Mice lacking adrenergic signaling have normal cochlear responses and normal resistance to acoustic injury but enhanced susceptibility to middle-ear infection

Abstract

The vasculature and neurons of the inner ear receive adrenergic innervation from the cervical sympathetic chain, and adrenergic receptors may be expressed by cells of the organ of Corti and stria vascularis, despite a lack of direct sympathetic innervation. To assess the functional role of adrenergic signaling in the auditory periphery, we studied mice with targeted deletion of the gene for dopamine beta-hydroxylase (DBH), which catalyzes the conversion of dopamine to noradrenaline; thus, these mutant mice have no measurable adrenaline or noradrenaline. Dbh (-/-) mice were more susceptible to spontaneous middle-ear infection than their control littermates, consistent with a role for sympathetics in systemic and/or local immune response. At 6-8 weeks of age, cochlear thresholds and suprathreshold responses assessed by auditory brainstem responses and distortion product otoacoustic emissions, as well as light-microscopic morphology, were indistinguishable from controls, if ears with conductive hearing loss were eliminated. Dbh (-/-) mice were no more susceptible to acoustic injury than controls, despite prior reports that sympathectomy reduces noise damage. Dbh (-/-) mice showed enhancement of shock-evoked olivocochlear suppression of cochlear responses, which may arise from the loss of adrenergic inputs to olivocochlear neurons in the brainstem. However, adrenergic modulation of olivocochlear efferents does not mediate the protective effect of contralateral cochlear destruction on ipsilateral response to acoustic overexposure.

FIG. 1

Immunostaining for DBH and/or TH reveals the profuse adrenergic innervation of the inner ear in a normal mouse. A Cross-section through the upper basal turn shows DBH-positive fibers (red) in the spiral ganglion cell (SGC) region and in the osseous spiral lamina (OSL), but not in the organ of Corti (OC). Double-immunostaining shows that the small, unmyelinated DBH-positive fibers (e.g., arrow) do not co-localize with the high molecular weight neurofilaments (NF) seen in the large myelinated cochlear nerve fibers (green). B cross-section through the upper basal turn shows co-localization of DBH (red) and TH (green) in all thin varicose fibers in the OSL. TH and DBH immunostaining often alternate in adjacent regions of the same fiber (arrow). C surface view of the upper basal turn shows the profuse sympathetic innervation (green) in the OSL, with many fibers ending blindly near the habenula (open arrows) or spiraling at the tympanic lip (filled arrow). For clarity, nuclear staining with TOPRO3 (red) has been erased in the OSL region.

FIG. 2

Dbh^−/− mice are more susceptible to middle-ear infections and conductive hearing loss than their heterozygous counterparts. In both Dbh^+/− (A) and Dbh^−/− mice (B), the distribution of DPOAE thresholds (for f₂ = 16 kHz) was bimodal, and all ears with a cloudy tympanic membrane (TM; n = 4 and n = 26, respectively) fell in the high-threshold peak. For each genotype, the number of ears with low vs. high thresholds (re 60 dB SPL) is given. C Umbo-velocity measurements were made in a selected group of Dbh^+/+ and Dbh^+/− mice with various DPOAE thresholds but normal-appearing TMs: the reduced ossicular motion in the high-threshold cases suggests that the threshold shift is of conductive origin. Umbo velocity is shown here at 11 kHz, the frequency near 2f₁–f₂ when the f₂ frequency is 16 kHz, as for panels A and B. Photomicrographs of one low- and one high-threshold ear (arrows) appear in Figure 3.

FIG. 3

When mice show high DPOAE thresholds, despite a normal-appearing TM, histological analysis shows cellular debris and/or fibrous tissue in the round-window niche. A Normal round-window niche from an ear with excellent DPOAE thresholds (27 dB SPL at 16 kHz). B and C Pathological appearance of the round-window niche in two cases with elevated DPOAE thresholds (64 and 75 dB SPL, respectively, at 16 kHz). All three ears were from a selected group of Dbh^+/+ and Dbh^+/− mice. The tissue orientation in panel C is different: the round-window niche communicates with the rest of the middle ear in another section.

FIG. 4

If ears with demonstrable or putative middle-ear disease are ignored (see Fig. 1), lack of DBH produces only subtle changes in cochlear thresholds (A, B) suprathreshold responses (C,D), or neural latencies (E). A, B Mean thresholds (±SEM) for 6–8 weeks Dbh^−/− and Dbh^+/− ears by ABRs or DPOAEs. ABR thresholds are statistically indistinguishable between the genotypes; DPOAE threshold differences are significant at 8 and 11.3 kHz (p < 0.001 by t test). C, D Mean amplitude-vs.-level functions from the same ears in A and B, as seen via ABRs (Wave 1) or DPOAEs, evoked by tones (ABR) or with f₂ (DPOAEs) at 16 kHz. E Mean latencies (±SEM) for Wave 1 of the ABR evoked at 16 kHz. For all these panels, group sizes for DPOAEs (Dbh^−/−n = 84; Dbh^+/−n = 46) are larger than those for ABR (Dbh^−/−n = 19; Dbh^+/−n = 37), because DPOAEs were usually recorded from both ears, whereas ABRs were recorded from only one side.

FIG. 5

All structures of the cochlear duct are histologically normal in Dbh^−/−mice, as seen in this light micrograph of osmium-stained plastic sections through the upper basal turn.

FIG. 6

Efferent-mediated suppression of cochlear responses is enhanced in Dbh^−/− mice. To assay efferent effects, DPOAE amplitudes evoked by low-level tones were measured before, during and after delivering a 70-s shock train to the olivocochlear bundle at the floor of the IVth ventricle. A Amplitude of DPOAE is plotted as deviation from the mean value seen before the shock-train onset: i.e., the difference in dB between the measured DPOAE (in dB SPL) and the mean amplitude (in dB SPL) of the pre-shocks DPOAE. Data are means of all runs from Dbh^+/− (n = 5) and Dbh^−/−(n = 3) for f₂ at 22.6 kHz. B Mean suppressive (during shocks) and enhancing (post-shocks) effects of efferent stimulation as a function of stimulus frequency. Time periods for measurement of these effects are indicated by the brackets in A.

FIG. 7

Noise vulnerability is similar in Dbh^−/− vs. Dbh^+/− mice. A, B Mean permanent threshold shifts (±SEM) measured by ABRs (A) or DPOAEs (B) 1 week after exposure to an 8–16 kHz noise band (gray bars) at 100 dB SPL for 2 h. There were 12 Dbh^+/− and 6 Dbh^−/− ears in the DPOAE measurement groups and half that many for the ABRs.

FIG. 8

Protective effect of contralateral cochlear destruction (CCD) on ipsilateral noise-induced threshold shift is not extinguished by removal of adrenergic signaling. Mean DPOAE threshold shifts (±SEM) were recorded 5–10 min after exposure to an 11-kHz pure tone (gray bar) at 102 dB SPL for 1 min in groups of Dbh^+/− (An = 11 animals) and Dbh^−/− (Bn = 5 animals) (circles). In a second experiment (triangles), groups of Dbh^+/− (An = 5) and Dbh^−/− (Bn = 7) animals underwent a contralateral cochlear destruction 24 h prior to the trauma-tone exposure. Protective effects of this surgical manipulation were similar in both genotypes.