Patterns of hair cell loss in the 23 human temporal bones analyzed in the present study. A, Age-related loss of hair cells, showing mean survival in each case, averaged over the 11 half-octave samples within the audiometric frequency range (0.25–8.0 kHz inclusive). OHC values represent the average of all three rows. Best-fit straight lines are shown in red, as indicated in the key, along with correlation coefficients and p values. B, C, Frequency pattern of hair cell loss, showing mean survival (±SEMs) of IHCs (B) and OHCs (C) at each of the 14 half-octave cochlear frequency locations sampled. Case patients are arbitrarily divided into three age groups, as illustrated in A. The significance of the intergroup differences is indicated by asterisks: *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001; or n.s. (not significant).

Figure 3.

Most of the ChAT-positive fibers in the osseous spiral lamina are unmyelinated. A, B, These images from two confocal z-stacks are both from a 78-year-old male. In each cochlear frequency region (A, 0.35 kHz; B, 4.0 kHz), a maximum projection in the xy plane is shown, along with selected xz and zy slices, positioned as indicated by the dashed yellow lines. Black-filled arrowheads in both xy projections point to the spiraling bundles of efferent fibers. Arrowhead in A zy points to a spiraling myelinated efferent (ChAT-positive) axon, while the arrowhead in A xz points to the unmyelinated radially directed portion of an efferent axon. Scale bar and orientation arrows in B apply to A. The x arrows point along the spiral toward the base, y arrows point radially toward the organ of Corti, and z arrows point toward scala tympani. NF, neurofilament.

Figure 4.

A–D, Confocal images of the MOC innervation of OHCs in a middle-aged (A, B) vs an older (C, D) subject. From each subject, two views of the same confocal z-stack are shown: B and D are maximum projections in the acquisition (xy) plane, while A and C are maximum projections of the entire stacks in the zy plane (i.e., rotated 90° to show the view along the axis of the cochlear spiral) with the approximate boundaries of the three OHC rows indicated in white dashed lines. White arrowheads in A and C point to MOC terminals at the bases, or along the sides of OHCs, while black-filled arrowheads point to MOC terminals within the spiraling bundles of type II fibers (e.g., at the arrowheads in B and D). Immunostaining key and scale bar in C apply to all panels. Orientation arrows in A and B apply to C and D, respectively. The x arrows point along the spiral toward the apex, y arrows point radially away from the modiolus, and z arrows point toward scala tympani. Dashed yellow boxes enclose the regions included in the digitizations used to quantify MOC terminal density without including tunnel-crossing fascicles. yrs, Years; NF, neurofilament; Myo, myosin VIIa.

Figure 5.

MOC innervation density decreases significantly with age. A, Each point shows the mean area of the ChAT-positive boutons in the OHC area, averaged over 11 half-octave samples within the audiometric frequency range (0.25–8.0 kHz inclusive) in each case. The correlation coefficient for the linear regression is shown along with the p value. B, C, Frequency pattern of MOC innervation density, showing mean values (±SEMs), for case patients arbitrarily divided into three age groups, as shown by the gray boxes in A. In all panels, MOC area is expressed in kilopixels, as extracted from maximum projections in the xy plane, such as those in Figure 4, B and D. The data in C are the same as those in B, except they have been normalized by dividing by the number of surviving OHCs in the same z-stack. Significance of the intergroup differences are indicated by asterisks, or n.s., as described for Figure 2: precise p values are in the text.

Figure 6.

A–D, Confocal images of the LOC innervation of IHCs from the apical (A, B) vs basal (C, D) half of the cochlea from one subject. At each locus, two views of the same confocal z-stack are shown: B and D are maximum projections in the acquisition plane (xy), while A and C are maximum projections of the entire stacks in the zy plane, with the approximate boundaries of the IHCs indicated in white dashed lines. White-filled arrowhead in all panels point to ChAT-positive boutons in the ISB, while the black-filled arrowhead in B points to a ChAT-positive bouton in the TSB. Immunostaining key and scale bar in D apply to all panels, and the orientation arrows in C and D also apply to A and B, respectively: the x arrows point along the spiral toward the apex, y arrows point radially away from the modiolus, and z arrows point toward scala tympani. yrs, Years; NF, neurofilament.

Figure 7.

LOC innervation density decreases with age, but not after correction for the loss of IHCs. A, Each point shows the mean area of the ChAT-positive boutons in the IHC area, averaged over 11 half-octave samples within the audiometric frequency range (0.25–8.0 kHz inclusive) in each case. The correlation coefficient and p value for the linear regression is shown. B, Frequency pattern of LOC area measures, showing mean values (±SEMs), for cases arbitrarily divided into three age groups, as shown by the gray boxes in A. C, Same data as in B, except the areas have been divided by the number of surviving IHCs in the same z-stacks. In all panels, LOC area is expressed in kilopixels, as extracted from maximum projections in the xy plane, such as those in Figures 3B and 2D. The ChAT immunostaining in LOC area for the youngest subject (0.1 years) was too dim and indistinct to be reliably measured. The intergroup differences are not statistically significant (n.s.): precise p values are in the text.

Figure 8.

In humans, the MOC innervation is less dense than in other mammals. Each micrograph is the maximum projection from the ChAT channel of a z-stack taken from the cochlear region of maximum innervation density in each of four species: human (4.0 kHz region of a 39-year-old female), rhesus macaque (2.0 kHz region of a 9-year-old male), guinea pig (4.0 kHz region of a 1.5-month-old female), and mouse (22.6 kHz region of a 1.5-month-old male). In each image, the locations of the three OHC rows are indicated, and the outlines of the first-row OHCs (cuticular plates) are schematized (to scale, but regularized and offset vertically for clarity) as extracted from the same z-stack. In each image, tunnel-crossing efferent bundles are noted by arrowheads: in the mouse image, the tunnel-crossing bundles are truncated, because the original image field was more restricted than the others. The scale bar in the human panel applies to all images.

Figure 9.

Quantification of MOC terminal area relative to OHC surface area in each of four species. A, The human data are from the middle-aged group in Figure 5, expressed relative to OHC surface areas extracted from the myosin-immunostaining channel in the same image stacks. Mouse, guinea pig, and rhesus data are from four ears in each species, immunostained, imaged, and analyzed exactly as for the human data and similarly expressed relative to the OHC surface areas extracted from the same set of image stacks. B, To demonstrate the functional validity of the MOC silhouette area metric, we reanalyzed image stacks from a prior study in which MOC feedback strength was measured after partial sectioning of the MOC bundle (Liberman et al., 2014). Each point represents a different case, comparing shock-evoked MOC suppression at the frequency region of maximal effect (22.6 kHz) with the MOC silhouette area in the appropriate cochlear region. The correlation was highly significant (p < 0.001).

Figure 10.

LOC innervation density in humans is comparable to that in mouse. A–D, Confocal projections from the cochlear region with maximal LOC innervation: 0.35 kHz in human (A, B) vs 8.0 kHz in mouse (C, D). The human case patient was a 59-year-old female. For each species, two views of the same confocal z-stack are shown: B and D are maximum projections in the acquisition plane (yx), while A and C are maximum projections of the entire z-stacks in the yz plane. Orientation arrows in A and B also apply to C and D, respectively: the x arrows point along the spiral toward the apex, y arrows point radially away from the modiolus, and z arrows point toward scala tympani. White arrowheads in A and B point to ChAT-positive LOC terminals at the bases of IHCs; black-filled arrowheads in B and D point to the ChAT-positive tunnel-crossing fibers that give rise to MOC terminals. Red-filled arrowhead in A points to IHC cuticular plates, and the rough outline of IHCs is shown as a dashed line. The immunostaining key in A applies to all panels. Scale bars in A and C apply to B and D, respectively.