Muscarinic signaling in the cochlea: presynaptic and postsynaptic effects on efferent feedback and afferent excitability

Abstract

Acetylcholine is the major neurotransmitter of the olivocochlear efferent system, which provides feedback to cochlear hair cells and sensory neurons. To study the role of cochlear muscarinic receptors, we studied receptor localization with immunohistochemistry and reverse transcription-PCR and measured olivocochlear function, cochlear responses, and histopathology in mice with targeted deletion of each of the five receptor subtypes. M2, M4, and M5 were detected in microdissected immature (postnatal days 10-13) inner hair cells and spiral ganglion cells but not outer hair cells. In the adult (6 weeks), the same transcripts were found in microdissected organ of Corti and spiral ganglion samples. M2 protein was found, by immunohistochemistry, in olivocochlear fibers in both outer and inner hair cell areas. M3 mRNA was amplified only from whole cochleas, and M1 message was never seen in wild-type ears. Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were unaffected by loss of Gq-coupled receptors (M1, M3, or M5), as were shock-evoked olivocochlear effects and vulnerability to acoustic injury. In contrast, loss of Gi-coupled receptors (M2 and/or M4) decreased neural responses without affecting DPOAEs (at low frequencies). This phenotype and the expression pattern are consistent with excitatory muscarinic signaling in cochlear sensory neurons. At high frequencies, both ABRs and DPOAEs were attenuated by loss of M2 and/or M4, and the vulnerability to acoustic injury was dramatically decreased. This aspect of the phenotype and the expression pattern are consistent with a presynaptic role for muscarinic autoreceptors in decreasing ACh release from olivocochlear terminals during high-level acoustic stimulation and suggest that muscarinic antagonists could enhance the resistance of the inner ear to noise-induced hearing loss.

Figure 1.

A–C, RT-PCR (A, B) and qRT-PCR (C) analysis of mAChR expression in wild-type mice and their compensatory upregulation/downregulation in knock-out mice (C). M₂, M₄, and M₅ receptors were consistently expressed in the following: A, inner hair cells (IHCs) and spiral ganglion neurons (SGNs) but not outer hair cells (OHCs) harvested from immature cochleas (P11–P13); B, both organ of Corti and spiral ganglion from microdissected adult cochleas (4–6 weeks); and C, whole adult cochleas (2 years). M₃ receptors were present only in whole-cochlear digests (C), and M₁ receptors were never amplified from wild types (A–C). All five mAChRs are expressed in adult brain (B, positive control). A, B, A glutamate receptor (GluR2), otoferlin, and oncomodulin (Oncomod.) primers were used as markers for spiral ganglion neurons, inner hair cells, and hair cells, respectively. All bands appear at the location expected for the size of the amplicon (see Table 1), as calibrated by the ladder lane in each gel: dashed lines are positioned at amplicon sizes of 100, 300, 500, and 700 bp. The 8-bit gel micrographs were adjusted digitally: after inversion, offset was set to 0 by subtracting the mean pixel value from an empty lane, and gain was optimized by setting to 256 the mean pixel value from the 500 bp ladder band. Primer bands (<100 bp) were cut from graphs. C, Mean expression levels for mAChR mRNA are normalized to 18S rRNA, after adjusting for primer efficiency, as described previously (Stankovic and Corfas, 2003). Error bars represent SEMs.

Figure 2.

A, M₂ receptor immunostaining (red) in adult (6–8 weeks old) wild-type cochleas is seen in olivocochlear fibers and their terminals in the inner spiral bundle under inner hair cells and under the three rows of outer hair cells (OHCs). Olivocochlear terminals also express VAT (green). B, Absence of M₂ immunostaining in M₂-null mice confirms the specificity of the antibody. Photomicrographs show the cochlear duct in the upper basal turn.

Figure 3.

The cochlear duct is histologically normal in adult mice (6–8 weeks old) lacking mAChRs, as seen in these light micrographs of osmium-stained plastic sections through the upper basal turn in an M₂/M₄ knock-out (A, B). Inner (IHCs) and outer (OHCs) hair cells are indicated by the open and filled arrows in B.

Figure 4.

A, B, Immunostaining for a cholinergic marker (VAT; green) and a neural marker (NF 200; red) reveals normal afferent and efferent innervation in an adult (6 weeks old) M₂/M₄ knock-out (A) compared with an age- and place-matched control (B). Each panel shows the x–y and y–z projections from a confocal z-stack through the organ of Corti in the 22–32 kHz region, from outer hair cells (OHCs) to the inner spiral bundle (ISB). In the z-dimension, each stack spans the region in which efferent terminals are found. Filled arrowheads (A) point to the efferent terminal clusters under each of the three rows of outer hair cells: the approximate outlines of the basal poles of the outer hair cells are indicated by the dashed lines. Open arrowheads (B) point to small VAT-positive puncta among the supporting cells under outer hair cells, indicating medial olivocochlear synapses with type II afferent fibers. Scale bar in A applies to both panels.

Figure 5.

A–D, Deletion of M₁, M₃, or M₅ mAChRs did not affect cochlear thresholds (A, B) or suprathreshold responses (C, D), as measured by DPOAEs (A, C) or ABRs (B, D). A, B, Mean DPOAE or ABR thresholds for groups of 6–8 week mutants and wild-type controls (numbers in each group are given in Table 2). C, D, Mean amplitude-versus-level functions for the same animals from A and B, as seen in DPOAEs or ABRs, evoked by tones at 16 kHz (for DPOAEs, f₂ = 16 kHz). For ABRs, only wave 1, the earliest negative–positive deflection, was measured. Error bars in all panels represent SEMs: the symbol key in A applies to all panels.

Figure 6.

A–D, Deletion of either M₂ or M₄ mAChRs or both elevated high-frequency DPOAE (A) and ABR (B) thresholds and reduced suprathreshold neural responses (D) even in midcochlear regions in which thresholds (A, B) and DPOAEs (C) were normal. A, B, Mean DPOAE or ABR thresholds for groups of 6–8 week mutants and wild-type controls (numbers in each group are given in Table 2). C, D, Mean amplitude-versus-level functions for the same animals from A and B, as seen in DPOAEs or ABRs evoked by tones at 16 kHz (for DPOAEs, f₂ = 16 kHz). For ABRs, only wave 1, the earliest negative–positive deflection, was measured. Error bars in all panels represent SEMs: the symbol key in A applies to all panels.

Figure 7.

Cochlear effects of activating the medial olivocochlear efferents are undiminished by deletion of mAChR receptors. To assay efferent effects, DPOAE amplitudes evoked by low-level tones are measured before, during, and after delivering a 70 s shock train to the olivocochlear bundle at the floor of the IVth ventricle. A, One run of the assay for each mAChR knock-out shows DPOAE amplitudes, normalized to the mean pre-shock value. Efferent effect is defined as the difference between the pre-shock mean and the mean DPOAE amplitude for the first three measures taken after shock-train onset (i.e., during shock window). B, Mean ± SEM efferent effect size for wild-type versus mAChR-null ears for each of the four lines tested.

Figure 8.

A–D, Vulnerability to temporary (A, C) and permanent (B, D) acoustic injury is reduced by conjoint loss of M₂ and M₄ mAChRs. A, C, Mean ± SEM threshold shift for wild types versus double knock-outs, as measured 6 h after exposure to an 8–16 kHz noise band (gray bars) at 95 dB for 15 min. B, D, Mean ± SEM threshold shift for wild types versus double knock-outs, as measured 1 week after exposure to the same noise band at 100 dB for 2 h. For both exposures, the threshold shifts are similar when measured by ABRs (A, B) or DPOAEs (C, D). Symbol key in D applies to all panels.

Figure 9.

Schematic showing the peripheral targets of the cholinergic terminals of efferent fibers from the lateral olivocochlear (LOC) and medial olivocochlear (MOC) divisions and of the possible locations of mAChRs (see key), according to the present results.